key: cord-023585-n3lr9z3u authors: phillpotts, robert title: interferon prophylaxis of the common cold date: 2003-01-06 journal: trends pharmacol sci doi: 10.1016/0165-6147(84)90509-1 sha: doc_id: 23585 cord_uid: n3lr9z3u interferon is a potential prophylactic agent for the common cold. but there are problems. the present levels of side-effects that have been observed don't justify its use in the long term. robert phillpotts describes the mechanism of interferon action and the future hopes and developments for its use in preventing colds. mrc common cold unit, coombe road, solisbuty, wil~chire sp2 8bw, uk, interferon is a potential prophylactic agent for the common cold. but there are problems. the present levels of side-effects that have been observed don't justify its use in the long term. l~'obert phillpotts describe~ the mechanism of interferon action and the future hopes and developments for its use in preventing colds. interferon appears to be the ideal antiviral drug for use in preventing colds; it is extremely potent, and active against a wide range of viruses. however, it is too toxic for systemic use in minor respiratory illnesses, and when taken in adequate doses intranasahy it causes a mild inflammation of the mucosa. current research is directed towards overcoming this problem. for most people the common cold is a mild, self-limiting illness. however, the high incidence of colds, and their ability to cause an exacerbation of chronic underlying respiratory disease leads to considerable morbidity. research has established that over 200 serologicaily distinct viruses can cause a cold, and there is little hope that infectica can be controlled by vaccination. therefore control by means of a broad-spectrmn antivizal agent is the most rational approach, and interferon is without doubt the most pote=t, and broadly active antiviral agent yet discovered. c~ course there are limitations upon the k~nd of medication which could be used to prevent a cold. for example it should be cheap and easy to manufacture, as well as being highly effective, and virtually free from side-effects. i what is interferen? i human interferons (hulfn) are pro-/ teins or glycoproteins of which there are: 3 principal types, a, 13, and "y. hulfn a and p: ~re both produced by cel~s after exposure to a virus: wn a by" leucocytes and lfn 13 by fibroblast,,,. hulfn ~ is also produced by fibroblasts exposed to double-stranded rna, while ifn ~/is produced by lymphocytes only in response to an antigenic or mitogenic stimulus. interferons do not directly inhibit virus growth, but exposure of :mittfected cells to interferon makes them resistant to attack by viruses, interferon is not internalized by cells but binds to cell surface receptors (there is one receptor for ifn a and [3, and another receptor for ifn y) triggering off a series of events within the cell. multiple effects have been observed in cells, including the inhibition of penetration and maturation of certain viruses. however, the principal effect of interferon treatrnent seems to be to inhibit viral protein synthesis t. a system of interferoninduced enzymes has been described, some of which are activated by doublestranded rna molecules such as those produced during the replication of rna 'druses. the effect of these enzymes is to inhibit the initiation of viral protein synthesis, and to increase the rate at which viral mrna is degraded within the cell (see fig. 1 ). future research will mldoubtedly disclose other mechanisms by which virus growth is prevented. "[here is already evidence to suggest that i[fn ~ has a different mechanism of ~k~tion from ifns a and 13, as combinations of ifn ~ with ifn ct or ifn 13 (but not ifn a with ifn 13) exhibit synergy. the potency of various interferon preparations may therefore be compared in terms of units of antiviral activity. there is species specificity in this process v ,'11, while not absolute, means in practice that human interferon must be used to treat human cells. human interferon may be produced from cells cultured in vitro. a partially purified human leucocyte (a) interferon is produced by the finnish red cross from huffy coat cells (derived from blood donations) which have been stimulated with sendal virus. however, this procedure is expensive, and the amount of interferon which can be produced is limited by the availability of buffy coat cells. more recently, dna copies of the mrnas coding of all 3 types of hulfn have been synthesized, and inserted into plasmid vectors downstream from a suitable prokaryotic promoter. such plasmis are introduced into bacteria by transfection, where they multiply, and high levels of expression of the interferon gene sequence can be cheap, costs will undoubtedly be further reduced. intranasal interferon prevents colds in an initial experiment at the common cold unit in wiltshire, uk, intranasal administration of partially purified human leucocyte interferon to volunteers was shown to prevent colds caused by rhinovirus type 4 (ref. 5). rhinoviruses cause approximately 50% of colds. however, leucocytes from 30-50 donations of blood were required to produce the 14 mu of interferon used to treat each volunteer. furthermore, the use of partially purified material in which only about 1% of the protein was interferon also raised the question of whether interferon itself was responsible for preventing rhinovirus infection. could the activity have been due to contaminating, biologically active molecules also derived from human leucocytes? this question was answered in a further experiment carried out in 1982, in which hulfn a, purified to virtual homogeneity on a monoclonal antibody affinity column was shown to prevent colds caused by another rhinovirus, type 9 (ref. 6). a total dose of 90 mu was given intranasally to each volunteer over a period of 4 days (12 doses, 3 doses daily). four doses were given before, and 8 after vixus challenge. there was a dramatic reduction in the frequency of colds in the interferon treated group (table 1) . this was accompanied by lesser reductions in the number of volunteers who shed virus, or who had an increase in serum neutralizing antibody to the challenge virus. in a further experiment conducted under an identical protocol, closely similar results were achieved using highly purified, hulfn a-2, pro-duced in escherichia coil 7 (manufactured by schering plough). this experiment clearly demonstrated that interferons produced in bacteria by recombinant dna techniques could be as active as those produced by human celk. how can interferon be used in prm'tke? in these experiments, interferon was given intranasally in large doses by a physidan. therefore ffinterferon is to find application as a prophylactic against the common cold. two questions have to be answered. firstly, could people treat themselves with interferon using a simple design of nasal spray? sc-condly, what is the minimum quantity of interferon necessary to protect against infection with cold viruses? answers to these questions were sought in a dose-ranging study, in which volunteers gave themselves various doses of hulfn a-2 using a finger-actuated nasal spray s . interferon was given for one day before, and 3 days after, challenge with virulent human rhinoviruses. not only was the dose of interferon varied, but also the time of virus challenge in relation to a dose, so that the period of maximum vulnerability to virus infection could be identified. the results of this study suggest that at least 3-4 mu of ifn a-2 self-administered intranasally 3 times daily are necessary to protect against experimental rhino~rus infection. subsequent experiments have shown that 3-4 mu of huifn a-a (a similar molecule to hulfn a-, produced by roche) can protect against experimental infect/on with a human respnratory coronavtrus (see fig. 2 )". coronaviruses are the ~econd most frequently encouatered cause of a l.oid. and are responsible for 15-20e/r of cases. however. one more requirement must be. met by a prophylactic against the common cold -almost complete freedom from unpleasant snde-effects, it is here that research has run into difficulties. further studies have shown that while regimens of ifn a similar to that proposed seem to he necessa~" for protection against natural colds, such doses are also toxi=, and produce a mild inflammation of the nasal mucosa. in a recent stuly from the university of virginia, prclorge.d intranasal administration of the hulf?,~ or-2 was associated with muxr~d irritation in 23% ot recipients"l histologically, marked epithelial acute t qfiammation with ulceration occurr,'d in 19%, and 58% had pronounced submucosal l.~anphoq,/tic and mononuclear cell ~filtrates. although these abnormalities re'solved within 8 weeks after stopping treatment, long-term administration of ifn a would not be acceptable. however. there has been little sign of irritation of volumeers wen interferon f~r 4 days. therefore interferon prophylaxis could be used when the time of exposure to virus (or fear of exposure to virus) can be predicted. examples of this situation would be contact with a cold sufferer, or before an examination or some other important event. there is ever' reason to believe that the problem of poor tolerance to intranasal interferon will be overcome. only a very. small number of interferons have been tested for antiviral activity and toxicity in the nose, and it is conceivable that a molecular subspecies of hulfn c~. huifn t~ or hulfn -¢, or a hybrid interferon molecule prepared from these, may have an improved therapeutic ratio. interferon could then be taken for prolonged periods as a prophylactic against the common cold by patients with underlying chronic respiratory disease, such as bronchitis or asthma. not surprisingly therefore, the discovery of inteffemns with this desirable property is one of the major goals in common cold research today. the question of whether interferon can be used to treat a cold also remains open. the relatively short course of the illness suggests that virus replication in the nose oomns rapidly, and may even be essentially complete by the time symptoms have begun. however, this pessimistic view has yet to be confirmed, and there is some evidence to suggest that even a relatively we~k antiviral agent, such as enviroxime (registered trade name, produced by eli lilly and company), administered locally after virus infection can affect the course of a cold tt. perhaps a suitable preparation of interferon, which could be over 1 000 times more potent than enviroxime in vitro, could prove clinically useful. as modulators of appetite. the intiuen~es of other neurotransmitters and the physicochemical properties of food will, therefore, receive less prominence. at the outset it should be stres~d that the complexities of appetite regulation make it extremely unlikely that any single agent will prove to be the holy grail of appetite suppressants. future pharmacological approaches will need to take cognizance of this fact and attempt to tailor the treatment to the individual rather than the individual to the treatment. this is particularly important in view of the fact that the majority of peptides appear to have multiple effects, making it likely that the indiscriminate use of these agents, in subjects in whom a deficit or an excess has not been demonstrated, will lead to an unacceptably high incidence of side effects. opuad reading m a large body of evidence has now accumulated supporting a role for opioid peptides in the modulation of feeding behavior t. in particular, the relatively specific opioid antagonist, naloxone, has been demonstrated to decrease fe~ding under a variety of conditions in many species including humans. in humans, however, the major effect o! opioid blockade with naloxone appears to be to reduce carbohydrate intake rather than to produce an absolute reduction in calories. this reduction is offset by an increase in fat intake. in addition, the first ion 8 term study in humans by atkinson 2 using the long-acting opiate antagonist, naltrexone, produced disap09s2) erin. ciples of gme nmnipulatwn key: cord-003855-so8xl199 authors: ebert, gregor; paradkar, prasad n.; londrigan, sarah l. title: virology downunder, a meeting commentary from the 2019 lorne infection and immunity conference, australia date: 2019-09-02 journal: virol j doi: 10.1186/s12985-019-1217-6 sha: doc_id: 3855 cord_uid: so8xl199 the aim of this article is to summarise the virology content presented at the 9th lorne infection and immunity conference, australia, in february 2019. the broad program included virology as a key theme, and the commentary herein highlights several key virology presentations at the meeting. the lorne infection and immunity conference is one of five scientific meetings held during each month of february in seaside town of lorne, on the great ocean road in victoria (australia). the specific aim of the meeting is to bring together basic, clinical and translational researchers -those who examine microbes and their impact on the innate or adaptive immune response, researchers who study the mechanisms that regulate immune responses, and those who apply this knowledge to preventing and treating infectious and inflammatory diseases. 2019 was the 9th lorne infection and immunity conference, convened by heidi drummer (burnet institute, melbourne, australia) and paul hertzog (hudson institute of medical research, melbourne, australia). the broad program included virology as a key theme, and the commentary herein highlights several key virology presentations at the meeting. the 'infection and inflammation' session of the meeting was opened with a well-received presentation by linfa wang (duke-nus medical school, singapore) entitled 'holy immune balance, batman', about the highly adapted immune response of bats to viral infections. bats have been characterized as an important reservoir of various zoonotic viruses including nipah, sars, mers, marburg and ebola viruses [1] , but remarkably are able to live asymptomatically with otherwise potentially lethal viruses [2] . wang and colleagues are interested in understanding the underlying immune mediated regulatory mechanisms that facilitate a highly effective balance between viral defense and tolerance in bats as viral hosts. during their evolutionary adaption to effective flight, bats not only developed elevated levels of basal alertness reflected in increased metabolic heart rate and body temperature, they also seem to have evolved a highly adapted immune defense against viral infections [3] . remarkably, bats have increased tolerance to viral infections by exhibiting higher basal levels of innate defence regulators but also a dampened innate immune response upon infection, substitutional to increased responsiveness to viral pathogens [4] . the bat innate immune response appears to be 'pre-activated' with higher basal levels of type i interferon expression, in contrast to humans, who are very quick responders to viral infections, but require a lot more dampening of their immune signals afterwards to get back to basal levels. current research also shows the absence of any aim2 mediated inflammasome activation [5] , dampened nlrp3 mediated inflammasome activation [6] and dampened sting activation [7] in bats upon infection, which are all mediators of a robust type i interferon response. overall, wang et al. demonstrated that bats' response to stress in form of viral infections is more targeted and thus potentially more effective by numerous adaptions and modifications of the innate immune system. in 'viruses and their hosts' session, vinod sundaramoorthy (csiro-australian animal health laboratories) discussed a novel defence mechanism in neurons against rabies virus (rabv). rabv is a neurotropic virus, which causes tens of thousands of deaths every year, despite available vaccines [8] . endemic dog rabies results in an ongoing risk to humans in many resource-limited countries, whereas rabies in wildlife is important in north america and europe [9] . requirement for an uninterrupted vaccine cold chain and the high cost of the immunoglobulin component of rabies prophylaxis therapies substantiate the unmet need for novel rabv-specific antivirals [10] . furthermore, the pathogenesis of rabv relating to viral replication in neurons is not fully understood [11] . although most of the infection with rabv manifests as the 'furious' form, where virus silently spreads through the neuronal axon without any damage, in 20 % of cases it can cause axonal damage in peripheral neurons leading to paralysis [12] . using a new in vitro microfluidics model for studying synaptically connected neurons, sudaramoorthy investigated the pathogenesis of different strains of rabies virus, showing distinct mechanisms at play in determining disease outcomes for each strain. future efforts to define a key molecular determinant in the viral replication pathway in neurons may provide a novel therapeutic target. in 2015, an association between zika virus (zikv) infection during pregnancy as a cause of microcephaly and other congenital abnormalities in the developing fetus and newborn, was made [13] . as such, there has been a plethora of recent zikv research focused on understanding the pathogenesis of disease, as well as immunity to the virus and the development of vaccines and effective therapeutics. novel findings pertaining to all of these areas were presented throughout the meeting. developments in understanding the structure of zikv particles was showcased by shee-mei lok (duke-nus, singapore). lok and colleagues have previously solved a thermally stable 3.7 å resolution cryo-electron microscopy structure of zikv [14] , and were able to now show high resolution structures of zikv at various stages of viral assembly. specifically, the organisation of the envelope protein of the virus and changes during assembly and maturation were presented. previously, lok's lab has also published structures of mature and immature dengue virus, providing insight into the viral maturation process [15] . the research from lok and colleagues will have future implications in designing novel therapeutics and vaccines for zikv. efforts to develop a zikv vaccine using virus like particles were presented by julio carrera, working with researchers at the monash university and the university of melbourne. in the 'pathogenesis and prevention of infection' session, rosa coldbeck-shackley working with michael beard at the university of adelaide, australia, and also colleagues at the hudson institute, presented findings on the importance of interferon-epsilon (ifn-ɛ) in the innate immune response to zikv infection. ifn-ɛ is a novel type i ifn, encoded within the type i ifn locus in mice and humans, whose function has recently been characterized [16] . like other type i ifns, it acts via ifn-α receptors 1 and 2, activating interferon stimulated genes (isgs). however, ifn-ɛ is preferentially expressed by epithelial cells of the female reproductive tract in both mice and humans, and in contrast to viral induced type i ifn expression, ifn-ɛ is hormonally regulated. other than mosquito-borne transmission, zikv is also sexually transmitted [17] . this makes the research presented by coldbeck-shackley significant, and concurs with previous studies where ifn-ɛ-deficient mice were more susceptible to infection with sexually transmitted pathogens [16] . thus, ifn-ε appears to be a potent antipathogen and immunoregulatory cytokine that may be important in combating sexually transmitted infections that represent a major global health and socioeconomic burden. also in the 'pathogenesis and prevention of infection' session, allison abendroth (university of sydney) presented 'disarming the killer: targeting of natural killer cells by varicella zoster virus'. varicella zoster virus (vzv), is known to infect numerous immune cell types such as t-cells and dendritic cells [18] . moreover, the virus is able to modulate and manipulate innate and adaptive immune responses to infection to its replicative benefit. vzv infection of dendritic cells dampens type i ifn responses [19] and leads to evasion of cd8 t cell recognition via downregulation of mhc class i expression [20] . in addition, vzv mediated delay in immune responses to infection facilitates establishment of initial primary infection and lifelong latency in neurons. most recently, abendroth and colleagues found that vzv also productively infects natural killer (nk) cells and that nk cells effectively transmit infection to other permissive cell types [21] . the group is now trying to understand, how nk cells, a cell type that normally demonstrates very effective direct and indirect antiviral capacity, is manipulated by vzv infection, leading to impaired cytotoxicity and cytokine responses upon infection and facilitating infection and spread of the virus. led by the observation that patients with impaired nk cell functionality are highly susceptible to severe and life threatening vzv infection, abendroth et al. found limited nk cell activation and efficacy upon vzv infection of target cells and characterised differential modulation of ligands normally recognised by activated nk cells [22] . the exact underlying mechanisms, how vzv infection prevents the release of proinflammatory cytokines during infection of nk cells to impair their general function and whether correlations can be drawn to vzv infection of monocytes and macrophages [23] , is currently under their investigation, abendroth stated. a key highlight at the conclusion of the meeting was a presentation from the victorian infection and immunity network young investigator prize winner, simone park (the university of melbourne). park, working alongside thomas gebhardt and laura mackay, has recently published seminal research investigating skin tissue-resident memory t cells (trm cells). specifically, park has demonstrated how these cells contribute to antiviral immune memory in peripheral tissues, using a herpes simplex model of infection [24] . using an epicutaneous melanoma model, park and colleagues also demonstrate that trm cells play protective role in tumor surveillance [25] , which has important implications for advancing anticancer immunotherapies. the organisers are looking forward to celebrating the 10th lorne infection and immunity meeting in 2020 and invite all researchers with an interest in infectious and inflammatory diseases and associated immune responses to participate. for more information: http://www.lor neinfectionimmunity.org/. viruses in bats and potential spillover to animals and humans bats and viruses: friend or foe? comparative analysis of bat genomes provides insight into the evolution of flight and immunity the egyptian rousette genome reveals unexpected features of bat antiviral immunity unique loss of the pyhin gene family in bats amongst mammals: implications for inflammasome sensing dampened nlrp3-mediated inflammation in bats and implications for a special viral reservoir host dampened sting-dependent interferon activation in bats rabies molecular virology, diagnosis, prevention and treatment status of antiviral therapeutics against rabies virus and related emerging lyssaviruses immunological aspects of rabies: a literature review human rabies: neuropathogenesis, diagnosis, and management increase in reported prevalence of microcephaly in infants born to women living in areas with confirmed zika virus transmission during the first trimester of pregnancy -brazil structure of the thermally stable zika virus immature and mature dengue serotype 1 virus structures provide insight into the maturation process interferon-epsilon protects the female reproductive tract from viral and bacterial infection leparc-goffart i. evidence of sexual transmission of zika virus varicella-zoster virus infection of human dendritic cells and transmission to t cells: implications for virus dissemination in the host impact of varicella-zoster virus on dendritic cell subsets in human skin during natural infection varicella-zoster virus productively infects mature dendritic cells and alters their immune function varicella zoster virus productively infects human natural killer cells and manipulates phenotype varicella -zoster virus and herpes simplex virus 1 differentially modulate nkg2d ligand expression during productive infection infection and functional modulation of human monocytes and macrophages by varicella-zoster virus local proliferation maintains a stable pool of tissue-resident memory t cells after antiviral recall responses tissue-resident memory cd8(+) t cells promote melanoma-immune equilibrium in skin publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations authors' contributions ge, pp and sl all contributed equally in the writing of this manuscript. all authors read and approved the final manuscript. the authors declare that they have no competing interests. key: cord-004685-qote5nx2 authors: vassão, r. c.; sant' anna, o. a.; pereira, c. a. title: a genetic analysis of macrophage activation and specific antibodies in relation to the resistance of heterogeneous mouse populations to mhv3 infection date: 1994 journal: arch virol doi: 10.1007/bf01310802 sha: doc_id: 4685 cord_uid: qote5nx2 the genetically selected high antibody responder mice (h(iii)) are susceptible and the low antibody responder mice (l(iii)) are resistant to the experimental infection with mouse hepatitis virus 3 (mhv3). the mortality rates of the f(1) hybrids and of the f(2) segregants showed the codominance of the susceptible and resistant characters. the direct individual intrapopulation correlation between the induction of antiviral state in macrophages activated by ifn gamma and the resistance to the virus infection, showed that an antiviral state could be induced in resistant mouse macrophages, whereas in susceptible mouse macrophages no restriction of virus replication could be observed. a direct interand intrapopulation correlation of pre-existing antibody titres against mhv3 with the mortality and a direct interpopulation correlation of those titres with the mean survival time of susceptible animals was shown. the data indicate, among the mechanisms of resistance against the virus infection, a role of ifn gamma macrophage-activation and of antibodies against mhv3 which may delay the mean survival time in susceptible animals. the mouse hepatitis virus (mhv) strains of coronavirus are responsible for well-known epizootics of enteritis that occur endemically in most mouse colonies. most of the animals in these colonies were found to have antibodies against different types of mhv, such as mhv3, which have been isolated from a variety of mouse strains under diverse conditions [3, 6, 12, 19, 22] . the mhv3 was isolated by dick et al. [3] and has been used as a model of viral infection in which resistance varies according to the genetic background of the mouse strain [-1, 5, 8, 10, 11, 24] . the resistance pattern of mouse strains has been shown to be not directly linked to the presence of antibodies in sera of animals from contaminated colonies [12] . the virus replication in target cells, the antiviral state induced by interferon (ifn) and the expression of a monokine with procoagulant activity (pca) have been implicated in the resistance/susceptibility of the genetic homogeneous or heterogeneous mouse populations to the mhv3 infection [t, 2, 4, 7, 17, 18, 23, 243 . we have shown that resistance to mhv3 infection can be a consequence of a t-cell-dependent mechanism, in which the production of ifn gamma and the sensitivity of macrophages to ifn gamma play an essential role [10, 12, 13] . the genetically defined high (h) and low (l) responder selected mice and their segregants and hybrids have been proved to be a useful tool for studying the intervention of multiple alleles in the polygenic control of infectious diseases such as salmonella typhimurium and rabies virus infection [16, 20] . moreover, the analysis performed in the f2 heterogeneous population allows the elucidation of the major specific and nonspecific traits implicated in resistance/susceptibility to a given pathogen, as well as that of the environmental and genetic factors that play in these characters. mice selected for high (h) and low (l) responsiveness, were obtained by bidirectional selective breeding, and the effect of the polygenic regulation of responsiveness to selection antigens is essentially multi-specific, i.e., selected genes regulate the antibody response to many complex immunogens unrelated to those used during the selective breeding, the high or low states resulting from distinct mechanisms, including the regulatory role of macrophages [21] . the hm and lm mice, although showing no mhv in their tissues, were chronically infected by a mhv strain, and had in their sera distinct levels of antibodies against mhv3. h m mice were shown to be fully susceptible and lm mice fully resistant to the experimental infection with mhv3. one of the mechanisms suggested to be involved in the resistance against this virus infection was the ability of macrophages to develop an antiviral state following ifn gamma activation [2, 233. the present study was undertaken in an attempt to investigate whether the sensitivity ofmacrophages to the induction of an antiviral state and the antibody responsiveness characters, correlated individually with the resistance to mhv3 and were influenced at least partially by the same genetic control. therefore the investigation was directed towards a co-inherited expression of the two traits, aiming to elucidate the complexity of the biological factors that intervene in resistance to infection. high (hm) and low (lm) antibody responder mice from selection ill [21] , from the laboratorio de imunogenetica, instituto butantan, were used in the experiments. mice of both sexes were analysed at 2 3 months of age. interline reciprocal crosses of (h m x ln~)f 1 hybrids, f2 segregants and backcrosses (bchh~ and bclh0, were analysed as well. virus mhv3 originally obtained from dr. j. l. virelizier, institut pasteur, paris, france, was cloned by limiting dilution. one plaque was selected and amplified on l929 cells to serve as the inoculum for future stocks 1-14] to limit spontaneous mutations. the stocks were always titrated by plaque assay on l929 cells as previously described [18] . aliquots containing 2 x l0 s plaque forming units per milliliter (pfu/ml) were stored at -8 0 °c and used in all experiments. for the study of the resistance to virus infection, the animals were subcutaneously inoculated with 103 pfu of mhv3, and mortality was recorded daily for 30 days. the peritoneal exudates were collected by peritoneal lavage with 5ml of rpmi, and centrifugated at 200 g for 10 minutes. the peritoneal exudate cells were re-suspended at a concentration of 1 x 10 6 cells/ml of rpmi containing 10% of fetal calf serum (fcs) and cultured on 96-well plates (100gl/well). the cells were incubated for 2h at 37°c in 5% co 2 and washed three times with medium after vigorous shaking to remove nonadherent cells. ninety percent of the cells were macrophages as determined by their ability to take up zymosan particles. peritoneal macrophages were treated with 100 u/ml of murine recombinant ifn gamma (holland biotechnology, leiden, holland). twenty-four hours later, activated or nonactivated cultures were infected with mhv3 at a multiplicity of infection (m.o.i.) of 0.01, in order to study the inhibition of mhv3 replication. the supernatants of cell cultures were collected at 24 h after infection and tested for the virus titre by plaque assay [18] . for the study of the individual correlation between in vivo resistance and in vitro inhibition of virus replication by ifn gamma, peritoneal macrophages from hm, lm and f 2 segregants were collected without sacrificing the animals, which were infected with mhv3 a week later. the macrophages were treated with ifn gamma, infected with mhv3 and the supernatants titrated, as described above. mice from the colony were bled from the retro-orbital venous plexus and the individual anti-mhv3 antibody titres in their sera, reported as log 2, were expressed by the rec]procal of the highest serum dilution producing a 100% inhibition of cytopathic effect induced by mhv3 on l929 cells [18] . the results in table 1 show the resistance/susceptibility of mice to m h v 3 infection, and indicate that the macrophages from lm or resistant f1 or f 2 mice were sensitive to the induction of an a n t i -m h v 3 state by i f n gamma. however, under the same conditions, no a n t i -m h v 3 effect could be induced in macrophages from the susceptible hin parental line nor from the susceptible r.c. vass~o et al. cultured macrophages were individually collected, treated for 18 h with ifn gamma (100 u/mt) before infection with 0.1 m.o.i, of mhv3. virus titres were determined in the supernatants collected 24 h after the infection. one week later, the same animals were infected subcutaneously with 103 p f u of mhv3, observed for 30days and the percent (~o) of mortality recorded. the mhv3 titres are the average +_ standard deviation fi or f2 mice. the in vitro treatment with i f n gamma, which induced an anti-mhv3 effect only in resistant mouse macrophages, correlated with the in vivo resistance observed after mhv3 infection. figure 1 shows the linear regression analysis of individual values of the induction of antiviral state in macrophages of resistant and susceptible f 2 phenotypes, indicating a significant direct correlation between the resistance and the induction of an antiviral state in the macrophages. the figure 3 shows the linear regression analysis of the correlation between the mean survival time (mst) and the antibody titres against mhv3 in groups which exhibited mortality. it indicates a significant direct correlation (r = 0.96, p < 0.05) between the mean survival time of susceptible mice infected with mhv3 and the anti-mhv3 antibody titres. the genetic studies based on the segregation analysis of resistant or susceptible inbred mouse lines to mhv3 infection showed that these characters are under the control of two major loci [4, 9] . more recently, a comparative study performed in genetically homogeneous and heterogeneous mouse populations confirmed these results, and demonstrated the codominance of the susceptibility and resistance characters. the same genes were shown to be present in the isogenic balb/c (susceptible) and a/j (resistant) lines, as well as in genetically selected hh~ (susceptible) and l m (resistant) antibody responder mice [2] . the use of hm and lm mice made it possible to analyse the relationship between distinct traits in segregation studies, and demonstrated a direct interpopulation correlation between antibody production to unrelated antigen and mortality to mhv3 infection [2] . the present studies go deep into the intra-and interpopulation analysis of the correlation between resistance to infection and antiviral macrophage action induced by ifn gamma or specific anti-mhv3 antibody production. by studying, individually, the mortality of the parental mouse lines, their f~ hybrids, f 2 segregants and backcrosses, the anti-mhv3 activity of their macrophages after ifn gamma activation, as well as the titre of antibodies against mhv3, we were able to show: i) a direct individual correlation between the resistance and the ability of macrophages to partially inhibit mhv3 replication after ifn gamma activation (table 1 and fig. 1) , ii) an inter-and intrapopulation inverse correlation between the mortality and the antibody titres against mhv3 (fig. 2) , iii) an interpopulation direct correlation between the mean survival time of hn~, f~, f 2 and bchm susceptible animals and the antibody titres against mhv3 (fig. 3) . the genetic analysis performed in this study, demonstrates that the resistance to mhv3 experimental infection and the ability of ifn gammaactivated macrophages from resistant mice to display an antiviral activity, are submitted to at least partially common genetic control. this brings further support to our previous suggestion that one of the main mechanisms involved in the resistance of mice to the mhv3 infection is based on the ability of their macrophages to restrict the virus replication upon ifn gamma activation [10 13, 153 . although the participation of antibodies in the resistance to mhv3 infection remains unclear, the data here shown indicate their possible role in resistance mechanisms and in prolongating the mean survival time of susceptible animals. adult mouse hepatocytes in primary monotayer culture express genetic resistance to mouse hepatitis virus type 3 a comparative study of resistance to mhv3 infection in genetically homogeneous and heterogeneous mouse populations a virus related to that causing hepatitis in mice (mhv) susceptibility/resistance in mouse hepatitis virus strain 3 and macrophage procoagulant activity are linked and controlled by two non-h-2 linked genes genetically determined resistance to mouse hepatitis virus type 3 is expressed in hematopoietic donor cells in radiation chimeras new strain of mouse hepatitis virus as the cause of lethal enteritis in infant mice immunopathology of mouse hepatitis virus type 3 infection. i. role of humoral and cell-mediated immunity in resistance mechanisms induction of monocyte procoagulant activity by murine hepatitis virus type 3 (mhv3) parallels disease susceptibility in mice genetic study of mouse sensitivity to mhv3 infection. influence of the h-2 complex a major role of macrophage activation by interferon gamma during mouse hepatitis virus type 3 infection. i. genetically dependent resistance a major role of macrophage activation by interferon gamma during mouse hepatitis virus type 3 infection. ii. age dependent resistance acquired immunity of a/j mice to mouse hepatitis virus 3 infection: dependence on interferon gamma synthesis and macrophage sensitivity to interferon gamma in vivo depletion of interferon gamma leads to susceptibility of a/j mice to mouse hepatitis virus 3 infection temperature sensitive mutants of mouse hepatitis virus type 3 (mhv3): isolation, biochemical and genetic characterization virus specificity of the antiviral state induced by ifn gamma correlates with resistance to mhv3 infection gennari m (t979) rabies virus resistance of heterogeneous mouse populations to mhv3 infection 425 immunity in genetically selected high and low responder lines of mice interaction between mouse hepatitis viruses and primary cultures of kupffer and endothelial liver cells from resistant and susceptible inbred mouse strains induction of natural killer cells and interferon during mouse hepatitis virus infection of resistant and susceptible inbred mouse strains mouse hepatitis virus infection as a highly contagious, prevalent, enteric infection in mice salmonella typhimurium infection in high and low antibody responder mice: inverse correlation between antibody responsiveness and resistance to infection selective breeding of mice for antibody responsiveness to flagellar and somatic antigens of salmonellae isolation of a routine hepatitis virus from swiss mice treated with antilymphocyte serum resistance of genetically selected mice to mhv3 infection is not dependent on the h20 2 release by macrophages role of interferon in the pathogenesis of viral diseases of mice as demonstrated by the use of anti-interferon serum. v. protective role in mouse hepatitis virus type 3 infection of susceptible and resistant strains of mice we thank d. mouton for critically reading the manuscript, f. souberbielle for editorial assistance and m.l. silva and a.c. barbosa for technical assistance. this work was supported in part by grants from the fapesp and cnpq. c.a. pereira and o.a. sant'anna are recipients of cnpq research fellowships, key: cord-001901-2s6hpakk authors: kwok, hoi-hin; poon, po-ying; fok, siu-ping; ying-kit yue, patrick; mak, nai-ki; chan, michael chi-wai; peiris, joseph sriyal malik; wong, ricky ngok-shun title: anti-inflammatory effects of indirubin derivatives on influenza a virus-infected human pulmonary microvascular endothelial cells date: 2016-01-06 journal: sci rep doi: 10.1038/srep18941 sha: doc_id: 1901 cord_uid: 2s6hpakk influenza a virus (iav) poses global threats to human health. acute respiratory distress syndrome and multi-organ dysfunction are major complications in patients with severe influenza infection. this may be explained by the recent studies which highlighted the role of the pulmonary endothelium as the center of innate immune cells recruitment and excessive pro-inflammatory cytokines production. in this report, we examined the potential immunomodulatory effects of two indirubin derivatives, indirubin-3′-(2,3-dihydroxypropyl)-oximether (e804) and indirubin-3′-oxime (e231), on iav (h9n2) infected-human pulmonary microvascular endothelial cells (hpmecs). infection of h9n2 on hpmecs induced a high level of chemokines and cytokines production including ip-10, rantes, il-6, ifn-β and ifn-γ1. post-treatment of e804 or e231 could significantly suppress the production of these cytokines. h9n2 infection rapidly triggered the activation of innate immunity through phosphorylation of signaling molecules including mitogen-activated protein kinases (mapks) and signal transducer and activator of transcription (stat) proteins. using specific inhibitors or small-interfering rna, we confirmed that indirubin derivatives can suppress h9n2-induced cytokines production through mapks and stat3 signaling pathways. these results underscore the immunomodulatory effects of indirubin derivatives on pulmonary endothelium and its therapeutic potential on iav-infection. scientific reports | 6:18941 | doi: 10.1038/srep18941 manufacturer's instruction. complementary dna was synthesized from dnase-treated total rna using superscript ii first-strand synthesis system (invitrogen). the relative expression of target gene was quantified by real-time rt-pcr using kapa sybr fast abi prism qpcr kit (kapa biosystems, woburn, ma, usa) and detected by a steponeplus real-time pcr system (applied biosystems, foster city, ca, usa). the relative expression of target gene was normalized by the level of glyceraldehyde-3-phosphate dehydrogenase (gapdh), and then calculated by the comparative ct method. plaque assay. the virus titers were determined by standard plaque assay on madin-darby canine kidney (mdck) cells. in brief, mdck cells were grown in mem and seeded onto 6-well plates. diluted cell culture medium from influenza virus-infected hpmecs were added to the confluent mdck cells monolayers for 1h. then, the inoculum was removed, and a mixture of agarose (2%, w/v) containing l-(tosylamido-2-phenyl) ethyl chloromethyl ketone (tpck) (1 μ g/ml) was added onto the mdck cells monolayers. after 72 h of incubation, the plate was fixed by formaldehyde (4%, v/v) overnight and then the agarose was discarded. the plaques were counted after staining with crystal violet (0.2%, w/v). were prepared by ne-per nuclear and cytoplasmic extraction reagents (thermo scientific, rockford, il, usa) according to the manufacturer's protocol. for extraction of whole-cell lysate, cells were lysed by cytobuster tm protein extraction reagent (novagen, madison, wi, usa) containing protease (0.5%, v/v) and phosphatase inhibitor cocktails (0.5%, v/v) (calbiochem, san diego, ca, usa). the cell lysate was collected after centrifugation. protein concentration of the sample was determined by the detergent-compatible protein assay (bio-rad, hercules, ca, usa). equal amounts of protein were loaded and separated by 10% sds-page followed by electroblotting onto nitrocellulose membrane. the membrane was soaked in blocking buffer (1% non-fat milk in tbs-t, w/v) and then incubated with specific primary antibodies overnight at 4 °c and secondary antibody for 1 h at room temperature. immunoreactive bands were visualized using supersignal west pico kit (thermo scientific). in vitro mitogen-activated protein kinases assay. to detect the activity of individual mapks after treatment with iav and indirubin derivatives, the non-radioactive in vitro protein kinase assay kit from cell signaling technology was used. in brief, the sepharose bead-immobilized antibody was used to immunoprecipitate active mapks from an equal amount of total cell lysate (200 μ g) overnight. the immunoprecipitate was washed twice with cell lysis buffer and kinase reaction buffer. the immunoprecipitate were then incubated with indirubin derivatives e804 or e231 (1 μ m) for 3 min before addition of atp. subsequently, kinase reactions using corresponding protein substrate were performed at 37 °c for 30 min. the kinase reaction was stopped with sds loading buffer. phosphorylation of protein substrate was detected by immunoblotting with specific antibody. immunofluorescence microscopy. hpmecs at a density 1 × 10 4 were seeded onto a glass coverslip in a 24-well plate. after treatment for the indicated time, cells were fixed with 4% paraformaldehyde for 15 min at room temperature. cells were incubated with primary antibody (1: 200 dilution rabbit anti-phospho-stat3 (tyr 705 ) antibody) overnight at 4 °c. the coverslip was washed and then incubated with fitc-conjugated goat anti-rabbit secondary antibody (1: 250 dilution) (invitrogen) for 2 h at room temperature. nuclei were visualized by staining with dapi (0.5 μ g/ml). the coverslip was washed and mounted on a slide using dako fluorescence mounting medium (carpinteria, ca, usa). fluorescence image was captured by the olympus fluoview fv1000 confocal laser-scanning microscope (tokyo, japan). small interfering rna (sirna) transfection. transfection of sirna was performed using lipofectamine rnaimax (invitrogen). non-targeting-sirna (50 nm) was used in parallel with stat3-specific sirna (50 nm) (ambion, austin, tx, usa). cells plated at 80% confluence were transfected in opti-mem medium (gibco brl, grand island, ny, usa) for 24 h. after transfection, cells were rinsed with opti-mem prior to further treatment. all results were expressed as mean ± standard derivation (s.d.) of at least 3 independent experiments. statistical significance between groups was determined by one-way anova with tukey's post hoc test. p < 0.05 was considered to be statistically significant. influenza a virus h9n2 is a potent inducer of cytokines production in pulmonary endothelial cells. recent studies suggested that lung endothelium is the central regulator of cytokine amplification during influenza a virus infection, while dysregulation of cytokines production may result in systemic inflammation 22 . in this study, we found that the infection of influenza a virus subtype a/quali/hong kong/g1/97 (h9n2) on hpmecs induced excessive production of various pro-inflammatory cytokines and chemokines, including ip-10 ( to examine the immunomodulatory effects of indirubin derivatives, hpmecs were infected with h9n2 for 1 h followed by incubation with indirubin derivatives e804 or e231 for another 24 h. we have tested the cytotoxicities of indirubin derivatives in hpmecs prior to the elisa. as shown in fig. 2 was observed at or below 10 μ m in hpmces. next, indirubin derivatives were found to suppress h9n2-induced ip-10 ( fig. 3a) , rantes (fig. 3b ) and il-6 ( fig. 3c ) expression in a concentration-related manner. e804 significantly inhibited cytokines expression at 1 μ m, a similar inhibitory effect was observed when a higher concentration of e231 (10 μ m) was used. both indirubin derivatives slightly induced and inhibited the basal level of rantes and il-6 respectively. it may be due to the regulatory effects of indirubins on innate immunity 45 . for 46 . similar to the results of direct virus infection, viral rna stimulated cytokines expressions, and the inhibitory effects of mapks inhibitors were very similar to the experiments in direct infection. this confirmed that the suppression effects of mapks inhibitors on cytokines expression were not due to their potential effects on viral load. as a result, the anti-inflammatory effects of indirubin derivatives are mainly due to its inhibitory effects on different kinases. in contrast, stat3-specific sirna had no effects on ip-10, rantes and il-6 production induced by h9n2 infection in hpmecs (fig. 5d-f) . time-course experiments showed that h9n2 rapidly induced p38 and jnk phosphorylation in 15 min after addition of the virus, then a second wave of p38 and jnk phosphorylation were induced at 24 h.p.i. and 6 h.p.i., respectively (fig. 6a,b) . no activation of erk was found after h9n2 infection. similar to the early phosphorylation of stress-related mapks, stat3 was phosphorylated early in 30 min after addition of the virus. however, the second wave of stat3 phosphorylation was found at 2 h.p.i. taken together, these results suggested that h9n2 infection on hpmecs could activate p38, jnk and stat3 signaling pathways rapidly, and the expression of cytokines including ip-10, rantes and il-6 were mainly due to the activation of p38 and jnk. indirubin derivatives suppress h9n2-induced cytokines expression through direct inhibition of p38 and jnk activity. to study the underlying mechanism of the anti-inflammatory effects of indirubin derivatives, hpmecs were treated with indirubin derivatives e804 or e231 after h9n2 infection. as shown in fig. 7 , e804 can significantly reduce h9n2-induced phosphorylation of p38 (fig. 7a) and jnk (fig. 7b ) at 24 and 6 h.p.i., respectively, and e804 demonstrated a more potent effect than e231. it has been suggested that indirubin and its derivatives are potent inhibitors of various kinases, including mapks. in vitro kinase assay on p38 and jnk showed that e804 but not e231 inhibited p38 and jnk kinases activity. this action was reflected by the reduced phosphorylation of their direct downstream substrates atf2 (fig. 7c ) and c-jun (fig. 7d) respectively. indirubin derivatives prevent h9n2-induced ifn-β expression through inhibition of stat3 phosphorylation and nuclear translocation. though we found no relationship between stat3 and h9n2-induced ip-10, rantes and il-6 expressions (fig. 5d-f) , stat signaling pathway is indispensable for the induction of interferons. we showed that knockdown of stat3 strongly inhibited h9n2-induced ifn-β (fig. 8b ) mrna expression. to elucidate the inhibitory effects of e804 on stat3, western blot analysis was performed. treatment with e804 or e231 inhibited h9n2-induced stat3 tyrosine phosphorylation (fig. 8c ). upon activation, stat3 forms homo-or heterodimers that translocate to the nucleus. hpmecs fractionation of nucleus and cytoplasm was obtained by means of subcellular fractionation followed by western blot analysis. the results showed that h9n2 infection increased phosphorylated stat3 in the nuclear fraction, while treatment with e804 significantly reduces the nuclear translocation (fig. 8d) . similar to the result of western blot analysis, the confocal image also showed that increased fluorescence signal was found in the nucleus after h9n2 infection in hpmecs (fig. 8e) . treatment with e804 reduced stat3 fluorescence signal in the nucleus. the emergence of iav poses a serious global threat to human health. besides regular epidemic outbreaks, severe pandemics like the 1918 spanish flu and the more recent 2009 swine flu had caused enormous social and economic burden. current treatment of iav infection by m2-ion channel inhibitors or na inhibitors emerged high frequency of resistance, and the efficacy and effectiveness of these antiviral drugs are limited by disappointing success rate 1 , so alternative or complementary therapies that modulate the signaling pathways utilized by iav came into focus. in this report, we demonstrated that indirubin derivatives, particularly e804 is a potent immunomodulatory compound for iav-infection in vitro by inhibiting intracellular signaling pathways in pulmonary endothelial cells. during the early stage of iav infection, innate immune cells are recruited to the site of infection and are associated with an overwhelming production of pro-inflammatory cytokines and chemokines. endothelial cells in the pulmonary vasculature form a barrier between the blood and interstitium. this strategic position suggests that pulmonary endothelial cells are prone to be affected by the cytokines and viral particles released from the iav-infected epithelial cells. a recent study by teijaro et al. identified endothelial cells as the central orchestrator which contribute to the aberrant pro-inflammatory cytokine and chemokine production during early iav infections 22 . concomitant with our in vitro data, we showed that h9n2 virus can efficiently infect hpmecs (fig. 4a,b) and induce a significant amount of ip-10, rantes, il-6, ifn-β and ifn-γ 1. ip-10 and rantes are the chemoattractants for leukocytes including t cells, nk cells, and granulocytes, while production of il-6 by endothelial cells initiates infiltration of neutrophils in the early phase of infection 47 . these cytokines have been found histopathologically in the lungs (including epithelial and endothelial cells) of h5n1 infected patients, who showed acute respiratory distress syndrome (ards) 48 , and ards can be characterized by progressive pulmonary endothelial damage. it has been suggested that treatment with antibodies against ip-10 in h1n1 infected mice can improve the survival rate and reduce acute lung injury 49 . furthermore, suppression of early innate cytokine and chemokine production in the pulmonary endothelium can significantly improve survival of mice infected with lethal h1n1 swine iav 22 . these studies suggested that inhibition of cytokines production of the pulmonary endothelium is an attractive therapeutic strategy against iav-induced cytokine storm. since severe infection of the influenza virus triggers the activation of the innate immune response and sometimes results in the induction to a cytokine storm. in this study, we demonstrated the immunomodulatory effects of indirubin derivatives, particularly e804 on iav-infected pulmonary endothelial cells. over the past two decades, many studies have identified that indirubin derivatives are potent inhibitors of various kinases, including mapks 35,36 , src kinase 37 , glycogen synthase kinase-3β (gsk-3β ) 50 and cyclin-dependent kinases (cdks) 51 . based on these findings, potential functions of indirubin derivatives have been proposed, including anti-inflammation 35, [40] [41] [42] , anti-leukemia 36 , antiviral 38, 39 and angiosuppression 52, 53 . crystal structure analysis revealed that indirubin can form three hydrogen bonds with the atp-binding pocket of cdks, thereby competitively inhibiting atp binding in the catalytic domain of cdks 36 . the results from our in vitro kinase assay also demonstrated that e804 is a potent inhibitor of p38 and jnk (fig. 7c,d) . the cytokine elisa data also suggest that h9n2-induced ip-10 expression of hpmecs was dependent on p38 and jnk activation, while rantes and il-6 were controlled by jnk and p38, respectively ( fig. 5a-c) . however, the western blot analysis showed that e804 could also inhibit phosphorylation of p38 (fig. 7a) and jnk (fig. 7b) , which mean upstream kinases of p38 and jnk may also be inhibited by e804. mapks are important mediators of influenza-induced cytokine expression. in fact, p38 has been shown to regulate the stability of il-6 mrna 54 . meanwhile, another study also indicated the critical function of p38 on ip-10 during viral infection 55 . furthermore, inhibition of p38 by specific inhibitor sb202190 in vivo can greatly diminish h5n1 lethal infection 28 . however, the role of jnk in iav infection has not been fully examined. nacken et al. elucidated that influenza viral rna induces jnk phosphorylation in an rig-i dependent manner, but the ns1 of iav has also an intrinsic jnk activating property 56 . taken together, the potent inhibitory effect of p38 and jnk signaling pathways by e804 strongly correlates with its anti-inflammatory function. ifns are pro-inflammatory cytokines crucial for antiviral responses to iav infection. stat1 and stat2 are predominant and essential transcription factors of type i and ii interferons signaling pathway, but the role of stat3 activation after ifn binding remains controversial. undeniably, stat3 is indispensable for downstream signaling pathway of many other cytokines like il-6, vegf or egf 57 . our data showed that h9n2 infection on hpmecs can significantly induce ifn-β and ifn-γ 1 expression. interestingly, stat3-specific sirna has no effect on h9n2-induced il-6 ( fig. 5f ) and ifn-γ 1 (fig. 8b ), but strongly inhibit ifn-β expression level, indicating the involvement of stat3 in ifn-β induction. the western blot data showed that iav-infection could activate stat3 in early stage (15 min after addition of virus) followed by another activation at 2 h.p.i. (fig. 6) . h9n2 was found to upregulate tlr-8 and myd88, which is critical to the induction of ifn-β 58 . in line with this finding, the early activation of stat3 may function together with the downstream signaling molecules of tlr-8, possibly the interferon regulatory factors (irfs), to induce rapid expression of ifn-β mrna at 2 h.p.i. (fig. 1h) . and then the autocrine effect of ifn-β induces a further amplification of ifn-β expression and result in a cytokine storm. a previous study suggested that e804 could inhibit stat3 dimerization and subsequent dna binding 37 . our translocation experiments clearly indicated that e804 could inhibit stat3 phosphorylation and nuclear translocation. since the induction of many pro-inflammatory cytokines requires type i interferon signaling, inhibition of ifn-β production by e804 may blunt the early induction of these cytokines. though ifn-β is a well-known antiviral cytokine, it is also involved in the pathogenesis of influenza infection. in vivo studies focusing on the s1p 1 receptor and p38 pathway also suggested that, even the iav-induced ifns were suppressed by an s1p 1 receptor agonist 22 or p38 inhibitor 28 , the survival rate of the infected mice could still be significantly improved if those strongly induced cytokines were suppressed. in many studies, increased viral load were concomitant of reduced interferons expressions. however, in the present study, viral titer did not further increased in indirubin derivatives treated cells (fig. 4b ) even ifnβ and ifn-γ 1 were suppressed, the results indicated that suppression of cytokines produced by the infected pulmonary endothelium could reduce iav pathogenicity independent of the viral clearance. in fact, many kinases including cdk 39 and mapks, which are suggested being involved in the influenza replication process are also the target of indirubin derivatives, this might explain the partial antiviral effect of indirubin derivatives in this model. meanwhile the phosphorylation and nuclear translocation of stat3 leaded to induction of ifn-β . indirubin derivatives particularly e804 is a potent inhibitor of p38 and jnk signaling pathways. e804 could also reduce the phosphorylation and nuclear translocation of stat3. by inhibition of these signaling pathways, e804 could significantly suppress h9n2-induced cytokine burst in hpmecs. scientific reports | 6:18941 | doi: 10.1038/srep18941 combination therapies coupling with antiviral and immunomodulatory drugs have been investigated intensively 29 . the encouraging results from in vitro and pre-clinical studies have led to an increased interest on this topic. however, to achieve the best clinical outcome, antiviral and immunomodulatory drugs should be administrated at the appropriate time during an infection. further understanding of the immune dynamic could allow us to design an optimum therapy strategy. in this report, we demonstrated for the first time, the potent immunomodulatory effects of indirubin derivatives on pulmonary endothelial cells and their therapeutic potential for iav-infection (fig. 9) . as a result, the combinational effects of indirubin derivatives and antiviral drug in animal model warrant further investigation. perspectives on influenza evolution and the role of research structure and assembly of the influenza a virus ribonucleoprotein complex pathogenesis of influenza virus infections: the good, the bad and the ugly innate immunity to influenza virus infection detrimental contribution of the toll-like receptor (tlr)3 to influenza a virus-induced acute pneumonia efficient influenza a virus replication in the respiratory tract requires signals from tlr7 and rig-i characterization of conserved viral leader rna sequences that stimulate innate immunity through tlrs toll-like receptor 10 is involved in induction of innate immune responses to influenza virus infection differential role of tlr-and rlr-signaling in the immune responses to influenza a virus infection and vaccination innate immune responses to influenza a h5n1: friend or foe? mitogen-activated protein kinases in innate immunity n-terminal kinases as potential therapeutic targets induction of proinflammatory cytokines in primary human macrophages by influenza a virus (h5n1) is selectively regulated by ifn regulatory factor 3 and p38 mapk targeting cell signalling pathways to fight the flu: towards a paradigm change in anti-influenza therapy rna viruses and the mitogenic raf/mek/erk signal transduction cascade influenza virus non-structural protein 1 (ns1) disrupts interferon signaling mammalian innate resistance to highly pathogenic avian influenza h5n1 virus infection is mediated through reduced proinflammation and infectious virus release human and avian influenza viruses target different cell types in cultures of human airway epithelium transmission of influenza a in human beings influenza a viruses target type ii pneumocytes in the human lung influenza h5n1 virus infection of polarized human alveolar epithelial cells and lung microvascular endothelial cells endothelial cells are central orchestrators of cytokine amplification during influenza virus infection h5n1 virus activates signaling pathways in human endothelial cells resulting in a specific imbalanced inflammatory response human pulmonary microvascular endothelial cells support productive replication of highly pathogenic avian influenza viruses: possible involvement in the pathogenesis of human h5n1 virus infection into the eye of the cytokine storm pathogenesis of influenza-induced acute respiratory distress syndrome dissecting influenza virus pathogenesis uncovers a novel chemical approach to combat the infection inhibition of p38 mitogen-activated protein kinase impairs influenza virus-induced primary and secondary host gene responses and protects mice from lethal h5n1 infection newer influenza antivirals, biotherapeutics and combinations new strategies to overcome the drawbacks of currently available flu vaccines the epidemiology and spread of drug resistant human influenza viruses targeting the "cytokine storm" for therapeutic benefit influenza neuraminidase: a druggable target for natural products dual functions of ginsenosides in protecting human endothelial cells against influenza h9n2-induced inflammation and apoptosis indirubin-3-monoxime exhibits anti-inflammatory properties by down-regulating nf-kappab and jnk signaling pathways in lipopolysaccharide-treated raw264.7 cells indirubin, the active constituent of a chinese antileukaemia medicine, inhibits cyclin-dependent kinases indirubin derivatives inhibit stat3 signaling and induce apoptosis in human cancer cells anti-inflammatory and antiviral effects of indirubin derivatives in influenza a (h5n1) virus infected primary human peripheral blood-derived macrophages and alveolar epithelial cells indirubin-3′ -monoxime, a derivative of a chinese antileukemia medicine, inhibits p-tefb function and hiv-1 replication 5′ -nitro-indirubinoxime inhibits inflammatory response in tnf-alpha stimulated human umbilical vein endothelial cells indirubin inhibits inflammatory reactions in delayed-type hypersensitivity inhibition of rantes expression by indirubin in influenza virus-infected human bronchial epithelial cells amino acid residues 253 and 591 of the pb2 protein of avian influenza virus a h9n2 contribute to mammalian pathogenesis the ginsenoside protopanaxatriol protects endothelial cells from hydrogen peroxide-induced cell injury and cell death by modulating intracellular redox status indirubin-3′ -(2,3 dihydroxypropyl)-oximether (e804) is a potent modulator of lpsstimulated macrophage functions the rac1 inhibitor nsc23766 exerts anti-influenza virus properties by affecting the viral polymerase complex activity understanding the roles of cytokines and neutrophil activity and neutrophil apoptosis in the protective versus deleterious inflammatory response in pneumonia pathological study of archival lung tissues from five fatal cases of avian h5n1 influenza in vietnam monoclonal antibody against cxcl-10/ip-10 ameliorates influenza a (h1n1) virus induced acute lung injury soluble 3′ ,6-substituted indirubins with enhanced selectivity toward glycogen synthase kinase -3 alter circadian period indirubins inhibit glycogen synthase kinase-3β and cdk5/p25, two protein kinases involved in abnormal tau phosphorylation in alzheimer's disease: a peoperty common to most cyclin-dependent kinase inhibitors an indirubin derivative, e804, exhibits potent angiosuppressive activity automated, quantitative screening assay for antiangiogenic compounds using transgenic zebrafish the p38 map kinase pathway signals for cytokine-induced mrna stabilization via map kinase-activated protein kinase 2 and an au-rich region-targeted mechanism innate immune response to h3n2 and h1n1 influenza virus infection in a human lung organ culture model activation of c-jun n-terminal kinase upon influenza a virus (iav) infection is independent of pathogen-related receptors but dependent on amino acid sequence variations of iav ns1 stats in cancer inflammation and immunity: a leading role for stat3 human intestinal epithelial cells are susceptible to influenza virus subtype h9n2 this work was supported by the area of excellence scheme of the university grants committee, hong kong sar government (aoe/m-12/06) and dr. gilbert hung ginseng laboratory fund. key: cord-103511-31njndob authors: broggi, achille; ghosh, sreya; sposito, benedetta; spreafico, roberto; balzarini, fabio; lo cascio, antonino; clementi, nicola; de santis, maria; mancini, nicasio; granucci, francesca; zanoni, ivan title: type iii interferons disrupt the lung epithelial barrier upon viral recognition date: 2020-05-05 journal: biorxiv doi: 10.1101/2020.05.05.077867 sha: doc_id: 103511 cord_uid: 31njndob lower respiratory tract infections are a leading cause of mortality driven by infectious agents. rna viruses such as influenza virus, respiratory syncytial virus and the new pandemic coronavirus sars-cov-2 can be highly pathogenic. clinical and experimental evidence indicate that most severe and lethal cases do not depend on the viral burden and are, instead, characterized by an aberrant immune response. in this work we assessed how the innate immune response contributes to the pathogenesis of rna virus infections. we demonstrate that type iii interferons produced by dendritic cells in the lung in response to viral recognition cause barrier damage and compromise the host tissue tolerance. in particular, type iii interferons inhibit tissue repair and lung epithelial cell proliferation, causing susceptibility to lethal bacterial superinfections. overall, our data give a strong mandate to rethink the pathophysiological roles of this group of interferons and their possible use in the clinical practice against endemic as well as emerging viral infections. 8 efficiently than mice that bear wt stromal cells, and phenocopy ifnlr1 -/à ifnlr1 -/chimeras (fig. 171 3g) . these data further support the direct activity of ifn-λ on epithelial cells. 172 interestingly, the gene that was most downregulated in ifnlr1 -/epithelial cells compared to 173 wt cells is the e3 ubiquitin-protein ligase makorin-1 (mrkn1) (fig. 3a) , 174 which controls p53 and p21 stability by favoring their proteasomal degradation (48) . under 175 oxidative stress condition and dna damage, a hallmark of severe viral infections (49), p53 is 176 stabilized by phosphorylation and p21 degradation, mediated by mkrn1, favors apoptosis over 177 dna repair (48). indeed, ifnlr1 -/epithelial cells, that express lower levels of mkrn1, have elevated 178 levels of p21 as measured by flow cytometry (fig. 3h , i). these data indicate that the capacity of 179 ifn-λ to reduce tissue tolerance stems from its capacity to inhibit tissue repair by directly 180 influencing epithelial cell proliferation and viability. also, that p21 degradation via mrkn1 181 upregulation is potently influenced by ifn-λ signaling. 182 rna viruses can use several strategies to modulate the immune response to their 183 advantage(33, 50), therefore it is crucial to understand the molecular pathways involved in the 184 maintenance of sustained ifn-λ production. moreover, the difference between mrna expression 185 and protein levels of interferons suggest that a low abundance cell type with high secretory 186 capacity may be responsible for long term ifn-λ production. we thus investigated the cellular 187 source and molecular pathways that drive ifn-λ production in our model. early after initial 188 influenza virus infection, ifn-λ is expressed by infected epithelial cells, however, at later time 189 points, dcs from the parenchyma of the lung start to express high levels of the ifn-λ transcript(5). 190 we thus hypothesized that lung dcs are the main producers of ifn-λ and are responsible for the 191 secretion of ifn-λ during viral infections. accordingly, sorted lung resident dendritic cells express 192 high levels of ifn-λ transcript after 5 days of poly (i:c) treatment, in contrast to epithelial cells, 193 alveolar macrophages and monocytes (fig. 4a) , which, instead, express inflammatory cytokines (fig. s8a, b) . moreover, diphtheria toxin (dt)-mediated depletion of 195 cd11c + cells in cd11c-dt receptor (dtr) mice was sufficient to completely abolish ifn-λ 9 transcript and protein upregulation upon 6 days of poly (i:c) treatment (fig. 4b, c) , while production remained unaltered (fig. s8c with the response measured in vivo, tlr7 stimulation did not induce ifn production while it 207 induced upregulation of pro-inflammatory cytokines, and intracellular delivery of poly (i:c) induced 208 high levels of ifn-i but not ifn-λ (fig.4d, fig. s9a , b). in agreement with the key role of tlr3, 209 ifn-λ production upon extracellular poly (i:c) encounter was abolished by genetic deletion of the 210 signaling adaptor trif (encoded by the gene ticam1) but not by deletion of the rig-i/mda5 211 adaptor mavs (mavs) (fig. 4d) . conversely, ifn-i production in response to intracellular delivery 212 of poly (i:c) was largely dependent on the signaling adaptor mavs (fig. s9a ). consistent with 213 our previous data, when the rig-i/mavs pathway was activated by transfection of the influenza 214 a virus derived pathogen-associated molecular pattern (pamp) 3-phosphate-hairpin-rna (3p-215 hprna), ifn-i but not ifn-λ, was efficiently induced in a mavs-dependent manner ( fig. s10a -216 e, poly (i:c) was used as a control). finally, inhibition of endosomal acidification by treatment with 217 the pharmacological agent chloroquine abolished ifn-λ induction in response to extracellular poly 218 (i:c), while it preserved ifn-i production upon intracellular poly (i:c) delivery (fig. s11a, b) . 219 these evidences clearly indicate that tlr3 stimulation potently induces ifn-λ production by dcs 220 in vitro. we, thus, explored the importance of the tlr3-trif pathway in vivo under our 221 experimental conditions. dendritic cells sorted from ticam1 -/mice treated with poly (i:c) for six 222 days did not express appreciable levels of ifn-λ transcripts while still produced type i interferons 223 ( fig. 4e, f) . moreover, poly (i:c) treated ticam1 -/mice were protected from s. aureus 224 superinfections (fig. 4g) , and the decrease in bacterial burden correlated with lower ifn-λ 225 transcript levels in the lung, although ifn-i levels remained similar to those of wt mice (fig. 4h , 226 i). confirming the crucial role of tlr3 signaling in dcs for ifn-λ production, chimeric mice in 227 which ticam1 -/bone marrow (bm) cells are transferred in a wt irradiated host (ticam1 -/ àwt) 228 phenocopied ticam1 -/animals ( fig. 4j-l) . 229 the immune system evolved to prevent and resist to pathogen invasion but doing so often 230 threatens host fitness and causes disease in the form of immunopathology (51). rna viruses are 231 the major cause of most severe lower respiratory tract viral infections (52, 53). while most virus 232 infections manifest as self-limiting upper respiratory tract infections, influenza viruses, sars-233 cov, sars-cov-2 and mers-cov can progress to severe lung disease with potentially lethal 234 outcomes (50, 54, 55) . although different viruses vary in their virulence and pathogenic potential, 235 the most severe cases of lung rna viral infections share similar features that suggest an immune 236 pathological etiology. in covid-19, sars, mers and flu, severe symptoms and death occur late 237 after the initial symptoms onset, and after the peak in viral load (56-61) further indicating a central 238 role for an immune etiology of the most severe forms. 239 while ifn-λ is uniquely equipped to induce a gentler immune response that favors viral 240 clearance in the lungs without inducing overt immune activation (1, 3, 62), its impact on epithelial 241 cell biology and its effect on the maintenance of tissue integrity and tolerance to pathogen invasion 242 is incompletely understood. in a system that allowed us to isolate the effect of immune activation 243 from resistance to viral infection, we demonstrate that sustained ifn-λ production in the lung in 244 response to viral pamps compromises epithelial barrier function, induces lung pathology and 245 morbidity and predisposes to lethal secondary infections by impairing the capacity of the lungs to 246 tolerate bacterial invasion. loss of lung barrier tolerance is sufficient to induce lethality upon 247 bacterial challenge independently of bacterial growth (39), and alteration of the repair response 248 11 in the lung can favor bacterial invasion independently from immune cell control (63). in our model 249 immune cell recruitment is not affected by ifn-λ and neutrophils are dispensable for the impaired 250 control of bacterial infections, while ifn-λ signaling on epithelial cells is necessary and sufficient 251 to cause heightened bacterial invasion. 252 under our experimental conditions, tlr3-trif signaling in conventional lung dcs is 253 responsible for the induction of ifn-λ. this is consistent with reports indicating that tlr3-deficient 254 mice are protected from influenza-induced immune pathology(64). moreover, tlr3 detects 255 replication intermediates from necrotic cells (35) and is, thus, insensitive to viral immune evasion. 256 this is of particular interest during highly pathogenic human coronavirus infections, whose 257 success in establishing the initial infection is partly due to their ability to dampen tlr7 and mavs 258 dependent early ifn responses (50) (2020) (available at https://clinicaltrials.gov/ct2/show/nct04331899). 328 clin. microbiol. rev. 19, 571-582 (2006) . intratracheal instillations (i.t.) were performed as previously described in (69) rectal temperature and body weights were monitored daily. mice were deemed to have reached endpoint at 75% of starting weight or after reaching body temperature of 25°c or lower. to generate mice with hematopoietic-specific deletion of ifnlr1 or ticam1, 6-week-old cd45.1+ mice were exposed to lethal whole-body irradiation (950 rads per mouse) and were reconstituted with 5 × 10 6 donor bone marrow cells from 6-week-old wild-type, ifnlr1 -/or ticam1 -/mice. mice were treated with sulfatrim in the drinking water and kept in autoclaved cages for 2 weeks after reconstitution. after 2 weeks, mice were placed in cages with mixed bedding from wild-type, and ifnlr1 -/or ticam1 -/mice to replenish the microbiome and were allowed to reconstitute for 2 more weeks. a similar procedure was used to generate bone-marrow chimeras with stromal cellsspecific deletion in ifnlr1. here, recipient wt or ifnlr1 -/mice underwent irradiation and were reconstituted with bm cells derived from cd45.1+ mice similarly as described above. to evaluate the percentage of chimerism, a sample of peripheral blood was taken from chimeric mice after 4 weeks of reconstitution and stained for cd45.1 and cd45.2 (antibodies as identified under 'reagents and antibodies') and were analyzed by flow cytometry. in order to deplete cd11c + cells, cd11c-dtr mice received 16μg/kg diphtheria toxin (dtx) intravenously starting one day before tlr ligand or saline administration and continuing every other day until day 6 post-treatment to maintain depletion. in vivo depletion of neutrophils was carried out by injecting anti-ly6g antibody (100μg/mouse) intraperitoneally, starting one day before treatments and then continuing every other day through the duration of the treatment. as controls for no depletion, mice were injected with rat igg isotype control. to assess lung permeability, treated mice were administered fitc-dextran (10μg/mouse) i.t. before or after s. aureus infection. after 1hr of dextran instillation, blood was collected from the retro-orbital sinus, and the plasma was separated by centrifugation. leakage of dextran in the bloodstream was measured as fitc fluorescence in the plasma compared to plasma from mocktreated mice. bal was collected as described in (70) briefly, the lungs of euthanized mice were lavaged through the trachea with 3ml pbs to collect the bal. samples were centrifuged and the supernatants were used for total protein measurement (pierce bca protein assay, thermo fisher scientific #23227) and ldh quantification (pierce ldh cytotoxicity assay, thermo fisher scientific #c20301). lungs were excised and used for rna extraction using tri reagent (zymo research #r2050-1-200). the left lobe of the lung was weighed and homogenized in 1ml of sterile d.i. water in a fisherbrand™ bead mill 24 homogenizer. to calculate bacterial load, homogenate was serially diluted and plated on tsb-agar plates in duplicate. colonies were counted after 16h incubation, and bacterial burden in the lungs was calculated as cfu normalized to individual lung weight. cytokines production in the lungs was measured in the supernatants collected after centrifuging the lung homogenates. lung cells were isolated as described in (71) briefly, mice were euthanized and perfused. 2 ml of warm dispase solution (5mg/ml) were instilled into the lungs followed by 0.5ml of 1% low-melt agarose (sigma #a9414) at 40°c, and allowed to solidify on ice. inflated lungs were incubated in dispase solution, for 30' at rt. the lungs were then physically dissociated, incubated 10' with dnase i 50 μg/ml and filtered through 100μm and 70μm strainers. red blood cells were lysed using ack buffer. single cell suspensions were stained for live/dead using zombie red or zombie violet, and then with antibodies against surface antigens diluted in pbs + bsa 0.2% for 20 minutes at 4°c. cells were then washed, fixed with 3.7% paraformaldehyde for 10 minutes at room temperature, washed again and resuspended in pbs + bsa 0.2%. samples were acquired on a bd lsrfortessa flow cytometer and data were analyzed using flowjo v.10 software (bd biosciences). countbright absolute counting beads (invitrogen #c36950) were used to quantify absolute cell numbers. purified rna was analyzed for gene expression on a cfx384 real-time cycler (bio-rad) using a taqman rna-to-ct 1-step kit (thermo fisher scientific) or sybr green (bio-rad). probes specific for ifnl2/3, ifnb1, il1b, rsad2, gapdh were purchased from thermo fisher scientific, and sybr-green primers for rsad2, cxcl10, gapdh were purchased from sigma. cytokine analyses were carried out using homogenized lung supernatants, and cell supernatants from stimulated flt3l-dcs. ifnλ2/3 elisa (r&d systems dy1789b) and mouse ifnβ, il1β, il-6, tnfα elisa (invitrogen) were performed according to manufacturer's instructions. bronchoalveolar lavages (bal) were obtained from five intensive care unit (icu)-hospitalized sars-cov-2-positive patients. in parallel, five naso-oropharyngeal swabs were collected from both sars-cov-2-positive and -negative subjects. among positive patients, two were hospitalized but without the need of icu support, whereas three out of five did not require any hospitalization. the negative swabs were obtained from subjects undergoing screening for suspected social contacts with covid-19 subjects. swabs were performed by using floqswabs® (copan) in utm® universal transport medium (copan). all samples were stored at -80°c until processing. the study involving human participants was reviewed and approved by san raffaele hospital irb in the covid-19 biobanking project. the patients provided written informed consent. rna extraction was performed by using purelink™ rna thermo fisher scientific according to manufacturers' instruction. in particular, 500 µl for each bal and swab analyzed sample were lysed and homogenized in the presence of rnase inhibitors. then ethanol was added to homogenized samples which were further processed through a purelink™ micro kit column for rna binding. after washing, purified total rna was eluted in 28 µl of rnase-free water. system (invitrogen™) protocol by using 8 µl of rna extracted from each bal and swab sample. qrt-pcr analysis for was then carried out for evaluating il6, il1b, ifnb1, ifna2, ifnl1 and ifnl2 expression. all transcripts were tested in triplicate for each sample by using specific primers. gapdh was also included. real-time analysis was then performed according to manufacturer instructions by using taqman® fast advanced master mix (applied biosystems™ by thermo fisher scientific). real-time pcr analysis was performed on abi 7900 by applied biosystems. statistical significance for experiments with more than two groups was tested with one-way anova, and dunnett's multiple-comparison tests were performed. two-way anova with tukey's multiple-comparison test was used to analyze kinetic experiments. two-way anova with sidak's multiple-comparison test was used to analyze experiments with 2 grouped variables (i.e. treatment, genotype). statistical significance for survival curves were evaluated with the log-rank (mantel-cox) test and corrected for multiple comparisons with bonferroni's correction. to establish the appropriate test, normal distribution and variance similarity were assessed with the d'agostino-pearson omnibus normality test using prism8 (graphpad) software. when comparisons between only two groups were made, an unpaired two-tailed t-test was used to assess statistical significance. to determine the sample size, calculations were conducted in nquery advisor version 7.0. primary outcomes for each proposed experiment were selected for the sample size calculation and sample sizes adequate to detect differences with an 80% power were selected. for animal experiments, four to ten mice per group were used, as indicated in the cells were gated on fsc and ssc to eliminate debris, on fsc-a -fsc-h to select single cells and cells negative for live/dead dye and lineage markers (cd3, cd19, ter119). epithelial cells were gated as cd45 -epcam + cd31 -. the epcamcells were sorted for immune cells as follows: amac were gated as cd45 + ly6g -cd11c hi siglec-f + , monocytes and monocyte-derived cells (mo) were gated as cd45 + ly6g -siglec-f -ly6c + , cdcs were gated as cd45 + ly6g -siglec-f -ly6c -cd11c + mhc-ii hi . estimates of the severity of coronavirus 457 disease 2019: a model-based analysis clinical progression and viral load in a community 462 outbreak of coronavirus-associated sars pneumonia: a prospective study of the n. t. u. (ntu) c. of m. hospital progression in patients with severe acute respiratory syndrome virological assessment 472 of hospitalized patients with covid-2019 influenza and rhinovirus viral load and 474 disease severity in upper respiratory tract infections innate and adaptive immune responses in patients with severe acute respiratory 481 materials and methods mice c57bl/6j jax 004509) mice were purchased from jackson labs. c57bl/6 il-28r -/-(ifnlr1 -/-) mice were provided by bristol-myers squibb. mice were housed under specific pathogen-free conditions at boston children's hospital. staphylococcus aureus infections were conducted in the biosafety level-2 facility at boston children's hospital. all procedures were approved under the institutional animal care and use committee (iacuc) and conducted under the supervision of the department of animal cd24 (m1/69), mhc-ii i-a/i-e (m5/114 r848 (tlr-r848) and 3p-hprna (tlrlhprna) were purchased from invivogen. for in vivo administration of type iii ifn, we used polyethylene glycol-conjugated ifn-λ2 (peg-ifn-λ) (gift from bristol-myers squibb). diphtheria toxin (unnicked) from corynebacterium diphtheriae was purchased from cayman chemical. anti-ly6g antibody, clone 1a8 (be0075-1) and rat igg2a isotype control (be0089) for in vivo administration was purchased from bioxcell. 2'-deoxy-5-ethynyl uridine (edu) was purchased from carbosynth (ne08701) epithelial cell proliferation') before being stained with antibodies against cell-surface antigens. intracellular staining of ki67 and p21 were carried out using foxp3 fix/perm buffer set (biolegend #421403) following the manufacturer's instructions. epithelial cell proliferation proliferation of lung after permeabilization cells were washed and incubated with 4 mm copper sulphate (millipore-sigma), 100 mm sodium ascorbate (millipore-sigma) and 5 μm sulfo-cyanine3-azide (lumiprobe #a1330) in tris buffered saline (tbs) 100mm, ph 7.6, for 30 min at room temperature. ion torrent for targeted transcriptome sequencing, 20 ng of rna isolated from sorted cells was retro barcoded libraries were prepared using the ion ampliseq transcriptome mouse gene expression kit as per the manufacturer's protocol and sequenced using an ion s5 system genes were called expressed (n=11,294) if they had average log2 expression of 2 or greater in either wt or ifnlr1 -/-. differentially expressed genes (degs) between wt and ifnlr1 -/-were selected by thresholding on fold change (+/-1.5) and p value (0.005). in heatmaps, degs were z-scaled and clustered (euclidean distance, ward linkage). pathway analysis was performed with the r package hyper cell culture flt3l-dcs were differentiated from bone marrow cells in iscove's modified dulbecco's media (imdm; thermo fisher scientific), supplemented with 30% b16-flt3l derived supernatant and 10% fetal bovine serum (fbs) for where indicated poly (i:c) stimulated cells were pre-treated with 10μg/ml chloroquine for 5 minutes prior to stimulations. qrt-pcr and elisa rna was isolated from cell cultures using a genejet rna purification kit (thermo fisher scientific #k0731) according to manufacturer's instructions. rna was extracted from excised lungs by homogenizing them in 1ml of tri reagent. rna was isolated from tri reagent samples using phenol-chloroform extraction or column-based extraction systems (direct-zol rna microprep and miniprep, zymo research #r2061 and #r2051) according to the manufacturer's protocol flow cytometric isolation of primary murine type ii alveolar epithelial cells for functional and molecular studies wt mice were treated daily with i.t. 0.5 mg/kg poly (i:c), 0.5 mg/kg r848 or saline for 6 days and infected i.t. with 0.5 x 10 8 cfu of s. aureus at day 6. (a) body temperature, (b) total protein in the bal and **p < 0.01 and ***p < 0.001 (one-way anova). each mouse represents one point a-h) wt mice were treated daily for 1, 3, 5 or 6 days with i.t. 0.5 mg/kg poly (i:c) or 6 days of saline, and infected with i.t. 0.5 x 10 8 cfu of s. aureus for 12h. total lung homogenates were analyzed by qpcr for bacterial burden was evaluated in total lung homogenate **p < 0.01 and ***p < 0.001 (one-way anova compared to saline treatment). each mouse represents one point wt and ifnlr1 -/-mice were treated daily with i.t. 0.5 mg/kg poly (i:c) for 6 days and infected with i.t. 0.5 x 10 8 cfu of s. aureus for 12h. (a) weight change, (b) total protein in the bal **p < 0.01 and ***p < 0.001 (two-way anova). each mouse represents one point lung resident dcs are the primary producers of ifn-β upon poly (i:c) treatment 5 mg/kg poly (i:c) or saline for 6 days were sorted for epithelial cells (ec), resident dc (resdc), monocyte-derived dc (modc), and alveolar macrophages (amac) and assessed for (a) il1b and (b) ifnb1 relative mrna expressions. cd11c-dtr mice depleted for cd11c + cells in vivo by dtx injections were treated daily with i.t. 0.5 mg/kg poly (i:c) or saline for 6 days. total lung lysates of the treated mice were analyzed for (c) ifnb1 relative mrna expression, and (d) ifn-β protein expression by elisa 05, **p < 0.01 and ***p < 0.001 (two-way anova) rig-i or tlr7 ligands. flt3l-dcs from wt, ticam1 -/-or mavs -/-mice were treated with 50μg/ml poly (i:c), 1μg/ml transfected poly (i:c) or 50μg/ml r848 for 3h. ifnb1 (a), and il1b (b) relative mrna expressions were evaluated by qpcr 05, **p < 0.01 and ***p < 0.001 (two-way anova) mean and sem of 3 independent experiments is depicted flt3l-dcs upregulate ifn-λ uniquely upon activation of tlr3 signaling and not in response to the rig-i specific ligand 3p-hprna. flt3l-dcs from wt mavs -/-mice were treated with 50μg/ml poly (i:c), or 1μg/ml transfected 3p-hprna for 3h or 6h ifnb1 (b), and il1b (c) relative mrna expressions were evaluated by qpcr after 3h and ifn-β (e) levels in the supernatants were evaluated by elisa after 6h the endosomal tlr inhibitor chloroquine inhibits poly (i:c) dependent ifn-λ expression in flt3l-dcs. flt3l-dcs from wt mice were treated with 50μg/ml poly (i:c), or 1μg/ml transfected poly (i:c) for 3h in the presence or absence of 10μg/ml chloroquine we thank dr. jc kagan for discussion, help and support. funding: iz is supported by nih grant 506 1r01ai121066, 1r01dk115217, and niaid-dait-nihai201700100. ab is supported by ccfa key: cord-000403-vzbh457k authors: zhang, weijun; lin, yan; bai, yu; tong, tiegang; wang, qun; liu, nihong; liu, guangliang; xiao, yihong; yang, tao; bu, zhigao; tong, guangzhi; wu, donglai title: identification of cd8(+ )cytotoxic t lymphocyte epitopes from porcine reproductive and respiratory syndrome virus matrix protein in balb/c mice date: 2011-05-30 journal: virol j doi: 10.1186/1743-422x-8-263 sha: doc_id: 403 cord_uid: vzbh457k twenty-seven nanopeptides derived from the matrix (m) protein of porcine reproductive and respiratory syndrome virus (prrsv) were screened for their ability to elicit a recall interferon-γ (ifn-γ) response from the splenocytes of balb/c mice following dna vaccination and a booster vaccination with recombinant vaccinia virus rwr-prrsv-m. we identified two peptides (amino acid residues k(93)fitsrcrl and f(57)gymtfvhf) as cd8(+ )cytotoxic t lymphocyte (ctl) epitopes. these peptides elicited significant numbers of ifn-γ secreting cells, compared with other m nonapeptides and one irrelevant nonapeptide. bioinformatics analysis showed that the former is an h-2k(d)-restricted ctl epitope, and the latter is an h-2d(d)-restricted ctl epitope. multiple amino acid sequence alignment among different prrsv m sequences submitted to genbank indicated that these two ctl epitopes are strongly conserved, and they should therefore be considered for further research on the mechanisms of cellular immune responses to prrsv. porcine reproductive and respiratory syndrome virus (prrsv) is one of the most important swine viral pathogens, and has caused significant economic losses to the swine industry worldwide. characterization of field isolates suggested that prrsv are genetically diverse, and this genetic variation increases the difficulty of developing effective vaccines. based on significant sequence differencesprrs viruses are grouped into two distinct genotypes, european isolate (lelystad virus, lv) and north american isolate (vr-2332) [1] prrsv has two major structural proteins, gp5 and m, encoded by orfs 5 and 6, respectively. gp5, the most important neutralizing antigen of prrsv, has the highest genetic diversity among isolates [2] . and, recent studies in yorkshire × landrace crossed and outbred pigs, showed that there are two immuno-dominant t-cell epitopes derived from the gp5 protein: l 117 aalicfvirlaknc and k 149 grlyrwrspvii/vek [3] . the m protein, which contains highly conserved amino acid sequences, also has very good immunogenicity and is associated with protection against prrsv infection. dna vaccinations have also revealed that m is the most potent inducer of t lymphocyte proliferation [4] . at present, effective vaccination strategies for the prevention and control of prrsv infection are not available. vaccines based on inactivated prrsv virus have been ineffective at inducing protective immune responses. live-attenuated prrsv vaccines can provide protection against this pathogen, but have been observed to revert to virulence [5] , restricting the application of this vaccination approach. the rational development of future prrsv vaccines will necessitate a systematic understanding of the protective humoral and cellular immune responses that occur during prrsv infection, and should aim to induce a broad immune responses that accommodates the plasticity of the major antigenic sites. recent research has indicated that cell-mediated immunity may play a very important role in the clearance of prrsv [6] . major histocompatibility complex (mhc) i restricted cytotoxic t lymphocytes (ctls) can kill virus-infected cells and eliminate potential sources of new virus [7] . hence, identification of ctl epitopes is crucial in the design of synthetic vaccines, and a number of studies have successfully identified pathogen-derived t cell epitopes [8] [9] [10] [11] . unlike many other pathogens, there is limited knowledge of the specific prrsv-derived peptides targeted by t-cells. in the current study, we report the identification of ctl epitopes from the prrsv (ch-1a strain) m protein in a mouse model. we screened peptides derived from the prrsv m protein for their ability to induce interferon (ifn)-γ in splenocytes harvested from balb/ c mice following dna vaccination and a booster vaccination with recombinant vaccinia virus expressing m protein. the screen identified two peptides that elicited ifn-γ production in cd3 + cd8 + splenocytes of vaccinated mice. a multiple amino acid sequence alignment among different prrsv m proteins indicates that these two peptides are strongly conserved across multiple prrsv strains and therefore should be considered for further research. the prrsv ch-1a strain, the vaccinia virus wr strain, and the akabane virus obe-1 strain were part of our laboratory collection. the former was propagated in marc-145 cells, and the latter two were propagated in bhk-21 cells, respectively, and these two cell lines were cultured in dmem (invitrogen) supplemented with 10% of fetal calf serum (fcs, invitrogen) in a humidified 37°c, 5% co 2 incubator. total rna was extracted from the prrsv ch-1a strain and the spleens of balb/c mice. full length cdnas were amplified based on the complete open reading frames (orfs) of m and ubiquitin (ub) following reverse transcription (genbank accession numbers ay032626 and az033428) using the m-f and m-r primers pair or the ub-f and ub-r primer pair ( table 1) . the full length cdnas were cloned into the pmd18-t vector (takara) and are referred to as pmd-m and pmd-u, respectively. an indirect enzyme-linked immunosorbent assay (ielisa) method was established to evaluate specific antibody against m protein. a truncated m protein (85-174aa), which was used as the coating antigen, was expressed and purified using a prokaryotic expression system. the truncated m gene cdna was amplified from pmd-m using the truncated-m-f and truncated-m-r primers (table 1) , ligated into pgex-6p-1 (ge healthcare), and subsequently named pgex-m. for protein expression, pgex-m was transformed into e.coli bl21 (de3) and recombinant protein expression was induced with 0.5 mm iptg. the samples were harvested by centrifugation and the pellets were resuspended with phosphate buffered saline (pbs, ph 8.0). after being analyzed by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (sds-page) and western blot with anti-prrsv-m monoclonal antibody (our laboratory collection), the fusion-expressed truncated m protein was purified by gst tag according to the manufacturer's instructions (ge healthcare). finally, the concentration of the purified protein was determined using a bradford kit (bio-rad) according to the manufacturer's instructions. the ub and m genes were fused using splicing by overlapping extension pcr (soe-pcr). the ub and m genes were amplified from pmd-u and pmd-m with the primer pairs ub-f and fusion-m-ub-r and fusion-m-ub-f and m-r, respectively ( table 1 ). the fusion gene product, referred to as ub-m, was amplified from the purified pcr products with ub-f and m-r and inserted into the eukaryocyte expression vector pvax1, and was named pvax1-ub-m. the transferring vector psc11 (our laboratory collection), which is composed of the early promoter p7.5 and late promoter p11 of vaccinia virus, and the lacz and amp genes controlled by the promoter p11 as the reporter genes, was used in this study. to construct the transferring vector psc11-m, the complete m gene was amplified with the psc11-m-f and psc11-m-r primer pair (table 1 ) and inserted into the psc11. the primer p7.5-f (table 1) , derived from promoter p7.5 sequence, was used for directional identification and the positive clones, which were subsequently named psc11-m. homologous recombination was performed by lipofectin-mediated co-transfection of the transferring plasmid psc11-m and the wr strain vaccinia virus into 80% confluent tk -143 cells cultured in mem medium containing 25 μg/ml brdu (sigma). the viruses were collected after the appearance of cytopathic effect (cpe), and recombinant vaccinia virus was purified according to the expression of lacz gene. western blot analysis was performed as described previously [12] . briefly, a bhk-21 cell layer was infected with rwr-prrsv-m recombinant vaccinia virus or the vaccinia virus wr strain at a multiplicity of infection (moi) of 0.1. cells were harvested 3 days post-infection, and total cell lysates were prepared with lysis buffer (10 mm tris-cl ph 7.4, 1 mm mgcl 2 , 0.5% np40, 20 μg/ml dnase i). cell lysates were separated by sds-page and were subsequently transferred to a membrane for western blot analysis using an anti-prrsv-m monoclonal antibody, hrp-conjugated goat anti-mouse igg secondary antibody (bio-rad), and dab substrate. the positive recombinant virus was named rwr-prrsv-m. the expression of m was further confirmed by ifa. briefly, bhk-21 cells were infected with either rwr-prrsv-m or the vaccinia virus wr strain when the bhk-21 cells reached 70-80% confluence in a 24-well plate. m protein expression was evaluated by ifa 3 days post infection using the anti-prrsv-m monoclonal antibody followed by a fitc-conjugated rabbit antimouse igg secondary antibody (sigma). specific fluorescence was observed using a fluorescence microscope (leica dm ire2). the sequences of m were screened for potential h2-k d / h2-d d /h2-l d 9-mer epitopes using the algorithms from the syfpeithi website [13] , the hla peptide binding prediction website (bimas) [14] and pred balb/c [15] . the 27 peptides (table 2) , with highest binding score (bs) as predicted by each algorithm, were synthesized by scilight biotechnology llc (beijing, china) and purified to a purity > 95% using high performance liquid chromatography (hplc). all animal experiments were performed according to national and institutional guidelines. one hundred and ninety female balb/c mice (vital river laboratory animal technology co., beijing, china) were maintained in isolation cages at the experimental animal center of harbin veterinary research institute (harbin, china). mice were divided into three groups: the pvax1-ub-m vaccination group (n = 120), the pvax1 control vaccination group (n = 35) and the pbs control vaccinated group (n = 35). the plasmid dna used for immunization was purified using the endofree mega plasmid preparation kit (qiagen). the pvax1-ub-m and pvax1 cohorts were intramuscularly (i.m.) vaccinated with 100 μg pvax1-ub-m or pvax1 plasmid dna in 100 μl pbs, respectively. the pbs control group received an i.m. injection of 100 μl pbs. each group was vaccinated four times at 3-week intervals. to enhance the specific ctl responses to m protein, the mice received an intraperitoneal (i.p.) injection containing 0.1 moi of rwr-prrsv-m on day 7 following the fourth dna vaccination. at the same time, five mice from the pvax1 and pbs vaccination groups were also inoculated intraperitoneally with 0.1 moi of rwr-prrsv-m on day 7 following the fourth vaccination in order to compare the specific antibody raised against m protein of different experimental groups following vaccination with rwr-prrsv-m. all procedures were conducted with the protocols approved by experimental animal center of harbin veterinary research institute (hvri) of the chinese academy of agricultural sciences (caas). to investigate the specific antibody response, serum samples was obtained from vaccinated mice 7 days after each dna vaccination and 3 days after boosting with rwr-prrsv-m. an ielisa was performed according to methods described previously [16] . the purified m protein was used as the coating antigen, the tested sera applied at a 1:10 dilution, and an hrp-conjugated goat anti-mouse igg antibody (bio-rad) used as the secondary antibody. the microplates were developed using orthophenylene diamine (opda, amersco) and h 2 o 2 for 10 min, after which the reaction was stopped by the addition of 1 m h 2 so 4 . finally, the optical density (od) was read at 492 nm. an anti-prrsv-m monoclonal antibody was used as a positive control. serum containing antibodies against akabane virus and the sera of control group mice served as negative controls. isolated splenocytes were added to u-bottomed 96-well plates (corning inc) at 10 6 cells/well in 100 μl complete rpmi-1640 medium supplemented with 10% fcs. the cells were then mixed with 100 μl media containing note: the 27 peptides with highest binding score (bs) as predicted by each algorithm are shown, starting with the peptide giving the highest score at the top for each protein. sequences of 9 mers are given from bimas, syfpeithi and pred balb/c predictions, respectively. the n-terminal amino acid position is indicated for epitopes predicted from the whole m protein. a nonamer peptide derived from prssv m protein at 20 μg/ml, or phorbol-12-myristate-13-acetate (pma 10 ng/ ml) and ionomycin (500 ng/ml) as a positive control. cells incubated in medium alone or with a peptide derived from the infectious bronchitis virus (ibv) h52 strain [17] were used as negative controls. following a 12 h incubation at 37°c, 10 um monensin (sigma) was added and the splenocytes were incubated for an additional 4 h at 37°c before staining. the number of cd3 + cd8 + t cells producing ifn-γ on days 3, 5 and 12 after boosting with rwr-prrsv-m was determined using flow cytometric analysis. ics analysis was performed using a facscalibur flow cytometer (bd) according to the methods described previously [11] . twenty mice were tested from the group vaccinated with pvax1-ub-m, and five mice from the groups vaccinated with pvax1 and pbs were tested at each time point. the splenocytes from each vaccination group were counted, pooled, and stimulated in vitro with m protein-derived peptides, as detailed in section 2.12. ten thousand cd3 + t lymphocytes were acquired per sample and the number of ifn-γ-secretion cd3 + cd8 + t cells were enumerated. the cd3 + cd8 + lymphocytes that expressed ifn-γ following peptide stimulation were considered to be peptide-specific ctls. specific ctl responses were evaluated as the increase in the number of cd3 + cd8 + ifn-γ + cells. reagents used include percp-conjugated cd3e, pe-conjugated cd8a and fitc-conjugated ifn-γ, and bd cytofix/cytoperm™, all purchased from becton, dickinson & co (bd). to further confirm the results of the ics, the other sixty mice from the group vaccinated with pvax1-ub-m and thirty mice from the groups vaccinated with pvax1 and pbs were tested by the ifn-γ elispot assay at three different time points (as detailed in section 2.13). data are expressed as the number of ifn-γ-secreting cells per two hundred thousand splenocytes. peptide-specific ifn-γ elispot responses were considered to be positive if the response (minus media background) was ≥ 3fold above the media response and ≥ 50 spot-forming cells (sfc)/2 × 10 5 splenocytes were registered. the ielisa results, the percentage of ifn-γ positive cd3 + cd8 + t lymphocytes and the number of spots per 2 × 10 5 splenocytes were analyzed using the analysis of variance (anova), and a probability value below 0.05 was considered significant. as the full-length m protein is difficult to express in vitro, we expressed a truncated version of m that lacks 84 hydrophobic amino acids at the n-terminus. sds-page analysis showed that cells transformed with the pgex-m expression vector produced a large amount of protein with a molecular mass of approximately 36 kda, consistent with the expected molecular weight of the truncated m protein fused with a gst tag (data not shown). western blot analysis using an anti-prrsv-m antibody confirmed the expression and identity of the truncated m protein fused with a gst tag (additional file 1, fig. s1 ). the ub proteasome pathway (upp) is the principal mechanism for protein catabolism in the mammalian cytosol and nucleus. in order to enhance the ub-mediated degradation efficiency of m protein, we expressed a ub-m fusion protein in bhk-21 cells using the eukaryotic expression plasmid pvax1-ub-m, in which the ub coding sequence was fused in-frame with the prrsv m coding sequence. following transient transfection of bhk-21 cells with the pvax1-ub-m plasmid, the ub-m fusion protein was expressed and accumulated predominantly in the cytoplasm (additional file 2, fig. s2 ). immunization strategies that prime and boost with recombinant dna vectors encoding antigens have been shown to elicit t-cell immunity against hiv in non-human primates [18] , and more recently, in humans [19] . therefore, we used a dna vector encoding the prrsv m protein to immunize mice in order to generate and characterize m protein-reactive cd8 + t cells. the recombinant vaccinia virus rwr-prrsv-m drove expression of an m protein with the expected molecular weight when transfected into bhk-21 cells (additional file 3, fig. s3 ). ifa confirmed expression of m protein following transfection of bhk-21 cells, and revealed m protein accumulation in the cytoplasm (additional file 4, fig. s4 ). m protein-specific serum antibody increased steadily from the second to the fourth dna vaccination with the pvax1-ub-m vector which drives m protein expression in vivo (additional file 5, fig. s5 ), indicating that dna vaccination elicited m protein-specific immune responses as expected. a subsequent booster vaccination with the recombinant vaccinia virus, rwr-prrsv-m, elicited a further increase in prrsv-specific antibody (p < 0.01, additional file 5, fig. s5 ). in contrast, control mice vaccinated with pvax1-only or pbs-only did not show significant increases in m protein-specific antibody titers after the booster vaccination with rwr-prrsv-m (p > 0.05, additional file 5, fig. s5 ). overall, mice vaccinated with pvax1-ub-m generated significantly higher m protein-specific antibody titers compared to mice vaccinated with pvax1 or pbs (p < 0.01, additional file 5, fig. s5 ). these results indicate that rwr-prrsv-m amplifies the protective effects of dna vaccination and reveals the advantage of this priming-boosting strategy. dna vaccination with pvax1-ub-m likely drives the differentiation of memory b cells which are subsequently activated by rwr-prrsv-m following the booster immunization, resulting in increased m-specific antibody titers. mice vaccinated with pbs and pvax1 would not be expected to generate m protein-reactive memory b cells, accounting for a less pronounced increase in m protein-specific antibody titers following rwr-prrsv-m innoculation. in this study we identified potential ctl epitopes in balb/c mice vaccinated with pvax1-ub-m and boosted with rwr-prrsv-m. the frequency and number of cd3 + cd8 + t cells that produced ifn-γ following stimulation with peptides derived from prssv m protein was enumerated using ics and elispot assays. using these approaches, we identified two peptides from prssv m protein that elicited ifn-γ production from the splenocytes of vaccinated mice. as shown in figure 1 , intracellular ifn-γ staining following stimulation of splenocytes from vaccinated mice with the peptide k 93 fitsrcrl and f 57 gymtfvhf revealed a population of ifn-γ producing cd8 + t cells comprising 4-5% of the total cd3 + splenocyte population. in contrast, unstimulated splenocytes and splenocytes exposed to an irrevelant peptide did not contain a population of ifn-γ producing cd8 + t cells (figure 1 , panel b and c). consistent with the ics data, peptides k 93 fitsrcrl and f 57 gymtfvhf elicited ifn-γ production from splenocytes of vaccinated mice when measured by elispot, whereas unstimulated splenocytes and splenocytes stimulated with an irrelevant peptide did not reveal ifn-γ producing cells (figure 2 ). the k 93 fitsrcrl and f 57 gymtfvhf prssv m protein peptides were identified bioinformatically as h-2k d and h-2d d restricted ctl epitopes (table 2) . specific increases in the number of cells producing ifn-γ following stimulation with the peptides "k 93 fitsrcrl" and "f 57 gymtfvhf" was observed by day 3 after the booster vaccination with rwr-prrsv-m (figure 1 and 2) . furthermore, splenocytes from mice vaccinated with pvax1-ub-m responded strongly to "k 93 fitsrcrl" and "f 57 gymtfvhf", but not other m protein-derived peptides, at all time points tested following the booster vaccination with rwr-prrsv-m. when the same pattern of reactivity on three different time points after boosting with rwr-prrsv-m within each vaccination group were analyzed statistically using anova analysis, statistically significant differences were noted for the peptides "k 93 fitsrcrl" and "f 57 gymtfvhf" when compared to the other peptides tested among mice vaccinated and boosted with pvax1-ub-m and rwr-prrsv-m, respectively (p < 0.01, figure 3a and 3b). conversely, and confirming the specificity of these responses, no difference among the stimuli was observed with mock-vaccinated (pvax1) mice and naïve mice (data not shown). it is important to recognize that the ics assay calculates the percentage of ifn-γ + cells among cd3 + t cells in the spleen (4-5%), whereas the elispot assay assesses the number of ifn-γ + cells among all splenocyte cell types (0.05%), and cannot definitively assign the production of ifn-γ to a particular cell type. thus, the two assays use different denominators in calculating the frequency of ifn-γ + production by splenocytes. importantly, each method clearly identified k 93 fitsrcrl and f 57 gymtfvhf as the only two peptides from a panel of 27 that elited significant ifn-γ production from the splenocytes of vaccinated mice. in this study we used balb/c mice as a model system to identify ctl epitopes in the m protein of prssv to circumvent limitations derived from shortages of inbred pigs and a paucity of reagents to evaluate porcine immune responses. the identification of prssv ctl epitopes in balb/c mice allow for the future generation of reagents, such as mhc class i: peptide staining reagents, that will enable the in-depth investigations of cd8 + t cell responses during prssv infection and immuinization. whether these two epitopes can bind the sla class i molecules of pigs remains to be determined. in some cases, specific peptide epitopes are known to be recognized by cytotoxic t cells in different animal species. for instance, the core region of hcv contains an epitope that is recognized by cytotoxic t cells of both mice and humans [19] . further research on the role of these peptide epitopes in different species is ongoing in our laboratory. in conclusion, we identified peptides "k 93 fitsrcrl" and "f 57 gymtfvhf" from the m protein of prssv as h-2k d and h-2d d restricted ctl epitopes, respectively. in this study, we also developed a mouse model of prrsv infection and this will undoubtedly contribute to our understanding of the cell-mediated immune responses to prrsv. porcine reproductive and respiratory syndrome virus current knowledge on the structural proteins of porcine reproductive and respiratory syndrome (prrs) virus: comparison of the north american and european isolates identification of immunodominant t-cell epitopes present in glycoprotein 5 of the north american genotype of porcine reproductive and respiratory syndrome virus t cell responses to the structural polypeptides of porcine reproductive and respiratory syndrome virus reversion of a live porcine reproductive and respiratory syndrome virus vaccine investigated by parallel mutations immunological responses of swine to porcine reproductive and respiratory syndrome virus infection hepatitis b virus immunopathogenesis understanding presentation of viral antigens to cd8+ t cells in vivo: the key to rational vaccine design identification of an h-2d(b)-restricted cd8+ cytotoxic t lymphocyte epitope in the matrix protein of respiratory syncytial virus characterization of a new h-2d(k)-restricted epitope prominent in primary influenza a virus infection dna immunization with 2c fmdv non-structural protein reveals the presence of an immunodominant cd8+, ctl epitope for balb/c mice immune responses of pigs inoculated with a recombinant fowlpox virus coexpressing gp5/ gp3 of porcine reproductive and respiratory syndrome virus and swine il-18 syfpeithi: database for mhc ligands and peptide motifs scheme for ranking potential hla-a2 binding peptides based on independent binding of individual peptide side-chains predbalb/c: a system for the prediction of peptide binding to h2d molecules, a haplotype of the balb/c mouse dna vaccination of pigs with open reading frame 1-7 of prrs virus localization of a t-cell epitope within the nucleocapsid protein of avian coronavirus a dna/mva-based candidate human immunodeficiency virus vaccine for kenya induces multi-specific t cell responses in rhesus macaques enhanced t-cell immunogenicity of plasmid dna vaccines boosted by recombinant modified vaccinia virus ankara in humans submit your next manuscript to biomed central and take full advantage of: • convenient online submission • thorough peer review • no space constraints or color figure charges • immediate publication on acceptance • inclusion in pubmed, cas, scopus and google scholar • research which is freely available for redistribution additional file 5: elisa antibody response in mice after immunization following dna vaccination and a booster vaccination with recombinant vaccinia virus. fig.s5 . m protein-specific antibody responses in mice immunized with pbs, or pvax1 or pvax1-u-m dna, and boosted with rwr-prrsv-m. serum samples was obtained from vaccinated mice 7 days after each dna vaccination and 3 days after boosting with rwr-prrsv-m, and were evaluated for reactivity to mprotein in an elisa based on coating with the truncated m protein fused with a gst tag. and, the day 0 represents the day of the first dna immunization. statistically significant differences are indicated by "*" or "**" for p-values < 0.05 or < 0.01, respectively, as determined by anova.authors' contributions dlw and gll gave me the idea of this study. wjz and yl participated in the design and conducted the majority of the experiments as well as drafted the manuscript. tgt helped revise the manuscript and participated in the first stage of the experiments. yb and qw participated in the prediction of ctl epitopes and analyzed the data. yhx, nhl and ty participated in the sequence alignment. zgb provided the expression system of recombinant vaccinia virus, and gzt provided the prrsv ch-1a strain. all the authors read and approved the final manuscript. the authors declare that they have no competing interests. key: cord-001834-6xf4o3oy authors: sung, pil soo; shin, eui-cheol; yoon, seung kew title: interferon response in hepatitis c virus (hcv) infection: lessons from cell culture systems of hcv infection date: 2015-10-07 journal: int j mol sci doi: 10.3390/ijms161023683 sha: doc_id: 1834 cord_uid: 6xf4o3oy hepatitis c virus (hcv) is a positive-stranded rna virus that infects approximately 130–170 million people worldwide. in 2005, the first hcv infection system in cell culture was established using clone jfh-1, which was isolated from a japanese patient with fulminant hcv infection. jfh-1 replicates efficiently in hepatoma cells and infectious virion particles are released into the culture supernatant. the development of cell culture-derived hcv (hcvcc) systems has allowed us to understand how hosts respond to hcv infection and how hcv evades host responses. although the mechanisms underlying the different outcomes of hcv infection are not fully understood, innate immune responses seem to have a critical impact on the outcome of hcv infection, as demonstrated by the prognostic value of ifn-λ gene polymorphisms among patients with chronic hcv infection. herein, we review recent research on interferon response in hcv infection, particularly studies using hcvcc infection systems. hepatitis c virus (hcv) is a positive-stranded rna virus in the family flaviviridae, and it is estimated 130-170 million people are infected with hcv worldwide [1] . acute hcv infection is spontaneously cured in 20%-30% of patients, but the majority of infected patients fail to clear the virus and develop chronic persistent infection [2] [3] [4] . in addition to a combination regimen of pegylated interferon (ifn)-α and ribavirin, direct acting antiviral drugs (daas) against hcv have been developed, and a high rate of sustained virological response (svr) has been achieved by using these antiviral drugs [5] . however, the high cost of these drugs results in limited access in developing nations where the disease burden is high; therefore, there is still a need for the development of a prophylactic vaccine. until now, the pathogenesis of hcv infection has not been clearly elucidated yet. importantly, the detailed mechanism of innate immune activation by hcv and its implications for viral persistence and treatment response have not been clearly explained. therefore, understanding hcv-host interactions and immune responses are important novel therapeutics with a higher barrier to viral resistance can be developed [6] . however, there is no established small animal model for the study of the entire life cycle of hcv infection and immunopathogenesis [7] . severe combined immunodeficiency mice grafted with human hepatocytes are the only small animals that can be infected with hcv, although they cannot exert adaptive immune responses [8] . recently, genetically-humanized mouse models are being developed to recapitulate the entire life cycle of hcv [9, 10] , but these models have restricted replication of hcv, limiting their utility. as an experimental tool, development of cell culture-derived hcv (hcvcc) systems has dramatically facilitated hcv research over the last 10 years. here, we review recent advances in the research on innate immune response in hcv infection and focus primarily on interferon response of host cells. it was not until 2005, more than 15 years after the discovery of hcv [11] , that the first efficient cell culture model of hcv became available. the identification of a clinical isolate (genotype 2a) that replicates efficiently in huh-7 hepatoma cells [12] made the first cell culture system possible. this isolate was obtained from a japanese patient with fulminant hcv infection and was called jfh-1 [13] [14] [15] . viral particles produced by the transfection of huh-7 cells with in vitro transcribed jfh-1 rna could infect naïve cells in cell culture and the liver of chimpanzees in vivo [14] . the hcv virion particles derived from the cell culture system were named "hcvcc" [13] . until now, only jfh-1 spontaneously replicates in huh-7 cells without adaptive mutations and releases infectious virus particles [14, 15] . after the discovery of jfh-1-based hcvcc system, other hcv cell culture systems with various genotypes were established. for genotype 2 cell culture systems, j6cc (genotype 2a) [16] and j8cc/dh8cc/dh10cc (genotype 2b) [16, 17] were developed. they replicated and propagated efficiently in huh-7.5 cells, although they had adaptive mutations to facilitate their replication [16, 17] . the first genotype 1a strain, h77-s, replicated and released infectious particles in huh-7 cells and immortalized human hepatocytes, although the amount of released virus was lower than jfh-1 [18, 19] . the con1 (genotype 1b) cell culture system was also reported, but a very low level of replication has also limited its utility [20] . recently, a new cell culture system of genotype 1a was developed. the tn genome with eight mutations (tncc) [21] and h77c recombinant harboring 19 mutations (h77ccc) replicated and spread efficiently in huh-7.5 cells [22] . recently, a cell culture system for infectious genotype 3a was also established by introducing adaptive mutations into the s310 strain [23] . hcvcc system has some limitations that should be considered. the most important limitation is the restricted availability of genotypes established in cell culture models. currently, hcvcc systems for genotypes 4, 5, and 6 are unavailable. for genotypes 1 and 2, only specific patient clones have been propagated in cell culture systems. it should be noted, however, that a new host factor, sec14l2 was recently reported to enable replication of non-adapted hcv in hepatoma cells [24] . new cell culture system utilizing sec14l2-expressing hepatoma cells may overcome the limited availability of hcvcc system. another limitation of the current hcvcc system is the non-polarized nature of huh-7-based cells [25, 26] . hepatocytes are highly polarized in the liver and cell-to-cell transmission takes an important part in the spread of hcv, but the current hcvcc system does not reflect the viral spread occurring in the infected liver. in addition, huh-7 cells are not fully differentiated [27] and, thus, have a defect in activation of the innate immune response by hcvcc infection [28] . in primary human hepatocytes (phhs), replication and virus production by hcvcc infection have been reported [27] , but it is difficult to obtain phhs for experimental use. immortalized human hepatocyte was reported to support hcv genome replication, virus assembly, and robust ifn response against the virus [19, [29] [30] [31] and, thus, can be used as an alternative. differentiated hepatocyte-like cells (dhcs) induced from pluripotent stem cells have also been used for hcvcc infection [32] [33] [34] . dhcs were found to mount an efficient innate immune response after hcvcc infection, including the production of chemokines and type iii ifns [33] . recently, dhcs from adipose tissue-derived human mesenchymal stem cells (at-hmscs) were used for hcvcc infection [35] , and the entry and replication of hcvcc were found to occur efficiently in dhcs from at-hmscs. hcvcc infection systems provide a unique opportunity to study innate immune responses to hcv infection. here, we focus mainly on recent advances in the study of interferon response in hcv infection. in hcv-infected cells, viral rna is sensed by retinoic acid-inducible gene i (rig-i) and melanoma differentiation-associated protein 5 (mda-5) in the cytoplasm and toll-like receptor 3 (tlr3) in the endosome, which leads to downstream signaling that results in the induction of type iii and i ifns and other inflammatory cytokines [28, [36] [37] [38] [39] . among these receptors, a role of mda-5 in hcv sensing has remained controversial for several years, and it was recently proven that mda-5 also participates in hcv sensing in the cytoplasm using hcvcc infection systems [28, 36, 40] . intracellular signals from rig-i, mda-5, and tlr3 are transmitted via mitochondrial antiviral signaling protein (mavs) and toll/il-1 receptor domain-containing adaptor inducing ifn- (trif), respectively, which leads to the interferon regulatory factor-3 (irf-3)-dependent induction of ifns and nf-κb activation in hcv-infected cells [38, 39] . similar to other viruses, hcv uses several mechanisms to interfere with the induction of ifns, particularly ns3/4a protease. ns3/4a cleaves mavs, which leads to the impairment of signaling and ifn production in response to hcv rna [41] . mavs cleavage by ns3/4a has also been confirmed in hcv-infected liver tissue [42] . ns5a also contributes to immune evasion from the host. ifn-γ expression is inhibited in ns5a-transgenic mice after adenoviral challenge, meaning that ns5a plays an important role toward establishment of chronic hcv infection [43] . despite hcv interference with the induction of ifns, ifns are endogenously produced by hcv-infected cells [28, 29, [44] [45] [46] . both genotype 2a [28, 29, [44] [45] [46] and genotype 1a [29] were reported to activate intracellular interferon signaling pathways. ifns are currently classified into three major classes: type i, type ii, and type iii. among them, type i and iii ifns are considered innate immune response ifns. among type i ifns, there are 13 ifn-αs, in addition to ifn-β, ifn-ω, ifn-ε, and ifn-κ [47] . ifn-λs (ifn-1 or il-29; -2 or il-28a; and -3 or il-28b) are a new family of ifns that have been designated as type iii ifns. since the discovery of ifn-λs in 2003, their functions have been considered to overlap with type i ifns because signaling via the ifn- receptor is similar to that via the ifn-α/β receptor. after binding to their receptors, type i and iii ifns initiate a signaling cascade through the janus kinase (jak)-signal transducer and activator of transcription (stat) pathways. the cellular actions are then mediated by the induction of interferon-stimulated genes (isgs) that have antiviral and/or immunomodulatory activity [48, 49] . recently, it was demonstrated that ifn-s are major ifns produced by hcv-infected cells [28, [44] [45] [46] . ifn-s activate the same jak-stat pathway as type i ifns [48] [49] [50] , thereby inducing a similar set of isgs. although the exact source of ifn-s in hcv-infected liver remains to be clarified, it seems that the production of ifn-s by hcv-infected hepatocytes results in the expression of isgs, presumably through autocrine and/or paracrine signaling via the ifn-λ receptor [28, [44] [45] [46] . although hcv interferes with the induction of ifns, continuous isg up-regulation in hcv-infected liver has been demonstrated in chimpanzee models [51, 52] and hcv-infected patients [53, 54] . interestingly, hcv rna and isg mrna are detected simultaneously in hepatocytes from patients with chronic hcv infection [55] . this finding suggests that hcv infection potently stimulates the production of endogenous ifns, which leads to isg up-regulation in infected liver [55] , and that hcv survives under the isg up-regulation perhaps due to the protein kinase r (pkr)-mediated suppression of isg protein translation [56, 57] . as a rapid response to type iii and i ifns, ifn stimulated gene factor 3 (isgf3), which consists of tyrosine-phosphorylated stat1 (py-stat1), tyrosine-phosphorylated stat2 (py-stat2), and irf9, mediates the induction of numerous isgs, including stat1, stat2, and irf9 themselves [47] . recently, it was demonstrated that prolonged induction of a set of isgs is mediated by unphosphorylated isgf3 (u-isgf3), which is composed of unphosphorylated stat1 (u-stat1), unphosphorylated stat2 (u-stat2), and irf9 [58, 59] . the u-isgf3 level is increased by sustained exposure to ifns, and u-isgf3 leads to enhanced expression of a set of isgs (u-isgf3-downstream isgs, u-isgs) [58] . in other words, there appear to be two phases of isg expression following type iii or i ifn stimulation. the initial rapid response is driven by the classical phosphorylated form of isgf3, which is followed by a second, more prolonged response driven by u-isgf3 [58] . in line with this report, we recently demonstrated that endogenous production of type iii and i ifns by hcv infection increases the levels of u-isgf3, composed of u-stat1, u-stat2, and irf9 proteins [60] . using hcvcc infection systems with immune-competent liver cells such as phhs and tlr3-transfected huh7 cells, we demonstrated that u-isgf3 induces the expression of u-isgs [60] . as mentioned above, isgs induced by endogenous type iii or i ifns are up-regulated in hcv-infected liver [53, 54, [60] [61] [62] . representative isgs that are maintained at high levels of expression include isg15, ifi27, ifi44, mx1, and oas-1 [53, 54, [60] [61] [62] . these isgs are regulated not only by isgf3 but also by u-isgf3, and they are mainly antiviral [58, 60] . we recently found that increased levels of u-stat1, u-stat2, and irf9 are able to inhibit hcv rna replication without exogenous ifn treatment [60] . this finding suggests that the sustained expression of isgs by u-isgf3 has antiviral activity against hcv in the infected liver but is insufficient to clear the virus. previously, it was demonstrated that patients with high levels of isgs in their liver at baseline respond poorly to combined therapy with pegylated ifn- and ribavirin [53, 54, [60] [61] [62] [63] [64] . moreover, it has been shown that increased isg expression at baseline is a stronger predictor of a poor response to pegylated ifn-/ribavirin therapy than is the il28b genotype [53] . some reports have emphasized usp18 as a critical factor conferring unresponsiveness to exogenous ifn-α treatment by suppressing intracellular signaling [65] [66] [67] [68] . however, the mechanism underlying the increase and maintenance of usp18 protein levels in hcv-infected liver has not been clearly elucidated. recently, we found that prolonged exposure to ifn- up-regulates u-isgf3 and u-isgs, including isg15, and that isg15 causes the refractoriness to exogenous ifn- treatment by stabilizing usp18 protein [60] . in 2013, the gene ifnl4 was first described [69] . ifnl4 expression is influenced by a germline dinucleotide frameshift variant located in exon 1 of ifnl4 [69] . the ifnl4-∆g allele generates the full-length ifn-λ4 protein, whereas the ifnl4-tt allele does not create ifn-λ4 due to a premature stop [69] . the ifnl4-∆g allele is associated with a poor response to pegylated ifn-α/ribavirin therapy [69, 70] , and a recent study concluded that the ifnl4-∆g/tt genotype is the primary polymorphism underlying poor treatment response in hcv-infected patients [71] . another study showed that ifnl4-∆g genotype is associated with high levels of isgs and that hepatic levels of isg15 in chronic hepatitis c are strongly associated with ifn-λ4 expression, suggesting that ifn-λ4 contributes to induction of isgs in hcv-infected liver [72] . forced expression of ifnl4 gene up-regulates isgs in phhs and hepg2 cells [69, 73] , and has antiviral effects against hcv [74] . recombinant ifn-λ4 protein activates the jak-stat pathway through binding to the ifn-λ receptor [75] , evokes similar gene expression pattern to ifn-λ3 [76] . future study using hcvcc infection systems will explain the mechanism of ifn-λ4 induction in hcv-infected cells and the effects of endogenous ifn-λ4 on both isg induction and the response to exogenous ifn-α treatment. after hcv infection, the expression of some genes is down-regulated, and the expression of those genes tends to be further down-regulated in the liver of non-responders to ifn treatment [61, 77] . one of the genes down-regulated in hcv-infected liver is dual specificity phosphatase 1 (dusp1), a mitogen-activated protein kinase phosphatase (mkp) that de-phosphorylates mitogen-activated protein kinases (mapks) [78] . we demonstrated that silencing dusp1 expression inhibits hcv replication in hcvcc-infected cells and hcv replicon cells by up-regulating antiviral isgs [77] . dusp1 silencing enhances the nuclear translocation of stat1 and causes the induction of isgs [77] . although the detailed mechanism of dusp1 down-regulation in hcv-infected liver remains to be elucidated, this serves as an example of how hosts regulate the expression of isgs and restrict viral infection while bypassing endogenous ifn production. in virus-infected cells, viral peptides are processed and loaded onto major histocompatibility complex (mhc) class i molecules and presented to viral peptide-specific cd8 + cytotoxic t cells [79] . recently, we demonstrated using an hcvcc system that ifn-induced up-regulation of mhc class i molecules is attenuated by hcv infection [57] . hcv rna activates pkr, which phosphorylates the translation initiation factor eif2α to block the translation of proteins, including isgs [56] and mhc class i [57] . the attenuated expression of mhc class i by hcv infection causes a reduction of the effector functions of hcv-specific cd8 + t cells [57] . before our study, several studies had investigated the effect of hcv proteins on mhc class i expression with conflicting results. the expression of mhc class i was not affected by overexpression of hcv proteins in one study [80] , whereas another study showedup-regulation of mhc class i expression by the hcv core [81] . by using an hcvcc model, we were able to evaluate the effect of the whole life cycle of hcv infection on mhc class i expression, and we demonstrated the attenuation of ifn-induced mhc class i expression by hcvcc infection. the isolation of the jfh-1 clone and the establishment of hcvcc infection systems made it possible to perform various studies on host-virus interactions and innate immune responses against hcv infection. now is the era of daas, and hcvcc systems have greatly contributed to the advent of the daa era. however, much remains to be resolved. above all, the precise mechanism of interferon response and its paradoxical contribution to viral persistence should be elucidated. novel cell culture models that closely mimic host responses with various clinical strains are the prerequisite for understanding the pathogenesis of hcv infection and clarifying the mechanism of viral persistence. epidemiology and natural history of hcv infection hepatitis c virus-induced hepatocellular carcinoma hepatitis c virus and hepatocarcinogenesis cd8(+) t-cell responses in acute hepatitis c virus infection current and future therapies for hepatitis c virus infection antiviral resistance and direct-acting antiviral agents for hcv animal models for the study of hepatitis c virus infection and related liver disease hepatitis c virus replication in mice with chimeric human livers completion of the entire hepatitis c virus life cycle in genetically humanized mice murine models of hepatitis c: what can we look forward to? isolation of a cdna clone derived from a blood-borne non-a, non-b viral hepatitis genome efficient replication of the genotype 2a hepatitis c virus subgenomic replicon complete replication of hepatitis c virus in cell culture production of infectious hepatitis c virus in tissue culture from a cloned viral genome robust hepatitis c virus infection in vitro robust full-length hepatitis c virus genotype 2a and 2b infectious cultures using mutations identified by a systematic approach applicable to patient strains highly efficient infectious cell culture of three hepatitis c virus genotype 2b strains and sensitivity to lead protease, nonstructural protein 5a, and polymerase inhibitors production of infectious genotype 1a hepatitis c virus (hutchinson strain) in cultured human hepatoma cells generation of infectious hepatitis c virus in immortalized human hepatocytes production of infectious genotype 1b virus particles in cell culture and impairment by replication enhancing mutations highly efficient full-length hepatitis c virus genotype 1 (strain tn) infectious culture system efficient infectious cell culture systems of the hepatitis c virus (hcv) prototype strains hcv-1 and h77 development of hepatitis c virus genotype 3a cell culture system sec14l2 enables pan-genotype hcv replication in cell culture the hcv life cycle: in vitro tissue culture systems and therapeutic targets hepatitis c virus envelope components alter localization of hepatocyte tight junction-associated proteins and promote occludin retention in the endoplasmic reticulum production of infectious hepatitis c virus in primary cultures of human adult hepatocytes hepg2 cells mount an effective antiviral interferon-lambda based innate immune response to hepatitis c virus infection hepatitis c virus infection induces the beta interferon signaling pathway in immortalized human hepatocytes hepatitis c virus genotype 1a growth and induction of autophagy hepatitis c virus infection impairs irf-7 translocation and alpha interferon synthesis in immortalized human hepatocytes human pluripotent stem cell-derived hepatocytes support complete replication of hepatitis c virus modeling hepatitis c virus infection using human induced pluripotent stem cells productive hepatitis c virus infection of stem cell-derived hepatocytes reveals a critical transition to viral permissiveness during differentiation microrna-27a modulates hcv infection in differentiated hepatocyte-like cells from adipose tissue-derived mesenchymal stem cells mda5 plays a critical role in interferon response during hepatitis c virus infection control of temporal activation of hepatitis c virus-induced interferon response by domain 2 of nonstructural protein 5a regulation of hepatic innate immunity by hepatitis c virus immune responses to hcv and other hepatitis viruses eftud2 is a novel innate immune regulator restricting hepatitis c virus infection through the rig-i/mda5 pathway cardif is an adaptor protein in the rig-i antiviral pathway and is targeted by hepatitis c virus cleavage of mitochondrial antiviral signaling protein in the liver of patients with chronic hepatitis c correlates with a reduced activation of the endogenous interferon system inhibition of intrahepatic gamma interferon production by hepatitis c virus nonstructural protein 5a in transgenic mice hepatitis c virus induces interferon-lambda and interferon-stimulated genes in primary liver cultures il-29 is the dominant type iii interferon produced by hepatocytes during acute hepatitis c virus infection hcv infection induces a unique hepatic innate immune response associated with robust production of type iii interferons interferons at age 50: past, current and future impact on biomedicine ifn-lambdas mediate antiviral protection through a distinct class ii cytokine receptor complex il28b inhibits hepatitis c virus replication through the jak-stat pathway interleukin-29 uses a type 1 interferon-like program to promote antiviral responses in human hepatocytes genomic analysis of the host response to hepatitis c virus infection stealth and cunning: hepatitis b and hepatitis c viruses interferon-induced gene expression is a stronger predictor of treatment response than il28b genotype in patients with hepatitis c interferon signaling and treatment outcome in chronic hepatitis c simultaneous detection of hepatitis c virus and interferon stimulated gene expression in infected human liver hepatitis c virus blocks interferon effector function by inducing protein kinase r phosphorylation hepatitis c virus attenuates interferon-induced major histocompatibility complex class i expression and decreases cd8+ t cell effector functions ifnβ-dependent increases in stat1, stat2, and irf9 mediate resistance to viruses and dna damage unphosphorylated stat1 prolongs the expression of interferon-induced immune regulatory genes roles of unphosphorylated isgf3 in hcv infection and interferon responsiveness hepatic gene expression discriminates responders and nonresponders in treatment of chronic hepatitis c viral infection cell-type specific gene expression signature in liver underlies response to interferon therapy in chronic hepatitis c infection hepatic gene expression during treatment with peginterferon and ribavirin: identifying molecular pathways for treatment response hepatic isg expression is associated with genetic variation in interleukin 28b and the outcome of ifn therapy for chronic hepatitis c protein isgylation modulates the jak-stat signaling pathway silencing of usp18 potentiates the antiviral activity of interferon against hepatitis c virus infection interferon induces long-lasting refractoriness of jak-stat signaling in the mouse liver through induction of usp18/ubp43 interferon-β and interferon-λ signaling is not affected by interferon-induced refractoriness to interferon-α in vivo a variant upstream of ifnl3 (il28b) creating a new interferon gene ifnl4 is associated with impaired clearance of hepatitis c virus ifn-λ4: the paradoxical new member of the interferon lambda family comparison of functional variants in ifnl4 and ifnl3 for association with hcv clearance hepatic expression levels of interferons and interferon-stimulated genes in patients with chronic hepatitis c: a phenotype-genotype correlation study prokunina-olsson, l. expression of interferon λ 4 is associated with reduced proliferation and increased cell death in human hepatic cells interferon-lambda4 is a cell-autonomous type iii interferon associated with pre-treatment hepatitis c virus burden interferon λ 4 signals via the ifnλ receptor to regulate antiviral activity against hcv and coronaviruses transcriptome analysis reveals a classical interferon signature induced by ifnlambda4 in human primary cells suppression of dual specificity phosphatase i expression inhibits hepatitis c virus replication dual-specificity phosphatases: critical regulators with diverse cellular targets mhc class i antigen presentation: learning from viral evasion strategies expression of hepatitis c virus proteins does not interfere with major histocompatibility complex class i processing and presentation in vitro upregulation of major histocompatibility complex class i on liver cells by hepatitis c virus core protein via p53 and tap1 impairs natural killer cell cytotoxicity the authors declare no conflict of interest. key: cord-001124-qcjbtflt authors: carrero, javier antonio title: confounding roles for type i interferons during bacterial and viral pathogenesis date: 2013-10-24 journal: international immunology doi: 10.1093/intimm/dxt050 sha: doc_id: 1124 cord_uid: qcjbtflt although type i interferons (ifn-i) were initially defined as potent antiviral agents, they can also cause decreased host resistance to some bacterial and viral infections. the many antiviral functions of the ifn-i include direct suppression of viral replication and activation of the immune response against viruses. in addition to their antiviral effects, ifn-i are also protective against several extracellular bacterial infections, in part, by promoting the induction of tnf-α and nitric oxide. in contrast, there is a negative effect of ifn-i on host resistance during chronic infection with lymphocytic choriomeningitis virus (lcmv) and acute infections with intracellular bacteria. in the case of lcmv, chronic ifn-i signaling induces adaptive immune system suppression. blockade of ifn-i signaling removes the suppression and allows cd4 t-celland ifn-γ-mediated resolution of the infection. during acute intracellular bacterial infection, ifn-i suppress innate immunity by at least two defined mechanisms. during francisella infection, ifn-i prevent il-17 upregulation on γδ t cells and neutrophil recruitment. following listeria infection, ifn-i promote the cell death of macrophages and lymphocytes, which leads to innate immune suppression. these divergent findings for the role of ifn-i on pathogen control emphasize the complexity of the interferons system and force more mechanistic evaluation of its role in pathogenesis. this review evaluates ifn-i during infection with an emphasis on work carried out ifn-i-receptor-deficient mice. the type i interferons (ifn-i) are an extensive family of pleiotropic cytokines that all signal through the ubiquitously expressed ifn-i receptor, termed the 'ifn-α receptor' (ifnar) (1) . the activity of these cytokines was discovered in the 1950s because of their ability to 'interfere' with viral infection (2) . molecular cloning techniques and genome sequencing have led to the identification of an extensive number of members of the ifn-i family. in mice, the two best analyzed members of this family are the cluster of 13 ifn-α subtypes and 1 ifn-β molecule. genetic ablation of ifnar (ifnar -/-) in the mouse is sufficient to prevent signaling by all members of the ifn-i family and is the biologically defining activity that groups the ifn-i molecules into one family (1) . ifnar is composed of two chains (ifnar1 and ifnar2) that coordinately activate the kinases jak1 and tyk2 (tyrosine kinase 2) upon ifn-i binding. jak1 (janus kinase 1) and tyk2 phosphorylate stat1 and stat2 that, together with interferon regulatory factor 9 (irf9), form the interferon-stimulated gene factor 3 (isgf3) complex (3) . isgf3 binds to interferonstimulated response elements to cause upregulation of over 300 genes. the type ii interferon receptor, ifngr1-ifngr2, which binds ifn-γ, the only type ii interferon, activates jak1 and jak2, leading to stat1 phosphorylation and homodimerization (4) . because of the similarities in signaling, many of the genes that are upregulated by ifn-i are also upregulated by ifn-γ, providing some degree of redundancy between the ifn-i and ifn-γ signaling pathways (5) . however, there are a large number of genes unique to ifn-i. although many of the ifn-i-specific genes have defined antiviral functions, the role of many remains unresolved. the antiviral activity of ifn-i was initially defined with conditioned supernatant inhibition of viral growth and then with purified cytokines. however, it was the generation of mice deficient in ifnar signaling that permitted examination of infections under more physiological conditions (6) . this review will focus on the role of ifn-i during murine infection with viral and bacterial pathogens. there is also extensive work on the role ifn-i in the pathogenesis of autoimmunity and cancer, and more recently, in fungal and protozoan infections (7) (8) (9) . although this work has contributed to our understanding of the biological activities of ifn-i, it is beyond the scope of this review. because of the extent of the ifn-i literature, this review will mostly limit itself to experiments that have examined viral and bacterial pathogenesis in ifnar -/mice with a particular emphasis on work that has examined lethality and pathogen burden. four major themes will be covered: first, there is a broad summary of the (generally) protective role of ifn-i in mice infected with viruses. second is the recent finding that ifn-i promote the suppression of adaptive immunity seen during chronic infection with lymphocytic choriomeningitis virus (lcmv). third, there is an examination of the divergent results that have been obtained using different bacterial pathogens. finally, there is a detailed examination of the role of ifn-i during listeria monocytogenes infection. the consensus in the field is that ifnar signaling is protective against most types of viral infection. table 1 summarizes some of the results obtained after infecting wild-type and ifnar -/mice with multiple species and strains of viruses. in most cases, the absence of systemic ifnar signaling by the mouse led to an increase in viral titer, lethality, or both compared with controls. ifn-i is important in handling all major genetic classes of viruses including single-stranded rna (ssrna; +/-stranded), double-stranded rna and double-stranded dna viruses, and acute retroviruses (ssrna-rt). the two exceptions to the strict requirement for ifnar are influenza and dengue virus infections. in the case of influenza and potentially other respiratory viruses, the type iii interferon system (which comprises ifn-λ subtypes and signals using il-10r2-ifnlr1) plays a dominant role in restricting acute epithelial cell infection, thereby limiting the requirement of ifn-i signaling (10, 11) . in dengue, ifnγ-mediated protection is dominant over ifn-i, although the combined ifnar -/-× ifngr -/mice are more susceptible than the ifngr -/mice (12) . the effects of ifn-i that limit viral infection are extensive, but several aspects are important to consider. ifn-i signaling enhances the susceptibility of virally infected cells to undergo programmed cell death, thereby limiting viral replication (4, 23) . dendritic cells (dcs) exposed to ifn-i become activated and secrete proinflammatory cytokines that lead to activation of the adaptive immune response (1) . nk cells become potent killers of virally infected cells after exposure to ifn-i (24, 25) . ifn-i has direct effects on adaptive αβ t cells and sensitizes them to activation via the tcr (26, 27) . ifn-i production by plasmacytoid dcs promotes b-cell activation and production of antiviral antibody (28, 29) . in general, these effects of ifn-i signaling are beneficial to the host, as they lead to control of viral replication and spread. the importance of host ifn-i signaling is further reinforced by viral evolution. viruses have evolved extensive immune-evasion strategies many of which center around inhibition of the host ifn-i response (30, 31) . however, the biological responses to ifn-i do not always lead to beneficial outcomes to the host. in the case of viral infections, the best studied example of the negative role of ifn-i are chronic viral infections, in particular infection with lcmv. a long-standing finding in the lcmv field is that small genetic changes can convert an acutely infective strain of lcmv (armstrong 53b) into a chronically infective strain (armstrong 53b clone 13; cl13) (32) . several hallmarks of the negative effects of chronic viral infection have been discovered using this system. mice become chronically infected with lcmv because of t-cell 'exhaustion' that prevents normal clearance (33) . several factors have been implicated in the suppression of t-cellmediated clearance of chronic lcmv; most salient among them are il-10 and pd-1 (programmed cell death 1). il-10 is known to antagonize inflammatory activation on multiple immune cell types and its neutralization prevents chronic infection with lcmv (34) . pd-1, a member of the cd28/ctla4 family of t-cell regulators, is upregulated on exhausted t cells found in chronically infected mice. its ligands, pd-1l and pd-2l, are broadly expressed and inducible by interferons (35) . the interaction of pd-1 with pd-l1 acts to limit t-cell activity during chronic infection. blockade of the pd-1-pd-l1 interaction using mabs derepresses cd8 t-cell activity and leads to enhanced adaptive immune responses to lcmv infection (36) . in two recent publications, the effects of il-10 and pd-1 in limiting the response to lcmv infection have been causally linked to ifn-i signaling (37, 38) . during the initial stages of infection with cl13, the absence of ifn-i signaling allows for an increased viral titer and delayed clearance during the acute phase of the response (6, 18) . wild-type mice control the primary infection well but become chronic carriers. ifnar -/mice also become chronic carriers albeit at higher viral loads. the cl13 strain induces higher levels of ifn-α and ifn-β than the acutely infective armstrong strain (37) . the major early producer of ifn-i are plasmacytoid dcs that are infected with the virus (37) . the presence of ifn-i is associated with a prolonged signature of interferon-inducible genes in spleen cells (38) . despite having higher early titers of lcmv, ifnar -/mice show reduced il-10 in serum and reduced pd-l1 expression on myeloid cells. in wild-type mice, blockade of ifnar signaling with neutralizing mabs replicates this effect, leading to reduction of il-10 and pd-l1. furthermore, neutralization of ifnar after the establishment of chronic infection leads to reduced viral burden (37) . therefore, ifnar plays a major role in the establishment of chronic infection with lcmv and neutralization of ifnar has therapeutic potential for patients harboring chronic viral infections. the response of ifnar -/mice to bacterial infections varies depending on the species and route of infection. table 2 summarizes some of the findings of ifnar -/mice infected with several important pathogenic bacteria. in streptococcus, escherichia coli, and helicobacter infections, ifnar -/mice have higher titers and/or lethality than wild-type controls. during brucella, francisella, salmonella, chlamydia, mycobacterium, or yersinia infections, ifnar -/mice control infection better than wild-type. the ifnar -/mice are more resistant to l. monocytogenes given systemically. a recent report has shown that during oral infection with listeria, ifnar signaling may be protective. based on these initial studies, the simplest conclusion is that ifnar signaling is beneficial during extracellular bacterial infection and detrimental during intracellular bacterial infection. type i ifn signaling provides increased protection during streptoccocus infection by promoting upregulation of tnf-α, ifn-γ and nitric oxide (49) . this is associated with restriction of bacterial growth. ifnar -/mice have bacteremia, increased systemic titers, and decreased survival. in vitro, streptococcus spp. induce ifn-β production through cell-type-specific signaling pathways (52) . strepcococcus can trigger ifn-i via the complex of irf3, stimulator of interferon genes (sting) and tank-binding kinase 1 (tbk1). in streptococcus-infected macrophages, ifn-β is induced through the irf3-sting-tbk1 complex and this is partially dependent on myd88 (ifn-i can also be produced in a myd88-independent pathway). in contrast, streptococcusinfected dcs induce ifn-β through irf5 and myd88. more mechanistic studies are still required to resolve the molecular triggers and in vivo cellular sources of ifn-i during streptoccocus infection. helicobacter pylori-infected ifnar -/mice have higher titers, but the mechanism of ifn-i action in this infection remains unresolved (50) . further work needs to be done on the extracellular bacterial infections to determine how ifn-i is protective and what distinguishes ifn-i from ifn-ii in these types of infections. following brucella infection, there is ifnar-dependent upregulation of tnf-related apoptosis-inducing ligand (trail) and splenic apoptosis that is associated with increased susceptibility to infection (39) . ifnar -/mice also express more ifn-γ and nitric oxide. in the case of salmonella enterica and chlamydia muridarum, ifn-i signaling sensitizes the infected macrophage to undergo cell death (47, 40) . prevention of macrophage cell death during s. enterica infection led to decreased bacterial titer. ifnar −/− mice infected with francisella have decreased titers and lethality compared with controls. this is attributed to the inhibitory effect of ifn-i signaling on il-17a/f expression (41) . ifnar -/mice express more il-17a/f, have an expansion of il-17 + γδ t cells and increased neutrophils at the site of infection. finally, treatment with ifn-i agonists such as poly(i:c) also promotes negative outcomes during bacterial infection. in the case of mycobacterium tuberculosis, intranasal delivery of poly(i:c) throughout the course of infection led to increased inflammatory infiltrates and necrosis of lung tissue that was dependent of ifnar signaling (53) . similar detrimental effects of poly(i:c) treatment are also seen following streptoccocus pneumoniae, staphylococcus aureus and l. monocytogenes infections (54) (see below). the first example of the detrimental effect of ifn-i during bacterial infection was discovered following l. monocytogenes infection. to date, it remains the best examined system, yielding information on the mechanisms of ifn-i induction, cellular sources and targets of ifn-i, and the nature of biological outcomes. macrophages infected with l. monocytogenes induce expression of ifn-i that is dependent on bacterial expression of the pore-forming toxin listeriolysin o (llo) (55, 56) . llo is important for the bacterial egress from the nascent phagosome to the cytosol (57) . llo alone does not induce strong levels of ifn-i production by the infected macrophage, suggesting that the presence of cytosolic bacteria is the driver of ifn-i production (56) . this is reinforced by experiments demonstrating that bacillus subtilis expressing llo gain access to the cytosol and also strongly induce ifn-responsive genes. the major molecular driver of ifn-i induction by l. monocytogenes is the cyclic dinucleotide c-di-amp (58) . the cyclic dinucleotides were initially discovered in bacteria as a second messenger system that also doubles as a pathogen-associated molecular pattern (59) . interestingly, c-di-amp is actively exported from the bacteria and the induced expression of a c-di-amp synthesizing enzyme (di-adenylate cyclase) increases ifnb1 gene expression by infected macrophages (58) . a sensor for cyclic dinucleotides has been identified as the helicase ddx41, which recruits sting, tbk1 and irf3 (see above) to drive upregulation of ifn-i genes (60) . splenic macrophages (cd11b + cd11c -pdca1 -b220 -) and tnf/inos-producing dcs (tip-dcs; cd11b + cd11c + ly6c + ) produce ifn-i following l. monocytogenes infection in vivo (61, 62) . mice lacking ccr2 expression, which do not recruit tip-dcs to the spleen, have reduced expression of ifn-α following l. monocytogenes infection (63) . to date, there is no clear demonstration that a l. monocytogenes-infected myeloid cell population is producing ifn-i in vivo. on the basis of experiments using immunofluorescent colocalization, tip-dcs appear not to be infected (62) . the work that identified ifn-i production by splenic macrophages did not evaluate the infected status of the cells (61) . therefore, at this time, the connection between the molecular mechanisms of induction and in vivo cellular sources of ifn-i cannot be definitely established. listeria monocytogenes causes apoptotic cell death of macrophages that is enhanced by ifnar signaling. within 2 h of infection, bone marrow-derived macrophages upregulate ifn-β and phosphorylate stat1 (64) . deletion of ifnar on macrophages raises their resistance to l. monocytogenesmediated killing significantly. the death induced by l. monocytogenes is dependent on bacterial expression of llo (64) . since llo is essential for virulence, it is unclear if it has a direct role in killing the infected macrophage or is only important for allowing egress of the bacteria to the cytosol. ifnar-dependent macrophage death is also found following infection of mice with l. monocytogenes (43) . a population of tnf-α-producing cd11b + macrophages is depleted following infection of wild-type mice. this population is maintained in ifnar -/mice, demonstrating a role for ifn-i in sensitization of macrophage death. it is not known at this time if the macrophages that die in vivo are infected by the bacteria. the most profound ifnar-dependent effect seen in mice infected with l. monocytogenes is the extensive depletion of white-pulp lymphocytes via apoptotic cell death (42) . in wildtype mice, apoptosis begins in the periarteriolar lymphoid sheath (t-cell area) and extends to the entire white pulp in a dose-dependent manner (65) . removal of ifnar significantly limits the number of apoptotic profiles and the extent of apoptotic death in any given white pulp (42) . treatment of t cells with ifn-α sensitizes them to llo-induced apoptosis suggesting that secreted llo may be a killer molecule in vivo (42) . additionally, ifn-i upregulate trail on nk cells and trail receptor (dr5) on the t cells and macrophages, providing a second potential mechanism for interferon-mediated lymphocyte and macrophage killing (66) . trail -/mice harbor lower bacterial burdens than wild-type counterparts, have decreased splenic lymphocyte apoptosis, and increased accumulation of myeloid cells in the spleen following l. monocytogenes infection. the reduction in lymphocyte death seen following l. monocytogenes infection is the major reason that ifnar -/mice are more resistant to infection (67) . several lines of evidence support this conclusion. first, mice deficient in lymphocytes (scid/ rag mice) are highly resistant to l. monocytogenes infection (68, 69) . second, mixed bone marrow chimeras that create mice that are ifnar + in all cells except lymphocytes are also resistant to l. monocytogenes infection (69) . this demonstrates the dominance of ifn-i signaling effects on lymphocytes. third, induction of ifn-i using poly(i:c) increases the susceptibility to infection of wild-type but not scid mice (69, 44) . finally, l. monocytogenes infection induces myeloid cell expression of il-10 that is dependent on ifnar expression by lymphocytes (69) . the upregulation of il-10 is a negative regulator of pathogen handling and il-10 -/mice are more resistant to infection despite having normal lymphocyte apoptosis (69, 70) . as an aside, the work on ifnar effects on l. monocytogenes infection was conducted on three different genetic backgrounds with different susceptibilities to infection. in all three strains-c57bl/6 (44), 129s6 (42) and balb/c (43)-ifnar signaling was detrimental to the outcome of infection. this demonstrates that ifnar effects are dominant over the genetic susceptibilities of the mouse strains to l. monocytogenes infection. further work needs to be done to determine if this applies to other bacterial infection models. the initial paradigm of the ifn-i system is that it provides antiviral protection that sometimes goes awry in certain autoimmunities. this simplified view has been replaced with a more complex and interesting role for the interferons in regulating immune responses. chronic viral infections are teaching us that while early ifn-i is important in controlling viremia, pathogens that can overcome this initial control benefit from the immune regulation that takes place following long-term interferon induction. the clinical relevance of this can be seen during infection of patients with hiv, where chronic ifn-i leads to trail-mediated t-cell death and poor disease outcome (71) . future work needs to be done to determine the applicability of ifn-i modulation as a therapeutic to important chronic human viral infections. another important area of research is the interface between viral and bacterial coinfections. the clinical importance of severe bacterial infections occurring after a primary viral infection is well established (72) . respiratory bacterial infections are more dangerous to patients when they occur following infection with viruses such as influenza and respiratory syncytial virus. this observation has been replicated in mouse models of infection (73, 74) . however, the interaction between viral and bacterial infection is not always deleterious. infection with herpesvirus induces prolonged ifn-γ production that leads to protection against infection with l. monocytogenes and yersinia pestis (75) . the main distinguishing feature between the two potential outcomes (acute versus chronic virus) and (detrimental versus beneficial) appears to center on the balance between ifn-i and ifn-γ effects. this reinforces the need to understand the molecular effects of these cytokines during bacterial infection. in the bacterial world, ifn-i were once believed to be relatively unimportant. this idea was reversed by research on l. monocytogenes. careful examination of additional bacterial infections has demonstrated that both route and tropism of bacterial infections matter in the requirement of ifn-i. future studies will be needed to determine cellular sources, molecular triggers and biological outcomes of ifn-i for many classes of bacterial pathogens. it will be interesting to see if bacteria have evolved mechanisms to manipulate the ifn-i system like some viruses do. recently, it has been shown that the balance of ifn-i and ifn-ii may be important in the outcome of human mycobacterial infections (76) . reminiscent of chronic lcmv infection, il-10 is also a key player in the ifn-i-mediated suppression of mycobacterial immunity. future work will be needed to determine if chronic ifn-i production is a common determinant of negative outcomes in infectious diseases. finally, we need a better understanding of how the genes specific for ifn-i lead to different outcomes from their close cousin, ifn-ii. national institute of allergy and infectious diseases (ai062832). type i interferons (alpha/beta) in immunity and autoimmunity the jak-stat pathway at twenty mechanisms of type-i-and type-ii-interferon-mediated signalling how cells respond to interferons systematic identification of type i and type ii interferon-induced antiviral factors functional role of type i and type ii interferons in antiviral defense type i interferon: friend or foe? type i interferons and the innate immune response-more than just antiviral cytokines interferons, immunity and cancer immunoediting lambda interferon renders epithelial cells of the respiratory and gastrointestinal tracts resistant to viral infections induction and function of type i and iii interferon in response to viral infection interferon-dependent immunity is essential for resistance to primary dengue virus infection in mice, whereas t-and b-cell-dependent immunity are less critical interferon function is not required for recovery from a secondary poxvirus infection effects of type i interferons on friend retrovirus infection alpha/beta interferons regulate murine gammaherpesvirus type i interferons and infection latent gene expression and reactivation from latency the role of alpha/beta and gamma interferons in development of immunity to influenza a virus in mice the role of interferon in influenza virus tissue tropism critical role for alpha/beta and gamma interferons in persistence of lymphocytic choriomeningitis virus by clonal exhaustion of cytotoxic t cells type i interferons are essential in controlling neurotropic coronavirus infection irrespective of functional cd8 t cells type i interferons produced by hematopoietic cells protect mice against lethal infection by mammalian reovirus theiler's virus infection of 129sv mice that lack the interferon alpha/beta or interferon gamma receptors alpha/beta interferon protects against lethal west nile virus infection by restricting cellular tropism and enhancing neuronal survival type i interferons in host defense type 1 interferons and the virus-host relationship: a lesson in detente coordinated and distinct roles for ifn-alpha beta, il-12, and il-15 regulation of nk cell responses to viral infection regulation of effector and memory t-cell functions by type i interferon type i interferon-mediated stimulation of t cells by cpg dna plasmacytoid dendritic cells promote rotavirusinduced human and murine b cell responses plasmacytoid dendritic cells induce plasma cell differentiation through type i interferon and interleukin 6 mechanisms of evasion of the type i interferon antiviral response by flaviviruses recent advances in understanding viral evasion of type i interferon molecular basis of viral persistence: a single amino acid change in the glycoprotein of lymphocytic choriomeningitis virus is associated with suppression of the antiviral cytotoxic t-lymphocyte response and establishment of persistence virus persistence in acutely infected immunocompetent mice by exhaustion of antiviral cytotoxic effector t cells interleukin-10 and the interleukin-10 receptor pd-1 and its ligands in tolerance and immunity restoring function in exhausted cd8 t cells during chronic viral infection persistent lcmv infection is controlled by blockade of type i interferon signaling blockade of chronic type i interferon signaling to control persistent lcmv infection myd88 and sting signaling pathways are required for irf3-mediated ifn-β induction in response to brucella abortus infection type i ifns enhance susceptibility to chlamydia muridarum lung infection by enhancing apoptosis of local macrophages type i ifn signaling constrains il-17a/f secretion by gammadelta t cells during bacterial infections type i interferon sensitizes lymphocytes to apoptosis and reduces resistance to listeria infection mice lacking the type i interferon receptor are resistant to listeria monocytogenes type i interferon production enhances susceptibility to listeria monocytogenes infection dynamic roles of type i and type ii ifns in early infection with mycobacterium tuberculosis the type i ifn response to infection with mycobacterium tuberculosis requires esx-1-mediated secretion and contributes to pathogenesis type i interferon induces necroptosis in macrophages during infection with salmonella enterica serovar typhimurium opposing roles for interferon regulatory factor-3 (irf-3) and type i interferon signaling during plague type i ifn signaling is crucial for host resistance against different species of pathogenic bacteria nod1 contributes to mouse host defense against helicobacter pylori via induction of type i ifn and activation of the isgf3 signaling pathway route of infection determines the impact of type i interferons on innate immunity to listeria monocytogenes type i interferon production induced by streptococcus pyogenes-derived nucleic acids is required for host protection intranasal poly-ic treatment exacerbates tuberculosis in mice through the pulmonary recruitment of a pathogen-permissive monocyte/macrophage population poly i:c enhances susceptibility to secondary pulmonary infections by gram-positive bacteria production of type i ifn sensitizes macrophages to cell death induced by listeria monocytogenes a specific gene expression program triggered by gram-positive bacteria in the cytosol listeriolysin o: a phagosome-specific lysin c-di-amp secreted by intracellular listeria monocytogenes activates a host type i interferon response innate sensing of bacterial cyclic dinucleotides: more than just sting the helicase ddx41 recognizes the bacterial secondary messengers cyclic di-gmp and cyclic di-amp to activate a type i interferon immune response characterization of the interferon-producing cell in mice infected with listeria monocytogenes a fluorescence reporter model defines "tip-dcs" as the cellular source of interferon β in murine listeriosis tnf/inos-producing dendritic cells mediate innate immune defense against bacterial infection ifn-beta increases listeriolysin o-induced membrane permeabilization and death of macrophages lymphocyte apoptosis during early phase of listeria infection in mice reduced apoptosis and ameliorated listeriosis in trail-null mice mechanisms and immunological effects of apoptosis caused by listeria monocytogenes regulation of macrophage ia expression in mice with severe combined immunodeficiency: induction of ia expression by a t cell-independent mechanism lymphocytes are detrimental during the early innate immune response against listeria monocytogenes both innate and acquired immunity to listeria monocytogenes infection are increased in il-10-deficient mice hiv-1 immunopathogenesis: how good interferon turns bad how do viral infections predispose patients to bacterial infections? type i interferon induction during influenza virus infection increases susceptibility to secondary streptococcus pneumoniae infection by negative regulation of γδ t cells viral infection augments nod1/2 signaling to potentiate lethality associated with secondary bacterial infections herpesvirus latency confers symbiotic protection from bacterial infection type i interferon suppresses type ii interferon-triggered human anti-mycobacterial responses i wish to thank dr emil r. unanue for his support and insightful discussions. the content is solely the responsibility of the author and does not necessarily represent the official views of the national institutes of health. key: cord-007013-tlvgyzft authors: chan, kok fei; carolan, louise a; korenkov, daniil; druce, julian; mccaw, james; reading, patrick c; barr, ian g; laurie, karen l title: investigating viral interference between influenza a virus and human respiratory syncytial virus in a ferret model of infection date: 2018-08-01 journal: j infect dis doi: 10.1093/infdis/jiy184 sha: doc_id: 7013 cord_uid: tlvgyzft epidemiological studies have observed that the seasonal peak incidence of influenza virus infection is sometimes separate from the peak incidence of human respiratory syncytial virus (hrsv) infection, with the peak incidence of hrsv infection delayed. this is proposed to be due to viral interference, whereby infection with one virus prevents or delays infection with a different virus. we investigated viral interference between hrsv and 2009 pandemic influenza a(h1n1) virus (a[h1n1]pdm09) in the ferret model. infection with a(h1n1)pdm09 prevented subsequent infection with hrsv. infection with hrsv reduced morbidity attributed to infection with a(h1n1)pdm09 but not infection, even when an increased inoculum dose of hrsv was used. notably, infection with a(h1n1)pdm09 induced higher levels of proinflammatory cytokines, chemokines, and immune mediators in the ferret than hrsv. minimal cross-reactive serological responses or interferon γ–expressing cells were induced by either virus ≥14 days after infection. these data indicate that antigen-independent mechanisms may drive viral interference between unrelated respiratory viruses that can limit subsequent infection or disease. viral interference is a phenomenon whereby infection with one virus limits or delays infection with a second virus. it has been described in human epidemiological studies observing viral epidemic peaks [1] [2] [3] [4] , vaccine efficacy studies [5] , studies assessing virus infections in clinical samples [6] [7] [8] , animal studies [9] [10] [11] [12] [13] and in vitro infectivity studies [14] . viral interference has been observed between a range of viruses, including between arboviruses, such as yellow fever and dengue virus [15] ; between different respiratory viruses [9, 13, 16] ; and between influenza viruses of different types [10] and subtypes/lineages [10, 11] . at a population level, respiratory virus infections may display distinct epidemic peaks. observational studies from the netherlands, france, and hong kong showed that emergence of 2009 pandemic influenza a(h1n1) virus (a[h1n1]pdm09) delayed infections with human respiratory syncytial virus (hrsv) [1, 3, 4] . influenza a virus infections also interrupted peak incidences of hrsv infections in japan during 2000-2002 [2] and in the netherlands during 2003-2012 [3] . negative associations between respiratory viruses have been reported when analyzing the proportion of coinfections with different respiratory viruses, using swab specimens from patients [6, 7, 17] . a(h1n1)pdm09 was least likely to be detected with any of the other respiratory viruses tested, including hrsv, in samples from all age groups [8, 18] . taken together, these data suggest that interference may occur between a(h1n1)pdm09 and hrsv. the ferret provides an ideal model of human influenza because animals can be directly infected with virus without adaptation and display similar disease symptoms to those in humans [19, 20] . historically, the ferret has also been used to study hrsv infection [21] [22] [23] , with recent studies assessing the pathogenesis, immunity, and transmission of hrsv [24, 25] . clinical symptoms are mild in ferrets infected with hrsv strains described to date [24, 25] . previously, we used the ferret model to demonstrate that viral interference can occur following infection with human influenza a and b viruses and will prevent, delay, or limit subsequent infection with an influenza virus of a different type, subtype, or lineage [10, 11] . notably, this effect depends on the virus combinations and the order and timing of sequential infections [10, 11, 26] . we have established complementary influenza viral dynamics models that explain these observations via the innate immune response [27] and cross-reactive adaptive immune responses [28] . ecological data suggest that infection with a(h1n1)pdm09 can prevent or delay infection with hrsv. using our ferret models of influenza and hrsv, we have systematically investigated this hypothesis. adult ferrets were housed at the peter doherty institute for infection and immunity bioresources facility. experiments were conducted with approval from the university of melbourne microbiology and immunology animal ethics committee, in accordance with the australian national health and medical research council code of practice for the care and use of animals for scientific purposes. all ferrets were seronegative for antibodies to currently circulating influenza viruses and hrsv (long and a2 strains) before use in experiments. a/tasmania/2004/2009 (a[h1n1]pdm09) virus was passaged allantoically in embryonated hen's eggs and stored at −80°c. the infectious influenza virus titer was measured by a 50% tissue culture infectious dose (tcid 50 ) assay [29] , read by hemagglutination with turkey red blood cells. hrsv long and a2 strains were passaged [24] . infectious hrsv titers were determined by plaque assay [24] . ferrets were infected intranasally with 10 3.5 tcid 50 a(h1n1) pdm09 in 500 µl and 10 5 plaque-forming units (pfu) of long hrsv or 10 6 pfu of long or a2 hrsv in 500 µl and monitored [24, 26] . ferrets were housed in pairs, by infection group. nasal wash specimens were collected and stored [24] . on the day of collection, viral rna was extracted from 140-µl nasal wash specimens for quantitative polymerase chain reaction (qpcr) analysis. blood samples were obtained from ferrets before primary virus infection and immediately before and 14 days after challenge, and serum was isolated. the proportional change in weight was calculated as the percentage difference from the weight on the day of challenge. four microliters of viral rna [24] was assayed by rt-qpcr with a(h1n1)pdm09 hemagglutinin-specific primers/probes from the cdc influenza virus rt-qpcr influenza a (h1/h3/h1pdm09) subtyping panel, obtained from the influenza reagent resource (available at: http://www.influenzareagentresource.org/) and hrsv n-specific primers/probes [24] . copy numbers for a(h1n1) pdm09 viral rna were calculated relative to plasmid phw2000-a/ tasmania/2004/2009 hemagglutinin; copy numbers for rsv rna were calculated relative to a hrsv rna standard [24] . mrna was isolated from nasal wash samples [30] . mrna expression of cytokines, chemokines, and housekeeping genes was quantified by qpcr [30, 31] . infectious hrsv in nasal wash samples was measured using the vs assay [24] . interferon γ (ifn-γ) enzyme-linked immunospot (elispot) assay ifn-γ-producing cells were detected by a ferret ifn-γ elispotplus assay (mabtech). single cell suspensions were prepared from ferret retropharyngeal lymph nodes [31] . a total of 5 × 10 4 lymph node cells were cultured with or without live influenza virus, hrsv, or 5 µg/ml concanavalin a (sigma) for 48 hours at 37 o c in 5% co 2 [11] . titers of antibodies to a/tasmania/2004/2009 were measured using hi assays [31, 32] . titers were expressed as the reciprocal of the highest dilution of serum for which hemagglutination was prevented. geometric mean titers (gmts) were calculated, with undetectable titers expressed as having a value of "5." seroconversion was defined as a titer of ≥40 at the end of the experiment and at least a 4-fold rise from baseline. titers of antibodies that neutralize hrsv long and a2 were measured using vs mn assays [24] . seroconversion was defined as a titer ≥160 at the end of the experiment and an increase of at least 4-fold from the baseline titer. antibodies that bind to the f glycoprotein of hrsv were detected by an elisa [24] . viral kinetics were assessed in viral rna from nasal wash specimens. for a(h1n1)pdm09, >10 6 copies of hemagglutinin/100 µl of nasal wash were positively correlated with replicating virus, based on the tcid 50 assay [10] and the level of infectious virus as measured by transmission in ferrets [33] . for hrsv, 10 3.8 copies of n/100 µl of nasal wash corresponded to a 50% chance of a sample being positive by the virospot assay, as determined using a probit regression model (supplementary figure 1) . accordingly, samples were considered to be infectious for hrsv when the amount of viral rna exceeded 10 3.8 copies/100 μl nasal wash and infectious for a(h1n1)pdm09 when viral rna exceeded 10 6 copies/100 µl of nasal wash for at least 1 measurement. clinical signs (ie, weight loss and fever) were assessed daily, and seroconversion was measured 14 days after challenge. statistical analysis was conducted using prism, version 6.0g, unless otherwise indicated and is described in the figure legends. ferrets were first infected with a(h1n1)pdm09 virus then challenged with hrsv 3, 7, or 11 days later, or vice versa ( figure 1a ). the intervals between inoculations spanned the times of peak titer and clearance of both virus infections [24, 30] and induction of humoral immunity ( figure 2a ). primary infection with a(h1n1)pdm09 prevented subsequent infection with hrsv in 3 of 4 ferrets when primary infection and challenge were separated by 3 days. shedding of hrsv was minimal in the single ferret infected, compared with control animals ( figure 1b and 1c) . no ferrets in this group seroconverted to hrsv (figure 2bi and 2bii ). primary infection with a(h1n1)pdm09 prevented infection with hrsv in 2 of 4 ferrets when infections were separated by 7 days ( figure 1d ). ferrets that did not shed virus did not seroconvert (figure 2bi and 2bii), while ferrets that shed virus seroconverted to hrsv (figure 2bi and 2bii). prior infection with a(h1n1)pdm09 did not prevent infection with hrsv 11 days later ( figure 1e ), with all ferrets showing a similar pattern of virus shedding ( figure 1b ) and antibody titers to control animals that received hrsv alone (figure 2bi and 2bii). the kinetics of hrsv shedding was examined in animals not protected from hrsv challenge. the peak of hrsv shedding was delayed in ferrets infected with a(h1n1)pdm09 followed by hrsv as compared to control animals infected with hrsv alone (median, 8 vs 6 days; p = .0091 by the mann-whitney test; figure 2ci ). there was no change in the duration of virus shedding ( figure 2cii ). clinical signs following hrsv challenge were minimal (supplementary figure 2) , consistent with our previous study [21] . all ferrets, except 1 control ferret infected with hrsv, maintained or gained weight (supplementary figure figure 2biv ). the median duration of a(h1n1)pdm09 shedding was increased in ferrets infected with hrsv followed by a(h1n1)pdm09 as compared to control animals infected with a(h1n1)pdm09 alone (8 vs 7 days; p = .0196 by the mann-whitney test; figure 2civ ). there was no change in the peak day of shedding (figure 2ciii ). prior infection with hrsv did reduce disease following infection with a(h1n1)pdm09. the mean maximum weight loss (±sd) among 8 control ferrets infected with a(h1n1)pdm09 was 10.6% ± 3.7% (supplementary figure 3a and 3d) . the mean maximum weight loss (±sd) for ferrets in all test groups (n = 12) was 4.1% ± 2.3% (supplementary figure 3b , 3c, and 3e). thus, prior infection with hrsv significantly reduced morbidity, as measured by weight loss, after challenge with a(h1n1) pdm09 (p = .0002 by the mann-whitney test). no fever was detected following a(h1n1)pdm09 infection (supplementary figure 3f -j). ferrets were infected (1) with an increased viral dose of the same hrsv strain, long, or (2) with an alternate hrsv strain, a2 (also at an increased viral dose), then challenged with a(h1n1) pdm09 3 days later. a2 is a laboratory-adapted strain that is shed at similar levels to long in ferrets and transmits between cohoused animals [24] . infection of ferrets with a 10-fold higher inoculum (ie, 10 6 pfu) of hrsv long led to a small increase in virus shedding on days 2-6 after infection, compared with animals infected with 10 5 pfu of hrsv long, although these differences were not significant (supplementary figure 4) . primary infection with 10 6 pfu hrsv long or a2 did not prevent infection with a(h1n1)pdm09 when infections were separated by 3 days (figure 4 ). all animals seroconverted to a(h1n1)pdm09 at similar levels ( figure 2bv and 2bvi). most ferrets lost weight after a(h1n1)pdm09 infection. the mean maximum weight loss (±sd) among 4 ferrets infected with a(h1n1)pdm09 alone was 7.1% ± 3.0%, whereas the mean maximum weight loss (±sd) for ferrets (n = 8) that received primary infection with hrsv prior to a(h1n1)pdm09 challenge was 4.9% ± 3.2% (p = .2828 by the mann-whitney test; supplementary figure 5a -c). there was no difference in fever (supplementary figure 5d -f) and no change to the kinetics of infection between animals that received a prior hrsv infection, compared with those that did not (data not shown). inflammation induced by viral infection may contribute to viral interference [10, 11] . we investigated the localized immune response following infection with hrsv or a(h1n1)pdm09. nasal wash specimens were collected early (day 2) and later (day 6/7) after infection, because the pattern of inflammatory mediators changes throughout h(h1n1)pdm09 [30] and hrsv [24] infections. expression of influenza virus matrix (m) mrna was highest on day 2 after infection, whereas expression of hrsv nucleoprotein (n) mrna was highest on day 6 ( figure 5a ). two days after infection, animals infected with a(h1n1)pdm09 had significantly higher levels of interferon β (ifn-β), granzyme b, ifn-γ, interleukin 6 (il-6), monocyte chemoattractant protein 1 (mcp-1), and tumor necrosis factor α (tnf-α) mrna as compared to animals infected with hrsv ( figure 5d, 5g-i, 5n , and 5o). expression of ifn-α and granzyme a mrna was increased but not significantly (ifn-α, p = .097; granzyme a, p = .18; figure 5e and 5f). on day 6/7 after infection, levels of granzyme closed circles and open circles indicate animals that did or did not, respectively, shed detectable challenge virus, as determined by quantitative reverse-transcription polymerase chain reaction analysis of viral rna from nasal wash (nw) samples. for statistical analysis, titers or fold changes were compared between test and control groups, using 1-way kruskal-wallis analysis of variance with the dunn multiple comparison test. *p < .05 and **p < .01. c, the kinetics of shedding was analyzed for all ferrets that shed challenge virus in figures 1 and 3 . data from ferrets obtained at the 3-day, 7-day, and 11-day intervals were pooled into the test group. the number of days from challenge inoculation to the peak level of challenge virus shedding (ci and ciii) and the number of days the challenge virus was shed (cii and civ) was determined for each ferret in the indicated groups. horizontal lines indicate median values. the number of days of virus shedding were compared between test and control groups, using the mann-whitney test. *p < .05 and **p < .01. a, granzyme b, ifn-γ, interleukin 17, and mcp-1 mrna were significantly higher in animals infected with a(h1n1)pdm09 as compared to those infected with hrsv ( figure 5f -h, 5m, and 5n). there was significant increase in expression of interleukin 8 (il-8) mrna 2 days after infection and of interleukin 1β, il-6, and il-8 mrnas 6/7 days after infection in ferrets infected with hrsv as compared to a(h1n1)pdm09 ( figure 5c, 5i, and 5j ). this suggests a localized inflammatory response was induced after hrsv infection, which coincided with the increase in hrsv virus replication ( figure 5a ). to directly compare the magnitude of expression of cytokines and chemokines induced by both virus infections, we assessed mrna expression on the day after infection at which the level of virus shedding was highest (ie, day 2 for a(h1n1) pdm09 and day 6 for hrsv). when assessed at these times, an equivalent fold change in mrna expression was observed for rsv n and influenza virus m ( figure 5a ). infection with a(h1n1)pdm09 induced significantly higher levels of ifn-β (p = .00021; figure 5d ), il-6 (p = .0088; figure 5i ), interleukin 12p40 (p = .0137; figure 5l ), mcp-1 (p = .0018; figure 5n ), and tnf-α (p = .00078; figure 5o ) mrna expression in ferrets, compared with hrsv. these data suggests there is increased inflammation in nasal tissues of animals infected with a(h1n1)pdm09 as compared to hrsv. there was no difference in expression of any cytokines or chemokines between ferrets infected with 10 5 or 10 6 pfu of hrsv long (data not shown). we have demonstrated that cross-reactive ifn-γ cellular responses can be detected between influenza b virus lineages and may contribute to viral interference [11] . thus, we assessed whether cellular immunity induced by infection with a(h1n1) pdm09 showed any cross-reactivity to hrsv. whereas retropharyngeal lymph node cells from a(h1n1)pdm09-infected ferrets were restimulated with a(h1n1)pdm09 ( figure 6b ), few cells produced ifn-γ when stimulated with hrsv ( figure 6a ). lymph node cells from hrsv-infected ferrets were restimulated with hrsv in vitro, although at much lower levels ( figure 6a ), and were not restimulated by a(h1n1)pdm09 ( figures 6b) . responses to concanavalin a were similar for all ferrets regardless of infection ( figure 6b ). moreover, there was limited serological cross-reactivity. animals infected with a(h1n1)pdm09 had high levels of influenza virus-specific neutralizing antibodies ( figure 6c ), yet minimal total serum or neutralizing antibodies to hrsv ( figure 6d and 6e) . similarly, infection with hrsv induced total serum and neutralizing antibodies to hrsv but few antibodies that were reactive with a(h1n1)pdm09 ( figure 6c-e) . discussion we have demonstrated that infection with a(h1n1)pdm09 can prevent infection and replication of hrsv in a ferret model of human disease for up to 7 days. infection with hrsv did not prevent subsequent infection with a(h1n1)pdm09; rather, animals were coinfected, albeit with reduced morbidity. infection with a(h1n1)pdm09 leads to increased levels of proinflammatory cytokines in the respiratory tract as compared to infection with hrsv. overall, these data support the ecological observation that viral interference induced by a(h1n1)pdm09 infection delayed infection with hrsv in the winter of 2009-2010. infection with a(h1n1)pdm09 induced higher expression of mcp-1, il-6, type i ifns, tnf-α, ifn-γ, and granzyme a/b mrnas as compared to hrsv infection. mcp-1 and tnf-α regulate the migration of macrophages/monocytes and natural killer (nk) cells into the respiratory tract. macrophages produce mcp-1, tnf-α, and il-6; thus, upregulation of these genes suggests an influx of macrophages and nk cells into the respiratory tissues [34] . nk cells produce ifn-γ, which activates macrophages and neutrophils and promotes t-cell proliferation and killing of virus-infected cells [34] . because cytotoxic t lymphocytes and nk cells also produce granzymes a/b, increased expression of ifn-γ and granzyme a/b mrnas on day 6 after infection suggests recruitment/activation of these cells to the site of infection. il-6 and type i ifns are produced by respiratory epithelial cells, monocytes/macrophages, and dendritic cells [34, 35] . il-6 is a proinflammatory cytokine, whereas type i ifns induce an antiviral state that may also limit replication and spread of hrsv [34, 35] it would be useful to explore the cellular infiltrate following a(h1n1)pdm09 and hrsv infections to gain further insight . ferrets were infected with 10 5 plaque-forming units (pfu) of hrsv strain long or 10 3.5 50% tissue culture infectious doses of a(h1n1)pdm09 (n = 4 ferrets/virus). nasal wash specimens were collected after challenge from ferrets on days 2 and 6 after infection, and mrna was assayed for the indicated genes, using quantitative polymerase chain reaction (qpcr) assays. for each graph, qpcr data are expressed as fold changes relative to values for nasal wash specimens from uninfected animals and normalized to atf4 and gapdh housekeeping genes. in panel a, expression of n is shown for hrsv-infected ferrets, and expression of m is shown for influenza virus-infected ferrets. for statistical analyses, inflammatory mediators were compared between (1) hrsv-infected and a(h1n1)pdm09-infected animals sampled on day 2 after infection, (2) hrsv-infected and a(h1n1)pdm09-infected animals sampled on day 6/7 after infection, and (3) a(h1n1)pdm09-infected animals sampled on day 2 and hrsv-infected animals sampled on day 6 after infection. fold changes were compared between viruses, using the mann-whitney u test. ifn, interferon; il-1, interleukin 1; il-6, interleukin 6; il-8, interleukin 8; il-10, interleukin 10; il-12p40, interleukin 12p40; il-17, interleukin 17; mcp-1, monocyte chemoattractant protein 1; tnf-α, tumor necrosis factor α. *p < .05, **p < .01, ***p < .001, and ****p < .0001. into potential differences in the level and cellular composition present in local inflammation. infection with a(h1n1) pdm09 induced a 7-fold higher cellular ifn-γ recall response as compared to infection with hrsv in our study. because there was no significant difference in ifn-γ responses to concanavalin a between the groups, this observation was not due to a difference in overall t-cell numbers but, instead, was due to an increase in the reactivation of a(h1n1)pdm09-specific cells. taken together, these data suggest that infection with a(h1n1)pdm09 induces a robust cytokine and chemokine response that strongly stimulates the adaptive and memory immune responses. conversely, infection with hrsv elicited a weaker and more limited cytokine and chemokine response that led to a reduced antigen-specific cellular response. however, it is possible that hrsv may not infect the ferret respiratory tract as efficiently as a(h1n1)pdm09 does, and this could result in reduced inflammatory responses. yet, infected animals seroconverted at titers consistent for sterilizing immunity, indicating a productive infection (data not shown) [24] . furthermore, increasing the inoculum of hrsv did not significantly affect the pattern or amount of virus shedding nor the expression of inflammatory mediators, suggesting that the hrsv level was already maximal in this ferret model. notably, increased expression of inflammatory mediators following infection with influenza virus as compared to hrsv has been observed in studies assessing human clinical samples and in vitro airway epithelial cell cultures [36] [37] [38] [39] . what is the mechanism of viral interference induced by a(h1n1)pdm09? the increased antiviral state and inflammation observed after a(h1n1)pdm09 infection has the potential to prevent subsequent infection or delay shedding of hrsv, as was observed here. both viruses predominantly infect ciliated airway epithelial cells, and we have shown that a(h1n1)pdm09 and hrsv long replicated in the upper and lower respiratory tracts of ferrets [24, 30] . infection with a(h1n1)pdm09 can also prevent infection with an influenza b/yamagata virus [10] . there are minimal shared epitopes between influenza a and b viruses [40] , and we showed that minimal cross-reactive ifn-γ-producing cells were induced between hrsv and influenza virus. these data suggest that short-lived mechanisms drive this effect, as no effect was detected in ferrets after one week or, as shown by others, in mice, when infections were separated by 35 days [41] . the timing of interference indicate that interactions between different viruses may also be important. it is possible that different mechanisms act on different virus combinations. gene expression analysis of early markers of the immune response (cona; b) . the number of interferon γ (ifn-γ)-producing cells was determined by an enzyme-linked immunospot (elispot) assay. c-e, sera were tested for antibodies to a(h1n1)pdm09, by a hemagglutination inhibition (hi) assay (c); for total serum antibody binding to hrsv f protein, by an enzyme-linked immunosorbent assay (elisa; d); and for neutralizing serum antibody to hrsv long or a2, by a virospot microneutralization (mn) assay (e). data were obtained from 2 ferrets per group. of respiratory epithelium infected with the virus strains used in these studies may provide further insight. it is notable that infection with hrsv reduced morbidity induced by a(h1n1)pdm09 infection. although virus loads were not decreased in nasal wash specimens, virus shedding may be reduced in the lower respiratory tract, limiting clinical disease. il-8 mrna expression was elevated in nasal wash samples of ferrets infected with hrsv as compared to a(h1n1) pdm09. increased il-8 expression has been associated with milder disease in ferrets infected with pathogenic influenza virus strains, potentially mediated by rapid recruitment of neutrophils, which assist in clearing virus [42] . analysis of the lung influenza virus loads in animals that have been infected with hrsv prior to challenge with a(h1n1)pdm09 would be of interest. epidemiological data reported in france described a 3-4week delay in the peak incidence of hrsv infections following the emergence of the a(h1n1)pdm09, compared with previous years [1] . similarly, a delay of 2-4 weeks of the expected peak of hrsv was reported following an early influenza a season in the netherlands. [3] . these population-level observations of viral interference arise from the interplay between (1) immunodynamics (ie, host-level viral interference), (2) heterogeneity between hosts (ie, differences in immunity to virus strains between individuals), and (3) transmission dynamics (ie, within or between different age groups) [43] . for influenza, these processes have been investigated in some detail. others have demonstrated that a short period (ie, days rather than weeks) of viral interference at the host level may result in substantial separation between epidemic waves at the population level [43] . our results provide the first hostlevel immunodynamic evidence in support of these processes driving the epidemiological interactions observed previously in europe [1, 2] . our study has limitations. we used a circulating strain of a(h1n1)pdm09 from early 2009 and laboratory strains of hrsv, long and a2. the long and a2 strains induce consistent infections and disease in ferrets, with characterized cytokine profiles [24] . use of a circulating clinical isolate of hrsv may provide more-realistic data but was not available for these experiments. interference between influenza virus and hrsv has been reported in epidemiological studies in various years [2, 3] , suggesting that influenza viruses other than a(h1n1)pdm09 may also prevent/limit infection and replication of hrsv. there are currently no licensed vaccines for hrsv. identification of host-and/or virus-encoded factors that contribute to viral interference provides a platform to facilitate development of novel prophylactic or therapeutic strategies to prevent or ameliorate respiratory infections, such as hrsv infection, that have a significant burden in the community, especially among young children. impact of the 2009 influenza a(h1n1) pandemic wave on the pattern of hibernal respiratory virus epidemics the clinical features of respiratory syncytial virus: lower respiratory tract infection after upper respiratory tract infection due to influenza virus early occurrence of influenza a epidemics coincided with changes in occurrence of other respiratory virus infections impact of the 2009 h1n1 pandemic on age-specific epidemic curves of other respiratory viruses: a comparison of pre-pandemic, pandemic and post-pandemic periods in a subtropical city increased risk of noninfluenza respiratory virus infections associated with receipt of inactivated influenza vaccine interference between respiratory syncytial virus and rhinovirus in respiratory tract infections in children epidemiology of multiple respiratory viruses in childcare attendees respiratory viral infections during the 2009-2010 winter season in central england, uk: incidence and patterns of multiple virus co-infections virus interference between h7n2 low pathogenic avian influenza virus and lentogenic newcastle disease virus in experimental co-infections in chickens and turkeys interval between infections and viral hierarchy are determinants of viral interference following influenza virus infection in a ferret model evidence for viral interference and cross-reactive protective immunity between influenza b virus lineages protective heterologous antiviral immunity and enhanced immunopathogenesis mediated by memory t cell populations specific history of heterologous virus infections determines anti-viral immunity and immunopathology in the lung inhibition of influenza a virus replication by influenza b virus nucleoprotein: an insight into interference between influenza a and b viruses viral interference and persistence in mosquito-borne flaviviruses dual infections of feeder pigs with porcine reproductive and respiratory syndrome virus followed by porcine respiratory coronavirus or swine influenza virus: a clinical and virological study rhinoviruses delayed the circulation of the pandemic influenza a(h1n1) 2009 virus in france protection of mice against lethal challenge with 2009 h1n1 influenza a virus by 1918-like and classical swine h1n1 based vaccines the ferret as a model organism to study influenza a virus infection ferret models of viral pathogenesis experimental infection with respiratory syncytial virus in several species of animals efficacy of adenovirus-vectored respiratory syncytial virus vaccines in a new ferret model the pathogenesis of respiratory syncytial virus infection in infant ferrets pathogenesis, humoral immune responses and transmission between co-housed animals in a ferret model of human rsv infection ferrets as a novel animal model for studying human respiratory syncytial virus infections in immunocompetent and immunocompromised hosts multiple infections with seasonal influenza a virus induce cross-protective immunity against a(h1n1) pandemic influenza virus in a ferret model innate immunity and the inter-exposure interval determine the dynamics of secondary influenza virus infection and explain observed viral hierarchies modelling cross-reactivity and memory in the cellular adaptive immune response to influenza infection in the host mdck-siat1 cells show improved isolation rates for recent human influenza viruses compared to conventional mdck cells characterisation of the localised immune response in the respiratory tract of ferrets following infection with influenza a and b viruses taqman real time rt-pcr assays for detecting ferret innate and adaptive immune responses world health organization. global influenza surveillance networkmanual for the laboratory diagnosis and virological surveillance of influenza antigenic drift of the pandemic 2009 a(h1n1) influenza virus in a ferret model a question of self-preservation: immunopathology in influenza virus infection viral interference between hrsv and influenza virus monocyte chemoattractant protein-1 (mcp-1): an overview respiratory syncytial virus infection: immune response, immunopathogenesis, and treatment interferon production in children with respiratory syncytial, influenza, and parainfluenza virus infections plasticity and virus specifcity of airway epithelial cell immune response during respiratory virus infection interferon in nasal secretions from infants with viral respiratory tract infections cross-reactive human b cell and t cell epitopes between influenza a and b viruses influenza virus lung infection protects from respiratory syncytial virus-induced immunopathology severe seasonal influenza in ferrets correlates with reduced interferon and increased il-6 induction does homologous reinfection drive multiple-wave influenza outbreaks? accounting for immunodynamics in epidemiological models supplementary materials are available at the journal of infectious diseases online. consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author.notes key: cord-007654-lchdm4xr authors: liu, yang; king, nicholas; kesson, alison; blanden, robert v.; müllbacher, arno title: flavivirus infection up-regulates the expression of class i and class ii major histocompatibility antigens on and enhances t cell recognition of astrocytes in vitro date: 2002-12-11 journal: j neuroimmunol doi: 10.1016/0165-5728(89)90171-9 sha: doc_id: 7654 cord_uid: lchdm4xr west nile virus (wnv) infection of astrocytes can up-regulate their expression of both class i and class ii major histocompatibility complex (mhc) antigens as determined by flow cytometry with monoclonal antibodies specific for class i and class ii mhc antigens. the up-regulation of class i mhc antigen expression could be partly caused by interferon secreted after wnv infection because the synthetic interferon inducer polyinosinic-polycytidylic acid (poly i : c) has similar effects. in contrast the up-regulation of class ii mhc antigen expression was not induced by poly i : c. the increased mhc antigen expression by wnv infection had significant effects on t cell recognition. thus, wnv and influenza virus a/wsn double-infected astrocytes but not astrocytes infected by a/wsn alone were lysed by influenza virus-immune cytotoxic t cells. similarly, wnv-infected astrocytes were better stimulators than normal astrocytes for a class ii mhc-reactive t cell line, both in terms of t cell proliferation and interleukin release. it is well established that the major histocompatibility complex (mhc) antigens form an essential part of antigenic structures recognised by t iymphocytes (reviewed by zinkernagel and in the case of experimental allergic encephalitis of rats, where transfer of syngeneic t cells specific for myelin basic protein (mbp) results in full expression of the disease (lipton et al., 1976; tourtellotte et al., 1978; kreth et al., 1982; booss et al., 1983; wekerle et al., 1985) . the paradox has been partly resolved by findings which indicate that at least some cells from the cns, such as astrocytes, ofigodendrocytes, brain endothelial cells and neurons can be induced to express class i and/or class ii mhc antigens by lymphokines such as "t-interferon (t-ifn) and by certain viral infections such as murine hepatitis virus and theiler's murine encephalomyelitis virus (hirsch et al., 1983; massa et al., 1986; suzumura et al., 1986; rodriguez et al., 1987) . additional data suggested that such elevation of the mhc antigens on astrocytes and/or brain endothelial cells can potentiate the antigen-presenting capacity of these cells male et al., 1987) . thus, the induction of expression of mhc antigens on the cells of the cns could be an important regulatory mechanism in the initiation, amplification and targeting of the local immune response. the etiology of a number of neurological disease, most notably, multiple sclerosis, still remains a matter of debate. the preferred hypotheses involve an autoimmune phenomenon in which cellular and/or humoral immune responses are directed against constituents of myelin. however, epidemiological data implicate some kind of acquired factor, most likely an infection, in the etiology of multiple sclerosis (waksman et al., 1986) . in recent years, evidence has accumulated that viral infections can result in the breakdown of immunological tolerance. thus both autoreactive b cells (haspel et al., 1983a, b) and autoreactive t cells (pfizenmaier et al., 1975; komatsu et al., 1978) have been observed during viral infection. in this context it would be of interest to study the possible effect of neurotropic viral infection on the local immune response in the cns. flaviviruses are one group of viruses which are responsible for a number of neurological diseases (monath, 1986) . in this study, we address the effect of west nile virus (wnv) infection on astrocytes in vitro. west nile virus sarafend strain (wnv) was obtained from dr. i.d. marshall and grown either in vero cells (wnv-v) or in new-born mouse brain (wnv-b). the stocks were prepared and titrated as described (reed and muench, 1938; taylor and marshall, 1975) . influenza virus a/wsn was prepared by a standard method (yap and ada, 1977) . mice were bred under pathogen-free conditions at the animal breeding establishment of the john curtin school of medical research. the strains used were: cba/h (h-2k), balb/c (h-2d), b10.t(6r) (kqiqdd), b10.aqr (kqlkdd). newborn mice were used at 1 or 2 days of age and adult female mice at 6-8 weeks. astrocytes were prepared in essentially the same way as has been described (mccarthy and de vellis, 1980) . briefly, 1-to 2-day-old cba/h mice were anesthetized at -20°c for 15 min. after careful removal of the meninges, the brains were broken down by pipetting and were then subjected to treatment with trypsin/dnase i solution (0.1~ trypsin and 0.001~ dnase i, w/v) for 20 min, shaken occasionally. the action of trypsin was stopped by adding equal volumes of neural medium (dulbecco's minimum essential medium (dmem) supplemented with l-arginine 12 mg/l, l-asparagine 36 mg/l, folic acid 6 mg/l, v-glucose 7 g/l, and non-essential amino acids 1~ v/v (flow laboratories, . the suspe-sion was centrifuged and the pellet was dispersed. these cells were cultures in 80 cm 2 nunclon plastic flasks at a density of 106/ml. at day 8, the cells were confluent and the medium was changed. one day later, the flasks were shaken for 15-18 h (37°c, 250 rpm). the medium was then sucked off and the cultures were washed with puck's saline, trypsinized into single-cell suspensions and cultured again. the secondary cultures were tested by indirect immunofluorescence microscopy with rabbit anti-human glial fibrillary acidic protein (gfap) serum (dako, code a561) and shown to he more than 95% gfap positive. the secondary cultures were used in this study. after astrocytes were grown to confluency (approximately l0 s cell/well in linbro 24-well plastic iissue culture plates), the medium was sucked off and west nile viruses (100 plaque-forming units (pfu)/cell of wnv-b and 20 pfu/cell wnv-v, respectively) were added in 100/li and incubated at 37 o c for i h. at different times after inoculation, cells were harvested and frozen at -20 ° c. free virus was obtained by freeze-thawing plus ultrasonication as described (taylor and marshall, 1975) . the protocol is essentially the same as described by de clercz (1981) . briefly, astrocytes were grown in 24-well tissue culture trays until confluency; the cultures were then exposed to poly i:c (sigma, 25/lg/ml) for 8 h and washed with dmem medium 3 times. fresh neural medium was added and the cultures were further incubated for 40 h. the supernatants were harvested and ifn activity determined. ifn was assayed by inhibition of viral rna synthesis in l929 cells as described by morris et al. (1987) . the titer of ifn was defined as reciprocal of the final dilution that gave 50% inhibition of semliki forest virus rna synthesis. 100/11 of 1 : 20 dilution of supernatants from 48 h wnv-b-infected astrocytes were incubated with an equal volume of medium or 100 units of rabbit antiserum to mouse ifn (a +/]) (lee biomedical research, cat. no. 21031) at 37°c for 1 h. the ifn activity of these supernatants was titered as described above. spleen cells from b10~t(6r) mice (kqlqd d) were stimulated with those from b10.aqr (kqlkd d) as described (king et al., 1986) for 159 three passages. at the fourth passage, an enriched medium for t cell lines (dmem supplemented with 2 x 10 -4 m 2-mercaptoethanol, 10~ fetal calf serum (fcs) and 5% concanavalin a (cona)-activated spleen cell supernatant (cs) prepared as described by sinickas et al (1985a) ) was added together with allogeneic stimulators. generation of secondary cytotoxic t cells against influenza virus a/wsn and primary allo-reactive cytotoxic t cells has been described in detail (yap and ada, 1977; king et al., 1986) . the method for the l929 target system has been described (yap and ada, 1977) . wnv-v-infected or uninfected astrocytes were cultured in flat-bottomed tissue culture plates at 37°c for 48 h. these cultures were then reinfected with influenza virus a/wsn (3 eid~o/cell) and labeled with 51cr by adding 25 /~1 medium containing 6 x 104 eids0/well influenza virus a/wsn and 1:10 dilution of na2slcr204 and incuba~,a at 37°c for 1 h. the rest of the assay was carried out in the same way as that for l929 targets. the proliferation of the h-2ik-reactive t cell line was determined by [3h]methyl-thymidine (amersham) incorporation as has been described (sinickas et al., 1985b) . both spleen cells and astrocytes were used as stimulators. after growth to confluency in 96-well tissue culture plates (approximately 2 × 104 cells/well), the astrocytes were irradiated with 6000 r from a e°co v-ray source. 10/~g/ml of indomethacin was added to the enriched medium of t cell lines and 150/tl of this medium per well was added together with 2 x 104 cells of an h-21k-reactive t cell line. 24 h later, 0.5 ;tci/well of the [3h]methyl-thymidine was added. after another 24 h, the cultures were harvested onto glass fiber filter strips and the incorporation of label into dna was determined by scintillation counting. wnv-infected or mock-infected astrocytes grown to confluency in 96-well plates (2 x 104/ well) were irradiated with 2000 r and were used as stimulators of 2 × 104 t cells in 0.2 ml dmem medium per well supplemented with 10 -4 m 2mercaptoethanol, 10 #g/ml indomethacin and 10% fcs. 24 h later the supernatants were collected and their interleukin activity was tested, as described by lafferty et al. (1980) . it is now known that this assay can measure interleukin-2 (il-2) and/or imerleukin-4 (il-4) activity (severinson et al., 1987) . moneclonal antibodies (mcabs) specific for d k, i-a k and i-a a antigens were obtained from the supernatants of cultures of hybridoma clones 15-5-5s, 11-5.2.1.9, and mk-d6 (oi et al., 1978; ozato et al., 1980; kappler et al., 1981) (atcc nos. hb-24, t1b-94 and hb-3), respectively, obtained from the american type culture collection. the supernatants were concentrated 10-fold on an american p10 membrane. in adoition, an mcab specific for k k (clone 11-4.1) was purchased from becton-dickinson. rabbit anti-mouse immunoglobulin (ramig) was raised in a new zealand white rabbit, separated from whole serum using a protein a-sepharose column (pharmacia) and conjugated to fluorescein-isothiocyanate (fitc) by standard methods ((3oding, 1976) . astrocvtes ~ ere infected with wnv or treated with recombinant ~-ifn (500 u/ml) or poly i : c for 48 h. s:,ngle-cell suspensions of astrocytes were obtained from the culture vessel by gentle trypsinization. the cells were counted and viability, as measured by trypan blue dye exclusion, was invariably > 95~. all subsequent steps of the labeling procedure were carried out at 4 ° c. samples of 3 × l0 s astrocytes were incubated in 100 /l1 of anti-mhc antibody for 60 rain. after the first antibody incubation the cells were centrifuged through a bed of 500 pl fcs, the supernatant removed, and the astrocytes resuspended in 100/~1 of ramig-fitc for a further 60 min at a dilution giving saturation labeling (determined by prior titration). following this incubation, the cells were centrifuged through an fcs bed and resuspended in 250 #1 dmem for flow cytometry. fluorescence was measured using a facs iv (beckton-dicldnson) equipped with an argon ion laser set at the standard excitation wavelength of 488 nm for fitc. emitted fluorescence between 515 and 540 nm was measured. 3 × 104 cells were analyzed from each labeled sample. in no experiment did any of the astrocyte sample populations show changes detectable by lowangle (cell size) or right-angle (membrane configuration) scatter analysis on the facs. thus we presume that differences in the mhc fluorescence distributions between astrocyte groups reflect a ~:hange in the cell surface mhc antigen concentration. astrocytes grown to confluency in 24-well tissue culture plates were infected with either 100 pfu/cell of wnv-b or 20 pfu/cell of wnv-v, and viral titers at different times after infection were determined. titers dropped about 100-fold within 12 h (table 1) . however, a clear increase in viral titers was observed by 36 h. the titers reached a plateau at 48 h after infection and remained at a similar level for at least 1 week. further experiments revealed that viral antigens became detectable after 24 h by immunofluorescence microscopy and by 40 h 50-60~ of the astrocytes were expressing detectable viral antigen. the proportion of viral antigen-positive cells remained at a similar level for at least 2 weeks. these data show that wnv can productively infect some cells in astrocyte populations. astrocyte cultures, either infected with wnv or treated with poly i:c, were tested for their ability to produce ifn. high titers of ifn were detected in the supernatant of astrocytes infected by wnv-b or wnv-v, or treated by poly i : c, all for 48 h ( table 2 ). the ifn titers were 4-fold 7.0 6.5 60 6.7 6.5 72 6.7 6.3 84 6.7 6.5 96 6.5 6.0 144 6.5 6.5 • west nile virus prepared from mouse brain. b west nile virus prepared from verb cell line. lower in the supernatant of poly l:c-treated astrocytes than that of wnv-infected astrocytes. furthermore, the ifn produced in wnv-b-infected astrocytes can be neutralized by antibody specific for ifn (a + ~8), hence the ifn produced is a-and/or p.ifn (fig. 1) . the expression of class i mhc antigens on cba/h astrocytes was investigated by flow cytometry. the single-cell suspensions were stained with mcabs specific either for h-2d k or h-2k k. after the astrocytes were infected by wnv-v for 48 h, the expression of h-2k ~ as determined by peak fluorescence increased by more than 2-fold compared to mock-infected astrocytes (fig. 2a) . the binding of the mcab to the h-2k k was specific as an isotype control mcab specific for h-21-a d (hb-3) did not bind significantly to either normal or wnv-infected astrocytes. similar enhancement was observed in h-2d k expression (fig. 2b) . as both wnv-b and wnv-v are potent ifn inducers (table 2) , the question arose as to whether the elevation in mhc antigen expression was due *.o the ifn produced. to investigate this question a synthetic ifn inducer, poly i:c was used. as shown in fig. 2c , poly i : c treatment can significantly enhance the expression of class i mhc antigens, which admits the possibility that the enhanced expression after wnv infection is at least partly the result of virus-induced ifn. as class i mhc antigens are the major restricin each case astrocytes were treated for 48 h. virus-immune tc (table 3 ). in contrast, astrocytes infected by influenza virus or wnv alone were not lysed by influenza virus-immune tc more than normal astrocytes. the activity of the tc was confirmed as *.hey lysed influenza virus-infected l929 (h-2 t) cells, but not mock-infected l929 cells. this result revealed that wnv infection enhances mhc-restricted virus-specific tc recognition on astrocytes. lysis of wnv-infected astrocytes by balb/c anti-cba/h alloreactive t cells was significantly higher than that of normal astrocytes while comparable to the lysis of y-ifn-treated astrocytes (fig. 3) , hence wnv infection enhances ulloreactive tc recognition on astrocytes. the expression of cell surface cl~s ii mhc antigens on cba/h astrocytes was also determined by flow cytometry. astrocytes were labeled with h-2i-ak-specifi¢ mcab tib94; control cells were treated with hb-3, an mcab specific for h-21-a d. normal cba/h (h-2 k) astrocytes 163 express little or no detectable class ii mhc antigens, as their binding to tib94 (h-21-ak-speeific) is not significantly higher than to hb-3 (h-2i-a dspecific) (fig. 4a) . however, after the astrocytes were infected by wnv-b for 48 h, a distinct increase in expression of h-21-a k antigen was detected fig. 4b) , although the amount of h-2i-a k expressed on wnv-infected astrocytes was not as , and astrocytes treated with "g-ifn (c). c: as for b, but normal astrocytes (a) and astrocytes treated with poly 1 : c (b). d: as above but astroq,'tes infected by wnv-v. ast.~ocytes were subject to pre-treatment with wnv or ifn or poly i : c for 48 h before labeling. much as that on y-ifn-treated astrocytes. the specificity of the labe~ng was ascertained in two ways: (a) hb-3 (anti-h-2i-a °) mcab dad not bind wnv.infected cba/h (h-2 k) astrocytes (fig. 2a) ; (b) as hb-3 is of the igg2a subclass, while tib~ is igg2b, another igg2b mcab specific for murine cd4 was tested and shown not to bind either wnv-infected or normal astrocytes (data not shown). it seems unlikely that material other than wnv in wnv-b (e.g. murine ifn) is responsible for the up-regulation of class ii mhc antigen, as wnv-v that was passaged 4 times in veto cells (a permanent cell line originating from monkey kidney that does not secrete murine ifn) also consistently up-regulated class ii mhc antigen, though to a lesser extent than wnv-b in this particular experiment (fig. 4d) . furthermore, poly i :c, which "nduced ifn production and enhanced class i mhc antigen expression in astrocyte cultures (fig. 2c , table 2 ) dad not induce class ii mhc ant/gen expression (fig. 4c) . the mechanism of the induction of class ii mhc antigen is at present unknown. an alloreactive b10.t(6r) anti-b10.aqr t cell line specific for h-2i k antigens was used to de-table 4 termine if the wnv-induced expression of class ii mhc antigens on astrocytes can be recognised by t cells. the t cell line was induced to proliferate by spleen cells from mice bearing h-2i k antigens, e.g. b10.aqr and cba/h mice, but not by antologous b10.t(6r) spleen cells (table 4 , expt. 1). wnv-infected cba/h astrocytes also stimulated the proliferation of the h-2ik-reactive t cell line (table 4 , expt. 2). the stimulation was comparable to cba/h spleen cells; mock-infected astrocytes had no stimulation effect. to test whether the induced stimulation effect of astrocytes was due to induced ¢lass ii mhc antigens on their surface, the h-2i-ak-specific mcab tib94 was added. tib94 blocked the proliferation of the h-21k-specific cell line stimulated by either b10.aqr spleen cells or by wnv-v-infected cba/h astrocytes ( wnv-infected and uninfected astrocytes were also compared for their ability to stimulate il-2 and/or il-4 release from the h-2ik-specific t cell line described above. wnv-infected cba/h astrocytes stimulated significant release of il-2 and/or 1l-4, whereas uninfected cba/h astrocytes were, much poorer stimulators (fig. 5) . the quantity of il-2 and/or il-4 released was about 25-fold higher with wnv-infected astrocytes than with normed astrocytes. specificity of the stimulawnv-infected cba/h astrocytes stimulated significantly il-2 and/or il-4 release from the primary h-2ik-reactive t cell population, while co!s the normal astrocytes induced essentially no il-2 and il.4 release (fig. 6 ). since it was established that mhc antigens formed an important part of the antigenic struc-165 ture recognised by t lymphocytes (zinkernagel and doherty, 1979; schwartz, 1986) , there "has been great interest in the regulation of mhc antigen expression on potentjal antigen-presenting cells. it is well documented that different virus infections may either inhibit or enhance the expression of mhc antigens, notably, infection by adenovirus (f~i~lbo et al., 1986) , herpes simplex virus type 2 (jennings et al, 1985) , ectromeiia virus (gardner et al., 1975) and measles virus (rager-zisman et al., 1981) can down-regulate class i mhc antigen expression of their host cells and may make the latter less susceptible to cytotoxic t cells. on the other hand, murine hepatitis virus (massa et al., 1986; suzumura et al., 1986 ), epstein-barr virus (mccune et al., 1975 , moloney murine leukemia virus (flyer et ai., 1985) and theiler's murine encephalomyelitis virus (rodriguez et al., 1987) can up-regulate class i and/or class ii mhc antigen expression. our results described here revealed that flavivirus infection can up-regulate the e:~pression of cell surface class i and class 1i i~,~hc antigens. it has been docuraented that interferon can up-regulate class ! and ii mhc antigen. all three classes of ifn l~own (a, t, ~,) have been reported to enhance ,class i mhc antigen expression, while only ~,-ifn can up-regulate the expression of class ii mhc ~mtigens ovong et al., 1985) . in our experiment, any treat~ent that can induce ifn production, e.,g. wi,4v-b and wnv-v infection or poly i : c treatmen~,, can up-regulate class i mhc antigens; it is possible ~hat the up-regulation of class i mhc antigen expression by wnv infection is at least patti3, the result of ifn produced after virus infection. in contrast, poly i:c treatment of astrocytes, which induces good ifn production and increases class i mhc antigen expression equal to or more than wnv infection, fails to induce class ii mhc antigen expression. this finding together with the data that ifn produced in wnv-b-infected astrocyte culture can be neutralized by anti-(a + fl)-ifn antibody strongly suggests that the induction of class ii mhc antigen by wnv does not depend on ifn produced. addition of antibodies that neutralize y-ifn to wnv-infected astrocytes culture would help to answer the question if free v-ifn was involved in class ii mhc antigen induction. however, the question as to what extent virus replication per se and induced interferon actually contribute to enhanced class i mhc expression is more problematic. some obviol:s approaches used by others (massa et al., 1986) , i.e. use of ultraviolet-inactivated virus and neutralizing mcab to the virus cannot provide definile information, because it is not known to what extent uv inactivation will inhibit viral protein and nucleic acid synthesis and the mechanism of viral neutralization is at present not well understood. furtherraore, addition of ifn antibody may not help to exclude the role of ifn due to the autocrine activity without the need of secretion (sanceau et al., 1987) . it seems unlikely that the binding of h-2i-a k-speo_p._c mcab to wnv-infected but not normal astrocytes of cba/h(h-2 k) was due to the molecular mimicry by a viral product of an epitope on the i k molecule, because an mcab specific for h-21-a d can bind to wnv-infected astrocytes from balb/c(h-2 d) mice but cannot bind to wnv-infected astrocytes from cba/h(h-2 k) mice; wnv can. repucate productively in astrocytes from both strains of mice (data not shown). a number of studies have shown that treatment which up-regulates the expression of cell surface class i mhc antigens increases the susceptibility of cells to lysis by appropriately sensitised tc cells, probably due to recognition by low-affinity clones that could not exhibit cytotoxic activity without the elevation of mhc antigen expression (shimoukevitz et al, 1985) . infection by viruses that down-regulate cell surface mhc antigen expression, on the other hand, results in reduced susceptibility of the infected cells to lysis by virus-specific cytotoxic t cells. thus it has l~een shown that herpes simplex virus type 2 infection, which down-regulates cell surface mhc expression, reduces the susceptibility of an sv40-and hsv2-double-infectezl targets to lysis by sv40specific tc cells, as compared to target cells infected with sv40 alone (jennings et al., 1985) . adenovirus type 5 infection inhibits the cell surface expression of class i mhc antigen via the e3 protein. this phenomenon results in less efficient recognition by adenovirus-specific tc cells of target cells infected by this wild-type virus compared to target cells infected by an e3 deletion mutant which has no effect on host mhc antigen expression (miillbacher and bellett, personal communication) and could be responsible for the tumorigenicity of the wild-type virus (eager et al., 1985) . a moloney murine leukemia virus that increases cell surface class i mhc antigen expression was also reported to increase the susceptibility of host cells to lysis by alloreactive tc cells (flyer et al., 1985) . however, no attempt was made to address the effect of up-regulated mhc antigen expression on the recognition of mhc-restricted, antigen-specific t ells. in this paper, we compared wnv.infected astrocytes with uninfected controls for their ability to act as targets of influenza virus-specific tc cells and as stimulators for an ik-reactive t cell line. the results showed that wnv infection has significant effects on t cell recognition of astrocytes. 'ilms, after infection by influenza virus, normal astrocytes are poor targets of influenza virus-specific tc, while doubly infected (with wnv and influenza) astrocytes can be well recognized by influenza-specific tc. wnv-infected astrocytes from cba/h mice can also stimulate ik-reactive t cells significantly better than the normal astrocytes, in terms of both t cell proliferation and interleukin release. the latter function was also enhanced in populations of primary anti-h-2i k t cells. these findings are particularly interesting because astrocytes are a large population of cells in the cns, presumably important both for the physiology and immunology of the cns (fontana e~ al., 1985) . elevated t cell recognition of astroeytes could have an important effect on the organism. these findings also raise interesting questions regarding self tolerance. if t cell tolerance to self antigens is determined quantitatively (blanden et al., 1987) the up-regulation of mhc antigens in the cns could possibly break t cell self tolerance in the cns. this may explain the autoimmune/virai etiology of some neurological diseases. quantitative considerations of t cell activation and self-tolerance immunohistological analysis of t lymphocyte subsets in the central nervous system in chronic progressive multiple sclerosis interferon induction by polynucleotides, modified polynucleotides, and polycarboxylates expression of histocompatibility antigens h-k. -d, and -l is reduced in adenovirus-12-transformed mouse cells and is restored by interferon ~1985) astrocytes as antigen-presenting cells. i. induction of la antigen expression on astrocytes t cells via in-anune interferon and its effect on antigen presentation retrovirus induced changes in major histocompatibility complex antigen expression influence susceptibility to lysis by cytotoxic t lymphocytes the endothelium-astrocyte immune control system cell mediated cytotoxicity against ectromelia virus-infected target cells conjugation of antibodies with fluorochromes: modrficat!ous to the standard methods viral induced autoimmnnity: monoclonai antibb.lies that react with endocrine tissues multiple organ-re.~ctive monoclonal autoantibodies expression of la antigens by cultured estrocytes treated with y-interferon quantitative variation in la antigen expression plays a central role in immune regulation effect of herpes simplex virus type 1 and 2 on surface expression of class ! major histecompatibility complex antigens on infected cells antigen-inducible, h-2-restricted interleukin-2-producing t cell hybridomas lack of independent antigen and h-2 re. cognition relationship between surface h-2 concentration, size of different target cells and lysis by cytotoxic t cells clones of cytotoxic lymphocytes can recognize uninfected cells in a primary response against influenza virus immunohistochemical identification of t lyn~lphocytes in the central nervous system of patients with multiple selerosis and subacute sclerusilag panencephalitis an improved assay for interleukin-2 (lymphocyte growth factor) produced by mitogen activated lymphocytes theiler's virus-induced demyelination: prevention by immunosuppression preparation of separate astre~ljial and ollgodendroglial cell cultures from rat cerebral tissue enhanced representation of hl-a antigens on human lymphocytes after mitogenesis induced by phytohemagglutinin of epstein-barr virus antigen presentation in brain: mhc induction on brain end~ thdium and astrocytes compared viral particles induce la antigen expression on astrocytes pathobiology of flaviviruses infection of cultured mnrine brain cells by semliki forest virus: effect of interferon-o.~ on viral replication, viral antigen display, major histocompafibility complex antigen display and lysis by cytotoxie t lymphocytes properties of mo~clonal antibodies to mouse ig allotypes, h-2 and ia antigens hybridoma cell lines secreting monoclonal antibodies to mouse h-2 and la antigens adenoviruses of subgenera b, c, d, and e modulate cell-surface expression of major histccompatibility complex class i antigens temporary pr~-~,ence of self-reactive cytotoxic t iymphocytes during murine lymphocytic choriomeningitis decreased expression of h-2 antigens following acute measles virus infection a simple method of estimating fifty percent end points hlunone response geue products (la antigens) on gliai and endothelial cells in virus induced demyelination intracellular v-interferon triggers an antiviral state in transformed l cells immune response (it) gene of the murine major histocompatibility complex interlenkin 4 (iggi induction factor): a multifunctional lymphokin acting also on t cells clonal analysis of cytolytic t lymphocyte-mediated lysis of target cells with inducible antigen expression: correlation between antigen density and requirement for ly-2/3 function l~e cytotoxic response to murine cytomegalovirus. i. parameters in vivo the cytotoxic response to murine cytomegalovirus. ii. in vitro requirement for generation of cytotoxlc t cells coronavirus infection induces h.2 antigen expression on oligodendrocytes and astrocytes adaption studies with ross river virus: laboratory mice and cell cultures multiple sclerosis: the blood-braln barrier and the measurement of de novo central nervous system of ig synthesis viral etiology of multiple sclerosis: where does the truth fie'? t lyraphocyte auto-inununity in experimental autoimmune encephalomyelitis dist~bution and quantitation of hla-abc and dr (ia) antigens on human kidney and other tissues inducible expression of h-2 and la antigens on brain cells interferon-v induces the expression of h-2 and la antigen on brain cells cytotoxic t cells specific for influenza virus infected target cells mhc-restricted cytotoxic t cells: studies on the role of polymorphic major transplantation antigens determining t cell restrictionspecificity, function and responsiveness we thank boehringer ingelheim for the kind gift of recombinant 7-ifn and barb geary for typing the manuscript. part of the work was carried out when one of us (y.l.) was a recipient of a world health organisation fellowship. key: cord-001546-ndz3oarf authors: ayithan, natarajan; bradfute, steven b.; anthony, scott m.; stuthman, kelly s.; bavari, sina; bray, mike; ozato, keiko title: virus-like particles activate type i interferon pathways to facilitate post-exposure protection against ebola virus infection date: 2015-02-26 journal: plos one doi: 10.1371/journal.pone.0118345 sha: doc_id: 1546 cord_uid: ndz3oarf ebola virus (ebov) causes a severe hemorrhagic disease with high fatality. virus-like particles (vlps) are a promising vaccine candidate against ebov. we recently showed that vlps protect mice from lethal ebov infection when given before or after viral infection. to elucidate pathways through which vlps confer post-exposure protection, we investigated the role of type i interferon (ifn) signaling. we found that vlps lead to accelerated induction of ifn stimulated genes (isgs) in liver and spleen of wild type mice, but not in ifnar(-/-) mice. accordingly, ebov infected ifnar(-/-) mice, unlike wild type mice succumbed to death even after vlp treatment. the isgs induced in wild type mice included anti-viral proteins and negative feedback factors known to restrict viral replication and excessive inflammatory responses. importantly, proinflammatory cytokine/chemokine expression was much higher in wt mice without vlps than mice treated with vlps. in ebov infected ifnar(-/-) mice, however, uninhibited viral replication and elevated proinflammatory factor expression ensued, irrespective of vlp treatment, supporting the view that type i ifn signaling helps to limit viral replication and attenuate inflammatory responses. further analyses showed that vlp protection requires the transcription factor, irf8 known to amplify type i ifn signaling in dendritic cells and macrophages, the probable sites of initial ebov infection. together, this study indicates that vlps afford post-exposure protection by promoting expeditious initiation of type i ifn signaling in the host. ebola viruses (ebovs) are enveloped, negative-sense rna filoviruses that can cause a severe hemorrhagic fever in humans and non-human primates (nhps) [1, 2] . mouse-adapted ebov causes similar acute disease in mice, offering a useful animal model to study ebov infection [3, 4] . ebov infection is characterized by rapid viral replication and dysregulated innate and adaptive immune responses. the disease follows profound suppression of type i ifn signaling and a contrasting excess inflammation that leads to mucosal hemorrhages and multi-organ failure resembling septic shock syndrome [5, 6] . virally encoded anti-ifn proteins, vp24 and vp35 play major roles in ebov virulence [7, 8] . vp35 blocks type i ifn induction in dendritic cells (dcs) and macrophages, and acts as a virulence factor necessary for a recombinant virus to attain infectivity in the host [9] [10] [11] [12] . vp24, on the other hand, blocks ifn signaling by interfering with ifn activated jak/stat pathways [7] . lines of evidence support the critical importance of type i ifn signaling in providing resistance against ebov infection; mice deficient in stat1, a transcription factor required for ifn induction, or ifnar1, encoding the membrane receptor for type i ifns, are susceptible to wild type zaire ebov, against which wild type mice are resistant [13] [14] [15] . a study of sudan ebov infection in humans showed that ifnα levels are significantly higher in surviving patients than those with fatal ebov infection, who had higher levels of proinflammatory cytokines/chemokines such as il-6, and mip-1β [16, 17] . high ifnα production is reported to correlate with increased resistance against ebov in mice as well [18] . administration of recombinant ifnα or ifnβ confers delayed time-to-death in nhps [19, 20] . furthermore, ifnα, used as an adjunct therapy for monoclonal antibody treatment, is shown to enhance protection in nhps [21] . ebov infection remains a potential threat to public health, which is compounded with the lack of effective prevention or treatment. to overcome this problem, various vaccine candidates have been developed, including various dna constructs, recombinant viruses, vlps, as well as treatment with anti-sense sirna [22] [23] [24] . vlps are subunit-based vaccines, extensively studied for a variety of infectious pathogens [25, 26] . vlps prepared from ebov and other filoviruses are composed of the matrix protein (vp40), glycoprotein (gp), and at times nucleoprotein (np) and represent a potentially promising candidate for ebov vaccine. ebov vlps have been shown to confer protection upon rodents and nhps when given prior to infection [27] [28] [29] . in the accompanying paper, we show that post-exposure administration of trivalent vlps protects mice from lethal ebov infection, further crediting the potential of vlps as a possible vaccine [30] . in that study, we show that vlp protection requires macrophages, dendritic cells (dcs) as well as b and either cd4 or cd8 t lymphocytes, indicating that both innate and adaptive immunity are involved in conferring protection. the aim of this study was to further investigate molecular bases of postexposure protection by vlps. based on our previous report that vlps stimulate type i ifn expression in dcs and macrophages, in vitro, we focused on the role of type i ifn signaling, and found that post-exposure vlp treatment leads to accelerated activation of ifn signaling, resulting in early induction of isgs. significantly, vlp stimulated isg induction coincided with the attenuation of proinflammatory cytokine surge in ebov infected mice. the reduced inflammatory responses was attributed to activation of type i ifn signaling, since vlp treated ifnar -/mice were unable to inhibit not only viral replication but proinflammatory responses, and succumbed to death. our results indicate that early type i ifn response is a major mechanism that contributes to vlp mediated protection against ebov infection. ifnar -/-, ifnar +/+ mice of balb/c background and irf8 -/and irf8 +/+ mice of c57bl/6 background were bred in the nichd animal facility and transferred to the facility of the united states army medical research institute of infectious diseases (usamriid) for ebov infection studies. research was conducted in compliance with the animal welfare act and other federal statutes and regulations relating to animals and experiments involving animals and adheres to principles stated in the guide for the care and use of laboratory animals, national research council, 1996. the facility where this research was conducted is fully accredited by the association for assessment and accreditation of laboratory animal care international. the iacuc committee approving this protocol is the united states army medical research institute of infectious diseases (usamriid) iacuc. animals were monitored at least once daily and their status was evaluated according to an intervention score sheet approved by usamriid iacuc. monitoring increased to three times daily if the animals were given a score of three or four. euthanization was by co 2 inhalation followed by confirmatory cervical dislocation. analgesics and anesthetics were not used in this study and animals were euthanized for humane purposes if they reached a score of five or more, which would be indicated if the animals exhibited ruffled fur, weakness, unresponsiveness, and/or difficulty walking. otherwise, animals were euthanized at the end of the study. vlps were composed of ebov gp, np and vp40 and were generated in mammalian 293t cells as reported previously [31] . vlp preparations used in this study contained <0.03 endotoxin u/mg. mice were infected with~1000 pfu (~3,000 ld 50 ) of mouse-adapted ebov via intraperitoneal (i.p.) route [32] . mice were injected with vlps (50 μg) diluted in pbs through i.p. 24 h after ebov infection. morbidity and mortality of ebov infected mice were monitored twice daily for up to 14 days. total rna from liver and spleen of ebov infected mice were extracted by trizol method (invitrogen) and cdna was synthesized from 1 μg total rna by superscript ii reverse transcriptase (invitrogen). qpcr amplification was done with 3 ng cdna in 5 μl sybr green pcr master mix (applied biosystems) with 3 μm of both reverse and forward primers used in the abi prism 7500 sequence detection system (applied biosystems). mrna of expression of indicated genes were analyzed as described in detail elsewhere [33] . the primer pairs were used for ebov gp, 5'-tgggctgaaaactgctacaatc-3' and 5'-ctttgtgcacataccggcac-3'; nlrp3, 5'-tgctcttcactgctatcaagccct-3' and 5'-acaagcctttgctccagaccctat-3'. all other gene primer sequences were followed from the previous publications [10, 33] . transcript levels were normalized with hprt, and expressed as relative expression. statistical analysis was carried out by excel software using two-tail paired student's t test. data represent the mean of at least three independent assay ± sem. a p value < 0.05 was considered significant. to assess the role of type i ifn signaling in vlp-mediated protection against ebov infection, we tested ifnar1 -/mice for protection by vlps. in fig. 1a , wild type (wt) and ifnar1 -/mice (both balb/c background, n = 10) were injected with 50 μg of vlps 24h after infection by the mouse adapted (ma) ebov, and the morbidity and mortality were checked daily for the subsequent 14 days. wt mice without vlp injection all died between day 5 and day 7, whereas 80% of mice that received vlps survived after ebov infection, confirming that vlps protect mice even when they were given post-infection. in contrast, ifnar -/mice that received vlps all died before or at day 5 as those without vlp injection (fig. 1a) . these results are in agreement with previous report on early death of ma-ebov infected ifnar -/mice [15] . ebovs are thought to initially infect dcs and macrophages in liver and spleen, making these tissues the major sites of ebov replication in the mouse, although the virus infects many other organs later [3, 34] . to ascertain whether vlps inhibit viral replication, we measured ebov glycoprotein (gp) mrna expression in liver and spleen from wt and ifnar -/mice with or without vlp administration. qrt-pcr analysis in fig. 1b and 1c, found that levels of gp mrna rose sharply in ifnar -/mice on day 2 of infection when gp mrna was still at background in wt mice. ifnar -/mice that received vlps also expressed considerable amounts of gp mrna, although levels varied between liver and spleen in the vlp treated group. thus, ebov appeared to replicate faster and to a greater extent in ifnar -/mice than wt mice. it should be noted here that in wt mice, gp mrna levels began to increase rapidly after day 2, peaking on day 3 to day 5, and that vlp injection inhibited gp mrna expression by more than half (s1 fig.) . 1000 pfu ma ebov, followed 24 h later by injection i.p. with 50 μg of ebov vlps. one group of ifnar +/+ (wt) mice infected with ebov without vlp served as control. mortality is expressed as percent survival of each group on indicated days. results are a representative of three independent experiments, which gave very similar outcomes. qrt-pcr detection of ebov gp mrna level on day 2 post-ebov infection with or without vlps from liver (b) and spleen (c) of wt or ifnar -/mice. gp transcripts were normalized by hprt and values represent the mean ± sem of duplicate samples from three independent experiments. asterisk denotes significant differences compared to wt controls (*p 0.05, **p 0.01). these results are in line with the results that ifnar -/and stat1 -/mice are more susceptible to ebov infection, suggesting the possibility that vlp mediated protection is linked to the activation of type i ifn signaling [13] [14] [15] . however, vlp injection may not have prevented ebov pathogenesis in ifnar -/mice, possibly because the disease manifests more severely in these mice than in wt mice. on the other hand, it has been recently shown that adenovirus based vaccine can protect ifnar -/mice from lethal evob infection presumably through antibody responses, which indicates that ifnar -/mice are not universally vulnerable, and anti-ebov resistance can be attained in some cases [35] . we recently reported that ebov vlps activate type i ifn transcription in dcs and macrophages in vitro, leading to induction of many isgs in these cells [33] . here we asked whether vlps stimulate isg induction in vivo. wt mice were infected with ma ebov and received vlps 24 h later, and induction of isg mrnas was tested on days 1.5 and 2. isgs encoding anti-viral proteins were first examined, as they may provide early protection against ebov infection. upper panels in fig. 2a and 2b compare induction of anti-viral isgs, ifit1, mx1, oas1a and stat1 with or without vlp injection in liver and spleen. in this early stage, levels of these isgs were consistently higher in the vlp-injected groups than those without vlps. at later stages of infection, however, the situation reversed, in that mice without vlps had higher levels of isgs, as seen on day 3 (s1 fig. for complete kinetics) . these results indicate that vlp administration accelerated type i ifn and isg induction, which presumably provide early anti-viral activity, not afforded without vlps. we next tested whether vlps induce other isgs, particularly those with negative regulatory activities. this question was of interest to us, since mice that did not receive vlps expressed higher levels of proinflammatory cytokines and chemokines, which raised the possibility that ifn signaling exerts negative regulatory activity towards proinflammatory responses, perhaps by controlling nf-κb activation [36] . shown in the lower panels in fig. 2a and 2b is induction of irgm1, usp18, trim21 and trim30. irgm1 is an ifn inducible gtpase that inhibits lps induced endotoxin shock in mice [37] . usp18 is an isg15 deconjugating factor that negatively regulates tlr signaling and resultant cytokine induction [38] . trim21 and trim30 are members of the tripartite motif family that downregulate tlr induced inflammatory responses [39] [40] [41] [42] . expression of these isgs was also higher in the vlp injected group than that without vlps both in liver and spleen. similar to anti-viral isgs, expression of these negative regulatory factors changed at the later stage (s1 fig). these data indicate that vlps accelerate induction of anti-viral and negative regulatory isgs, which may help suppress ebov's anti-ifn antagonism (see discussion). to confirm that vlp induction of isg is dependent on type i ifn signaling, we next tested isg induction in ifnar -/mice. as expected, none of the isgs tested in fig. 2 were induced in ifnar -/mice after vlp treatment or ebov infection (s2 fig). vlps lower expression of proinflammatory cytokines in ebov infected mice ebov pathophysiology such as severe hemorrhagic symptoms and tissue damage is thought to be associated with dysregulated inflammatory cytokine production [2, 43] . given that vlps accelerated induction of negative regulatory isgs, we next evaluated whether vlps modulate expression of proinflammatory genes. in fig. 3 , expression of tnfα, il-6 and il-1β, chemokines such as mcp-1 (ccl2), mip-1α (ccl3), mip-1β (ccl4), kc (cxcl1) and inflammasome gene nlrp3 was measured in ebov infected mice with or without vlps. these genes were all strongly induced upon ebov infection and peaked on day 3 with a gradual decline on days 5 in all cases, their expression was significantly attenuated in the vlp-treated group as compared to the group without vlps. the difference was most dramatic in the early stage on day 3, where the expression was reduced at least by 50%. in agreement with these results, we noted that serum levels of some of these proinflammatory cytokines were higher in ebov infected mice that were treated with vlps as compared those without vlps [30] . these results support the view that limiting superfluous inflammatory responses contribute to vlp mediated protection. ifnar -/mice increasing evidence indicates that type i ifns antagonize inflammatory responses in a variety of settings [44] [45] [46] . in light of the results that vlps stimulate those isgs known to suppress proinflammatory responses, it was of importance to determine whether type i ifn signaling by and of itself affects ebov induction of proinflammatory cytokines and chemokines. results in fig. 4 and s3 fig compare expression of the above proinflammatory factors in ifnar +/+ and ifnar -/mice infected with ebov. all cytokines and chemokines tested were induced after ebov infection in both strains. importantly, their levels were much higher in ifnar -/mice than ifnar +/+ mice. these results indicate that type i ifn signaling downregulates ebov stimulated induction of proinflammatory factors, possibly through isgs with negative regulatory activities. the above results indicated that type i ifns attenuate proinflammatory responses during ebov infection. to explore whether type i ifns have a similar activity in settings other than ebov infection, we next tested lps and ifnβ induced inflammatory responses in macrophages in vitro. lps activates nf-κb mediated proinflammatory cytokine induction, which can result in endotoxin shock [36] . as shown in fig. 5 , combined treatment with lps and ifnβ led to hyper induction of tnfα, il-6, il-1β and a chemokine kc in ifnar -/macrophages as compared to wt cells. lps and ifnβ also induced negative feedback factors, trim21 and trim30, with much lower expression in ifnar -/cells than ifnar +/+ cells. these results support a model in which type i ifns negatively regulate proinflammatory cytokine/chemokine responses at least in some situations. we found that mip-1α, mip-1β and mcp-1 were not hyperinduced in ifnar -/cells, suggesting that some proinflammatory genes are regulated not only by type i ifns but other factors (data not shown). alternatively, these differences may reflect variances between in vivo and in vitro conditions. to further define pathways downstream of ifnar activity, important for vlp protection, we directed our attention on irf8, a transcription factor expressed in macrophages and dcs [47] [48] [49] . irf8 is induced by ifns and tlr ligands in a stat1 dependent manner, and plays a pivotal role in facilitating innate immune responses. although irf8 is not involved in initial triggering of type i ifn induction, it amplifies ifn transcription in dcs and macrophages [50] . irf8 promotes induction of multiple anti-microbial factors and is required for innate resistance against a variety of pathogens [50] [51] [52] . irf8 stimulates expression of mhc and costimulatory molecules to boost antigen presentation [48, 50] . we thus tested whether irf8 disruption affects vlp-mediated protection against ebov. survival data in fig. 6a show that approximately 80% of irf8 -/mice that received vlps died between day 6 and 8, which is nearly identical to the mortality curve of wt mice without vlps. as expected, the majority of wt mice that received vlps survived against ebov infection. it is of note that ifnar -/mice died 1 to 2 days earlier than irf8 -/mice, which may be attributed to the difference in the mouse background. correlating with the lack of protection, ebola gp mrna levels were much higher role of type i ifn in vlp-mediated protection against ebov infection in ebov infected irf8 -/mice than irf8 +/+ mice with or without vlps (fig. 6b) . we next examined whether induction of anti-viral and negative feedback isgs is dependent on irf8. data in fig. 6c illustrate that induction of these isgs was very meager in irf8 -/mice, in contrast to robust induction in irf8 +/+ mice. importantly, vlps did not rescue isg induction in irf8 -/mice. results were similar in liver and spleens ( fig. 6c and s4 fig). these results indicate that vlps, upon initial activation of type i ifn cascade, rely subsequently on the activation of downstream pathways represented by irf8 to confer protection against ebov. to gain insight into the pathways through which vlps confer resistance against ebov infection, we investigated the role of type i ifn signaling in vivo and found that it significantly contributes to vlp-mediated protection. this conclusion is supported by the observation that post-exposure vlp treatment accelerated isg induction in ebov infected mice, leading to reduced viral replication and inflammatory gene expression. further supporting the critical role of type i ifn signaling in the protection, vlps did not induce isgs in ifnar -/mice, and did not protect the mice from lethal ebov infection. these results are consistent with the report that post-exposure ifnβ or ifnα treatment increases protection against ebov infection in nhps [1, 6, 15, 19] . it is likely that vlps initially stimulated type i ifn genes, which in turn led to early induction of isgs. in line with this notion, we recently showed that exogenous vlps stimulate transcription of ifnα and ifnβ in dcs and macrophages in vitro, an event coupled with immediate and robust isg induction [33] . it may be reasonable to assume that ifnar -/mice were not protected by vlps primarily because isg induction was absent. however, ifnar -/mice may be susceptible to infection due to additional defects in innate immunity that are a secondary consequence of defective ifn signaling, which obliterates vlps protection. contouring this notion however, it is of note that ifnar -/mice can be protected against ebov by an adenovirus-based vaccine, indicating that ifnar -/mice are not totally without defense [35] . rather, it is possible that ifnar -/mice are not protected by vlps that rely on isg induction for protection, whereas they are protected by the adenovirus vaccine that depends on antibody response. vlp-induced isgs included anti-viral proteins known to inhibit replication of rna viruses such as ifit1, mx1 and oas1a, as well as negative feedback factors that curb excess inflammatory responses, such as irgm1, usp18, trim21 and trim30. although the question of which antiviral isgs are effective in inhibiting ebov replication awaits further research, it is anticipated that some of anti-viral isgs induced by vlps may interfere with ebov life cycle [53] . what is the significance of accelerated ifn response in vlp mediated protection? available evidence suggests that vlps may overcome ebov's anti-ifn antagonism. the virally encoded vp24 and vp35 disable the entire ifn system in the host; while vp24 blocks the jak/stat pathway of ifn signaling, vp35, an ebov virulence factor, inhibits type i ifn induction in many cell types [6, 7, 9, 11] . we previously showed that vp35 inhibits type i ifn induction in murine dcs by premature sumoylation and inactivation of irf7 [10] . it is thought that vp24 and vp35 have a decisive effect on the subsequent host resistance, since abated ifn signaling would impair proper innate immune responses, leading to deficiency in dc maturation, defective antigen presentation and aberrant inflammation. compromised innate immunity would consequently undermine development of adaptive immunity [6] (see a model in fig. 7) . it is remarkable that in the vlp treated mice, isg induction began early within 1.5 to 2 days after ebov infection (which was only 0.5 to 1 days after vlp treatment), when little to no isg induction was seen in mice without vlps. the delayed isg induction in ebov infected mice is reminiscent of the reports showing that influenza virus delays isg induction in lung epithelial cells through ns1, an influenza anti-ifn protein that is linked to disease pathology [54, 55] . an influenza virus strain deficient in ns1 is shown to induce isgs earlier than wild type virus, although the wild type strain does stimulate isgs later on [54, 55] . supporting the view that viral anti-ifn factors stall isg induction, rather than completely abrogate the induction, we also observed isg induction on day 3 and later in mice without vlps. it may be envisaged that vlps trigger ifn activation early on, thereby eluding the activity of the ebov anti-ifn proteins (a model in fig. 7) . the most striking observation made in this study is the vlp-dependent suppression of proinflammatory responses. this suppression was a result of type i ifn signaling, as ifnar -/mice expressed higher levels of proinflammatory cytokines and chemokines, observed not only after ebov infection but also by ifnβ and lps stimulation. these results are in accordance with the growing recognition that type i ifns are linked to attenuation of inflammatory responses [6, 44] . for example, pinto et al., reported, in the west nile virus infection model that ifnar -/mice express excess proinflammatory cytokines, including those found in this study, as compared to wt mice, which correlated with increased disease pathology. in this system, the overt inflammatory responses were attributed to ifn signaling in macrophages and dcs [46] . induction of proinflammatory cytokines and chemokines may be negatively regulated by ifn signaling through a series of negative feedback factors [37, [39] [40] [41] [42] [56] [57] [58] . irgm1, induced by ifn signaling restricts lps induced endotoxin shock without limiting ifnβ expression [37] . trim21 and trim30 inhibit proinflammatory cytokine induction, at least in part by interfering with the nf-κb dependent arm of transcription [39] [40] [41] . in addition, these factors may act by post-transcriptional mechanisms, affecting inflammasome activation [42] . in this regard, guarda et al. [45] reported that type i ifns inhibit production of il-1 by inhibiting activity of the nlrp1 and nlrp3 inflammasomes and by il-10 induction. thus, isgs with negative regulatory activity may preferentially attenuate proinflammatory pathways, while sparing ifn induction pathways. given our earlier observations that vp35 does not grossly affect nf-κb activation, while strongly inhibiting type i ifn activation, ebov may promote proinflammatory pathways at least in part through vp35 [10] . lastly, we show that the transcription factor irf8 is required for vlp mediated post-exposure protection. our results offer an added mechanistic insight into the pathways through which vlps provide protection. irf8 is expressed predominantly in macrophages and dcs, and helps to amplify type i ifn gene induction and boosts ifns biological activities [51] . given that macrophages and dcs are the putative early sites of ebov infection, vlps may exert a major impact on these cells to facilitate early innate immunity, in an irf8 dependent manner. in conclusion, vlps confer post-exposure protection upon ebov infected mice by rapidly inducing isgs, thereby permitting timely establishment of anti-viral and anti-inflammatory states in the host. vlps may act primarily by relieving ebov's antagonism against type i ifns, resulting in reduced systemic inflammation and subsequent enhancement in acquired immune responses (a model in fig. 7 ). pathogenesis of ebola hemorrhagic fever in primate models: evidence that hemorrhage is not a direct effect of virus-induced cytolysis of endothelial cells a compendium of 40 years of epidemiological, clinical, and laboratory studies mouse models for filovirus infections a mouse model for evaluation of prophylaxis and therapy of ebola hemorrhagic fever monocyte-derived human macrophages and peripheral blood mononuclear cells infected with ebola virus secrete mip-1alpha and tnf-alpha and inhibit poly-ic-induced ifn-alpha in vitro how ebola and marburg viruses battle the immune system evasion of interferon responses by ebola and marburg viruses the ebola virus vp35 protein functions as a type i ifn antagonist molecular determinants of ebola virus virulence in mice ebola zaire virus blocks type i interferon production by exploiting the host sumo modification machinery whole-genome expression profiling reveals that inhibition of host innate immune response pathways by ebola virus can be reversed by a single amino acid change in the vp35 protein inhibition of irf-3 activation by vp35 is critical for the high level of virulence of ebola virus filovirus infection of stat-1 knockout mice lethality and pathogenesis of airborne infection with filoviruses in a129 alpha/beta -/-interferon receptor-deficient mice the role of the type i interferon response in the resistance of mice to filovirus infection human fatal zaire ebola virus infection is associated with an aberrant innate immunity and with massive lymphocyte apoptosis cytokine and chemokine expression in humans infected with sudan ebola virus protection from lethal infection is determined by innate immune responses in a mouse model of ebola virus infection interferon-beta therapy prolongs survival in rhesus macaque models of ebola and marburg hemorrhagic fever evaluation of immune globulin and recombinant interferon-alpha2b for treatment of experimental ebola virus infections mabs and ad-vectored ifn-alpha therapy rescue ebola-infected nonhuman primates when administered after the detection of viremia and symptoms current ebola vaccines ebolavirus vaccines for humans and apes effective postexposure treatment of ebola infection virus-like particles as a highly efficient vaccine platform: diversity of targets and production systems and advances in clinical development protection against filovirus infection: virus-like particle vaccines ebola virus vp40 drives the formation of virus-like filamentous particles along with gp effect of ebola virus proteins gp, np and vp35 on vp40 vlp morphology ebola virus-like particle-induced activation of nf-kappab and erk signaling in human dendritic cells requires the glycoprotein mucin domain mechanisms of immunity in post-infection vaccination against ebola virus infection induction of humoral and cd8+ t cell responses are required for protection against lethal ebola virus infection correlates of immunity to filovirus infection ebola virus-like particles stimulate type i interferons and proinflammatory cytokine expression through the toll-like receptor and interferon signaling pathways intracellular events and cell fate in filovirus infection vaccination with recombinant adenoviruses expressing ebola virus glycoprotein elicits protection in the interferon alpha/ beta receptor knock-out mouse role of type i ifn in vlp-mediated protection against ebov the ifn-inducible gtpase lrg47 (irgm1) negatively regulates tlr4-triggered proinflammatory cytokine production and prevents endotoxemia ubp43 is a novel regulator of interferon signaling independent of its isg15 isopeptidase activity trim30 alpha negatively regulates tlr-mediated nfkappa b activation by targeting tab2 and tab3 for degradation trim family proteins and their emerging roles in innate immunity gene disruption study reveals a nonredundant role for trim21/ro52 in nf-kappab-dependent cytokine expression in fibroblasts tripartite-motif protein 30 negatively regulates nlrp3 inflammasome activation by modulating reactive oxygen species production filovirus research: knowledge expands to meet a growing threat anti-inflammatory property of type i interferons type i interferon inhibits interleukin-1 production and inflammasome activation deficient ifn signaling by myeloid cells leads to mavs-dependent virus-induced sepsis the irf family transcription factors in immunity and oncogenesis the small ubiquitin-like modifier-deconjugating enzyme sentrin-specific peptidase 1 switches ifn regulatory factor 8 from a repressor to an activator during macrophage activation essential role of the irf8-klf4 transcription factor cascade in murine monocyte differentiation innate immunity to intraphagosomal pathogens is mediated by interferon regulatory factor 8 (irf-8) that stimulates the expression of macrophage-specific nramp1 through antagonizing repression by c-myc the feedback phase of type i interferon induction in dendritic cells requires interferon regulatory factor 8 irf8 mutations and human dendritic-cell immunodeficiency distinct patterns of ifitm-mediated restriction of filoviruses, sars coronavirus, and influenza a virus cellular transcriptional profiling in influenza a virus-infected lung epithelial cells: the role of the nonstructural ns1 protein in the evasion of the host innate defense and its potential contribution to pandemic influenza overcoming ns1-mediated immune antagonism involves both interferon-dependent and independent mechanisms negative regulation of cytokine signaling influences inflammation negative regulation of toll-like receptor-mediated immune responses socs proteins, cytokine signalling and immune regulation the content of this publication does not necessarily reflect the views or policies of the us department of defense or the united states army medical institute of infectious diseases. we thank members of pgd for in depth discussions and critical reading of the manuscript. we also thank ms. monica gupta for breeding ifnar -/mice for this study. key: cord-003382-v3w1wi5c authors: rahmatpanah, farah; agrawal, sudhanshu; jaiswal, natasha; nguyen, hannah m.; mcclelland, michael; agrawal, anshu title: airway epithelial cells prime plasmacytoid dendritic cells to respond to pathogens via secretion of growth factors date: 2018-10-02 journal: mucosal immunol doi: 10.1038/s41385-018-0097-1 sha: doc_id: 3382 cord_uid: v3w1wi5c plasmacytoid dendritic cells (pdcs) are critical for defense against respiratory viruses because of their propensity to secrete high levels of type i interferons (ifn). the functions of pdcs in the lung can be influenced by airway epithelial cells. we examined the effect of human primary bronchial epithelial cells (pbecs) on pdc functions by performing rna-sequencing of pdcs after co-culture with air liquid interface differentiated pbecs. functional analysis revealed that pdcs co-cultured with pbecs displayed upregulation of type i ifn production and response genes. upregulated transcripts included those encoding cytosolic sensors of dna, zbp-1,irf-3, and nfkb as well as genes involved in amplification of the ifn response, such as ifnar1, jak/stat, isg15. in keeping with the rna-seq data, we observe increased secretion of type i ifn and other cytokines in response to influenza in pdcs co-cultured with pbecs. the pdcs also primed th1 responses in t cells. the enhanced response of pdcs co-cultured with pbecs was due to the action of growth factors, gmcsf, gcsf, and vegf, which were secreted by pbecs on differentiation. these data highlight possible mechanisms to enhance the production of type-i ifn in the airways, which is critical for host defense against respiratory infections. plasmacytoid dendritic cells (pdcs) are a subset of dendritic cells, which are found in circulation and other organs. they are distinguished by their capacity to produce copious amounts of type i interferons (ifn) 1,2 via stimulation of toll like receptor 7 and 9 with nucleic acids or viruses. because of this property, they play a crucial role in fighting viral infections. 1, 2 however, the cytokine secretion by pdcs is not restricted to ifns as they can also secrete other cytokines and chemokines including il-6, il-12, cxcl8, cxcl10, ccl3, and ccl4. similar to conventional myeloid dcs, they also express mhc class ii and costimulatory molecules and migrate to prime t cell responses. 3, 4 production of type i ifns along with il-12 by pdcs primes cd8+ t cells and ifn-γ secreting th1 cd4+ t cells. pdcs can also induce t regulatory cells via induction expression of ido. 3, 4 in addition, type i ifn production by pdcs enhances b cell activation, plasma cell generation and antibody secretion. tnf-related apoptosis inducing ligand (trail) and granzyme b serve as immunoregulatory factors that endow pdcs with the capacity to kill tumor cells, induce apoptosis of infected cd4 + t cells and suppress t cell proliferation. 3, 4 pdcs also play an important role in pulmonary immune responses. pdcs in the lung have been reported to be immature, expressing low levels of mhc ii and costimulatory molecules but high levels of pdl-1. 5 these characteristics make them poor inducers of t cells and that is why they are thought to play an immune-regulatory role in the lung. depletion of pdcs in a mouse model of asthma enhanced the inflammation against harmless antigens. similarly, in acute lung injury (ali) and acute respiratory distress syndrome (ards), activated pdcs are protective and have been shown to prevent recruitment of pro-inflammatory monocytes into the lung. 6 in contrast, pdcs were reported to be key drivers of immune-inflammatory cascade during asthma exacerbations via boosting of th2 responses. 7 thus, pdcs can be inflammatory or tolerogenic depending on the context. activated pdcs are major drivers of protection against respiratory infections such as influenza and rsv by virtue of production of type i ifns and other cytokines. 4 the functions of lung dc are influenced by many soluble factors such as growth factors, chemokines, and cytokines that are released from different cells within the lung. these soluble factors regulate the intensity and duration of immune response by stimulating or inhibiting activation and function of dc. for example, previous studies from our group and others have demonstrated that pbecs enhance the inflammatory and immune-surveillance capacity of mdcs. [8] [9] [10] however, their effect on pdcs is not elucidated. this knowledge is essential for manipulation of pdc response to respiratory pathogens. here we examined the changes introduced in human pdcs by primary bronchial epithelial cells at gene and functional level. peripheral blood samples were obtained from healthy volunteers (22-52 years) , approved by the institutional review board of the university of california (irvine, ca, usa). written, informed consent was obtained. obtained from lonza inc. (basel, switzerland) and were differentiated at the ali on transwell plates as described. 8 briefly, 5 × 10 4 pbecs per insert were seeded into 24-well transwell plate with apical chamber coated with the rat tail collagen (bd biosciences, san jose, ca, usa). cells were seeded in 100 µl b-ali™ growth medium onto the collagen coated trans well inserts; 500 µl of b-ali™ growth medium was added to the basal chamber of wells containing the inserts. on day 3 after seeding, once the monolayer of pbec was confluent, media were removed from the apical chamber and allowed for air-liquid differentiation. the media in the bottom chamber were changed every two days. approximately 28 days post-differentiation, pbecs were tested for the presence of cilia by staining for beta-tubulin and mucus secretion (data not shown). isolation and culture of human plasmacytoid (pdcs) with pbecs for rna sequencing pdcs were purified from the peripheral blood mononuclear cells (pbmcs) of young subjects by negative selection using a dc enrichment kit (stemcell sep, vancouver). enriched dc population was subsequently sorted using facsaria (bd biosciences) to obtain a pure pdc population. purity was above 92%. (cd123+/cd11c−) pdcs from different donors were added to the bottom chamber of the transwell with ali-differentiated pbecs for 24 h. subsequently, the pdcs were collected for rna-seq and other studies. pdcs cultured in pbec medium without pbecs were used as controls. rna isolation from pdcs total rna was isolated from cultured pdc cells from four individuals using ffpe rna/dna purification plus kit (cat # 54300, norgen biotek corp) with minor modifications. this kit is suitable for challenging samples such as formalin fixed paraffin embedded tissue in, which the recovery of quality rna is difficult. we applied this kit to isolate rna from pdcs cells since the numbers of cells were small. the rna quality and quantity was assessed using agilent bioanalyzer and qubit fluorimeter respectively. high yield and quality rna was obtained. the illumina truseq rna access library prep kit was used to study gene expression profiling in stimulated and unstimulated pdcs. the access method of illumina is based on sequence specific capture of coding rna. this method is suitable for wide range of rna quality and low rna input. briefly, 50 ng of rna was used for library preparation. the total rna is fragmented into small pieces using divalent cation at 98 o c. then cdna is synthesized using the cleaved rna fragments using random priming during the first and the second synthesis. sequencing adaptors are ligated to the dscdna fragments. the quality of libraries was assessed after the first pcr amplification. the coding regions of the transcriptome are then captured from the library using sequence specific probes to create the final libraries (as described by the manufacturer). samples were assayed after the second pcr amplification on the agilent 2100 bioanalyzer. qualification assay (qpcr) was performed using kapa sybr fast library quantification kit (illumina) universal qpcr mix ref # 0796014001. samples were sequenced at 2 × 100 cycle at the uci high throughput genomic core facility (https://biochemghtfuci.wordpress.com/). sequencing reads were aligned to genome reference files from ensembl using strand ngs 3.1 (http://www.strand-ngs.com/) sequencing analysis package. approximately 70-90% of mapped reads were aligned to protein coding regions as shown by the pie chart plot (supplementary fig.1 ). reads were filtered on duplicates to remove pcr artifacts. reads with better mapping quality and base average were retained (we set the number of duplicated reads to be permitted at "0") (excel supplementary data). deduplicated sequencing reads for each sample was quantified for gene expression, which is the measurement of the association of reads to genes and exons "raw reads". to normalize the raw reads we used reads per kilo million (rpkm) method in strand ngs program. we measured the variations in 5′ to 3′ end coverage along each transcript using gene body coverage analysis in strand ngs. uniform transcript coverage was observed among all samples. pooled analysis was performed using the audic claverie test (ac), which pools together raw counts across four pdcs and pdcs co-cultured with epithelial cells, separately, and tests the differences between them based on a poisson assumption on distribution of counts. multiple testing correction: benjamini hochberg fdr of 0.05 and the maximum p value cut off 0.05 was used (excel supplementary data). the genes of ac test were ranked based on p value adjusted (fdr corrected) and cut off was set at p adj <0.001 to select for statistically significant differentially expressed genes between the pdcs co-cultured with epithelial cells and pdcs. the methods used for rna isolation and rna seq library construction worked with samples with small number of cells. for co-culture experiments purified pdcs were cultured without or with pbec differentiated at ali. after overnight co-culture, pdcs from both groups were collected and put together with purified cd4 t cells at a ratio of 1:10 (pdc:cd4 t) in serum free aim v medium (invitrogen) for 6 days. subsequently, the cells were collected and stained with specific antibodies for cd4, cd25 and foxp3 (rnd systems). gated cd4 cells were analyzed for the expression of cd25 and foxp3 using flow jo. supernatant collected was assayed for ifn-γ and il-10 by elisa. for co-culture experiments purified pdcs were cultured without or with pbec differentiated at ali. after overnight co-culture, pdcs from both groups were collected and stimulated with influenza (heat killed influenza a strain a/pr/8/34-(flu, charles river, north franklin, ct) 11 at 1 µg/ml) for 24 h. 12 culture supernatants were collected 24h post stimulation and run on multiplex kit. the specific kit used was milliplex human 30-plex cytokine panel 1 (cat# hcytmag-60k-px30), which contains the following analytes: gm-csf, g-csf, ifnγ, il-1α, il-1rα, il-1β, il-2, il-3, il-4, il-5, il-6, il-7, il-8, il-10, il-12p70, il-12p40, il13, il15, il-17, mcp-1, tnfα, tnfβ, eotaxin, type i ifn, ip-10, mip-1α, mip-1β, egf, vegf, and rantes. the procedure followed was according to manufacturer's protocol. briefly, supernatant was mixed with premixed beads (30 cytokines) overnight and after incubation with detection antibodies and streptavidin-pe for 1 h each, the plate was run on magpix to identify specific cytokines. culture of pdcs with pbecs/growth factors purified pdcs were cultured with and without gmcsf (10 ng/ml), g-csf (10 ng/ml) or vegf (10 ng/ml), il-6(10 ng/ml), il-8(10 ng/ml), mcp-1 (10 ng/ml) (rnd systems) or a cocktail of these in various combinations and stimulated with influenza for 24 h. supernatant collected was assayed for inflammatory mediators with multiplex as described above. culture of pdcs with blocking antibodies pdcs cultured without or with pbec were stimulated with influenza in the presence or absence of specific blocking antibodies against gmcsf (invitrogen), gcsf, vegf (rnd systems) or a mixture of the three antibodies at 1 µg/ml. after 24 h supernatant was collected and assayed for type i ifn, tnf-α, il-1β, il-6 using multiplex. percent inhibition in the antibody treated groups was calculated compared to control group without antibody treatment. pbecs from three different subjects were used for this experiment. pbecs were allowed to differentiate at ali. the conditioned basal airway epithelial cells prime plasmacytoid dendritic cells to respond to. . . f rahmatpanah et al. medium in the bottom chamber was collected once the aec differentiation was complete. pbec differentiation media alone was used as control. multiplex detection of 30 factors (same as above) was performed to determine the factors secreted by pbecs using milliplex. statistical analysis for cell culture experiments was performed using graphpad prism (graphpad inc., san diego, ca, usa). one way anova followed by tukeys multiple comparison test was used for the analysis. a p-value of <0.05 was considered statistically significant. the effect of pbecs on pdcs gene expression changes was investigated using the transwell co-culture system. to investigate the effect of aecs in the form of pbecs on pdcs, the pbecs were cultured at ali till differentiation was achieved. pdcs purified from the blood were then added to the basal side of the monolayer to model the airways. 13, 14 an aliquot of the pdcs was also cultured without pbecs but with the media. twenty-four later pdcs were collected and rna was extracted. pdcs from four different individuals were used. the description is provided in supplementary table 1 . the viability was comparable between pdcs cultured with and without pbecs (data not shown). because of the low quantity of rna, we used the access method of library construction from illumina, which is based on sequence specific capture of coding rna. this method is suitable for wide range of rna quality and low rna input. paired end rna sequencing (rna-seq) was performed to determine the gene expression differences between pdc and pdcs co-cultured with epithelial cells. we observed a uniform reads coverage in the gene body for all rna seq data plot. we did not observe very large increases or decreases in transcript abundance, which was to be expected because pdcs were exposed to pbecs and there was no stimulation with a pathogen. pdcs in the airways are reported to induce tolerance via the expression of ido. 15, 16 we found increased expression of ido-1 (fig. 1a) in the pdcs cultured with pbecs compared to pdc alone. in addition, the expression of costimulatory molecules, cd80 and cd86 was decreased. however, expression of other costimulatory molecules including, cd40, ox40, icos, icam was upregulated. the expression of the inhibitory costimulatory molecule, pdl-1 was also decreased. the pdcs therefore displayed a mixed phenotype in terms of costimulatory molecules. we performed functional analysis to confirm whether the pdcs were tolerized by pbecs. pdcs exposed to pbecs induced significantly increased levels of cd4 + cd25 + foxp3 + t regulatory cells to controls (p = 0.02), fig. 1b, c) . the secretion of ifn-γ was significantly decreased (p = 0.04) while il-10 was increased (p = 0.03) in the pdcs exposed epithelial cell group compared to control. these data suggest that pbecs enhance the tolerogenic capacity of pdcs at homeostasis. rna-seq data indicates pbecs enhance the production and response of type i ifn in pdcs pdcs in the airways are also important for host defense against infections because of their capacity to secrete high levels of type i ifns. our results indicate that the major pathways altered in pdcs in response to pbecs are the type i ifn related pathways such as the type i ifn signaling, interferon stimulated gene 15 (isg15), cytosolic sensors of dna as well as jak-stat (fig. 2a-d) . in the type i ifn pathway, the expression of ifnar1 as well as jak-stat genes such as jak1, stat2, stat3, which signify activation, are upregulated while socs1-and 3, which inhibit the pathway, are downregulated ( fig. 2a-d) . 17 furthermore pathways involved in the interferon response such as isg15 also displayed alterations after co-culture (fig. 2b) . among the innate sensors, the expression of cytosolic dna sensor, zbp-1 18 was upregulated on pdcs after co-culture with apcs (fig. 2c) . the expression of irf-3 and nfkb2, which are required for signaling via this receptor, was also upregulated. in contrast, the negative regulators of the pathway, nfkb1a, ikbkb are downregulated. interestingly, the majority of the genes in the prostaglandin pathway which is a major negative regulator of ifn production were downregulated (fig. 2e) . in addition to ifn related pathways, g-protein coupled receptor (gpcr) signaling pathway also displayed major changes in pdcs co-cultured with pbecs compared to pdcs (fig. 2f) . several of the chemokine receptor genes, such as ccr4, ccr5, ccr7, ccr10, cx3cr1, cxcr4 were upregulated indicating an activated state of pdcs. genes in the il-3 pathway also displayed changes (fig. 2g) . il-3 is known to promote pdc activation and survival. 19 in summary, rna seq data suggests that the capacity of pdcs to produce and amplify type i ifn production is upregulated in pdcs after co-culture with pbecs. pbecs enhance the cytokine and chemokine response of pdcs to influenza to confirm the rna-seq data we performed functional assays to determine the alterations in the capacity of pdcs to produce type i ifn after exposure to pbecs. briefly, pdcs co-cultured with/without pbecs differentiated at ali were stimulated with influenza and cytokines and chemokines were assayed in the supernatants. conditioned pbec medium was used as control. pdcs exposed to pbecs secreted significantly higher levels of type i ifns (p = 0.0001) on stimulation with influenza as compared to pdcs cultured alone (fig. 3a) , which is in keeping with the rna-seq data. tnf-α secretion was also significantly increased (p = 0.007) in influenza stimulated aec-exposed pdcs as compared to unexposed pdc (fig. 3a) . secretion of il-1α (p = 0.004) and il-1β (p = 0.01) was also increased significantly in pdcs co-cultured with pbecs with and without stimulation with influenza (fig. 3a) . no type i ifn, tnf-α, il-1α, and il-1β was detected in the conditioned medium. secretion of other mediators including anti-inflammatory cytokine, il-10 was below the limit of detection (data not shown). these data indicate that pbecs enhance the inflammatory response of pdcs to pathogens as suggested by the rna-seq data. to confirm that the aec-exposed pdcs were indeed primed to induce enhanced responses to influenza, the pdcs co-cultured with/without pbecs and influenza were cultured with purified t cells for five days and secretion of t cell specific cytokines was determined by multiplex. culture of t cells with pdc exposed to pbecs and stimulated with influenza induced significantly higher level of il-2 (p = 0.01) as compared to pdcs stimulated with influenza without aec exposure (fig. 3b) . enhanced il-2 secretion indicated increased proliferation. in keeping with the increased proliferation, the levels of tnf-α (p = 0.002) and ifn-γ (p = 0.001) secretion by these t cells was also significantly enhanced (fig. 3b) . other cytokines such as il-10, il-17, il-4, il-5 were not induced by influenza primed pdcs and were comparable between the t cells cultured with pdc with and without pbecs (data not shown). these results suggest that pbecs enhance the inflammatory response of pdcs to influenza, which leads to increased induction of th1 responses. mechanisms used by pbecs to prime pdcs, a multiplex analysis was performed to determine the soluble factors in the conditioned medium from pbecs differentiated at the ali. the medium for differentiation was used as control. remarkably, out of 30 mediators tested (see methods) we observed only six factors, which were significantly upregulated in the medium of ali differentiated pbecs (fig. 4) . differentiated pbecs secreted significant levels of growth factors g-csf (p = 0.01), gmcsf (p = 0.01) and vegf (p = 0.04). secretion of cytokines, il-6 (p = 0.02) as well as chemokines, cxcl-8 (il-8, p < 0.0001), ccl-2 (p = 0.03) was also upregulated. ali differentiated pbecs might be priming the pdcs via secretion of the growth factors and the cytokines, chemokines. to identify the specific mechanism that leads to pdc priming in presence of pbecs, the effect of the factors found to be significantly enhanced in differentiated pbecs were explored. initial experiments were performed with three cocktails, growth factor and cytokine, chemokine cocktail (gmcsf, vegf, gcsf, il-6, il-8, ccl-2), growth factor cocktail (gmcsf, vegf and gcsf) and cytokine, chemokine cocktail (il-6, il-8, ccl-2). purified pdcs were cultured in the presence or absence of these cocktails and activated with influenza. culture of pdcs with combination cocktail as well as growth factor cocktail enhanced the secretion of type i ifn while the cytokine, chemokine cocktail had no significant effect (supplementary fig. 2 ). further experiments were then done with growth factors. we tested the individual effects of the growth factors to determine whether this was due to a single factor or a synergistic effect of all 3 factors. both gcsf (p = 0.03), gmcsf (p = 0.04) led to increase in type i ifn secretion by pdcs in the response to influenza (fig. 5a) . the increase by vegf was not significant (p = 0.1). the secretion of type i ifn was maximal when a cocktail of all three growth factors was used indicating a synergistic effect. both gmcsf and gcsf were also able to induce significantly increased (p = 0.03-gmcsf; p = 0.04-gcsf; fig. 5a ) levels of tnf-α. tnf-α was also significantly increased in the cocktail (p = 0.005). il-1β displayed no significant increase with the growth factors as well as the cocktail suggesting that the growth factors do not induce il-1β in pdcs. to further confirm the action of the growth factors, experiments using blocking antibodies against gmcsf, vegf and gcsf as well as a combination of all three antibodies were performed. pdcs cultured in the presence of pbec conditioned medium were stimulated with influenza in the presence of the antibodies and percent inhibition of type i ifn, tnf-α and il-1β secretion by pdcs was calculated. as is evident from fig. 5b conditioned media and stimulated with influenza (data not shown). these data suggest that though gmcsf and gcsf can enhance the induction of type i ifn and tnf-α in pdcs, the synergistic action of all three factors is really what is responsible for the increase of these cytokines observed in pdc cocultured with pbecs. type i ifn secretion by pdcs plays a critical role in fighting viral infection at the respiratory mucosa. here we demonstrate for the first time that the capacity of pdcs to secrete type i ifn is enhanced by growth factors secreted by pbecs. previous studies suggest that pdcs may play a tolerogenic role in the respiratory mucosa via induction of t regulatory cells. 5,20 a very recent study in mice by lynch et al. 21 has demonstrated that depletion of pdcs early in life enhanced features of asthma such as airway hyper-responsiveness, allergic inflammation and airway remodeling due to an impaired ability to expand tregs and maintain tolerance in the airways. our data indicates that exposure to pbecs induces tolerance in pdcs at homeostasis but concomitantly also primes pdcs to enhance their response to infections (figs. 1, 2) . the induction of tregs by pdcs is enhanced which could be due to the increased expression of ido. the factors which lead to the increase in tolerogenic capacity of pdcs remain to be explored. in keeping with the activation genes in rna-seq, we also observed increased secretion of type i ifn from influenza stimulated pdcs co-cultured with pbecs. the expression of tlr9 and zbp1 was upregulated. zbp1 has recently been shown to be the innate sensor of influenza virus. 22 it signals via irf3 and nfkb both of, which were upregulated in pdcs exposed to pbecs. the rna-seq data did not display significant changes in pathways known to induce ifn secretion in pdcs such as the irf7. 23 however, increased nfkb related genes may play a role in tlrdependent ifn production. 24, 25 interestingly, pge2 has been reported to be a negative regulator of type i ifn secretion by human dcs upon stimulation with tlrs 26 and prostaglandin pathway was downregulated in pdcs exposed to pbecs (fig. 2e) . the expression of another gene, fcɛriγ/fce1a is significantly downregulated in pdcs co-cultured with pbecs and it has been demonstrated to inhibit tlr mediated type i ifn production in human pdcs via itam-mediated signals. 27 similarly socs1, a key negative regulator of myd88-dependent type i ifn signaling 28, 29 is also downregulated in pbecs exposed pdcs. in addition to negative regulators, pdcs co-cultured with also pbecs displayed increased expression of genes involved in type-i-ifn signaling. type i ifn can act both in an autocrine and a paracrine manner. 30 it can amplify its own secretion via autocrine actions. increased expression of ifnar1 would enhance the autocrine action of ifns. therefore, pbecs seem to prime pdcs to enhance type i ifn secretion. this is supported by the increase in the genes of the isg15 pathway. isg15 is not only an interferon response pathway 31 but is also reported to bind to irf-3 and enhance ifn responses. 32, 33 furthermore, isg15 pathway can also affect important signaling pathways such as ifn, nfκb, and jnk pathways, all of, which are upregulated in pdcs after exposure to pbecs. 34 together the gene expression changes suggest that pbecs enhance the capacity of pdcs to produce type i ifn via inhibiting the negative regulators as well as by enhancing the expression of molecules, which can amplify the production of type i ifns. we had observed similar priming of mdcs by pbecs earlier. 8, 35 even though rna-seq data suggests that pdcs cocultured with pbecs express type i ifn at homeostasis, we were unable to detect type i ifn in our supernatants without stimulation with influenza. this could be due to lack of sensitivity of the elisa which may not be able to detect the various type i ifn subtypes. it is also possible that the type i ifn is bound to the receptor and therefore not detectable. high level of type i ifn secretion in the absence of infection is not desirable since its upregulation under such conditions is usually associated with autoimmunity. the tolerogenic nature of pdcs at homeostasis may therefore prevent secretion of the type i ifn to prevent inflammation. it is also possible that the changes visible at gene level are not transcribed to proteins as it is well established that only about 40% of genes are transcribed to proteins. this is especially true for cytokines. we also find that growth factors, gmcsf, gcsf, and vegf secreted by pbecs help prime pdc responses to influenza (fig. 5) . interestingly, these factors signal via the jak-stat pathway, which displays significant upregulation in the rna-seq data (fig. 2d) . some of the growth factors have already been reported to modulate dc function. for example, knockdown of gcsf receptor in dcs has been reported to reduce the expression of at least proinflammatory cytokines and antigen presenting molecules, while the expression of pdl1 and pdl2 was increased. 36 gmcsf is also considered as a dc activation agent 37 and has been demonstrated to enhance the ifn-α production from pdcs in lupus patients. 38 moreover, gmcsf secretion by kidney epithelial cells has also been reported to enhance maturation and type i ifn production in pdcs. 39 additionally, gmcsf has been shown to synergize with tlr-dependent microbial stimuli to upregulate of proinflammatory cytokines. 40 interestingly, bdca-4, which functions as a marker for pdcs is a receptor on endothelial and tumor cells for vascular endothelial growth factor (vegf-a). 41 studies examining the effect of vegf on lung dc subtypes in vegf-transgenic mice have reported an increase in lung mdc and pdc populations. 42 an increase in inflammatory responses of mdc was also reported in the same study. the effect of vegf on pdc responses has not been examined. our results indicate a possible synergistic effect of vegf with other growth factors on type i ifn production by pdcs. the present study only examines effect of soluble factors from pbecs on pdc functions. information regarding cell to cell contact between pbecs and pdc is missing which can also play an important role in modulation the functions of pdcs. this is because existing technology only allows the differentiation of pbecs at ali on a transwell insert which prevents contact with pdcs. development of systems which can allow pbec differentiation on surfaces which are capable of providing cell to cell contact would be very valuable in studying such interactions. in summary, rna-seq data indicates that exposure of pdcs to pbecs both tolerizes and activates pdcs. pbecs exposed pdcs induce increased levels of tregs at homeostasis. however, the pdcs are also primed by pbecs to respond to infections. in keeping with the rna-seq data, we observe increased secretion of type i ifn and other cytokines in response to influenza in pdcs cocultured with pbecs. the pdcs also primed th1 responses in t cells. the enhanced response of pdcs co-cultured with pbecs was due to the action of growth factors, gmcsf, gcsf and vegf, which were secreted by pbecs on differentiation. to our knowledge this is the first report, which investigates the crosstalk between human pdcs and pbecs. these data highlight possible mechanisms to enhance the production of type-i ifn in the airways, which is critical for host defense against respiratory infections. plasmacytoid dendritic cells: sensing nucleic acids in viral infection and autoimmune diseases unraveling the functions of plasmacytoid dendritic cells during viral infections, autoimmunity, and tolerance the dendritic cell lineage: ontogeny and function of dendritic cells and their subsets in the steady state and the inflamed setting the multifaceted biology of plasmacytoid dendritic cells essential role of lung plasmacytoid dendritic cells in preventing asthmatic reactions to harmless inhaled antigen plasmacytoid dendritic cells control lung inflammation and monocyte recruitment in indirect acute lung injury in mice plasmacytoid dendritic cells drive acute asthma exacerbations airway epithelial cells enhance the immunogenicity of human myeloid dendritic cells under steady state airway epithelial cells condition dendritic cells to express multiple immune surveillance genes airway epithelial cells regulate the functional phenotype of locally differentiating dendritic cells: implications for the pathogenesis of infectious and allergic airway disease impaired secretion of interferons by dendritic cells from aged subjects to influenza: role of histone modifications age-associated impaired plasmacytoid dendritic cell functions lead to decreased cd4 and cd8 t cell immunity macrophages and dendritic cells express tight junction proteins and exchange particles in an in vitro model of the human airway wall a three-dimensional cellular model of the human respiratory tract to study the interaction with particles tolerogenic plasmacytoid dendritic cells control paracoccidioides brasiliensis infection by inducting regulatory t cells in an idodependent manner ido-orchestrated crosstalk between pdcs and tregs inhibits autoimmunity regulation of type i interferon responses zbp1/dai ubiquitination and sensing of influenza vrnps activate programmed cell death the enigmatic plasmacytoid t cells develop into dendritic cells with interleukin (il)-3 and cd40-ligand tolerogenic plasmacytoid dc plasmacytoid dendritic cells protect from viral bronchiolitis and asthma through semaphorin 4a-mediated t reg expansion zbp1/dai is an innate sensor of influenza virus triggering the nlrp3 inflammasome and programmed cell death pathways irfs: master regulators of signalling by toll-like receptors and cytosolic pattern-recognition receptors compartment-specific control of signaling from a dnasensing immune receptor transcriptional networks in plasmacytoid dendritic cells stimulated with synthetic tlr 7 agonists prostaglandin e2 inhibits ifn-alpha secretion and th1 costimulation by human plasmacytoid dendritic cells via e-prostanoid 2 and eprostanoid 4 receptor engagement plasmacytoid dendritic cell-specific receptor ilt7-fc epsilonri gamma inhibits toll-like receptor-induced interferon production socs1 regulates the ifn but not nfkappab pathway in tlrstimulated human monocytes and macrophages cross-regulation of two type i interferon signaling pathways in plasmacytoid dendritic cells controls anti-malaria immunity and host mortality self-priming determines high type i ifn production by plasmacytoid dendritic cells proteomic identification of proteins conjugated to isg15 in mouse and human cells positive regulation of interferon regulatory factor 3 activation by herc5 via isg15 modification beyond isglylation: functions of free intracellular and extracellular isg15 isg15 enhances the innate antiviral response by inhibition of irf-3 degradation dendritic cell-airway epithelial cell cross-talk changes with age and contributes to chronic lung inflammatory diseases in the elderly gcsf receptor regulates antigen uptake and expression of cytokines and costimulatory molecules in dendritic cells systematic cytokine receptor profiling reveals gm-csf as a novel tlr-independent activator of human plasmacytoid predendritic cells regulation of the interferon-alpha production induced by rna-containing immune complexes in plasmacytoid dendritic cells human plasmacytoid dendritic cells acquire phagocytic capacity by tlr9 ligation in the presence of soluble factors produced by renal epithelial cells cutting edge: granulocyte-macrophage colony-stimulating factor is the major cd8+t cell-derived licensing factor for dendritic cell activation bdca-2, bdca-3, and bdca-4: three markers for distinct subsets of dendritic cells in human peripheral blood lung vascular endothelial growth factor expression induces local myeloid dendritic cell activation this study was supported by grant from the nih ag045216 (to aa), and from the national center for research resources and the national center for advancing translational sciences # ul1 tr000153. the content is solely the responsibility of the authors and does not necessarily represent the official views of the nih. we are grateful to icts uc irvine for providing the blood samples. the online version of this article (https://doi.org/10.1038/s41385-018-0097-1) contains supplementary material, which is available to authorized users.competing interests: the authors declare no competing interests. key: cord-025181-eg108wcd authors: zheng, zhihang; li, min; liu, zhihua; jin, xia; sun, jin title: establishment of murine infection models with biological clones of dengue viruses derived from a single clinical viral isolate date: 2020-05-25 journal: virol sin doi: 10.1007/s12250-020-00229-y sha: doc_id: 25181 cord_uid: eg108wcd dengue virus (denv) is a single-stranded rna virus transmitted by mosquitoes in tropical and subtropical regions. it causes dengue fever, dengue hemorrhagic fever and dengue shock syndrome in patients. each year, 390 million people are estimated to be infected by four serotypes of dengue virus, creating a great burden on global public health and local economy. so far, no antiviral drug is available for dengue disease, and the newly licensed vaccine is far from satisfactory. one large obstacle for dengue vaccine and drug development is the lack of suitable small animal models. although some denv infection models have been developed, only a small number of viral strains can infect immunodeficient mice. in this study, with biologically cloned viruses from a single clinical isolate, we have established two mouse models of denv infection, one is severe lethal infection in immunocompromised mice, and the other resembles self-limited disease manifestations in balb/c mice with transient blockage of type i ifn responses. this study not only offers new small animal models of dengue viral infection, but also provides new viral variants for further investigations on dengue viral pathogenesis. dengue virus (denv) is an arbovirus transmitted by aedes aegypti and aedes albopictus mosquitoes. it belongs to flavivirus genus of flaviviridae family and contains a positive single-stranded rna genome. denv can be antigenically classified into four serotypes (denv-1-4), which are often co-circulating in the endemic regions with similar clinical manifestations. though the majority of denv infections are asymptomatic, a minority of which can result in self-limited disease including dengue fever (df), and less than 1% may develop into dengue hemorrhagic fever (dhf) and dengue shock syndrome (dss). because of the global warming, increased air travel, and the lack of an efficacious vaccine, denv has become the most prevalent mosquito-borne viral pathogen in recent decades (guzman et al. 2010) . each year, denv is estimated to infect 390 million people globally, affecting nearly half of the world population. asia accounts for 75% of the dengue disease burden, followed by latin america and africa (bhatt et al. 2013) . as an imported pathogen in china, dengue virus had caused sporadic outbreaks in southern china before 2013. however, an unexpected large dengue outbreak attacked guangzhou in 2014, resulting in 46,864 national reported cases that year (jin et al. 2015) , in which the majority was caused by dengue virus serotype 1 (denv-1), and the others by serotype 2 (denv-2) (zhao et al. 2016; li et al. 2017) . since then, 2000-5000 confirmed cases were reported to china cdc (chinese centers for disease control and prevention) each year. in nature, non-human primates and human are the only mammalian hosts for denv infection. naturally acquired dengue virus does not infect any outbred or inbred mice, unless viruses are inoculated with extremely high dose (e.g. 10 -9 pfu of denv-2 16681 in 4-week old c57/bl6) (chen et al. 2007) or through intracerebral injection. in some other models, mouse-brain adapted strains are used. but such infection of adapted strain or intracerebral infection usually results in neurological manifestations, which are not routine clinical signs of human diseases. and, after passaging in mice, characteristics of these mouse brain-adapted viral strains may differ from strains naturally acquired, attenuation in their ability to cause human disease was documented (sabin and schlesinger 1945; hotta 1952; sarathy et al. 2015) . thus, the inability of dengue virus to replicate in murine model had hampered the development of dengue vaccine and antiviral therapy. monkey models, while more useful, are not suitable for routine preclinical testing of vaccine candidates or drug prototypes because of their high cost, limited animal availability and ethical concerns. the species specificity of denv infection may be partially attributed to the differences of innate immunity between primates and mice. interferon (ifn) signaling plays an essential role in the protection against dengue disease in humans, nevertheless denv has also evolved many mechanisms to antagonize ifn signaling and ifn production in vivo (green et al. 2014) . one of them is the binding and degradation of human stat2 by dengue viral protein ns5 (ashour et al. 2010) . another is the cleavage of human sting molecules by dengue viral protease ns2b3 (aguirre et al. 2012; yu et al. 2012) . interestingly, both of these two antagonistic mechanisms only exist in human. denv ns5 does not bind to murine stat2, neither does ns2b3 cleave murine sting proteins, because the target sequences on these two molecules are highly species specific (ashour et al. 2010; aguirre et al. 2012; stabell et al. 2018) . but deletion of mstat2 or msting could enhance the replication of denv in mouse or mouse derived primary cells (ashour et al. 2010; aguirre et al. 2012) . these observations also imply the necessity of ifn signaling and ifn production in restricting denv infection in mice. therefore, immunocompromised mice without complete ifn responses or humanized mice are presumed more susceptible to denv infection. as humanized mice are laborious to prepare, and often introduce mouse-to-mouse variation, they are less suitable for application in vaccine development (akkina et al. 2011) . in contrast, knockout mice are easier to manipulate and a series of ifn deficient mice have been tested in the development of dengue infection model, including those of ifna/br -/-, ifncr -/-, ifna/b/cr -/-, mavs -/-, stat1 -/-, stat2 -/-, irf3 -/and irf7 -/(shresta et al. 2006; perry et al. 2011; prestwood et al. 2012 ). among them, type i/ii ifn receptors double knock out mice usually develop lethal diseases when challenged with suitable denv strains, thus are used more frequently. however, only a limited number of dengue virus strains are able to replicate in mice and reproduce the severe symptoms (e.g., vascular leakage) of human disease. these viruses are usually based on clinical isolates (denv-3 c0360/94, denv-3 703-4, denv-4 tvp-376), obtained through alternate passage between mosquito c6/36 cells and ag129 mice (denv-2 d2s10), or isolated after plaquepurification (denv-2 d2y98p-pp1, denv-2 s221) (sarathy et al. 2015) . such mouse models are valuable in testing vaccine efficacy and studying viral pathology or transmission. notably, there are an increasing number of investigations that suggest t cell immunity is an essential component for flavivirus vaccine (st john and rathore 2019). but, type i interferon are vital for cd4 ? and cd8 ? t cell generation and maturation (crouse et al. 2015) . and ifn-c is also one of the major cytokines mediating th1/ctl function and t cell development. therefore, ifn deficient transgenic mice are not ideal for studies of t cell immunity or t cell vaccines in an active immunization model, for the lacking of cross-talk between ifn pathway and adaptive immunity. in this study, we have purified two dengue virus strains of serotype 2 exhibiting different plaque sizes from one clinical isolate denv-2 gz. through subcutaneous injection of low doses of these viruses to type i/ii ifn receptor knock-out mice, we successfully established mouse models of lethal infection. the pathogenicity of the two variants in ag6 was demonstrated to be different. further, we compared the infectivity of these two viral variants in a self-limited infection model, in which type i ifn receptor of wild-type balb/c mice had been transiently blocked before infection, and found only the virus strain exhibiting larger plaque size caused infectious viral particles in sera. our research here provides two kinds of dengue virus infection mouse models, which reproduce both severe and self-limited manifestations of human diseases, expanding the current choice of dengue viral infection small animal models. dengue virus serotype 2 clinical strain, denv-2 gz, was originally isolated from a patient in guangzhou in 2012, and was kindly provided by dr. xiaoyan che of zhujiang hospital in guangzhou, china. the virus was propagated in mosquito c6/36 cells for no more than three passages in our lab. virus titer was determined in vero cells by foci forming assay. vero cells were passaged in dulbecco's modified eagle's medium (dmem, gibco, ny, usa) supplemented with 10% fetal bovine serum (fbs, gibco, auckland, new zealand) and 1% penicillin/streptomycin (p/s, gibco, ny, usa), and cultured at 37°c in a 5% co 2 incubator. mosquito c6/36 cells were cultured in minimum essential medium (mem, gibco) supplemented with 10% fbs, 1% penicillin/streptomycin and 1% non-essential amino acids (gibco, ny, usa) in a 28°c incubator under 5% co 2 atmosphere. to purify viruses of different plaque morphology from the original clinical isolate, virus stock of denv-2 gz was serially diluted, and used to infect c6/36 cell at 8, 4, 2, 1, and 1/2 pfu per well in a 96-well plate. seven days post infection, supernatant of each well was harvested. the foci size of virus that produced in each well was determined subsequently in vero cells by foci forming assay in 48-well plate. only supernatant containing virus with uniform foci size was kept for further propagation. after three passages, two viral clones maintained different plaque sizes, herein named as denv-2 1d4-5-sp (variant with small plaque size) and denv-2 8h2-7-lp (variant with large plaque size), were stored in a -80°c freezer. titer of virus stock and infectious viral particle in serum of mice were determined using foci forming assay in vero cells. specifically, vero cells were seeded at 7 9 10 4 cells/ well in a 48-well tissue culture plate and incubated overnight at 37°c, 5% co 2 . on the second day, sera of infected mice or viral stocks were serially diluted in dmem and added into wells containing vero cells for a 2 h incubation period. subsequently, the viral inoculum was removed and dmem supplemented with 1.5% fbs, 1% p/s, 1.5% carboxymethylcellulose (cmc, sigma, mo, usa) was added. after incubation for 96 h at 37°c, cells were fixed with 4% paraformaldehyde (pfa). following the removal of pfa, infected cells were stained with antidengue monoclonal antibody d1-11 (1:500, santa cruz biotechnology, ca, usa) for 2 h, followed by addition of biotin labeled anti-mouse igg (santa cruz biotechnology, ca, usa), and then streptavidin-alkaline phosphatase (sigma, mo, usa). finally, the foci of dengue virus were visualized by adding bcip/nbt substrate (beyotime biotechnology, jiangsu, china). neutralizing activity of mouse sera was evaluated using foci reduction neutralization test (frnt) as previous described (sun et al. 2017 ). genome information of two viruses was acquired through sanger sequencing. briefly, viral genomic rna was extracted from the virus stocks. after synthesis of viral cdna with specific primers, 14 fragments covering the whole coding region of genome were amplified using phusion high-fidelity pcr polymerase (thermofisher, lithuania). when the pcr product concentration was high enough, they were sent directly for sequencing. otherwise, fragments were inserted into the peasy-blunt plasmid (transgene, beijing, china), which was then used to transform e. coli bacteria. three colonies containing each of the fragments were sequenced. full-length sequence of the single open reading frame was achieved based on the consensus sequence of each fragment, and then stringed together by overlapping them with each other. after that, sequence information of these two viruses was submitted to genbank, and the access numbers are mn952966 (denv-2 8h2-7-lp) and mn952967 (denv-2 1d4-5-sp) respectively. ifn-c elispot assay was performed according to manufacturer's protocol (mabtech, nacka strand, sweden). splenocytes of infected or control mice were isolated by homogenizing and filtering through 40 lm cell strainers. after depleting erythrocytes, cells were re-suspended in 10% fbs/prmi-1640 and added to elispot plates (millipore, co. cork, ireland) pre-coated with ifn-c antibodies. then pbs, 10 lg/ml peptide, ns1 or e80 protein or 5 lg/ ml cona was added to each well to stimulate splenocytes, and the plates were incubated at 37°c for 40-48 h. after being washed with pbs for 5 times, plates were stained with biotin conjugated antibody, and subsequently streptavidin-horse radish peroxidase (hrp). finally, immune spots were visualized with the addition of tmb substrate. the image of spots was captured and analyzed by immu-nospot analyzer (cellular technology ltd. usa). balb/c mice were purchased from beijing vital river laboratory animal technology. type i and type ii ifn receptor double knock-out c57bl/6 mice (ag6) were originally provided by dr. guangxun meng of institut pasteur of shanghai, and raised in our laboratory. all mice were bred and maintained in specific pathogen-free barrier facilities and used at 6-8 weeks of age. for virus infection experiments, all procedures were completed under bsl-2 and absl-2 laboratory conditions as approved by the biosafety committee of institut pasteur of shanghai. female balb/c mice of 6-8 weeks old were injected with 2 mg anti-type i ifn receptor monoclonal antibody (mar1-5a3, bioxcell, usa) intraperitoneally (i.p.) at one day prior to challenge with 1.8 9 10 6 pfu parental strain, 8h2-7-lp or 1d4-5-sp through subcutaneous (s.c.) injection. blood samples were collected though retro-orbital bleeding daily from the 1st day post infection (1 dpi) to 7th day post infection (7 dpi). whole blood was lysed and homogenized within trizol-ls reagent (invitrogen, ca, usa) for measuring viral rna copy. meanwhile, for further detection of infectious viral particles in serum, sera were isolated from the blood through centrifugation. female ag6 mice of 6-8 weeks old were infected with 1 9 10 3 pfu, 1 9 10 4 pfu or 1 9 10 5 pfu denv viruses through subcutaneous (s.c.) injection on day 0. body weight and survival rate were monitored from 1 dpi to 15 dpi, during which moribund mice with weight loss over 20% were euthanatized and recorded as death. vascular leakage was determined following intravascular administration of evans blue dye (sigma, louisiana, usa). briefly, 200 ll evans blue dye (1% in pbs) was intravenously injected to mice on the 7th days post-infection of the two viral variants. after 1 h dissemination, the mice were anaesthetized and perfused with 50 ml pbs. digestive tracts and livers were collected and photographed. to quantify viral genomic copy in blood, rna was extracted following the user's guide of trizol ls reagent. a two-step quantitative rt-pcr (qrt-pcr) was then performed. first, cdna was synthesized using the fas-tquant rt kit (with gdnase) (tiangen biotech, beijing, china). second, amplifications were carried out using the fastfire qpcr premix (probe) kit (tiangen biotech, beijing, china) with the primer pairs designed according to the conserved sequence in denv 3 0 utr: (forward, 5 0 -tgayaagcartcagacac-3 0 ; reverse, 5 0 -tcac-carrctccctttgc-3 0 ; fluorescence probe 5 0 -cca-gagatcctgctgtctc-3 0 ). amplification cycles consisted of an initial incubation step of 95°c for 1 min, 40 cycles at 95°c for 5 s, 55°c for 10 s, and 72°c for 20 s. standard rna were in vitro transcribed from a dna template that contains a t7 promoter and the target polynucleotide fragment for the primers indicated above. to obtain the standard curve, serially diluted standard rna (10 2 -10 9 copy) were used as templates in parallel with sample rnas. copy of viral rna was calculated according to standard curves. all data were analyzed using graphpad prism software. t test or two-way anova were used to analyze the significance as indicated in legends. all results were expressed as means plus and minor standard errors of mean (sem). p values of 0.05 or less were considered statistically significant (*p \ 0.05; **p \ 0.01, ***p \ 0.001; ****p \ 0.0001). during in vitro propagation of a clinical strain of denv-2 obtained from guangzhou, china, heterogenous foci of different plaque sizes were observed in vero cell cultures (fig. 1a) . through limiting dilution and culture in mosquito c6/36 cells, two viral variants were separated. after subsequent passage for three rounds in c6/36 cells, two virus strains with stable foci morphology were obtained. one of them forms larger foci of about 1.25 mm diameter after 80 h culture, and it was termed as denv-2 8h2-7-lp (fig. 1a) ; the other forms smaller foci of about 0.25 mm diameter and was termed as denv-2 1d4-5-sp (fig. 1a) . we next compared the replication abilities of these two viruses in both vero cells and c6/36 cells using the onestep growth curve. denv-2 8h2-7-lp had a higher replicating efficiency in vero cells than denv-2 1d4-5-sp during the first 72 h after infection, followed by a decline afterwards, due to the probability of running out target cells for infection; in comparison, denv-2 1d4-5-sp rose slowly to a peak at 96 h post infection (hpi), and kept its plateau until 120 hpi (fig. 1b) . distinct from what was observed in vero cells, there was no obvious growth difference between the two viruses in mosquito cells (fig. 1c) . taken together, these data demonstrated that two viral variants with different foci sizes are isolated from one clinical virus isolate. and these two variants have different replication abilities in vero but not mosquito c6/36 cells. to determine whether the different replication phenotypes reflect underlying genetic divergence between the lp and sp viruses, we sequenced the whole coding sequence of denv-2 8h2-7-lp and denv-2 1d4-5-sp, and aligned their envelope sequence with those of representative viruses belonging to different denv-2 genotypes, including asian genotype, american asian genotype, cosmopolitan genotype and sylvatic genotype. additionally, denv-2 strains isolated in guangdong, china, from 2005 to 2014 were also included for comparison. the phylogenetic analysis showed that denv-2 8h2-7-lp and denv-2 1d4-5-sp belonged to the cosmopolitan genotype and were clustered together with many strains isolated in west pacific regions, with percentages of homology ranging from 99.5% to 100% (fig. 2) . further comparison of genomic information between these two viruses also indicated that they had sequence variations in coding regions as illustrated in table 1 . collectively, we found 14 sequence variations in e, ns3, ns4b and ns5 regions, including 7 synonymous mutations and 7 amino acid changes. among the 7 nonsynonymous mutations, two are in domain i/ii regions of e protein, two are in peptidase domain and helicase domain of ns3 respectively, another is located in a transmembrane helix of ns4b, and the rest two are in ns5 rdrp domain. all of these domains are components of viral binding, replication or processing machinery, and highly related to viral antagonisms to host immunity. the conservation of mutations was also analyzed in comparison with representative strains from different genotypes, interestingly, though most synonymous mutations were also observed in other dengue-2 viruses, the seven nonsynonymous mutations were unique, and these sites seem more conserved in dengue-2 viruses (table 1) . these genomic characteristics may contribute to the different infectivity both in vitro and in vivo. as type i/ii interferon deficient mice are more susceptible to dengue virus infection, we determined the in vivo infectivity of these two viruses in type i and type ii ifn receptors double knock-out ag6 mice. ag6 mice between 6 and 8 weeks old were infected through s.c. injection with 10 3 pfu, 10 4 pfu and 10 5 pfu denv-2 8h2-7-lp or denv-2 1d4-5-sp (fig. 3) . it is observed that infection of either virus led to 100% mortality within 7-11 days, even at a dose as low as 10 3 pfu, though denv-2 8h2-7-lp caused more weight loss and faster death than denv-2 1d4-5-sp at the same dosage. specifically, weight loss began on 3 dpi, 4 dpi and 5 dpi in mice infected with 10 5 , 10 4 , and 10 3 pfu denv-2 viruses were used to infect cells at moi 2.5, supernatant of infected cells were collected at the indicated timepoints. titers of infectious virus in supernatant were measured using foci forming assay in vero cells. detection limits of infectious virus titer were indicated with dashed lines. significance of the differences was calculated with two-way anova test, ***p \ 0.001, n.s., not significant. 8h2-7-lp, respectively (fig. 3a , 3b, 3c), and death began on 7 dpi, 8 dpi and 9 dpi correspondingly (fig. 3d, 3e, 3f ). in contrast, weight loss was first observed 1-3 days later in mice infected with denv-2 1d4-5-sp, on 5 dpi or 6 dpi with inoculation of 10 4 -10 5 pfu or 10 3 pfu, respectively. the death was also delayed, began on 8 dpi or 9 dpi correspondingly. as a mixture of two types of viral variants, the parental strain was also able to cause weight loss and lethal diseases in ag6 mice. comparing to the two purified variants, the parental quasispecies presented intermediate level of pathology, which was most obvious in the dose groups of 10 5 pfu. through evans blue staining, we also detected plasma leakage in sick mice on day 7 post infection by either denv-2 8h2-7-lp or denv-2 1d4-5-sp viruses (fig. 4) . consistent with the progress of weight loss and survival curves, leakage of evans blue in liver or digestive tract was more severe in mice infected with 8h2-7-lp on 7 dpi. the evidence above demonstrated that the two viral isolates show differences on the pathology and disease kinetics, though they both have capabilities for causing lethal infection in ifn deficient mice. although both viruses can cause lethal infection in type i and type ii double knock-out mice, whether the lp and sp viruses behave differently in type ii ifn competent host is unknown. we have recently developed a zikv infection model in balb/c mice with transient blockage of type i ifn fig. 2 phylogenetic analysis of denv-2 1d4-5-sp and denv-2 8h2-7-lp with representative serotype-2 dengue viruses of different genotypes isolated from different geographical regions. the phylogenetic tree was obtained after analyzing envelope sequences of denv-2 with mega x software, using the maximum likelihood method and kimura 2-parameter model. members of six reported genotypes of denv-2 were included. denv-2 1d4-5-sp (mn952967) and denv-2 8h2-7-lp (mn952966) were denoted by red dots in the tree. the scale bar denotes an evolutionary distance of 0.02 nucleotides per position in the sequence. virologica sinica receptor (ifnar) (liang et al. 2018) , we thus used the same strategy to examine these two dengue viruses for further comparison. wt balb/c mice were injected with antibody specific for ifnar at one day prior to s.c. infection with 10 6 pfu of either viral strain, and monitored for seven days. the viral rna of denv-2 8h2-7-lp could be detected throughout the week after infection, and the peak rna level of about 10 6 genomic copy per microgram total rna was reached on day 3 post infection, followed by gradually dropping to 10 4 copy on the 7th day (fig. 5a) . in contrast, viral rna of denv-2 1d4-5-sp was not detected until day 4 post infection, when merely 10 4 copy per microgram of total rna was transiently detected for 2 days. consistently, infectious virus was detected in sera of denv-2 8h2-7-lp infected mice from 2 to 4 dpi, during which peak viremia of 10 3 pfu/ml appeared on day 3 post infection. meanwhile, infectious virus was not detectable in mice infected by denv-2 1d4-5-sp (lower than detection limit of 20 pfu/ml) (fig. 5b) . moreover, 8h2-7-lp viral rna was also detected in liver, spleen and eyes on 3 dpi (fig. 5c ), further confirming replication of 8h2-7-lp in susceptible organs. the parental strain also replicated in balb/c mice ( fig. 5a and 5b) , and the kinetics of viremia were closer to that of 8h2-7-lp, except for the delay of peak viremia to the 4th day post infection in both assays. to examine whether the above observed different virological properties of the two denv variants affect adaptive immunity in host, we evaluated adaptive immune responses induced by their infection in balb/c mice. denv-2 8h2-7-lp infection elicited antibodies crossneutralizing the reference strain denv-2 16681 (fig. 6a ) and it also stimulated t cell responses specific to peptides of denv-2 ns1 and zikv ns3 (fig. 6b) . similarly, neutralizing antibody and t cell responses were also detected in mice which were previously infected by denv-2 1d4-5-sp, though both responses were weaker than those elicited by denv-2 8h2-7-lp. considering genome similarity between the two variants, such different magnitudes of adaptive immune responses are mainly related to the different viral replication of two variants and different expression level of immunogens in vivo. in summary, we isolated from the same viral quasispecies two denv variants that showed distinct in vitro replication phenotype in vero cells, and different in vivo infectivity and pathogenicity in mice. using either of these two viral variants, we have established a lethal infection model in immune deficient ag6 mice. with the variant denv-2 8h2-7-lp, we have also developed a self-limited infection model in wt mice pretreated with type-i ifn receptor antibodies. these two closely-related viruses not only offer new tools for in vivo studies, but also provide table 1 sequence variation between 8h2-7-lp and 1d4-5-sp. in this study, we have successfully separated two viral variants that show different foci sizes in vero cells from a common serotype-2 clinical isolate of dengue virus. using these two viral variants, we established mouse infection models that reproduce severe manifestations of dengue diseases in ag6 mice. such lethal model with 8h2-7-lp or 1d4-5-sp viruses in ag6 mice can be used in pathology study, for example, to explore the viral and host factors that mediate hemorrhagic manifestation of dengue viruses. on the other hand, denv-2 8h2-7-lp was used in another infection model which resembles self-limited infection of dengue virus in balb/c mice. and this transient infection model is more suitable for study of t cell responses, since the immune cell lineages are intact and the ifn-c responses are normal during the development of adaptive immunity. additionally, this self-limited infection model is also suitable for investigating how murine host resolves dengue infection. in previous investigations of dengue infection, only a limited number of viral strains were found to infect immune deficient mice, in which human illness without neurological disease can be studied, and lethal doses of these viruses ranged from 10 4 to 10 7 pfu (sarathy et al. 2015) . one often used virus is dengue serotype-2 strain d220, which was obtained from a neuropathic strain pl046 through alternative passage between c6/36 mosquito cells and ag129 mice. the ld50 of this virus in ag129 is 10 5 -10 6 pfu (orozco et al. 2012) . another is d2y98p-pp1, a double-plaque purified clone from a laboratory strain d2y98p, which had been passaged in c6/36 cell for 20 times. and the lethal dose of d2y98p-pp1 in ag129 mice fig. 3 weight loss and survival curve of ag6 mice after infection with denv-2 8h2-7-lp and denv-2 1d4-5-sp. ag6 mice between 6-8 weeks old were infected with two viral variants and the parental isolate through s.c. injection. three different dose of viruses, 10 3 pfu (a, d; n = 3), 10 4 pfu (b, e; n = 3), and 10 5 pfu (c, f; lp and sp n = 4, parental n = 3), were inoculated into mice. mice injected with pbs were included within each panel for control (ct, n = 6). weight change was recorded and presented as a ratio to the initial weight on day 0. moribund mice with weight loss over 20% were euthanatized and recorded as death. two-way anova was used in statistical analysis of differences among groups, *p \ 0.05, **p \ 0.01, ***p \ 0.001,**** p \ 0.0001, n.s., not significant. was documented higher than 10 5 pfu (tan et al. 2011) . in contrast, the virus strains in this study had 100% mortality within 11 days after inoculation in ag6 mice of 6-8 weeks old even when the dose was reduced to 10 3 pfu, i.e., approximately 100 times more lethal than the d220 and d2y98p-pp1 viral strains, and thus making denv-2 8h2-7-lp virus a unique tool for studying the pathogenesis of dengue virus. through evans blue staining, we also confirmed the manifestation of plasma leakage in infected mice, which is a characteristic of severe dengue diseases in human. collectively, we show the highly virulent dengue strain 8h2-7-lp is ideal for establishing lethal infection models at low inoculum. to increase the utility of the new dengue viral variants in the context of relatively normal immune system, we have also adapted a recently developed model that transiently blocks ifnar with antibodies in balb/c mice. high level of viral rna was maintained within the first week after infection by denv-2 8h2-7-lp, but only low level of transient viral rna was detected at 3-4 dpi in mice administered with denv-2 1d4-5-sp. though no clinical symptom was observed, in mice infected with denv-2 8h2-7-lp, we successfully detected infectious virus in sera, and viral rna in multiple organs including liver, spleen and eyes. the viruses might have been eliminated from mice after 7 days of infection, accompanied by the maturation of denv adaptive immunity. thus, such characteristic and progress of infection is consistent with what was observed in patients with mild disease (who 2009). in previous animal models, denv infection of adult wt mice required extremely high inoculum, intracranially injection, or mouse adapted viruses. a portion of these models led to neurological symptoms or additional clinically irrelevant manifestations, limiting their use for pathology and therapeutic study (plummer and shresta 2014; sarathy et al. 2015) . to our knowledge, this is the first study which developed a denv infection model in wt mice, using a moderate dose of a non-adapted virus strain through physiologically more relevant s.c. injection route. it is often observed that many viruses form heterogenous plaques of different sizes in vitro, indicating that viruses exist as quasispecies during transmission or passaging (kato et al. 2017; moser et al. 2018 ), but the virological differences among various phenotypic variants have not been carefully examined. clinically, such viral diversity may assist arbovirus to survive under different selection pressure from two fig. 4 vascular leakage in ag6 mice induced by infection of denv-2 8h2-7-lp and denv-2 1d4-5-sp. ag6 mice of 8 weeks old were infected with 10 5 pfu denv-2 8h2-7-lp or 1d4-5-sp through s.c. injection. seven days post infection, mice were injected intravenously with evans blue and denv-induced vascular leakage was visualized in different tissues. pbs infected mice were used as negative controls. female balb/c mice of 6-8 weeks old were injected through i.p. with 2 mg antibody to type i ifn receptor (mar1-5a3) at one day prior to infection. on the next day, 10 6 pfu of viruses were inoculated into mice through s.c. route. then, blood samples were collected daily after infection. a viral rna copies in blood were measured using qrt-pcr, two-way anova was used in statistical analysis of the differences among groups, **p \ 0.01, ****p \ 0.0001, and b titers of infectious virus in serum samples were determined through foci forming assay in vero cells; n = 5. t test was used in statistical analysis of the differences among groups, *p \ 0.05, **p \ 0.01, *** p \ 0.001, ****p \ 0.0001. c eye, liver and spleen were collected from 8h2-7-lp virus-infected mice, and viral rna loads were determined, n = 4. detection limits of rna copy and infectious virus titer were indicated with dotted lines. n.d. not detected. distinct hosts, invertebrate and vertebrate. denv-2 8h2-7-lp exhibits better replication capability than denv-2 1d4-5-sp in vero cells, whereas their growth in mosquito cells is similar, reflecting possibility that the large-plaque variants in the original isolate contribute more to viral fitness in mammalian host. consistently, in mouse model, denv-2 8h2-7-lp presents higher pathogenicity in ag6 mice and better replication in balb/c mice. comparing the coding sequence of denv-2 8h2-7-lp and denv-2 1d4-5-sp, we found 7 amino acid variations on e, ns3, ns4b and ns5 proteins. e protein is responsible for virus attachment and entry; ns3, ns4b and ns5 are all the components of replication complex and essential antagonists for type i ifn responses (green et al. 2014 ). thus, further investigation of the underlying mechanism for the different phenotypes of these two viruses is valuable for understanding the molecular basis of dengue virulence and denv-host interaction. denv inhibits type i ifn production in infected cells by cleaving human sting humanized rag1-/-gammac-/-mice support multilineage hematopoiesis and are susceptible to hiv-1 infection via systemic and vaginal routes mouse stat2 restricts early dengue virus replication the global distribution and burden of dengue both virus and tumor necrosis factor alpha are critical for endothelium damage in a mouse model of dengue virus-induced hemorrhage regulation of antiviral t cell responses by type i interferons innate immunity to dengue virus infection and subversion of antiviral responses dengue: a continuing global threat experimental studies on dengue. i. isolation, identification and modification of the virus at 40 days post infection, frnt (foci reduction neutralization test) a and ifn-c elispot assay (b) were performed with mouse sera and splenocytes, respectively. reference strain denv-2 16681 was used in frnt assay, peptides and proteins derived from denv-2 16681 (d2-ns1 265, d2-ns1, d2-e80) and zika virus (z-ns3 43, z-ns3 291) were included in elispot assay to measure specific and crossreactive responses dengue fever in china: an emerging problem demands attention characterization of large and small-plaque variants in the zika virus clinical isolate zikv/hu/s36/chiba/2016 molecular epidemiology demonstrates that imported and local strains circulated during the 2014 dengue outbreak in guangzhou recombinant zika virus envelope protein elicited protective immunity against zika virus in immunocompetent mice growth and adaptation of zika virus in mammalian and mosquito cells characterization of a model of lethal dengue virus 2 infection in c57bl/6 mice deficient in the alpha/beta interferon receptor stat2 mediates innate immunity to dengue virus in the absence of stat1 via the type i interferon receptor mouse models for dengue vaccines and antivirals gamma interferon (ifngamma) receptor restricts systemic dengue virus replication and prevents paralysis in ifn-alpha/beta receptor-deficient mice production of immunity to dengue with virus modified by propagation in mice mouse models of dengue virus infection for vaccine testing murine model for dengue virus-induced lethal disease with increased vascular permeability adaptive immune responses to primary and secondary dengue virus infections dengue viruses cleave sting in humans but not in nonhuman primates, their presumed natural reservoir elaboration of tetravalent antibody responses against dengue viruses using a subunit vaccine comprised of a single consensus dengue envelope sequence subcutaneous infection with non-mouse adapted dengue virus d2y98p strain induces systemic vascular leakage in ag129 mice in: dengue: guidelines for diagnosis, treatment, prevention and control, new edn. who guidelines approved by the guidelines review committee dengue virus targets the adaptor protein mita to subvert host innate immunity epidemiological and virological characterizations of the author contributions js and xj designed and supervised the study. zhz and js carried out the experiments. zhz, js and xj analyzed the data and wrote the paper. zhl and ml assisted with animal experiments on ag6 mice. all authors read and approved the final manuscript. conflict of interest all authors declare that they have no conflict of interest. key: cord-012497-n5pu1yeu authors: rogers, meredith c.; miranda-katz, margot; zhang, yu; oury, tim d.; uccellini, melissa b.; garcía-sastre, adolfo; williams, john v. title: stat2 limits host species specificity of human metapneumovirus date: 2020-07-04 journal: viruses doi: 10.3390/v12070724 sha: doc_id: 12497 cord_uid: n5pu1yeu the host tropism of viral infection is determined by a variety of factors, from cell surface receptors to innate immune signaling. many viruses encode proteins that interfere with host innate immune recognition in order to promote infection. stat2 is divergent between species and therefore has a role in species restriction of some viruses. to understand the role of stat2 in human metapneumovirus (hmpv) infection of human and murine tissues, we first infected stat2(−/−) mice and found that hmpv could be serially passaged in stat2(−/−), but not wt, mice. we then used in vitro methods to show that hmpv inhibits expression of both stat1 and stat2 in human and primate cells, but not in mouse cells. transfection of the murine form of stat2 into stat2-deficient human cells conferred resistance to stat2 inhibition. finally, we sought to understand the in vivo role of stat2 by infecting hstat2 knock-in mice with hmpv, and found that mice had increased weight loss, inhibition of type i interferon signaling, and a th2-polarized cytokine profile compared to wt mice. these results indicate that stat2 is a target of hmpv in human infection, while the murine version of stat2 restricts tropism of hmpv for murine cells and tissue. human metapneumovirus (hmpv) is a negative sense single-stranded rna virus and a member of the pneumoviridae family with its closest related human pathogen respiratory syncytial virus (rsv) [1] . hmpv is a leading cause of severe respiratory illness in children and adults [2] [3] [4] [5] [6] , and causes significant morbidity and mortality in immunocompromised hosts [4, [7] [8] [9] . even though virtually all humans are infected with hmpv by the age of 5 years [10, 11] , reinfection with hmpv is common, which can lead to particularly poor outcomes in immunocompromised and elderly patients [2] [3] [4] [7] [8] [9] . due to the wide prevalence and burden of severe disease in multiple risk groups, it is essential to better understand the virus and host factors that are involved in promoting and restricting hmpv infection. many viruses encode proteins that interfere with interferon (ifn) signaling or inhibition of ifn stimulated genes (isgs) [12] . pneumoviruses and the related paramyxoviruses antagonize host ifn signaling, specifically stat1 (signal transduction and activator of transcription) and/or stat2. paramyxoviruses utilize alternate transcripts of the phosphoprotein [13] , while rsv encodes the nonstructural proteins ns1 and ns2 that suppress stat1/stat2 signaling [14] . despite being related to rsv and paramyxoviruses, none of the nine proteins encoded by the hmpv genome have homology with known pneumovirus or paramyxovirus inhibitors of stat1 or stat2 [15, 16] . in spite of this, hmpv was shown to inhibit phosphorylation of stat1 in cell lines and primary human epithelial cells [17] . recent work in our lab identified the hmpv small hydrophobic (sh) protein as necessary and sufficient to inhibit phosphorylation of stat1 [18] . others found no inhibition of stat2 by hmpv; however, these experiments used a relatively low multiplicity of infection (moi) in cell culture [17, 19] . other groups have also shown roles for sh [20, 21] as well as the hmpv proteins g and m2-2 [22] [23] [24] in the inhibition of innate immunity. viruses have tropism for cell types, tissues, and host species, which explains why natural infections only cause certain symptoms in certain species. tropism can be caused by a host cell lacking a viral entry receptor, or by the virus' ability to circumvent host immunity only in certain cells or species. mouse models for hmpv have been developed, but mice are only semi-permissive for hmpv, requiring a high viral inoculum for productive in vivo infection ( [25] and unpublished observations), which may be due to species differences in the innate immune proteins antagonized by the virus. for example, human and mouse stat1 have~95% amino acid similarity, while human and mouse stat2 are relatively divergent, with only~70% similarity [26, 27] . as a consequence of this sequence divergence, murine stat2 restricts the related human viruses rsv, hpiv2, and hpiv5 in cell culture [28, 29] . we sought to test the hypothesis that hmpv may similarly be restricted by stat2 in a species-specific manner. in this study, we used in vitro and in vivo approaches to determine the effect of hmpv on human and murine stat1/2. we found that hmpv antagonized expression and nuclear localization of both stat1 and stat2 in primate cells but not murine cells. transfection of stat2-deficient u6a cells with hstat2 or mstat2 revealed that suppression of both hstat1 and hstat2 were prevented by mstat2. in vivo, hmpv infection of hstat2 knock-in (hstat2 ki) mice led to greater weight loss, inhibition of interferon stimulated genes, and a th2-skewed cytokine profile compared to wt mice. these data indicate that mstat2 restricts hmpv's ability to inhibit both stat1 and stat2 and suggest that productive infection of humans by hmpv is partly due to inhibition of hstat2 antiviral signaling. the following cell lines were used: beas2b (atcc crl-9609), vero e6 (atcc ccl-81), nih/3t3 (atcc crl-1658), u3a (ecacc) (atcc: manassas, va, usa; ecacc: salisbury, uk). cmt64/61 (ecacc) and u6a (ecacc) cells were purchased from sigma (st. louis, mo, usa). all cell lines were maintained, infected, and transfected in dmem supplemented with 10% fbs. puno1-hstat2 and puno1-mstat2 plasmids were purchased from invivogen (san diego, ca, usa). hmpv clinical strain tn/94-49 (subtype a2) was grown in llc-mk2 cells (atcc ccl-7) and virus titers measured by plaque assay in llc-mk2 cells as previously described [25] . for cell experiments, cells were inoculated with hmpv strain tn/94-49 at an moi of 1-10 in a 24-well plate. mock-infected cells were inoculated with media or llc-mk2 cell lysate, which had an equivalent effect on stat1 and stat2 protein levels and phosphorylation [30] . then, 16-24 h post-infection, cells were treated with 1000 u/ml human ifnα (alpha 2a) (pbl; piscataway, nj, usa) for human and primate cells, or 1000 u/ml murine ifnβ (pbl) for mouse cells for 30-40 min. after treatment, media were aspirated from the tissue culture dish and cells were lysed in ripa buffer (thermofisher; waltham, ma, usa) for western blotting or fixed in 4% paraformaldehyde for immunofluorescence. transfections were performed using lipofectamine 2000 (life technologies; carlsbad, ca, usa) following the manufacturer's protocol with some exceptions: for each well in the 24-well plates, 2.5 µl lipofectamine 2000 was diluted into 37.5 µl opti-mem (not supplemented) and was mixed with 1 µg plasmid in 37.5 µl opti-mem for a total volume of~75 µl. this was incubated for 15 min before being added to wells. fifty percent of media was replaced after 6 h. then, 16-24 h post-transfection, cells were treated with ifnα/β as above and lysed for western blotting. cells were lysed in ice-cold ripa buffer (thermo scientific, waltham, ma, usa) containing halt protease and phosphatase inhibitors (thermo scientific). samples were centrifuged at 14,000× g for 15 min to pellet debris, and supernatant was used for protein analysis. total protein from cell lysate was quantified by bca assay (thermo scientific) and protein was normalized between samples. a total of 11-20 ug of protein was loaded into each well for each western blot experiment. after total protein quantification, samples were normalized and more concentrated samples were diluted in ripa buffer to achieve equal concentrations across samples. samples were diluted in 4× lds sample buffer (invitrogen; carlsbad, ca, usa) and 10× sample reducing agent (invitrogen: carlsbad, ca, usa) and boiled for 8 min at 95 • c. proteins were separated on a 4-12% bis-tris polyacrylamide gel before transfer to a pvdf membrane. membranes were blocked in 5% bsa in tris-buffered saline with 0.1% tween-20 (tbs-t) or 5% nonfat dry milk in tbs-t. primary antibodies against stat1 (cell signaling technologies (cst, danvers, ma, usa), d1k9y), pstat1 (cst, 58d6), stat2 (cst, d9j7l), pstat2 (for human, cst d3p2p; for mouse (polyclonal), emd millipore (burlington, ma, usa)), actin (hrp conjugated, abcam (cambridge, uk), and gfp (invitrogen, a-11122)) were used in a 1:1000 dilution (or a 1:10,000 dilution for actin) overnight with rocking at 4 • c. after tbs-t wash, hrp-conjugated secondary antibodies against rabbit or mouse were added in 5% bsa-tbs-t or 5% milk-tbst for 1 h. blots were washed with tbs-t and put in tbs until imaging. western blots were developed using west femto (thermo scientific) and imaged on a chemidoc xrs+ (biorad, hercules, ca, usa). band quantification was performed using image lab v5.2 (biorad). after infection and ifn treatment, cell supernatant was removed from beas2b cells and cells were fixed with 4% paraformaldehyde, then permeabilized with 100% methanol at −20 • c. cells were blocked with 5% goat serum and 0.3% triton-x100 in pbs. primary and secondary antibodies were diluted in 1% bsa and 0.3% triton-x100 in pbs. dapi (5 µg/ml) was added to distinguish nuclei. antibodies used were stat1 (cst, d1k9y) stat2 (cst, d9j7l), hmpv anti-fusion protein 54g10 [31] , secondary alexa fluor 488-conjugated anti-human and alexa fluor 568-conjugated anti-rabbit (invitrogen). all fluorescent images were equally color enhanced (increased brightness and contrast) for better visibility in publication. for quantification of nuclear stat1 or stat2 signal, original (non-color enhanced) fluorescent images were analyzed using fiji imagej [32] [33] [34] . nuclei were selected from the dapi viruses 2020, 12, 724 4 of 18 channel, then the mean intensity was measured in the stat1 or stat2 channel. for quantification of cytosolic protein, the cytosol was selected from the transmitted light image and the mean intensity was measured in the stat1 or stat2 channel. c57bl/6 mice were obtained from jackson laboratories (bar harbor, me, usa). stat1 −/− and stat2 −/− mice were from dr. david levy (new york university; new york, ny, usa) [35] and dr. christian schindler (columbia university; new york, ny, usa) [36] , courtesy of dr. john alcorn. hstat2 ki mice were from adolfo garcia-sastre (icahn school of medicine at mount sinai; new york, ny, usa) [37] . male and female 6-14-week-old mice were used for all experiments, with control groups age and sex matched. all mice were bred and maintained in specific pathogen free conditions in accordance with the institutional animal care and use committee of university of pittsburgh (protocol #18032376, approved 1 march 2018). for all animal experiments, mice were anesthetized with ketamine-xylazine (mouse passaging experiments) or isofluorane (hstat2 ki experiments) and intranasally (for mouse passaging) or intratracheally (for hstat2 ki) infected with 1-5 × 10 6 pfu/ml hmpv tn 94-49, or lung homogenate from a previously infected mouse, in a 100-µl volume. for serial passage, mice were euthanized at day 5 post-inoculation. lungs were harvested and homogenized in 1-2 ml 0% opti-mem in a glass tenbroeck homogenizer as previously described [25] . lung homogenates were clarified by centrifugation at 1200 rpm (300× g) for 10 min. clarified lung homogenate was aliquoted into cryovials and snap-frozen in an ethanol dry ice bath before storage at −80 • c. for serial passage, clarified lung homogenate was pooled from 2-3 mice before inoculation into recipient mice. the left lobe of the lung was inflated and fixed in formalin, then subsequently sectioned and stained with h&e, with groups combined into a single slide. all fields of each h&e-stained slide were scanned at 200× magnification, and each field was scored as follows: 0: normal lung tissue, 1: 0% to 25% of tissue area with inflammation, 2: 25-50% of tissue area with inflammation, 3: 50-75% of tissue area with inflammation, or 4: 75-100% of tissue area with inflammation [38] . the frequency of each inflammation score was combined by group. inflammation scores were combined as follows to allow for statistical analysis by fisher's exact test: scores of 0-2 were categorized as mild/moderate inflammation and scores of 3-4 were categorized as severe inflammation. lung homogenate from the whole lung was frozen at −80 • c until use for quantitative rt-pcr (qpcr). rna was extracted from the lung homogenate with the purelink rna mini kit (thermo fischer scientific, waltham, ma, usa) according to manufacturer instructions and stored at −80 • c until further use. five microliters of extracted rna were used for qrt-pcr in 25-µl reaction mixtures on an abi steponeplus real-time pcr system (thermo fisher scientific) using the agpath-id one-step rt-pcr kit (thermo fisher scientific). taqman primers and probes were used according to manufacturer's instructions (all thermo fisher scientific). all values were normalized to the housekeeping gene hprt, and fold change in chemokine was measured in infected groups compared to mock-infected controls using the ∆∆ cycle threshold method. lung homogenate from the whole lung was used for cytokine analysis by cytokine & chemokine 36-plex mouse procartaplex panel 1a (invitrogen) according to manufacturer's instructions. data were analyzed using prism version 8.0 (graphpad software; san diego, ca, usa). a one-tailed student's t test was used to analyze fold change from western blots. fisher's exact test was used to analyze differences in histology inflammation scores. a two-tailed unpaired student's t test was used to analyze comparisons between two groups. for comparisons between multiple groups, a one-or two-way anova was performed. error bars on graphs represent sd. some respiratory viruses, including influenza, sars, and mers, have been serially passaged in mice to generate mouse-adapted virus strains, which have provided important tools for studying viral and host determinants of disease and tropism [39] [40] [41] [42] [43] . we initially sought to generate mouse-adapted hmpv by serially passaging infected lung homogenate directly into a recipient mouse. however, viral titers in lung homogenate of infected c57bl/6 mice were not sufficiently high enough to productively infect a recipient mouse, despite repeated attempts, and serial passage in rag-2 −/− or ifnar −/− mice was also unsuccessful [30] . however, hmpv replicates to significantly higher titer in ifnar −/− mice [44] , indicating that type 1 interferon signaling restricts hmpv in mice in vivo. we therefore inoculated stat2 −/− mice with hmpv strain tn/94-49 and found that the virus grew to significantly higher titer in stat2 −/− mice compared to stat1 −/− or wt mice ( figure 1a ). hmpv could be passaged mouse to mouse at low level detectable by qpcr in stat1 -/mice, but we chose to pursue stat2 based on our data showing higher replication in stat2 −/− mice and the fact that the stat2 protein is less conserved between human and mouse compared to stat1. we subsequently inoculated stat2 −/− and wt mice with clarified lung homogenate from infected stat2 −/− and wt mice, respectively, and found that hmpv could be serially passaged mouse to mouse in stat2 −/− but not wt mice ( figure 1b ). these data show that serial passage of hmpv is restricted by stat2 in vivo, indicating that this protein and/or the ifn signaling pathway in general is a barrier to murine infection by hmpv. viruses 2020, 12, 724 6 of 18 inoculated stat2 −/− and wt mice with clarified lung homogenate from infected stat2 −/− and wt mice, respectively, and found that hmpv could be serially passaged mouse to mouse in stat2 −/− but not wt mice ( figure 1b) . these data show that serial passage of hmpv is restricted by stat2 in vivo, indicating that this protein and/or the ifn signaling pathway in general is a barrier to murine infection by hmpv. since stat2 appeared to be important in restricting hmpv infection of mice, we next explored how hmpv infection affects stat1 and stat2 in human cells. previously, it had been shown by us and by others that hmpv can specifically inhibit stat1 phosphorylation and expression [17, 18] . to further understand the effects of hmpv on stat1 and stat2, we used a human bronchoepithelial cell line, beas2b, to perform imaging studies of stat1 and stat2 in the presence or absence of hmpv. after infection with hmpv for 24 h, beas2b cells were treated with ifn to induce phosphorylation and nuclear translocation of stat1 and stat2. after treatment with ifn, stat1 and stat2 translocated to the nucleus in mock-infected cells, whereas nuclear import was inhibited in hmpv-infected cells but not in uninfected cells in the same dish ( figure 2 ). these data indicate that hmpv infection inhibits nuclear translocation of the stat1/2 heterodimer in vitro, suggesting that antagonism of stat1/2 may be a key step to promote hmpv infection. hmpv grew to significantly higher titer in stat2 −/− mice than in wt mice, leading us to hypothesize that hmpv inhibits stat1 and stat2 in primate cells but fails to inhibit these in murine cells. to test this hypothesis, we infected both primate and murine cell lines with hmpv and measured expression as well as phosphorylation of stat1 and stat2 after ifn treatment. we found that hmpv infection of veroe6 cells (primate cells that are ifn-responsive but cannot produce endogenous ifn) led to decreased total and phosphorylated stat1 and stat2 (figure 3) . the decreased levels of pstat1 and pstat2 appeared to be driven by the decreased protein abundance of total stat1 and stat2 in vero cells, as quantified by the relative fold change in phosphorylated compared to total stat proteins. in contrast, when murine cell lines cmt64/61 (c57bl/6 lung adenocarcinoma) and nih/3t3 (murine fibroblast, deficient in some steps of ifn signaling [45] ) were infected with hmpv, we saw increased total and phosphorylated stat1 and stat2 (figure 4) . these data indicate that hmpv antagonizes stat1 and stat2 in primate cells but fails to achieve this inhibition when introduced to murine cells. cells. to test this hypothesis, we infected both primate and murine cell lines with hmpv and measured expression as well as phosphorylation of stat1 and stat2 after ifn treatment. we found that hmpv infection of veroe6 cells (primate cells that are ifn-responsive but cannot produce endogenous ifn) led to decreased total and phosphorylated stat1 and stat2 (figure 3 ). the decreased levels of pstat1 and pstat2 appeared to be driven by the decreased protein abundance of total stat1 and stat2 in vero cells, as quantified by the relative fold change in phosphorylated compared to total stat proteins. in contrast, when murine cell lines cmt64/61 (c57bl/6 lung adenocarcinoma) and nih/3t3 (murine fibroblast, deficient in some steps of ifn signaling [45] ) were infected with hmpv, we saw increased total and phosphorylated stat1 and stat2 (figure 4) . these data indicate that hmpv antagonizes stat1 and stat2 in primate cells but fails to achieve this inhibition when introduced to murine cells. studies in hpiv2 and hpiv5 showed that degradation of stat2 or stat1, respectively, required the expression of both stat1 and stat2 [46] . to understand whether hmpv inhibition of stat1 and stat2 is dependent on expression of both proteins, we used u3a and u6a cells, which are specifically deficient in stat1 and stat2, respectively [47] . we found that hmpv infection of stat2-deficient u6a cells led to reduction of stat1 and pstat1 in a dose-dependent manner with increasing moi (figure 5a,c) . hmpv infection of stat1-deficient u3a cells also reduced stat2 and pstat2, but only at a moi of 3 ( figure 5b,d) . at moi = 1 in u3a cells, hmpv did not inhibit stat2 expression or phosphorylation. these data indicate that hmpv has the capacity to target stat1 and stat2 independently of each other; however, antagonism of stat1 by hmpv appears to be a more efficient process than stat2 inhibition. viruses 2020, 12, x for peer review 10 of 18 studies in hpiv2 and hpiv5 showed that degradation of stat2 or stat1, respectively, required the expression of both stat1 and stat2 [46] . to understand whether hmpv inhibition of stat1 and stat2 is dependent on expression of both proteins, we used u3a and u6a cells, which are specifically deficient in stat1 and stat2, respectively [47] . we found that hmpv infection of stat2-deficient u6a cells led to reduction of stat1 and pstat1 in a dose-dependent manner with increasing moi (figure 5a,c) . hmpv infection of stat1-deficient u3a cells also reduced stat2 and pstat2, but only at a moi of 3 ( figure 5b,d) . at moi = 1 in u3a cells, hmpv did not inhibit stat2 expression or phosphorylation. these data indicate that hmpv has the capacity to target stat1 and stat2 independently of each other; however, antagonism of stat1 by hmpv appears to be a more efficient process than stat2 inhibition. we next asked whether the species-specific stat2 inhibition was specifically due to differences in the human and murine forms of stat2, or whether hmpv fails to inhibit some other step in the innate immune response in murine cells. u6a cells were transfected with either human or murine stat2 (hstat2 and mstat2, respectively), infected with hmpv, treated with ifn, and analyzed for stat1/2 expression and phosphorylation. we found that cells transfected with mstat2 were more resistant to inhibition of stat1 and 2 than those transfected with hstat2 (figure 6 ). at an moi of 3, a significant reduction of stat2 expression was seen in hmpv-infected cells that had been transfected with hstat2, but not those transfected with mstat2. however, with an increased moi of 10, reduced stat2 protein levels were seen regardless of the transfection type ( figure 6d ). interestingly, we found that even though stat2 was not required for stat1 inhibition (figure 5) , the presence of mstat2 in u6a cells constrained inhibition of stat1 by hmpv ( figure 6b,c) . overall, these data suggest that species-specific differences in stat2 limit the inhibition of both stat proteins by hmpv. to establish whether hmpv antagonizes the human form of stat2 compared to murine stat2 in vivo, we used mice engineered to express human stat2 in place of murine stat2 (hstat2 ki) [37] . we infected hstat2 ki and wt mice with either a low (2 × 10 6 pfu/ml) or high (5 × 10 6 pfu/ml) inoculum of hmpv. at both low and high inoculum, hstat2 ki mice lost significantly more weight compared to wt controls ( figure 7a ). this correlated with a greater degree of lung inflammation ( figure 7b) , where hstat2 ki mice had a significantly increased frequency of inflammation scores that were categorized as severe (scores of 3 or 4, p = 0.0024). the weight loss and increased inflammation was not associated with higher viral titers in infected hstat2 ki mice; in contrast, at day five of lower inoculum infection, hstat2 ki mice had modestly lower viral burden compared to wt mice, though this was not replicated at higher titer ( figure 7c ). these data suggest that, in vivo, stat2 antagonism worsens disease severity but does not contribute to increased virus replication in mice. viruses 2020, 12, x for peer review 12 of 18 to establish whether hmpv antagonizes the human form of stat2 compared to murine stat2 in vivo, we used mice engineered to express human stat2 in place of murine stat2 (hstat2 ki) [37] . we infected hstat2 ki and wt mice with either a low (2 × 10 6 pfu/ml) or high (5 × 10 6 pfu/ml) inoculum of hmpv. at both low and high inoculum, hstat2 ki mice lost significantly more weight compared to wt controls ( figure 7a ). this correlated with a greater degree of lung inflammation ( figure 7b) , where hstat2 ki mice had a significantly increased frequency of inflammation scores that were categorized as severe (scores of 3 or 4, p = 0.0024). the weight loss and increased inflammation was not associated with higher viral titers in infected hstat2 ki mice; in contrast, at day five of lower inoculum infection, hstat2 ki mice had modestly lower viral burden compared to wt mice, though this was not replicated at higher titer ( figure 7c ). these data suggest that, in vivo, stat2 antagonism worsens disease severity but does not contribute to increased virus replication in mice. figure 7 . hstat2 ki mice have greater disease severity and inhibition of isgs compared to wt mice after infection by hmpv, despite no increase in virus titer. hstat2 ki mice and c57bl/6 mice were intratracheally inoculated with hmpv at a low (2 × 10 6 pfu/ml) or high (5 × 10 6 pfu/ml) titer and weighed daily over the course of infection (a). (b) lung specimens were taken at day five postinoculation with hmpv at low titer, stained with h&e, and scored by a pathologist. histology images were captured at 100×. histological scoring was calculated by percentage of inflammation per field of view, with scores of 0, 1, 2 (mild/moderate) representing 0, 1-25, and 26-50%, and 3 or 4 (severe) representing 51-75 or 76-100%, respectively. the difference between wt and hstat2 ki mice in the frequency of mild/moderate and severe inflammation scores was statistically significant by fisher's to better understand the ability of hmpv to antagonize interferon signaling in vivo, we quantified levels of ifnα and the isgs mx1 and socs1 in hstat2 ki and wt mice after hmpv infection. ifnα is produced via a feedback amplification loop of isgs during the type i ifn response [48] , while mx1 and socs1 were selected for their roles as known isgs that have differential functions in the type i ifn response. we found that mrna expression of mx1 and socs1 were reduced in hstat2 ki mice compared to wt mice at 24 h post-hmpv infection ( figure 7d) . additionally, protein level of ifnα declined in hstat2 ki mice compared to wt mice during hmpv infection ( figure 7e ). overall, these data indicate that hmpv is indeed able to antagonize type i ifn signaling when mstat2 is replaced by hstat2 in vivo, though this did not lead to increased virus replication. we hypothesized that the increased weight loss and inflammation despite slightly decreased viral titer in hstat2 ki mice was due to aberrant stat signaling in these mice. to better understand the global consequences of substituting human stat2 into mice in vivo, we performed multiplex cytokine analysis on lung homogenates of hmpv-infected hstat2 ki and wt mice at day five after high-dose inoculation, as day five represents a time point that bridges innate and adaptive immunity. overall, hstat2 ki mice had reduced expression of the characteristic th1 cytokines ifnγ and tnfα ( figure 8a ) and adopted a th2-skewed cytokine profile demonstrated by increased concentrations of il-4, il-5, il-13, eotaxin, and il-31 ( figure 8b ). altogether, our data suggest that the substitution of hstat2 for murine stat2 leads to skewing of both the innate and the adaptive immune response to hmpv. to better understand the ability of hmpv to antagonize interferon signaling in vivo, we quantified levels of ifnα and the isgs mx1 and socs1 in hstat2 ki and wt mice after hmpv infection. ifnα is produced via a feedback amplification loop of isgs during the type i ifn response [48] , while mx1 and socs1 were selected for their roles as known isgs that have differential functions in the type i ifn response. we found that mrna expression of mx1 and socs1 were reduced in hstat2 ki mice compared to wt mice at 24 h post-hmpv infection ( figure 7d) . additionally, protein level of ifnα declined in hstat2 ki mice compared to wt mice during hmpv infection ( figure 7e ). overall, these data indicate that hmpv is indeed able to antagonize type i ifn signaling when mstat2 is replaced by hstat2 in vivo, though this did not lead to increased virus replication. we hypothesized that the increased weight loss and inflammation despite slightly decreased viral titer in hstat2 ki mice was due to aberrant stat signaling in these mice. to better understand the global consequences of substituting human stat2 into mice in vivo, we performed multiplex cytokine analysis on lung homogenates of hmpv-infected hstat2 ki and wt mice at day five after high-dose inoculation, as day five represents a time point that bridges innate and adaptive immunity. overall, hstat2 ki mice had reduced expression of the characteristic th1 cytokines ifnγ and tnfα ( figure 8a ) and adopted a th2-skewed cytokine profile demonstrated by increased concentrations of il-4, il-5, il-13, eotaxin, and il-31 ( figure 8b ). altogether, our data suggest that the substitution of hstat2 for murine stat2 leads to skewing of both the innate and the adaptive immune response to hmpv. figure 8 . hmpv-infected hstat2 ki mice adopt a th2-skewed cytokine profile. hstat2 ki mice and c57bl/6 mice were intratracheally inoculated with hmpv at 5 × 10 6 pfu/ml. at day five post-infection, lungs were homogenized and cytokines were measured by multiplex cytokine assay. (a) measurement of prototypical th1 cytokines ifnγ and tnfα in lung homogenates of hstat2 ki and c57bl/6 mice. (b) concentration of th2-related cytokines il-4, il-5, il-13, eotaxin, and il-31 in murine lung homogenates. * p < 0.05, ** p < 0.01, unpaired t test. n = 5/group, one experiment. animal models are essential tools for studying infections by human viruses. the differences in host responses between humans and animals, both in vitro and in vivo, help reveal important mechanisms of how viruses interact with the immune system. we showed that hmpv can be serially passaged in stat2-deficient mice, whereas serial passage in wt mice was not possible, suggesting a role for stat2 in mediating in vivo restriction. hmpv inhibited expression and nuclear localization of stat1 and stat2 in primate airway epithelial cells, while the virus failed to inhibit either stat in murine cells. stat inhibition occurred in a hstat2-dependent manner, as transfection of mstat2 into human cells reduced stat1 and stat2 antagonism. furthermore, the knock-in of hstat2 into mice enabled hmpv to inhibit type i ifn signaling and alter the cytokine profile of infected mice. collectively, these data suggest that stat2 in mice represents a significant barrier to murine infection by hmpv. we observed that a relatively high moi of hmpv (three or more) was needed to inhibit stat2, whereas inhibition of stat1 occurred at a moi of one. these effects at different mois suggest that stat1 inhibition by hmpv is more efficient than stat2 inhibition. hmpv proteins could have a higher affinity for stat1 compared to stat2, or perhaps stat2 inhibition is achieved with a viral protein that is transcribed at a lower abundance. further research to elucidate which hmpv protein is responsible for antagonizing stat2 will increase our understanding of how hmpv inhibits different steps of the innate immune response. a limitation of the in vitro component of this work is that cell lines may have inherent differences in the strength of their innate immune signaling, in addition to the differences in the efficiency of infection and transfection between cell lines. previous work in our lab showed that autocrine and paracrine effects of ifn signaling on neighboring uninfected cells could confound the effects of hmpv infection on stat1 expression and phosphorylation [18] . here, we attempted to control for this effect in primate cells by using vero e6 cells, which cannot produce endogenous ifn. however, no equivalent murine cell line currently exists, though we opted to use the murine nih/3t3 cell line for experiments in figure 4 , as this line has some known impairments in endogenous ifn signaling [45, 49] . several prior studies have described species-specific differences in stat2 inhibition by the flaviviruses dengue, zika, and yellow fever viruses [50] [51] [52] . consequently, hstat2 ki mice infected with mouse-adapted zika virus exhibited increased viral replication, broader tissue spread, and enhanced transplacental spread [37] . in contrast, we found that despite the clear preference for inhibition of hstat2 over mstat2 in vitro, hmpv-infected hstat2 ki mice had similar virus replication in the lung compared to wt mice. nevertheless, the presence of hstat2 resulted in reduced expression of ifnα and mx1 in these mice, consistent with inhibition of stat1/2 signaling, although not to a sufficient level to result in a significant increase in viral replication in lung. interestingly, hstat2 ki mice had worse disease than wt mice when infected with hmpv, as measured by weight loss and lung inflammation. this phenotype was associated with an altered cytokine profile that favored th2 cytokines over th1. since ifnα was significantly reduced in hstat2 ki compared to wt mice, it is likely that either direct or indirect inhibition of stat2 by hmpv drove the th2 phenotype in this study. ifns and il-4 have opposing roles in the immune response, with ifns promoting an antiviral th1 response and il-4 driving a th2 response characterized by asthma and allergy. this response is driven in part by ifn-mediated inhibition of il-4 via increased socs1 expression [53] ; our data here are consistent with this previous work, as we saw increased il-4 and other th2 cytokines while ifns and socs1 were reduced (figures 7 and 8) . due to the dramatic skewing of the th1/th2 axis in the presence of hstat2, we hypothesize that antagonism of stat2 by hmpv in human infection may contribute to the association of hmpv and asthma in susceptible individuals [54] [55] [56] . in addition, stat2 has immunoregulatory functions that may have been disrupted in the hstat2 ki mice [57] [58] [59] . overall, these data indicate that hmpv targets expression of both stat1 and stat2 in a host-specific manner. future studies to determine the specific hmpv protein(s) and molecular interactions involved in stat inhibition will reveal mechanisms of productive infection and pathogenesis in humans. while mouse models of human diseases have strengths and limitations, these data highlight how the innate immune response to hmpv in mice may be profoundly different than it is during natural infection of humans, an important consideration for the development of future therapeutics and vaccines. taxonomy of the order mononegavirales: update human metapneumovirus and lower respiratory tract disease in otherwise healthy infants and children the role of human metapneumovirus in upper respiratory tract infections in children: a 20-year experience virological features and clinical manifestations associated with human metapneumovirus: a new paramyxovirus responsible for acute respiratory-tract infections in all age groups an outbreak of severe respiratory tract infection due to human metapneumovirus in a long-term care facility human metapneumovirus (hmpv) infection in immunocompromised children rates of hospitalizations for respiratory syncytial virus, human metapneumovirus, and influenza virus in older adults a prospective study comparing human metapneumovirus with other respiratory viruses in adults with hematologic malignancies and respiratory tract infections seroepidemiologies of human metapneumovirus and respiratory syncytial virus in young children, determined with a new recombinant fusion protein enzyme-linked immunosorbent assay ten strategies of interferon evasion by viruses paramyxovirus evasion of innate immunity: diverse strategies for common targets human metapneumovirus: lessons learned over the first decade analysis of the genomic sequence of a human metapneumovirus genomic analysis of four human metapneumovirus prototypes human metapneumovirus inhibits ifn-alpha signaling through inhibition of stat1 phosphorylation human metapneumovirus small hydrophobic (sh) protein downregulates type i ifn pathway signaling by affecting stat1 expression and phosphorylation human metapneumovirus inhibits ifn-β signaling by downregulating jak1 and tyk2 cellular levels human metapneumovirus small hydrophobic protein inhibits interferon induction in plasmacytoid dendritic cells human metapneumovirus small hydrophobic protein inhibits nf-kappab transcriptional activity human metapneumovirus glycoprotein g inhibits innate immune responses human metapneumovirus glycoprotein g inhibits tlr4-dependent signaling in monocyte-derived dendritic cells human metapneumovirus m2-2 protein inhibits innate immune response in monocyte-derived dendritic cells the cotton rat (sigmodon hispidus) is a permissive small animal model of human metapneumovirus infection, pathogenesis, and protective immunity murine stat2 is uncharacteristically divergent stat2: a shape-shifting anti-viral super stat respiratory syncytial virus nonstructural proteins ns1 and ns2 mediate inhibition of stat2 expression and alpha/beta interferon responsiveness stat2 acts as a host range determinant for species-specific paramyxovirus interferon antagonism and simian virus 5 replication unpublished observations a broadly neutralizing human monoclonal antibody exhibits in vivo efficacy against both human metapneumovirus and respiratory syncytial virus imagej2: imagej for the next generation of scientific image data an open-source platform for biological-image analysis image to imagej: 25 years of image analysis targeted disruption of the mouse stat1 gene results in compromised innate immunity to viral disease immune response in stat2 knockout mice an immunocompetent mouse model of zika virus infection il-10 restrains il-17 to limit lung pathology characteristics following pulmonary infection with francisella tularensis live vaccine strain a mouse-adapted sars-coronavirus causes disease and mortality in balb/c mice mouse-adapted mers coronavirus causes lethal lung disease in human dpp4 knockin mice adaptation of pandemic h1n1 influenza viruses in mice studies on the mechanism of adaptation of influenza virus to mice the pathogenesis of infections of the mouse caused by virulent and avirulent variants of an influenza virus role of type i interferon signaling in human metapneumovirus pathogenesis and control of viral replication a mouse cell line, which is unprotected by interferon against lytic virus infection, lacks ribonuclease f activity selective stat protein degradation induced by paramyxoviruses requires both stat1 and stat2 but is independent of alpha/beta interferon signal transduction use of a selectable marker regulated by alpha interferon to obtain mutations in the signaling pathway interferon-inducible antiviral effectors the cgas-sting signaling pathway is required for the innate immune response against ectromelia virus mouse stat2 restricts early dengue virus replication zika virus targets human stat2 to inhibit type i interferon signaling host-specific ns5 ubiquitination determines yellow fever virus tropism interferons inhibit activation of stat6 by interleukin 4 in human monocytes by inducing socs-1 gene expression human metapneumovirus bronchiolitis in infancy is an important risk factor for asthma at age 5 human metapneumovirus infection plays an etiologic role in acute asthma exacerbations requiring hospitalization in adults human metapneumovirus infection in children hospitalized for wheezing stat2 is an essential adaptor in usp18-mediated suppression of type i interferon signaling selective loss of type i interferon-induced stat4 activation caused by a minisatellite insertion in mouse stat2 the combination of ifn beta and tnf induces an antiviral and immunoregulatory program via non-canonical pathways involving stat2 and irf9 the hstat2 ki mice used for this study were produced by the mouse genetics and gene targeting center of research excellence (core), which is supported by the icahn school of medicine at mount sinai and a cancer center support grant (1p30ca196521) from the national cancer institute/national institutes of health. committee for glaxosmithkline, neither activity involved with this publication. key: cord-007783-z1vv63u2 authors: nielsen, claus h.; bendtzen, klaus title: immunoregulation by naturally occurring and disease-associated autoantibodies: binding to cytokines and their role in regulation of t-cell responses date: 2012-08-17 journal: naturally occurring antibodies (nabs) doi: 10.1007/978-1-4614-3461-0_9 sha: doc_id: 7783 cord_uid: z1vv63u2 the role of naturally occurring autoantibodies (nabs) in homeostasis and in disease manifestations is poorly understood. in the present chapter, we review how nabs may interfere with the cytokine network and how nabs, through formation of complement-activating immune complexes with soluble self-antigens, may promote the uptake and presentation of self-molecules by antigen-presenting cells. both naturally occurring and disease-associated autoantibodies against a variety of cytokines have been reported, including nabs against interleukin (il)-1α, il-6, il-8, il-10, granulocyte-macrophage colony-stimulating factor, interferon (ifn)-α, ifn-β, ifn-γ, macrophage chemotactic protein-1 and il-21. nabs against a variety of other self-antigens have also been reported, and using thyroglobulin as an example we discuss how nabs are capable of promoting uptake of immune complexes via complement receptors and fc-receptors on antigen-presenting cells and thereby regulate t-cell activity. knowledge of the influence of nabs against cytokines on immune homeostasis is likely to have wide-ranging implications both in understanding pathogenesis and in treatment of many immunoinflammatory disorders, including a number of autoimmune and autoinflammatory diseases. naturally occurring antibodies play an essential role in our defense against invading microorganisms by directly neutralizing viruses and bacteria, by activating the complement system and by enhancement of phagocytosis, as described elsewhere in this book and in reference 1. in contrast, naturally occurring autoantibodies (nabs) are characterized by binding to self-molecules, and their primary function may involve clearance of senescent cells and metabolic waste products, but they also appear to play important roles in immunoinflammatory processes. [2] [3] [4] [5] [6] in the present chapter, we shall focus on the immunomodulatory effects that self-reactive nabs may have as a result of interactions with cytokines or formation of complement-activating immune complexes (ics) with other soluble self-antigens and targeting these self-antigens to b cells and presumably other antigen-presenting cells (apcs). nabs have been shown to bind a variety of self-molecules, ranging from cytokines and other plasma proteins to tissue-specific antigens, structural proteins, metabolic enzymes, heat shock proteins and dna (tables 1 and 2 ). some nabs, including those reacting with various cytokines and thyroglobulin (tg), are relatively specific, while others, e.g., igm anti-dna antibodies of all species, tend to be polyreactive. 7 nabs exist as igm, iga and igg isotypes. the occurrence of the igg isotype among nabs is indicative of t-cell involvement in shaping of the autoreactive b-cell repertoire. [8] [9] [10] igm nabs are already synthesized in newborns, and different babies produce igm nabs to a similar set of self-molecules. 10 several reports have documented the presence of autoantibodies against cytokines both in healthy individuals (nabs) and in patients with various immunoinflammatory disorders (disease-associated autoantibodies, dabs) ( table 1 ). in most cases, the physiological and/or pathological significance of these antibodies is unclear. however, recent evidence suggests that certain anti-cytokine nabs have significant, if not decisive, pathogenic roles in rare disorders (reviewed in refs. 9, 11, 12) . the biological roles of nabs against cytokines are poorly understood. fab fragments of some of these nabs bind in a saturable and highly specific manner to their respective cytokine. as for example in the cases of nabs to il-1 , il-6 and gm-csf. 9 in some cases, these antibodies are found at levels at or above 50 nm, for example in healthy blood donors and apparently without untoward effects. 13 why and how high-affinity nabs to some cytokines and not to others are induced in healthy individuals is unknown. also obscure is whether these in vitro neutralizing antibodies neutralize their respective cytokines in vivo too, or whether they exhibit carrier-or cytokine-regulatory, or even cytokine-protective functions. 5 low levels of igg and igm capable of neutralizing ifn-, ifn-and ifn-have been detected in blood of apparently healthy individuals (as reviewed in refs. 14, 15). igg nabs against ifn-are especially frequent, and are easily detectable in pharmaceutical preparations of normal human igg (ivig). nabs against ifn-have been found in approximately 10% of healthy caucasians, but the exact frequency is likely to be higher, as these nabs are often difficult to detect in plasma because they are complexed with native ifn-. 16 igg nabs against ifn-are of high avidity and specific, as they do not cross-bind ifn-or other cytokines. nabs to ifn-have been demonstrated in vivo in bioactive form after treatment with ivig. 17 as these nabs neutralize ifn-, ivig therapy suppresses both antiviral and other effects of endogenous ifn-, which may explain some of the many therapeutic effects of ivig. 5, 14, 17, 18 nabs against interleukin-1 nabs to interleukin (il)-1 were first found by direct binding of igg from normal individuals to radiolabeled, human, recombinant il-1 and by igg-mediated competitive interference with il-1 binding to cellular il-1 receptors. 19 subsequently, human ivig, cord blood and sera of patients with various immunoinflammatory disorders were found to contain high-avidity autoantibodies that bind to and neutralize il-1 both in vitro and in vivo. 5, 9, 17, 18, [20] [21] [22] they bind il-1 in a saturable fashion and through the fab fragments of the igg isotypes igg1, igg2 and igg4. the occurrence of detectable anti-il-1 igg in sera of healthy individuals vary with age and sex with male preponderance and markedly increased frequency with age (up to 75% positives among elderly males). 16, 19, 22 a human anti-il-1 autoantibody has been cloned. 23 it is an igg4/ monoclonal antibody which reacts with il-1 , but not with il-1 , the il-1-receptor antagonist (il-1ra) or several other cytokines. it binds with high affinity (k d »10 -10 m), and the presence of somatic mutations in the variable regions suggests antigen-driven affinity maturation. as il-1 from antigen presenting cells is an important co-activator of t cells, particularly in its membrane-bound form, it is important to note that anti-il-1 nabs not only neutralize il-1 in lysates of human blood monocytes, but also membrane-associated il-1-activity. 21 igg nabs to il-6 were first reported in sera of normal individuals. 24 since then, the presence of nabs to il-6 and similar antibodies in patients with immunoinflammatory and fibrotic diseases has been confirmed (reviewed in refs. 9, 11, 14, 15) . high-affinity igg nabs to il-6 have been detected in up to 15% of normal danish blood donors with 1% having titers ranging from 64 to greater than 10,000 and 0.1% having exceedingly high titers. 13 the anti-il-6 nabs bind to il-6 through their fab fragments and they effectively inhibit binding of il-6 to il-6 receptors and, hence, neutralize the bioactivity of il-6. nab-positive donors with high antibody titers have no overt signs of pathology even though they are likely to be functionally il-6-deficient. nabs to il-6 are detectable in ivig and are found in bioactive form, binding il-6 in the circulation following ivig administration. 17 igg nabs against il-10 have been reported in normal sera and in preparations of pooled normal human igg. 15, 25 indeed, 0.4% of healthy danish blood donors present with these nabs at such high concentrations and avidity that these blood donors are functionally il-10-deficient. 26 these nabs are of the igg isotype and of polyclonal origin. they prevent il-10 from binding to its receptor thereby neutralizing il-10 bioactivity. anti-il-10 nabs are highly specific in that they fail to bind viral forms of il-10 and other members of the human il-10 family including il-19, il-20, il-22, il-24, il-26, il-28a, il-28b, and il-29. using a direct radioligand binding assay, high-affinity nabs to granulocytemacrophage colony-stimulating factor (gm-csf) were reported at very high titers in 4 of 1,238 (0.3%) apparently healthy blood donors. 15 later, all of 72 tested, apparently healthy japanese individuals were shown to possess low levels of these nabs such that more than 99% of gm-csf were bound and neutralized by these antibodies. 27 anti-gm-csf nabs have also been found in human ivig. 9, 18 nabs against xxc-and cc chemokines nabs to various chemokines have also been reported. for example, il-8-and macrophage chemotactic protein (mcp)-1-containing igg-immune complexes have been demonstrated in sera from healthy individuals, where the chemokines themselves are usually not detected. it is hypothesized that circulating igg nabs to chemokines may play a role as a sink for the spill-over of chemokines produced in local tissues. 28 igg nabs to other cytokines, for example il-2, il-3, granulocyte colony-stimulating factor (g-csf), nerve growth factor (ngf), leukemia inhibitory factor (lif), tumor necrosis factor (tnf)-, and soluble tnf receptors have also been reported in normal and diseased individuals, while ige antibodies to il-4, tnf-, tnf-and various chemokines have been reported in sera of aids patients. some of both isotypes, however, cannot be regarded as nabs in that they bind the relevant mediator(s) with low avidities and in some cases bind only to cytokines denatured by adsorption to nitrocellulose membranes or plastic surfaces (using elisa-technologies). anti-cytokine dabs have been reported in a wide range of pathological disorders, suggesting that pathogenetically obscure diseases may eventually be at least partly explained by the presence of anti-cytokine dabs (reviewed in refs. 9, 11, 12). anti-type 1 interferon (ifn) dabs, preferentially of igg type, were first reported in patients with varicella-zoster and hepatitis virus infections, in patients with autoimmune and neoplastic diseases, and in a patient with chronic graft-vs.-host disease (reviewed in refs. l 5, 14, 15, 29, 30) . later reports have documented dabs that bind to other ifn species, usually in patients with various infectious diseases or severe immunodeficiencies. neutralizing dabs against ifn-/ / have been demonstrated primarily in patients with thymoma and/or myasthenia gravis. 31 however, patients with thymic malignancy/ myasthenia gravis have also been reported to express dabs to a variety of other cytokines including, il-1 , il-12 p35 and il-12p40, and il-17a. 31 high-titer dabs against ifn-2 and ifn-have been reported in 100% of european patients with autoimmune polyendocrinopathy syndrome type i (aps-i). 32 this disease is a result of mutations in the autoimmune regulator gene (aire), which impairs thymic self-tolerance induction in developing t cells. the ensuing autoimmunity particularly targets ectodermal and endocrine tissues, but chronic candidiasis is a frequent and early manifestation. although the underlying immunodeficiency of aps-1 is unclear, neutralizing anti-ifn dabs and, most recently, anti-il-17a and anti-il-22 dabs appear to be implicated directly in the pathogenesis, as they appear before development of candidiasis in all informative cases. 33, 34 anti-ifn-dabs have been reported in patients with viral infections and in cerebrospinal fluids from patients with multiple sclerosis and guillain barré syndrome (reviewed in ref. 15 ). circulating dabs to ifn-have also been positively correlated with the severity of both tuberculous and nontuberculous mycobacterial infections (reviewed in ref. 11 ). it is characteristic that patients with extremely high antibody titers had rapidly progressive disease and severe immunodeficiency, most likely due to dab-induced blockade of the crucial macrophage-activating effect of ifn-. although il-1 is secreted from cells producing the cytokine, the dominant form of il-1 appears to be the cell-associated precursor form that is found both intracellularly and on the surface of many cell types, including keratinocytes and 'professional' apcs such as macrophages and b cells. on these cells il-1 is thought to be involved as a juxtacrine co-activator of t cells. 9 il-1 is usually absent in the circulation, if present then only at low concentrations. during infection and inflammation, however, substantial amounts of il-1 may be found in the blood, most likely released from dying cells. cell-associated il-1 is biologically active, and its biological activities are neutralized by antibodies to il-1 , including igg anti-il-1 nabs, but not by antibodies against il-1 . 21 the prevalence of dabs to il-1 in immunoinflammatory disorders vary considerably. for example, anti-il-1 dabs have been found more commonly and at higher levels in patients with non-destructive forms of arthritis. 35 progression of joint destruction in patients with rheumatoid arthritis was negatively associated with the occurrence of circulating il-1 dabs, but patients who seroconverted more than two years after the onset of ra showed the most aggressive development of joint erosion. interestingly, transgenic mice overexpressing the membrane form of human il-1 in macrophage-like and fibroblast-like synoviocytes develop severe arthritis, correlating with the degree of membrane expression of il-1 , but not circulating il-1 . 36 taken together, this suggests that il-1 and/or the lack of il-1 dabs play a role in the erosive processes of rheumatoid arthritis. along with dabs to other cytokines, anti-il-1 dabs have also been demonstrated in patients with juvenile chronic arthritis, thymoma and myasthenia gravis. 22, 31 dabs against interleukin-6 there is an increased prevalence of high-avidity igg dabs to il-6 in patients with rheumatoid arthritis and systemic sclerosis, and the presence of these antibodies signals a poor survival in patients with alcoholic cirrhosis, possibly because of an increased risk for recurrent infections. 9,37,38 a 2.5-fold increase in anti-il-6 dab-positivity has recently been reported in type 2 diabetic patients, and mice vaccinated with il-6 develop obesity and impaired glucose tolerance. 39 these data suggest that an autoimmune reaction against il-6 may be involved in a subset of type 2 diabetics. anti-gm-csf dabs are of clinical interest not only because of gm-csf's growth-potentiating effect on macrophages and granulocytes, but also because gm-csf appears to be a central mediator affecting bronchial epithelial cells, possibly through its marked effect on eosinophils, both as a chemoattractant, growth promoter and stimulator. in accordance with results obtained in gm-csf knockout mice, anti-gm-csf dabs have been associated with pulmonary alveolar proteinosis (pap), a rare disease in which surfactant lipids and proteins accumulate in pulmonary alveolar macrophages and alveoli, resulting in respiratory insufficiency and failure. 40 recently, isolated anti-gmcsf dabs from a patient with pap were shown to reproduce the pathologic manifestations of the human disease in previously healthy primates. 41 these findings may have therapeutic implications for the potential use of gm-csf not only to treat pap, but also other immunoinflammatory respiratory disorders such as asthma. igg dabs to granulocyte colony-stimulating factor (g-csf) have been demonstrated in felty's syndrome, a relatively rare complication in rheumatoid arthritis, and in some patients suffering from systemic lupus erythematosus (sle) with accompanying neutropenia. 42 igm antibodies were found in 6 neutropenic and 3 normocytic sle patients. interestingly, anti-g-csf antibodies were associated with an exaggerated serum level of g-csf and a low neutrophil count. this may suggest that exposure to high levels of intrinsic g-csf (not known whether bioinactive) may trigger the production of g-csf dabs and, further, that these dabs may have a carrier function in vivo thus slowing the elimination of g-csf from the circulation. 5 using radioimmune and radioreceptor assays, dabs against macrophage-inhibitory protein (mip)-1 and mip-1 have been demonstrated in about 1% of patients, suffering from hiv infection. 43 these antibodies specifically inhibited receptor binding of both chemokines; there was no association between the presence of antibodies and disease stage, or hiv progression rate. igg dabs against il-2, il-10 and transforming growth factor-(tgf-) have recently been demonstrated at relatively high concentrations in up to 33% of patients with inflammatory bowel diseases using elisa and western blot assays. 44 neutralizing dabs against il-12 p35 and il-12p40 have been demonstrated primarily in patients with thymoma and/or myasthenia gravis. 31 indeed, patients with thymic malignancy/myasthenia gravis express dabs to a variety of other cytokines including ifn-/ , ifn-, il-1 and il-17a. 31, 45 interestingly, among these patients those with opportunistic infections possess multiple anti-cytokine dabs, suggesting that these antibodies may be important in the pathogenesis of infections in patients with thymic malignancy. most patients with autoimmune polyendocrine syndrome type i suffer from chronic mucocutaneous candidiasis, and this immunodeficiency was recently associated with high titers of dabs against il-17a, il-17f, and/or il-22. 46 the dabs against il-17a, il-17f and il-22 neutralized these cytokines, but not a host of other cytokines. as these dabs were not found in healthy controls nor in 102 patients with other immunoinflammatory disorders, dabs against il-17a, il-17f and il-22 may have a causative relationship with the development of chronic mucocutaneous candidiasis in patients with this polyendocrine syndrome. anti-il-8 dabs, often complexed with endogenous il-8, have been shown to be an important prognostic indicator for the development and outcome of acute lung injury/ acute respiratory distress syndrome (ards). 47 the il-8/anti-il-8 complexes purified from lung edema fluids activate and trigger chemotaxis of neutrophils and regulate neutrophil apoptosis via igg receptor fc riia. these ics promote an inflammatory phenotype of human umbilical vein endothelial cells, and they upregulate the expression of intercellular adhesion molecule (icam)-1 on the cell surface. lung tissues from patients with ards also express high levels of icam-1. hence, il-8/anti-il-8 complexes may contribute to pathogenesis of lung inflammation by inducing activation of endothelial cells through engagement of igg receptors. anti-cytokine tabs may develop in response to prolonged therapies with natural and recombinant-derived human cytokines. it is unclear whether preexisting anti-cytokine nabs play a role in this regard, but they are probably not without importance, considering the high avidity and high titers of some of these nabs (discussed above). in general, however, tabs develop over time as a result of repeated 'inoculations' with cytokines. they may wax and wane, change in affinity, give rise to side-effects, and/or influence the primary intended therapy. the development of neutralizing tabs was first noted in scattered patients undergoing therapies with human fibroblast-derived ifn-and human recombinant ifn-. [48] [49] [50] [51] this finding received little attention at that time, but the increased use of recombinant human cytokines and cytokine constructs for therapeutic purposes have sharpened the awareness of the clinical importance of anti-cytokine tabs. [52] [53] [54] thus, induction of tabs has now been reported in patients treated with several human recombinant cytokines and growth factors including ifn-, ifn-, ifn-, il-2, gm-csf, often resulting in therapeutic failure and in rare cases even in serious side-effects (reviewed in refs. 9, 15, 55) . while the above-mentioned anti-cytokine nabs are immunoregulatory by nature, there is evidence to suggest that nabs against other self-antigens may also be immunoregulatory, as we shall describe in the following. nabs against a broad variety of self-antigens has been demonstrated ( table 2 ), but their physiological functions, if any, has not been fully elucidated. while some of these nabs have been suggested to play a role in the clearance of senescent cells and metabolic waste products, their role in maintenance (or breakage) of tolerance toward self remains obscure. [2] [3] [4] one mechanism by which nabs against soluble self-antigens may play a regulatory role is by targeting self-antigens to apcs and thereby affecting self-antigen presentation to t cells. it is likely that nabs of igg isotype are capable of directing captured self-antigens to fc -receptor expressing apcs. 56 similarly, nabs of igm or iga isotype may direct self-antigens to b cells and macrophages, expressing the fc / receptor. 57 all these events may promote presentation of self-antigens to autoreactive t cells. one of the self-antigens against which nabs have been most frequently described is thyroglobulin (tg). in a serum-free environment, a subset comprising 2-4% of normal, circulating b cells is capable of binding tg (this subset presumably represents b cells with polyreactive surface immunoglobulins), while tg binds to the entire b-cell population in the presence of autologous serum. 58 the binding can be substantially inhibited by immunoabsorption of tg-reactive autoantibodies from serum or by heat inactivation of serum complement, suggesting that formation of complement-activating ics promoted the binding of tg to the b cells. in accordance with this hypothesis, blockade of complement receptors 1 (cr1/cd35) and 2 (cr2/cd21) reduced the binding of tg to b cells by > 90%, and addition of tg to preparations of normal mononuclear cells suspended in sera from healthy individuals lead to increased deposition of c3-fragments on b cells. 58, 59 in accordance with these findings using tg as model self-antigen, thornton et al. showed that normal serum contains nabs with reactivity against the primary antigen keyhole limpet hemocyanin, and that ics formed with these nabs are capable of fixing fragments of complement component 3 (c3) and bind to b cells via cr1 and cr2 (fig. 1b) . 60, 61 they further demonstrated that expression of the t-cell co-stimulatory molecule cd80 by b cells required a secondary ligation of ic-associated igg to fc rii. 60 it is widely accepted that b cells bearing specific antigen-receptors efficiently take up and present antigens to t cells (fig. 1a) . 62 as outlined, non-specific b cells may also engage in antigen presentation provided for the antigen in question is incorporated in complement-opsonized ics and thereby targeted to cr1, cr2 and fc rii (fig. 1b) . 60, 61, 63, 64 this recruitment of non-specific b cells increases the number of antigen-presenting b cells from a few antigen-specific ones to the entire b-cell population. however, only b cells bearing specific antigen receptors are eventually stimulated by t-helper (th) cells for antibody production. 61 thornton et al. also showed that ic3b-containing tg/nab complexes were also taken up by neutrophils, via complement receptors cr1 and cr3 (cd11b/cd18), indicating that the formation of ics with nabs may also be similarly important for the uptake of self-antigens by myeloid cells (of relevance for dendritic cells). 60 taken together, these findings suggest that antigen presentation is strongly promoted by incorporation of antigens into complement-activating ics with nabs. correspondingly, the presentation of tg on b cells is proportional to the anti-tg nab content of the surrounding serum, and is thus considerably higher in the presence of sera from patients with hashimoto's thyroiditis (ht) or graves' disease (gd) with a high content of anti-tg, as compared with sera from healthy individuals. in analogous studies using myelin basic protein (mbp) as self-antigen, we found that sera from patients with multiple sclerosis (ms) and sera from healthy individuals contained approximately equal concentrations of mbp-reactive igm. 65 upon addition of mbp to normal mononuclear cells suspended in patient or control sera, mbp co-deposited with igm, igg and c3-fragments on monocytes. while the deposition of igm and c3 was approximately similar in patient and control sera, the igg deposition was increased 9-fold in the presence of ms sera, presumably due to the presence of circulating high-affinity igg dabs in the patients. notably, the deposition of c3 fragments as well as that of igm and mbp on monocytes was abrogated by disruption of the tertiary structure of mbp by boiling, as would be expected if complex formation depends upon the interaction of antibodies with conformational epitopes on mbp. this, together with co-stimulatory signals mediated through the cd80/cd86-cd28 pathway and others, activates the th cells. b) antigen-nonspecific b cells, however, may also contribute to presentation of a given antigen, provided that the antigen is found in complement-activating immune complexes (ics) generated by preexisting nabs and/or dabs. attached to the ics, the final degradation product of complement component 3 (c3dg) may bind to complement receptor 2 (cr2) on b cells, which then take up antigen, process it, and present antigen peptides on mhc class ii molecules. c3-opsonized nab/dab-containing ics may also be taken up by fc receptor iib (fc riib). this latter type of interaction stimulates the cd80-dependent binding to th cells. in the presence of untreated serum, even cd4 + th cells from healthy individuals respond to a challenge with tg at high concentrations ( 10 g/ml), although tg induces increased responses by th cells (and b cells) from patients with autoimmune thyroid disease. 58, 66 inactivation of serum complement, immunoabsorption of tg-reactive nabs or disruption of the tertiary structure of tg by boiling.significantly inhibits the tg-induced th cell proliferation and production of t-cell cytokines such as il-2 and il-5. this suggests that th cell responses to self-antigens are strongly influenced by formation of ics between the self-antigens and nabs. presumably, nabs target self-antigens to apcs, as has been shown for polyclonal antibodies to foreign antigens, as well as for dabs to the thyroid self-antigen, thyroid peroxidase (tpo). [67] [68] [69] moreover, recruitment of t cells to the site of infection may also be influenced by nabs and complement. askenase and tsuji demonstrated that t-cell-dependent contact sensitivity responses to hapten and subsequent rises in local ifn-levels were absent in pan b-cell-and antibody-deficient mice, but could be restored by adoptive transfer of purified normal peritoneal b-1 cells, or by i.v. injection of antigen-specific igm monoclonal antibodies. 70 they concluded that the contact sensitivity response was initiated by the formation of complement-activating ics between hapten and naturally occurring igm antibodies, followed by complement activation and c5a-mediated release of vasoactive substances by mast cells, facilitating the recruitment of t cells. it remains to be clarified whether ic-formation with nabs contributes to maintenance of tolerance, or whether it promotes breakage of tolerance. in mononuclear cell cultures from healthy individuals, grown in media containing autologous serum (30% v/v), tg induces immediate production of tnf-and il-10 by mononuclear cells, followed by an almost exclusive production of il-10, a regulatory cytokine with a protective role in autoimmune diseases. 71, 72 a subset of t cells with a cd45ro memory phenotype seems to orchestrate this il-10 production, suggesting that the tg-driven t-cell response in healthy individuals is protective and contributes to the maintenance of tolerance. 72 by comparison, the foreign antigen tetanus toxoid induces no il-10 production, but instead a mixed pro-inflammatory th1/th2-response (il-2, ifn-, il-4 and il-5) under similar conditions. 72 an absolute requirement for an intact tertiary structure of tg and mbp for induction of an il-10 release by mononuclear cells supports a role for nabs in maintaining tolerance. 65, 73 nabs and dabs against cytokines and other self-antigens as discussed here, differ from one another in terms of isotype distribution and epitope recognition patterns. for example, in autoimmune thyroid disease most of the anti-tg activity is associated with igg, with only ~1% in the form of igm, whereas tg-reactive nabs are predominantly of igm isotype. nevertheless, as much as 0.3% of igg in ivig preparations for intravenous use are reactive with tg or cytokines such as il-1 and il-6. 9, 13, 74, 75 there is also evidence to suggest that the antibody recognition patterns of nabs differ from those of dabs. for example, the latter are more restricted in idiotypes and less polyreactive than nabs. 76 a certain idiotype, t44 id, is associated with autoimmune thyroid disease and recognizes one of at least six epitopic clusters on human tg, designated region ii. 77, 78 dabs derived from sera of patients with hashimoto's thyreoiditis and graves' disease recognize primarily region ii and occasionally another region (region iv). 79 by contrast, nabs frequently react with region v and rarely with region ii. 80 thus, recognition of region v may reflect the normal homeostatic recognition of tg. interestingly, the reactivity against particular epitopes commonly recognized by both nabs and dabs seems to change with aging, without affecting the total igg anti-tg autoreactivity. 80 we have recently demonstrated that nabs to thyroid peroxidase (tpo) show a quantitatively different recognition pattern than dabs from patients with ht. anti-tpo nabs recognize an immunodominant region involving two conformational, overlapping epitopes on tpo, referred to as immunodominant regions a (idr-a) and -b (idr-b). 81, 82 in ht, approximately 50% of anti-tpo dabs are directed to the idr-b epitope, while dabs against the idr-a and non-a/non-b regions are approximately equally distributed. 83 tpo-reactive nabs, on the other hand, contain a significantly lower proportion of antibodies to idr-a. 84 interestingly, the propensity to produce autoantibodies directed against the idr-a epitope of tpo seems to be inherited. we recently demonstrated that ht patients and their healthy, monozygotic co-twins had higher proportions of idr-a-reactive anti-tpo antibodies (medians 19% and 18%, respectively) than healthy ordinary siblings to ht patients (9%) and euthyroid controls with no family history of ht (0%). 85 these data confirmed the findings by jaume et al. based on family studies that idr-recognition patterns were genetically transmitted. 86 in other words, the propensity to produce certain dab reactivities may be inherited. further studies are required to determine whether this applies to dabs in general. we have reviewed the immonoregulatory role of nabs with special focus on autoantibodies against cytokines and other soluble self-antigens. based on numerous publications, we and others believe that nabs against cytokines and other self-molecules may in many cases contribute to homeostasis, and that dabs may contribute to disease manifestations, in some instances perhaps as causative pathogenetic factors. these diseases likely include both autoimmune and autoinflammatory conditions. moreover, tabs may neutralize the effect of a number of "biologic" drugs and give rise to side-effects control of early viral and bacterial distribution and disease by natural antibodies auto-antibodies and immunological theories: an analytical review naturally occurring autoantibodies to exoplasmic and cryptic regions of band 3 protein, the major integral membrane protein of human red blood cells naturally occurring anti-band-3 antibodies and complement together mediate phagocytosis of oxidatively stressed human erythrocytes autoantibodies to cytokines -friends or foes? natural autoantibodies: from "horror autotoxicus" to "gnothi seauton natural recognition repertoire and the evolutionary emergence of the combinatorial immune system natural antibodies in childhood: development, individual stability, and injury effect indicate a contribution to immune memory newborn humans manifest autoantibodies to defined self molecules detected by antigen microarray informatics high avidity cytokine autoantibodies in health and disease: pathogenesis and mechanisms anticytokine autoantibodies in infectious diseases: pathogenesis and mechanisms high levels of neutralizing il-6 autoantibodies in 0.1% of apparently healthy blood donors high-avidity autoantibodies to cytokines natural and induced anti-cytokine antibodies detection of autoantibodies to cytokines increased in vivo antibody activity against interferon a, interleukin-1alpha, and interleukin-6 after high-dose ig therapy neutralizing antibodies to granulocyte-macrophage colony-stimulating factor, interleukin-1alpha and interferon-alpha but not other cytokines in human immunoglobulin preparations igg autoantibodies against interleukin 1a in sera of normal individuals distribution and characterization of autoantibodies to interleukin 1a in normal human sera effects of human anti-il-1alpha autoantibodies on receptor binding and biological activities of il-1 autoantibodies to il-1alpha in sera from umbilical cords, children, and adults, and from patients with juvenile chronic arthritis generation and characterization of a human monoclonal autoantibody that acts as a high affinity interleukin-1alpha specific inhibitor anti-interleukin-6 antibodies in normal human serum naturally occurring autoantibodies to interleukin-1alpha, interleukin-6, interleukin-10 and interferon-alpha a state of acquired il-10 deficiency in 0.4% of danish blood donors granulocyte/macrophage-colony-stimulating factor autoantibodies and myeloid cell immune functions in healthy subjects plasma chemokine and chemokine-autoantibody complexes in health and disease interferon-alpha antibodies in autoimmune diseases natural autoantibodies to interferons anti-cytokine autoantibodies in autoimmunity: preponderance of neutralizing autoantibodies against interferon-alpha, interferon-omega and interleukin-12 in patients with thymoma and/or myasthenia gravis autoantibodies against type i interferons as an additional diagnostic criterion for autoimmune polyendocrine syndrome type i interferon autoantibodies associated with aire deficiency decrease the expression of ifn-stimulated genes chronic mucocutaneous candidiasis in apeced or thymoma patients correlates with autoimmunity to th17-associated cytokines autoantibodies against interleukin 1alpha in rheumatoid arthritis: association with long-term radiographic outcome membrane-associated il-1 contributes to chronic synovitis and cartilage destruction in human il-1 alpha transgenic mice anti-interleukin-6 autoantibodies in plasma are associated with an increased frequency of infections and increased mortality of patients with alcoholic cirrhosis autoantibodies against interleukin-6 in rheumatoid arthritis interleukin-6 autoantibodies are involved in the pathogenesis of a subset of type 2 diabetes gm-csf autoantibodies and neutrophil dysfunction in pulmonary alveolar proteinosis patient-derived granulocyte/macrophage colony-stimulating factor autoantibodies reproduce pulmonary alveolar proteinosis in nonhuman primates autoantibodies against granulocyte colony-stimulating factor in felty's syndrome and neutropenic systemic lupus erythematosus low prevalence of antibodies and other plasma factors binding to cc chemokines and il-2 in hiv-positive patients patients with inflammatory bowel disease may have a transforming growth factor-beta-, interleukin (il)-2-or il-10-deficient state induced by intrinsic neutralizing antibodies anti-cytokine autoantibodies are associated with opportunistic infection in patients with thymic neoplasia autoantibodies against il-17a, il-17f, and il-22 in patients with chronic mucocutaneous candidiasis and autoimmune polyendocrine syndrome type i anti-chemokine autoantibody:chemokine immune complexes activate endothelial cells via igg receptors interferon-neutralizing antibodies in a patient treated with human fibroblast interferon high titer of interferon (ifn)-neutralizing antibody in a patient with glioblastoma treated with ifn-alpha. case report antitumor activity of recombinant-derived interferon alpha in metastatic renal cell carcinoma development of neutralizing and binding antibodies to interferon (ifn) in patients undergoing ifn therapy natural and therapy-induced antibodies to cytokines immunogenicity of recombinant human proteins: causes and consequences critical review: assessment of interferon-beta immunogenicity in multiple sclerosis antibodies against erythropoietin and other protein-based therapeutics: an overview a role for autoantibodies in enhancement of pro-inflammatory cytokine responses to a self-antigen, thyroid peroxidase fc alpha/mu receptor mediates endocytosis of igm-coated microbes natural autoantibodies and complement promote the uptake of a self antigen, human thyroglobulin, by b cells and the proliferation of thyroglobulin-reactive cd4+ t cells in healthy individuals autoantibodies in autoimmune thyroid disease promote immune complex formation with self antigens and increase b cell and cd4+ t cell proliferation in response to self antigens natural antibody and complement-mediated antigen processing and presentation by b lymphocytes function of c3 in a humoral response: ic3b/c3dg bound to an immune complex generated with natural antibody and a primary antigen promotes antigen uptake and the expression of co-stimulatory molecules by all b cells, but only stimulates immunoglobulin synthesis by antigen-specific b cells antigen-specific interaction between t and b cells antigen-bound c3b and c4b enhance antigen-presenting cell function in activation of human t-cell clones complement opsonization is required for presentation of immune complexes by resting peripheral blood b cells autoantibodies to myelin basic protein (mbp) in healthy individuals and in patients with multiple sclerosis: a role in regulating cytokine responses to mbp self-reactive cd4(+) t cells and b cells in the blood in health and autoimmune disease: increased frequency of thyroglobulin-reactive cells in graves' disease antibodies to hepatitis b surface antigen potentiate the response of human t lymphocyte clones to the same antigen presentation by peritoneal macrophages: modulation by antibody-antigen complexes effect of antigen/antibody ratio on macrophage uptake, processing, and presentation to t cells of antigen complexed with polyclonal antibodies b-1 b cell igm antibody initiates t cell elicitation of contact sensitivity interleukin-10 and the interleukin-10 receptor the self-antigen, thyroglobulin, induces antigen-experienced cd4 t cells from healthy donors to proliferate and promote production of the regulatory cytokine, interleukin-10, by monocytes production of interleukin (il)-5 and il-10 accompanies t helper cell type 1 (th1) cytokine responses to a major thyroid self-antigen, thyroglobulin, in health and autoimmune thyroid disease subpopulations of thyroid autoantibody secreting lymphocytes in graves' and hashimoto thyroid glands autoantibodies to cytokines in ivig polyreactivity is a property of natural and disease-associated human autoantibodies normal immunoglobulin g (igg) for therapeutic use (intravenous ig) contain antiidiotypic specificities against an immunodominant, disease-associated, cross-reactive idiotype of human anti-thyroglobulin autoantibodies evidence for a restricted idiotypic and epitopic specificity of anti-thyroglobulin autoantibodies in patients with autoimmune thyroiditis antigenic domains on the human thyroglobulin molecule recognized by autoantibodies in patients' sera and by natural autoantibodies isolated from the sera of healthy subjects significance of the recognition of certain antigenic regions on the human thyroglobulin molecule by natural autoantibodies from healthy subjects genetic and epitopic analysis of thyroid peroxidase (tpo) autoantibodies: markers of the human thyroid autoimmune response human thyroid peroxidase: mapping of autoantibodies, conformational epitopes to the enzyme surface proportion of antibodies to the a and b immunodominant regions of thyroid peroxidase in graves and hashimoto disease epitope recognition patterns of thyroid peroxidase autoantibodies in healthy individuals and patients with hashimoto's thyroiditis monozygotic twin pairs discordant for hashimoto's thyroiditis share a high proportion of thyroid peroxidase autoantibodies to the immunodominant region a. further evidence for genetic transmission of epitopic "fingerprints thyroid peroxidase autoantibody epitopic 'fingerprints' in juvenile hashimoto's thyroiditis: evidence for conservation over time and in families natural antibodies against tubulin, actin myoglobin, thyroglobulin, fetuin, albumin and transferrin are present in normal human sera, and monoclonal immunoglobulins from multiple myeloma and waldenstrom's macroglobulinemia may express similar antibody specificities naturally occurring antibodies against nine common antigens in human sera. i. detection, isolation and characterization comparative study of natural autoantibodies in the serum and cerebrospinal fluid of normal individuals and patients with multiple sclerosis and other neurological diseases polyreactive antigen-binding b cells are the predominant cell type in the newborn b cell repertoire igm class autoantibodies in human cord serum t-cell autoimmunity in type 1 diabetes mellitus naturally occurring autoantibodies to skeletal proteins from human red blood cells natural polyreactive iga and igm autoantibodies in human colostrum autoantibodies to factor viii antibodies to a conserved region of hla class i molecules, capable of modulating cd8 t cell-mediated function, are present in pooled normal immunoglobulin for therapeutic use autoantibodies to heat shock protein 90 in the human natural antibody repertoire production and characterization of monoclonal igm autoantibodies specific for the t-cell receptor a monoclonal anti-idiotypic antibody against the antigen-combining site of anti-factor viii autoantibodies defines and idiotope that is recognized by normal human polyspecific immunoglobulins for therapeutic use (ivig) establishment of reference distributions and decision values for thyroid antibodies against thyroid peroxidase (tpoab), thyroglobulin (tgab) and the thyrotropin receptor (trab) b-cell depletion with rituximab in the treatment of autoimmune diseases: graves' ophthalmopathy the latest addition to an expanding family key: cord-002079-jne14jqf authors: macparland, sonya a.; ma, xue-zhong; chen, limin; khattar, ramzi; cherepanov, vera; selzner, markus; feld, jordan j.; selzner, nazia; mcgilvray, ian d. title: lipopolysaccharide and tumor necrosis factor alpha inhibit interferon signaling in hepatocytes by increasing ubiquitin-like protease 18 (usp18) expression date: 2016-05-27 journal: j virol doi: 10.1128/jvi.02557-15 sha: doc_id: 2079 cord_uid: jne14jqf inflammation may be maladaptive to the control of viral infection when it impairs interferon (ifn) responses, enhancing viral replication and spread. dysregulated immunity as a result of inappropriate innate inflammatory responses is a hallmark of chronic viral infections such as, hepatitis b virus and hepatitis c virus (hcv). previous studies from our laboratory have shown that expression of an ifn-stimulated gene (isg), ubiquitin-like protease (usp)18 is upregulated in chronic hcv infection, leading to impaired hepatocyte responses to ifn-α. we examined the ability of inflammatory stimuli, including tumor necrosis factor alpha (tnf-α), lipopolysaccharide (lps), interleukin-6 (il-6) and il-10 to upregulate hepatocyte usp18 expression and blunt the ifn-α response. human hepatoma cells and primary murine hepatocytes were treated with tnf-α/lps/il-6/il-10 and usp18, phosphorylated (p)-stat1 and myxovirus (influenza virus) resistance 1 (mx1) expression was determined. treatment of huh7.5 cells and primary murine hepatocytes with lps and tnf-α, but not il-6 or il-10, led to upregulated usp18 expression and induced an ifn-α refractory state, which was reversed by usp18 knockdown. liver inflammation was induced in vivo using a murine model of hepatic ischemia/reperfusion injury. hepatic ischemia/reperfusion injury led to an induction of usp18 expression in liver tissue and promotion of lymphocytic choriomeningitis replication. these data demonstrate that certain inflammatory stimuli (tnf-α and lps) but not others (il-6 and il-10) target usp18 expression and thus inhibit ifn signaling. these findings represent a new paradigm for how inflammation alters hepatic innate immune responses, with usp18 representing a potential target for intervention in various inflammatory states. importance inflammation may prevent the control of viral infection when it impairs the innate immune response, enhancing viral replication and spread. blunted immunity as a result of inappropriate innate inflammatory responses is a common characteristic of chronic viral infections. previous studies have shown that expression of certain interferon-stimulated genes is upregulated in chronic hcv infection, leading to impaired hepatocyte responses. in this study, we show that multiple inflammatory stimuli can modulate interferon stimulated gene expression and thus inhibit hepatocyte interferon signaling via usp18 induction. these findings represent a new paradigm for how inflammation alters hepatic innate immune responses, with the induction of usp18 representing a potential target for intervention in various inflammatory states. i nterferon (ifn) is a key endogenous mediator of viral clearance by the innate immune response. one excellent example of this is hepatitis c virus (hcv) infection of the liver (1) . interferon signaling drives the expression of multiple interferon-stimulated genes (isgs), which mediate viral clearance. isgs, however, can also be induced by other factors, including inflammatory stimuli such as tumor necrosis factor alpha (tnf-␣) (2) . hcv infection, which induces chronic inflammation of the liver, is associated with high serum tnf-␣ (3, 4) . interestingly, anti-tnf-␣ treatment has been shown to lead to an improved virologic response to ifn-␣/ribavirin antiviral therapy for hcv (5) , while other data show safety of combined treatment but no effect on hcv viral loads of hcv treatment-naive individuals (6) . the purpose of the present study is to define an important causal link between inflammation and the host hepatic innate immune response, with broad viral relevance. our recent work in the host innate immune response to chronic hcv infection suggested an association between hepatocytes and the liver resident immune cells that drive liver inflam-mation (7) (8) (9) . there is a dichotomous response to chronic hcv infection in the liver, and this response predicts who will and who will not respond to exogenous ifn-␣ treatment. in patients that do not respond to therapy with ifn/ribavirin, hepatocytes have strong preactivation of the ifn-response, with high expression of a subset of isgs (7) (8) (9) . in patients exhibiting a sustained virologic response on therapy with ifn/ribavirin, tissue macrophages show a high expression of isgs (7, 9) . the expression of isgs in macrophages or hepatocytes is more predictive of treatment outcome than il28b polymorphisms (7) ; this finding suggests a link between hepatic inflammatory cell activation and viral clearance. liver kupffer cell inflammatory responses are induced by and play a role in the control and clearance of multiple viral infections of the liver (10, 11) . although the mechanisms underlying these patterns are undoubtedly complex, the association between liver macrophages and hepatocytes raises the question of how the hepatocytes react to the cytokines produced by the macrophages (and other inflammatory cells). if isg expression in hepatocytes reflects a response to inflammatory stimuli, then how the liver responds to a viral infection will be modulated by the inflammatory milieu. in hcv infection, we have found that the isg15/usp18 pathway is an important regulatory pathway. both isg15 and usp18 are isgs that are strongly upregulated in the livers of ifn treatment-resistant patients (7) (8) (9) . isg15 is a ubiquitin-like protein that is strongly upregulated by ifn and conjugates to multiple cellular proteins (12, 13) . usp18 is an isg15-specific protease that is also upregulated by ifn-␣ (12, 13) . both isg15 and usp18 have been suggested to blunt type 1 ifn signaling (12, (14) (15) (16) . in addition to its ability to remove isg15 from its conjugated proteins, usp18 has been shown to bind to the type 1 ifn receptor and blunt ifn signaling (15) . upregulation of usp18 may represent a negative-feedback loop counteracting the effects of type 1 ifn. furthermore, knockdown of usp18 increases both isg induction and anti-hcv activity of ifn-␣ (14) , and data have shown that ifn-␣ treatment given to mice in vivo increases hepatic usp18 and blunts the effect of a subsequent dose of ifn-␣ (12) . the mechanisms controlling usp18 expression in the liver are poorly understood but will have relevance to understanding innate immune mechanisms in chronic viral infection. although the majority of work on usp18 has centered on its upregulation by type 1 ifn, interferon stimulated genes have multiple upregulatory stimuli other than ifn-␣ (2, 17) . for example, inflammatory stimuli, e.g., endotoxin and lipopolysaccharide (lps), have been shown to upregulate usp18 in peritoneal exudate macrophages (18) . if similar stimuli lead to increased usp18 expression in hepatocytes, then this pathway could represent a novel link between inflammatory and innate immune responses in the liver. the present study is based on the hypothesis that liver inflammation will directly impact hepatocyte expression of usp18 and therefore will impact ifn signaling. this link will have relevance to multiple diseases, since inflammatory stimuli such as tnf-␣ and lps have been shown to play roles in many liver diseases, including viral hepatitis (19) (20) (21) . the link may also help to explain our observations in chronic hcv infection, since chronic hcv infection is characterized by increased serum tnf-␣ (3, 4), increased hepatocyte usp18, and impaired ifn responsiveness (8) . quite apart from hcv, the importance of the link between hepatic inflammation and innate immune response lies in the downstream effects of the impairment of innate immunity. we speculate that by blocking ifn-␣ signaling, usp18 expression may lead to an enhanced susceptibility to infection with interferon-sensitive viruses and enhanced viral proliferation. support for this notion is found in a mouse study in which expression of usp18 in macrophages led to lower ifn responsiveness, leading to locally restricted replication of vsv (22) . in the present study, treatment of hepatic cells with lps and tnf-␣, but not il-6 or il-10, led to upregulated usp18 expression in hepatocytes. the enhanced usp18 expression was associated with decreased ifn-␣-stimulated expression of p-stat1 and isgs, a phenomenon reversed by usp18 knockdown. as an in vivo correlate of our in vitro findings, experimentally induced hepatic ischemia/reperfusion injury induced usp18 mrna expression in liver and enhanced lymphocytic choriomeningitis (lcmv) replication, an effect not seen in usp18 ϫ/ϫ mice. these data demonstrate that certain inflammatory stimuli (tnf-␣ and lps), as well as ischemic injury, but not other cytokines (il-6 and il-10) can lead to enhanced hepatocyte usp18 expression and thereby inhibit ifn signaling. these findings lend new knowledge to our understanding of how inflammation can modulate hepatic innate immune responses, with usp18 representing a potential target for intervention to reverse any proviral effect of inflammation. phorylated-stat1 (p-stat-1; cell signaling technology, boston, ma), rabbit polyclonal anti-stat-1 (santa cruz biotechnology), goat polyclonal anti-ube1 antibody (santa cruz biotechnology), or mouse monoclonal anti-actin (sigma) antibodies, followed by anti-mouse or anti-rabbit igg conjugated to horseradish peroxidase (calbiochem, billerica, ma). an enhanced chemiluminescence detection kit (amersham pharmacia biotech, uppsala, sweden) was used to determine the levels of protein expression. flow cytometric quantification of usp18 expression in huh7.5 cells. usp18 expression on huh7.5 cells treated with lps or tnf was quantified by using flow cytometry as previously described (28) . briefly, cells were fixed, permeabilized, and stained with mouse anti-human usp18 polyclonal antibody (abnova) or a relevant isotype-matched control antibody. staining was detected with a secondary goat anti-mouse igg labeled with fluorescein isothiocyanate (fitc; santa cruz biotechnology). the cells were acquired using a facscalibur cytometer (bd biosciences, san jose, ca). a minimum of 10,000 events were collected. the resulting data were analyzed using flowjo software (tree star, inc.). the experiments were repeated four times. sirna targeting murine usp18 and ube1l. usp18 small interfering rna (sirna) is a pool of three target-specific 19-to 25-nucleotide (nt) sirnas designed to knock down usp18 gene expression that was obtained from santa cruz biotechnology. the ube1l sirna used was a pool of three to five target-specific 19-to 25-nt sirnas designed to knockdown mouse ube1l gene expression and was obtained from santa cruz. usp18 and ube1l sirna was transfected into 5 ϫ 10 5 primary murine hepatocytes according to the manufacturer's instructions using the santa cruz sirna reagent system sc-45064 (santa cruz) and as previously described (14) . inhibition of inflammatory signaling in primary mouse hepatocytes. to inhibit lps-or tnf-␣-stimulated nf-b activation, primary mouse hepatocytes were incubated with the following inhibitors for 30 min prior to 6 h of stimulation with lps or tnf-␣: p6062, a protein kinase a inhibitor (29); nsc33994, a jak2 inhibitor (30); p3115, a protein kinase c inhibitor (31); pd098059, a mitogen-activated protein kinase kinase inhibitor (32); silibinin, an inhibitor of ikk␣ (33) ; and wortmannin, a phosphatidylinositide 3-kinase inhibitor (34) . the concentrations, sources, and main targets of these inhibitors are described in table 1 . after lps or tnf-␣ stimulation, hepatocytes were harvested and usp18 and il-1␤ (a surrogate of nf-b activation) mrna expression was evaluated as described below. rna isolation and quantitative real-time pcr analysis. huh7.5 cells and primary murine hepatocytes were treated with ifn-␣ (100 u/ml), tnf-␣ (20 ng/ml), lps (100 ng/ml), il-6 (100 ng/ml), or il-10 (10 ng/ ml) over a 24-h time course, and usp18 expression was determined by quantitative pcr (qpcr) as previously described (8, 9) . briefly, total rna was prepared from cells using the trizol reagent (invitrogen) according to the manufacturer's instructions. the reverse transcription reactions of the extracted rna were performed with a first-strand cdna synthesis kit (amersham pharmacia biotech) according to the manufacturer's directions. first, 1 g of extracted rna was added in a total volume of 15 l of combined cdna reaction reagents with random hexamer oligonucleotides as the first-strand primer in a 1.5-ml reaction tube. samples were heated to 65°c for 10 min, chilled on ice for 5 min, and incubated at 37°c for 1 h, followed by a 10-min incubation at 80°c. the specific primers for all of the detected genes for the pcrs were based on genbank-published sequences: mus musculus ubiquitin-specific peptidase 18 (usp18; nm_011909) forward primer (5=-tacagcagagagcagcagga) and reverse primer (5=-cacatgtcggagcttgctaa); mus musculus myxovirus (influenza virus) resistance 1 (mx1; nr_003520) forward primer (5=-tctgaggagagccagacaat-3=) and reverse primer (5=-actctggtccccaatgacag); mouse hypoxanthine guanine phosphoribosyltransferase (hprt; nm_013556) forward primer (5=-tcagt caacgggggacataaa) and reverse primer (5=-ggggctgtac tgcttaaccag); mus musculus il-1␤ (nm_008361) forward primer (5=-gaaatgccaccttttgacagtg) and reverse primer (5=-tgg atgctctcatcaggacag); homo sapiens ifn-␣2b (ifna2b; ay255838) forward primer (5=-gcttgggatgagaccctccta) and reverse primer (5=-cccaccccctgtatcacac); homo sapiens ifn-␥ (ifng; nm_000416) forward primer (5=-tcggtaactgacttgaatg tcca) and reverse primer (5=-tcgcttccctgttttagctgc); homo sapiens actin, beta-like 2 (actbl2; nm_001017992) forward primer (5=-gtctgccttggtagtggataatg) and reverse primer (5=-tcgagg acgccctatcatgg); homo sapiens ubiquitin specific peptidase 18 (usp18; nm_017414) forward primer (5=-aggagaagcgtccctt tcca) and reverse primer (5=-tggtccttaatcaggttccagag); and homo sapiens myxovirus (influenza virus) resistance-1 (mx1; nm_001144925) forward primer (5=-ggtggtccccagtaatgtgg) and reverse primer (5=-cgtcaagattccgatggtcct). quantitative real-time pcr was performed on an abi prism 7900ht machine (applied biosystems, foster city, ca) with sybr green realtime pcr master mix (applied biosystems) according to the directions provided by the manufacturer. all of the rna samples and controls were assayed in duplicate. real-time pcr conditions were as follows: 10 min at 95°c, followed by 45 cycles of 15 s at 95°c and 15 s at 60°c monitor fluorescence in sybr channel during a 60°c annealing/extension step. the results were analyzed by using applied biosystems sds2.2 software (applied biosystems). experimental hepatic ischemia/reperfusion injury model. hepatic ischemia/reperfusion injury (hiri) was simulated as before (35) . partial (70%) hepatic ischemia was induced for 60 min in mice, after which the surgical clamps were removed. control (sham) animals underwent anesthesia and laparotomy alone. animals were euthanized 6 or 24 h after ischemia/reperfusion, the affected liver segments were removed, and target gene expression was determined by qpcr in whole liver tissue. lcmv strain we was propagated in l929 cells (atcc ccl-1) as previously described (36) . in additional experiments, after 60 min hiri or sham laparotomy, mice were infected with 2 ϫ 10 6 pfu of lcmv we by intravenous tail vein injection. the ischemic liver lobes were harvested at day 7 postinfection. snap-frozen liver tissue samples were homogenized in ␣-mem (multicell, usa) supplemented with 5% fbs, l-glutamine, and 100 u of penicillin/ml plus 100 g of streptomycin/ml using the tissuelyser lt system (qiagen, netherlands). viral titers were examined on mc57 cells (atcc crl-2295) using a focus-forming assay as previously described (37) . statistical analysis. all data were analyzed using prism version 5.0 software (graphpad software, san diego, ca). statistically significant differences in fig. 1e were calculated by using a two-tailed student t test (prism software). the impact of hiri on lcmv replication (see fig. 6a and b) was evaluated by one-way analysis of variance with the tukey's post hoc test. usp18 is induced in hepatocytes by lps and tnf-␣ but not by il-6 and il-10. we have previously shown that usp18 can modulate the type 1 ifn response (14) . we sought to determine whether inflammatory stimuli could increase usp18 expression in hepatocytes. liver tissue inflammation is mediated by both proand anti-inflammatory stimuli, acting through diverse pathways. we therefore compared the effects of three proinflammatory stimuli (tnf-␣, lps, and il-6) and one anti-inflammatory stimulus (il-10), all four signaling through independent receptors and signaling cascades (38, 39) . in a 24-h time course experiment, usp18 mrna expression was measured after stimulation with lps (100 ng/ml), tnf-␣ (20 ng/ml), il-6 (100 ng/ml), or il-10 (10 ng/ml). usp18 mrna expression was induced by tnf-␣ and lps but not by il-6 or il-10 ( fig. 1a) . meanwhile, neither tnf-␣ nor lps treatments had a marked effect on mx1 mrna expression (fig. 1b) , indicating that the effects we observe are not due to a generalized upregulation of isgs or to type 1 ifn signaling. tnf-␣ and lps treatment also led to augmented usp18 protein expression by western blotting (fig. 1c ) and by intracellular staining (fig. 1d to f). treatment of huh7.5 cells with tnf-␣ or lps induced expression of usp18 in 74.1% ϯ 8.9% and 70.5% ϯ 5.2%, respectively, compared to untreated controls in which usp18 expression was 31.0% ϯ 4.7% (fig. 1fi) . usp18 induction was also demonstrated with a significant shift in the mean fluorescence intensity of usp18 staining after tnf-␣ or lps simulations (fig. 1fii ). in these assays, the degree of protein expression did not correlate completely with mrna expression, a finding that is consistent with many genes in the liver (40) in that the degree of mrna induction was higher than the observed usp18 protein expression. however, the functional effects we observed in terms of ifn-␣ responses were robust. tnf-␣ and lps block ifn-␣ signaling in huh7.5 hepatoma cells. to determine whether inflammatory stimuli such as tnf-␣ and lps can interfere with type 1 ifn signaling, we sought to determine whether pretreatment of hepatoma cells with tnf-␣ or lps blocked ifn-␣ signaling. huh7.5 cells were treated with tnf-␣ (20 ng/ml) or lps (100 ng/ml) for 24 h. the cells were subsequently treated with ifn-␣ for 6 h. we chose 6 h to measure mx1 expression based on previous descriptions of ifn-stimulated mx1 induction in the literature (41) and based on our observations with primary mouse hepatocytes. control cells were left untreated, followed by a 6-h ifn-␣ treatment at 24 h. after the 6-h ifn-␣ treatment, the cells were harvested, and mx1 expression was measured by qpcr as a measure of ifn-inducible gene expression. as expected, a 6-h exposure to ifn-␣ strongly induced mx1 expression, whereas a 24-h exposure to tnf-␣ and lps did not (fig. 2, columns 2, 3, and 5 ). both tnf-␣ and lps pretreatment inhibited the effect of 6 h of exposure to ifn-␣ (fig. 2, columns 2, 4, and 6 ), indicating that ifn-␣ signaling was impaired in the presence of these inflammatory stimuli. having shown that certain inflammatory stimuli both increase hepatocyte usp18 and blunt ifn-␣ signaling, we next sought to determine whether the blunting of ifn-␣ signaling is mediated via increased usp18. we addressed this question by selective knockdown of usp18 mrna. thus, huh7.5 cells transfected with usp18 sirna or control sirna were treated with lps (100 ng/ml), tnf-␣ (20 ng/ml), and/or ifn-␣ (100 iu/ml) for 2 h or pretreated with lps (100 ng/ml) or tnf-␣ (20 ng/ml) for 24 h and then either left untreated or treated with ifn-␣ (100 iu/ml) for 2 h (a 2-h time point was selected for optimal expression of pstat1 in huh7.5 cells). the expression of pstat1, stat1, and isg15 conjugates (as a marker of usp18 knockdown), usp18, and actin was detected by western blotting. as seen in fig. 3a , usp18 expression was largely knocked down in cells transfected with usp18 sirna. ifn-␣-induced phosphorylation of stat-1 was inhibited by pretreatment (for 24 h) with tnf-␣ or lps (fig. 3a , sirna control lanes f and h versus lane d), but this effect is reversed by knockdown of usp18 (fig. 3a , sirna usp18, lanes f and h versus lane d). of note, usp18 knockdown did not increase pstat1 in response to ifn-␣ before 24 h; these findings are consistent with previous observations (12) . we next wanted to confirm whether our findings from hepatoma cells would hold true in primary mouse hepatocytes. with this in mind, primary murine hepatocytes from usp18 ϩ/ϩ mice were isolated, transfected with usp18 sirna or control sirna, and then exposed to ifn-␣ (100 iu/ml), lps (100 ng/ml), or tnf-␣ (20 ng/ml) for 24 h. after a washing step, the cells were treated with ifn-␣ for an additional 6 h. mx1 isg mrna expression was measured by qpcr (normalized to expression of the hprt housekeeping gene) as an index of downstream ifn-␣ effect. ifn-␣, lps, and tnf-␣ treatment blocked the effect of the final dose of ifn-␣ (fig. 3b, columns 2, 3, 5, and 7) . however, knockdown of usp18 reversed this effect and augmented the effect of ifn-␣ (fig. 3b, columns 9, 10, 12, and 14) . the data from this experiment are consistent with those obtained with human huh7.5 cells (data not shown). thus, the ability of tnf-␣ and lps to block ifn signaling is seen in both primary and immortalized hepatocytes. as noted earlier, usp18 has dual roles: it both strips isg15 from its target proteins and impairs type 1 ifn signaling independent of its protease activity (15) . to determine whether the usp18-dependent blunting of hepatocyte ifn signaling was due to its ability to strip isg15 from its conjugates, we blocked the process of isgylation by knockdown of the isg15 e1 enzyme, ube1l. murine hepatocyte ube1l was knocked down via transfection with sirna specific for ube1l. usp18 ϩ/ϩ murine hepatocytes transfected with ube1l sirna or control sirna were stimulated with ifn-␣ (100 iu/ml), lps (100 ng/ml), or tnf-␣ (20 ng/ml) for 24 h and then treated with an additional dose of ifn-␣ for 6 h. knockdown was measured in the hepatocytes transfected with ube1l sirna by western blotting probing for ube1l expression and isg15 conjugate formation (fig. 4a) . next, to examine the effect of this knockdown on isg induction, wild-type murine hepatocytes transfected with ube1l sirna or control sirna were stimulated with tnf-␣ (20 ng/ml) and lps (100 ng/ml) for 24 h and then restimulated with ifn-␣ (100 iu/ ml) for 6 h, at which time mx1 isg mrna expression was measured by qpcr. as seen in fig. 4a , ube1l knockdown was achieved, resulting in a decrease in isg15 conjugates. however, as seen in fig. 4b , we observed that neither the presence nor the relative absence of isg15 conjugation impacted the ability of tnf-␣ and lps to block ifn-␣ signaling since there was no change in isg expression after 6 h of ifn-␣ treatment when usp18 ϩ/ϩ hepatocytes transfected with anti-ube1l sirna were compared to hepatocytes transfected with irrelevant sirna (fig. 4b , sirna control, columns 2, 5, and 7, and ube1l sirna, columns 9, 12, and 14). these results are consistent with there being no role for isgylation (and, thus, for the ability of usp18 to strip isg15 from its protein conjugates) in the ability of tnf-␣ and lps to block type 1 ifn signaling in hepatocytes, and this finding is in agreement with previous data showing that usp18 blocks ifn signaling independent of its enzymatic activity (15) . we next sought to determine whether tnf-␣ and lps could induce usp18 expression in hepatocytes via the induction of ifn-␣. as observed in fig. 3a , stat1 phosphorylation shows distinct activation profiles with ifn-␣ stimulation compared to tnf-␣ or lps stimulation (fig. 3a , sirna control and sirna usp18, lanes b and c compared to lane d). although these data strongly suggest that the effect of lps and tnf-␣ stimulation are a result of direct induction of isgs and not secondary to the induction of ifn-␣, we wanted to confirm whether lps and tnf-␣ could induce type 1 or type 2 ifn (ifn-␣ or ifn-␥) in primary murine hepatocyte. thus, primary murine hepatocytes were treated with ifn-␣ (100u/ml), lps (100 ng/ml), or tnf-␣ (20 ng/ml) for 6, 12,18, or 24 h; they were then lysed and assessed for ifn-␣, ifn-␥, and usp18 expression by qpcr. we observed that neither ifn-␣, lps, nor tnf-␣ induce much, if any, hepatocyte expression of ifn-␣ or ifn-␥, although the same doses induce strong expression of usp18 (fig. 5) . thus, the induction of isgs by lps and tnf-␣ is very unlikely to reflect the induction of hepatocyte type 1 or type 2 ifn, which suggests that the observed usp18 induction is not due to type 1 or type 2 ifn secretion and autocrine stimulation of the ifn-␣ receptor. experimental hepatic ischemia/reperfusion induces usp18 expression and enhances lcmv replication. we then assessed whether tissue-wide hepatic inflammatory stress increases liver usp18 expression, as an in vivo confirmation of our in vitro findings. this was approached by inducing partial (70%) hepatic ischemia/reperfusion injury (hiri) for 60 min in mice, euthanizing the animals at 6 or 24 h (time points relevant to our in vitro time course) and measuring induction of usp18 by qpcr. we chose hiri as a very well-characterized inflammatory stress, known to be driven both by tnf-␣ and lps (35) . as seen in fig. 6a , hiri alone induced usp18 mrna expression in whole livers by ͼ10fold that of untreated animals. thus, in vivo liver inflammation leads to increased usp18 expression at the organ level. after determining that hiri induces usp18 expression, we next wanted to examine the impact of hiri on viral control. thus, we induced 70% hiri for 60 min prior to lcmv we infection of usp18 ϩ/ϩ and usp18 ϫ/ϫ mice. as seen in fig. 6b , hiri led to a significant increase in lcmv viral titers in usp18 ϩ/ϩ mice, an effect that was not observed in usp18 ϫ/ϫ mice. having shown that lps/ tnf-␣ stimulation increases hepatocyte usp18 expression, we then sought to determine whether we could pharmacologically inhibit the induction of usp18 by lps and tnf-␣. we focused on small-molecule agents that have been linked to the nf-b signaling pathway, because of the central role of this pathway in inflammatory activation in response to a large number of inflammatory mediators, including lps and tnf-␣ (42) . we were particularly interested in inhibitors, such as silibinin, that in addition have been linked to clinical suppression of hepatic viral production (in the case of silibinin and hcv) (43, 44) . thus, we preincubated primary mouse hepatocytes for 30 min with various inhibitors of lps and tnf-␣ signaling and measured their impact on usp18 induction, while simultaneously measuring expression of proinflammatory cytokine il-1␤ mrna, since nf-b is also known to promote il-1␤ transcription (45, 46) . as seen in fig. 7a and as table 1 , tnf-␣ induced expression of usp18 was potently downregulated by all inhibitors tested, and this inhibition coincided with impaired il-1␤ mrna expression (fig. 7b) . meanwhile, only two inhibitors potently (ͼ80%) inhibited usp18 expression as well as il-1␤ mrna expression in response to lps stimulation; nsc33994 (an inhibitor of jak2) and silibinin (an inhibitor of ikk␣) (fig. 7a and b and table 1 ). these data suggest that the induction of usp18 by tnf-␣ and lps, and pos-sibly other inflammatory stimuli, is promoted by nf-〉 signaling and that hepatocyte usp18 expression in particular-compared to il-1␤-may be an attractive target for pharmacologic manipulation in the setting of liver inflammation. in this study we examined the role of various inflammatory stimuli in the induction of usp18 and the downstream establishment inflammatory stimuli, we examined the induction of stat-1 phosphorylation in huh7.5 cells with or without usp18 knockdown (a) and the expression of isg mrna (mx1) in primary mouse hepatocytes and the ability of usp18 knockdown to restore isg induction after lps and tnf-␣ stimulation (b). (a) huh7.5 cells were transfected with anti-usp18 sirna or control irrelevant sirna. huh7.5 cells were then pretreated with lps (100 ng/ml), tnf-␣ (20 ng/ml), or ifn-␣ (100 u/ml) or left untreated for 24 h and then exposed to ifn-␣ or left untreated for an additional 2 h. as controls, huh7.5 cells were treated with lps, tnf-␣, and ifn-␣ for 2 h only. the expression of usp18, pstat1, stat1, and isg15 conjugates and actin proteins was measured by western blotting. (b) primary murine hepatocytes from usp18 ϩ/ϩ mice were isolated, transfected with anti-usp18 sirna or control irrelevant sirna, and pretreated with ifn-␣ (100 iu/ml), lps (100 ng/ml), or tnf-␣ (20 ng/ml) for 24 h (ifn-␣ pre, lps pre, or tnf-␣ pre, respectively). after being washed, the cells were cultured in the presence or absence of ifn-␣ for 6 h (ifn-␣ final). mx1 isg mrna expression was measured by qpcr and normalized to the expression of the hprt housekeeping gene. the data were generated from pooled triplicate experiments analyzed in duplicate. error bars represent the sem for duplicate pcrs. of an ifn-␣ refractory state. we used multiple methods to show that certain inflammatory stimuli, including lps and tnf-␣ are able to induce the expression of usp18, which results in downstream downregulation of ifn-␣-induced isg expression. the role of usp18 in the ifn-␣ refractory state has been previously demonstrated by human and mouse usp18 knockdown studies (12, (14) (15) (16) . other inflammatory stimuli, including il-10 and il-6, did not induce usp18 or impair expression of isgs, including usp18. in vivo, hepatic inflammatory stress (ischemia/reperfusion injury) led to increased hepatic usp18 gene expression that was associated with poor control of lcmv infection. thus, the hepatic inflammatory milieu, contributed to by individual inflammatory cytokines and stimuli, modulates usp18 expression. these results demonstrate one mechanism by which liver inflammation directly impacts the hepatocellular innate immune response. usp18 is known to be induced by multiple inflammatory stimuli in macrophages (18) and lymphocytes (47) . our findings in hepatocytes are consistent with work done by other groups in immune cells. for example, lps treatment of a murine macrophage cell line upregulates usp18 in an irf3-dependent manner (18) . the cytokine specificity of our results is also consistent with finding that il-6 alone is not able to induce usp18 in murine t cells (47) . only when t cells were treated with il-6 and another proinflammatory cytokine, such as transforming growth factor ␤, il-1␤, and il-23, was usp18 expression induced (47) . these data point out that usp18 can be induced by inflammatory stimuli in multiple cell types in the absence of ifn. usp18 is therefore well positioned to act as the mediator of "cross talk" between innate immunity and inflammatory responses. in the present study, we show that increased usp18 expression following exposure of hepatocytes by inflammatory stimuli blunts the ifn response. the binding of usp18 with the ifn-␣ receptor has been shown to inhibit the interaction of stat-1 with the ifn-␣ receptor and thus block downstream ifn signaling (12, 15) . our data are consistent with this mechanism, since the blunting of hepatocyte ifn signaling after exposure to inflammatory stimuli is independent of usp18-mediated removal of isg15 from its target proteins. the degree of tnf-␣ and lps-induced ifn-␣ refractoriness was not changed by ube1l knockdown, although isgylation was considerably reduced. however, other mechanisms may also be at play. recent work has demonstrated that usp18 has the ability to deubiquitinate the transforming growth factor-activated kinase 1 (tak1) complexes required for nf-b activation in t cells and that overexpression of usp18 leads to decreased nuclear activation and impaired formation of tak1 complexes (47) . usp18-mediated nf-b inhibition may be of importance not only for t cell adaptive immunity but also for liver inflammation. the role of tak1 in innate and adaptive immunity has been previously demonstrated (48) , and studies have also shown that tak1 deletion interferes with hepatocyte homeostasis and leads to hepatic injury (49) . thus, increased hepatocyte usp18 in response to inflammatory stimuli may constitute a negative-feedback cycle with relevance to multiple aspects of the liver's response to infection. our in vivo experiments demonstrated that inflammation resulting from ischemia/reperfusion injury induces usp18 expression, which coincides with diminished lcmv control in mouse livers. the general finding that liver inflammation promotes lcmv production is in agreement with work showing that polymicrobial sepsis, characterized by high tnf-␣ and inflammation, leads to increased susceptibility to lcmv infection (50) . in our present study, hepatic ischemia/reperfusion injury did not enhance lcmv production in usp18 knockout mice. these data are intended as proof-of-concept but do suggest that the role of usp18 in hepatic viral infection and inflammation deserves further investigation. our in vitro and in vivo findings suggest a new model for how inflammation alters hepatic innate immune responses. in this model, liver inflammation leads to increased hepatocyte usp18, which in turn makes the liver more susceptible to infections targeting the hepatocyte, such as hcv. this mechanism may help to explain the clinical observation that hcv infection of a transplanted, hcv-naive liver graft (that has gone through an ischemia/reperfusion cycle) is more aggressive than the original infection and that the severity of the reinfection correlates with the severity of the ischemia/reperfusion injury suffered during the transplant (51, 52) . however, we have also found that usp18 is necessary but not sufficient on its own to induce an ifn-␣ refractory state (53) . with this in mind, we hypothesize that the innate immune response and its ability to control ifn-sensitive viruses will depend on cumulative effect of multiple intrahepatic signals, including the induction of usp18. if these results are to be translated into a clinical setting, then one approach is to target usp18 induction pharmacologically. using multiple inhibitors of tnf-␣/lps signaling, we found that two inhibitors, silibinin (an inhibitor of ikk␣) and nsc33994 (a jak2 inhibitor) possess the ability to inhibit tnf-␣ and lps-induced usp18 expression and proinflammatory effects. these findings raise the possibility of pharmacologically targeting usp18 expression during inflammatory events to prevent the establishment of an ifn-␣ refractory state. previously, inhibition of jak2 signaling led to a protection of mouse livers from ischemic insult (54) . as well, silibinin has been shown to reduce of hcv liver graft reinfection (43) and enhance hcv clearance in ifn-␣ nonresponders (55) via multiple mechanisms, including by direct inhibition of hcv ns5b rna polymerase (44) . usp18 modulation may be one mechanism by which silibinin exerts its anti-hcv effects. the present study focuses on a relatively small number of inflammatory stimuli; while tnf-␣ and lps are important to a large number of liver diseases, they are far from being the only drivers of the hepatic inflammatory response and tissue levels of usp18 are likely influenced by other stimuli as well. for example, we previously demonstrated that increased usp18 expression in the liver is and enhances lcmv replication. to examine the in vivo effect of an inflammatory stimulus on usp18 expression and viral replication, a hepatic ischemia/reperfusion model was used. (a) partial (70%) hepatic ischemia was induced for 60 min, after which the portal vascular clamp was removed. control animals (sham) underwent anesthesia and a laparotomy alone. animals were euthanized 6 and 24 h after ischemia/reperfusion (i/r), and the affected liver segments were removed. usp18 mrna expression was determined by qpcr in whole liver tissue and normalized to hypoxanthine-guanine phosphoribosyltransferase (hprt) expression. data are expressed as means ϯ the sem with n ϭ 3 to 4 mice/group. a p value of ͻ0.05 was considered significant. (b) after 60 min of hiri or sham laparotomy, mice were infected with 2 ϫ 10 6 pfu of lcmv we. liver lobes were harvested at day 7 postinfection. viral titers were examined on mc57 cells using a focus-forming assay. data are expressed as means ϯ the sem with n ϭ 3 to 10 mice/group. a p value of ͻ0.05 was considered significant. inhibition of nf-b activation impairs lps-and tnf-␣-stimulated usp18 induction. to investigate the link between lps/tnf-␣ stimulation and usp18 induction, inhibitors of nf-b activation were added to primary mouse hepatocytes for 30 min prior to stimulation with 20 ng of tnf-␣/ml or 100 ng of lps/ml for 6 h (the inhibitor names, targets, and concentrations used are given in table 1 ). inhibitor-treated cells were also left unstimulated as a control. as a control for usp18 induction, hepatocytes were stimulated with 100 u of ifn-␣/ml for 6 h. usp18 (a) and il-1␤ (b) mrna expression levels were evaluated by pcr and normalized to the hprt housekeeping gene expression prior to determining the fold increase versus mock-treated samples. the data were generated from pooled triplicate experiments analyzed in duplicate. error bars represent the sem for duplicate pcrs. predictive of patients with chronic hcv infection who will not respond to ifn-based anti-hcv therapy (8) . although we found that tnf-␣ hepatic mrna expression was increased in treatment nonresponders, it was relatively more increased in treatment responders (9) . we suspect that whereas increased tnf-␣ does contribute to usp18 expression, there are other stimuli, for example, lps and perhaps the recently described interferon-lambda 4 (56) , that also modulate hepatic usp18 expression. we propose that the ultimate usp18 expression is due to the liver's coordinate response to multiple stimuli. the finding that hepatic usp18 expression is modulated by inflammatory stimuli is a new paradigm for the interaction of the liver inflammatory microenvironment and viral infection. taken together, these data may suggest that usp18 represents a good target for intervention in numerous inflammatory states and in the clinical setting of hcv-related liver transplantation. hiri-induced upregulation of usp18 could lead to worsened outcomes posttransplant, including a quicker, more aggressive reinfection of the new organ with higher hcv rna titers. we suggest that the finding that ifn responses and usp18 expression are tightly linked to the hepatic inflammatory response helps to explain the finding that tnf-␣ antibody treatment improves treatment outcomes of chronic hcv with treatment combinations, including ifn-␣ (5). mechanism of action of interferon and ribavirin in treatment of hepatitis c interferon, mx, and viral countermeasures hepatitis c virus infection induces inflammatory cytokines and chemokines mediated by the cross talk between hepatocytes and stellate cells tumor necrosis factor alpha gene expression and the response to interferon in chronic hepatitis c etanercept as an adjuvant to interferon and ribavirin in treatment-naive patients with chronic hepatitis c virus infection: a phase 2 randomized, double-blind, placebocontrolled study safety of anti-tumor necrosis factor-alpha therapy in patients with rheumatoid arthritis and chronic hepatitis c virus infection hepatic celltype specific gene expression better predicts hcv treatment outcome than il28b genotype hepatic gene expression discriminates responders and nonresponders in treatment of chronic hepatitis c viral infection cell-type-specific gene expression signature in liver underlies response to interferon therapy in chronic hepatitis c infection tissue macrophages suppress viral replication and prevent severe immunopathology in an interferon-idependent manner in mice the role of kupffer cells in hepatitis b and hepatitis c virus infections alpha interferon induces long-lasting refractoriness of jak-stat signaling in the mouse liver through induction of usp18/ubp43 the isg15/usp18 ubiquitin-like pathway (isgylation system) in hepatitis c virus infection and resistance to interferon therapy silencing of usp18 potentiates the antiviral activity of interferon against hepatitis c virus infection ubp43 is a novel regulator of interferon signaling independent of its isg15 isopeptidase activity usp18-based negative feedback control is induced by type i and type iii interferons and specifically inactivates interferon alpha response gene induction pathways mediated by distinct irfs during viral infection lipopolysaccharide activates the expression of isg15-specific protease ubp43 via interferon regulatory factor 3 tumor necrosis factor-alpha in liver ischemia/reperfusion injury the role of intestinal endotoxin in liver injury: a long and evolving history viral and host factors induce macrophage activation and loss of toll-like receptor tolerance in chronic hcv infection enforced viral replication activates adaptive immunity and is essential for the control of a cytopathic virus complete replication of hepatitis c virus in cell culture tnf-alpha-induced sphingosine 1-phosphate inhibits apoptosis through a phosphatidylinositol 3-kinase/ akt pathway in human hepatocytes oncostatin m is a potent inducer of hepcidin, the iron regulatory hormone lipopolysaccharide, immune activation, and liver abnormalities in hiv/hepatitis b virus (hbv)-coinfected individuals receiving hbv-active combination antiretroviral therapy protein interferon-stimulated gene 15 conjugation delays but does not overcome coronavirus proliferation in a model of fulminant hepatitis hepatitis c virus persisting after clinically apparent sustained virological response to antiviral therapy retains infectivity in vitro protein-kinase inhibitor-(6-22)-amide peptide analogs with standard and nonstandard amino-acid substitutions for phenylalanine-10: inhibition of campdependent protein-kinase bone marrow-derived myofibroblasts promote colon tumorigenesis through the il-6/jak2/stat3 pathway nonmuscle myosin is regulated during smooth muscle contraction pd-098059 is a specific inhibitor of the activation of mitogen-activated protein-kinase kinase in-vitro and in-vivo silibinin inhibits constitutive and tnf alpha-induced activation of nf-b and sensitizes human prostate carcinoma du145 cells to tnf alpha-induced apoptosis wortmannin is a potent phosphatidylinositol 3-kinase inhibitor: the role of phosphatidylinositol 3,4,5-trisphosphate in neutrophil responses protective strategies against ischemic injury of the liver interleukin-10 determines viral clearance or persistence in vivo quantification of lymphocytic choriomeningitis virus with an immunological focus assay in 24-well or 96-well plates dubbing down innate immunity malignant pirates of the immune system a comparison of selected mrna and protein abundances in human liver kinetic differences in the induction of interferon stimulated genes by interferon-alpha and interleukin 28b are altered by infection with hepatitis c virus nf-b in the liver-linking injury, fibrosis, and hepatocellular carcinoma successful prevention of hepatitis c virus (hcv) liver graft reinfection by silibinin mono-therapy differential in vitro effects of intravenous versus oral formulations of silibinin on the hcv life cycle and inflammation dexamethasone inhibits il-1␤ gene expression in lps-stimulated raw 264.7 cells by blocking nf-b/rel and ap-1 activation nf-b regulates il-1␤ transcription through a consensus nf-b binding site and a nonconsensus cre-like site usp18 inhibits nf-b and nfat activation during th17 differentiation by deubiquitinating the tak1-tab1 complex essential function for the kinase tak1 in innate and adaptive immune responses disruption of tak1 in hepatocytes causes hepatic injury, inflammation, fibrosis, and carcinogenesis polymicrobial sepsis increases susceptibility to chronic viral infection and exacerbates cd8 ϩ t cell exhaustion recipient age affects long-term outcome and hepatitis c recurrence in old donor livers following transplantation prolonged rewarming time during allograft implantation predisposes to recurrent hepatitis c infection after liver transplantation the ubiquitin specific protease usp18 is necessary but not sufficient for a hepatocyte ifn refractory state: variable roles in type i and type iii ifn responsiveness blockade of janus kinase-2 signaling ameliorates mouse liver damage due to ischemia and reperfusion silibinin is a potent antiviral agent in patients with chronic hepatitis c not responding to pegylated interferon/ribavirin therapy a variant upstream of ifnl3 (il28b) creating a new interferon gene ifnl4 is associated with impaired clearance of hepatitis c virus we thank dong er zhang for the gift of the usp18 ϫ/ϫ mice. s.a.m. thanks the casl/cihr hepatology fellowship program and the national cihr research training program in hepatitis c for financial support. key: cord-002670-rjrw875d authors: chen, jidang; ly, hinh title: immunosuppression by viral n proteins date: 2017-06-22 journal: oncotarget doi: 10.18632/oncotarget.18597 sha: doc_id: 2670 cord_uid: rjrw875d nan arenaviruses and coronaviruses (cov) are enveloped rna viruses. the nucleoproteins (n) of these viruses (termed nucleocapsid in cov) play a key role in the formation of the viral ribonucleoprotein (rnp) complexes and in causing type i interferon (ifn) suppression. the primary function of the n proteins of cov, such as the severe acute respiratory syndrome coronavirus (sars-cov), and of arenaviruses, such as lassa virus that can cause severe and lethal hemorrhagic fever infections in humans, is to package the viral single stranded genomic rna(s) into rnp complexes [1, 2] . another important function of these proteins is to suppress type i interferons (ifns) in infected cells, the molecular mechanisms of which have only recently been elucidated and will be discussed in this article. the host innate immune system presents a significant barrier and defense against viruses. the host pattern recognition receptors (prrs), including retinoic acid-inducible gene i (rig-i), melanoma differentiationassociated protein 5 (mda5) and laboratory of genetics and physiology 2 protein (lgp2), can specifically recognize virus-specific components, such as rna, dna or glycoproteins. following virus recognition by the prrs, important cellular signaling pathways are activated to lead to production of cytokines, chemokines and type i ifns, which play a critical role in the eradication of the virus. arenaviruses and cov both have evolved unique mechanisms to evade recognition by prrs, which lead to type 1 ifn inhibition. immune stimulatory dsrna generated during virus replication is important for rig-i recognition and subsequently mediating type-i ifn production. we have previously discovered that arenaviruses encode a 3'-5'exoribonuclease function in the c-terminal domain of the n protein (np) that acts as a type i ifn-antagonist [3] . arenavirus np exoribonuclease, a rnase member of the deddh superfamily, degrades dsrna which is the important signal for rig-i activation. we have shown that recombinant arenaviruses carrying np rnase-defective mutations induce strong ifn responses to inhibit virus replication early in infection in vivo through the activation of the rig-i pathway [3] . coronaviruses are the only other family of viruses known to encode the deddh exoribonuclease in the n-terminal domain of their non-structural protein 14 (nsp14), which has been attributed to its important proofreading role during viral rna genome replication [4] . it has recently been shown that nsp14 exoribonuclease can also inhibit type i ifn production [5] . recombinant viruses with mutations in one of the zinc-finger motifs of the nsp14 exoribonuclease domain of the transmissible gastroenteritis coronavirus (tgev) were found to only mildly affect genome replication and transcription, but they appeared to result in less dsrna accumulation in virus-infected cells. the reduction in dsrna levels correlates with a decrease in the levels of type i ifns and of some representative interferon-stimulated genes (isgs). taken together, coronaviral nsp14 exoribonuclease, similar to that of the arenaviral np exoribonuclease, has the potential to degrade immune stimulatory dsrna during virus replication and thereby suppress type i ifn production. the n protein of cov has recently been found to employ a different way to mediate suppression of type 1 ifn production. hu et al. [6] showed that the c-terminus of the sars-cov n protein could bind to the spry domain of the cellular trim25 e3 ubiquitin ligase. this tripartite motif protein 25 (trim25) e3 ligase plays important role in post-translational modification of the n terminal caspase recruitment domains (cards) of rig-i by ubiquitination [7] . the interaction between the c-terminus of n protein of sars-cov with the spry domain of the trim25 e3 ubiquitin ligase interferes with the association between trim25 and rig-i and thereby inhibiting trim25-mediated rig-i ubiquitination and activation and type 1 ifn production [6] . similarly, gack et al. have reported that influenza a virus (iav) contains a novel domain in its ns1 protein that can also block trim25 multimerization and rig-i card domain ubiquitination via the interaction between iav ns1 protein with the coiled-coil domain of trim25 [7] . taken together, the molecular mechanism of inhibition of type 1 ifn production via rig-i's ubiquitination by direct protein-protein interaction appears to have been exploited by different viruses that include but are not necessarily limited to sarv-cov and iav. therefore, an in-depth understanding of the conserved molecular mechanisms of innate immune evasion by different viruses may lead to the development of novel broad-spectrum antivirals against many medically significant human viruses. this is an open-access article distributed under the terms of the creative commons attribution license 3.0 (cc by 3.0), which permits unrestricted use, distribution, and reproduction in any medium key: cord-007689-0vpp3xdl authors: schlee, m.; barchet, w.; hornung, v.; hartmann, g. title: beyond double-stranded rna-type i ifn induction by 3prna and other viral nucleic acids date: 2007 journal: interferon: the 50th anniversary doi: 10.1007/978-3-540-71329-6_11 sha: doc_id: 7689 cord_uid: 0vpp3xdl production of type i ifn is the key response to viral infection. since the discovery of type i ifns in 1957, long double-stranded rna formed during replication of many viruses was thought to be responsible for type i ifn induction, and for decades double-stranded rna-activated protein kinase (pkr) was thought to be the receptor. recently, this picture has dramatically changed. it now became evident that not pkr but two members of the toll-like receptor (tlr) family, tlr7 and tlr9, and two cytosolic helicases, rig-i and mda-5, are responsible for the majority of type i ifns induced upon recognition of viral nucleic acids. in this review, we focus on the molecular mechanisms by which those innate immune receptors detect viral infection. based on the recent progress in the field, we now know that tlr7, tlr9, and rig-i do not require long double-stranded rna for type i ifn induction. type i ifns (ifn-α isoforms and ifn-β) are regarded as the dominant mediators of antiviral defense in vertebrates. since their initial discovery half a century ago as acid-stable, soluble factors "interfering" with viral proliferation in cultured cells (isaacs and lindenmann 1957; nagano and kojima 1958) , intense research has focused on type i ifn receptor signaling and the plethora of type i ifn-mediated effects (theofilopoulos et al. 2005) . for the host, an intact type i ifn response is critical for the survival of many viral infections (gresser et al. 1976; muller et al. 1994) . sensing of viral replication has been proposed to be responsible for triggering the production of type i ifns by infected host cells. however, the specific host immune receptors and their respective molecular ligands remained elusive until very recently. moreover, to mount an appropriate antiviral response, the innate immune system must distinguish viruses from bacteria, fungi, and multicellular parasites. charles janeway was the first to propose that the detection of highly conserved pathogen-associated molecular patterns (pamps) may be mastered by a limited number of germline-encoded pattern recognition receptors (prrs) (janeway 1989) . a few years later, the first experimental evidence of such a receptor came from the fruit fly (lemaitre et al. 1996) . shortly afterwards, a member of the family of toll-like receptors (tlr), the mammalian homolog of drosophila toll, was demonstrated to be responsible for detecting lipopolysaccharides (lps), a characteristic component of the cell walls of gram-negative bacteria (medzhitov et al. 1997; poltorak et al. 1998 ). this observation was confirmed by the subsequent generation of tlr4-deficient mice (hoshino et al. 1999) . parasites, bacteria, and fungi rely on a multitude of molecules that are distant in evolutionary terms from the mammalian organism, and are thus readily discernible as non-self by members of the toll-like receptor (tlr) and nodlike receptor (nlr) families (reviewed in meylan et al. 2005) . in sharp contrast, all components of viruses are produced within the infected host cell, and therefore lack distinguishable non-self molecular patterns. nevertheless, viruses are promptly recognized by the innate immune system and elicit pronounced antiviral type i interferon and cytokine responses. shortly after the discovery of type i interferons, it was proposed that viral nucleic acids could be stimulating the type i ifn response (isaacs et al. 1963) . many viruses synthesize double-stranded rna (dsrna) during their replication cycle (baltimore et al. 1964; montagnier and sanders 1963) , whereas dsrna was thought to be absent in uninfected cells. therefore dsrna formed during viral infection was postulated to be the molecular signature of viral infection. in support of this hypothesis, the enzymatically generated double-stranded rna polynucleotide polyinosinic:polycytidylic acid (poly i:c) was found to be a potent inducer of type i ifn (field et al. 1967) . although the authors carefully emphasized that all other double-stranded polynucleotides were inactive, the notion that long viral double-stranded rna elicits type i ifn became commonplace, and poly i:c has been used as an interferon-inducing mimic of viral dsrna ever since. in early attempts to uncover the inducers of interferon and of other mediators of antiviral activity, ifn-α and poly i:c-treated or reticulocyte extracts were analyzed. chromatographic separation of lysates revealed proteins that were increased by preincubation with ifn-α, and whose enzymatic activity depended on the presence of dsrna (usually poly i:c) (farrell et al. 1978; hovanessian et al. 1977; zilberstein et al. 1978) . two proteins, interferon inducible double-stranded rna-activated protein kinase (pkr) and the 2′,5′ oligoadenylate synthetase (oas) could be affinity purified using poly i:c-cellulose (farrell et al. 1978; hovanessian et al. 1977) . both activated pkr and oas were found to block translation of viral rna by distinct mechanisms. in the presence of poly i:c, oas catalyzes the synthesis of 2′,5′ oligomers of adenosine (2-5as) (hovanessian et al. 1977; zilberstein et al. 1978) , which activate rnase l (farrell et al. 1978) . rnase l in turn degrades single-stranded viral and cellular rnas (farrell et al. 1978 ) in a sequence-independent manner (minks et al. 1979) . consequently rnase l-deficient mice displayed a reduced antiviral activity of ifn-α, as well as impaired apoptosis (zhou et al. 1997) . in contrast, the serine threonine kinase pkr was found to more specifically block the translation of viral rna (farrell et al. 1978 ) by phosphorylation of the eukaryotic translation initiation factor eif2a. besides its function in limiting translation of viral protein, pkr was also reported to activate nf-κb (kumar et al. 1994) . pkr was therefore proposed as a key receptor mediating virus-and dsrna-induced production of type i interferons (kumar et al. 1994 ). however, these findings remained controversial, as other studies that examined pkr-deficient mice and cells (chu et al. 1999; iordanov et al. 2001; maggi et al. 2000; smith et al. 2001; yang et al. 1995) found no defects in the induction of interferon in response to poly i:c or viral infection that could not be overcome with type i ifn pretreatment. further analysis revealed that pkr is not only activated by poly i:c but is able to interact with dsrna as short as 11 bp. however, at least 30 bp are required to activate pkr kinase activity (manche et al. 1992) . in another study (zheng and bevilacqua 2004) , recombinant pkr could also be activated by rna oligonucleotides containing a 16-bp dsrna stem loop in combination with a more than 11bp-long single-stranded rna part at the 5′ or 3′ end. all these studies question the often quoted requirement of a dsrna molecule longer than 30 bp; furthermore, it became evident that the translational shut-down by pkr is not linked to the induction of type i ifn synthesis and secretion. the finding that pkr -/cells still produce type i ifns spurred further research on receptors capable of recognizing long double-stranded rna. such investigations led to a member of the toll-like receptor (tlr) family, tlr3, which was proposed to bind to long dsrna and to induce ifn-β (alexopoulou et al. 2001) . tlrs are transmembrane receptors that were shown to recognize a variety of conserved pathogenassociated molecular patterns (pamps) of bacterial, fungal, and parasitic origin. the study of alexopoulou et al. was the first to demonstrate a role for tlrs in the recognition of viruses. tlr9 was found to be the receptor for unmethylated cpg motifs in dna (hemmi et al 2000) ; however, cpg-dna at first was thought to be characteristic for bacterial dna, and the role of tlr9 in detecting dna viruses was only proposed later (krug et al. 2004a (krug et al. , 2004b tabeta et al. 2004) . upon engagement with their specific ligands, tlrs trigger signaling pathways that lead to the activation of nf-κb and irfs (signaling of tlrs reviewed in moynagh 2005) . tlr3 was found to induce type i ifns upon poly i:c stimulation by activation of the kinase tbk1, which phosphorylates the transcription factor irf3, resulting in the induction of ifn-β (doyle et al. 2002; fitzgerald et al. 2003; sharma et al. 2003) . another group reported that tlr3 is activated by ssrna (kariko et al. 2004b ); however, tlr3-deficient mice and mice deficient in the signaling adapter trif (gitlin et al. 2006; kato et al. 2006 ) still responded to poly i:c. moreover, dendritic cells derived from tlr3-deficient mice were still stimulated by dsrna transfected into the cytosol (diebold et al. 2003 ). unlike tumor cell lines, which were examined in early studies on type i ifn and dsrna, primary immune cells such as peripheral blood mononuclear cells (pbmcs) express a wide spectrum of functional tlrs. different immune cell subsets express distinct patterns of tlrs (hornung et al. 2002) . plasmacytoid dendritic cells (pdcs) (reviewed in colonna et al. 2004 ) are the major producers of early type i ifn production upon viral infection. pdcs express tlr7 and tlr9 but not tlr3. both tlr7 and tlr9 are located in the endosomal compartment and signal via the adaptor molecules myd88, irak1, and traf6, leading to activation of irf7 and the induction of type i interferons as reviewed by moynagh (2005) . in addition, recent studies show that traf3 plays a crucial role in the myd88-dependent signaling cascade (hacker et al. 2005; oganesyan et al. 2005) . single-stranded dna and the small antiviral compound r848 had been shown to induce ifn in pdcs dependent on tlr9 and tlr7, respectively (hemmi et al. 2000 (hemmi et al. , 2002 jurk et al. 2002; krug et al. 2001b; rothenfusser et al. 2002) . tlr9 detects unmethylated so-called cpg motifs in single-stranded dna (hemmi et al. 2000) . different classes of synthetic cpg oligodeoxynucleotides (odn) were developed based on the distinct effects on the two tlr9-expressing immune cell types: pdcs and b cells (hartmann et al. 2003; hartmann and krieg 2000; krug et al. 2001a) . in contrast to tlr3, both tlr7 and 9 depend on the signaling adapter myd88. accordingly, pdcs derived from tlr9-or myd88-deficient mice are unable to produce type i ifn in response to dna viruses such as herpes simplex viruses (hsv) and murine cytomegalovirus (mcmv) (krug et al. 2004a (krug et al. , 2004b lund et al. 2003; tabeta et al. 2004) . while tlr9 was responsible for detecting viral dna, tlr7 was shown to recognize rna: tlr7 detects synthetic short (20-27 bases) single-stranded rna (diebold et al. 2004; heil et al. 2004 ) and short interfering double-stranded rna (sirna) (hornung et al. 2005; judge et al. 2005; sioud 2005 ; reviewed in schlee et al. 2006 ). the amount of type i interferon induction was dependent on the rna sequence. ironically, hornung and colleagues came across a very potent type i interferon inducing small rna sequence core motif (5′-guccuucaa-3′) in the attempt to knock down the interferon inducer tlr9 in pdcs using the sirna technology (hornung et al. 2005) . it was demonstrated that these sirnas induce systemic immune activation in mice, and that the immunological activity required tlr7. of note, the same sirna did not induce type i interferon in immortalized human embryonic kidney cells (hek293), which produced type i interferon in response to poly i:c. in subsequent studies, similar findings were reported by judge et al. (2005) (identifying a core motif 5′-ugugu-3′) and sioud (2005) . in all three studies, transfection with cationic lipids (e.g., dotap, lipofectamine) or cationic polymers (e.g., pei, polyethylenimine) was essential for the immunological activity of sirna. the same applies for the immunological activity of single-stranded rna (diebold et al. 2004; heil et al. 2004; scheel et al. 2005 ). while for short rna oligonucleotides the immunological activity is clearly sequence dependent (hornung et al. 2005; judge et al. 2005) , for long rna molecules such as mrna, sequence specificity of immunological activity is less prominent (scheel et al. 2005) . this raises the question of how the immune system is able to distinguish between self and non-self (for example viral) rna. this question was addressed recently by kariko and colleagues (2005) who showed that human mitochondrial rna, when transfected into monocyte-derived dendritic cells, provoked secretion of tnf-α at similar quantities compared to total rna isolated from escherichia coli . in contrast, rna of other cellular compartments showed no immunological activity. the authors proposed that mammalian rna is masked by naturally occurring nucleoside modifications that are expected to be similar in closely related species. according to this concept, mitochondrial rna is stimulatory since it resembles bacterial rather than mammalian rna. in healthy cells, mitochondrial rna will not be released. in contrast to other self-rna, mitochondrial rna never enters the cytosolic compartment. as a consequence, mitochondrial rna under healthy conditions is not detected by cytosolic mechanisms of detection. only if the cell is lysed can mitochondrial rna enter the endosomal compartment of immune cells via phagocytosis. indeed, the stimulatory effect of in vitro rna transcripts composed of unmodified nucleotides in their study could be abrogated by incorporation of modified nucleosides such as pseudouridine, 5-methylcytidine, n6-methyladenosine, inosine, and n7methylguanosine. in order to examine modification sensitivity of different tlrs, hek293 cells expressing tlr3, tlr7, tlr8, or tlr9 were transfected with rna containing modified nucleosides. transfection of unmodified rnas stimulated il-8 production (sensitive readout for immunoactivation of hek293 cells) in hek293 cells overexpressing tlr3, 7, and 8. interestingly, rna recognition by tlr3, tlr7, and tlr8 is suppressed by the presence of different types of modified nucleotides within the rna ligand. tlr3 was the least sensitive receptor with regard to suppression by nucleoside modifications. furthermore, the authors showed that in monocyte-derived dendritic cells, 5%-10% of modified nucleosides were sufficient to inhibit tnf-α secretion by 75%-90%. together, these results show that rna modification contributes to the distinction of self versus non-self rna by the immune system. further insight into the properties that render rna molecules stimulatory to the immune system is driven by sirna technology. based on studies by tuschl and colleagues (elbashir et al. 2001) , sirna is now used worldwide as a robust tool for target-specific gene silencing in cell lines and human primary cells. however, depending on the mode of synthesis and the sequences used to generate sirna, also nonspecific, so-called nonspecific off-target effects of sirnas were observed. to overcome limitations with the transfection of synthetic sirna, vectorbased (e.g., lentiviral) expression systems for the introduction of short hairpin sirnas (shrna) mimicking sirnas were developed (brummelkamp et al. 2002; harborth et al. 2003; paddison et al. 2002) . the most commonly used shrna expression system consists of a rna-polymerase iii dependent promoter driving the expression of two complementary 19-to 29-bp rna sequences linked by a short loop of 4-10 nt. the resulting transcript is exported to the cytoplasm and processed by dicer. lentiviral vectors haboring the pol iii-shrna expression cassette (li et al. 2003; rubinson et al. 2003; tiscornia et al. 2003) allow rnai-mediated gene silencing via sirna in cells that are otherwise difficult to transfect. sequence specificity of gene silencing by such shrna was questioned by bridge et al. (bridge et al. 2003) , who demonstrated that infection of human lung fibroblasts with pol iii-shrna containing lentivirus directed against the gene morf4l1 not only silenced morf4l1 but also stimulated interferoninducible genes such as 2′,5′-oas, an indicator of type i interferon. the ifninducing effect was dependent on the sequence and the dose of the vector; seven of 23 shrnas targeting different genes exhibited ifn induction. in contrast, transfection of synthetic sirna with the same putative ifn-inducing sequences led to sequence-specific silencing without triggering an ifn response. northern blot analysis of shrna showed that the majority of shrna transcripts were correctly processed to 20 nt transcripts. the authors speculated that remaining unprocessed transcripts could be detected by cytosolic rna sensing receptors. in the follow-up paper, the group of iggo (pebernard and iggo 2004) , further correlated the u6 promoter sequence with oas induction. this study revealed that the region between -2 (the end of the promoter) and +2 (the start of rna transcript) is crucial for the immune stimulatory effect, which was lost when they used the endogenous human sequence (ccga). further mutations leading to a partial mismatch in the shrna (predicted to create a 14-bp duplex) suggested that stimulation required more than a 14-bp duplex. william´s group (sledz et al. 2003 ) described the induction of ifn target genes by transfection of synthetic sirnas into a human glioblastoma cell line (t98g) or a renal carcinoma cell line (rcc). when comparing the two studies from bridge and colleagues and from sledz and colleagues, it is important to note that different cell lines (bridge, human lung fibroblasts; sledz, rcc and t98g) and different ways of sirna generation (bridge, synthetic sirnas and shrnas; sledz, synthetic sirna and t7-phage-polymerase sirna) were used. using mouse embryonic fibroblasts (mefs) with different gene deficiencies related to the ifn response system, sledz et al. proposed that pkr was the interferon-inducing receptor for sirnas. later, the same group postulated a different sirna receptor (rig-i, see below) in t98g cells (marques et al. 2006) . of note, in the two studies published by bridge et al. (2003) and sledz et al. (2003) , type i interferon was not analyzed at the protein level. kariko et al. (2004a) suggested that tlr3 was responsible for the induction of type i ifn by sirna. these data are based on keratinocyte (hacat) and hek 293 cells, which responded to synthetic sirna but not to the singlestranded components (ssrna) by secretion of low amounts of ifn-β that was comparable to stimulation with poly i:c. overexpression of tlr3 in hek 293 cells resulted in fourfold higher induction of type i ifn secretion in response to transfected sirna. however, overexpression of nf-κb-inducing receptors such as tlr3 may also contribute indirectly to the enhanced type i ifn response induced by sirna, for example by upregulating ifn-inducible cytosolic rna receptors. for example, tlr3 overexpressing hek 293 cells secrete more il-8 than empty vector or tlr9 overexpressing hek 293 cells (kariko et al. 2005) ; consequently, such studies do not necessarily provide evidence for a direct interaction between sirna and tlr3 . kim et al. (2004) showed that the induction of type i ifn by sirna depended on the use of t7-rna polymerase (t7 rnap) for sirna generation. in contrast to bridge et al. (2003) and sledz et al. (2003) , in the study by kim and colleagues, type i ifn was measured at the protein level, which is less sensitive than measuring ifn-dependent responses on the transcriptional level and thus underscores the magnitude of the ifn response they reported. in their study, kim and colleagues examined sirnas targeting the early icp4 gene of hsv-1. only t7 rnap-derived transcripts but not synthetic sirna elicited a potent antiviral activity when transfected into hek 293 cells. the same antiviral activity was observed by transfection of t7 transcripts with unrelated sequences. analysis of supernatants revealed the presence of substantial amounts of ifn-α and ifn-β protein. these results were reproduced in hela cells, as well as k562, cem, and jurkat cells. it is well known that unlike capped mammalian mrna, the 5′ ends of t7 transcripts harbor a triphosphate gtp-nucleotide. treatment of t7 transcripts with rnase t1 (with the 5′ end p-ggg removed, which was single-stranded in their case) and alkaline phosphatase was sufficient to completely abrogate interferon -inducing activity. additional experiments using t3 and sp6 phage rna polymerases demonstrated similar induction of type i ifn. the examination of multiple cell lines by kim et al. (2004) pointed to a powerful ubiquitously expressed sensor for short triphosphate rna. yoneyama and colleagues identified the interferon-inducing cytoplasmic dexd/ h box rna helicase rig-i, containing a caspase recruitment domain (card) (yoneyama et al. 2004) . expression of the card domain sensitized cells to activate the transcription factor irf3, leading to the induction of the ifn-β promoter. later on it was shown that this pathway involves the irf3 kinase tbk1, which is activated by the newly characterized adaptor protein ips-1, also known as cardif, mavs, or visa (kawai et al. 2005; meylan et al. 2005; seth et al. 2005; xu et al. 2005 ; reviewed in sen and sarkar 2005) . in overexpression experiments, rig-i was shown to bind poly i:c. however, overexpression of a dominant negative mutant of rig-i impaired irf3 activation by newcastle disease virus (ndv), a negative-strand rna virus, while irf3 activation by poly i:c was not inhibited. subsequent studies with rig-i -/mice and mefs showed no defect in the response to poly i:c. hornung and colleagues (2006) demonstrated that rig-i detects in vitro transcribed rna. rna with a triphosphate at the 5′ end (now termed 3prna), which is generated during in vitro transcription, was identified to be the ligand for rig-i. the minimal length of 3prna was 19 nucleotides. the activity of 3prna was independent of double-strand formation. both exogenous 3prna transfected into the cell and endogenously formed 3prna (expression of t7 rna polymerase) activated rig-i. genomic rna prepared from a negative-strand rna virus and rna prepared from virus-infected cells, but not rna from noninfected cells, triggered a potent ifn-α response in a 5′-triphosphate-dependent manner. binding studies of rig-i and 3prna revealed a direct molecular interaction. the 5′ capping or incorporation of modified nucleotides such as pseudouridine, 2-thiouridine, and 2′-o-methylated uridine in place of uridine in short 3prna strongly diminished ifn-α induction. in a parallel study, pichlmair et al. (2006) attributed the inhibitory effect of the influenza virus protein ns1 to its binding and inhibition of the rig-i triphosphate rna complex. these results provide evidence that uncapped unmodified 3prna is detected by rig-i in the cytosol of eukaryotic cells. of note, all primer-independent rna transcripts in a normal uninfected cell initially contain a 5′-triphosphate end. however, most if not all self-rna species entering the cytosol lack a free 5′triphosphate end. before self-rna leaves the nucleus, rna is further processed, which applies to rna transcripts of all three dna-dependent rna polymerases (pol) in eukaryotes. pol i transcribes a large polycistronic precursor of ribosomal rna (rrna) that contains the sequences for the mature rrnas (18, 5.8s, 25-28s rrna), two external transcribed spacers, and two internal transcribed spacers. this primary transcript is subjected to endo-and exonucleolytic processing steps to produce the mature rrnas. the net result of this maturation process is a monophosphate group at the 5′ end of all pol i transcribed rrnas (fromont-racine et al. 2003) . messenger rnas (mrnas) and small nuclear rnas (snrnas), which are transcribed by pol ii, receive a 7-methyl guanosine group that is attached to the 5′-triphosphate of the nascent rna by a process called capping (shatkin and manley 2000) . thus, upon export into the cytoplasm, no free triphosphate groups are found in pol ii transcripts. all mature trnas (pol iii) have a 5′-monophosphate (xiao et al. 2002) , as it is likely to apply to 5s rrna. u6 rna receives a γ-monomethylphosphate cap structure following transcription. however, 7sl rna (pol iii) has a triphosphate at the 5′ end, and is present at high copy numbers in the cytosol. therefore, the presence or absence of a 5′ triphosphate might not be the only structural feature of rna responsible for the distinction of self and viral rna. it is well known that eukaryotic rna undergoes significant modifications to its nucleosides and its ribose backbone. among all nucleoside modifications, pseudouridinylation is one of the most common post-transcriptional modifications of rna that appears to be universal among rrnas and small stable rnas such as splicing small nuclear rnas (snrnas), trnas, and small nucleolar rnas (snornas). however, the frequency and location of pseudouridinylated nucleotides vary phylogenetically. intriguingly, eukaryotes contain far more nucleoside modifications within their rna species. human ribosomal rna, for example, the major constituent of cellular rna, contains ten times more pseudouridine and 25 times more 2-o-methylated nucleosides than e. coli rrna (rozenski et al. 1999) . the same applies to eukaryotic trnas, the most heavily modified subgroup of rna with up to 25% of modified nucleosides. the host machinery that guides nucleoside modifications and 2′-o-methylation of the ribose backbone is located in the nucleolus, and consists of rna-protein complexes containing snornas and several associated proteins (snornps) (decatur and fournier 2003) . information on nucleolus-specific nucleoside modifications or ribose 2′-o-methylation of viral rna genomes is limited. since most rna viruses do not replicate in the nucleus and modification is tightly confined to the sequence and structure of their target, extensive modification of viral rna seems unlikely. altogether, post-transcriptional modifications of eukaryotic rna such as 5′ processing or capping, as well as nucleoside modifications or ribose backbone methylation, provide the molecular basis for the distinction of self-rna generated in the nucleus from viral rna of cytosolic origin containing 5′-triphosphate (3prna). the mrnas of viruses infecting eukaryotic cells also commonly contain 7-methyl guanosine cap-structures at their 5′ ends and poly(a) tails at their 3′ends (furuichi and shatkin 2000) . some viruses make use of the host transcription machinery to acquire caps and poly(a) tails. rna viruses that do not rely on the host transcriptional machinery produce their own capping enzymes or utilize other mechanisms such as snatching the 5′terminal regions of host mrnas. despite these adaptations of viruses to the host transcriptional system, viral rna synthesis leads to transient cytosolic rna intermediates with an uncapped 5′-triphosphate end. with notable exceptions such as the picornavirus family (see below), viral rna-dependent rna polymerases (rdrp) initiate polymerase activity de novo, without a specific primer (kao et al. 2001) . as a consequence, these rdrp-dependent transcripts start with an uncapped 5′-triphosphate. this has been studied in great detail for the replication of positive-strand rna viruses of the family of flaviviridae (including the genera flavivirus , pestivirus , and hepacivirus ); members of all of these virus genera were reported as being recognized via rig-i (honda et al. 1998; kato et al. 2006; sumpter et al. 2005) . segmented nsv rely on a cap-snatched primer for mrna transcription, yet initiate genomic and the complementary antigenomic rna replication by a primer-independent de novo mechanism resulting in a 5′-triphosphateinitiated transcript (honda et al. 1998; neumann et al. 2004) . nsv with a nonsegmented genome (order mononegavirales), including the paramyxoviruses and rhabdoviruses, initiate both replication and transcription de novo leading to 5′-triphosphate rna in the cytosol. both the full-length replication products, vrna and crna, and a short leader rna, which is abundantly synthesized during initiation of transcription, maintain their 5′triphosphate (colonno and banerjee 1978; whelan et al. 2004 ), while the virus-encoded mrna transcripts are further modified at their 5′ ends by capping and cap methylation. consequently, genomic rna from nsvs per se is expected to trigger an ifn-response without the need for replication and presumed dsrna formation. consistent with this notion, not only live virus but also rna purified from nsv virions (vsv) has been shown to trigger strong type i interferon responses depending on rig-i ). hornung and colleagues confirmed and extended these observations by demonstrating that dephosphorylation of the viral rna isolates completely abolished the ifn-response, thereby indicating that the 5′-triphosphate moiety is strictly required for recognition . a notable exception are the viruses in the picornavirus-like supergroup (picornavirus, potyvirus, comovirus, calicivirus, and other viruses), which exclusively employs a protein known as viral genome-linked protein (vpg) as a primer for both positive-and negative-strand rna production. this protein primer is part of the precursor rdrp and is cleaved off as elongation of the initial complex occurs, usually to become a 5′-genome-linked protein (lee et al. 1977) . thus during the life-cycle of picornaviruses uncapped, triphosphorylated 5′ ends are absent. consequently, based on our studies, rig-i is expected to be involved in the detection of flaviviridae and nsv but not picornaviruses. this is confirmed in a recent study . a number of studies suggested that the helicases mda-5 and rig-i recognize dsrna (andrejeva et al. 2004; rothenfusser et al. 2005; yoneyama et al. 2004) . the results in the work of hornung and colleagues (2006) demonstrated that double-strand formation of rna is not required for rig-i-rna interaction, and that dsrna is not sufficient for rig-i activation. these results further demonstrate that mda-5 is not involved in 5′-triphosphate rna recognition. although there is convincing evidence that mda-5 is activated by the long dsrna mimic poly i:c, activation of mda-5 by natural long dsrna is still controversial . taken together, tlr3 is so far the only receptor that induces type i ifn upon binding of the natural molecule long dsrna, but the contribution of tlr3 to type i ifn induction and viral clearance in vivo seems to be weak (rudd et al. 2006) . there is good evidence that short dsrna such as sirna generated by dicermediated cleavage of long dsrna does not elicit a type i ifn response in nonimmune cells (elbashir et al. 2001; hornung et al. 2005; kim et al. 2004) . a recent study suggests that the two-nucleotide overhang at the 3′ end of dicer cleavage products are essential for the lack of immunorecognition of short dsrna (marques et al. 2006) . the same study proposed that synthetic blunt-end short dsrna is recognized via rig-i. the conclusion that rig-i is the receptor for blunt end short dsrna is based on experiments using rig-i overexpression and using anti-rig-i sirna (short dsrna with two-nucleotide 3′overhangs) on top of stimulation with blunt end short dsrna stimulation. rig-i-deficient cells have not been examined in this study. this experimental design does not provide clear-cut evidence for the primary involvement of rig-i in type i ifn induction by blunt-end short dsrna. furthermore, in the study by hornung and colleagues, 5′ triphosphate blunt-end rna and 5′ triphosphate 2-nt overhang rna showed identical rig-i ligand activity, suggesting that the molecular feature 2-nt overhang does not inhibit rig-i-mediated recognition ). mda-5 is structurally related to rig-i, as it also contains two card domains and a helicase domain. mda-5 was originally identified as a type i ifn-inducible molecule mediating cell cycle arrest and apoptosis in melanoma cells (hence the name melanoma differentiation antigen 5) (kang et al. 2002 (kang et al. , 2004 kovacsovics et al. 2002) . a first indication of a role for mda-5 in virus recognition came from the observation that a paramyxoviral protein that mediated immune evasion bound to mda-5 (andrejeva et al. 2004 ). in overexpression experiments, mda-5 was shown to bind poly i:c, and enhanced the interferon response to poly i:c as well as several viruses. conversely, sirna mediated knock-down blocked type i ifn induction in response to these stimuli . mda-5 was then shown to play an essential role in the detection of picornaviruses such as encephalomyocarditis virus (emcv) or theiler's virus (gitlin et al. 2006; kato et al. 2006 ). in addition, mice deficient in mda-5 were found to be highly susceptible to emcv. although the nature of the natural rna ligand that engages mda-5 has so far remained obscure, a surprising observation was that cells derived from mda-5-deficient mice, as well as mda-5 -/mice stimulated in vivo were found unable to mount a type i ifn response to poly i:c, establishing mda-5, rather than the several other receptors that bind, or have been shown to be activated by poly i:c, as the dominant receptor mediating the interferon response to poly i:c (gitlin et al. 2006; kato et al. 2006 ). however, the natural viral ligand for mda-5 has not yet been identified. in addition to rig-i and mda-5, another cytosolic receptor may exist for detecting dna. until recently tlr9 was the only innate sensor for detecting microbial dna. recent studies indicate that dna is detected in the cytosol independently of tlr9 (okabe et al. 2005; stetson and medzhitov 2006) , but the receptor has not been identified yet. the cytosolic receptor mediating recognition of b-form dna, unlike rig-i and mda-5, signals independently of ips-1. as discussed in the previous sections, immune and nonimmune cell types express characteristic patterns of nucleic acid receptors (table 1 ) . for example, melchjorsen and colleagues reported that activation of innate defense against a paramyxovirus is mediated by rig-i, tlr7, and tlr8 in a cell-type-specific manner (melchjorsen et al. 2005) . they found that nonimmune cells relied entirely on rna recognition through rig-i for activation of an antiviral response. in contrast, immune cells such as myeloid cells utilized tlr7 and tlr8. unlike modifications that have been shown to prevent detection by the receptors indicated and that are frequently found in mammalian nucleic acids in nonimmune cells, rna sensing in paramyxovirus-infected myeloid cells was independent of rig-i, tlr3, and pkr. kato and colleagues also found celltype specific involvement of rig-i in antiviral immune response. in their study type i ifn induction in both fibroblasts and myeloid dendritic cells was rig-i-dependent, while type i ifn induction in pdc was rig-i-independent . it is important to note that the mechanisms used for rna sensing may not only be cell-type-dependent but may also depend on the type of virus and its strategy to enter the target cell and to evade immune recognition. in contrast, recognition of synthetic rna or of rna transcribed from vector systems is more predictable because there is no immune evasion and because the mode of delivery is known. of note, the use of cationic lipids and polycationes leads to both endosomal and cytosolic delivery (almofti et al. 2003; boussif et al. 1995) and thus both tlr-and rig-i-mediated rna sensing is triggered, provided these receptors are expressed in the cell type examined, and the appropriate rna ligand is delivered. of note, subcellular localization of tlr3 is cell-type-specific (matsumoto et al. 2003) : in fibroblasts, tlr3 is located on the cell surface, and the tlr3-mediated activity can be blocked by anti-tlr3 antibodies. in myeloid dendritic cells, tlr3 is found in the cytosolic compartment. a more detailed analysis in tlr3-transfected b cells revealed that tlr3 is detectable in multivesicular bodies, a subcellular compartment situated in the endocytic trafficking pathway (matsumoto et al. 2003) . bacteria, fungi, or cellular parasites are recognized via conserved molecules typical for the respective type of pathogen. in contrast, all virus components are formed within the infected host cell; consequently, a virus-specific detection system is more difficult to achieve. it is now evident that host cells are equipped to detect viral nucleic acids. for viral infection in vivo, the following picture is evolving: large parts of the early type i ifn response upon viral infection are due to tlr7 and tlr9 expressed in pdcs; in fact, pdcs are the only considerable source of tlr7-and tlr9-induced type i ifn production upon viral infection. the major advantages of this pdc response are that the presence of viral particles is sufficient for recognition, that viral infection of cells is not required for detection, and that viruses are recognized before viral proteins have a chance to mediate immune evasion. this first wave of type i ifn production plays an important role in limiting viral spread by pdc-derived direct antiviral mechanisms early on, and by sensitizing yet uninfected cells for cytosolic recognition of viral nucleic acid via strong upregulation of the two cytosolic helicases, rig-i and mda-5. these two cytosolic receptors are then responsible for the second and prolonged wave of type i ifn production and for the induction of apoptosis of virally infected cells. for all four receptors, distinction of self from viral nucleic acid is based on a combination of localization and molecular structure. in this sophisticated system of virus detection, the following situations signal viral danger: 1. appearance of unmodified rna in the endosomal compartment of pdcs 2. appearance of dna containing unmethylated cpg motifs in the endosomal compartment of pdcs 3. unmodified rna with a triphosphate group at the 5′ end (3prna) in the cytosol of any cell type 4. dna in the cytosol of any cell type it is still unclear whether long dsrna in the cytosol is sufficient to elicit an antiviral response via one of the receptors known to date. although poly i:c is a ligand for mda-5, long double-stranded rna seems insufficient as a ligand, and the natural ligand still needs to be identified. in addition to rnadetecting receptors, the cytosolic receptor for dna may add new perspectives in therapeutic viral mimicry. with regard to viruses that perform inside the nucleus such as hbv and hiv, uncovering molecular mechanisms of sensing viral nucleic acids in the nucleus appears on the radar of scientific challenges. recognition of double-stranded rna and activation of nf-kappab by toll-like receptor 3 cationic liposome-mediated gene delivery: biophysical study and mechanism of internalization the v proteins of paramyxoviruses bind the ifn-inducible rna helicase, mda-5, and inhibit its activation of the ifn-beta promoter virus-specific double-stranded rna in poliovirus-infected cells dendritic cells respond to influenza virus through tlr7-and pkrindependent pathways a versatile vector for gene and oligonucleotide transfer into cells in culture and in vivo: polyethylenimine induction of an interferon response by rnai vectors in mammalian cells a system for stable expression of short interfering rnas in mammalian cells control of coronavirus infection through plasmacytoid dendritic-cell-derived type i interferon jnk2 and ikkbeta are required for activating the innate response to viral infection plasmacytoid dendritic cells in immunity complete nucleotide sequence of the leader rna synthesized in vitro by vesicular stomatitis virus rna-guided nucleotide modification of ribosomal and other rnas innate antiviral responses by means of tlr7-mediated recognition of single-stranded rna viral infection switches non-plasmacytoid dendritic cells into high interferon producers irf3 mediates a tlr3/tlr4-specific antiviral gene program duplexes of 21-nucleotide rnas mediate rna interference in cultured mammalian cells interferon-beta is required for interferon-alpha production in mouse fibroblasts interferon action: two distinct pathways for inhibition of protein synthesis by double-stranded rna inducers of interferon and host resistance. ii. multistranded synthetic polynucleotide complexes lps-tlr4 signaling to irf-3/7 and nf-kappab involves the toll adapters tram and trif ribosome assembly in eukaryotes viral and cellular mrna capping: past and prospects essential role of mda-5 in type i ifn responses to polyriboinosinic:polyribocytidylic acid and encephalomyocarditis picornavirus role of interferon in the pathogenesis of virus diseases in mice as demonstrated by the use of antiinterferon serum. i. rapid evolution of encephalomyocarditis virus infection specificity in toll-like receptor signalling through distinct effector functions of traf3 and traf6 sequence, chemical, and structural variation of small interfering rnas and short hairpin rnas and the effect on mammalian gene silencing rational design of new cpg oligonucleotides that combine b cell activation with high ifn-alpha induction in plasmacytoid dendritic cells mechanism and function of a newly identified cpg dna motif in human primary b cells species-specific recognition of single-stranded rna via tolllike receptor 7 and 8 small anti-viral compounds activate immune cells via the tlr7 myd88-dependent signaling pathway a toll-like receptor recognizes bacterial dna identification of the 5′ terminal structure of influenza virus genome rna by a newly developed enzymatic method role of a transductional-transcriptional processor complex involving myd88 and irf-7 in toll-like receptor signaling 5′-triphosphate rna is the ligand for rig-i sequence-specific potent induction of ifn-alpha by short interfering rna in plasmacytoid dendritic cells through tlr7 quantitative expression of toll-like receptor 1-10 mrna in cellular subsets of human peripheral blood mononuclear cells and sensitivity to cpg oligodeoxynucleotides cutting edge: toll-like receptor 4 (tlr4)-deficient mice are hyporesponsive to lipopolysaccharide: evidence for tlr4 as the lps gene product synthesis of low molecular weight inhibitor of protein synthesis with enzyme from interferon-treated cells activation of nf-kappab by doublestranded rna (dsrna) in the absence of protein kinase r and rnase l demonstrates the existence of two separate dsrna-triggered antiviral programs foreign nucleic acids as the stimulus to make interferon virus interference. i. the interferon approaching the asymptote? evolution and revolution in immunology sequencedependent stimulation of the mammalian innate immune response by synthetic sirna human tlr7 or tlr8 independently confer responsiveness to the antiviral compound r-848 expression analysis and genomic characterization of human melanoma differentiation associated gene-5, mda-5: a novel type i interferon-responsive apoptosisinducing gene mda-5: an interferon-inducible putative rna helicase with double-stranded rna-dependent atpase activity and melanoma growth-suppressive properties de novo initiation of viral rna-dependent rna synthesis small interfering rnas mediate sequence-independent gene suppression and induce immune activation by signaling through toll-like receptor 3 suppression of rna recognition by toll-like receptors: the impact of nucleoside modification and the evolutionary origin of rna cell type-specific involvement of rig-i in antiviral response differential roles of mda5 and rig-i helicases in the recognition of rna viruses interferon-alpha induction through tolllike receptors involves a direct interaction of irf7 with myd88 and traf6 ips-1, an adaptor triggering rig-i-and mda5-mediated type i interferon induction interferon induction by sirnas and ssrnas synthesized by phage polymerase overexpression of helicard, a card-containing helicase cleaved during apoptosis, accelerates dna degradation tlr9-dependent recognition of mcmv by ipc and dc generates coordinated cytokine responses that activate antiviral nk cell function herpes simplex virus type 1 activates murine natural interferon-producing cells through toll-like receptor 9 identification of cpg oligonucleotide sequences with high induction of ifn-alpha/beta in plasmacytoid dendritic cells toll-like receptor expression reveals cpg dna as a unique microbial stimulus for plasmacytoid dendritic cells which synergizes with cd40 ligand to induce high amounts of il-12 double-stranded rnadependent protein kinase activates transcription factor nf-kappa b by phosphorylating i kappa b a protein covalently linked to poliovirus genome rna the dorsoventral regulatory gene cassette spatzle/toll/cactus controls the potent antifungal response in drosophila adults inhibition of hiv-1 infection by lentiviral vectors expressing pol iiipromoted anti-hiv rnas toll-like receptor 9-mediated recognition of herpes simplex virus-2 by plasmacytoid dendritic cells recognition of single-stranded rna viruses by toll-like receptor 7 potential role of pkr in double-stranded rna-induced macrophage activation interactions between doublestranded rna regulators and the protein kinase dai differential viral induction of distinct interferonalpha genes by positive feedback through interferon regulatory factor-7 a structural basis for discriminating between self and nonself double-stranded rnas in mammalian cells subcellular localization of toll-like receptor 3 in human dendritic cells a human homologue of the drosophila toll protein signals activation of adaptive immunity activation of innate defense against a paramyxovirus is mediated by rig-i and tlr7 and tlr8 in a cell-type-specific manner cardif is an adaptor protein in the rig-i antiviral pathway and is targeted by hepatitis c virus structural requirements of doublestranded rna for the activation of 2' ,5'-oligo(a) polymerase and protein kinase of interferon-treated hela cells replicative form of encephalomyocarditis virus ribonucleic acid tlr signalling and activation of irfs: revisiting old friends from the nf-kappab pathway functional role of type i and type ii interferons in antiviral defense inhibition of vaccinia infection by a liquid factor in tissues infected by homologous virus orthomyxovirus replication, transcription, and polyadenylation critical role of traf3 in the toll-like receptor-dependent and -independent antiviral response toll-like receptor-independent gene induction program activated by mammalian dna escaped from apoptotic dna degradation short hairpin rnas (shrnas) induce sequence-specific silencing in mammalian cells determinants of interferon-stimulated gene induction by rnai vectors rig-i-mediated antiviral responses to single-stranded rna bearing 5'-phosphates defective lps signaling in c3h/hej and c57bl/10sccr mice: mutations in tlr4 gene the rna helicase lgp2 inhibits tlrindependent sensing of viral replication by retinoic acid-inducible gene-i plasmacytoid dendritic cells: the key to cpg the rna modification database: 1999 update a lentivirus-based system to functionally silence genes in primary mammalian cells, stem cells and transgenic mice by rna interference deletion of tlr3 alters the pulmonary immune environment and mucus production during respiratory syncytial virus infection positive feedback regulation of type i ifn genes by the ifn-inducible transcription factor irf-7 toll-like receptor-dependent activation of several human blood cell types by protamine-condensed mrna sirna and isrna: two edges of one sword hitching rig to action identification and characterization of mavs, a mitochondrial antiviral signaling protein that activates nf-kappab and irf 3 triggering the interferon antiviral response through an ikk-related pathway the ends of the affair: capping and polyadenylation induction of inflammatory cytokines and interferon responses by double-stranded and single-stranded sirnas is sequence-dependent and requires endosomal localization activation of the interferon system by short-interfering rnas irf3 and irf7 phosphorylation in virus-infected cells does not require double-stranded rna-dependent protein kinase r or ikappa b kinase but is blocked by vaccinia virus e3l protein recognition of cytosolic dna activates an irf3-dependent innate immune response regulating intracellular antiviral defense and permissiveness to hepatitis c virus rna replication through a cellular rna helicase, rig-i toll-like receptors 9 and 3 as essential components of innate immune defense against mouse cytomegalovirus infection type i interferons (alpha/ beta) in immunity and autoimmunity a general method for gene knockdown in mice by using lentiviral vectors expressing small interfering rna transcription and replication of nonsegmented negative-strand rna viruses eukaryotic ribonuclease p: a plurality of ribonucleoprotein enzymes visa is an adapter protein required for virus-triggered ifn-beta signaling deficient signaling in mice devoid of double-stranded rnadependent protein kinase shared and unique functions of the dexd/h-box helicases rig-i, mda5, and lgp2 in antiviral innate immunity the rna helicase rig-i has an essential function in double-stranded rna-induced innate antiviral responses activation of the protein kinase pkr by short double-stranded rnas with single-stranded tails interferon action and apoptosis are defective in mice devoid of 2' ,5'-oligoadenylate-dependent rnase l isolation of two interferon-induced translational inhibitors: a protein kinase and an oligo-isoadenylate synthetase key: cord-001273-plz1ja2e authors: dussurget, olivier; bierne, hélène; cossart, pascale title: the bacterial pathogen listeria monocytogenes and the interferon family: type i, type ii and type iii interferons date: 2014-04-28 journal: front cell infect microbiol doi: 10.3389/fcimb.2014.00050 sha: doc_id: 1273 cord_uid: plz1ja2e interferons (ifns) are secreted proteins of the cytokine family that regulate innate and adaptive immune responses to infection. although the importance of ifns in the antiviral response has long been appreciated, their role in bacterial infections is more complex and is currently a major focus of investigation. this review summarizes our current knowledge of the role of these cytokines in host defense against the bacterial pathogen listeria monocytogenes and highlights recent discoveries on the molecular mechanisms evolved by this intracellular bacterium to subvert ifn responses. listeria monocytogenes is a pathogenic gram-positive bacillus responsible for a foodborne disease in humans and animals called listeriosis (vázquez-boland et al., 2001) . this highly versatile bacterium can be isolated from multiple sources such as human and animal feces, soil, water, plants and food. as a common contaminant of fruits, vegetables, seafood, meat and cheese, it represents a major economic problem for the food industry. infection usually originates from ingestion of contaminated food (schlech et al., 1983) and may cause febrile gastroenteritis in otherwise healthy persons (ooi and lorber, 2005) . in contrast, in immunocompromised individuals it leads to a severe invasive disease, which manifests itself as septicemia, meningitis and encephalitis. in the specific case of pregnant women, infection may cause fetal loss or neonatal bacteremia and meningitis. in the united states, incidence of listeriosis ranged from 2.5 to 3.2 cases per million population between 2004 and 2009 (cartwright et al., 2013) . in france, incidence was 3.9 per million between 2001 and 2008, and increased risk of listeriosis was noticed in people with underlying diseases, such as chronic lymphocytic leukemia (goulet et al., 2012) . while relatively rare, listeriosis is among the most deadly foodborne diseases with mortality rates reaching up to 50% depending on the clinical manifestations (lorber, 1997) . in addition to the immunological status of the patient, the clinical outcome of the disease depends on the pathogenic potential of the infecting bacteria. l. monocytogenes strains of serovars 1/2a, 1/2b, and 4b account for 95% of human cases and serovar 4b alone is associated to most outbreaks (swaminathan and gernersmidt, 2007) . the capacity of l. monocytogenes to survive and multiply within the gastrointestinal tract is critical for the initial infection, persistence and transmission. l. monocytogenes is well adapted to this environment and produces multiple factors to compete with microbiota and counteract antimicrobial peptides, acidity, hyperosmolarity, hypoxia, bile and iron deprivation (gahan and hill, 2005) . crossing of the intestinal epithelium is thought to occur by invasion of enterocytes, in particular goblet cells and m cells of the peyer's patches. invasion of enterocytes requires the specific interaction between the listeria surface protein inla and its cellular receptor e-cadherin (lecuit et al., 2001; disson et al., 2008) , which can take place at sites of cell extrusion at the tip and other locations of intestinal villi (pentecost et al., 2006; nikitas et al., 2011) . indeed, as recently shown, upon interaction with e-cadherin, listeria preferentially crosses the intestinal barrier by transcytosis through goblet cells (nikitas et al., 2011) . entry through ileal peyer's patches via m cells does not rely on inla. it has been reported to require listeria invasion protein inlb (chiba et al., 2011) . after translocation, bacteria reach lymph nodes, the liver and spleen and finally secondary target sites of infection, including the central nervous system and the placenta. a remarkable feature of l. monocytogenes is its capacity to invade non-professional phagocytic cells such as enterocytes, hepatocytes and trophoblast cells. the exceptional repertoire of virulence factors necessary for entry, survival and multiplication has been extensively studied cossart, 2011) . expression of many virulence genes relies on the transcriptional activator prfa, whose role is pivotal for l. monocytogenes transition from saprophytic to intracellular lifestyle (freitag et al., 2009; toledo-arana et al., 2009) . elimination of l. monocytogenes is mostly based on the capacity of the host to mount an efficient cellular immune response to infection (mackaness, 1962; shi and pamer, 2011) . in particular, the fate of infection depends on the level of macrophage activation and on listeria ability to counteract bactericidal mechanisms of host cells (shaughnessy and swanson, 2007; corr and o'neill, 2009; stavru et al., 2011) . bacterial escape from the phagosome and avoidance of autophagy for intracytosolic replication and cell-cell spread have been well characterized. they have been shown to depend on five major virulence factors: the secreted pore-forming toxin listeriolysin o (llo) and two phospholipases c (plca and plcb) for vacuolar escape, the surface protein acta for actin-based motility and both acta and a surface protein of the internalin family, inlk, for autophagy evasion (cossart, 2011; dortet et al., 2012) . other strategies of immune escape that lead to the modulation of cytokine expression occur through a variety of mechanisms. modifications of l. monocytogenes peptidoglycan by the n-deacetylase pgda and the o-acetyl transferase oata prevent lysozyme-dependent release of microbe-associated molecular patterns (mamps), activation of pathogen-recognition receptors (prrs) and subsequent production of pro-inflammatory cytokines (boneca et al., 2007; aubry et al., 2011; rae et al., 2011) . the toxin llo induces dephosphorylation of histone h3 and deacetylation of histone h4, which correlate with decreased expression of pro-inflammatory genes, such as the chemokine gene cxcl2 (hamon et al., 2007) . the secreted internalin inlc inhibits inflammation by interacting with ikk-α, a component of the iκb-kinase complex, which is essential for nf-κb activation and expression of proinflammatory genes (gouin et al., 2010) . other evasion mechanisms remain to be characterized, such as the control of the expression of il-6 by the surface internalin inlh (personnic et al., 2010) . l. monocytogenes also has the capacity to modulate interferon (ifn) production during infection. type i ifn production by infected cells can be controlled by listeria multidrug efflux pumps mdrm and mdrt, via the secretion of the second messenger cyclic-di-amp (crimmins et al., 2008; woodward et al., 2010; schwartz et al., 2012) . synthesis of type iii ifn has also recently been shown to be tuned by listeria nucleomodulin lnta (lebreton et al., 2011) . our knowledge concerning the role of the ifn cytokine family during listeriosis has rapidly expanded in the last few years and will be the focus of this review. ifns form a family of proteins secreted by many cell types in response to infection. they were originally named for their capacity to interfere with viral proliferation (isaacs and lindemann, 1957) . this diverse family is composed of three groups of cytokines, namely type i-, type ii-, and type iii-ifns, which are important components of innate immune responses ( table 1) . type i-ifns consist of ifn-α, ifn-β, ifn-δ, ifn-ε, ifn-ζ, ifnκ, ifn-ν, ifn-τ, and ifn-ω (levy et al., 2011) . type ii-ifn is composed of a single cytokine, ifn-γ (pestka et al., 2004) . type iii-ifns are ifn-λ1, ifn-λ2, and ifn-λ3 (formerly il-29, il-28a, and il-28b) and ifn-λ4 (kotenko, 2011; prokunina-olsson et al., 2013) . type i-and type iii-ifns have similar signal transduction systems (see below) and are phylogenetically closer from each other than type ii-ifn (pestka et al., 2004) . sequence conservation and chromosome location suggest that type i-ifn genes evolved from a single ancestor through duplication. however, the extent of type i-ifn gene diversification varies greatly depending on the species (pestka et al., 2004) . generally, a single gene encodes the type i-ifn in fish. in contrast, multiple gene duplications and diversification led to the emergence of sub-types of type i-ifns in mammals ( table 1) . gene duplication varies also within each sub-type. a single ifn-β gene is found in the human and mouse genomes (decker et al., 2005; honda et al., 2006; durbin et al., 2013) . in contrast 13 ifn-α genes and one pseudogene and 14 ifn-α genes and three pseudogenes are found in the human and mouse genomes, respectively (van pesch et al., 2004; durbin et al., 2013) . a single gene encodes type ii-ifn and four genes encode type iii-ifns in human (decker et al., 2005; levy et al., 2011) . of note, ifn-λ1 and ifn-λ4 are pseudogenes in mice, which prevents the study of these cytokines in this animal model ( table 1 ) (fox et al., 2009 ). ifns are important components of the immune system, which generally trigger cellular protective defenses in response to infection or tumor formation. type-ii ifn (ifn-γ) is a paradigm for this, being an important mediator of innate and adaptive immune responses with a key role in clearance of viral and bacterial pathogens and in tumor control. ifn-γ was first described as an antiviral protein (wheelock and sibley, 1965) , but is now known to exhibit broader biological activities, non-redundant with that of other types of ifns. the crucial role of ifn-γ in immunity to infection is reflected by the phenotype of mice lacking the ifn-γ receptor or the ifn-γ gene, which are highly susceptible to mycobacterium bovis bcg infection (dalton et al., 1993; kamijo et al., 1993) . genetic deficiencies resulting in the loss of ifn-γ production or signaling in mice lead to increased susceptibility to infections by other intracellular pathogens, such as l. monocytogenes (see below), salmonella typhimurium and some viruses harty and bevan, 1995; jouanguy et al., 1999) . these defects also lead to the loss of tumor control (kaplan et al., 1998) . patients with deficiencies in the ifn-γ pathway, for instance by mutation in the gene for the ifn-γ receptor 1, are characterized by severe infections with viruses and intracellular bacteria including l. monocytogenes, salmonella sp. and mycobacteria (jouanguy et al., 1996; newport et al., 1996; roesler et al., 1999; van de vosse et al., 2009) . ifn-γ mediates macrophage activation, i.e., increased phagocytosis and production of pro-inflammatory cytokines, of microbicidal reactive oxygen and nitrogen species, leading to clearance of intracellular pathogens (schoenborn and wilson, 2007) . in addition, ifn-γ controls differentiation of t cells in th1 effector cells, antigen processing and presentation by antigen-presenting cells, which participate to cellular immunity against intracellular pathogens (schroder, 2003; hu and ivashkiv, 2009 ). the immunostimulatory and immunomodulatory properties of ifn-γ have therapeutic implications. indeed, ifn-γ is used in patients with chronic granulomatous disease to reduce infection and mortality, **protein length in amino-acids and protein modifications (g: glycosylation, p: phosphorylation). ***13 genes and a pseudogene in the human genome, 14 genes and three pseudogenes in the mouse genome. although the clinical benefit has not been demonstrated in all studies (holland, 2010) . type i-ifns are produced in responses to viruses, many bacteria and parasites. however, in contrast to type ii ifns, these cytokines are not always protective against bacterial infections. indeed, the role of type i-ifns in response to bacterial infection is complex and depends on the microorganism (decker et al., 2005; monroe et al., 2010; carrero, 2013) . they contribute to resistance of the host against infection by extracellular bacteria, such as escherichia coli, helicobacter pylori, streptococcus agalactiae and s. pneumoniae (mancuso et al., 2007; watanabe et al., 2010) . in contrast, they are associated with suppression of innate immune responses and increased susceptibility of the host to infection by l. monocytogenes (see below), brucella abortus, chlamydia muridarum, francisella novicida, salmonella enterica, staphylococcus aureus, and yersinia pestis (auerbuch et al., 2004; carrero et al., 2004; o'connell et al., 2004; qiu et al., 2008; martin et al., 2009; henry et al., 2010; de almeida et al., 2011; patel et al., 2012; robinson et al., 2012; archer et al., 2014) . these different effects on infection are likely linked to the wide range of cellular responses induced by their downstream effectors, the products of ifn-stimulated genes (isgs) (schoggins et al., 2011) . although type i-ifns have long been known to induce antiviral response in the infected host (isaacs and lindemann, 1957) , they can also induce apoptosis, autophagy, differentiation and migration, inhibit proliferation as well as angiogenesis and mediate cellular damage, inflammation or autoimmunity (trinchieri, 2010) . as a result, type i-ifns have a therapeutic potential that can be used to treat tumors and viral infections (pestka, 2007; heim, 2013; wilson and brooks, 2013) , while being detrimental for the host in response to a subset of pathogens. type iii-ifns have been discovered in 2003 (kotenko et al., 2003; sheppard et al., 2003) and their activities have been less extensively characterized than those of type i-and type ii-ifns. however, several studies suggest that type i and type iii-ifns share common biological activities (levy et al., 2011; zheng et al., 2013) . although type iii-ifns respond to different stimuli, use different receptors and are not always expressed by the same cells as type i ifns (see below), engagement of type i-and type iii-ifn receptors leads to similar transcriptional responses. like type i-ifns, type iii-ifns have been involved in antiproliferative and antiviral responses (iversen and paludan, 2010; mordstein et al., 2010; durbin et al., 2013; hamming et al., 2013) . recently, type iii-ifns have been shown to be induced in response to bacterial pathogens, but their downstream effects are not yet characterized (pietilä et al., 2010; lebreton et al., 2011; bierne et al., 2012) . transcription of ifn genes is induced rapidly in response to microbial infection. type i-ifns can be produced by all cells, while type iii-ifns are secreted by specific cell types, including dendritic and epithelial cells. type i-and type iii-ifns activation is initiated by detection of mamps by prrs such as endosomal transmembrane toll-like receptors and cytosolic receptors (stetson and medzhitov, 2006; monroe et al., 2010) . upon recognition of mamps, prrs trigger diverse signaling pathways that involve adaptor proteins and cytosolic or organelle-bound protein scaffolds activating kinases converging to phosphorylation of transcription factors and their subsequent translocation into the nucleus (figure 1) . irf1, irf3, irf4, irf5, irf7, and irf8 are important for transcription of the ifn-α genes, with irf7 considered as the master regulator of ifn-α response (honda et al., 2005; tailor et al., 2007; levy et al., 2011) . regulation of the ifnβ gene is more complex. activated irf3, irf7, ap-1, and nf-κb bind to the enhancer/promoter regions of the ifn-β gene and participate to the formation of the enhanceosome, which alters chromatin structure and allows transcription (panne et al., 2007; panne, 2008) . in contrast, irfs and nf-κb independently activate transcription of type iii-ifn genes (iversen and paludan, 2010) . regulation of the ifn-γ gene expression is different from that of type i and type iii-ifn genes. nk cells and nkt cells are effectors of the innate immune response and primary sources of ifn-γ. mature nk and nkt cells quickly react to infection by inducing ifn-γ secretion. upon recognition of ligands expressed on infected cells, nk cell activating-receptors trigger signaling cascades involving adaptor proteins and protein tyrosine kinases leading to activation of ras/sos, plc-γ and mapk pathways and induction of ifn-γ production (schoenborn and wilson, 2007) . in addition to receptors, il-2, il-15, il-12, il-18, and type i-ifns also contribute to induction of ifn-γ production by nk cells (newman and riley, 2007; schoenborn and wilson, 2007; marçais et al., 2013) . similarly, il-12 and il-18 induce ifn-γ production by nkt cells (godfrey and berzins, 2007) . in nk and nkt cells, the ifn-γ gene locus is transcriptionally permissive within accessible chromatin and allows rapid ifn-γ expression upon activation of transcription factors, such as ap-1, nf-κb, stat4, and t-bet (glimcher et al., 2004; schoenborn and wilson, 2007; lazarevic et al., 2013) . in addition, naive cd4 and cd8 t cells can differentiate into th1 cd4 effector t cells and cd8 cytotoxic t lymphocytes capable of ifn-γ secretion (wilson et al., 2009) . ifn-γ production by cd4 and cd8 t cells depends on il-12, il-18 and ifn-γ itself and share many signaling pathways with nk cells. multiple transcription factors act at the ifn-γ promoter, e.g., ap-1, atf-2/c-jun, c/ebp, eomes, ets-1, nfat, nf-κb, runx3, stats and t-bet (schoenborn and wilson, 2007; samten et al., 2008; wilson et al., 2009; lazarevic et al., 2013) . moreover, distal regulatory elements modify the chromatin and remodel the ifn-γ gene locus to facilitate ifn-γ production (wilson et al., 2009 ). ifns are rapidly secreted upon infection and then bind to their receptors on the surface of target cells (table 1) . type i-ifns bind the ubiquitous ifnar receptor, which consists of two chains, ifnar1 and ifnar2 (piehler et al., 2012) . type iii-ifns bind and signal through a different receptor complex, made of two chains: ifnlr1 (also known as il-28rα) and il10r2. this receptor is expressed primarily by epithelial cells and hepatocytes (iversen and paludan, 2010) . thus, the physiological roles of type i-and type iii-ifns are distinct because of the different distribution of their receptors in tissues, type iii-ifns acting predominantly at mucosal surfaces (mordstein et al., 2010; durbin et al., 2013) . type i-and type iii-ifns use different receptors but trigger the same jak-stat signal transduction cascade involving tyk2, jak1, stat1, and stat2 albeit with different kinetics (figure 2 ) (marcello et al., 2006) . ultimately, stat1, stat2, and irf9 form a transcription factor complex, referred to as isgf3, which translocates to the nucleus and binds to ifn-stimulated responsive elements (isre) in the promoter of isgs (schindler et al., 2007) . type ii-ifn, uses a heterodimeric receptor consisting of ifnγr1 and ifnγr2 chains, expressed by many cell types (bach et al., 1997) . ifn-γ activates jak1, jak2 and stat1, leading to transcription of genes bearing a γ-activation sequence (gas) in their promoter (figure 2 ) (schindler et al., 2007) . mamps activate prrs of host cells such as epithelial cells and macrophages (figure 1) . infection induces a robust type i-ifn response. in mice, macrophages have been identified as the major source of ifn-β (stockinger et al., 2009) . in vitro, ifn-β production by bone-marrow-derived murine macrophages has been shown to require bacterial escape from the phagosome and activation of cytosolic surveillance pathways (o'riordan et al., 2002) . induction of ifn-β depends on the adaptor protein sting and the cytosolic prr ddx41, which are activated by bacterial secondary messengers c-di-amp and c-di-gmp and by bacterial dna (woodward et al., 2010; burdette et al., 2011; sauer et al., 2011; parvatiyar et al., 2012; archer et al., 2014) . sting is a direct receptor for cyclic-dinucleotides, including the cellular second messenger cyclic gmp-amp (cgamp) which is produced by the cytosolic sensor cgamp synthase (cgas) upon interaction with microbial dna (ablasser et al., 2013; gao et al., 2013; wu et al., 2013; sun et al., 2013; schoggins et al., 2014) . interestingly, type i-ifn production requires activation of the rig-i helicase by listeria rna in non-immune cells lacking a functional sting signaling pathway (abdullah et al., 2012; hagmann et al., 2013) . another cytosolic prr, the leucine-rich repeat-containing protein lrrfip1, has also been implicated in ifn-β production by mouse primary peritoneal macrophages in response to listeria infection, possibly by sensing double stranded dna and rna (yang et al., 2010) . while production of ifn-β in response to listeria infection is independent from tlrs in bone-marrowderived macrophages (mccaffrey et al., 2004; stockinger et al., 2004; o'connell et al., 2005) , tlr-2 contributes significantly to ifn-β secretion by peritoneal macrophages, suggesting that specific macrophage populations have evolved different recognition strategies in response to listeria infection (aubry et al., 2012) . listeria infection has recently been shown to induce type iii-ifn gene expression in cells of epithelial origin, such as intestinal and trophoblast cells and hepatocytes (lebreton et al., 2011; bierne et al., 2012) . similar to type i-ifn, type iii-ifn induction is triggered by intracellular listeria . listeria infection also triggers a rapid and robust ifn-γ response. after intravenous infection of mice with l. monocytogenes, nk and t cells are the main sources of ifn-γ (thale and kiderlen, 2005; bou ghanem et al., 2009) . ifn-γ producing v1δ + -γδ t cells are other murine immune cells induced at an early stage of listeria infection in mice inoculated intraperitoneally (hamada et al., 2008) . using oral infection of mice, the natural route of infection in permissive hosts, l. monocytogenes has been shown to induce ifn-γ production by intraepithelial lymphocytes of the small intestine (okamoto, 1994) . more recently, human e-cadherin (hecad) expressing mice, a mouse line permissive for listeria oral infection (lecuit et al., 2001) , were used to study cells involved in intestinal mucosal immunity. infection induced ifn-γ production in nk cells of the small intestine (reynders et al., 2011) . the production of ifn-γ by immune cells promotes bacterial clearance and is thus critical in controlling primary l. monocytogenes infections (zenewicz and shen, 2007) . injection of neutralizing monoclonal anti-ifn-γ antibodies in mice infected intraperitoneally with l. monocytogenes inhibits macrophage activation and increases the mortality rate (buchmeier and schreiber, 1985) . in addition, resistance of ifn-γ gene or ifn-γ receptor knock-out mice infected intravenously with l. monocytogenes is frontiers in cellular and infection microbiology www.frontiersin.org april 2014 | volume 4 | article 50 | 6 severely impaired harty and bevan, 1995) . recent work using cell-type specific inactivation of stat1 in mice elegantly demonstrated the key role of ifn-γ and stat1 in macrophage activation and clearance of listeria (kernbauer et al., 2012) . interestingly, the role of stat1 was extremely different after infection of immunized mice. stat1 signaling in t cells and dendritic cells was critical for adaptive immunity to listeria, while ifn-γ-activated macrophages were not essential anymore once memory cells were produced. upon oral infection of hecad mice with listeria, ifn-γ contributes to the control of bacterial burden in the intestine and of bacterial dissemination to other organs. for instance, blocking ifn-γ with neutralizing antibodies increases listeria load in the small intestine, the mesenteric lymph nodes and in the spleen of mice infected orally (reynders et al., 2011) . in contrast to ifn-γ, type i-ifn is beneficial to l. monocytogenes. mice lacking type i-ifn receptor or irf3 are more resistant to listeria intraperitoneal or intravenous infection (auerbuch et al., 2004; carrero et al., 2004; o'connell et al., 2004; garifulin et al., 2007; jia et al., 2009) . the role of type i-ifns in increasing host susceptibility could be explained by modulation of components of the immune response involved in controlling bacterial growth such as induction of t cell apoptosis, resulting in greater il-10 secretion by phagocytic cells, in turn dampening the innate immune response (carrero and unanue, 2006) , the downregulation of ifn-γr (rayamajhi et al., 2010; kearney et al., 2013) , or neutrophil recruitment (brzoza-lewis et al., 2012) . as shown recently, sting-dependent activation of type i-ifn reduces the adaptive immune response to l. monocytogenes (archer et al., 2014) . in contrast, recent studies showed that type i-ifns can also play a beneficial role for the host during listeria infection, pointing to the infection route and the timing of type i-ifn production as determinative factors (pontiroli et al., 2012; kernbauer et al., 2013) . interestingly, different strains of l. monocytogenes have been shown to vary greatly in their capacity to induce ifn-β (reutterer et al., 2008; schwartz et al., 2012) . the lo28 strain hyperinduces ifn-β (reutterer et al., 2008) . this strain bears a nonfunctional brta (also named tetr), the transcriptional repressor of the multidrug efflux pump mdrt (schwartz et al., 2012; yamamoto et al., 2012) . in listeria, mdrt allows secretion of cdi-amp, which triggers ifn-β. thus, derepression of mdrt in the lo28 strain promotes ifn-β production. of note, high expression of mdrt in lo28 correlates with both induction of ifn-β and lower virulence. another listeria multidrug resistance transporter, mdrm, has been involved in the stimulation of ifn-β production, possibly by secreting c-di-amp (crimmins et al., 2008; woodward et al., 2010; witte et al., 2012) . the role of type iii-ifns during listeriosis remains to be determined. since listeria colonizes several tissues of epithelial origins, such as the liver, intestine and placenta, it is tempting to speculate that ifn-λs play a role in the interaction of listeria with epithelia. however, a prerequisite to address this question is the establishment of a new animal model, i.e., a mouse line expressing a human e-cadherin, thus permissive for listeria infection of epithelia (lecuit et al., 2001) and impaired in type iii-ifn responses, such as il28rα knockout mice (mordstein et al., 2010) . one should keep in mind that the mouse model is not optimal to address the role of type iii-ifn in human listeriosis. indeed, ifn-λ1 is a pseudogene in mice, while human cells produce this cytokine upon infection with l. monocytogenes. in addition, the type iii-ifn receptor is expressed at very low levels in the mouse liver and the ifn-λ response of the mouse liver is very weak (mordstein et al., 2010) . in line with this, it has been recently shown that mouse hepatocytes, in contrast to human hepatocytes, are not responsive to ifn-λ (hermant et al., 2014) . the beneficial or detrimental effects of ifns on listeria infection rely on the functional properties of their downstream effectors. indeed, ifns elicit expression of hundreds of interferonstimulated genes (isgs), which encode proteins involved in a broad range of cellular functions (reviewed in macmicking, 2004) . however, while about 2,000 isgs have been identified so far (rusinova et al., 2012) , their functions in immunomodulation remain to be characterized. to date, the contribution of interferon-induced proteins on listeria infection has mostly been studied in the context of the ifn-γ pathway. the antilisterial activity of ifn-γ in phagocytic cells involves induction of oxidative and nitrosative defences, via increased expression of enzymes that control production of reactive oxygen and nitrogen species, such as nox2/cybb, duox2, and inos/nos2 (macmicking, 2012). these enzymes play an important role in protecting infected cells against listeria cytoinvasion (myers et al., 2003; lipinski et al., 2009) . the assembly of these enzymes requires ifn-γ-inducible guanosine triphosphatases (gtpases) of the gbp (guanylate binding protein) family (boehm et al., 1998) , which not only participate to oxidative pathways but also regulate autophagy (kim et al., 2011) . several gbps have been shown to protect cells from listeria infection by coordinating a potent oxidative and vesicular trafficking program (kim et al., 2011) . ifn-γ also induces the expression of many nuclear genes encoding mitochondrial respiratory chain machinery, via activation of the nuclear receptor errα (estrogen-related receptor α). errα contributes to mitochondrial ros production and efficient clearance of l. monocytogenes (sonoda et al., 2007) . a family of ifn-γ-induced chemokines (cxcl9, cxcl10, cxcl11) displays direct antimicrobial activity against l. monocytogenes (cole et al., 2001) . in dendritic cells, one of the ifn-γ-associated isgs is the immunoregulatory enzyme indoleamine 2,3-dioxygenase (ido), a key enzyme of the tryptophan metabolism. ido is proposed to play a role in the containment of listeria within granulomatous structures, thus avoiding massive t cell activation (popov et al., 2006) . the function of type i ifn-associated isgs in listeria infection is less documented. zwaferink et al. have observed that upregulation of inos/nos2 by ifn-β promotes necrotic death of macrophages (zwaferink et al., 2007) . additionally, several interferon-inducible proteins belong to inflammasomes; thus, type i ifn may potentiate inflammasome activation and cell death by pyroptosis (malireddi and kanneganti, 2013 ). yet, the link between these effectors and the observed harmful effects of type i ifns on the host is still unclear. likewise, the role of of interest, a subset of isgs is amongst the most induced genes in the intestinal tissue of gnotobiotic humanized mice infected orally with l. monocytogenes (archambaud et al., 2012) . however, which type of ifns triggers this response and for which function on the intestinal mucosa remain to be explored. in addition, ifn-independent pathways may contribute to expression of these isgs. listeria has evolved several mechanisms to avoid immune detection and evade ifn responses. it has been demonstrated that deacetylation of listeria peptidoglycan by the deacetylase pgda confers resistance to host lysozyme, thus preventing release of mamps, such as dna, rna and lipopeptides, that trigger ifnβ production (boneca et al., 2007) . listeria pgda mutants are rapidly killed in murine macrophages, which produce lysozyme, and induce a strong secretion of ifn-β compared to wildtype listeria. the role of pgda is not limited to the control of type i-ifn production as a pgda mutant hyperinduces proinflammatory cytokines as well. modification of peptidoglycan by pgda is an extremely efficient mechanism of immune escape used by listeria, which correlates with its critical role in virulence. remarkably, listeria has evolved a sophisticated strategy to modulate, either negatively or positively, the expression of isgs in epithelial cells, by targeting a chromatin-repressive complex, bahd1 (bierne et al., 2009; lebreton et al., 2011 lebreton et al., , 2012 . indeed, listeria infection promotes, albeit via an unknown mechanism, the targeting of bahd1 at the promoter of a set of isgs, thereby downregulating type i-and type iii-ifn responses. on the other hand, listeria can produce a nucleomodulin, lnta, which when secreted by intracellular bacteria, enters the nucleus of infected cells, binds bahd1 and inhibits its function (lebreton et al., , 2014 . thus, lnta stimulates ifn responses. consistent with the presence of hdac1/2 in the bahd1-associated complex, the level of acetylation of lysine 9 on histone h3, which is a mark of active chromatin, increases at the promoters of isgs in the presence of lnta. when, in which host conditions, and how lnta targets bahd1 specifically at isgs remains an open question. the lnta-mediated stimulation of type iii-ifn responses might support localized pro-bacterial conditions, as was proposed for ifn-i responses. we have an extensive knowledge of the molecular and cellular mechanisms involved in listeria-host interactions. yet, our understanding of the immune response to listeria, and more specifically the role ifns and of their downstream effectors, is far from complete and often relies on studies performed in cultured cells or in mice. however, murine and human listeriosis differ in many aspects (lecuit, 2007; hoelzer et al., 2012) . for instance, e-cadherin, the major receptor for listeria in epithelial cells, is not functional for listeria uptake in the mouse. thus, the route of entry of listeria is not strictly the same in mice and humans. moreover, isgs induced in response to infection are not identical in mice and humans. additionally, murine hepatocytes do not respond to type iii-ifns (hermant et al., 2014) , precluding the study of these ifns during infection by human hepatotropic pathogens, such as l. monocytogenes. altogether, species-specific differences provide limits to the use of mouse models in characterizing ifn pathways engaged during listeria infection in humans, especially in key epithelial organs such as the gut, liver and placenta. it will be important to perform future studies using adapted animal models, such as humanized mice permissive to oral infection or transgenic mice with human xenografts (walters et al., 2006) , since the effect of type i-ifn on listeria infection depends on the route and time of infection (pontiroli et al., 2012; kernbauer et al., 2013) and type iii-ifn requires bacterial interaction with epithelia . finally, numerous isgs are induced upon listeria infection in vitro, but the relevant isgs and their cellular functions remain to be identified. validation of isgs identified in cultured cells in adequate in vivo models or deduced from analyses of patient samples, will be required to address the complex role of ifns and bacterial subversion strategies and provide new insights into listeria pathogenesis. rig-i detects infection with live listeria by sensing secreted bacterial nucleic acids cgas produces a 2 -5 -linked cyclic dinucleotide second messenger that activates sting impact of lactobacilli on orally acquired listeriosis sting-dependent type i ifn production inhibits cell-mediated immunity to listeria monocytogenes both tlr2 and trif contribute to interferon-β production during listeria infection oata, a peptidoglycan o-acetyltransferase involved in listeria monocytogenes immune escape, is critical for virulence mice lacking the type i interferon receptor are resistant to listeria monocytogenes the ifn gamma receptor: a paradigm for cytokine receptor signaling human bahd1 promotes heterochromatic gene silencing activation of type iii interferon genes by pathogenic bacteria in infected epithelial cells and mouse placenta two families of gtpases dominate the complex cellular response to ifn-gamma a critical role for peptidoglycan n-deacetylation in listeria evasion from the host innate immune system multiple mechanisms contribute to the robust rapid gamma interferon response by cd8+ t cells during listeria monocytogenes infection type i interferon signaling regulates the composition of inflammatory infiltrates upon infection with listeria monocytogenes requirement of endogenous interferon-gamma production for resolution of listeria monocytogenes infection sting is a direct innate immune sensor of cyclic di-gmp the arsenal of virulence factors deployed by listeria monocytogenes to promote its cell infection cycle lymphocyte apoptosis as an immune subversion strategy of microbial pathogens confounding roles for type i interferons during bacterial and viral pathogenesis type i interferon sensitizes lymphocytes to apoptosis and reduces resistance to listeria infection listeriosis outbreaks and associated food vehicles listerial invasion protein internalin b promotes entry into ileal peyer's patches in vivo cutting edge: ifn-inducible elr − cxc chemokines display defensinlike antimicrobial activity listeria monocytogenes infection in the face of innate immunity illuminating the landscape of host-pathogen interactions with the bacterium listeria monocytogenes listeria monocytogenes multidrug resistance transporters activate a cytosolic surveillance pathway of innate immunity multiple defects of immune cell function in mice with disrupted interferon-gamma genes myd88 and sting signaling pathways are required for irf3-mediated ifn-β induction in response to brucella abortus infection the yin and yang of type i interferon activity in bacterial infection conjugated action of two species-specific invasion proteins for fetoplacental listeriosis listeria and autophagy escape: involvement of inlk, an internalin-like protein interferon induction and function at the mucosal surface the role of genomic data in the discovery, annotation and evolutionary interpretation of the interferon-lambda family listeria monocytogenesfrom saprophyte to intracellular pathogen gastrointestinal phase of listeria monocytogenes infection cyclic gmp-amp synthase is an innate immune sensor of hiv and other retroviruses irf3 polymorphism alters induction of interferon beta in response to listeria monocytogenes infection recent developments in the transcriptional regulation of cytolytic effector cells control points in nkt-cell development the listeria monocytogenes inlc protein interferes with innate immune responses by targeting the iκb kinase subunit ikkα incidence of listeriosis and related mortality among groups at risk of acquiring listeriosis rig-i detects triphosphorylated rna of listeria monocytogenes during infection in non-immune cells importance of murine vdelta1 gammadelta t cells expressing interferon-gamma and interleukin-17a in innate protection against listeria monocytogenes infection interferon lambda 4 signals via the ifn-λ receptor to regulate antiviral activity against hcv and coronaviruses histone modifications induced by a family of bacterial toxins specific immunity to listeria monocytogenes in the absence of ifn gamma 25 years of interferon-based treatment of chronic hepatitis c: an epoch coming to an end type i ifn signaling constrains il-17a/f secretion by gammadelta t cells during bacterial infections human but not mouse hepatocytes respond to interferon-lambda in vivo animal models of listeriosis: a comparative review of the current state of the art and lessons learned chronic granulomatous disease type i interferon gene induction by the interferon regulatory factor family of transcription factors irf-7 is the master regulator of type-i interferon-dependent immune responses cross-regulation of signaling pathways by interferon-gamma: implications for immune responses and autoimmune diseases immune response in mice that lack the interferon-gamma receptor virus interference. i. the interferon mechanisms of type iii interferon expression myd88 and type i interferon receptor-mediated chemokine induction and monocyte recruitment during listeria monocytogenes infection interferon-gamma-receptor deficiency in an infant with fatal bacille calmette-guérin infection il-12 and ifn-gamma in host defense against mycobacteria and salmonella in mice and men mice that lack the interferon-gamma receptor have profoundly altered responses to infection with bacillus calmette-guérin and subsequent challenge with lipopolysaccharide demonstration of an interferon gamma-dependent tumor surveillance system in immunocompetent mice type i ifns downregulate myeloid cell ifn-gamma receptor by inducing recruitment of an early growth response 3/ngfi-a binding protein 1 complex that silences ifngr1 transcription route of infection determines the impact of type i interferons on innate immunity to listeria monocytogenes conditional stat1 ablation reveals the importance of interferon signaling for immunity to listeria monocytogenes infection a family of ifn-gamma-inducible 65-kd gtpases protects against bacterial infection ifn-λs ifn-λs mediate antiviral protection through a distinct class ii cytokine receptor complex t-bet: a bridge between innate and adaptive immunity bacteria tune interferon responses by playing with chromatin structural basis for the inhibition of the chromatin repressor bahd1 by the bacterial nucleomodulin lnta a bacterial protein targets the bahd1 chromatin complex to stimulate type iii interferon response human listeriosis and animal models a transgenic model for listeriosis: role of internalin in crossing the intestinal barrier induction and function of type i and iii interferon in response to viral infection duox2-derived reactive oxygen species are effectors of nod2-mediated antibacterial responses listeriosis cellular resistance to infection ifn-inducible gtpases and immunity to intracellular pathogens interferon-inducible effector mechanisms in cellautonomous immunity role of type i interferons in inflammasome activation, cell death, and disease during microbial infection type i ifn signaling is crucial for host resistance against different species of pathogenic bacteria regulation of mouse nk cell development and function by cytokines interferons α and λ inhibit hepatitis c virus replication with distinct signal transduction and gene regulation kinetics staphylococcus aureus activates type i ifn signaling in mice and humans through the xr repeated sequences of protein a a specific gene expression program triggered by gram-positive bacteria in the cytosol induction of type i interferons by bacteria what have we learned from the il28 receptor knockout mouse? localized reactive oxygen and nitrogen intermediates inhibit escape of listeria monocytogenes from vacuoles in activated macrophages whatever turns you on: accessory-celldependent activation of nk cells by pathogens a mutation in the interferon-gamma-receptor gene and susceptibility to mycobacterial infection transcytosis of listeria monocytogenes across the intestinal barrier upon specific targeting of goblet cell accessible e-cadherin type i interferon production enhances susceptibility to listeria monocytogenes infection immune activation of type i ifns by listeria monocytogenes occurs independently of tlr4, tlr2, and receptor interacting protein 2 but involves tnfr-associated nf kappa b kinase-binding kinase 1 host defense and endogenous interferon-gamma in the intestines during an oral infection with listeria monocytogenes gastroenteritis due to listeria monocytogenes innate recognition of bacteria by a macrophage cytosolic surveillance pathway the enhanceosome an atomic model of the interferon-beta enhanceosome the helicase ddx41 recognizes the bacterial secondary messengers cyclic di-gmp and cyclic di-amp to activate a type i interferon immune response opposing roles for interferon regulatory factor-3 (irf-3) and type i interferon signaling during plague listeria monocytogenes invades the epithelial junctions at sites of cell extrusion the stress-induced virulence protein inlh controls interleukin-6 production during murine listeriosis the interferons: 50 years after their discovery, there is much more to learn interferons, interferon-like cytokines, and their receptors structural and dynamic determinants of type i interferon receptor assembly and their functional interpretation inhibition of dynamin-dependent endocytosis interferes with type iii ifn expression in bacteria-infected human monocyte-derived dcs the timing of ifnβ production affects early innate responses to listeria monocytogenes and determines the overall outcome of lethal infection indoleamine 2,3-dioxygenase-expressing dendritic cells form suppurative granulomas following listeria monocytogenes infection a variant upstream of ifnl3 (il28b) creating a new interferon gene ifnl4 is associated with impaired clearance of hepatitis c virus type i ifns enhance susceptibility to chlamydia muridarum lung infection by enhancing apoptosis of local macrophages mutations of the listeria monocytogenes peptidoglycan n-deacetylase and o-acetylase result in enhanced lysozyme sensitivity, bacteriolysis, and hyperinduction of innate immune pathways antagonistic crosstalk between type i and ii interferons and increased host susceptibility to bacterial infections type i ifn are host modulators of strain-specific listeria monocytogenes virulence identity, regulation and in vivo function of gut nkp46(+)rorγt(+) and nkp46(+)rorγt(-) lymphoid cells type i interferon induces necroptosis in macrophages during infection with salmonella enterica serovar typhimurium listeria monocytogenes and recurrent mycobacterial infections in a child with complete interferon-gamma-receptor (ifngammar1) deficiency: mutational analysis and evaluation of therapeutic options interferome v2.0: an updated database of annotated interferon-regulated genes creb, atf, and ap-1 transcription factors regulate ifn-gamma secretion by human t cells in response to mycobacterial antigen the enu-induced goldenticket mouse mutant reveals an essential function of sting in the in vivo interferon response to listeria monocytogenes and cyclic-di-nucleotides jak-stat signaling: from interferons to cytokines epidemic listeriosis: evidence for transmission by food regulation of interferon-γ during innate and adaptive immune responses pan-viral specificity of ifn-induced genes reveals new roles for cgas in innate immunity a diverse range of gene products are effectors of the type i interferon antiviral response interferon-gamma: an overview of signals, mechanisms and functions hyperinduction of host beta interferon by a listeria monocytogenes strain naturally overexpressing the multidrug efflux pump mdrt the role of the activated macrophage in clearing listeria monocytogenes infection il-28, il-29 and their class ii cytokine receptor il-28r monocyte recruitment during infection and inflammation nuclear receptor err-alpha and coactivator pgc-1beta are effectors of ifn-gamma-induced host defense cell biology and immunology of listeria monocytogenes infections: novel insights type i interferons in host defense characterization of the interferon-producing cell in mice infected with listeria monocytogenes ifn regulatory factor 3-dependent induction of type i ifns by intracellular bacteria is mediated by a tlr-and nod2-independent mechanism cyclic gmp-amp synthase is a cytosolic dna sensor that activates the type i interferon pathway the epidemiology of human listeriosis. microbes infect the feedback phase of type i interferon induction in dendritic cells requires interferon regulatory factor 8 sources of interferon-gamma in early immune response to listeria monocytogenes the listeria transcriptional landscape from saprophytism to virulence type i interferon: friend or foe? genetic deficiencies of innate immune signalling in human infectious disease characterization of the murine alpha interferon gene family listeria pathogenesis and molecular virulence determinants host-specific response to hcv infection in the chimeric scidbeige/alb-upa mouse model: role of the innate antiviral immune response nod1 contributes to mouse host defense against helicobacter pylori via induction of type i ifn and activation of the isgf3 signaling pathway circulating virus, interferon and antibody after vaccination with the 17-d strain of yellow-fever virus decoding the complexity of type i interferon to treat persistent viral infections epigenetic control of t-helpercell differentiation innate immune pathways triggered by listeria monocytogenes and their role in the induction of cell-mediated immunity c-di-amp secreted by intracellular listeria monocytogenes activates a host type i interferon response cyclic gmp-amp is an endogenous second messenger in innate immune signaling by cytosolic dna listeria monocytogenes strain-specific impairment of the tetr regulator underlies the drastic increase in cyclic di-amp secretion and beta interferoninducing ability the cytosolic nucleic acid sensor lrrfip1 mediates the production of type i interferon via a β-catenin-dependent pathway innate and adaptive immune responses to listeria monocytogenes: a short overview interferon-λs: special immunomodulatory agents and potential therapeutic targets stimulation of inducible nitric oxide synthase expression by beta interferon increases necrotic death of macrophages upon listeria monocytogenes infection conflict of interest statement: the authors declare that the research was con the authors declare no conflict of interest. the authors' work has been supported in part by the institut pasteur, inserm, inra, université paris-diderot, labex ibeid, anr (grant epilis), french ligue nationale contre le cancer, fondation louis-jeantet, fondation le roch, and european research council (advanced grant award 233348 to pascale cossart). pascale cossart is an international research scholar of the howard hughes medical institute. key: cord-000018-amvlm09p authors: pauli, eva-k.; schmolke, mirco; wolff, thorsten; viemann, dorothee; roth, johannes; bode, johannes g.; ludwig, stephan title: influenza a virus inhibits type i ifn signaling via nf-κb-dependent induction of socs-3 expression date: 2008-11-07 journal: plos pathog doi: 10.1371/journal.ppat.1000196 sha: doc_id: 18 cord_uid: amvlm09p the type i interferon (ifn) system is a first line of defense against viral infections. viruses have developed various mechanisms to counteract this response. so far, the interferon antagonistic activity of influenza a viruses was mainly observed on the level of ifnβ gene induction via action of the viral non-structural protein 1 (ns1). here we present data indicating that influenza a viruses not only suppress ifnβ gene induction but also inhibit type i ifn signaling through a mechanism involving induction of the suppressor of cytokine signaling-3 (socs-3) protein. our study was based on the observation that in cells that were infected with influenza a virus and subsequently stimulated with ifnα/β, phosphorylation of the signal transducer and activator of transcription protein 1 (stat1) was strongly reduced. this impaired stat1 activation was not due to the action of viral proteins but rather appeared to be induced by accumulation of viral 5′ triphosphate rna in the cell. socs proteins are potent endogenous inhibitors of janus kinase (jak)/stat signaling. closer examination revealed that socs-3 but not socs-1 mrna levels increase in an rnaand nuclear factor kappa b (nf-κb)-dependent but type i ifn-independent manner early in the viral replication cycle. this direct viral induction of socs-3 mrna and protein expression appears to be relevant for suppression of the antiviral response since in socs-3 deficient cells a sustained phosphorylation of stat1 correlated with elevated expression of type i ifn-dependent genes. as a consequence, progeny virus titers were reduced in socs-3 deficient cells or in cells were socs-3 expression was knocked-down by sirna. these data provide the first evidence that influenza a viruses suppress type i ifn signaling on the level of jak/stat activation. the inhibitory effect is at least in part due to the induction of socs-3 gene expression, which results in an impaired antiviral response. influenza a viruses are negative-stranded rna viruses that belong to the family of orthomyxoviruses. the segmented genome of influenza a virus encodes for up to 11 viral proteins. as many other viruses, influenza viruses have evolved strategies to counteract cellular antiviral responses, especially to circumvent the type i ifn system as a first line of defense against the pathogenic invader. among the influenza viral proteins, the ns1 has been identified as the main type i ifn antagonistic factor. so far two major mechanisms have been described by which ns1 suppresses the initial expression of ifnb. on the one hand ns1 inhibits vrnamediated induction of the transcription factors interferon regulatory factor-3 (irf-3), activating protein-1 (ap-1) and nf-kb that target the ifnb promoter. this most likely occurs via binding to the rna-sensor retinoic acid inducible gene (rig-i) and inhibition of rig-i-mediated signaling in response to viral rna [1, 2] . on the other hand ns1 inhibits maturation [3, 4] and nuclear export of host mrnas [5] . other functions of the multifunctional protein include block of activation of the dsrnaactivated protein kinase pkr by direct interaction [6] or activation of the phosphatidylinositol-3 kinase pi3k/akt pathway to prevent premature apoptosis induction [7, 8] . while the ns1-mediated antagonistic activities of influenza viruses mainly affect the induction of genes such as ifnb, so far no viral suppression of ifn signaling has been described. ifn are among the first molecules synthesized in response to viral infections [9] . the ifn family includes three classes. type i comprises the well known ifna and ifnb. the only member of type ii ifn is ifnc. type iii ifn comprises ifnl1, -l2, and -l3. all classes of ifn bind to different receptors and are structurally not related [10, 11] . type i ifn belong to the key cytokines produced by influenza a virus-infected epithelial cells [12, 13] . the antiviral activity of type i ifn is mediated by a set of ifn-induced genes (isgs). binding of ifna/b to its receptor is the initial step in this signaling process, followed by activation of the jak family and subsequent activation of stat proteins [14] . ligand binding leads to dimerisation of the type i ifn receptor subunits ifnar1 and ifnar2 and causes their conformational change. the jak kinase tyk2, which is constitutively bound to ifnar1, phosphorylates the receptor at tyrosine residues and creates a docking site for stat2. subsequently, tyk2 phosphor-ylates stat2 at y690. at the same time the receptor-bound jak1 phosphorylates stat1 at y701 [15, 16] . the phosphorylated transcription factors dimerise and bind to irf-9 [17] . the newly formed heterotrimer, called ifnstimulated gene factor 3 (isgf3), translocates into the nucleus and binds to ifn-stimulated response elements (isre), to initiate gene transcription of isgs. treatment of cells with type i ifn upregulates expression of an array of genes including sp110, irf-1 and many others [18] . among these isgs the 29, 59oligoadenylate synthetase 1 (oas1), the mx proteins and the dsrna-activated protein kinase (pkr) are described to directly interfere with viral replication [19] . both, pkr and the oas1/ rnasel system are capable of inhibiting cellular and viral translation. ifn-induced jak/stat signaling can be inhibited at different levels by several viral and cellular factors through various mechanisms. the large t-antigen of murine polyomavirus (mpyv) binds to jak1 and inhibits downstream signaling [20] , whereas the vp24 of ebola virus (ebov) binds to karyopherina-1 thereby blocking nuclear accumulation of stat1 [21] . endogenous cellular key regulators, capable of negatively regulating jak/stat-mediated signal transduction, include suppressor of cytokine signaling (socs) proteins, protein tyrosine phosphatases (ptp) and protein inhibitor of activated stats (pias). the family of socs proteins comprises eight members (cytokine-inducible sh2 domain-containing protein (cis) and socs1-7). all members contain a central sh2 domain, an nterminus of variable length and sequence and a c-terminal 40 amino-acid module called socs box [22] . the socs box is necessary for recruitment of the ubiquitin transferase system and for stabilization and/or degradation of socs proteins [23] [24] [25] . the n-terminus contains a kinase inhibitory region (kir), which functions as pseudo substrate for the jak [26] . socs-1 and socs-3 differ in their mode of action. for inhibition of the kinase activity of jaks, socs-1 binds directly to the activation loop of jaks [26] [27] [28] . in contrast, socs-3 first binds to the receptor [29, 30] . induction of socs-3 gene transcription by viruses was reported for hsv-1, hcv [31] [32] [33] and for respiratory viruses, such as sars and rsv [34, 35] . the level of induction of socs-3 by hsv-1 seems to determine whether infection turns to acute or persistent progression [31] . for hcv it has been suggested that upregulation of socs-3 may contribute to the non-responsiveness of hcv patients to ifn therapy [33, [36] [37] [38] . elevated socs-3 mrna levels during rsv infection were linked to th2 cell-mediated immune disease as atopic dermatitis and asthma [39, 40] . in the present study we show that influenza a virus can be added to the list of viruses that induce socs-3 expression. the protein functionally interferes with viral replication by providing a virus-supportive ifn-antagonistic activity on the level of type i ifn-signaling that has not been described so far. phosphorylation of stat1 and stat2 by members of the jak tyrosine kinase family is a prerequisite for activation of these transcription factors to drive type i ifn-induced gene expression. therefore, we analyzed whether stat phosphorylation patterns are altered in influenza a virus infected cells that were stimulated with ifn at different time points post infection (p.i.). the human alveolar epithelial cell line a549 was infected with the influenza a virus strain a/puerto-rico/8/34 (h1n1) (pr8) ( figure 1a ). cells were subsequently stimulated with ifnb at given time-points p.i. and stat phosphorylation was assessed in western blots. both stat1 and stat2 were readily phosphorylated upon cytokine stimulation in uninfected cells or in infected cells up to 4 h p.i. ( figure 1a ). furthermore, virus infection alone resulted in a significant induction of stat phosphorylation 4-6 h p.i., presumably caused by virus-induced ifn expression. however, at later time points (6-10 h p.i.), in a549 cells both virus-and ifn-induced stat1 and stat2 phosphorylation was markedly reduced ( figure 1a ). similar patterns were observed upon stimulation of cells with ifna or upon infection with other viruses, such as the human influenza virus a/victora/3/75 (h3n2) (data not shown). in addition, this phenomenon could also be detected in other epithelial cells such as the human embryonic kidney cell line hek293 ( figure 2e ) or the human umbilical vein endothelial cells (huvec) ( figure s1b ). inhibition was not caused by indirect disturbing effects on cellular metabolism or enzyme activities due to ongoing virus replication, since ifnc-induced stat1 phosphorylation was not affected at all ( figure 1c ). finally, involvement of any auto-or paracrine action of virus-induced type i ifn could be ruled out, as the inhibitory effect was also observed in vero cells lacking functional type i ifn genes ( figure 1e ). with regard to the molecular basis of impaired ifna/b-induced stat phosphorylation in infected cells it was striking that the inhibitory effect correlated with the accumulation of viral proteins, as monitored in pb1 western blots ( figures 1a and 1e) . thus, the question arose whether individual expression of viral proteins may result in the interference with stat1 phosphorylation. out of the 11 viral proteins of pr8 we choose the nucleoprotein (np), the ns1 protein, the matrix protein (m1) (figure 2a ) and the subunits of the viral polymerase, pa, pb1 and pb2 ( figure 2c ), for a representative experiment. these proteins are known to bind to vrna/rnps or to interfere with the rna-mediated innate immune response. for efficient transfection of the expression the type i interferon (ifn) system is one of the most powerful innate defenses against viral pathogens. most rna viruses are sensitive to the action of type i ifn. therefore, these pathogens have evolved strategies to evade this response. for example, influenza viruses express a viral protein, the non-structural protein 1 (ns1), that suppresses production of ifnb by lowering cellular sensitivity to viral nucleic acid as a pathogen pattern. here we present data indicating that influenza a viruses are not only capable of suppressing production of the ifnb gene but also inhibit action of this antiviral cytokine on cells. this occurs by viral induction of a cellular protein, the suppressor of cytokine signaling (socs)-3, a potent endogenous inhibitor of ifn signaling. this is a novel mechanism by which influenza viruses inhibit the antiviral response of the host and paves the path to efficient virus replication. this may be especially relevant for influenza viruses that induce high cytokine responses (cytokine burst), such as highly pathogenic avian influenza viruses of the h5n1 subtype. induction of socs-3 expression would allow efficient replication despite high ifn and cytokine levels. constructs we used the highly susceptible cell line hek293 that also exhibits impaired ifnb-induced stat1 phosphorylation at later stages of infection ( figure 2e ). 24 h post transfection cells were stimulated with ifnb and stat phosphorylation was monitored in western blots (figures 2a and c) . expression of none of the viral proteins resulted in a significant decrease of ifnb-induced stat1 or stat2 phosphorylation (figures 2a and 2c ). similar results were obtained in the human bronchial epithelial cell line h1299 when expressing m1, ns1 or np alone or in different combinations (data not shown). thus, we concluded that viral proteins most likely do not play a prominent role as blockers of ifna/b-induced jak/stat signaling. decrease of stat phosphorylation might also be due to the action of virus-induced phosphatases. on the one hand these enzymes may cause direct dephosphorylation of stat proteins. on the other hand phosphatases could act via an indirect mechanism by dephosphorylation and inactivation of jaks resulting in an attenuated phosphorylation of stats. several protein tyrosine phosphatases (ptps) are known to mediate dephosphorylation of both, jaks and stats [41] . in order to investigate whether influenza a virus activates phosphatases that subsequently target jaks or stats, we treated infected or uninfected a549 cells with the well-known tyrosine phosphatase inhibitor sodium vanadate [42, 43] . uninfected cells or cells infected with pr8 for 10 h were incubated with increasing amounts of this compound 10 min prior to stimulation with ifnb. this time point of infection was chosen since we observed considerable inhibition of ifn-induced stat1 phosphorylation in the course of infection ( figures 1a and 1e ). increasing concentrations of vanadate lead to a gradual shift of the steady state balance of phosphorylation/dephosphorylation. accordingly, a gradual increase of stat1 phosphorylation was observed that was similar in both infected and uninfected cells, albeit starting from different basal levels of phospho-stat1 ( figures 3a and b ). this is illustrated by an almost identical slope of the regression line in the graphical analysis of the band intensities of the ifnb stimulated samples ( figure 3b ). if the blockade of ifnb-induced stat1 phosphorylation would be mediated by specific virus-activated phosphatases, a much steeper slope for vanadate-treated infected cells would be expected. however, the result in figure 3b indicates that the virus-induced suppression of phosphorylation is not compensated by phosphatase inhibition and consequently no virusactivated phosphatase appears to be involved. in support of these data, phosphatase assays revealed that the overall activity of tyrosine phosphatases in infected cells was not elevated compared to uninfected cells. this is indicated by constant levels of free phosphates released from two different phospho-peptides that represent common tyrosine phosphatase substrates ( figure 3c ). thus, involvement of phosphatases in influenza virus-induced alteration of stat1 phosphorylation can be greatly ruled out. phosphorylation of stats in the ifnb signaling cascade may not only be counter-regulated by phosphatases but also by other cellular factors, such as proteins of the suppressors of cytokine signaling (socs) family. action of these proteins is mainly controlled on the level of transcriptional activation. socs proteins are described to have high affinity for jak and stat proteins and to inhibit the transmission of ifna and ifnb induced signaling [44, 45] . to examine whether expression of socs genes is induced in influenza virus infected cells, a549 cells were infected with pr8 for different time points. subsequently total rna was analyzed for the amount of socs-1 and socs-3 mrna by means of quantitative real time-pcr (qrt-pcr). the mrna table 1 for accession numbers of viral genes) using l2000 according to manufacturer's instructions. note that the pol ii constructs in use also give rise to expression of second reading frames in the ns, m and pb1 genes (ns2, m2, pb1-f2). 48 h post transfection cells were stimulated with human ifnb (500 u/ml) for 15 minutes. total protein lysates were subjected to western blot analysis using anti-phospho-stat1, anti-phospho-stat2, anti-stat1 antibodies. expression of influenza viral proteins was monitored with antibodies against np, m1, ns1, pa, pb1 or pb2. (e) hek293 cells were infected with the human influenza virus pr8 (h1n1) (moi = 5) for the indicated time points and were subsequently stimulated for 15 min with either human ifnb at a concentration of 100 u/ml. cell lysates were subjected to western blots as described. (b, d, f) quantification of relative pstat1 and pstat2 band intensities in a, c and e using aida software and 2d densitometry (fuji). doi:10.1371/journal.ppat.1000196.g002 levels of socs-1 and socs-3 differed notably in the time course ( figure 4a ). while socs-3 mrna is strongly and transiently elevated in the early phases of infection, socs-1 gene transcription is only significantly induced 15 h p.i.. elevated socs-3 mrna levels were also observed in other host cell types, such as huvec starting 3 h p.i. ( figure s1a ). although elevation of socs-3 mrna levels in infected cells was rather transient, there appears to be a robust induction on protein level ( figure 4c ). first detected at 4 h p.i., socs-3 protein levels increased and stayed on a high level throughout the observation period. strikingly, expression kinetics of the socs-3 protein perfectly matched the kinetics of virus-induced inhibition of stat1 phosphorylation ( figure 4c ), indicating that both processes are functionally linked. virus mediated socs-3 gene induction at early stages of infection ( figures 4a, 4c and s1a) appeared to occur concomitant with an immediate and strong induction of ifnb ( figures 4b and s1d) . this prompted us to analyze whether socs-3 transcription might be induced due to an auto-or paracrine action of ifnb expressed during infection. a549 cells were stimulated with ifnb for different time points and socs-3 gene induction was measured by qrt-pcr ( figure 4d ). as a control we monitored expression of 29, 59oligoadenylate synthetase (oas1) and mxa, genes that are typically induced by ifnb. while oas1 and mxa mrnas were readily upregulated upon ifnb treatment socs-3 mrna was not significantly elevated ( figure 4d ). similar results were obtained from huvec stimulated with ifnb ( figure s1e ). to further confirm these results we knocked down the ifnar in a549-cells by an sirna approach. although the knock down was efficient and leads to more than 60% inhibition of ifnb induced stat1 phosphorylation ( figure 4e ), the induction of socs-3 expression was not impaired ( figure 4f ). socs-3 levels in the knock down cells were similar compared to wild type cells and even higher than in the vector control ( figure 4f ). these results are consistent with data gained from previous experiments in vero cells ( figure 1e ) and indicate that neither induction of socs-3 mrna nor inhibition of stat phosphorylation is dependent on virus-induced type i ifn expression. since accumulation of viral rna in infected cells is a potent inducer of antiviral gene expression we investigated its ability to induce socs-3 gene transcription. as a source for viral rna, a549 cells were infected with influenza a virus for 10 h and total rna from these cells was isolated. rna from uninfected a549 cells served as a negative control. different amounts of these rnas were used for stimulation of a549 cells for 3 h (figures 5a, 5b and 5c). transfection of rna from uninfected cells did not result in an increase of socs-1 or socs-3 gene transcription ( figure 5a ) or ifnb induction as a control ( figure 5b ). however, transfection of rna from virally infected cells led to strongly elevated socs-3 mrna amounts while socs-1 mrna is only induced weakly ( figure 5a ). this dose dependent induction of socs-3 by stimulation with increasing amounts of rna from infected cells corresponds with a gradual decrease in the ability of this rna to induce or potentiate stat1/2 phosphorylation ( figure 5c ). in contrast to cellular rna, influenza viral rna carries a triphosphate group at its 59 terminus that was previously shown to be a major pathogen pattern that triggers cellular signaling [46] . to verify that indeed the viral 59 triphosphate rna in the pool of rnas from infected cells is the major trigger for induction of socs-3 expression, rna from infected or uninfected cells was treated with phosphatase to remove the 59 triphosphate termini prior to stimulation of a549 cells ( figure 5e and 5f). the dephosphorylated viral rna was only poorly capable to induce socs-3 ( figure 5e ) or ifnb ( figure 5f ) mrna expression. in addition, poly(i:c) was transfected to mimic action of double-stranded (ds) rna ( figure 5e and 5f, right bars). however, the dsrna analog showed surprisingly little effects on socs-3 and ifnb mrna induction. since viral rna is able to induce ifnb gene transcription ( figure 5b and 5f) we again wanted to rule out that induction of socs-3 by viral 59 triphosphate rna is mediated by auto-or paracrine action of de novo synthesized ifnb. in order to do so, cells were stimulated with viral rna after treatment with the protein synthesis inhibitor anisomycin at two different concentrations ( figure 5g ). socs-3 mrna was still induced to the same extent in the presence of the protein synthesis inhibitor, providing the ultimate proof that de novo protein synthesis is not required for socs-3 induction. so far, our data suggest that influenza virus-induced transcriptional upregulation of the socs-3 gene is not mediated by the . equivalent mrna amounts were normalized to gapdh mrna levels and calculated as n-fold of the levels of untreated cells that were arbitrarily set as 1. to detect socs-3 protein expression (c) cells were infected for time points indicated or left uninfected. total cell lysate was subjected to western blot analysis using anti-socs-3 antibody. to allow better comparison of socs-3 protein expression and stat1 phosphorylation phospho-stat1 and stat1 western blots from figure 1a are shown again here. (e) to functionally test effective knock down of the ifnar, a459 wild type, a549 vector control cells or a549 cells stably expressing ifnar ii-1specific shrna were stimulated with human ifnb (100 u/ml) for 15 min. subsequently cells were lysed and levels of phospho-stat1 were determined by western blotting using specific antibodies. in addition, the relative pstat1 band intensities were quantified. doi:10.1371/journal.ppat.1000196.g004 autoregulatory action of type i ifns ( figure 4d and 4f) but is directly induced through accumulation of viral rna during infection. this raises the question, which rna-induced signaling pathways are responsible for socs-3 expression. the mkk/p38 mitogen activated protein kinase (mapk) pathway [47] [48] [49] as well as the ikb kinase (ikk)/nuclear factor of kb (nf-kb) cascade [50] [51] [52] are both known to be activated by rna or influenza virus infection and to be involved in the control of socs-3 expression. to assess whether the mkk6/p38-or the ikk/nf-kb-module is required for socs-3 gene induction, we generated a549 cell lines expressing dominant negative forms of either mkk6 (mkk6ala) or ikk2 (ikk2kd) (figure 6a to 6d). these mutants have been previously shown to efficiently block p38 or nf-kb signaling, respectively [52] [53] [54] . to monitor socs-3 gene induction, wild type, vector or mutant expressing cell lines were infected with pr8 ( figure 6a ) or stimulated with rna from virally infected or uninfected a549 cells ( figure 6c ). induction of ifnb mrna was monitored as a control ( figure 6b and 6d) . while mkk6ala expression did not result in significant reduction of socs-3 in either infected ( figure 6a ) or rna-stimulated cells ( figure 6c ), transcription is markedly reduced in ikk2kd expressing cell lines. to obtain independent evidence for nf-kb dependence of socs-3 gene transcription, a549 wild type cells were incubated with the nf-kb specific inhibitor bay 11-7085 prior to stimulation with rna from virally infected or uninfected a549 cells ( figure 6e ). again, ifnb mrna levels were assessed for control purposes ( figure 6f ). both, socs-3 and ifnb mrna levels were strongly reduced in bay 11-7085 treated cells. this indicates that virus-induced socs-3 expression strongly depends on ikk2 and nf-kb activation, while the mkk6/p38 appears not to play a prominent role. to further verify that influenza virus induces socs-3 via an rna sensory pathway and in an nf-kb dependent manner we infected cells with the influenza a virus mutant deficient for ns1 (dns1) (figure 6g and 6h) . the ns1 protein is known to block rna dependent signaling and nfkb activation [55] . accordingly, infection of cells with the mutant virus resulted in a more pronounced and sustained, albeit delayed induction of socs-3 ( figure 6g ) if compared to infection with the isogenic wild type, that is a very poor inducer of socs-3 but still reasonably well induces ifnb. noteworthy, this isogenic wild type strain differs from the pr8 wild type virus used in the other experiments shown here (see materials and methods for details). to analyze whether nf-kb activation is sufficient for socs-3 gene induction we stimulated cells with il-1b ( figure s2a ) or tnfa ( figure s2b ) that are both strong activators of the transcription factor. while mrna levels of il-6, a strictly nf-kb dependent cytokine, are strongly elevated, socs-3 gene transcription is not significantly induced. under the assumption that these cytokines do not additionally induce counteracting processes one can conclude that nf-kb is required, yet not sufficient for the induction of socs-3. thus viral induction of socs-3 may require additional factors that are only active in virus-infected cells. furthermore, these results rule out a potential role of virus-induced il-1b or tnfa in the induction of socs-3. this is supported by the observation that neither expression of il to further assess a functional role of socs-3 in virus-induced suppression of stat1 phosphorylation we analyzed mouse cells with a targeted deletion of the socs-3 gene [56] . wild type and socs-3 deficient mouse embryonic fibroblasts (mef) were infected for different time points with pr8. the time of infection was prolonged in comparison to the infection of a549 cells because the human pr8 replicates less efficiently in mouse than in human cells. following infection lysates of these cells were assessed for stat1 phosphorylation ( figure 7a ). both cell types showed no phosphorylation of stat1 in the uninfected state. in contrast, infection of socs-3 knock out cells resulted in strongly elevated phosphorylation of stat1 in a sustained fashion. to rule out that this stat1 phosphorylation is due to altered secretion of ifnb or figure 7 . enhanced stat1 phosphorylation in infected socs-3 deficient mef correlates with elevated induction of ifnb-stimulated genes. wild type mef and socs-3 knock out mef were infected with pr8 (moi = 5) for the indicated times. subsequently, cell lysates were analyzed for stat1 phosphorylation (a). for control of productive virus replication, cell lysates were analyzed for viral protein pb1 expression. in (e, f, g) wild type and knock out cells were lysed at indicated time-points of infection. subsequently rna was subjected to reverse transcription. cdna was analyzed in quantitative real time pcr to assess mrna amounts of three prototype type i ifn-stimulated genes, sp110 (e), interferon regulatory factor-1 (irf-1) (f) and oas1 (g). equivalent mrna amounts were normalized to gapdh mrna levels and calculated as n-fold of the levels of untreated cells that were arbitrarily set as 1. in (c) wild type mef and knock out mef were infected with pr8 (moi = 5) or left uninfected. supernatants were taken 6 p.i. and used for stimulation of wild type mef for 15 minutes. as control wild type mef were stimulated with 500 u/ml mouse ifnb for 15 minutes. cells were harvested and analyzed for the amount of stat1 and phospho-stat1 in western blot analysis by specific antibodies. in (b) and (d) the relative band intensities of phospho-stat1 of the blots in (a) and (c) were quantified as described. doi:10.1371/journal.ppat.1000196.g007 other stat1-activating cytokines in socs-3 deficient cells, we performed conditioned medium experiments ( figure 7c ). mef wild type and mef socs-3 deficient cells were infected for 6 h and supernatants were subsequently harvested. stimulation of mef wild type cells with these different supernatants for 15 min. revealed no differences in stat1 phosphorylation, indicating that both infected cell types secrete similar amounts of ifnb and other stat1 activating cytokines. this is a strong indication that the observed differences in virus-induced stat phosphorylation are directly due to the presence or absence of socs-3 in wild type and knock out mef, respectively. to answer the question whether enhanced stat phosphorylation in socs-3 deficient cells would also lead to enhanced expression of isgs, total rna was isolated at different time points p.i. from infected wild type and knock out cells and monitored for induction of sp110, irf-1 and oas1 ( figure 7e, 7f and 7g ). these genes are described as type i ifn-induced genes [18] . indeed mrna levels of all three representative isgs were elevated in socs-3 knock out versus wild type cells at almost every time point during the course of infection. this indicates that enhanced stat1 phosphorylation and activation in socs-3 deficient cells results in elevated expression of isgs. the remaining question was, whether the elevated ifn-induced gene response in knock out cells might also affect propagation of influenza a viruses. thus, both wild type and knock out cells were infected with pr8 ( figure 8a ) or the strain a/victoria/3/75 (h3n2) ( figure 8b ). virus titers were assessed at different time points post infection. progeny virus titers from socs-3 knock out cells were significantly reduced compared to titers from infected wild type cells. to independently confirm these results and to verify that the observed effects are really due to the lack of socs-3, we used an sirna approach to specifically knock down socs-3 mrna in a549 cells. cells were transfected with 150 nm sirna for 48 h and socs-3 protein levels were compared to control transfected samples ( figure 8c, right) . subsequently, cells were infected and progeny virus titers were determined by plaque assay (figure 8c, left) . similar to the results gained from infected knock out cells, knock down of socs-3 resulted in decreased virus titers. on the contrary, over-expression of socs-3 resulted in elevated virus titers ( figure 8d ) concomitant with an inhibition of ifnb-or virus-induced stat1 phosphorylation ( figure 8e) . taken together the data indicate that in the absence of socs-3, infection leads to a stronger activation of stat1, resulting in enhanced expression of isgs and reduced virus titers. vice versa, over-expression of socs-3 leads to an inhibition of stat1 activation and elevated virus titers, probably due to inhibited expression of isgs. this highlights the important role of virus induced socs-3 to limit the type i ifn-induced antiviral response program. the type i interferon (ifn) system is one of the most powerful innate defenses of vertebrate cells, which limits replication and spread of viral pathogens including avian and human influenza viruses. influenza virus propagation is sensitive to ifn activities and therefore, like other viral pathogens, these viruses do not only induce type i ifn but also antagonize the production and effects of these cytokines at the same time [55] . for influenza a and b viruses, this is accomplished through their non-structural ns1 proteins that are structurally related polypeptides of 26 kda (a/ ns1) and 32 kda (b/ns1), which are abundantly expressed in infected cells [55] . ns1 proteins predominantly act on the level of ifn gene induction in infected cells by obstructing rig-idependent signaling through interaction with cellular factor(s) and/or sequestration of rnas generated during virus replication [1, 2, 57] . some ns1 proteins were also described to inhibit the maturation of cellular pre-mrnas raising the possibility that this activity additionally reduces production of ifna/b in infected cells [58, 59] . while ns1 also interferes with the activity of some isgs, such as the dsrna dependent kinase pkr [5, 60] , so far no type i ifn antagonistic mechanism was described for influenza viruses that act on the level of ifn signaling rather than gene induction. here we present data, showing that rna-induced expression of socs-3 in early phases of infection leads to a functional inhibition of ifn-induced stat activation and gene expression. this is a novel mechanism by which influenza virus suppresses the antiviral response of the host and paves the path to efficient virus replication. while it was reported in the literature that expression of socs proteins can be induced upon stimulation with ifn [61] we could not detect any significant gene induction by ifnb in a549 cells. instead we observed a significant up-regulation of socs-3 by viral 59 triphosphate rna, indicating that gene induction occurs via accumulation of vrna during infection. this appears to occur through the rna-mediated activation of the ikk/nf-kb pathway, most likely activated through engagement of the rna sensor rig-i. at a first sight, this might appear controversial since nf-kb activation is among the rig-i-induced signaling responses and ns1 was reported to inhibit this signaling pathway. however, despite the action of ns1 it is well known that nf-kb is still significantly activated upon influenza virus infection and many nf-kb and ifnb dependent genes are still expressed. we hypothesized previously that the incomplete inhibition conferred by ns1 is an indication that the virus exploits the remaining signaling activities for efficient replication [52, 62, 63] . the findings described here, namely nf-kb dependent induction of socs-3 and limitation of type i ifn signaling responses, provide yet another example how influenza viruses take advantage of nf-kb activity. while the data show that nf-kb is required for viral socs-3 induction, the factor appears not to be sufficient, since prototype inducers of nf-kb, such as il-1b or tnf-a would not induce socs-3. thus there seems to be the need of additional virus or rna-induced transcription factors. the most likely candidate would be the constitutively expressed interferon regulatory factor 3 (irf-3), that is known to be simultaneously activated with nf-kb upon virus infection directly via the rig-i rna sensing pathway without the need of type i ifn. furthermore irf-3 is a factor suppressed by the ns1 protein. recently it was reported that ifn-induced gene expression responses are potentiated in cells, which lack the nf-kb factors p50 or p65 [64] . although these authors described an inhibitory binding of nf-kb transcription factors to some ifn-induced gene, this mechanism might be cell type dependent since we could not observe similar effects in the cell types used here (data not shown). thus, the underlying molecular mechanisms appear to be not fully clear. it is striking that the effects described for p50 and p65 knock out cells in these studies fully correlate with our observations in socs-3 deficient cells. while in the latter case cells lack the ifnb signaling inhibitor socs-3, the p50 and p65 knock out cells are deficient for the factors required for socs-3 induction. thus, given the nf-kb dependent induction of socs-3 described in the present manuscript, we provide an additional molecular mechanism that may explain the phenomenon described by wei et al. [64] . first indications for beneficial effects of socs-3 gene expression on viral replication came from studies using the hcv core protein as a replacement for the influenza a viral ns1 in the context of infections with a ns1 deficient influenza virus [33] . one of the hallmark responses of hcv core expression is a rapid induction of socs-3 expression. given the role of socs-3 described here, it was not surprising that hcv core could partially rescue growth of the ns1 deficient virus [33] . while this manuscript was in preparation it was demonstrated by pothlichet et al. that influenza a virus-induced socs-1 and socs-3 upregulation requires a tlr-3-independent, rig-i/ mavs-dependent pathway [65] . moreover, over-expression of socs-1 and socs-3 in infected cells revealed that both molecules inhibit antiviral responses. these studies are perfectly complemented by our findings. here we confirm involvement of rig-i/mavs by showing that 59 triphosphate rna, the ligand for rig-i, is a major inducer of socs-3. furthermore, the finding that dsrna is only a weak inducer of socs-3 is also consistent with the independence from the dsrna sensor tlr-3. the only discrepancy of this work and the study of pothlichet et al. is that they show a dependence on the type i ifn receptor. this may be due to the different virus-strains and cell types used. it is well known that the capability of type i ifns to induce socs proteins is strongly cell type specific [31] . while in some cell types socs-3 expression appears to be type i ifn dependent (e.g. fetal liver cells) [31] it is clearly independent of ifn in other cell types [66] . recently it was shown that socs-3 is not significantly induced by ifna in a549 cells [18] , the major cell type used in our study. evidence that cell type specificities may be the cause of discrepancy is additionally provided by the fact that pothlichet et al. show identical induction kinetics of socs-1 and socs-3. in contrast the kinetics of the two proteins differ clearly in the cells we used, with socs-3 being induced much earlier than socs-1 on mrna and protein level. finally, it should be stated that regardless whether socs-3 is additionally induced by type i ifns at a later stage of infection, it is important that it can be induced earlier and in parallel to ifnb directly by vrna accumulation. this is supported by the finding that ifnb and socs-3 induction occurs in parallel kinetics ( figure 4a and 4b) while ifn-induced genes such as oas1 and mxa are only up-regulated later in a delayed and more sustained fashion ( figure 4d ). this makes a qualitative difference since the blocking effect of socs-3 on ifnb signaling already kicks-in during the first wave of ifnb action. taken together we describe here for the first time that at least some influenza a virus strains are able to suppress type i ifn signaling by a mechanism involving nf-kb dependent activation of socs-3 expression, which negatively affects stat phosphorylation. this adds a new aspect to our knowledge of the strategies used by influenza a virus to antagonize type i ifn responses. human influenza a/puerto-rico/8/34 (h1n1) (pr8) (giessen variant) and a/victora/3/75 (h3n2) (victoria) were originally taken from the strain collection of the institute of virology, giessen, germany. the human ns1 deficient influenza virus mutant dns1 and its isogenic wild type variant were propagated and used as described earlier [7, 67] . it should be noted that this isogenic wild type strain as described by garcia-sastre et al. [68] is different from the pr8 (giessen variant) used in the other experiments and in many previous studies [52, 67] . the supernatant was aspirated and cells were incubated with specific medium containing 0.2% bsa and antibiotics. to score for production of viral plaques the overlay was stained for 1 h using 1 ml neutral red in pbs per well [69] . to trigger jak/stat signaling cells were stimulated using human ifna/b or c as well as mouse ifnb. for stimulation of a549 cells or huvecs 100 u/ml human ifna or human ifnb was used. for stimulation of the green monkey epithelial cell line vero or hek 293 cells 500 u/ml human ifnb was applied. ifnc was always used in the concentration of 500 u/ml. mouse embryonic fibroblasts (mef) were incubated with 100 u/ml mouse ifnb. the different ifn were diluted in infection medium. for stimulation after infection, viral supernatants were aspirated and diluted cytokine was incubated for 15 minutes at 37uc. to investigate the potential of other cytokines to induced socs-3 gene expression a549 cells were stimulated with 100 u/ml il1b or 20 ng/ml tnfa at 37uc for times indicated. after stimulation cells were lysed and subjected to immune blotting. to block the activity of phosphatases after infection with influenza virus, sodium vanadate was used. dilutions were prepared using infection medium. sodium vanadate was added to the virus-containing infection medium at the time points indicated. after 10 minutes of incubation ifnb, diluted in infection medium, was added to the medium containing virus and sodium vanadate. the cells were stimulated with ifnb for 15 minutes. incubation with sodium vanadate started 25 min before cells were lysed and subjected to western blotting as described. for conditioned medium experiments wild type and socs-3 knock out mef were infected with pr8 (moi = 5) for 10 h or left uninfected. supernatants were used for stimulation of mef wild type for 15 minutes. cell lysates were subjected to western blot analysis. to investigate the induction of socs-3 expression by viral rna, rna isolated from infected or uninfected cells (control) was used. a549 cells were infected with pr8 (moi = 5) or left mock infected. 10 h post infection rna was isolated using the rneasy mini kit from qiagen according to manufacturer's instructions. to dephosphorylate viral 59 triphosphate rna, calf intestine alkaline phosphatase (ciap) (fermentas) was used. briefly, rna was isolated using trizol according to manufacturer's instructions. for dephosphorylation the reaction mix was set up in a 50 ml volume with 50 mg rna, 25 u ciap and 80 u ribolock rnase inhibitor (fermentas) and was incubated for 3 h at 42uc. thereafter the rna was isolated using the rneasy mini kit from qiagen. rnas used as control were mock-treated replacing ciap by glycerol. for stimulation, the different rna species and analogues were transfected using lipofectamine 2000 (l2000) according to manufacturer's instruction (invitrogen). in brief, l2000 was incubated with opti-mem for 5 minutes at room temperature; different amounts of rna were added and incubated for additional 15 minutes. for stimulation of cells with cellular or viral rna 400 ml rna-l2000 mix were added to 2 ml serum-free medium. cells were stimulated for 3 hours and subjected to either western blot analysis or quantitative real time pcr. for silencing socs-3 mrna, a549 cells were transfected with 150 nm human socs-3 sirna 48 h before infection using hiperfect (qiagen) according to manufacturer's instructions. in brief, 150 nm sirna was added to a mixture of d-mem without fcs/antibiotics and hiperfect and incubated for 10 min at room temperature. for transfection 400 ml of this mixture were added to the cells. subsequently cells were subjected to plaque assay analysis or western blot analysis. control sirna was purchased from qiagen. the sequences for the human socs-3 sirna in use are: human socs-3 sirna sense 59-cca aga acc ugc gca ucc adtdt-39, human socs-3 sirna anti-sense 59 -ugg aug cgc agg uuc uug gdtdt-39 ) (see table 1 for accession number of the human socs-3 gene). to determine whether tyrosine phosphatases become activated upon infection with influenza virus a phosphatase assay using the tyrosine phosphatase assay system (promega) was performed. a459 cells were infected for 10 h (moi = 5) or left uninfected. cells were harvested in assay buffer (100 mm tris-hcl ph 5.2, 100 mm cacl 2 , 100 mm mgcl 2 , 0.02% b-mercapto ethanol), cracked by a single freeze/thaw step at 280uc and disrupted by ultrasonic pulsing. lysates were precleared from cell debris and residual free phosphates according to the manufacturer's instruction. tyrosine phosphatase activity was measured by enzymatic release of free phosphate of two given pseudosubstrates (phosphorylated peptides representing target sequences for the most common tyrosine phosphatases). quantification was performed in comparison to a given standard according to the manufacturer's instruction. for western blot analysis cells were lysed with ripa [25 mm tris/hcl, ph 8.0, 137 mm nacl, 10% glycerol, 0.1% sds, 0.5% nadoc, 1% igepal, 2 mm edta, ph 8.0, pyrophosphate 5 mg ml 21 aprotinin; 5 mg ml 21 leupeptin; 1 mm sodium vanadate and 5 mm benzamidine] on ice for a minimum of 30 minutes. supernatants were cleared by centrifugation in a standard tabletop centrifuge (eppendorf) at maximum speed. protein concentration was determined by bradford assay. the phosphorylated and unphosphorylated forms of stat1 were detected using anti-stat1 (y701) antibody and anti-stat1 (bd bioscience). an antibody directed against y690 of stat2 was used for detection of the phosphorylated form of stat2 (upstate). antibodies to detect influenza viral proteins were purchased from serotec (np, m1), santa cruz (pb1, pb2). the anti-pa antibody was kindly provided by j. ortin (madrid/ spain). a monoclonal antibody directed against the viral ns1 was generated at the imv, muenster, germany [70] . a monoclonal anti-myc-tag antibody to detect myc-m1 was kindly provided by viktor wixler. imv, muenster, germany. all secondary antibodies were purchased from amersham and diluted 1:2500 in tbs-t. secondary antibodies were incubated for a minimum of 60 minutes at room temperature. to synthesize cdna from cells, rna was isolated using qiagen rneasy mini kit according to manufacturer's instruction. in brief, cells were lysed in the presence of b-mercaptoethanol and lysates were loaded to a column, washed and eluted in rnase-free water. for reverse transcription 3 mg total rna, 0.5 mg oligo dt primer in a total volume of 12 ml were heated for 10 minutes at 70uc. enzyme mix was prepared (56 enzyme buffer (fermentas), water and 500 mm dntps) and pre-warmed at 42uc for 2 minutes before adding 535 u/100 ml revertaid h 2 m-mulv (fermentas). reverse transcription was performed at 42uc for 1 hour. the enzyme was inactivated at 70uc for 10 minutes. samples were stored at 220uc or directly used in quantitative real-time pcr. for analysis of gene expression relative quantification of the dna amount was applied. in order to do that gene expression of the housekeeping gene gapdh was determined. to ascertain changes in expression of the gene of interest the differences between expression of gapdh and the gene of interest was calculated using the 2 2ddct method [71] . for quantitative real time brilliant qpcr sybr green mastermix (stratagene) was used according to manufacturer's instructions. the fragment of interest was amplified in 40 cycles. the following primers were used (see table 1 the pcfg5-egz retroviral vector used for transfection [72] as well as the constructs to express dominant negative mkk6 (mkk6ala) or ikk2 (ikk2kd) have been described earlier [52, 73] . the phoenix amphotropic retroviral producer cells (a gift from g. nolan, stanford, ca) [74] were cultured in dulbecco's modified eagle's medium containing 10% fetal bovine serum, 100 units/ml penicillin and 100 mg/ml streptomycin. generation of mkk6ala or ikkkd expressing producer cells as well as transduction of a549 cells to stably express these transgenes was performed as previously described [52, 53] . figure s1 infection of huvec results in inhibition of stat1 phosphorylation and ifnb independent socs-3 gene transcription. huvec were infected with pr8 (moi = 5) (a, b, d) or stimulated with 100 u/ml ifnb (e) for time points indicated. to assess the mrna levels of socs-3 (a, e), ifnb (b) and mxa (e) rna was reverse transcribed and cdna was subjected to quantitative real time pcr. equivalent mrna amounts were normalized to endogenous gapdh and calculated as n-fold of untreated cells that were arbitrarily set as 1. to assess the amount of phosphorylated stat1 (b) a549 cells were infected with pr8 (moi = 5) for time points indicated. total cells lysate was subjected to western blot analysis using anti-phospho-stat1, anti-stat1 antibodies. to assess effective viral replication viral ns1 was detected using an anti-ns1 antibody. (c) quantification of relative band intensities of (b) using aida software and 2d densitometry (fuji). figure s2 il1b and tnfa do not affect induction of socs-3 gene transcription. a549 wt cells were stimulated with 100 u/ml il1b (a), 20 ng/ml tnfa (b) or infected with pr8 (moi = 5) (c) for time points indicated. cells were lysed, and rna was subjected to reverse transcription. cdna was analyzed in quantitative real time pcr to assess mrna amounts of socs-3 and il6 (a and b) or il1b (c). equivalent mrna amounts were normalized to gapdh mrna levels and calculated as n-fold of the levels of untreated cells that were arbitrarily set as 1. found at: doi:10.1371/journal.ppat.1000196.s002 (4.87 mb tif) ifnbeta induction by influenza a virus is mediated by rig-i which is regulated by the viral ns1 protein inhibition of retinoic acid-inducible gene i-mediated induction of beta interferon by the ns1 protein of influenza a virus the 39-end-processing factor cpsf is required for the splicing of single-intron pre-mrnas in vivo influenza virus ns1 protein interacts with the cellular 30 kda subunit of cpsf and inhibits 39end formation of cellular pre-mrnas binding of the influenza virus ns1 protein to double-stranded rna inhibits the activation of the protein kinase that phosphorylates the elf-2 translation initiation factor biochemical and genetic evidence for complex formation between the influenza a virus ns1 protein and the interferon-induced pkr protein kinase influenza a virus ns1 protein activates the pi3k/akt pathway to mediate antiapoptotic signaling responses influenza a virus ns1 protein binds p85beta and activates phosphatidylinositol-3-kinase signaling interferons at age 50: past, current and future impact on biomedicine interferons, interferon-like cytokines, and their receptors ifn-lambdas mediate antiviral protection through a distinct class ii cytokine receptor complex activation of ifn-alpha, ifn-gamma, mxa, and ifn regulatory factor 1 genes in influenza a virusinfected human peripheral blood mononuclear cells influenza a virusinduced ifn-alpha/beta and il-18 synergistically enhance ifn-gamma gene expression in human t cells the jak-stat signaling pathway: input and output integration stats: transcriptional control and biological impact the receptor of the type i interferon family acetylationdependent signal transduction for type i interferon receptor differential gene induction by type i and type ii interferons and their combination interferon signalling network in innate defence the polyoma virus t antigen interferes with interferon-inducible gene expression ebola virus vp24 binds karyopherin alpha1 and blocks stat1 nuclear accumulation suppressors of cytokine signaling and immunity the elongin bc complex interacts with the conserved socs-box motif present in members of the socs, ras, wd-40 repeat, and ankyrin repeat families vhl-box and socs-box domains determine binding specificity for cul2-rbx1 and cul5-rbx2 modules of ubiquitin ligases the conserved socs box motif in suppressors of cytokine signaling binds to elongins b and c and may couple bound proteins to proteasomal degradation the jak-binding protein jab inhibits janus tyrosine kinase activity through binding in the activation loop a three-dimensional model of suppressor of cytokine signalling 1 (socs-1) three distinct domains of ssi-1/socs-1/jab protein are required for its suppression of interleukin 6 signaling socs-3 inhibits il-12-induced stat4 activation by binding through its sh2 domain to the stat4 docking site in the il-12 receptor beta2 subunit suppressors of cytokine signalling: socs induction of suppressor of cytokine signaling-3 by herpes simplex virus type 1 confers efficient viral replication induction of suppressor of cytokine signaling-3 by herpes simplex virus type 1 contributes to inhibition of the interferon signaling pathway ifn-alpha antagonistic activity of hcv core protein involves induction of suppressor of cytokine signaling-3 cytokine regulation in sars coronavirus infection compared to other respiratory virus infections respiratory syncytial virus inhibits interferon-alpha-inducible signaling in macrophage-like u937 cells non-response to antiviral therapy is associated with obesity and increased hepatic expression of suppressor of cytokine signalling 3 (socs-3) in patients with chronic hepatitis c, viral genotype 1 defective hepatic response to interferon and activation of suppressor of cytokine signaling 3 in chronic hepatitis c suppressor of cytokine signaling 3 (socs3) expression and hepatitis c virus-related chronic hepatitis: insulin resistance and response to antiviral therapy socs-3 regulates onset and maintenance of t(h)2-mediated allergic responses role of endogenous inhibitors of cytokine signaling in allergic asthma regulation of jak-stat signalling in the immune system mechanism of inhibition of protein-tyrosine phosphatases by vanadate and pervanadate inhibition of membrane phosphotyrosyl-protein phosphatase activity by vanadate the suppressor of cytokine signaling (socs) 1 and socs3 but not socs2 proteins inhibit interferon-mediated antiviral and antiproliferative activities socs-1 and socs-3 inhibit ifn-alpha-induced expression of the antiviral proteins 2,5-oas and mxa 59-triphosphate rna is the ligand for rig-i the mitogen-activated protein (map) kinase p38 and its upstream activator map kinase kinase 6 are involved in the activation of signal transducer and activator of transcription by hyperosmolarity the mkk6/ p38 mitogen-activated protein kinase pathway is capable of inducing socs3 gene expression and inhibits il-6-induced transcription ringing the alarm bells: signalling and apoptosis in influenza virus infected cells dual function of interleukin-1beta for the regulation of interleukin-6-induced suppressor of cytokine signaling 3 expression ikkalpha, ikkbeta, and nemo/ikkgamma are each required for the nf-kappab-mediated inflammatory response program nf-kappab-dependent induction of tumor necrosis factor-related apoptosisinducing ligand (trail) and fas/fasl is crucial for efficient influenza virus propagation activation of nf-kappa b via the ikappa b kinase complex is both essential and sufficient for proinflammatory gene expression in primary endothelial cells multiple signaling pathways regulate nf-kappab-dependent transcription of the monocyte chemoattractant protein-1 gene in primary endothelial cells type 1 interferons and the virus-host relationship: a lesson in detente il-6 induces an anti-inflammatory response in the absence of socs3 in macrophages ns1 protein of influenza a virus inhibits the function of intracytoplasmic pathogen sensor, rig-i intracellular warfare between human influenza viruses and human cells: the roles of the viral ns1 protein regulation of a nuclear export signal by an adjacent inhibitory sequence: the effector domain of the influenza virus ns1 protein binding of the influenza a virus ns1 protein to pkr mediates the inhibition of its activation by either pact or double-stranded rna suppressors of cytokine signalling (socs) in the immune system influenza viruses and map kinase cascades -novel targets for an antiviral intervention exploited defense: how influenza viruses take advantage of antiviral signaling responses nfkappab negatively regulates interferon-induced gene expression and antiinfluenza activity cutting edge: innate immune response triggered by influenza a virus is negatively regulated by socs1 and socs3 through a rig-i/ifnar1-dependent pathway interferon-gamma, but not interferon-alpha, induces socs 3 expression in human melanoma cell lines rac1 and pak1 are upstream of ikk-epsilon and tbk-1 in the viral activation of interferon regulatory factor-3 influenza a virus lacking the ns1 gene replicates in interferon-deficient systems caspase 3 activation is essential for efficient influenza virus propagation activation of phosphatidylinositol 3-kinase signaling by the nonstructural ns1 protein is not conserved among type a and b influenza viruses analysis of relative gene expression data using real-time quantitative pcr and the 2(-delta delta c(t)) method a1 expression is stimulated by cd40 in b cells and rescues wehi 231 cells from anti-igm-induced cell death transcriptional profiling of ikk2/nf-kappa b-and p38 map kinasedependent gene expression in tnf-alpha-stimulated primary human endothelial cells expression vectors and delivery systems we would like to thank akihiko yoshimura, kyushu university, fukuoka, japan, for providing socs3 2/2 mef via fred schaper, aachen, germany. we also would like to thank viktor wixler, imv, muenster, germany, for providing anti-myc and anti-ns1 monoclonal antibodies. key: cord-021872-rhi7hi9m authors: wilkes, rebecca p.; hartmann, katrin title: update on antiviral therapies date: 2015-12-04 journal: august's consultations in feline internal medicine, volume 7 doi: 10.1016/b978-0-323-22652-3.00007-4 sha: doc_id: 21872 cord_uid: rhi7hi9m nan update on antiviral therapies rebecca p. wilkes antiviral chemotherapy use is still relatively uncommon in veterinary medicine. controlled studies evaluating the efficacy of antiviral drugs in cats are lacking, or, if studies have been done, in many cases, the data are insufficient to determine effective dosing for these drugs. with the exception of the recombinant feline interferon (rfeifn)-omega, so far no antiviral drugs are specifically licensed for veterinary medicine, which leaves the veterinary community with the option to use off-label antivirals made for humans to combat viral diseases in feline patients. the goal of research in antiviral chemotherapy is the discovery of antiviral agents that are specific for the inhibition of viral multiplication without affecting normal cell division; however, because viruses are dependent on host cell machinery for replication, drug targets are often nonspecific. this makes antivirals inherently more toxic than antimicrobials are because the antiviral drugs are damaging to not only the virus but also the host cells as well. in addition, agents considered safe for human use are not always safe when administered to cats. 1 antivirals made for systemic use often require host and/ or viral metabolism to be active. therefore, agents designed for use in humans are neither reliably nor predictably metabolized by cats or their viruses. thus antiviral agents should always be tested first in vitro for efficacy and safety, and then followed by pharmacokinetic studies in cats. 1 systemic antivirals often have a relatively narrow safety margin, and special considerations should always be given to patients with reduced hepatic or renal function. well-designed blinded, placebo-controlled studies in client-owned animals should follow studies in laboratory-bred, experimentally infected cats to confirm results in genetically diverse cats. 1 most of the human antivirals are specifically intended for treatment of human immunodeficiency virus (hiv) or human herpesvirus infections. therefore, feline immunodeficiency virus (fiv) and feline herpesvirus type 1 (fhv-1) infections have been the most important indications for antiviral chemotherapy in veterinary medicine. topical antiviral therapy has been mainly used for herpetic ocular disease, but studies have evaluated a systemic antiviral compound (famciclovir) for treatment of multiple clinical syndromes associated with fhv-1 infections. even though combination antiviral therapy has been successful in slowing disease progression in people with hiv, similar therapy has not been thoroughly evaluated in cats. 2 recent studies have focused on combination therapy and evaluation of additional hiv drugs that have not been previously evaluated in feline cells. it is hoped that expanding the number of drugs that are shown to be effective for fiv will lead to effective combination therapy for feline patients. some additional feline infections that have been the focus of current antiviral studies are feline leukemia virus (felv) infection and feline infectious peritonitis (fip). some of the hiv antivirals, such as raltegravir, are nonspecific, and they display activity against additional retroviruses, including felv, in in vitro studies. identification of the human coronavirus that causes severe acute respiratory syndrome (sars) has led to evaluation of antivirals for treatment of various coronaviruses, including the feline coronaviruses (fcovs) that cause fip, although testing is mainly in in vitro stages. several studies have also evaluated the use of rfeifn-omega for treatment of multiple feline viruses. a review of the literature for antiviral treatment in cats, including current recommendations for drug dosages and use, is given in table 7 -1. feline immunodeficiency virus infects lymphocytes, cells of the monocyte-macrophage lineage, and cells of the central nervous system causing a variety of clinical signs (figure 7-1) . the viral replication cycle of fiv is highly similar to hiv. feline immunodeficiency virus binds to host cells by an initial interaction of the fiv envelope (env) glycoprotein with the cd134 molecule on the host cell, resulting in subsequent interaction with the co-receptor cxcr4 on the host cell, followed by viral envelope fusion with the host cell membrane. this allows entry of the viral nucleocapsid into the cytoplasm. the viral rna is released into the cytoplasm and transcribed to complementary dna (cdna) by the reverse transcriptase (rt) enzyme, which is specific to retroviruses. the cdna is subsequently synthesized to double-stranded dna, transported to the nucleus, and integrated into the host genome by another virus-specific enzyme, the integrase. viral messenger ribonucleic acid (mrna) and genomic rna are then transcribed and transported to the cytoplasm. viral proteins are translated and processed by a third virus-specific enzyme, the protease. the immature virion moves to the cell membrane and acquires the viral envelope and glycoproteins and then is finally released from the cells. 2 antiretroviral drugs studied extensively in hiv infection have targeted the three virus-specific enzymes (protease, rt, and integrase), as well as some additional targets, interfering with different steps of the virus replication cycle. 3 as of 2014, approximately 30 compounds are approved by the u.s. food and drug administration (fda) for treatment of different stages of hiv infection. 2 some of these drugs can also be used for fiv, and steps that can be inhibited include: (1) virus entry into susceptible cells by blocking attachment to the host cell co-receptor cxcr4; (2) reverse transcription of viral genomic rna; (3) viral dna integration into host genomes; and (4) proteolytic processing of precursor viral proteins into mature viral proteins (figure 7 -2). 2,3 dose indication close similarities exist between the rt of hiv and fiv, and it has been shown that several rt-targeted antiviral compounds active against hiv are also effective in inhibiting fiv replication in vitro. 4 the rt of hiv is actually the target for three classes of inhibitors: nucleoside rt inhibitors (nrti), nucleotide rt inhibitors (ntrti), and nonnucleoside rt inhibitors (nnrti). nucleoside rt inhibitors and ntrti interact with the catalytic site (the substrate-binding site) of the rt enzyme, whereas nnrti interact with an allosteric site located at a short distance from the catalytic site. for the nrti and ntrti to interact with the substrate-binding site, they need to be phosphorylated. 3 all , and emtricitabine) can be considered as nucleoside analogues, and they act in a similar fashion. after they have been taken up by the cells, they are phosphorylated three times to the active triphosphate form, and they act as competitive inhibitors of the normal deoxynucleoside triphosphate (dntp) substrates, which are used by the cell to make dna. unlike dntp substrates, nrti lack a 3′-hydroxyl group on the deoxyribose moiety. once incorporated into the dna chain, the absence of a 3′-hydroxyl group, which normally forms the 5′-to 3′-phosphoester bond with the next nucleic acid, blocks further extension of the dna by rt, resulting in dna chain termination. the analogues cannot be cleaved from the active center and thus block the rt enzyme. 3 nucleoside analogues are not only accepted as false substrates by viral enzymes, but also by cellular enzymes, and infectious diseases mononuclear cells. 4 six of these drugs (abc, ddi, emtricitabine, 3tc, d4t, and azt) had been previously evaluated in feline cells, and three (amdoxovir, racivir, and dexelvucitabine) had not. significant differences among the drugs were not found, but based on the data obtained, amdoxovir, dexelvucitabine, and racivir appear to be options for future studies investigating their potential use in fiv-infected cats. though pharmacological data for cats are not available for these drugs, cytotoxic properties of these compounds suggest they could likely be used in vivo at dosages comparable to that for azt. 4 nucleotide rt inhibitors are distinguished from nrti as they are nucleotide analogues (not nucleoside analogues), which means that they only need two (not three) phosphorylation steps to be converted to their active form. most importantly, they contain a phosphonate group that cannot be cleaved by hydrolases (esterases), which would make it more difficult to cleave off these compounds, once incorporated at the 3′-terminal end, compared with their regular nucleotide counterparts. use of these compounds also results in dna chain termination. one of these drugs, cidofovir, is active against virtually all dna viruses, including polyoma-, papilloma-, adeno-, herpes-, and poxviruses. cidofovir has been used for treatment of fhv-1 (see feline herpesvirus type 1). adefovir (9-(2-phosphonylmethoxyethyl)adenine [pmea]) has a spectrum of activity that partially overlaps with cidofovir, in that both are active against herpesviruses, but adefovir is also active against hepadnaviruses (hepatitis b) and retroviruses, including fiv and felv. the antiviral activity spectrum of tenofovir (pmpa) is narrower than that of pmea, in that it no longer extends to herpesviruses but is confined to hepadna-and retroviruses. 6 this drug has been tested in vitro against felv (see feline leukemia virus). adefovir has been tested in fiv-infected cats in a 6-week placebo-controlled, double-blinded, clinical trial; 10 cats received adefovir (10 mg/kg subcutaneously [sc] twice weekly) and 10 cats received a placebo. 7 there was no decrease in the proviral or viral loads in treated cats, and the cats developed a progressive, life-threatening anemia. this is a common adverse effect of some nucleotide analogues. 7 adefovir was also tested in combination with the co-receptor inhibitor plerixafor (see co-receptor inhibitors) in the same study, producing the same outcome as seen with use of the adefovir alone. 7 a related drug, (r)-9-(2-phosphonylmethoxypropyl)-2,6diaminopurine (pmpdap), has been shown previously to be a potent inhibitor of fiv replication in cell culture and has reduced the viral load in three of four cats experimentally infected with fiv when treated at 20 mg/kg sc three times per week for 6 weeks. there were no changes in the red blood cell counts or hemoglobin values with treatment. 8 a recent study evaluated the efficacy of this drug in a placebocontrolled, double-blind study with a population of 20 cats naturally infected with fiv. 8 no significant differences were found between pmpdap-treated (25 mg/kg sc twice weekly for 6 weeks) and placebo-treated cats, although cats treated with pmpdap showed a tendency for improvement this is the major cause of their toxicity. zidovudine is the nrti most studied in cats, including in vivo studies evaluating the clinical response of experimentally and naturally fivinfected cats treated with the drug. zidovudine can increase the cd4+/cd8+ ratio and improve clinical condition scores in fiv-infected cats; however, it can result in adverse effects, such as dose-dependent nonregenerative anemia and neutropenia. 4, 5 in addition, mutations producing resistance against the drug can develop. 4,5 therefore, a study evaluated nine nrti to inhibit fiv replication in feline peripheral blood receptor and co-receptor proteins dna a significant decrease in the provirus load but did not lead to improvement of clinical or immunological variables. a statistical decrease in serum magnesium levels was observed in the treatment group, without clinical consequences. no development of resistance of fiv isolates to plerixafor was found during the treatment period, making it a potential treatment for fiv-infected cats. 7 limited oral bioavailability and short half-life preclude clinical use of plerixafor in hiv infection, 2,7 but additional cxcr4 antagonists are under development and should be tested for efficacy against fiv when available. integrase catalyzes strand transfer (3′-end joining), which inserts both viral dna ends into a host cell chromosome. 3 integrase inhibitors are used to treat hiv infection. one of the integrase inhibitors (raltegravir) has been shown to be effective for inhibition of felv (see feline leukemia virus). administration of a combination of drugs from different classes, termed highly active antiretroviral therapy (haart), to hiv-infected patients has turned an invariably fatal disease into a chronic but manageable condition. 2, 3 the goals associated with the use of combinations of three (or more) anti-hiv compounds are: (1) to obtain synergism among different compounds acting at different molecular targets; (2) to lower the individual drug dosages to reduce their adverse side effects; and (3) to diminish the likelihood of development of drug resistance. 3 combination therapy has not been thoroughly investigated for treatment of fiv infection in cats, 2 and use of multiple classes of drugs is more difficult in cats because some of the drug classes that are effective for hiv do not work for fiv. 2, 4 however, the need for combination antiretroviral therapy for feline patients has been the focus of recent studies. the goal of antiviral therapy should be improvement of the cat's clinical status. this is not always correlated with virus replication, as measured by a plasma viral load. 9 it has been suggested that antiretroviral therapy should be administered to fiv-infected cats in the later stages of the asymptomatic phase of infection, during which the cat does not show clinical signs and the immune system is relatively normal and more likely to respond to treatment. 5 after experimental infection, when the cd4+/cd8+ ratio decreases, viral load increases markedly, and clinical signs of immunosuppression begin to appear. however, the situation in naturally infected cats is different, and the quality of life is not associated with the viral load. 11 therefore, it is debated at which time point antiviral therapy should be started and whether it should be administered to asymptomatic cats. in a recent study, antiretroviral therapy was initiated during the later stages of the asymptomatic phase of infection in naturally infected cats. the cats were defined as being in the later stages of the in their clinical signs and cd4+/cd8+ ratios. mild hematological side effects (slight decline in packed cell volume and hemoglobin values) were seen in the treatment group. compared with other ntrti, pmpdap seems to be slightly less toxic. 8 unlike the nrti and ntrti, nnrti are an active form, with no dependence on intracellular metabolic pathways. nnrti inhibit the rt by binding to the enzyme in a hydrophobic pocket that is located away from its catalytic site. the interaction of the compounds with the rt induces conformational changes that affect the catalytic activities of the enzyme. 9 nonnucleoside rt inhibitors are considered highly specific inhibitors of hiv-1, and thus not active against other retroviruses, including fiv. 9 this is due to differences in the structure and/or flexibility of fiv rt that prevent nnrti from interacting with the fiv rt. 10 protease inhibitors are based on the "peptidomimetic" principle, that is, they contain a hydroxyethylene scaffold that mimics the normal peptide linkage (cleaved by the hiv protease) but which itself cannot be cleaved. they thus prevent the hiv protease from carrying out its normal function, which is the proteolytic processing of precursor viral proteins into mature viral proteins. 3 despite similarities between the hiv and fiv proteases, all but one of the currently available hiv protease inhibitors have failed to inhibit the protease of fiv. the one compound of interest, tipranavir, has only been tested against fiv in vitro so far. 4 however, studies have demonstrated that these compounds can be used to inhibit fcov replication (see feline coronavirus). co-receptor inhibitors block viral attachment by binding to receptors on the host cell membrane to obscure the site of interaction of env with the receptor. 2 most of the receptor homologues or antagonists are highly selective for hiv and not useful for veterinary medicine. one exception can be used in cats with fiv infection, the class of bicyclams (e.g., plerixafor). plerixafor (1,1′-[1,4-phenylenbismethylene]bis(1,4,8,11-tetraazacyclotetradecane)-octachloride dehydrate, [amd3100], [ jm3100]), is the prototype compound among the bicyclams. bicyclams are dimeric low-molecular weight nonpeptidic compounds that bind selectively to the chemokine receptor cxcr4. this is the cell surface co-receptor used by both hiv and fiv for attachment and infection of susceptible cd4+ lymphocytes, and the amino acid sequences of human and feline cxcr4 are highly similar. drug binding inhibits attachment of the viral envelope to the host cell. the efficacy of plerixafor against fiv was recently investigated in naturally fiv-infected cats that were treated in a placebo-controlled, double-blind clinical trial. 7 plerixafor was administered at 0.5 mg/kg sc every 12 hours. treatment of fiv-infected cats with plerixafor caused infectious diseases agriculture (usda) as a treatment aid for cats infected with fiv or felv. the primary therapeutic effect is activation of progenitor cd4 t-cells to mature cells, which then produce cytokines, including interleukin (il)-2 and interferon (ifn). a few studies performed by the manufacturer are highlighted in a review article. 13 the studies suggest reduced virus load, improved clinical signs, and improved hematological parameters with treatment. however, the data for placebocontrolled studies were not shown, and a field study with naturally infected cats lacked a control group. independent placebo-controlled, blinded studies are warranted. additional information about immunomodulators and immunostimulants is provided in the feline herpesvirus type 1 and feline coronavirus sections. feline leukemia virus, like fiv, is a member of the family retroviridae, but unlike fiv, felv is a gammaretrovirus and not a lentivirus. feline leukemia virus causes a wide variety of clinical signs in infected cats (figure 7 -3). structural differences affect the susceptibility of gammaretroviruses to anti-hiv drugs, but the similarities in mechanism of replication suggest that some of these drugs can also inhibit felv. this is true of most nrti. 14 zidovudine effectively inhibits felv replication in vitro, and in vivo in experimental infections. however, in naturally felv-infected cats, it did not reduce plasma virus load, improve immunological and clinical status, increase quality of life, nor prolong life expectancy. 15 its bone marrow toxicity can also cause adverse side effects (e.g., nonregenerative anemia) that are more pronounced in felv-infected cats than in fiv-infected cats. therefore, it is not recommended as a first line of therapy for felv infection. 16 asymptomatic phase of infection when the cd4+/cd8+ ratio reached 0.9, because at this stage of infection, the viral load increased markedly, and clinical signs of immunosuppression began to appear. the ratios were calculated every 4 months for 2 to 5 years prior to initiation of the antiviral therapy, and viral loads of all cats were quantified once a year. the cats were randomly assigned to treatment groups of eight cats each. treatment included combination therapy, but no placebo group was used, and the study was not blinded. 5 the follow-up was performed over 1 year, through clinical evaluation and the determination of viral loads and cd4+/cd8+ ratios. comparisons of pretreatment and post-treatment values from the cats were performed, as well as comparison of values between treatment groups. a combination of two nrti (azt + 3tc, 25 mg/kg every 12 hours orally [po]) was compared to treatment with azt alone (5 mg/kg every 12 hours po). the combination of azt and 3tc is often used in hiv-infected patients, given that both drugs show a synergistic effect. treatment with azt alone or in combination with 3tc induced a significant increase in the cd4+/ cd8+ ratio and a significant decrease in viral load within and among groups, with an even greater reduction with combination therapy than with azt alone. only mild side effects, including vomiting in one of eight cats, anorexia in two of eight cats, and anemia in one of eight cats, were seen with this treatment combination, but therapeutic interventions resolved the problems, and treatment did not have to be stopped. 5 however, the lack of a control group and lack of blinding make the results of the study very difficult to interpret. therefore, treatment of asymptomatic fiv-infected cats with antivirals cannot be generally recommended based on the currently available data. an earlier in vivo study was performed in experimentally fiv-infected cats that were treated with a high-dose azt and 3tc combination (100 or 150 mg/kg/day po for each drug). the combination had no anti-fiv activity in these chronically infected cats. severe side effects, which included fever, anorexia, and marked hematologic changes, were observed in some of the cats with such high-dose dual-drug treatment, but the toxic effects were reversed when the dose was lowered to 20 mg/kg every 24 hours. 12 ideally, combination therapy for feline patients will contain at least two to three drugs from at least two different classes, as recommended for human patients. 9 as previously mentioned, pmea (an ntrti) was tested in combination with the co-receptor inhibitor plerixafor; however, because of the toxicity associated with the pmea, this combination cannot be recommended. 7 therefore, use of plerixafor in combination with other ntrti that are less toxic than pmea or compounds of other drug classes are should be further investigated in the future. lymphocyte t-cell immunomodulator (ltci), a protein produced by a bovine-derived thymic stromal epithelial cell line, is conditionally licensed by the united states department of alphaherpesviruses, such as herpes simplex virus 1 (hsv-1). they have been investigated for treatment of fhv-1. members of this group of antiviral agents include acyclovir (and its prodrug, valacyclovir), ganciclovir, and penciclovir (and its prodrug, famciclovir). all require three phosphorylation steps for activation. the first of these steps must be catalyzed by the fhv-1 viral enzyme, thymidine kinase. this makes the drugs less toxic in vivo compared to many of the other antiviral drugs. however, the activity of the thymidine kinase in fhv-1 is not equivalent to the enzyme of human herpesviruses. the second and third phosphorylation steps must be performed by host enzymes, which are not as effective in cats as they are in humans. this knowledge helps explain why the acyclic nucleoside antiviral agents developed for humans infected with hsv-1 are not predictably effective when administered to cats infected with fhv-1 and why pharmacokinetic and efficacy studies are always needed to establish appropriate dosing in cats. 1 acyclovir has been adequately tested in cats for the treatment of fhv-1, but it has a relatively low antiviral potency and poor bioavailability. a very high dose is required for effective treatment, which is associated with unacceptable toxicity, with signs related to bone marrow suppression and nephrotoxicity. 1 a prodrug of acyclovir, valacyclovir, was developed for increased bioavailability in humans, but use for fhv-1 treatment in experimentally infected cats induced fatal renal and hepatic necrosis and bone marrow suppression, and did not reduce viral shedding or clinical disease severity. 1, 19 therefore, despite its superior pharmacokinetics, valacyclovir should not be used in cats. 1 ganciclovir appears to be at least 10-fold more effective against fhv-1 than acyclovir in vitro. ganciclovir is available for systemic as well as topical use in the form of a 0.15% ophthalmic gel formulation in humans. ganciclovir holds promise for feline fhv-1 infection and currently available formulations warrant safety and efficacy studies in cats. 1 tenofovir, an ntrti used for treatment of hiv, has been shown to be effective against felv in vitro. 14 the anti-felv mechanism of tenofovir is probably similar to what has been described for hiv-1. tenofovir is given in the form of a prodrug, which is converted to an acyclic nucleoside phosphate. once converted to the active diphosphate form, tenofovir is incorporated by rt into viral dna, where it acts as a chain terminator to inhibit further elongation of the viral dna. 14 however, in vivo studies in felv-infected cats are lacking. another compound currently used for human hiv therapy, raltegravir, could be considered for the treatment of felv-infected cats. 14 the high degree of conservation across lentiviruses, betaretroviruses, gammaretroviruses, and alpharetroviruses of integrase active sites suggests that felv might be highly sensitive to integrase inhibitors. 16 the mechanism of action against felv is the same as for fiv, inhibition of integration of the viral dsdna that is produced by reverse transcription of the viral rna genome. 14 an in vitro study evaluated the effective 50% inhibitory concentration (ec 50 ) for felv inhibition of raltegravir in several feline cell lines and found these values are in the range of that observed for hiv and a related gammaretrovirus, xenotropic murine leukemia virus, and are well below the minimal plasma concentrations found in humans. 16 the effective concentration of raltegravir had no appreciable effect on cell viability nor induced apoptosis, suggesting that this could be an effective and safe drug also in vivo. 16 however, raltegravir is partly eliminated as glucuronide, a metabolic pathway that is not very efficient in cats, and it would increase the risk of toxicity resulting from drug accumulation. 16 as of 2014, no in vivo studies have been published. feline herpesvirus type 1 is a member of the subfamily alphaherpesvirinae, order herpesvirales. herpes simplex viruses 1 and 2 and varicella zoster virus are also members of this subfamily, and antivirals developed for the treatment of these human viruses have been used for treatment of fhv-1 in cats. feline herpesvirus type 1 typically infects epithelial and mucosal surfaces and travels retrograde along sensory axons to establish latency in the trigeminal ganglia. reactivated virus travels down those same axons to infect similar tissues to those that were originally infected, potentially resulting in recurrent or chronic sequelae, including keratitis, conjunctivitis, rhinosinusitis, dermatitis (figure 7-4) , and potentially blindness. 17 whereas drug combinations have become standard procedure for the treatment of hiv infections, the treatment of other virus infections, including herpesviruses, is routinely based on the use of a single antiviral drug. 18 a group of antiviral drugs known as acyclic nucleoside analogues are used for the systemic treatment of human effective against fhv-1. 23 this is potentially an alternative therapy to the use of topical drugs, the majority of which require multiple daily applications. 23 however, an implantable silicone polymer device impregnated with penciclovir has been developed that holds promise for long-term, steadystate subconjunctival delivery of the drug for the treatment of ocular herpetic disease. 17 although herpetic ocular disease is commonly treated with topical antiviral ophthalmic solutions or ointments (including idoxuridine, vidarabine, or trifluridine), 1 these antivirals do not require a virus-specific phosphorylation step for activation. moreover, they damage host cells, specifically resulting in bone marrow suppression. therefore, they should not be used systemically. 1 for good reviews of these topical drugs, see the reports of maggs 1 and gould. 24 cidofovir, a member of the ntrti class of drugs, has been tested for topical treatment of fhv-1 ocular disease but not for systemic use. it appears to be efficacious topically and is a newer drug (therefore it is included in this section). cidofovir requires the typical two host-mediated phosphorylation steps without virally mediated phosphorylation. 1 its safety when given topically arises from its relatively high affinity for hsv dna polymerase compared with human dna polymerase. it is commercially available only in injectable form in the united states for treatment of a human betaherpesvirus. when applied topically as a 0.5% solution twice daily to cats experimentally infected with fhv-1, it led to reduced viral shedding and improvement of clinical disease compared to the placebo group. 25 its efficacy with only twice daily administration (despite being virostatic) is believed to be due to the long tissue half-lives of the metabolites of this drug. there are reports of its experimental topical use in humans and rabbits being associated with stenosis of the nasolacrimal duct, but this has not been shown in cats. the fact that a twice-daily topical treatment is sufficient, whereas all other topical antivirals require application every 3 to 4 hours, makes cidofovir a useful alternative for ocular topical treatment. 1, 24, 25 small interfering rna small interfering rnas (sirnas) designed to target the fhv-1 dna polymerase 26 and glycoprotein d 27 have been used in vitro to induce rna interference in an immortalized cell line and in primary feline corneal epithelial cells to inhibit fhv-1 replication. rna interference is a posttranscriptional, rna-guided gene-silencing mechanism present in eukaryotes. 28 interference of the fhv-1 essential genes resulted in reduction of virus replication up to 98 ± 1%. this type of therapy is intended for topical treatment of chronic herpetic disease. however, a preliminary in vivo study evaluating topical delivery of sirnas to feline corneas was unsuccessful. 29 the lack of delivery was likely the result of sirna dilution and rapid removal by tear film and blinking. studies are ongoing to identify a means of increasing the most promising systemic drug for the treatment of fhv-1 is famciclovir, a prodrug of the active compound penciclovir, which has been shown to be highly efficacious in inhibiting fhv-1 replication in vitro. penciclovir is absorbed poorly when given orally, so the oral form famciclovir was developed with increased bioavailability and uptake from the intestinal tract. 1 famciclovir requires di-deacetylation, mainly in the blood, and oxidation by a hepatic aldehyde oxidase for conversion to the active compound penciclovir. unfortunately, hepatic aldehyde oxidase activity is basically absent in cats, which makes the pharmacokinetics of this drug complex and results in lower than expected plasma penciclovir concentrations despite administration of relatively high doses of famciclovir. 20, 21 despite this, studies evaluating famciclovir in vivo have shown it to be safe and efficacious for use in feline patients. 20, 22 cats experimentally infected with fhv-1 and receiving famciclovir 90 mg/kg po three times daily for 21 days had significantly improved outcomes for systemic, ophthalmic, clinicopathologic, virologic, serologic, and histologic variables when compared with placebo-treated cats. treatment was initiated on day zero, the same day the cats were infected. 20 even though this study did not mimic how cats with natural infection would be treated, results from a clinical case study suggested this drug is likely effective for treatment of clinical cases, though it was not blinded and placebo controlled. 22 clinical cases with primary ocular disease, rhinosinusitis, and dermatitis each attributed to fhv-1 (though not definitively diagnosed), were treated with famciclovir at doses of 62.5 mg po once or twice daily for ocular herpetic disease or rhinosinusitis or up to 125 mg po three times daily for dermatitis. famciclovir was well tolerated with each dose and had a positive effect on each clinical condition. 22 a definitive dose rate has not been established for famciclovir. however, penciclovir has no appreciable in vitro effect if present for 24 hours prior to infection, suggesting that famciclovir should be administered more frequently than once every 24 hours to ensure exposure to penciclovir as additional epithelial cells become exposed to viral infection. 21 current pharmacokinetic data suggest that dosing three times daily is required, 21 and 40 mg/kg po three times daily has been suggested for treatment of cats infected with fhv-1, based on effective concentrations obtained in in vivo studies 20, 22 and determination of new in vitro 50%-inhibitory concentrations. 21 the most commonly reported adverse effects of famciclovir treatment in humans include urticaria, hallucinations, headaches, and confusion (especially in elderly humans), which would likely be more difficult to detect in animals. for these reasons, judicious use of this drug is recommended in client-owned cats, especially those with preexisting hepatic or renal insufficiency. 21 pharmacokinetic studies have also evaluated the concentration of penciclovir in tears, and treatment with an oral dose of 40 mg of famciclovir/kg three times daily achieves a penciclovir concentration at the ocular surface likely to be a bolus and not added to food. any benefit from lysine therapy is likely only possible with daily, lifelong treatment of cats with chronic herpetic disease, rather than use of lysine as a treatment during acute or recrudescent episodes. potentially, daily therapy would reduce episodes of viral recrudescence. however, clinical studies in pet cats are lacking. the cost of this therapy should be weighed against the potential benefits. owners should be made aware that this is only an adjunctive therapy and that administration of antiviral drugs might be necessary to gain better control of signs. polyprenyl immunostimulant (pi) is an immunomodulator that has a conditional license in the united states for treatment of fhv-1 infection. in blinded, placebo-controlled, experimental challenge studies, pi started on the day of virus exposure significantly reduced the severity and duration of rhinitis and conjunctivitis associated with acute fhv-1 disease (legendre and kuritz, manuscript in preparation). according to the manufacturer, pi upregulates the innate immune system and modulates the immune response toward a cellular response. this activity was attributed to positive effects associated with treatment of fhv-1, which requires a cell-mediated immune response for control. viral titers were not compared between treatment and control groups in the studies, but based on the reduced signs associated with treatment, clinical studies are warranted. feline infectious peritonitis is associated with clinical signs that can affect almost any body system (figure 7-5) . currently, there is no effective treatment for fip despite its importance as the leading infectious cause of death in young cats. 33 following the discovery that sars is caused by a coronavirus (sars-cov), efforts to find an antiviral drug for coronaviruses increased. a few antiviral agents that target different steps in the replication cycle have been tested against feline fcov. coronavirus spike proteins on the viral envelope initially bind to receptors on the host cell membrane. 33 the spike protein mediates fusion of the viral envelope with host cell membranes. during this process, heptad repeats 1 and 2 (hr1 and hr2) of the spike protein assemble to form a complex, resulting in a conformational change that is necessary for fusion. 34 peptides have been used as antivirals by inhibiting the hr1-hr2 interaction, thus preventing membrane fusion. 34 the spike protein must be cleaved for entry of the virus into the cytoplasm. feline coronavirus infection is dependent on cathepsin b, a host cysteine protease found within the cell, making this the likely protease responsible for spike protein cleavage. therefore, cathepsin b can serve as a potential target for the development of therapeutic drugs against fcov. following entry into the cell, fcov produce viral polyproteins that are processed into contact time between the corneal cells and sirnas to allow delivery. twice-daily oral l-lysine bolus administration, initiated prior to experimental infection, reduced the severity of conjunctivitis in cats undergoing primary infection. l-lysine bolus administration also reduced viral shedding in latently infected cats experimentally infected with fhv-1, following changes in husbandry and housing but not following corticosteroid administration. in vitro, lysine supplementation led to reduction of fhv-1 replication. 1 arginine exerts a substantial growth-promoting effect on fhv-1 and is an essential amino acid for viral protein synthesis, and lysine antagonizes this effect. lysine and arginine competitively inhibit transport of each other by using a common transport system, and lysine induces arginase, an enzyme that causes the degradation of arginine. arginine deficiency inhibits synthesis of infectious viral particles and downregulates synthesis of viral proteins. however, unlike the protocol for hsv-1-infected humans, owners of cats receiving lysine for fhv-1 should not be advised to restrict their cat's arginine intake 1 because feeding a diet lacking l-arginine is associated with a severe risk of hyperammonemia and encephalopathy. 30 it has been suggested that the ratio of l-lysine to l-arginine, rather than the concentration of each amino acid, is critical in achieving an inhibitory effect on viral replication. dietary supplementation increases mean plasma concentrations of l-lysine without reducing l-arginine concentrations and has been shown to be safe for use in cats, up to 86 g/kg of diet. supplementation with higher doses has been shown to result in reduced food intake. 30, 31 despite promising initial in vitro data and in vivo results from experimental studies, current studies question whether viral inhibition with increased lysine concentrations, in the absence of decreased arginine concentrations, can be biologically important. a new study evaluating the effect of various ratios of l-lysine and l-arginine on fhv-1 dna replication in vitro demonstrated only a modest reduction in viral dna (less than 1 log) at ratios considered difficult to obtain in vivo in healthy cats. 31 a lack of efficacy of l-lysine supplementation has also been demonstrated in vivo in shelter settings. 1, 32 dietary supplementation was unsuccessful, likely because the cats were anorexic during peak disease and were not ingesting the lysine when they needed it the most. bolus administration was also unsuccessful, likely because of stress associated with the lysine administration. 1, 32 the stress of bolus administration in shelter situations could negate its effects and even cause transfer of pathogens among cats by shelter workers administering the lysine. however, data do not support dietary supplementation. 1, 32 unfortunately, no studies to date have been conducted on client-owned cats; however, anecdotal evidence suggests that there is a benefit from administration of lysine in individuals. dosing is 500 mg po twice daily, which should be given as infectious diseases ment to the host cell. the antiviral effects were concentration dependent, and nelfinavir displayed cellular toxicity at higher doses. gna was a better inhibitor of fcov, and when the two agents were added together, a synergistic antiviral effect was produced. the results suggest that the combined use of gna and nelfinavir could have therapeutic potential in the treatment of cats with fip. 35 viral fusion has also been targeted effectively with a synthetic peptide based on the putative hr2 sequence of fcov. virus replication was significantly inhibited in vitro compared to controls, and the peptide was nontoxic. 34 this peptide was also used in combination with human ifn-alpha. the two displayed a synergistic effect, but the cells were pretreated with ifn prior to infection by the virus. 34 see the section on interferon for further information about interferon treatment for fip. immunomodulators have been considered because fip is an immune-mediated disease. 35 a drug that has shown promise for immunomodulation is pi. 36 this drug has a conditional license in the united states for treatment of fhv-1 infection. in a case series of three cats, pi was associated with prolonged survival in cats with noneffusive fip. 36 no placebo group was included for comparison, so definitive conclusions about the effectiveness of this drug for treatment of fip cannot be drawn. additional immunostimulants such as immunoregulin (propionibacterium acnes), an inactivated bacterin, and a t-cell receptor peptide (manufactured by imulan biotherapeutics), have been suggested for treatment of fip. these are not antiviral drugs; instead, each of these products is reported to stimulate the immune response toward a cell-mediated response or to reduce an overactive type 2 helper t-cell (th2) response. an imbalance in t-cell versus b-cell immune response has been suggested to contribute to the development of fip. it has been proposed that a strong cell-mediated immune response protects a cat from the development of fip, whereas the production of antibodies is counterproductive, enhancing the uptake and replication of feline infectious peritonitis virus (fipv) in macrophages and contributing to the pathology by producing a type iii hypersensitivity vasculitis. however, this hypothesis has been questioned. 37 therefore, even though the use of these types of drugs for stimulation of a cell-mediated response might seem a logical approach for the treatment of fip, there is currently no data to support their use. an additional non-antiviral drug that has been evaluated for treatment of fip is propentofylline. this drug appears to downregulate proinflammatory cytokines, which in turn can reduce vasculitis. vasculitis, as stated earlier, is responsible for pathology associated with fip. however, in a placebocontrolled, double-blind study in cats with late stage fip, mature proteins by viral-specific proteases, the main protease (3c-like [3cl] protease) and the papain-like protease. because the cleavages of viral polyproteins are an essential step for virus replication, blockage of viral protease is also an attractive target for therapeutic intervention. 33 in an in vitro experiment in crandell-rees feline kidney cells, 3cl protease inhibitors and cathepsin inhibitors were tested for their ability to inhibit fcov replication. 33 both types of drugs produced effective inhibition with ec 50 values in the nanomolar range and each drug tested was nontoxic to the cells at effective concentrations. the 3cl protease inhibitors were more effective than the cathepsin inhibitors and when used in combination, these drugs had strong synergic effects. there have not been any in vivo studies with these drugs in cats to date. in one in vitro study, 16 antiviral compounds, including nucleoside analogues used to treat herpesviruses, nrti used for hiv, and protease inhibitors also used for hiv, were tested for their ability to inhibit fcov in cell culture. 35 among the 16 drugs tested, two showed significant inhibition of fcov when compared to the untreated cells. these were nelfinavir and galanthus nivalis agglutinin (gna). nelfinavir is a hiv protease inhibitor that has been shown to target the 3cl protease of sars-cov by interacting with 18 residues of the protease. the drug was slightly less effective against fcov than against sars-cov, likely because only seven of the corresponding residues of the 3cl protease of fcov are identical to the sars-cov protease. 35 gna, a carbohydrate-binding agent, exhibits its antiviral effect by binding to coronavirus glycosylated envelope glycoproteins, thereby inhibiting viral attachineffective within a few weeks because of the development of neutralizing antibodies that limit its activity. 40, 41 rhuifn-α can be given orally for a longer period because no antibodies will develop during oral treatment. unlike rhuifn, rfeifnomega, being a feline recombinant product does not induce neutralizing antibodies when administered sc. this means that the high-dose parenteral protocol can be used safely and efficiently even if repeat administration is required. this is an important factor to consider in a condition where management needs to be lifelong. 42 given po, ifns are inactivated by gastric acid and destroyed by trypsin and other proteolytic enzymes in the duodenum and therefore are not absorbed and cannot be detected in the blood after oral administration. direct antiviral effects are unlikely after oral application; however, ifn still seem to have immunomodulatory activity. type i ifns likely bind to mucosal receptors in the oral cavity, stimulating the local lymphoid tissue, leading to cytokine release from lymphatic cells in the oropharyngeal lymphoid tissues, triggering a cascade of immunologic responses that act systemically. 43 interferons have been used for the treatment of feline retrovirus infections. treatment with ifn improved the clinical condition scores of cats infected with felv and fiv, but not because of a reduction in viral load. this suggests that the improved clinical condition seen with treatment is not specific to an antiviral effect, at least not for fiv and felv, but instead is a result of immunomodulation, potentially associated with the innate immune response. 40, 42, 44 some clinical signs in fiv-infected cats are caused by immunopathological reactions, such as gingivostomatitis and uveitis. immunomodulation might be the cause of improvement of some clinical signs associated with ifn treatment, probably the result of an effective control over inflammatory cytokines in diseased organs. 44 it has been suggested that a nonspecific stimulation of the immune system with ifn therapy might be contraindicated in fiv-infected cats because it could lead to a rise in viral replication produced by the activation of lymphocytes and macrophages harboring latent infections and therefore accelerate disease progression in these cats, and the use of ifn in hiv-infected humans is controversial. 5 however, use of low-dose oral huifn (natural, not recombinant in this study) in ill fiv-infected cats (50 iu per kg on the oral mucosa daily for 7 days on alternating weeks for 6 months, followed by a 2-month break, and then repetition of the 6-month treatment) resulted in improvement of clinical signs in a placebo-controlled, doubleblind study. 44 parenteral rfeifn-omega used according to the licensed protocol (table 7 -1) resulted in decreased mortality rates in felv-infected cats, compared with the control group in another placebo-controlled study. 40, 45 in another study evaluating fiv-and/or felv-infected cats housed in a shelter, hematologic values remained within reference ranges, and there were no biochemical abnormalities there was no statistically significant difference in the survival time, the quality of life, or any clinical or laboratory parameter in cats treated with this drug versus cats receiving a placebo. 38 of the cats in the study, 21 of 23 cats displayed effusion at the start of the study. the drug might be more useful in cats without effusion because it may have a chance to prevent vasculitis and therefore effusions, but studies are lacking. interferons are molecules produced by vertebrate cells in response to viral infections or certain inert substances, such as double-stranded rna, and other microbial agents. there are three types of ifns. type i ifns comprise the largest subfamily and include ifn-α, ifn-β, and ifn-omega. type i ifns are produced by various cell types, such as leukocytes and fibroblasts, in direct response to virus infection. 39 there is only one member of the type ii ifn subfamily, ifn-γ, that is an immunomodulatory cytokine, produced in response to recognition of infected cells by t lymphocytes and natural killer cells of the host's immune system. 39 type iii ifns, which contain three ils, (il-28a, il-28b, and il-29), are identified. this subfamily also has the ability to interfere with virus replication and has been suggested to be the ancestral antiviral system of vertebrates. 39 interferons are not virucidal; rather, they trigger expression of various antiviral proteins and thus induce an antiviral state within the host cell to limit replication and spread of viruses. further, type i ifns have been shown to potently enhance innate and adaptive immune responses in vivo, through various immunomodulatory effects, such as activation of dendritic cells (dcs), amplification of antibody response, and enhancement of t-cell and natural killer cell cytotoxicity. 40 viruses causing lysis of their target cell are most effectively inhibited by ifn through their antiviral activity in noninfected cells. therefore, ifns have their highest utility in the prophylaxis or early postexposure management for virus infections. given that ifns are not specific for a particular virus, they have been tested for the treatment of multiple feline viruses, including fhv-1, fiv, felv, feline calicivirus (fcv), and fcov. 40 two molecules of type i ifns are currently being used for therapy in cats: human recombinant interferon alpha (rhuifn-α), and rfeifn-omega, which is licensed for use in cats and dogs in europe, australia, and some asian countries. ifns are not strictly species-specific in their effects; however, their biologic activity and toleration are greater in cells of genetically related species. in vitro results suggest that rfeifn-omega would likely be more effective than rhuifn-α in vivo, although both ifns have been shown to have therapeutic value in cats. 40 there are two common treatment regimens for use of rhuifn-α in cats: injection of a high dose (10 4 to 10 6 international unit per kg sc every 24 hours) or oral application of a low dose (1 to 50 international unit [iu] per kg every 24 h). when given parenterally to cats, rhuifn-α becomes feline coronavirus shedding was reduced but not significantly, and there was not enough fpv detected in the population to draw any significant conclusions. 41 however, there was no placebo group used for this study, and without a placebo group, it is difficult to determine definitively if the results are due to antiviral effects of ifn or are just consistent with the natural resolution of viral shedding. in a separate study, 36 cats with naturally acquired upper respiratory tract disease housed in a humane society facility were treated with one drop of rfeifn-omega solution (10 6 unit/ml), rhuifn-α solution (10 6 iu/ml), or saline (0.9% nacl) solution (12 cats/group) in each eye twice daily for 14 days for the treatment of keratoconjunctivitis. 46 there was no statistical difference between the treatment groups and the placebo group with regard to clinical scores or viral shedding (fhv-1 and fcv), determined by real-time quantitative pcr from oropharyngeal and conjunctival swabs. feline herpesvirus type 1 shedding was lower, though not statistically significant, on day 14 compared with day 0 for all groups (including the placebo group), and clinical scores were significantly decreased on day 14 compared to day 0, again for all groups including the placebo group. 46 therefore, comparing results between days 0 and 14 in the treated cats without the inclusion of the placebo group would have resulted in a different conclusion for this study. these cats were not infected with felv, and even though the fiv status was unknown for all the cats, the ones that were tested were negative. however, this study highlights the need for a placebo group for accurate evaluation of the effect of ifn therapy on fhv-1 and fcv shedding and associated disease. oral and sc ifn therapy has been associated with an improvement in oral ulcers and gingivitis and gingivostomatitis in cats infected with fiv, 40,41 a condition that is common in cats with fcv infections. 41 feline calicivirus is also associated with chronic gingivostomatitis in cats not infected with fiv or felv, 43 and a study evaluated the efficacy of rfeifnomega (10 5 iu/day for 90 days by topical oromucosal administration) for the treatment of fcv-associated feline chronic gingivostomatitis (fcgs) and caudal stomatitis in fiv-/ felv-negative cats. 43 cats were included in the study if they continued to show persistence of clinical signs of fcgs at least 2 months after periodontal treatment (scaling, subgingival débridement, and polishing), tooth extraction, and 3 weeks of antibiotics with analgesic and anti-inflammatory drugs as needed. twenty-four cats were treated with the ifn, and the effect was compared with a positive control group that received a standard corticosteroid therapy. the results suggested that the ifn therapy was as effective as the corticosteroid treatment for this condition for improvement in clinical signs. 43 feline calicivirus viral loads were not evaluated in this study, and there was no placebo group used for comparison. however, assuming the ifn therapy was the cause of the improvement seen in these cats, the results add to the hypothesis that improvement in oral lesions in fivand/or felv-infected cats likely is associated with the effect of ifn on opportunistic viral infections. differences in the outcome of the different studies could be due to different associated with rfeifn-omega treatment used according to the licensed protocol. 41 these findings suggest that ifn treatment is safe for treatment of fiv-and felv-infected cats, 40, 41, 44, 45 but further studies are required to clearly demonstrate its efficacy against fiv and felv in vivo. a recent study evaluated the use of oral administration of rfeifn-omega for the treatment of symptomatic naturally infected, client-owned fiv-infected cats. 42 the treatment protocol was 10 5 iu/cat po every 24 hours for 90 consecutive days, administered by the cats' owners. a historical group that was treated sc with the licensed protocol 41 was used as a control for comparison, but no placebo group was included. treatment resulted in significant improvement of clinical scores between pretreatment and post-treatment values, and there was no significant difference between the sc historical control group and the po group, suggesting that po administration of rfeifn-omega could be used effectively as an alternative to the licensed protocol, at a significantly reduced cost. 42 an additional benefit of using ifn therapy for fiv and felv treatment could be the effect of ifn on opportunistic infections by other viruses, including fhv-1 and fcv. 41 in fact, the effect of ifn on these additional viral infections might be the cause of the improved clinical scores associated with ifn treatment. 40, 41 both fiv and felv replicate in lymphoid and monocytoid cell subsets and cause immunosuppression. in fiv-infected cats, most of the clinical signs are not directly caused by the fiv itself but are the result of secondary infections, as well as neoplasia. 40, 46 although felv causes more severe clinical syndromes than fiv does, diseases secondary to immunosuppression account for a large portion of the syndromes seen in felv-infected cats as well. 11 considering that ifn therapy seems to have no effect on fiv and felv virus load but it is immunomodulatory, it would seem advisable to treat retrovirus-infected cats with ifn when they have clinical signs, as they would benefit from its effects in improving their clinical condition. 40 a recent study attempted to evaluate the hypothesis that improvement in clinical scores with ifn treatment in fivand felv-infected cats might be a reflection of reduction in viral shedding of secondary viruses in these cats. 41 sixteen naturally infected fiv-and/or felv-infected cats (seven fiv, six felv, and three coinfected) were followed during rfeifn-omega therapy (used according to the licensed protocol) to monitor clinical signs and to correlate them with excretion of concomitant viruses (fcv, fhv-1, fcov, and feline parvovirus [fpv] ). shedding of these viruses was evaluated by real-time quantitative polymerase chain reaction (pcr) (fhv-1 and fcov) or conventional pcr (fcv and fpv). pretreatment and post-treatment samples were compared. feline calicivirus shedding was detected in 13 of 16 cats on day 0 and not detected on day 65. the amount of fhv-1 shedding was significantly reduced in the cats at the end of the study (day 65), compared with the beginning. before euthanasia with a mean survival time of 18 days. there was only one long-term survivor (>3 months) in the rfeifnomega group. interferon treatment might be more effective if started earlier, but this is not of relevance in the treatment of cats with fip in the field. 47 however, ifn therapy might be useful for treatment of cats with chronic fcov shedding, but further studies are required. as previously mentioned, treatment with rfeifn-omega (licensed sc protocol) was associated with a decrease in fcov shedding in fiv-and/ or felv-infected cats; however, the results were not compared with a placebo group. 41 in conclusion, antivirals are still in their infancy for the treatment of feline diseases. however, as new drugs are produced for human viral diseases that can be used for feline patients, and testing of currently available drugs continues, it is hoped that determination of effective protocols for treatment of feline viral diseases will be possible in the future. application methods (e.g., ocular versus oral); however, definitive conclusions cannot be drawn without additional studies that also evaluate viral load in naturally infected cats that are randomized, placebo-controlled, and double-blinded. ifn has also been evaluated for treatment of fip. in a randomized placebo-controlled, double-blinded treatment trial, 37 cats with fip were treated with rfeifn-omega or placebo. 47 in all cats, fip was confirmed by histology and/or immunohistochemical or immunofluorescence staining of fcov antigen in effusion or tissue macrophages. all cats received glucocorticoids, either as dexamethasone in case of effusion (1 mg/kg intrathoracic or intraperitoneal injection every 24 hours) or prednisolone (2 mg/kg po every 24 hours). cats also received either a placebo or rfeifn-omega at 10 6 iu/kg sc every 24 hours for 8 days and subsequently once a week. there was no statistically significant difference in the mean survival time of cats treated with rfeifn-omega versus the placebo. cats survived for a period of 3 to 200 days antiviral therapy for feline herpesvirus infections pharmacological inhibition of feline immunodeficiency virus (fiv) anti-hiv drugs: 25 compounds approved within 25 years after the discovery of hiv antiviral efficacy of nine nucleoside reverse transcriptase inhibitors against feline immunodeficiency virus in feline peripheral blood mononuclear cells evaluation of different antiretroviral drug protocols on naturally infected feline immunodeficiency virus (fiv) cats in the late phase of the asymptomatic stage of infection acyclic nucleoside phosphonates: past, present and future bridging chemistry to hiv, hbv, hcv, hpv, adeno-, herpes-, and poxvirus infections: the phosphonate bridge efficacy and adverse effects of the antiviral compound plerixafor in feline immunodeficiency virus-infected cats -phosphonylmethoxypropyl) -2,6-diaminopurine (pmpdap) in the treatment of feline immunodeficiency virusinfected cats their discovery, development, and use in the treatment of hiv-1 infection: a review of the last 20 years susceptibility of feline immunodeficiency virus/ human immunodeficiency virus type 1 reverse transcriptase chimeras to non-nucleoside rt inhibitors clinical aspects of feline immunodeficiency and feline leukemia virus infection is azt/3tc therapy effective against fiv infection or immunopathogenesis? lymphocyte-cell immunomodulator (ltci): review of the immunopharmacology of a new veterinary biologic discovery of drugs that possess activity against feline leukemia virus a trial with 3′-azido-2′,3′-dideoxythymidine and human interferon-a in cats naturally infected with feline leukaemia virus inhibition of feline leukemia virus replication by the integrase inhibitor raltegravir controlled release delivery of penciclovir via a silicone (med-4750) polymer: kinetics of drug delivery and efficacy in preventing primary feline herpesvirus infection in culture a 40-year journey in search of selective antiviral chemotherapy effects of valacyclovir in cats infected with feline herpesvirus 1 evaluation of orally administered famciclovir in cats experimentally infected with feline herpesvirus type-1 in vitro cytotoxicity and antiviral efficacy against feline herpesvirus type 1 of famciclovir and its metabolites treatment of feline herpesvirus-1 associated disease in cats with famciclovir and related drugs pharmacokinetics of famciclovir and penciclovir in tears following oral administration of famciclovir to cats: a pilot study feline herpesvirus-1: ocular manifestations, diagnosis and treatment options effect of topical ophthalmic application of cidofovir on experimentally induced primary ocular feline herpesvirus-1 infection in cats evaluation of the effects of small interfering rnas on in vitro replication of feline herpesvirus-1 use of interfering rnas targeted against feline herpesvirus 1 glycoprotein d for inhibition of feline herpesvirus 1 infection of feline kidney cells therapeutic sirna: principles, challenges, and strategies evaluation of delivery agents used for introduction of small interfering rnas into feline corneal cells excess dietary lysine does not cause lysinearginine antagonism in adult cats effects of physiologic concentrations of l-lysine on in vitro replication of feline herpesvirus 1 oral supplementation with l-lysine did not prevent upper respiratory infection in a shelter population of cats potent inhibition of feline coronaviruses with peptidyl compounds targeting coronavirus 3c-like protease peptides corresponding to the predicted heptad repeat 2 domain of the feline coronavirus spike protein are potent inhibitors of viral infection synergistic antiviral effect of galanthus nivalis agglutinin and nelfinavir against feline coronavirus effect of polyprenyl immunostimulant on the survival times of three cats with the dry form of feline infectious peritonitis an update on feline infectious peritonitis: virology and immunopathogenesis randomized, placebo controlled study of the effect of propentofylline on survival time and quality of life of cats with feline infectious peritonitis interferons: signaling, antiviral and viral evasion use of recombinant interferon omega in feline retrovirosis: from theory to practice relevance of feline interferon omega for clinical improvement and reduction of concurrent viral excretion in retrovirus infected cats from a rescue shelter oral recombinant feline interferon-omega as an alternative immune modulation therapy in fiv positive cats: clinical and laboratory evaluation comparative efficacy of a recombinant feline interferon omega in refractory cases of calicivirus-positive cats with caudal stomatitis: a randomised, multi-centre, controlled, doubleblind study in 39 cats lowdose interferon-treatment for feline immunodeficiency virus infection therapeutic effects of recombinant feline interferon-omega on feline leukemia virus (felv)-infected and felv/feline immunodeficiency virus (fiv)-coinfected symptomatic cats effects of topical ocular administration of high doses of human recombinant interferon alpha-2b and feline recombinant interferon omega on naturally occurring viral keratoconjunctivitis in cats effect of feline interferon-omega on the survival time and quality of life of cats with feline infectious peritonitis key: cord-009813-o8ai730r authors: wang, wei; xiong, liang; wang, pengyun; wang, fubing; ma, qingfeng title: major vault protein plays important roles in viral infection date: 2019-11-26 journal: iubmb life doi: 10.1002/iub.2200 sha: doc_id: 9813 cord_uid: o8ai730r viral replication and related protein expression inside the host cells, and host antiviral immune responses can lead to the occurrence of diverse diseases. with the outbreak of viral infection, a large number of newly diagnosed and died patients infected with various viruses are still reported every year. viral infection has already been one of the major global public health issues and lead to huge economic and social burdens. studying of viral pathogenesis is a very important way to find methods for prevention, diagnosis, and cure of viral infection; more evidence has confirmed that major vault protein (mvp) is closely associated with viral infection and pathogenesis, and this review is intended to provide a broad relationship between viruses and mvp to stimulate the interest of related researchers. viruses are acellular that cannot naturally reproduce outside of the living host cells and only assemble themselves depending on the host cellular metabolism. 1 virion, known as the complete viral particle, consists of nucleic acid surrounded by capsid, which is enveloped with lipids in some viruses. virion is less than 300 nm in diameter, and its self-assembly is very fast, viral replication inside of the host cells may manipulate and damage the host cells, and the antiviral immune response of the host can damage tissue simultaneously. under the effort of viral toxicity and host immunity, the host is prone to get many kinds of diseases. hepatitis b virus (hbv) and hepatitis c virus (hcv) can cause chronic infection, which can lead to liver cirrhosis and subsequently develop hepatocarcinoma, the patients with viral hepatitis serve as reservoirs of infectious virus. 2 some viruses, including hepatitis a virus (hav), human enterovirus, ebola virus, sars virus, and avian influenza, can cause an outbreak of epidemic infection. [3] [4] [5] [6] the typical antibiotics are not effective of antiviral infection, antigenic drift of viruses can make effective treatments ineffective, 7 and treatment of viral infection is still one of challenges for humanity. abbreviations: aids, acquired immunodeficiency syndrome; atf, activating transcription factors; c/ebpβ, ccaat-enhancer-binding protein β; egf, endothelial growth factor; eif4a, eukaryotic initiation factor 4a; erk, extracellular signal-related kinase; irf7, interferon regulatory factor 7; mapk, mitogen-activated protein kinase; mda5, melanoma differentiation-associated protein 5; mdm, monocytederived macrophages; mvp, major vault protein; myd88, myeloid differentiation primary response 88; nf-kb, nuclear factor kappa-lightchain-enhancer of activated b cells; pbmc, peripheral blood mononuclear cells; pkm2, pyruvate kinase isozyme m2; prrs, pattern recognition receptors; pten, phosphatase and tensin homolog deleted on chromosome 10; srsfs, serine/arginine-rich splicing factors; stat-1, signal transducer and activator of transcription-1 . recent studies have shown that many host-encoded proteins are associated with viruses: heat shock protein 70 is incorporated into the virions of human immunodeficiency virus type 1 (hiv-1) 8 ; serine/arginine-rich splicing factors (srsfs) are related to viral replication, srsf2 promotes anogenital tumorigenesis by maintaining the stability of e6e7 mrnas of human papillomavirus 16 (hpv16), which is the pathogen of anogenital cancer; hiv-1 replication is increased by srsf1, srsf4, and srsf10 within the host cells 9 ; 36 host-encoded proteins are presented in influenza virions 10 ; mvp is involved in antiviral immune response 11 ; and the study of hostencoded proteins in relation to viruses contributes to finding novel targets for antiviral drugs. vaults, the large ribonucleoprotein particles, are composed with mvp, poly (adp-ribose) polymerase, telomerase-associated protein-1 (tep1), and one or more noncoding rna. 12, 13 the human mvp, encoded by mvp gene that is located in chromosome 16p11.2, 14 is highly conserved during evolution 15, 16 and predominant component of vaults. [17] [18] [19] [20] the expression of mvp is very strong and widespread, 21 the mvp is mainly located in the cytoplasm and associated with the cytoskeleton, and a small amount is localized at or around the nuclear membrane and the nuclear pore complex. 22, 23 current studies have confirmed that mvps are associated with multidrug resistance in treatment of non-small lung cancer, 24 human colon cancer, 25 and mesial temporal lobe epilepsy with hippocampal sclerosis. 26 mvp/vaults play important roles in several signal transduction pathways, suppress c-jun-mediated ap-1 transactivation by associating with cop1, 27 participate the phosphoinositide 3-kinase pathway by interacting with endogenous phosphatase and tensin homolog deleted on chromosome 10 (pten) with the help of ca2+ modulation, 28 act as a signaling scaffold protein of extracellular signal-related kinase (erk)/mitogen-activated protein kinase (mapk) pathway by interacting with src in response to endothelial growth factor (egf), 29 and affect the jak-stat signaling pathway by responding and interfering the interferon (ifn)-gamma-mediated stat1 signals. 30 growing evidences also confirmed that mvp is closely associated with other multiple cellular processes, such as nuclear-cytoplasmic transport, 31 malignant transformation, 32 senescence/ aging, 33 autophagy, 34 and innate immunity. 35 interestingly, mvp has been linked to several types of viral infectious diseases as well as to virus-mediated immune responses. 29, 36 here, we focus on the roles of mvp in the intracellular viral replication and host immune responses. the innate immune response, including the production of ifn-1, is the first barrier of eliminating invaded pathogens early. 37 in host cells, tlrs, rig-1 (rig-i-like receptor dsrna helicase enzyme), and mda5 (melanoma differentiation-associated protein 5) act as pattern recognition receptors (prrs), ifn-stimulated proteins, and sensors for viral infection. [38] [39] [40] the interferon regulatory factor 7 (irf7) plays the master transcriptional role in viral infection-induced ifn production and immune responses, [41] [42] [43] activates ifn-β production mediated by myd88 (myeloid differentiation primary response 88)independent rig-1/mda5 pathway, also activates ifn-α production mediated by the myd88-dependent tlrs pathway. [44] [45] [46] the ifn-1 inhibits viral replication (including hcv, influenza a virus [iav], and hiv) by the production of ifn-stimulated effective proteins. 11, 47, 48 after host cells or tissues are infected by hcv, prrs of host cells recognize stimulation signals of products of hcv processing, the interaction between prrs and stimulation signals activates ikba kinase to phosphorylate ikbα, 49 which is associated with nf-kb protein complex in the cytoplasm, phosphorylated ikbα is released from nf-kb complex and degraded by ubiquitin-proteasome pathway, 50 free nf-kb complex translocates to the nucleus, and subsequently activates mvp transcription under coactivators including hcv protein ns5a. 11 hcv infection also induces mvp expression through the sp1 signal pathways, and the infection of vesicular stomatitis virus (vsv), iav, and enterovirus 71 (ev71) has the same effect with hcv infection. 11 inducible mvp is helpful for the nuclear translocation of irf7 and nf-kb, and performs antiviral activity by promoting endogenous ifn-1 production and expression of the ifn-stimulated genes. the production of ifn is the critical step in an innate immune response, and mvp plays strong antiviral activity in an ifn-1-dependent manner. with the advent of effectively prophylactic vaccines and antiviral drugs, hbv infection remains a global public health problem, 51,52 an estimated 240 million people with chronic hbv infection are hbv carriers, 53 deadly complications of hbv chronic infection (including cirrhosis and hepatocellular carcinoma) result in approximately 600,000 deaths per year, 54 and hbv infection brings heavy economic pressure for individuals and heavy social burden for the world. as a type of pathogen, hbv causes host cells to produce ifns to increase protective defense of host immune system, 55 ifns play important roles of antivirus by regulating the host immune system, and have been used to treat some cancers 56 and hbv infection. 57, 58 hbv virus infection leads to the production of type 1 ifns by two main pathways. toll-like receptors 3/4 (tlr 3/4) recognize viral nucleotides and glycolipids and recruit the adaptor protein trif (tir-domaincontaining adapter-inducing ifn-β), trif interacts with traf6 (tumor necrosis factor [tnf] receptor-associated factor 6) to activate nf-kb (nuclear factor kappa-lightchain enhancer of activated b cells), and activated nf-kb provokes ifnb production. 59, 60 another pathway is triggered by tlr7/8 and tlr9, tlrs recognized viral nucleotides in the endosome recruit myd88, 61 in turn recruit irak1/4 (interleukin-1 receptor-associated kinase 1/4) 62 to the complex and interact with traf6 (tnf receptorassociated factor 6), 63, 64 and then activate irf5/7 (ifn regulatory factor 5/7) to induce ifnα expression. mvp is a virus-induced protein, and the level of mvp in peripheral blood mononuclear cells (pbmcs), sera, and liver tissue derived from patients with chronic hepatitis b (chb) is higher than healthy individuals; mvp expression is also increased in hbv stable expression cell lines (hepg2.2.15 and huh7.37) and hbv-infected hepatocarcinoma cell lines (hepg2 and huh7). 11 during hbv infection, tlrs recruit and activate myd88, which interacts with irak1/4, irf5/7, and traf6 to form a complex, 62 the middle domain (aa 310-620) of mvp can interact with myd88, high expressed mvp joins the myd88-mediated complex by interacting with myd88 to promote ifn-1 production through translocation of irf7 and nf-kb from the cytoplasm to the nucleus. 11, 65 however, hbsag and hbeag competitively bind the myd88-binding region of mvp and suppress the ifn-1 production by disrupting mvp/myd88 interaction; the ifn-1 increment effect induced by mvp is counterattacked through hbeag and hbsag binding to mvp 11 (figure 1 ). evidence suggests that hbv has other strategies to suppress the host immune response. hbv polymerase (pol) may inhibit ifn-ɑ-induced myd88 induction, 66 hbeag suppresses tlr-induced ifn-β, 67 hbsag can block the irf-7 mediated ifn-ɑ production pathway, 68 and multiple mechanisms lead to hbv immune escape. when the host is attacked by harmful pathogens including viral infection, one of protective immune response is inflammation to eliminate damage. 69 ifn to interfere viral replication, 55 interleukin 6 (il-6) acted as a proinflammatory cytokine, 70 and interleukin 8 (il-8) served as a chemokine for neutrophils and monocytes 71 are important mediators of immune response, and activation of il-6 and il-8 gene expression is regulated by transcription factors. 72, 73 activator protein 1 (ap-1), composed of proteins belonging to c-fos, c-jun, activating transcription factors (atf) and maf families, 74 is a heterodimeric complex and acts as a transcription factor. 75 the function of ap-1 complex is heavily dependent on the c-fos and c-jun subunits, 76 ap-1 complex binds dna at ap-1 specific sites at the promoter and enhancer regions of target genes and increases target gene expression, 77, 78 and researchers had confirmed that the ap-1 complex is involved in il-6 and il-8 regulation. 79, 80 ccaatenhancer-binding protein β (c/ebpβ) is a member of the c/ebp transcription factor family, the gene of c/ebpβ can be translated into three polypeptides: the 38 kda and 34 kda liver-enriched transcriptional activating proteins (laps), and the 20-kda liver-enriched transcriptional inhibitory protein (lip). 81 c/ebp proteins interact with certain gene promoters containing ccaat box motif, then recruit co-activators to promote gene expression. 81 the promoters of il-6 and il-8 consists of the ccaat box motif region, wherein c/ebpβ can bind and affect il-6 and il-8 expression. 82 f i g u r e 1 hbsag and hbeag weaken the effect of mvp on promoting ifn production in order to restrict the spread of infected virus, some activated transcription factors contribute to the production of inflammatory-related cytokines and chemokines. iav, as a kind of negative single-stranded rna viruses (ssrna), produces replicative intermediate double-stranded rna (dsrna) in the infected cells, 83 dsrna and the synthetic dsrna analog polyinosinic-polycytidylic acid (poly[i:c]) are recognized by tlr3, 29, 84 then activate the tlr3-ifn production pathway to robustly express type i ifns. 59, 60 mvp, as a regulator in the proinflammatory response and an effector in ifn signaling pathway, increases to against viral replication during viral infection. 65 mvp has been proven to be a nuclear-cytoplasmic transport protein 30 and interacts with c-fos of the ap-1 complex components and c/ebpβ-laps. 48 the interaction promotes the ap-1 complex and c/ebpβ-laps translocation from the cytoplasm to nucleus and follows to activate the il-6 and il-8 expression by the ap-1 complex and c/ebpβ-laps binding to the il6 and il8 promoters, and mvp plays a synergistic role in the expression of il-6 and il-8. 48 the expression of mvp, il-6, and il-8 increases simultaneously in iavinfected a549 or dsrna-stimulated pbmcs, and the expression of il-6 and il-8 is impaired in mvp knockdown cells and knockout mice 48 ; mvp plays a pivotal role in virus-triggered proinflammatory response by mediating the ap-1 and c/ebpβ signaling pathways. the model of mvp functions for proinflammatory response is summarized in figure 2 . hepatitis e virus (hev), belonged to the genus hepevirus, is classified as a positive-strand rna virus ([+] ssrna virus), 85 and hev infection is an important public health problem. hev is mostly transmitted via the fecal-oral route in developing countries under poor sanitary conditions, 86, 87 and often spread in many countries by food borne, 88 blood transfusion, 89, 90 and zoonotic origin. 91 hev can cause chronic infection in immunosuppressed patients, pegylated ifn-alpha-2b is used in the treatments for chronic hepatitis e (che) virus infection in liver transplant patients, 92 pegylated ifn-alpha-2a is used in the treatments for che virus infection in a hemodialysis patient, 93 and ribavirin as monotherapy may be effective in the treatment for che virus infection in solid-organ transplant patients. 94 silvestrol is a natural cyclopenta(b)benzofuran and acts as an inhibitor of the eukaryotic initiation factor 4a (eif4a) via hindering translation initiation from the 5 0capped and 5 0 -utr of mrnas. 95 the hev is a (+) ssrna virus containing 5 0 -cap and 5 0 -utr structure, 96 the released hev particles from persistently hev-infected a549 cells treated with silvestrol are robustly reduced, which are caused by the decrease of the intracellular hev capsid protein. 97 silvestrol also affect the expression and localization of antiviral host factor mvp, the mvp amount of the cytoplasm is reduced after treating with silvestrol in hev-infected cells, and the mvp transfers from the cytoplasm to the perinuclear area 97 that affects mvp-mediated ifn production. 98 the translation of mvp is highly activated to play an antiviral role by hev infection; however, the change of translation and cytoplasmic localization affected by the silvestrol treatment counteracts part of antiviral effect, mvp plays a complex interplay between the anti-hev replication and the effect of treating with silvestrol for hev infection. the infection of hiv is the pathogenesis of acquired immunodeficiency syndrome (aids) and one of major global public health issues. 99 hiv infected immune cells, including monocytes, lymphocytes, and macrophages, act as stable rival reservoirs, 100 and are main barrier to f i g u r e 2 mvp plays a pivotal role in the proinflammatory response caused by (−) ssrna viral infection eradicate virus by antiviral therapy. 101 the level of cystatin b, a cysteine protease inhibitor, is higher in blood monocyte-derived macrophages (mdm) than in placental macrophages, which are more resistant to hiv-1 infection than mdm. 102, 103 the expression of cystatin b is upregulated in hiv-1-infected mdm, 104 and cystatin b promotes hiv-1 replication by interacting with pyruvate kinase isozyme m2 (pkm2), 105 which is associated with the cocaine enhancement of hiv-1 replication. 106 in hiv-infected mdm, upregulated cystatin b interacts with mvp 105 and signal transducer and activator of transcription-1 (stat-1). 103 mvp, as an ifn-responsive protein, directly inhibits tyrosine phosphorylation of stat-1 to weaken ifn-induced antiviral response by interfering the jak/stat signal pathway, 29 then promote hiv replication. cystatin b directly interacts and decreases tyrosine phosphorylation of stat-1, and inhibits ifn-β response and stat-1 translocation from the cytoplasm to nucleus to reduce jak/stat signal pathway activity, and ultimately promote hiv replication. 105 under the cooperation of the cystatin b and mvp, hiv replication is activated by the damage of jak/stat signal pathway activity mediated by the low tyrosine phosphorylation of stat-1. mvp is involved in the diversely cellular processes, including multiresistant cancers, 24-26 signal transmission pathways, [27] [28] [29] [30] and immune response associated with viral infection and treatment. 11, 48, 65, 97, 105 viruses with divergent virulence and spreadways can cause diverse human diseases with different types and degrees of damage, as a response of viral infection, studies have confirmed that mvp is enhanced in diverse viral infection, including hbv, hcv, hiv, iav and vsv, and so on. the infection of (−) ssrna viruses (including hcv, vsv, iav, and ev71) or dsrna stimulation activates proinflammatory response by inducing the expression of mvp, il-6, and il-8, enhanced mvp further increase the expression of il-6 and il-8 by translocating transregulatory elements (ap-1 protein complex and c/ebpβ-laps) to the nucleus, 65 and lipopolysaccharide synthesized during viral replication also activates the tlr4 signaling pathway to induce cytokines, chemokines, and ifn-1 against iav replication 107 ; however, the value of mvp in the diagnosis, treatment, and prognosis of viral infection remains unclear and additional studies are still required. hbsag and hbeag compete to bind with mvp, facilitate hbv replication and survival by attenuating the effect of mvp-induced ifn, 48 and ifn and nucleotide analogs (nas) are used for the treatment of patients infected with hbv, the stage of liver diseases is important in guiding antiviral therapy 108 ; however, the effect of mvp on the severity of liver disease and the efficacy of different treatments is unclear. silvestrol, as a potent antiviral compound, inhibits hev assembly by interfering hev capsid protein translation, but deactivates the antiviral effect of mvp by translocating mvp to the perinuclear membrane 97 ; cystatin b, as a cysteine protease inhibitor, increases hiv replication by interacting with mvp and pkm2 to inhibit ifn response and tyrosine phosphorylation of stat-1. 105 mvp plays an opposite role in hiv infection by comparing with iva and hbv infection, weakens the antiviral efficacy of silvestrol in the treatment of hev infection, and additional studies are necessary to clarify the role of mvp more clearly in viral infection. i would like to thank my collaborators for their kind help to organize the thoughts and concepts. synthetic viruses: a new opportunity to understand and prevent viral disease significance of hbv dna by pcr over serological markers of hbv in acute and chronic patients. indian outbreak of infection with hepatitis a virus (hav) associated with a foodhandler and confirmed by sequence analysis reveals a new hav genotype ib variant survey of enterovirus infections from hand, foot and mouth disease outbreak in china who ebola response team. ebola virus disease in west africa -the first 9 months of the epidemic and forward projections cross-species virus transmission and the emergence of new epidemic diseases the influenza viruses specific incorporation of heat shock protein 70 family members into primate lentiviral virions physiological and pathological function of serine/arginine-rich splicing factor 4 and related diseases cellular proteins in influenza virus particles human hepatitis b virus surface and e antigens inhibit major vault protein signaling in interferon induction pathways isolation and characterization of a novel ribonucleoprotein particle: large structures contain a single species of small rna the vault complex the lrp gene encoding a major vault protein associated with drug resistance maps proximal to mrp on chromosome 16: evidence that chromosome breakage plays a key role in mrp or lrp gene amplification vaults. ii. ribonucleoprotein structures are highly conserved among higher and lower eukaryotes cryoelectron microscopy imaging of recombinant and tissue derived vaults: localization of the mvp n termini and vparp the drug resistance-related protein lrp is the human major vault protein the 193-kd vault protein, vparp, is a novel poly(adp-ribose) polymerase characterization of mvp and vparp assembly into vault ribonucleoprotein complexes vaults and telomerase share a common subunit, tep1 vaults are upregulated in multidrugresistant cancer cell lines evidence that vault ribonucleoprotein particles localize to the nuclear pore complex characterization of the sea urchin major vault protein: a possible role for vault ribonucleoprotein particles in nucleocytoplasmic transport mechanisms underlying lung resistance-related protein (lrp)-mediated doxorubicin resistance of non-small cell lung cancer cells molecular basis for the expression of major vault protein induced by hyperosmotic stress in sw620 human colon cancer cells major vault protein (mvp) gene polymorphisms and drug resistance in mesial temporal lobe epilepsy with hippocampal sclerosis major vault protein, in concert with constitutively photomorphogenic 1, negatively regulates c-jun-mediated activator protein 1 transcription in mammalian cells pten associates with the vault particles in hela cells crosstalk between src and major vault protein in epidermal growth factordependent cell signalling the major vault protein is responsive to and interferes with interferon-gamma-mediated stat1 signals phosphatase and tensin homologue deleted on chromosome 10 (pten) has nuclear localization signal-like sequences for nuclear import mediated by major vault protein lung resistance-related protein as a predictor of clinical outcome in advanced testicular germcell tumours on the role of major vault protein in the resistance of senescent human diploid fibroblasts to apoptosis listeria and autophagy escape: involvement of inlk, an internalin-like protein host resistance to lung infection mediated by major vault protein in epithelial cells evaluation of mdr1, lrp, mrp, and topoisomerase iialpha gene mrna transcripts before and after interferonalpha, and correlation with the mrna expression level of the telomerase subunits htertand tep1 in five unselected human melanoma cell lines innate immunity to virus infection pathogen recognition and innate immunity rig-i-mediated antiviral responses to single-stranded rna bearing 5 0 -phosphates length-dependent recognition of double-stranded ribonucleic acids by retinoic acid-inducible gene-i and melanoma differentiation-associated gene 5 irf-7 is the master regulator of type-i interferon-dependent immune responses irf family of transcription factors as regulators of host defense convergence of the nf-kappab and irf pathways in the regulation of the innate antiviral response toll-like receptors in innate immunity the immunobiology of the tlr9 subfamily pattern recognition receptors and control of adaptive immunity a diverse range of gene products are effectors of the type i interferon antiviral response inducible major vault protein plays a pivotal role in double-stranded rna-or virus-induced proinflammatory response nf-kappab regulation in the immune system ikk-1 and ikk-2: cytokine-activated ikappab kinases essential for nf-kappab activation global epidemiology of hepatitis b virus infection: new estimates of agespecific hbsag seroprevalence and endemicity time trends of chronic hbv infection over prior decades-a global analysis estimations of worldwide prevalence of chronic hepatitis b virus infection: a systematic review of data published between hepatitis b virus infection an overview of the immune system the role of interferon in cancer therapy: a current perspective the role of interferon therapy in hepatitis b combination therapy (interferon alfa and ribavirin) in the treatment of chronic hepatitis c: a rapid and systematic review distinct poly(i-c) and virus-activated signaling pathways leading to interferonbeta production in hepatocytes the host type i interferon response to viral and bacterial infections the family of five: tir-domaincontaining adaptors in toll-like receptor signalling the ikappab kinase complex regulates the stability of cytokineencoding mrna induced by tlr-il-1r by controlling degradation of regnase-1 sequential control of toll-like receptor-dependent responses by irak1 and irak2 traf6 is a signal transducer for interleukin-1 major vault protein: a virusinduced host factor against viral replication through the induction of type-i interferon hepatitis b virus polymerase impairs interferon-alpha-induced stat activation through inhibition of importin-alpha5 and protein kinase c-delta hepatitis b virus suppresses toll-like receptor-mediated innate immune responses in murine parenchymal and nonparenchymal liver cells hbsag inhibits tlr9-mediated activation and ifn-alpha production in plasmacytoid dendritic cells chronic inflammation: importance of nod2 and nalp3 in interleukin-1beta generation the pro-and anti-inflammatory properties of the cytokine interleukin-6 tumor-produced interleukin-8 attracts human myeloid-derived suppressor cells and elicits extrusion of neutrophil extracellular traps (nets) activation of il-8 gene expression by helicobacter pylori is regulated by transcription factor nuclear factor-kappa b in gastric epithelial cells transcription factors nf-il6 and nf-kappa b synergistically activate transcription of the inflammatory cytokines, interleukin 6 and interleukin 8 fos/ap-1 proteins in bone and the immune system ap-1 subunits: quarrel and harmony among siblings the role of jun, fos and the ap-1 complex in cell-proliferation and transformation the c-fos protein interacts with c-jun/ap-1 to stimulate transcription of ap-1 responsive genes translational regulation mechanisms of ap-1 proteins effects of il-10 and il-4 on lps-induced transcription factors (ap-1, nf-il6 and nf-kappa b) which are involved in il-6 regulation. leukemia human cytomegalovirus induces interleukin-8 production by a human monocytic cell line, thp-1, through acting concurrently on ap-1 and nf-kappab-binding sites of the interleukin-8 gene ccaat/enhancer binding proteins are critical components of the transcriptional regulation of hematopoiesis the c/ebpb isoform 34-kda lap is responsible for nf-il-6-mediated gene induction in activated macrophages, but is not essential for intracellular bacteria killing cutting edge: influenza a virus activates tlr3-dependent inflammatory and rig-i-dependent antiviral responses in human lung epithelial cells detrimental contribution of the toll-like receptor (tlr)3 to influenza a virus-induced acute pneumonia hepatitis e virus (hev): molecular cloning and sequencing of the full-length viral genome hepatitis e virus hepatitis e: discovery, global impact, control and cure public health risks associated with hepatitis e virus (hev) as a food-borne pathogen seroprevalence of hepatitis e virus (hev) and detection of hev rna with a transcriptionmediated amplification assay in blood donors from catalonia hepatitis e virus in blood components: a prevalence and transmission study in southeast england chronic hepatitis e with cirrhosis in a kidney-transplant recipient treatment of chronic hepatitis e in liver transplant recipients with pegylated interferon alpha-2b three-month pegylated interferon-alpha-2a therapy for chronic hepatitis e virus infection in a haemodialysis patient ribavirin for chronic hepatitis e virus infection in transplant recipients antitumor activity and mechanism of action of the cyclopentabbenzofuran, silvestrol hepatitis e virus (hev) protease: a chymotrypsinlike enzyme that processes both non-structural (porf1) and capsid (porf2) protein inhibition of hepatitis e virus spread by the natural compound silvestrol nuclear localization of the major vault protein in u373 cells emerging concepts in the immunopathogenesis of aids quantification of latent tissue reservoirs and total body viral load in hiv-1 infection the latent reservoir for hiv-1: how immunologic memory and clonal expansion contribute to hiv-1 persistence proteomic analyses associate cystatin b with restricted hiv-1-1 replication in placental macrophages cystatin b associates with signal transducer and activator of transcription 1 in monocytederived and placental macrophages restricted hiv-1 replication in placental macrophages is caused by inefficient viral transcription inhibition of interferon response by cystatin b: implication in hiv replication of macrophage reservoirs enhancement of hiv-1 replication by opiates and cocaine: the cytokine connection the tlr4-trif pathway protects against h5n1 influenza virus infection update on prevention, diagnosis, and treatment of chronic hepatitis b: aasld 2018 hepatitis b guidance the authors declare no potential conflict of interest.orcid pengyun wang https://orcid.org/0000-0001-6801-0705 fubing wang https://orcid.org/0000-0002-5971-2622 qingfeng ma https://orcid.org/0000-0002-1676-3235 key: cord-002630-5616n73p authors: dargahi, narges; katsara, maria; tselios, theodore; androutsou, maria-eleni; de courten, maximilian; matsoukas, john; apostolopoulos, vasso title: multiple sclerosis: immunopathology and treatment update date: 2017-07-07 journal: brain sci doi: 10.3390/brainsci7070078 sha: doc_id: 2630 cord_uid: 5616n73p the treatment of multiple sclerosis (ms) has changed over the last 20 years. all immunotherapeutic drugs target relapsing remitting ms (rrms) and it still remains a medical challenge in ms to develop a treatment for progressive forms. the most common injectable disease-modifying therapies in rrms include β-interferons 1a or 1b and glatiramer acetate. however, one of the major challenges of injectable disease-modifying therapies has been poor treatment adherence with approximately 50% of patients discontinuing the therapy within the first year. herein, we go back to the basics to understand the immunopathophysiology of ms to gain insights in the development of new improved drug treatments. we present current disease-modifying therapies (interferons, glatiramer acetate, dimethyl fumarate, teriflunomide, fingolimod, mitoxantrone), humanized monoclonal antibodies (natalizumab, ofatumumab, ocrelizumab, alemtuzumab, daclizumab) and emerging immune modulating approaches (stem cells, dna vaccines, nanoparticles, altered peptide ligands) for the treatment of ms. in the early 1900s, only a few cases of multiple sclerosis (ms) were reported, which quickly became a common occurrence for admission to neurological wards. today, ms accounts over 2.5 million affected individuals with an estimated cost of us$2-3 billion per annum [1] . the distribution of ms varies according to geographic location. for example, the further north or south from the equator the higher the prevalence of ms; countries that lie on the equator have extremely low prevalence compared to scotland, norway, and canada. the prevalence of ms has increased progressively over time with 30/100,000 diagnosed in 2008 to 33/100,000 diagnosed in 2013 globally. in fact, in a norwegian cohort over 53 years (1961-2014) , the prevalence increased from 20 to 203/100,000 and the incidence increased from 1.9 to 8/100,000 [2] . it is possible that the increase in prevalence is due to improved diagnostic procedures and reporting and changes in lifestyle (lack of vitamin d and increased smoking) [1] . ms is commonly diagnosed between 20 years and 40 years of age although it can affect younger and older individuals [3] , and most commonly affects those with a genetic predisposition (major histocompatibility complex (mhc) class ii phenotype, human leukocyte antigen (hla)-dr2 and hla-dr4 most commonly affected). in fact, the incidence of ms is increased 10-fold in monozygotic information is gathered, and transferred to the rest of the body [25, 27] . ms involves 2 main steps, (i) myelin sheath damage resulting in formation of lesions in the cns and (ii) inflammation, which together destroy the neuron tissue [25, 28] . in ms, damage of oligodendrocytes and destruction of myelin sheath leads to breakdown of the nerve axon and loss of neuronal function [28] . demyelination increases the inflammatory activation processes leading to damage of bbb and stimulation of macrophage activation and oxidative stress pathways [29] . the white matter lesions include myelin breakdown together with infiltration of monocytes, b cells, t cells and dc [30] . microglia and macrophages are the main innate immune cells present in ms lesions where they either act together with t and b cells, or directly cause neuroinflammatory tissue damage [31] . cells involved in the inflammatory process include those that are both in the innate and adaptive immune systems and are described below (figure 1 ). nkt cells share properties of both t cells and nk cells and recognize glycolipid antigens presented in complex with the mhc class i-like molecule, cd1d. two subsets of nkt cells have been identified (type i, invariant nkt (inkt) cells and type ii, variant nkt (vnkt) cells) and are implicated in the pathogenesis of ms in humans and in the murine model of ms, experimental autoimmune encephalomyelitis (eme). inkt cells express cell surface markers characteristic of activated or memory t cells (cd25, cd44, cd69) with the majority being cd4 + as well as markers characteristic of nk cells (nk1.1 or cd161, ly49). following activation of inkt cells (via binding to α-galcer-cd1d complex) an array of cytokines is secreted that are associated with both pro-and anti-inflammatory immune responses and play a role in both innate and acquired immunity. as such, inkt cells, (i) secrete interleukin (il)-4 and il-13 which stimulate cd4 + t cells to differentiate into anti-inflammatory th2 cells (il-4, il-10 producers) which inhibit th17, th1, cd8 + t cells in the nkt cells share properties of both t cells and nk cells and recognize glycolipid antigens presented in complex with the mhc class i-like molecule, cd1d. two subsets of nkt cells have been identified (type i, invariant nkt (inkt) cells and type ii, variant nkt (vnkt) cells) and are implicated in the pathogenesis of ms in humans and in the murine model of ms, experimental autoimmune encephalomyelitis (eme). inkt cells express cell surface markers characteristic of activated or memory t cells (cd25, cd44, cd69) with the majority being cd4 + as well as markers characteristic of nk cells (nk1.1 or cd161, ly49). following activation of inkt cells (via binding to α-galcer-cd1d complex) an array of cytokines is secreted that are associated with both pro-and anti-inflammatory immune responses and play a role in both innate and acquired immunity. as such, inkt cells, (i) secrete interleukin (il)-4 and il-13 which stimulate cd4 + t cells to differentiate into anti-inflammatory th2 cells (il-4, il-10 producers) which inhibit th17, th1, cd8 + t cells in the cns; (ii) secrete il-2 brain sci. 2017, 7, 78 4 of 27 and tumor growth factor (tgf)-beta which stimulate the production of t regulatory (treg) cells (il-10, tgf-beta producers) which inhibit th17, th1 and cd8 + t cells in the cns; and (iii) secrete il-4, il-10, il-13, interferon (ifn)-gamma and gm-csf which activate suppressive myeloid derived suppressor cells (mdcs), dc and macrophages which in turn secrete il-10 to activate treg cells and suppress th17, th1 and cd8 + t cells in the cns [32] . due to the pleiotropic properties of inkt cells, they play a role in protecting the host against pathogens, tumors, autoimmunity and are involved in tissue rejection, ischemia reperfusion injury and obesity related diabetes [32] ; deficiency or dysfunction of inkt cells has been shown to be linked to the development of autoimmune diseases. indeed, inkt cell numbers are decreased in patients with ms [32] and are restored in patients in remission [33] . analysis of inkt cells in ms patients in remission showed a th2 cytokine profile, suggesting an immunoregulatory effect of inkt cells in ms [34] . similarly, in the eae mouse model, protection of eae development is associated with high levels of inkt cells and suppression of th1 and th17 cells [35] . interestingly, injections of α-galactosylceramide (α-galcer), and analogues thereof, have potent activities in protecting mice against, cancer, infections, inflammatory conditions and autoimmune disorders. hence, it is possible to develop inkt cell based modulating therapies against ms [36, 37] . like inkt cells, variant nkt (vnkt) cells also share properties of both t cells (cd4 + ) and nk cells (nk1.1) and recognize β-linked glycolipid antigens in complex with cd1d. they are less common in mice compared to inkt cells but are more abundant in humans. of interest, vnkt cells recognize the self-glycolipid, sulphatide, which is abundantly expressed within the myelin sheath suggesting a role in ms although not yet established [38] . likewise, vnkt cells recognizing sulphatide self-myelin ligand are present in high levels in mice with eae suggesting their role in disease progression [38] . mait cells are a subset of t cells of the innate immune system to defend against microbial infections. they are present in the liver, lungs, mucosa and blood and make up to 25% of cd8 t cells in healthy individuals; they also support adaptive immune responses in that they have a memory like phenotype [39] . the mhc class i-like molecule, mri, presents microbial antigens and vitamin b metabolites to mait cells, leading to their activation [39, 40] . however, mait cells have also been implicated in autoimmune diseases such as ms, inflammatory bowel disease and rheumatoid arthritis where they are often noted at the site of autoimmune attack. recently, it was reported that in ms, mait cells are highly present at the sites of demyelination and secrete pro-inflammatory th1 cytokines (ifn-gamma and tnf-alpha) and activate th17 cells (il-17 and il-22 cytokines) [22] ; the major cytokines in the pathogenesis of chronic inflammatory and autoimmune diseases. in addition, mait cell have been noted in white matter inflammatory lesions [41] as well as transcription over expression of mr1 in ms lesions. conversely, it has been reported that mait cells are decreased in blood of patients with rrms [42] . it is not clear whether mait cells exert a protective or a non-protective role, thus a better understanding of how mait cells are involved in ms and of their interactions would aid in a better understanding of the pathogenesis of ms and development of therapeutic strategies. regulatory t cells (tregs; originally known as suppressor t cells) are a subset of cd4 + t cells that modulate immunity, maintain tolerance against self-antigens and prevent autoimmunity. tregs are primarily characterized as foxp3 + cd25 + cd4 + and are anti-inflammatory (secrete il-10). one of the first evidence of the role of treg cells in ms was in mouse eae models, where adoptive transfer of treg cells from control mice into mog or plp induced eae mice prevented the onset and progression of eae [43, 44] . adoptive transfer of treg cells recovering from eae into mog-induced active eae mice resulted in resolution of eae [45] . in addition, induction of treg cells by estradiol or by monocytes under glatiramer acetate treatment reduced clinical signs of mog-eae [46, 47] . furthermore, injection anti-cd28 monoclonal antibody in lewis rats results in treg cell expansion and reduction in eae disease severity [48] . interestingly, injection of anti-cd25 monoclonal antibody, which blocks the effects of treg cells into c57bl/6 mice increased susceptibility to eae induction [45] . in patients with ms however, the frequency of foxp3 + cd25 + cd4 + treg cells does not differ to those in healthy individuals, although the function of such cells are impaired (maturation and migration) [49] . in addition, mrna and protein levels of foxp3 are impaired in treg cells of patients with ms especially in rrms and are normalized during spms [49] . hence, impaired functionality of treg cells is primarily observed in the early stages of ms but not in their chronic stage, suggesting a causative role [50] . further studies of treg cells in ms may aid in the understanding for why tolerance against self-antigens is broken, leading to disease. however, it is not clear whether the impaired function of treg cells is a direct cause of ms or whether such impairment is a general outcome for all autoimmune disorders. macrophages are divided into m1 or m2 based on their pro-or anti-inflammatory cytokine secretion phenotype [51] . m1 macrophage phenotype of mice (f4/80 + cd11b + cd11c + inos + ) and human (cd40 + cd86 + cd64 + cd32 + ) is induced in the presence of interferon (ifn)-gamma and/or toll-like receptor (tlr) ligands such as lipopolysaccharide (lps). m1 macrophages are pro-inflammatory and primarily secrete il-1, il-6, il-12, tnf-alpha, inos and mcp-1 [51] . in general, they stimulate adaptive immune responses. the m2 macrophage phenotype of mice (f4/80 + cd11c − cd301 + arg1 + cd206 + ) and humans (cd163 + cd206 + ) is induced in the presence of il-4, il-10, il-13 and arg1 that blocks inos activity [51] . m2 macrophages are anti-inflammatory and primarily secrete il-1 receptor antagonist, il-4, il-10, transforming growth factor (tgf)-beta1. macrophages play a crucial role in the pathogenesis of ms. in fact, in active demyelinating and early re-myelinating lesions, macrophages are highly present compared to inactive, demyelinated or late re-myelinated lesions [52] . however, a distinction of m1 vs m2 macrophages in human brain tissues is not so clear, with both m1 macrophages and an intermediate subtype (m1/m2, cd40 + cd206 + ) being present [53] . like macrophages, microglia cells are divided into m1-and m2-polarized microglia cells. m1 microglia cells are pro-inflammatory and express cd40, cd74, cd86 and ccr7, whereas, m2 microglia cells are anti-inflammatory and express mannose receptor (cd206) and ccl22. in ms brain lesions however, like macrophages, an intermediate microglia phenotype is present expressing cd40, cd74, cd86 and ccl22 but not cd206 markers [54] . interestingly, in an eae model it was shown that suppression of ccl22 decreased m1 macrophage accumulation in the cns, thus therapies designed to suppress ccl22 have the potential to decrease demyelination and progression of disease. in addition, in mice m1 microglia cells have been found to switch to m2 microglia cells during remyelination, hence m2 polarization is necessary for efficient remyelination [55] . indeed, fasudil (a selective rho kinase inhibitor), injected into eae bearing mice shifted m1 to m2 macrophages and ameliorated the clinical severity of eae [56] . cd4 t cells or t helper (th) cells, recognize short 9-17 amino acid peptides presented on the surface of antigen presenting cells (apc) in complex with mhc class ii. cd4 t cells differentiate into distinct th cells depending on the cytokine secretion profiles [57] . (i) th1 cells are pro-inflammatory and produce high levels of il-2, il-12, tnf-alpha and ifn-gamma; (ii) th2 cells are anti-inflammatory and secrete il-4, il-5, il-6, il-10, il-13, il-25; (iii) th17 cells are pro-inflammatory and secrete high levels of il-17a, il-17f, il-21, il-22, il-24, il-26 and low levels of il-9 and ifn-gamma; (iv) th22 cells which are a combination of th1, th2, th17 phenotype and secrete il-13, il-22 and tnf-alpha and (v) the newest addition to the th subset, th9, was identified for its potent secretion of il-9. th1, th9, th17 cells are key contributors to ms by increasing inflammation within the milieu of the myelin site. th1 cells and their pro-inflammatory cytokine products are present in high levels within the demyelinating axon and cns lesions of humans and in mog, plp or mbp induced eae in mice. th1 cells recognize mog, plp and mbp peptide epitopes presented in the context of mhc class ii, hla-drb1*1501 (hla-dr2, hla-dr15) and hla-drb1*04 (hla-dr4) alleles. as a result cd4 t cells become activated, cross the blood brain barrier and induce cns autoimmunity. some drug therapeutics target the mhc class ii-peptide-t cell receptor (tcr) complex in an attempt to modulate or divert th1 responses to therapeutic th2 responses. indeed, it was recently shown that dimethyl fumarate (dmf) injection in rrms patients reduced th1, th17 and cd8 t cells and increased th2 cells; this resulted in high levels of il-4 and decreased levels of ifn-gamma and il-17 [58] . in addition, we have shown that mannan conjugation of self-mbp, plp or mog native peptides or altered peptide ligands, are able to divert th1 responses to th2 responses in human pbmc from ms patients, in immunized mouse spleen cells and are able to ameliorate eae in mice [59] [60] [61] [62] [63] [64] [65] [66] [67] [68] [69] [70] [71] [72] [73] . the role of th9 cells in ms is not as clear although in mice, il-9 and th9 cells induce eae and inflammation and il-9 knockout mice are protected from developing eae [74] . th17 cells play a crucial role in the pathogenesis of ms in both mice and humans by inducing an inflammatory milieu. in fact, il-17a is present at high levels in cns lesions, cerebrospinal fluid and in the serum of patients with ms [75] . th17 cells express high levels of ccr6 which binds to the ligand ccl20 on vascular endothelial cells, enabling their entry through the blood brain barrier where they secrete pro-inflammatory cytokines including il-17a. in addition, il-17 interferes with the remyelination process. of interest, anti-il-17a humanized neutralizing monoclonal antibody (ain457 or secukinumab) injected in patients with ms showed reduction of lesions compared to placebo-treated control subjects [75] . in addition, th22 cells are highly present in the peripheral blood and cerebral spinal fluid of patients with active rrms [76] , and il-22 mrna and th22 cells are increased in relapsing ms compared to remitting ms patients [77] . furthermore, th22 cells specifically recognize mbp and are resistant to ifn-beta therapy [76] . il-27, a member of the il-6/il-12 cytokine family, is secreted by macrophages, dendritic cells and microglia cells, with pleiotropic roles in immunomodulation being either pro-or anti-inflammatory. il-27 also stimulates or inhibits t cell differentiation. th1 cells are induced by il-27 whereas th2, th17 and treg cells are inhibited by il-27. in addition, tr1 cells a specialized subset of t cells which secrete il-10 are induced in the presence of il-27 [78] . in 40 patients with rrms, circulating plasma il-27 levels were significantly higher compared to healthy control subjects [79] . likewise, il-27 and il-27r are elevated in post-mortem ms brain lesions compared to non-ms control brains. macrophages and microglia were identified to be the source of il-27 and triggering infiltration of cd4 and cd8 t cells [80] . in addition, the effects of il-27 on microglia cells showed that nitric oxide, tnf-alpha and il-6 were secreted, promoting th1 polarization, suggestive that il-27 enhances microglia neuroinflammation [81] . hence, suppressing il-27 may be a strategy to modulate inflammatory responses in patients with ms. , recognize short antigenic 7-9-mer peptide epitopes presented on the surface of apc in complex with mhc class i. in ms there is a genetic association with hla-a3 [82] ; hla-a2 has been shown to reduce the risk of ms in individuals that also express mhc class ii, hla-drb1*1501. the antigen specificity of cd8 tc1 cells isolated from patients with ms, has been suggested to be against mog, mbp and plp with cytolytic activity against neuronal cells in vitro [83] although their pathogenic role in ms is still not clear. more recently other subsets of cd8 t cells have been identified and are grouped into different subsets based on their cytokine profile. in as such, classical tc1 cells secrete ifn-gamma, tc2 secrete il-4, tc10 secrete il-10, tc17 secrete il-17, tc21 secrete il-21, tc22 secrete il-22 and another subset is characterized by secreting tnf-alpha. in ms, regardless of the stage and activity of disease cd8 t cells are noted in high numbers, much higher than cd4 t cells at a ratio of 10:1 cd8:cd4 t cells. mhc class i is highly expressed within ms lesions and astrocytes, oligodendrocytes, neurons in addition to the classical apc, dcs and macrophages. in fact, cd8 t cells are found in great abundance within cns tissues and cerebrospinal fluid of patients with ms. cd8 t cells present in both acute and chronic ms lesions secrete high levels of il-17 (classed as, tc17 cd8 t cells) [84] . tc17 cells secrete il-17 and tnf-alpha and low ifn-gamma and are negative for granzyme b, perforin and cytolytic activity unlike the classical cd8 tc1 cells. in peripheral blood of patients with spms and rrms elevated levels of tc1 and tc17 cells are noted as well as a high percentage of tnf-alpha secreting cd8 t cells [85] ; tc21 cells are increased in the remission phase of rrms compared to spms. in addition, higher levels of cd8 + ifn-gamma + tnf-alpha + il-17 + t cells in the relapsing phase of rrms compared to remission phase, spms and controls [85] . it is clear that cd8 t cells contribute to the pathogenesis of ms, and it is important to understand how such cells escape t cell tolerance and induce cns autoimmunity in order to design and develop new therapeutics against ms. although there is a presence of t cells in ms plaques, b cells also contribute to the pathogenesis of ms where they secrete autoantibodies and cytokines and being apc they activate t cells. in patients with ms the presence of oligoclonal bands (ocb) in cerebrospinal fluid and brain parenchyma is a consistent finding in over 95% of patients. ocb is a product of clonally expanded b cells and igg synthesis. in ms plaques plasma cells are noted in large numbers where antigen uptake, processing and presentation takes place as well as synthesis of igg. interestingly, over 50 antibodies isolated from cerebrospinal fluid from patients with ms did not react to mbp, plp or mog [86] but some groups reporting that they bind to intracellular proteins such as, mknk1/2, fam84a, akap12a and glial potassium channel kir4.1, or, against intracellular lipid determinants [87, 88] . moreover, anti-mog autoantibodies is a hallmark of childhood ms as well as in some patients with neuromyelitis optical spectrum disorder. it is clear, that abnormal activation of b cells within the cns of patients with ms, suggests that b cells play a role in the pathophysiology of the disease. further studies are required to ascertain whether b cell depletion is able to restore immune function and hence, be used as a therapeutic target against ms. dc are professional apc which process and present antigenic peptide epitopes on their surface in complex with mhc class i or class ii, resulting in cd4 or cd8 t cell stimulation respectively. even though ms is generally associated with predominant auto-reactive t cells, emerging evidence indicates that dcs play an important role in the pathophysiology of ms, primarily due to their t cell activating and cytokine secreting properties. following activation of dcs in the periphery, t cells specific to myelin epitopes are activated inducing pro-inflammatory cytokines aiding their entry through the bbb into the cns. in the cns resident apc and t cells are further activated leading to demyelination and motor deficits. in patients with ms, dcs are abundantly present within inflamed lesions, cerebrospinal fluid and in the circulation and produce high levels of tnf-alpha, ifn-gamma and il-6 [89] . in addition, the expression of co-stimulatory molecules, cd40 and cd80 on dcs are increased in rrms and spms patients, suggesting an activated pro-inflammatory state of dcs, hence their contributing role in the pathogenesis of ms. myeloid-derived suppressor cells (mdsc) are myeloid progenitors, the same lineage to that of macrophages, dc and neutrophils. however, mdsc have strong immunosuppressive properties rather than immune-stimulatory properties as noted with macrophages, dc and neutrophils [90] . their major role is in tumor development and chronic inflammation having immune suppressive effects [90] . as such, it was recently shown following mbp 1-11 peptide immunization in mice, that mdscs were increased adopting a suppressive phenotype, inhibiting the activation of cd4 + t cells via arginase-1 and inducible nitric oxide synthase; such approach inhibited the development of eae in mice [91] . in addition, mdsc secrete inhibitory enzyme indoleamine 2,3-dioxygenase and th2 cytokine, il-10 [92] . it is not clear whether the number of mdscs are reduced or whether their functionality is altered in patients with ms, leading to the failure of mdscs to suppress autoimmune t cells, as a result of disease progression. the use of ex vivo cultured mdscs could be a viable strategy to develop new improved treatments against ms. the majority of the treatments for ms are long term mainly suppressing the immune system however, such immune-suppressants pose increased risks for infections and cancer [27] . alternative treatment options involve disease-modifying therapies such as, interferons, glatiramer acetate, monoclonal antibodies and sphingosine-1-phosphate receptor modulators (table 1, figure 2 ). these therapies have dramatically reduced the number of attacks and decreased disease progression. in fact, interferons are effective in the early relapsing phases of ms but not in the advanced phases of the disease [27] . ultimately, induction of tolerance against self-antigens and re-establishing immune homeostasis can effectively "cure" the disease; such strategies have been the focus of recent research. figure 2 ). these therapies have dramatically reduced the number of attacks and decreased disease progression. in fact, interferons are effective in the early relapsing phases of ms but not in the advanced phases of the disease [27] . ultimately, induction of tolerance against self-antigens and re-establishing immune homeostasis can effectively "cure" the disease; such strategies have been the focus of recent research. patients with ms who present with a relapse are generally treated with corticosteroids intravenously, plasma exchange or adrenocorticotropic hormone injections [50, 93] . although effective in reducing the duration of the relapse and patients recovery faster there are no long-term neuroprotective benefits [27, [94] [95] [96] [97] . the treatment of ms has been a challenge with treatment options being limited mainly to corticosteroids, the potent alkylating agent cyclophosphamide and potent immunosuppressant methotrexate (table 1, figure 2 ). however, with the advent of immunomodulatory drugs in mid-1990s, a big shift was carried to treatment options for the first time [50] . the first disease-modifying drug for rrms, interferon beta-1(ifnβ-1) was the primary key breakthrough for the treatment of ms [98, 99] . disease-modifying agents intend to modify the course of the disease rather than improving symptoms. until the approval of the first oral treatment in 2010 [11] , all ms treatments consisted of either intramuscular or subcutaneous injectable drugs. to date, 13 fda approved disease-modifying drugs are available for rrms, and several more agents are in different developmental stages [9, 11, 65, 66, 69] . in the last 20 years there has been an evolving trend in novel treatments for ms and the global progress of therapies for ms has been quite promising. in general treatments consist of ampyra ® , aubagio ® , avonex ® , betaseron ® , copaxone ® , extavia ® , gilenya ® , lemtrada ® , novantrone ® , plegridy ® , rebif ® , tecfidera ® and tysabri ® [100] . such treatment options consist of alemtuzumab (depletes lymphocytes), daclizumab (blocks the cytokine receptor il-2), dimethylfumarate (combines features of immunomodulatory and immunosuppressive actions), fingolimod (modulates the sphingosine-receptor system), natalizumab (inhibits the migration of lymphocytes) and teriflunomide (inhibits activated t and b cells) [9, 27, 50] . examples of current interferons include, schering ag's betaferon/betaseron (ifnβ-1b), biogen's avonex (ifnβ-1a) and serono/pfizer's rebif (ifnβ-1a). in addition, immune modulating agents include, teva's copaxone ® (copolymer glatiramer acetate), amgen/serono's (novantrone ® ; mitoxantrone), azathioprine, cyclophosphamide (endoxan ® ) and natalizumab ® an a 4 -integrin antagonist [101] [102] [103] . disease-modifying agents have commonly been shown to reduce the rate of relapses, reduce mri lesions and stabilize or delay ms disability. the key therapeutic features of disease-modifying drugs are their anti-inflammatory effects in the relapsing phase of ms, although demyelination leading to chronic disability still remains a major hurdle [27, [104] [105] [106] . some studies, however, have shown that early intervention of disease-modifying drugs to patients with rrms can reduce acute disability or death [27, [107] [108] [109] [110] . in general, disease-modifying drugs main action is by suppressing or altering the immune system. hence, based on this theory that ms is, at least in part, a result of altered or abnormal immune response that results in attack of the myelin sheath. current available drugs and their actions are described below (table 1, figure 2 ). interferon (ifn) type 1 consist of a group of ifns (ifn-α, -β, -ε, -κ, -τ, -δ, -ζ, -ω, -ν) which help regulate the immune system. ifn-β is primarily produced by fibroblasts but other cells such as nk cells, b cells, t cells, macrophages also secrete ifn-β. ifn-β has anti-viral and anti-tumor activity as well as being effective in reducing the relapse rate in patients with ms [106] . the mechanism by which ifn-β acts, is that it balances the expression of pro-and anti-inflammatory cytokines in the brain and decreases the number of inflammatory cells crossing the blood brain barrier. as a consequence, there is decreased inflammation of neurons, increases nerve growth factors and improves neuronal survival. moreover, ifn-β reduces th17 population and il-17 cytokine which are known to be involved in the immunopathophysiology of ms [111] . ifn-β injection subcutaneously or intramuscularly to patients with rrms aims to decrease the relapse rate, duration and severity, however, there is lack of efficacy to long-term disability. avonex was approved in 1996, the first fda approved treatment for rrms. to date there are 3 approaches using ifn-β; ifn-β1a low dosage (avonex ® ), ifn-β1a (rebif ® ) high dosage, and, ifn-β1b (betaseron ® , extavia ® ) high dosage. furthermore, pegifn-β-1a (plegridy ® ) has polyethylene glycol linked to ifn-β-1a allowing it to be active for longer in the body, hence fewer injections are required compared to avonex ® , rebif ® , betaseron ® and extavia ® . the first large scale human clinical trial in patients with rrms using ifn-β was published in 1993 and showed that relapse rates were reduced by 34% in high dose ifn-β1b and by 8% in lower dose compared to placebo group and severity of relapses were also reduced [112] . subsequent 5 year follow-up data showed that ifn-β1a and ifn-β1b decreased lesions up to 30% and reduced the formation of new lesions up to 50%, however, the study failed to show any reduction in disability progression in patients [113] . ifns have no direct neuroprotective effects, however, through their direct effect on cd4 + th1 cells and altering their profile results in decreased demyelination of neurons, which prevents further neuronal damage [114] . despite the impact of ifn-β in disease progression in patients with rrms there are limitations in their use, with side effects ranging from local body aches, skin reactions (swelling, redness), fever, myalgia, flu-like symptoms to more serious side effects such as suicidal thoughts, hallucinations, seizures and heart and liver problems [9] . as a result, many patients have stopped treatment and overall the benefit of using ifns is relatively small. glatiramer acetate (ga) is a synthetic 4-mer peptide (l-glutamic acid, lysine, alanine, and tyrosine) mimic of mbp, which competes with short antigenic mbp peptides in complex with mhc class ii. initially, ga was designed to induce eae but instead it suppressed eae, which was quickly translated into human trials with ms in order to prevent disease progression, as it bound to mhc class ii and inhibited the activation of encephalitogenic t cells [115] [116] [117] [118] . ga diverts th1 cells to th2 cells that suppress inflammatory responses and activate tregs in the periphery [119] . in patients, ga significantly reduced disease symptoms and development of new lesions by up to 30% in rrms, although it showed no improvement in long-term efficacy on progression of disability [120] . ga injection in patients results in side effects ranging from minor (fever, chills) to more serious (cardiovascular, digestive, muscular, respiratory issues). dimethyl fumarate (bg-12) is a methyl ester of fumaric acid that modulates immune responses and was approved by the fda in 2013. bg-12 was shown in phase iii clinical trials to reduce relapse rate and increase the time to disability progression in patients with rrms [121] . bg-12 reduces the migration of inflammatory cells through the blood brain barrier and activates nuclear factor erythroid 2-related factor (nrf2) [122] . nrf2 regulates anti-oxidative proteins that protect cells against oxidative damage and inflammation. in fact, bg-12 protects neuronal cells from oxidative stress by increasing glutathione levels and suppressing pro-inflammatory cytokines from splenocytes in vitro [123] . side effects of bg-12 include diarrhea, abdominal pain, nausea, abnormal liver enzymes and decreased lymphocyte counts. teriflunomide is an active metabolite of leflunomide (an immunosuppressive disease-modifying drug used for rheumatoid arthritis) which inhibits the enzyme dihydroorotate dehydrogenase [124] and inhibits the proliferation of b and t cells. in addition, teriflunomide exerts anti-inflammatory properties by inhibiting ifn-gamma producing t cells while il-4 and il-10 producing t cells are unaffected [125] . in ms, oral administration of teriflunomide reduced relapse rates, ms lesions and decreased disability progression [126] [127] [128] [129] [130] [131] . moreover, permanent discontinuation due to side effects was substantially less common in ms patients who received teriflunomide compared to ifn-β-1a. side effects include, reduced white blood cell count, alopecia, hepatic effects, nausea, diarrhea, numbness in hand and feet, allergic reactions, breathing issues and increased blood pressure. teriflunomide was approved by the fda in 2012 and by ema in 2013 for use in patients with rrms. fingolimod was granted fda approval in 2010 and was the first oral therapy (0.5 mg once daily) available for patients with relapsing forms of ms. fingolimod is a sphingosine 1-phosphate (s1p) receptor modulator, which acts as a super agonist of s1p receptor causing receptor internalization and leading to reduced infiltration of potentially auto-reactive lymphocytes into the cns, and as such, they remain localized in the lymph nodes [132] [133] [134] . in addition, a secondary beneficial effects of fingolimod is that it targets sip receptors on glia cells in the cns, activating signaling pathways within the cns [132, 135] . based on phase iii human clinical trials in patients with rrms (transforms, freedoms and freedoms ii), fingolimod was more effective compared to first line treatment ifnβ-1a and placebo, in reducing the frequency of flare-ups (clinical exacerbations), disability progression, mri outcome measures, including brain volume loss and was associated with clearly identified adverse events [103, 136, 137] . more than 180,000 patients have been treated with fingolimod in clinical trials and post-marketing settings globally, and the total patient exposure now exceeds 395,000 patient-years. side effects include bradycardia (within 6 h after treatment initiation), blurred vision, diarrhea, back pain, headache, cough and vomiting. with reasonable data showing its long-term safety and disease improvement, fingolimod is a great alternative choice for patients with highly active rrms and who prefer the oral treatment option. 3.2.6. mitoxantrone (novantrone ® , immunex/amgen, thousand oaks, ca, usa) mitoxantrone is primarily used to treat certain types of cancers, in particular, non-hodgkin's lymphoma, acute myeloid leukemia, breast and prostate cancer. mitoxantrone is a type-ii topoisomerase inhibitor, which disrupts dna synthesis and dna repair of cancer cells, however, normal cells are also affected. it is a potent immune suppressant, suppressing t cells, b cells and macrophages and reduces pro-inflammatory cytokines (ifn-γ, tnf-α, and il-2) [138, 139] . in patients with spms, intravenous injection of 12 mg/m 2 mitoxantrone every 3 months up to 2 years resulted in reduced disability progression by 84% [140, 141] . however, several side effects are associated with mitoxantrone which range from nausea, vomiting, hair loss, to, cardiotoxicity, leukemia, infertility, infection, leukopenia and thrombocytopenia [11] . as a result, its use has significantly been reduced over time. furthermore, due to the risk of cardiotoxicity and leukemia, there is a limit on the cumulative lifetime dose to be administered to patients [11, 142] . natalizumab is a humanized monoclonal antibody against the cellular adhesion molecule α4integrin. integrins are transmembrane receptors that enable cell-extracellular matrix adhesion activating cell signaling which regulate cell growth, division, survival, differentiation and migration. integrins are expressed on t cells, b cells, monocytes, macrophages, nk cells, dc, neutrophils and eosinophils. interfering or blocking α4-integrin affects immune cell migration across the blood brain barrier, thus, by blocking the interaction between α4-integrin and vascular endothelial adhesion molecule-1, inhibits transendothelial migration to the cns [143] . natalizumab is administered intravenously once a month [144] which reduces activated t cells within the cns, resulting in anti-inflammatory responses and hence, neuroprotective effects [114] . in a phase iii clinical trial natalizumab reduced brain lesions and the rate of disability progression up to 24 months [12, 145] . in addition, natalizumab decreased by 92% of contrast-enhancing lesions, by 83% of new or expanding t2-weighted lesions, and by 76% in new t1-weighted hypointense lesions [146, 147] . natalizumab, was approved by the fda in 2004, but was withdrawn due to 3 cases of rare brain infection, progressive multifocal leukoencephalopathy (pml; that usually leads to death or severe disability), but was re-introduced in 2006 under a special prescription program. however, by 2012 a further 212 cases (or 2.1/1000) of pml were reported to be attributed to natalizumab [148] . despite these reports the fda has not withdrawn natalizumab from the market as the clinical benefits outweigh the risks involved. other side effects include, hepatotoxicity, allergic reactions and increased risks of infection. due to the risks involved with natalizumab, there are reservations over its use as a preferred treatment option. ofatumumab (omb157) is the first fully human type 1 igg1 kappa (igg1κ) monoclonal antibody and is currently licensed for the treatment (of patients with chronic lymphocytic leukemia (intravenously (iv), arzerra ® ). it has also been shown to be beneficial to patients with rheumatoid arthritis, follicular non-hodgkin's lymphoma, diffuse b cell lymphoma and ms. b cells play a role in the pathogenesis of ms. b cells have essential functions in regulating immune response, by activating cd4 + t-cells and regulating t-cell responses via the secretion of cytokines and antibodies. b cells are present at demyelinating areas and in cerebrospinal fluid of patients with ms [149] . cd20 is a marker and present on the cell surface of all b cells. in an attempt to reduce the number of b cells including autoreactive b cells, the use of anti-cd20 antibodies would conceivably improve ms relapses and progression. in fact, there are several humanized anti-cd20 antibodies, such as rituximab [150] , ocrelizumab [151] and ofatumumab [152] , which have shown high efficacy in patients with rrms. in 2015, novartis acquired the rights from glaxosmithkline for the development of ofatumumab in oncology and other autoimmune indications. ofatumumab binds to 2 unique novel epitopes on the cd20 molecule, induces b-cell depletion via complement dependent cytotoxicity and antibody-dependent cell-mediated cytotoxicity causing b cell apoptosis [153] . ofatumumab has demonstrated high efficacy in hematologic malignancies and in rheumatoid arthritis. based on 2 phase ii dosing human clinical studies, ofatumumab demonstrated high efficacy in reducing new mri lesion activity more than 90% and was well tolerated in patients with ms [152] . currently, ofatumumab is a few months ago (march 2017), the fda approved ocrelizumab to be used in ppms, the first drug approved by the fda for this form of ms and phase iv clinical trials were a requirement of the fda to be conducted in order to determine the safety of ocrelizumab in younger patients with ms, ie, risk of cancer and effects on pregnancy (study outcomes due by 2024); although clinical trials in patients with lupus and rheumatoid arthritis were halted due to high rates of infections and increased risk of progressive multifocal leukoencephalopathy [155] . in addition, in patients with ms, there was an increased risk of breast cancer (6/781 females with ms on ocrelizumab compared to 0/668 females with ms in other trials) [155] alemtuzumab is a humanized monoclonal antibody against cd52, a cell surface molecule expressed on b and t cells; mature nk cells, plasma cells, neutrophils and importantly, hematological stem cells do not express cd52. in phase iii clinical trials in patients with rrms, alemtuzumab showed significantly lower annualized relapse rates and mri measures (gadolinium-enhancing lesions, new or enlarging t2 lesions and brain atrophy) and were free of clinical disease longer, compared to ifnβ-1a [156, 157] . alemtuzumab can cause serious side effects including, immune thrombocytopenia, kidney problems, serious infusion problems (trouble breathing, swelling, chest pain, irregular heart beat), certain cancers (blood cancers, thyroid cancer), cytopenia and serious infections. it was approved by the fda in 2014 to be used in rrms patients, but due to the frequent and significant adverse events of alemtuzumab, it is generally used in patients with rrms who have used 2 or more ms drugs and have failed to work. daclizumab is a humanized monoclonal antibody against cd25, the il-2 receptor expressed on the surface of t cells. the mechanism by which daclizumab works is that it blocks the il-2 receptor on t cells, preventing the activation of t cells. it was originally approved by the fda in 1997 to prevent acute kidney transplants (together with corticosteroids and cyclosporine) however its use was halted due to low market demand. in recent years its use has re-emerged to treat patients with rrms, it is injected subcutaneously, once a month [158] . in human clinical trials, daclizumab showed 45% reduced annualized relapse rates and 54% lower in the number of new lesions [158] . the side effects associated with daclizumab are relatively minor compared to other ms drugs, and include infections, skin rashes and liver complications. antigen/peptide specific immunotherapy or using immune cells (i.e., stems cells), aim to restore tolerance while avoiding the use of non-specific immunosuppressive drugs as describe in section 3, is a promising approach to fight autoimmune diseases including ms. as such, a number of approaches have been utilized. multipotent hematopoietic stem cells (hsc) are cells isolated either from the bone marrow, umbilical cord blood or peripheral blood and are transplanted into the recipient. more commonly used for hematological malignancies (leukemia, multiple myeloma) its application has also expanded into autoimmune diseases. the first report of a bone marrow transplant in 1997 in a chronic myelogenous leukemia patient with ms which showed marked improvements in ms brain lesions [159] quickly led to the use of hsc transplantation (hsct) in ms patients. hsct in patients with active rrms, reduce progression in about 70% of patients, decrease relapses dramatically and suppresses inflammatory mri activity [160] . ms patients who have not responded to conventional therapy, who's disease is aggressive with relapsing-remitting course and who are not presenting with high level of disability, are considered appropriate candidates for such treatment [161] . although the clinical efficacy of hsct long term has not been established. the mechanism by which hsct works is that hsct "reboots" the immune system and thus, prevents inflammation associated with the disease. mesenchymal stem cells (msc) are isolated from an adult's bone marrow, are differentiated in vitro for 2-3 weeks and re-injected back into the patient. in recent years a vast amount of research has been conducted in mscs to treat ms with most studies being in mice and eae models, and more recently in human clinical trials. in fact, in a pilot study in advanced ms patients, msc transplantation improved expanded disability scale score with stabilization in 1/7 and disease progression in 1/7 patients and vision and low contrast sensitivity test showed improvement in 5/6 patients with 1/6 showing worsening effects [162] . in a phase ii randomized double-blind, placebo-controlled crossover clinical trial showed lower mean cumulative number of lesions in patients receiving mscs compared to placebo [163] . no serious adverse events were reported. the mechanism of action of msc includes immunomodulation, neuroprotection and neuroregeneration [162] . the use of mscs that reduce mri parameters is a new and emerging research focus to develop new improved treatments for ms. bht-3009, a dna vaccine that encodes the full-length human mbp, was developed with the aim to tolerize patients with ms against mbp [9, 164, 165] . in fact, in 30 patients with rrms or spms who received 4 injections of bht-3009 on weeks 1, 3, 5, 9 with escalating doses of 0.5 mg, 1.5 mg or 3 mg was reported to be safe and conferred positive changes on brain mri and reduced the number of cd4 + t cells [9, 164, 166] . in addition, in a retrospective, randomized double blind, phase ii study in 155 ms patients, bht-3009 had no impact on the risk for persistent black holes (axonal loss and disability progression). however, there was a correlation to those who had generated high anti-igm mbp antibodies to reduced risk of persistent black holes [167] . nanoparticles have extensively been characterized and used as vaccine formulations in pre-clinical models of cancer and infectious diseases [168, 169] . polymeric biodegradable lactic-glycolic acid (plga) nanoparticles loaded with mog 35-55 peptide together with recombinant il-10, were partially endocytozed by dendritic cells, secreted both mog peptide and il-10 in culture media for several weeks in vitro [170] . in mice, plga nanoparticles (mog 35-55 + il-10) showed significant amelioration of eae and reduction of il-17 and ifn-gamma secretion by splenic t cells in vitro [170] . recently, poly(ε-caprolactone) nanoparticles loaded with recombinant human mbp reduced ifn-gamma cytokines, reduced the clinical score and showed only mild histological changes of the myelin sheath [171] . hence, nanoparticles as a delivery method of self-antigens are a promising tool to treat ms. altered peptide ligands (apl) are peptides closely related to the native (agonist) peptide with defined 1-2 substituted amino acid residues which interact with the t cell receptor (tcr) yet retains its binding ability to the mhc [65] . in phase i/ii clinical trial by neurocrine biosciences inc, used an apl of mbp 83−99 , where l-amino acids were changed to d-amino acids at positions 83, 84, 89, 91 (nbi-5788) [172] . however, this mode of apl induced t cell cross reactivity between the apl and the wild-type/agonist mbp 83−99 peptide and adverse events in some patients resulted [173] . a subsequent multi-center double-blinded phase ii clinical trial with nbi-5788 was suspended-th2 responses were induced (il-5, il-13), however, 13/142 patients developed immediate-type hypersensitivity, who also generated anti-nbi-5788 antibodies which cross-reacted with native agonist mbp [83] [84] [85] [86] [87] [88] [89] [90] [91] [92] [93] [94] [95] [96] [97] [98] [99] peptide [172, 174] . national institute of neurological disorders and stroke sponsored trial, cgp77116, was used in a mri-controlled phase ii clinical trial. cgp77116, has ala d-amino acids of mbp 83-99 peptide at positions 83, 84, 89, 91 (cgp77116) of mbp [83] [84] [85] [86] [87] [88] [89] [90] [91] [92] [93] [94] [95] [96] [97] [98] [99] peptide, in order to enhance stability [174] . however, this peptide was poorly tolerated at the dose tested, and the trial had to be discontinued. three patients showed exacerbations to disease of which two were linked to cgp77116 injection with high ifn-gamma and low il-4 (th1-skewing) were secreted by activated cd4 + t cells. these cd4 + t cells also cross reacted with the native agonist mbp 83-99 peptide [175] . accordingly, the problems noted with both nbi-5788 and cgp77116 were likely due to inadequate pre-screening of apl effects on the many clonotypes against the targeted epitopes. thus, although the apl was highly effective at blocking or switching some clones, it activated others. thus, further pre-clinical testing is required and new modified peptides need to be designed, or a carrier needs to be used which further changes the resulting immune response. cyclization of peptides increases the stability, since linear peptides are sensitive to proteolytic enzymes. in addition, cyclic peptides are an important intermediate step and a useful template towards the rational design and development of non-peptide mimetics. while mimetic strategy is a challenging perspective it is worth pursuing in particular for mbp epitope-based ms therapy as it is still in its infancy. efforts to design semi-mimetics of mbp 72-85 epitope by combining non-natural amino acids as spacers and mbp epitope immunophores (ser, arg, glu, ala, gln), led to substances that were effective to some extent in inducing the onset of eae. cyclic peptides are not only as a step towards non-peptide mimetics but also as putative therapeutics in ms [66] . structure activity studies of the immunodominant agonist peptide mbp [87] [88] [89] [90] [91] [92] [93] [94] [95] [96] [97] [98] [99] [83] [84] [85] [86] [87] [88] [89] [90] [91] [92] [93] [94] [95] [96] [97] [98] [99] showed significantly reduced mechanical pain hypersensitivity compared to cyclic mbp [83] [84] [85] [86] [87] [88] [89] [90] [91] [92] [93] [94] [95] [96] [97] [98] [99] peptide. this was associated with reduced t cell and macrophage infiltration to injured nerves of the spinal cord of animals [176] [177] [178] . in addition, these apl decreased cd4 + t cell line proliferation raised from a patient with ms, increased il-10 cytokine secretion, bound to hla-dr4 and were more stable to lysosomal enzymes (cathepsin b, d, h) compared to their linear counterparts [70] . double mutation of k 91 , p 96 to a 91 , a 96 in either linear or cyclic forms were also shown to be active, with suppression of eae in sjl/j mice, higher th2 over th1 cytokines produced, bound to hla-dr4, the cyclic forms were more stable to lysosomal enzymes and induced high levels of il-10 of peripheral blood mononuclear cells from patients with ms [61] . recently, cyclic native agonist mog peptide was shown to ameliorate clinical and neuropathological features of eae in mice compared to its linear counterpart [179] . thus, cyclic peptides, which offer greater stability and are able to modulate immune responses, are novel leads for the immunotherapy of many diseases, such as ms [66] . mannan, a polymannose, isolated from the wall of yeast cells has been shown to bind to the mannose receptor on dendritic cells as well as being a ligand for toll-like receptor 4 [180, 181] . mannan conjugated to muc1 cancer protein induces immune responses in mice and protects mice against tumor challenge. this work was translated into human phase i, ii and pilot iii clinical trials; mannan-muc1 induces protection against cancer recurrence at 18 years follow-up [182] [183] [184] [185] . furthermore, ex vivo cultured dendritic cells pulsed with mannan-muc1 (cvac tm ) and re-injection into patients induces strong cellular and clinical responses in ovarian cancer patients [186, 187] . due to the immunomodulatory properties of mannan, its effects as a carrier to ms peptides were determined. mutations of mbp 83 [139] [140] [141] [142] [143] [144] [145] [146] [147] [148] [149] [150] [151] peptides stimulated ifn-gamma secreting t cells [64] . furthermore, mannan conjugated to the immunodominant agonist mog 35-55 peptide primes non-pathogenic th1 and th17 cells and ameliorates eae in mice [73] ; a phase i human clinical trial is planned using mannan conjugated to mog peptide later this year. it is clear that, mannan is able to divert immune responses from th1 to th2 and is a promising carrier for further studies for the development of immunotherapeutics against ms. dalfampridine (ampyra/fampyra ® , acorda therapeutics) dalfampridine is not intended to delay symptoms or change the course of disease, but rather, to improve motor symptoms such as walking. dalfampridine, is a potassium channel blocker, resulting in improved potassium currents and nerve conductance. dalfampridine is used in patients who have had ms for more than 3 years and it was approved by the fda in 2010. common side effects include nausea, nervousness and dizziness, which are relatively minor compared to other ms drugs. ms is an autoimmune disorder of the cns with an array of immune cells being either activated or suppressed leading to demyelination and disease progression. in addition, genetic predisposition, viral mimicry, vitamin and mineral deficiency, geographical location are also etiological factors that contribute to disease. more recently, citrulination of myelin peptides have been shown to contribute to disease activation [59, 60] . a number of treatment options are available to patients with ms, in particular those with active disease, however due to side effects, limited long term effectiveness and inability to reverse disease, new improved treatment options are required. as described here a number of new and upcoming promising therapeutic candidates are becoming available, although their effectiveness in human clinical trials remains to be determined. recently, it was reported that non-peptide mimetics mapping the mbp 83-96 t cell epitope can function as t cell receptor antagonists, hence such an approach may pave the way to developing alternative and improved immunotherapeutics against ms [189] . with the plethora of information regarding the immunopathophysiology of ms and availability of treatment options and new upcoming treatments, the future holds promise for managing and treating the disease. multiple sclerosis time trends in the incidence and prevalence of multiple sclerosis in norway during eight decades primary progressive multiple sclerosis: part of the ms disease spectrum or separate disease entity? evidence for genetic basis of multiple sclerosis interleukin-10 plays a crucial role in suppression of experimental autoimmune encephalomyelitis by bowman-birk inhibitor immunopathogenesis and immunotherapy of multiple sclerosis immunology of multiple sclerosis improving ms patient care towards immunotherapeutic drugs and vaccines against multiple sclerosis defining the clinical course of multiple sclerosis: results of an international survey currently approved and emerging oral therapies in multiple sclerosis: an update for the ophthalmologist diagnostic criteria for multiple sclerosis: 2010 revisions to the mcdonald criteria the value of conventional high-field mri in ms in the light of the mcdonald criteria: a literature review the diagnostic criteria for multiple sclerosis: from charcot to mcdonald pathological mechanisms in progressive multiple sclerosis blood-brain barrier disruption in multiple sclerosis multiple sclerosis: a coordinated immunological attack against myelin in the central nervous system blood-brain barrier disruption and enhanced vascular permeability in the multiple sclerosis model eae emerging immunopharmacological targets in multiple sclerosis axonal damage is t cell mediated and occurs concomitantly with demyelination in mice infected with a neurotropic coronavirus phenotype and function of regulatory t cells in the genital tract invariant natural killer t cells and mucosal-associated invariant t cells in multiple sclerosis the role of il-17 and th17 lymphocytes in autoimmune diseases th17 cells, but not th1 cells, from patients with early rheumatoid arthritis are potent inducers of matrix metalloproteinases and proinflammatory cytokines upon synovial fibroblast interaction, including autocrine interleukin-17a production multiple sclerosis: therapeutic applications of advancing drug delivery systems promoting remyelination in multiple sclerosis-recent advances therapeutic strategies in multiple sclerosis: a focus on neuroprotection and repair and relevance to schizophrenia multiple sclerosis: new insights and trends. asian pac immune-inflammatory and oxidative and nitrosative stress biomarkers of depression symptoms in subjects with multiple sclerosis: increased peripheral inflammation but less acute neuroinflammation prostaglandins in pathogenesis and treatment of multiple sclerosis nadph oxidase expression in active multiple sclerosis lesions in relation to oxidative tissue damage and mitochondrial injury natural killer t cells in multiple sclerosis and its animal model, experimental autoimmune encephalomyelitis innate immunity modulates autoimmunity: type 1 interferon-beta treatment in multiple sclerosis promotes growth and function of regulatory invariant natural killer t cells through dendritic cell maturation th2 bias of cd4+ nkt cells derived from multiple sclerosis in remission cutting edge: v alpha 14-j alpha 281 nkt cells naturally regulate experimental autoimmune encephalomyelitis in nonobese diabetic mice alpha-galactosylceramide therapy for autoimmune diseases: prospects and obstacles invariant nk t cells: potential for immunotherapeutic targeting with glycolipid antigens prevention of autoimmunity by targeting a distinct, noninvariant cd1d-reactive t cell population reactive to sulfatide the role of mucosal associated invariant t cells in antimicrobial immunity mr1 presents microbial vitamin b metabolites to mait cells non-myeloablative autologous haematopoietic stem cell transplantation expands regulatory cells and depletes il-17 producing mucosal-associated invariant t cells in multiple sclerosis mucosal-associated invariant t cells regulate th1 response in multiple sclerosis cutting edge: cd4+cd25+ regulatory t cells suppress antigen-specific autoreactive immune responses and central nervous system inflammation during active experimental autoimmune encephalomyelitis il-10 is involved in the suppression of experimental autoimmune encephalomyelitis by cd25+cd4+ regulatory t cells natural recovery and protection from autoimmune encephalomyelitis: contribution of cd4+cd25+ regulatory cells within the central nervous system estrogen treatment induces a novel population of regulatory cells, which suppresses experimental autoimmune encephalomyelitis type ii monocytes modulate t cell-mediated central nervous system autoimmune disease selective targeting of regulatory t cells with cd28 superagonists allows effective therapy of experimental autoimmune encephalomyelitis the role of regulatory t cells in multiple sclerosis immunological treatment of multiple sclerosis exploring the full spectrum of macrophage activation macrophages in multiple sclerosis macrophages in inflammatory multiple sclerosis lesions have an intermediate activation status activation status of human microglia is dependent on lesion formation stage and remyelination in multiple sclerosis m2 microglia and macrophages drive oligodendrocyte differentiation during cns remyelination targeting the shift from m1 to m2 macrophages in experimental autoimmune encephalomyelitis mice treated with fasudil the complex immunological and inflammatory network of adipose tissue in obesity dimethyl fumarate selectively reduces memory t cells and shifts the balance between th1/th17 and th2 in multiple sclerosis patients cyclic citrullinated mbp87-99 peptide stimulates t cell responses: implications in triggering disease citrullination of linear and cyclic altered peptide ligands from myelin basic protein (mbp(87-99)) epitope elicits a th1 polarized response by t cells isolated from multiple sclerosis patients: implications in triggering disease properties of myelin altered peptide ligand cyclo(87-99) (ala91,ala96)mbp87-99 render it a promising drug lead for immunotherapy of multiple sclerosis design of novel cyclic altered peptide ligands of myelin basic protein mbp83-99 that modulate immune responses in sjl/j mice design and synthesis of a cyclic double mutant peptide (cyclo(87-99)[a91,a96]mbp87-99) induces altered responses in mice after conjugation to mannan: implications in the immunotherapy of multiple sclerosis immune responses of linear and cyclic plp139-151 mutant peptides in sjl/j mice: peptides in their free state versus mannan conjugation the good, the bad and the ugly: how altered peptide ligands modulate immunity round and round we go: cyclic peptides in disease a double mutation of mbp83-99 peptide induces il-4 responses and antagonizes ifn-γ responses mannosylation of mutated mbp83-99 peptides diverts immune responses from th1 to th2 altered peptide ligands of myelin basic protein (mbp87-99) conjugated to reduced mannan modulate immune responses in mice design and synthesis of a novel potent myelin basic protein epitope 87-99 cyclic analogue: enhanced stability and biological properties of mimics render them a potentially new class of immunomodulators antagonistic effects of human cyclic mbp(87-99) altered peptide ligands in experimental allergic encephalomyelitis and human t-cell proliferation synthesis and study of the electrophoretic behavior of mannan conjugates with cyclic peptide analogue of myelin basic protein using lysine-glycine linker mannan-conjugated myelin peptides prime non-pathogenic th1 and th17 cells and ameliorate experimental autoimmune encephalomyelitis th9 cells and il-9 in autoimmune disorders: pathogenesis and therapeutic potentials advances in t helper 17 cell biology: pathogenic role and potential therapy in multiple sclerosis th22 cells are expanded in multiple sclerosis and are resistant to ifn-beta il-22, gm-csf and il-17 in peripheral cd4+ t cell subpopulations during multiple sclerosis relapses and remission. impact of corticosteroid therapy interleukin-27 in t cell immunity il-27 plasma level in relapsing remitting multiple sclerosis subjects: the double-faced cytokine production of il-27 in multiple sclerosis lesions by astrocytes and myeloid cells: modulation of local immune responses interleukin-27 promotes inflammatory and neuroprotective responses in microglia the major histocompatibility complex and multiple sclerosis: a smoking gun? brain autoantigen recognition by human cd8 t cell clones: enhanced agonist response induced by altered peptide ligands interleukin-17 production in central nervous system-infiltrating t cells and glial cells is associated with active disease in multiple sclerosis differential frequency of cd8+ t cell subsets in multiple sclerosis patients with various clinical patterns the evidence for a role of b cells in multiple sclerosis b cells in multiple sclerosis antibodies in multiple sclerosis oligoclonal bands target debris multiple sclerosis is associated with high levels of circulating dendritic cells secreting pro-inflammatory cytokines myeloid derived suppressor cells and their role in diseases myeloid-derived suppressor cells mediate tolerance induction in autoimmune disease myeloid-derived suppressor cells suppress antitumor immune responses through ido expression and correlate with lymph node metastasis in patients with breast cancer corticosteroids or acth for acute exacerbations in multiple sclerosis randomized study of interferon beta-1a, low-dose azathioprine, and low-dose corticosteroids in multiple sclerosis high dose oral steroids commonly used to treat relapses in canadian ms clinics. can corticosteroids in the treatment of multiple sclerosis lack of interferon-beta bioactivity is associated with the occurrence of relapses in multiple sclerosis effect of early versus delayed interferon beta-1b treatment on disability after a first clinical event suggestive of multiple sclerosis: a 3-year follow-up analysis of the benefit study treatment with interferon beta-1b delays conversion to clinically definite and mcdonald ms in patients with clinically isolated syndromes challenges in randomized controlled trials and emerging multiple sclerosis therapeutics induction and escalation therapies in multiple sclerosis thalamus pathology in multiple sclerosis: from biology to clinical application safety and efficacy of fingolimod in patients with relapsing-remitting multiple sclerosis (freedoms ii): a double-blind, randomised, placebo-controlled, phase 3 trial interferon beta use and disability prevention in relapsing-remitting multiple sclerosis impact of diagnosis and early treatment on the course of multiple sclerosis association between use of interferon beta and progression of disability in patients with relapsing-remitting multiple sclerosis management of secondary progressive multiple sclerosis: prophylactic treatmentpast, present, and future aspects real-life impact of early interferonβ therapy in relapsing multiple sclerosis cause of death in ms: long-term follow-up of a randomised cohort, 21 years after the start of the pivotal ifnβ-1b study survival in ms a randomized cohort study 21 years after the start of the pivotal ifnβ-1b trial new pieces in the puzzle: how does interferon-beta really work in multiple sclerosis? interferon beta-1b is effective in relapsing-remitting multiple sclerosis. i. clinical results of a multicenter, randomized, double-blind, placebo-controlled trial the ifnb multiple sclerosis study group; the university of british columbia ms/mri analysis group. interferon beta-1b in the treatment of multiple sclerosis: final outcome of the randomized controlled trial experimental models of neuroprotection relevant to multiple sclerosis glatiramer acetate in primary progressive multiple sclerosis: results of a multinational, multicenter, double-blind, placebo-controlled trial copolymer 1: a most reasonable alternative therapy for early relapsing-remitting multiple sclerosis with mild disability mechanisms of action of glatiramer acetate in multiple sclerosis long-term therapy with glatiramer acetate in multiple sclerosis: effect on t-cells glatiramer acetate improves regulatory t-cell function by expansion of naive cd4(+)cd25(+)foxp3(+)cd31(+) t-cells in patients with multiple sclerosis copolymer 1 reduces relapse rate and improves disability in relapsing-remitting multiple sclerosis: results of a phase iii multicenter, double-blind placebo-controlled trial placebo-controlled phase 3 study of oral bg-12 for relapsing multiple sclerosis fumaric acid and its esters: an emerging treatment for multiple sclerosis effects of dimethyl fumarate on neuroprotection and immunomodulation teriflunomide, an inhibitor of dihydroorotate dehydrogenase for the potential oral treatment of multiple sclerosis modulation of effector cell functions in experimental autoimmune encephalomyelitis by leflunomide-mechanisms independent of pyrimidine depletion randomized trial of oral teriflunomide for relapsing multiple sclerosis novel therapeutic approaches to autoimmune demyelinating disorders current therapeutic options in pediatric multiple sclerosis a phase ii study of the safety and efficacy of teriflunomide in multiple sclerosis with relapses oral teriflunomide for patients with relapsing multiple sclerosis (tower): a randomised, double-blind, placebo-controlled, phase 3 trial teriflunomide versus subcutaneous interferon beta-1a in patients with relapsing multiple sclerosis: a randomised the immune modulator fty720 targets sphingosine 1-phosphate receptors alteration of lymphocyte trafficking by sphingosine-1-phosphate receptor agonists lymphocyte egress from thymus and peripheral lymphoid organs is dependent on s1p receptor 1 fty720 (fingolimod) efficacy in an animal model of multiple sclerosis requires astrocyte sphingosine 1-phosphate receptor 1 (s1p1) modulation oral fingolimod or intramuscular interferon for relapsing multiple sclerosis a placebo-controlled trial of oral fingolimod in relapsing multiple sclerosis combined use of etanercept and mtx restores cd4 + /cd8 + ratio and tregs in spleen and thymus in collagen-induced arthritis mechanism of action, antitumor activity, pharmacokinetics, efficacy in the treatment of solid tumors and lymphomas, and toxicity mitoxantrone in progressive multiple sclerosis: a placebo-controlled, double-blind, randomised, multicentre trial therapeutic effect of mitoxantrone combined with methylprednisolone in multiple sclerosis: a randomised multicentre study of active disease using mri and clinical criteria mitoxantrone: benefits and risks in multiple sclerosis patients the role of alpha-4 integrin in the aetiology of multiple sclerosis: current knowledge and therapeutic implications anti-α4 integrin therapy for multiple sclerosis: mechanisms and rationale diagnosis of multiple sclerosis 2010 revision of the mcdonald criteria diagnosis and therapy of multiple sclerosis-update a controlled trial of natalizumab for relapsing multiple sclerosis natalizumab: new drug. multiple sclerosis: risky market approval multiple sclerosis-the plaque and its pathogenesis b-cell depletion with rituximab in relapsing-remitting multiple sclerosis ocrelizumab in relapsing-remitting multiple sclerosis: a phase 2, randomised, placebo-controlled, multicentre trial safety and efficacy of ofatumumab in relapsing-remitting multiple sclerosis: a phase 2 study estimation of dose requirements for sustained in vivo activity of a therapeutic human anti-cd20 antibody identifier: nct02792218 and nct02792231. available online drugs@fda: fda approved drug products alemtuzumab versus interferon beta 1a as first-line treatment for patients with relapsing-remitting multiple sclerosis: a randomised controlled phase 3 trial alemtuzumab for patients with relapsing multiple sclerosis after disease-modifying therapy: a randomised controlled phase 3 trial monoclonal antibody therapies for the treatment of relapsing-remitting multiple sclerosis: differentiating mechanisms and clinical outcomes allogeneic bone marrow transplant for chronic myelogenous leukemia in a patient with multiple sclerosis autologous haematopoietic stem-cell transplantation in multiple sclerosis autologous hematopoietic stem cell transplantation in multiple sclerosis: a meta-analysis bone marrow mesenchymal stem cell transplantation in patients with multiple sclerosis: a pilot study randomized placebo-controlled phase ii trial of autologous mesenchymal stem cells in multiple sclerosis bht-3009, a myelin basic protein-encoding plasmid for the treatment of multiple sclerosis treg cell resistance to apoptosis in dna vaccination for experimental autoimmune encephalomyelitis treatment induction of antigen-specific tolerance in multiple sclerosis after immunization with dna encoding myelin basic protein in a randomized evolution of ms lesions to black holes under dna vaccine treatment pathogen recognition and development of particulate vaccines: does size matter? methods delivery of dna vaccines: an overview on the use of biodegradable polymeric and magnetic nanoparticles subcutaneous inverse vaccination with plga particles loaded with a mog peptide and il-10 decreases the severity of experimental autoimmune encephalomyelitis development and pre-clinical evaluation of recombinant human myelin basic protein nano therapeutic vaccine in experimental autoimmune encephalomyelitis mice animal model nbi-5788, an altered mbp83-99 peptide, induces a t-helper 2-like immune response in multiple sclerosis patients purely systemically active anti-inflammatory treatments are adequate to control multiple sclerosis induction of a non-encephalitogenic type 2 t helper-cell autoimmune response in multiple sclerosis after administration of an altered peptide ligand in a placebo-controlled, randomized phase ii trial encephalitogenic potential of the myelin basic protein peptide (amino acids 83-99) in multiple sclerosis: results of a phase ii clinical trial with an altered peptide ligand active immunization with myelin-derived altered peptide ligand reduces mechanical pain hypersensitivity following peripheral nerve injury effects of active immunisation with myelin basic protein and myelin-derived altered peptide ligand on pain hypersensitivity and neuroinflammation effects of vaccination with altered peptide ligand on chronic pain in experimental autoimmune encephalomyelitis, an animal model of multiple sclerosis cyclic mog 35-55 ameliorates clinical and neuropathological features of experimental autoimmune encephalomyelitis oxidative/reductive conjugation of mannan to antigen selects for t1 or t2 immune responses cell-mediated immune responses to muc1 fusion protein coupled to mannan pilot phase iii immunotherapy study in early-stage breast cancer patients using oxidized mannan-muc1 dendritic cell immunotherapy: clinical outcomes antibody and t cell responses of patients with adenocarcinoma immunized with mannan-muc1 fusion protein up to 15-year clinical follow-up of a pilot phase iii immunotherapy study in stage ii breast cancer patients using oxidized mannan-muc1 mannan-muc1-pulsed dendritic cell immunotherapy: a phase i trial in patients with adenocarcinoma a phase 2, single-arm study of an autologous dendritic cell treatment against mucin 1 in patients with advanced epithelial ovarian cancer mannosylated linear and cyclic single amino acid mutant peptides using a small 10 amino acid linker constitute promising candidates against multiple sclerosis design and synthesis of non-peptide mimetics mapping the immunodominant myelin basic protein (mbp 83-96 ) epitope to function as t-cell receptor antagonists this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license key: cord-001359-c1uom5f7 authors: oslund, karen l.; zhou, xu; lee, boram; zhu, lingxiang; duong, trang; shih, robert; baumgarth, nicole; hung, li-yin; wu, reen; chen, yin title: synergistic up-regulation of cxcl10 by virus and ifn γ in human airway epithelial cells date: 2014-07-17 journal: plos one doi: 10.1371/journal.pone.0100978 sha: doc_id: 1359 cord_uid: c1uom5f7 airway epithelial cells are the first line of defense against viral infections and are instrumental in coordinating the inflammatory response. in this study, we demonstrate the synergistic stimulation of cxcl10 mrna and protein, a key chemokine responsible for the early immune response to viral infection, following treatment of airway epithelial cells with ifn γ and influenza virus. the synergism also occurred when the cells were treated with ifn γ and a viral replication mimicker (dsrna) both in vitro and in vivo. despite the requirement of type i interferon (ifnar) signaling in dsrna-induced cxcl10, the synergism was independent of the ifnar pathway since it wasn’t affected by the addition of a neutralizing ifnar antibody or the complete lack of ifnar expression. furthermore, the same synergistic effect was also observed when a cxcl10 promoter reporter was examined. although the responsive promoter region contains both isre and nfκb sites, western blot analysis indicated that the combined treatment of ifn γ and dsrna significantly augmented nfκb but not stat1 activation as compared to the single treatment. therefore, we conclude that ifn γ and dsrna act in concert to potentiate cxcl10 expression in airway epithelial cells via an nfκb-dependent but ifnar-stat independent pathway and it is at least partly regulated at the transcriptional level. influenza pneumonia remains a major cause of morbidity and mortality worldwide. airway epithelial cells are the first line of defense against viral infections in the lung and are instrumental in coordinating the early inflammation leading to an adaptive immune response. cxcl10 (ifn gamma inducible 10 kda protein, ip10) is a non-elr cxc chemokine with potent biological effects including monocyte stimulation, natural killer and activated t cell migration, modulation of adhesion molecule expression, inhibition of angiogenesis [1] as well as antimicrobial effects at high concentrations [2] . the role of cxcl10 in viral pneumonia has not been thoroughly characterized but evidence suggests it is important for the migration of nk cells, macrophages, t cells, neutrophils and plasmacytoid dendritic cells into the lung [3, 4] . in a mouse model of rsv infection, antibody-mediated neutralization of cxcl10 resulted in a significant increase in disease symptoms including impaired viral clearance, reduced pulmonary dendritic cell numbers and maturation and a reduction in viral specific cd8(+) t cells [5] . synergistic up-regulation of cxcl10 has been described in vitro in several cell types and in response to different pro-inflammatory molecules but always in conjunction with ifn c. tnfa and ifn c induce synergistic levels of cxcl10 in a human endothelial cell line hmec-1 via an erk dependent pathway [6] , il-1b and ifn c in human intestinal epithelial cell lines [7] , pdgf and ifn c in blood derived macrophages [8] , tnfa and ifn c in primary human airway smooth muscle cells and gastric epithelial cells [9, 10] , prolactin and ifn c [11] as well as substance p and ifn c in human keratinocytes [12] and hyaluronan fragments with ifn c in mouse macrophages [13] . interestingly, synergistic induction of cxcl10 has been described with ifn c in conjunction with hiv-1 in human astrocytes and macrophages and thought to play a role in hiv induced encephalopathy [14, 15] . this marked variety of pro-inflammatory molecules that elicit a synergistic response of cxcl10 in a wide variety of cell types is indicative of a highly conserved and likely biologically important cellular response. however, synergistic induction of cxcl10 in response to influenza and ifn c in airway epithelial cells has not been previously reported. in this study, we demonstrate synergistic induction of cxcl10 in well differentiated primary human bronchial epithelial (hbe) cells following influenza virus infection and the treatment with ifn c. we further demonstrate that this synergy was mediated by the interaction between dsrna (an intermediate of viral replication) and ifn c in vitro and in vivo. in the follow-up mechanistic study, this synergism was found to be transcriptionally regulated as demonstrated by a chimeric promoter reporter gene assay. in addition, it was not dependent of the ifnar pathway as neither the neutralizing antibody to ifnar nor the ifnar deficiency affected the synergism. finally, nfkb, but not stat1, appeared to mediate this synergism. culture and treatments of primary human bronchial epithelial (hbe) cells human bronchial tissues were purchased from the commercial source (national disease research interchange, philadelphia, pa) as described before [16, 17] . no tissues from patients diagnosed with lung-related diseases were used. all those lungs were either autopsy leftovers or were rejected for transplant. they were sent to us with arbitrary numerical code. no identity link to the actual patient can be identified. protease-dissociated hbe cells were plated on transwellh chambers (corning) and were maintained in immersed culture conditions until they reached confluence when they were transferred to an air-liquid interface (ali) culture condition [16, 17] . at air-liquid interface, the cells were maintained in a ham's f12/dmem (1:1) with the addition of transferrin (5 mg/ml), insulin (5 mg/ml), cholera toxin (10 ng/ml), epidermal growth factor (10 ng/ml), dexamethasone (0.1 mm), bovine hypothalamus extract (15 mg/ml), bsa (0.5 mg/ml) and all-transretinoic acid (30 nm). cells were maintained at ali for 7 days and were placed in basal media devoid of the additives, with the exception of retinoic acid, overnight prior to the experiments. recombinant ifn c was purchased from r&d systems and synthetic double stranded (ds) rna (i.e. poly i:c) from emd biosciences. ifn c was used at 50 ng/ml and poly i:c at 25 mg/ ml. a neutralizing antibody to ifnar was obtained from us biologics and used at 2.5 mg/ml [18] . both ikb kinase and jak inhibitors were purchased from emd biosciences and used at 5 mm. for influenza infection, cells were infected with the influenza virus strain mem at 1200 hau. for in vivo study, balb/c mice were purchased from charles rivers laboratories and used at 6-8 weeks of age for intratracheal instillations. for primary airway epithelial cell cultures, ifnar null mice were a kind gift from dr. nicole baumgarth (ucd) and the wild-type controls mice were c57bl/6j. mice were used at 8-10 weeks of age. all protocols described were approved by the university of california, davis and university of arizona, which were responsible for the proper care and use of experimental animals. the protocol was performed as described previously [19] . briefly, at the time of necropsy, the chest and cervical region were exposed. a small puncture was placed in the proximal trachea to allow cannulation with sterile 0.86 mm polyethylene tubing (intramedic clay adams) which was secured in place with a 3.0 silk suture. a loose suture was placed at the distal end of the trachea just proximal to the carina. the trachea was dissected free and immediately placed in dmem at 4uc. and each trachea gently inflated with 0.2% protease through the tracheal cannula after tightening the distal suture. dissociated epithelial cells were gently harvested by injecting 5 milliliters of cell culture media through the trachea. mte cells from all tracheas were pooled and re-suspended in cell culture media prior to plating on transwellß chambers (corning) coated with purcolß (advanced biomatrix). mte cells were maintained in submerged conditions until confluent at which time they were kept in air-liquid interface conditions for one week in the presence of 100 nm retinoic acid. for the experiments, mte cells were treated with 50 ng recombinant murine ifn c (r and d systems) and/or 25 mg dsrna for 24 hours. the protocol was performed as described previously [20, 21] . briefly, balb/c mice were anesthetized with isoflorane. in dorsal recumbancy, the tongue was gently pulled out, and a pipette tip was gently inserted into their laryngeal region through the oral cavity. 2 mg of dsrna and/or 5 mg of recombinant murine ifn c in 50 ml of lps free pbs was deposited and breathed in. lps free pbs was used as the control. mice were kept in an upright position until recovery from anesthesia. twenty four hours later, mice were euthanized, and lungs were lavaged with 1 ml pbs. all lungs were flash frozen in liquid nitrogen for subsequent rna isolation. total rna was extracted with trizolß reagent (invitrogen) and cdna was generated from an equal amount of rna (1 mg per reaction) by moloney's murine leukemia virus-reverse transcriptase (applied biosystems) using random hexamers (invitrogen). sybr green master mix (roche applied science) and the abi7900ht detection system (applied biosystems) were used following the manufacturer's protocol for real-time pcr analysis. the relative mrna amount of each sample was calculated based on its threshold cycle, ct, in comparison to the ct of the housekeeping gene, beta actin or glyceraldehyde 3-phosphate dehydrogenase (gapdh). the results are presented as 2 2(ct cxcl10-ct of housekeeping gene) as fold induction over the control condition. the purity of the amplified product was determined as a single peak of the dissociation curve. throughout the study, there was no observable fluctuation in the ct values of the housekeeping genes from different treatment conditions. primers to human and murine cxcl10 were purchased from sa biosciences. the primer sequences for human beta actin are as follows: forward: tgtgtccgtcgtggatctga and reverse: cctgcttcaccaccttcttgat. the primer sequences for murine gapdh are as follows: forward: tcctccaccttt-gacgctg and reverse: accaccctgttgctgtagcc. in preparation for collecting conditioned media from primary human airway epithelial cells, the cells were washed twice with sterile pbs. fresh media containing ifn c and/or poly i:c was placed basally with 100 ml of media placed apically. following a 24 hour incubation, the media was harvested, centrifuged to remove any cellular debris and stored at 280uc. bronchoalveolar lavage fluid was used for the murine cxcl10 elisa. commercially available elisa kits were used according to the manufacturer's directions (r&d systems). results were normalized to the amount of conditioned media or lavage fluid and expressed as ng/ ml or pg/ml. the construct contains 735 bp upstream of the transcription start site of cxcl10 was cloned into pgl3 luciferase reporter vector (promega). the construct was confirmed by dna sequencing. early differentiated hbe cells (7 days) were transfected with cxcl10 promoter-luciferase construct and prl-tk using lipofectamine 2000 (invitrogen) according to the manufacturer's specifications. prl-tk was used as the internal control for normalizing transfection efficiency. the empty vector, pgl3 basic was used as a negative control. in brief, cells were plated onto 12 well plates at 80-90% confluency. the next day, cells were washed twice with opti-mem (invitrogen) before transfection. the cells were incubated at 37uc with the mixture of dna constructs and lipofectamine 2000 in opti-mem for 5-6 hours at which time cell culture media was also added to the cells. transfected cultures were treated with 50 ng/ml ifn c and/or 25 mg poly i:c for 24 hours. a dual luciferase reporter assay kit (promega) was used following the manufacturer's protocol. for each transfection, the relative firefly luciferase activity was normalized to the renilla luciferase activity. neutralization of ifnar 2.5 mg/ml of a mouse monoclonal antibody to chain 2 of the human alpha, beta, omega interferon receptor (ifnar) was added to culture media of cells for 1 hour prior to the start of the experiment. this antibody has previously been documented to neutralize the extracellular domain of the human type i interferon receptor with high affinity at the dose used in this study [18] . ifn c and/or poly i:c were added to the culture media following the pre-incubation period and with the ifnra antibody still present. mouse igg was used as the control. following a 24 hour incubation, rna isolation and qpcr were performed as previously described. data are expressed as mean 6 se. experiments were conducted in triplicate with at least three independent cultures. group differences were calculated using an analysis of variance (anova) followed by a bonferroni multiple comparison test. differences were considered significant for p values less than 0.05. as shown in fig. 1a , well differentiated hbe cells demonstrated significant synergistic induction of cxcl10 mrna following infection with the mem influenza virus and treatment with ifn c. cxcl10 induction was also significantly elevated following the combined treatment of mem and ifn c treatment as compared to the treatment with ifn c alone. consistently, potentiation of cxcl10 protein production by the combined treatments was detected in the cell culture supernatant from hbe cells (fig. 1b) . apical release of cxcl10 was enhanced in all treatment conditions with more pronounced levels detected in the combined mem infection and ifn c treatment (fig. 1b) . interestingly, significant levels of cxcl10 were also released basally except that they were much lower than the apical secretion. these results demonstrate that influenza virus in combination with ifn c synergistically induce cxcl10 mrna and protein production from primary human airway epithelial cells. because viral replication and its intermediate-double stranded (ds) rna have been shown to mediate many influenza induced phenotypes. we then examined the possibility if dsrna could synergize with ifn c in the induction of cxcl10. as shown in fig. 2a , hbe cells demonstrated synergistic induction of cxcl10 mrna in a time dependent manner starting as early as 3 hours after treatment. treatment with ifn c and dsrna resulted in significant synergistic induction of cxcl10 mrna levels at all the time points. combined treatment for 24 hours resulted in an over 8000 fold induction of cxcl10 mrna. consistently, fig. 2b demonstrates potentiation of cxcl10 protein in cell culture supernatant from hbe cells treated for 24 hours. apical release of cxcl10 was enhanced in all treatment conditions with more pronounced levels detected in the combined ifn c and dsrna treatment. significant levels of cxcl10 were also released basally, although the levels were much lower than the apical secretions. these results suggest that the synergy between influenza infection and ifn c treatment may be caused by the interaction between dsrna-and ifn c-mediated signaling pathways. to determine whether ifn c and dsrna synergistically induce cxcl10 in vivo, we performed intra-tracheal delivery of ifn c and dsrna in balb/c mice and examined cxcl10 mrna and protein levels. as shown in fig. 3a , dsrna alone induced a significant increase in cxcl10 mrna, and ifn c and dsrna treatment together synergistically induced cxcl10 mrna 54 fold in mouse lung. likewise, fig. 3b demonstrates a significant induction of cxcl10 protein in bronchoalveolar lavage fluid after treatment with dsrna and synergistic induction following ifn c and dsrna treatment. these results demonstrate synergistic induction of cxcl10 mrna and protein in vivo following ifn c and dsrna treatment. because dsrna is known to induce the type i interferon pathway, we investigated the involvement of this pathway by using a neutralizing antibody to human ifnar. fig. 4a demonstrates that the ifnar neutralizing antibody did not affect synergistic induction of cxcl10 mrna in hbe cells, although it did repress dsrna-induced cxcl10. to confirm this result, we isolated mte cells from ifnar null mice, which were completely lack of ifnar expression. fig. 4b demonstrates that the lack of ifnar did not affect the synergistic induction of cxcl10. interestingly, induction of cxcl10 by dsrna alone was significantly impaired in the mte cells from ifnar null mice as compared to the cells from wild-type mice. taken together, these results demonstrate that synergistic induction of cxcl10 mrna was independent of the type i interferon receptor in both human and mouse airway epithelial cells. in contrast, dsrna-induced cxcl10 appeared to depend on the type i interferon receptor, suggesting that the synergy may be mediated via a different pathway. because of the rapid elevation of cxcl10 by ifn c and/or dsrna ( fig. 2a) , we tested the possibility if this synergy occurred at transcriptional level by transient transfection of the hbe cells with a cxcl10 promoter/luciferase chimeric construct containing 735 bp of the upstream regulatory region of cxcl10. fig. 5 demonstrates the combined treatment of ifn c and dsrna significantly increased the luciferase reporter gene activity as compared with the treatment of ifn c or dsrna alone. these results support that the synergism occurred at least partially through a direct transcriptional mechanism and the proximal 735 bp region of the cxcl10 promoter was critical for the induction. because both nfkb and isre sites were present in this cloned cxcl10 promoter region, we further tested nfkb and stat pathways in this synergism as both pathways have been shown to be responsible for cxcl10 gene expression. indeed, the specific inhibitor targeting the kinase either upstream of nfkb (ikb kinase) or stat1 (jak) significantly blocked cxcl10 induction by ifn c, by dsrna, or by the combined treatment (fig. 6a) , which confirms the indispensable role of both pathways in . the synergism between ifn c and dsrna(dsr) was independent of ifnar. a) hbe cells were treated with 2.5 mg/ml of either an isotype control antibody (black bar) or a monoclonal neutralizing antibody ifnar (grey bar) for one hour prior to the addition of 50 ng/ml ifn c and/ or 25 mg/ml dsrna for 24 hours. rna was isolated followed by qpcr. ns: not significant. $: p,0.05. isotype control antibody vs. ifnar. b) primary mte cells isolated from either wild-type (black bar) or ifnar deficient (grey bar) mice were grown in vitro and treated with 50 ng/ml ifn c and/or 25 mg/ml dsrna for 24 hours. rna was isolated followed by qpcr. ns: not significant. $: p,0.05. wild-type vs. ifnar deficient. triplicate wells were used for each experiment and the experiment was repeated at least three times. doi:10.1371/journal.pone.0100978.g004 regulating cxcl1 expression. when the signaling pathway was further examined, the treatment of ifn c or dsrna alone was found to induce robust degradation of ikb, a surrogate of the activation of nfkb pathway which is initiated by the reaction catalyzed by ikb kinase. and the combined treatment resulted in much greater ikb degradation as compared to the single treatment (fig. 6b) . in contrast, despite the activation of stat1a and stat1b by these treatments, no synergism was observed (fig. 6b) . therefore, the synergism of ifn c and dsrna on cxcl10 expression was mediated by nfkb pathway. airway epithelial lining is the first defense against respiratory viral infection. in this study, we seek to understand the modulation of an important chemokine-cxcl10 by the combined effects of ifn c and viral infection. this is the first report documenting the observation and mechanism underlying the synergism between ifn c and viral infection (or dsrna treatment) in airway epithelial cells. ifn c is a type ii interferon and oftentimes elevated in the context of viral infection. it is generally accepted that direct innate antiviral defense is mediated by type i ifn. indeed, we and others have shown that dsrna (or virus)-induced epithelial derived type i ifn plays critical role in the airway antiviral defense [16, 21, 22] . in the present study, we demonstrate the synergy between dsrna-and ifn c-induced signaling in the regulation of cxcl10. interestingly, although dsrna-induced cxcl10 depended on type i ifn pathway, the synergy between dsrna and ifn c was completely independent of it in both human and mouse epithelial cells. this lack of dependence was not due to the remaining residue of the ifnar functionality in the antibody neutralization assay, because the epithelial cells from ifnar deficient mice used in the fig. 4a still preserved the synergistic response despite the loss of entire ifnar expression. thus, in the presence of ifn c, dsrna (or virus)-induced signaling is very likely to be altered, which emphasizes the importance of studying the pathway crosstalk as demonstrated in the present study. previous studies have demonstrated that ifn c in conjunction with different pro-inflammatory molecules leads to a synergistic and dramatic induction of cxcl10 in a variety of cell types including keratinocytes, macrophages, endothelial cells and smooth muscle cells [6, 8, 9, 12] . several of these studies have demonstrated that the transcription factor, nf-kb, is involved in cxcl10 induction in a variety of systems [7] [8] [9] . in contrast, very few studies have demonstrated that cxcl10 induction is dependent on an isre site in the promoter region. kanda and coworkers found substance p and ifn c induced synergism of cxcl10 in human keratinocytes was dependent on an isre site and two nf-kb sites in the cxcl10 promoter located 2210 to 2 221 of the transcription start site [12] . in addition, majumder and co-workers found evidence that ifn c and tnf a act in synergy via p48 complexes with stat-1a binding to this same isre site in the cxcl10 promoter of human fibrosarcoma lines [23] . evidence that similar synergism occurs at the transcriptional level in mouse cells has been demonstrated in a small number of in vitro studies [13, 24] . using the murine fibroblast nih 3t3 cell line, ohmoir and co-workers established that an isre site and one of two nf-kb sites in a 243 bp fragment flanking the transcription start site of the murine cxcl10 gene were critical for ifn c and tnf a induced synergy [24] . our study has further extended these studies to the airway epithelial system in the context of viral infection, and the results apparently support the importance of nfkb. figure 6 . nfkb-dependent signaling is responsible for the synergism between ifn c and dsrna (dsr). a) well differentiated primary hbe cells were treated with 50 ng/ml ifn c and/or 25 mg/ml dsrna for 24 hours in the presence or absence of ikb kinase or jak inhibitor. rna was isolated followed by qpcr. solvent control: black bar; ikb kinase inhibitor: grey bar; jak inhibitor: light grey bar. $: p,0.05. inhibitor treatment vs. solvent control. b) cells were treated with 50 ng/ml ifn c and/or 25 mg/ml dsrna for 3 hours. cellular protein was collected and analyzed by western blot analysis. actin was used as a loading control. pstat1: phosphorylated stat1. this is the representative image from three replicates. doi:10.1371/journal.pone.0100978.g006 the present study demonstrates the significant contribution of cxcl10 from epithelial cells, which is consistent with the emerging role of active defense by these cells in the respiratory viral infection. cxcl10 secretion by hbe cells appeared to be polarized. the treatment with ifn c, dsrna and combined treatments resulted in a progressively enhanced secretion of cxcl10 mainly from apical surface. in contrast, the basal secretions of cxcl10 protein in these cultures were much less. the gradient between the apical and basal compartments may have important implications in vivo leading to enhanced emigration of cxcr3 positive effector cells (primarily cd8+ t cells) to the airway epithelial microenvironment. although dsrna has been used extensively as a synthetic dsrna to model viral infections, its stabilized derivatives are also being investigated for clinical use as an immunomodulatory agent for use as vaccine adjuvants in anti-viral treatment and cancer therapies [25] [26] [27] [28] . while dsrna was used in this study to simulate a viral infection, it is important to note that it, in and of itself, can also be involved in synergistic induction of cxcl10 in airway epithelial cells. this fact could be an important consideration in the treatment of patients with pre-existing diseases in which ifn c is part of the pathogenesis such as viral infections. in summary, we demonstrate ifn c and the influenza virus synergistically induce cxcl10 in human airway epithelial cells. this synergism is likely to be mediated by dsrna-induced signaling both in vitro and in vivo, which is independent of the type i interferon receptor pathway. furthermore, we demonstrate the 735 bp proximal region of the cxcl10 promoter plays a critical role in regulating this synergistic induction. this region of the cxcl10 promoter contains punitive isre and nfkb transcription factor binding sites. therefore, further study has been carried out to demonstrate the involvement of nfkb, but not stat1, in this synergism. this capacity for airway epithelial cells to markedly up-regulate cxcl10 likely has important consequences for cd8+ t cell and nk cell migration to the airway epithelial microenvironment following influenza virus infection. the immunobiology of interferongamma inducible protein 10 kd (ip-10): a novel, pleiotropic member of the c-x-c chemokine superfamily cutting edge: ifn-inducible elr-cxc chemokines display defensin-like antimicrobial activity cellular immune responses to severe acute respiratory syndrome coronavirus (sars-cov) infection in senescent balb/c mice: cd4+ t cells are important in control of sars-cov infection ip-10 mediates selective mononuclear cell accumulation and activation in response to intrapulmonary transgenic expression and during adenovirus-induced pulmonary inflammation cxcl10/cxcr3-mediated responses promote immunity to respiratory syncytial virus infection by augmenting dendritic cell and cd8(+) t cell efficacy molecular mechanisms underlying the pro-inflammatory synergistic effect of tumor necrosis factor alpha and interferon gamma in human microvascular endothelium nf-kappab-dependent synergistic regulation of cxcl10 gene expression by il-1beta and ifn-gamma in human intestinal epithelial cell lines pdgf synergistically enhances ifn-gamma-induced expression of cxcl10 in blood-derived macrophages: implications for hiv dementia regulation of tnf-alpha-and ifn-gamma-induced cxcl10 expression: participation of the airway smooth muscle in the pulmonary inflammatory response in chronic obstructive pulmonary disease ifngamma synergizes with tnf-alpha but not with viable h. pylori in up-regulating cxc chemokine secretion in gastric epithelial cells prolactin enhances interferon-gamma-induced production of cxc ligand 9 (cxcl9), cxcl10, and cxcl11 in human keratinocytes substance p enhances the production of interferon-induced protein of 10 kda by human keratinocytes in synergy with interferon-gamma hyaluronan fragments synergize with interferon-gamma to induce the c-x-c chemokines mig and interferon-inducible protein-10 in mouse macrophages molecular mechanism(s) involved in the synergistic induction of cxcl10 by human immunodeficiency virus type 1 tat and interferon-gamma in macrophages proinflammatory cytokines and hiv-1 synergistically enhance cxcl10 expression in human astrocytes rhinovirus induces airway epithelial gene expression through double-stranded rna and ifndependent pathways characterization of human mucin 5b gene expression in airway epithelium and the genomic clone of the aminoterminal and 59-flanking region identification of a novel subunit of the type i interferon receptor localized to human chromosome 21 stimulation of airway mucin gene expression by interleukin (il)-17 through il-6 paracrine/ autocrine loop vanadium pentoxide (v(2)o(5)) induced mucin production by airway epithelium rhinovirus-induced major airway mucin production involves a novel tlr3-egfr-dependent pathway negative control of tlr3 signaling by ticam1 down-regulation ) p48/ stat-1alpha-containing complexes play a predominant role in induction of ifn-gamma-inducible protein, 10 kda (ip-10) by ifn-gamma alone or in synergy with tnf-alpha the interferon-stimulated response element and a kappa b site mediate synergistic induction of murine ip-10 gene transcription by ifn-gamma and tnf-alpha treatment of viral and neoplastic diseases with double-stranded rna derivatives and other new agents a north american brain tumor consortium phase ii study of poly-iclc for adult patients with recurrent anaplastic gliomas synthetic double-stranded rna poly(i:c) combined with mucosal vaccine protects against influenza virus infection antiviral role of toll-like receptor-3 agonists against seasonal and avian influenza viruses key: cord-001515-x11t9pbv authors: kosinska, anna d.; liu, jia; lu, mengji; roggendorf, michael title: therapeutic vaccination and immunomodulation in the treatment of chronic hepatitis b: preclinical studies in the woodchuck date: 2014-12-23 journal: med microbiol immunol doi: 10.1007/s00430-014-0379-5 sha: doc_id: 1515 cord_uid: x11t9pbv infection with hepatitis b virus (hbv) may lead to subclinical, acute or chronic hepatitis. in the prevaccination era, hbv infections were endemic due to frequent mother to child transmission in large regions of the world. however, there are still estimated 240 million chronic hbv carriers today and ca. 620,000 patients die per year due to hbv-related liver diseases. recommended treatment of chronic hepatitis b with interferon-α and/or nucleos(t)ide analogues does not lead to satisfactory results. induction of hbv-specific t cells by therapeutic vaccination or immunomodulation may be an innovative strategy to overcome virus persistence. vaccination with commercially available hbv vaccines in patients with or without therapeutic reduction of viral load did not result in effective immune control of hbv infection, suggesting that combination of antiviral treatment with new formulations of therapeutic vaccines is needed. the woodchuck (marmota monax) and its hbv-like woodchuck hepatitis virus are a useful preclinical animal model for developing new therapeutic approaches in chronic hepadnaviral infections. several innovative approaches combining antiviral treatments using nucleos(t)ide analogues, with prime-boost vaccination using dna vaccines, new hepadnaviral antigens or recombinant adenoviral vectors were tested in the woodchuck model. in this review, we summarize these encouraging results obtained with these therapeutic vaccines. in addition, we present potential innovations in immunostimulatory strategies by blocking the interaction of the inhibitory programmed death receptor 1 with its ligand in this animal model. more than 240 million people worldwide are persistently infected with hepatitis b virus (hbv) and are at risk of developing chronic liver disease, cirrhosis and hepatocellular carcinoma (hcc) [1] . an effective and affordable therapy to achieve sustained suppression of hbv replication and remission of liver disease is urgently needed. pegylated interferon-alpha 2a (ifn-a) is recommended for the treatment of chronic hepatitis b (chb) in the current consensus guidelines of many countries. compared with conventional recombinant ifn-a, however, pegylated ifn-a alone or in combination with nucleoside analogues does not significantly increase the rate of sustained response [2, 3] . nucleos(t)ide analogues, such as, entecavir and tenofovir, suppress hbv replication and result in the improvement of liver architecture. however, these agents cannot eradicate hbv genomes from the liver and may further limited by the development increasingly select drug-resistant mutants with prolonged use [4, 5] . therapy with additional antiviral drugs targeting other steps in the viral life cycle, in combination with immunomodulatory options, might be more beneficial and effective. more than 90 % of acutely infected adults resolve clinical symptoms and maintain lifelong protective immunity by mounting a vigorous, multi-specific immune response abstract infection with hepatitis b virus (hbv) may lead to subclinical, acute or chronic hepatitis. in the prevaccination era, hbv infections were endemic due to frequent mother to child transmission in large regions of the world. however, there are still estimated 240 million chronic hbv carriers today and ca. 620,000 patients die per year due to hbv-related liver diseases. recommended treatment of chronic hepatitis b with interferon-α and/ or nucleos(t)ide analogues does not lead to satisfactory results. induction of hbv-specific t cells by therapeutic vaccination or immunomodulation may be an innovative strategy to overcome virus persistence. vaccination with commercially available hbv vaccines in patients with or without therapeutic reduction of viral load did not result in effective immune control of hbv infection, suggesting that combination of antiviral treatment with new formulations of therapeutic vaccines is needed. the woodchuck (marmota monax) and its hbv-like woodchuck hepatitis virus are a useful preclinical animal model for developing new therapeutic approaches in chronic hepadnaviral infections. several innovative approaches combining antiviral treatments using nucleos(t)ide analogues, with primeboost vaccination using dna vaccines, new hepadnaviral antigens or recombinant adenoviral vectors were tested in the woodchuck model. in this review, we summarize these encouraging results obtained with these therapeutic vaccines. in addition, we present potential innovations this article is part of the special issue "therapeutic vaccination in chronic hepatitis b-approaches, problems and new perspectives". to hbv proteins. by contrast, patients with chronic hepatitis b tend to have delayed, transient or narrowly focused t cell responses [6] [7] [8] . patients who spontaneously recover from hbv infection might experience reactivation of hbv under immunosuppressive treatments. thus, the specific immune responses to hbv remain crucial for the long-term control of hbv infection even after resolution of the acute infection. for chronically infected patients, immunostimulatory and immunomodulatory strategies to boost or to broaden the weak virus-specific t cell response have been proposed to reach an effective control of viral infection. since more than 20 years, numerous clinical trials exploited the conventional prophylactic vaccine based on the hepatitis b surface antigen (hbsag) for therapeutic vaccination (table 1) . these studies demonstrated reductions in viremia, seroconversion of the hepatitis b "e" antigen (hbeag) to anti-hbe and hbv-specific t cell responses in some patients after vaccination. however, the antiviral effect was only transient and did not lead to an effective control of the hbv [9] [10] [11] [12] [13] [14] [15] [16] [17] . a more sophisticated therapeutic vaccination based on hbsag complexed with human anti-hbs was proposed by the group of wen et al. [18] . immunogenic complexes (ic) stimulate robust t cell responses by increasing uptake of hbsag through fc receptors on antigen-presenting cells (apc) and, therefore, enhance hbsag processing and presentation. it was demonstrated that this vaccine administered to hbeag-positive patients led to decrease of hbv dna in serum and hbeag seroconversion in some subjects [19] . in a phase ii b clinical trial, hbeag seroconversion was observed in about 21.6 % of treated patients. moreover, a moderate decrease in serum hbv dna and hbsag levels was observed after treatment [20, 21] . very recently, a large phase iii clinical trial with 12 injections of ic complex failed to show any therapeutic efficacy when compared to the placebo control injected only with alum [22] . overstimulation with ic-based vaccine did not increase but decreased efficacy of the therapeutic vaccination. these results underline that an appropriate immunization protocol is crucial for the efficacy of therapeutic vaccination. dna vaccines using plasmids expressing viral proteins have gained popularity given their ability to induce strong cellular and humoral immune responses. several phase i clinical studies investigated the therapeutic efficacy of plasmid dna vaccines expressing hbsag in chronic hbv carriers. these studies showed evidence for the safety of hbv dna vaccination (for details see below), but t cell responses were restored or activated at only a low level. furthermore, dna vaccines expressing only hbsag did not result in significant suppression of viremia in chronic carriers of hbv [23, 24] . from results of these studies, it can be concluded that the therapeutic vaccination alone is not sufficient to achieve pre-s1/pre-s2/s, hbcag, polymerase yoon et al. [27] the control over hbv. high load of virus particles and large amounts of hbsag in the liver and peripheral blood may be responsible for the immune tolerant status in the patients. therefore, pretreatment with nucleos(t)ide analogues has been proposed to achieve better cd8 t cell response and subsequent therapeutic efficacy after administration of dna vaccines. recently, the results of the trial of this combination therapy have been published. in a large double-blind study, 70 patients were treated effectively with nucleos(t)ide analogues for a median of 3 years resulting in undetectable levels of hbv dna and thereafter randomized into two groups: one received five intramuscular injections of dna vaccine expressing hbsag and one received placebo. nucleos(t)ide analogues were stopped. although this combination therapy was fairly well tolerated, the hbv dna vaccine did not decrease the risk of relapse in hbv-treated patients and did not restore the anti-hbv immune response despite effective viral suppression by analogues [25, 26] . during a study in korea, 27 patients randomly received either adefovir (adv) alone or adv in combination with hbsag-expressing dna vaccine. therapeutic vaccination was safe and tolerable in chb patients. vaccine-induced hbv-specific t cell responses and hbeag seroconversion were weaker in korean patients than in caucasian patients [27] . asian patients, who are generally infected via vertical transmission, may have a higher level of immune tolerance than caucasians who are usually infected later in life. improved vaccines for breaking immune tolerance may be needed to develop effective therapeutic hbv dna vaccines. the aim of a study in vietnam was to evaluate viral suppression following combined treatment with a new vaccine containing all three envelope proteins of hbv (pre-s1/pre-s2/s) and lamivudine in chb patients. the enhanced suppression of viremia in the combination group was reversed after the discontinuation of vaccine treatment, suggesting that booster doses are required for a sustained viral response. anti-hbs was detected in 55/120 vaccine recipients, but only three patients demonstrated hbsag loss, indicating that the vaccine-induced anti-hbs was unable to completely neutralize hbsag in the serum [28]. the eastern woodchuck (marmota monax) is naturally infected by woodchuck hepatitis virus (whv) which was discovered in 1978 [29] . whv was found to be closely related to hepatitis b virus (hbv) [30] and classified as the second member of the genus ortho-hepadnavirus, family hepadnaviridae. in contrast to hbv-associated hcc in patients without a preferred integration site of hbv dna, a frequent integration of the whv genome close to the n-myc and c-myc gene has been observed in woodchucks developing hcc [31] . infections of woodchucks with whv have been shown to be endemic in the mid-atlantic states of the usa, whereas in the state of new york and new england woodchucks are rarely infected with whv. recently, a chinese marmot marmota himalayana was found to be susceptible to whv infection [32] (fig. 1 this review is focusing on the characterization of woodchuck genes related to innate and adaptive response, the recent development of new tools to determine virusspecific t cell response, therapeutic vaccines, and finally immunostimulatory and immunomodulatory approaches to treat chronic whv infection. these new findings in this preclinical model will help the development of new strategies to treat chronic hbv infection in patients. in recent years, many efforts have been devoted to cloning and characterization of components of the woodchuck immune system. a number of immune function-related genes including cytokines and their receptors, immune cell surface markers and other immune function-related proteins have been cloned and characterized. so far, important woodchuck cytokines and their receptors such as tnf-α, ifn-α, ifn-γ, il-12, il-15, gmcsf, lymphotoxin (lt)-α and il-10r have been cloned and tested for their biological activities [54] [55] [56] [57] [58] [59] [60] [61] . in patients, ifn has been used in the treatment of chb for many years. therefore, the ifn system has also been characterized in woodchucks. woodchuck ifn-α was shown to reduce whv surface antigen expression in a dose-dependent fashion in whv-infected woodchuck hepatocytes [62] . the woodchuck ifn-α/β system and their expression in peripheral blood lymphocytes (pbls) from naïve and whv-infected woodchucks have also been studied [63] . the woodchuck ifn-α genes could be classified into ten subtypes and three pseudotypes. poly(i:c) stimulation on naïve woodchuck pbls could induce ifn-α subtypes one, four and five production, indicating a selective expression of woodchuck ifn-α subtypes. moreover, pbls from chronically whv-infected woodchucks showed a reduced ability to produce woodchuck ifn when stimulated with poly(i:c). the complete or partial sequences of the type i ifn receptors (ifnars) of woodchucks were also obtained and analysed by fan et al. [64] . ifn-α or ifn-γ stimulation significantly upregulated ifnar2 expression in primary woodchuck hepatocytes. a decreased ifnar1 and ifnar2 expression was observed in woodchucks chronically infected with whv. these data are essential for studying type i ifn-related innate immunity and therapy in hepadnaviral infection in the woodchuck model. il-10 is a pleiotropic cytokine acting on a variety of immune cells through its cell surface receptor (il-10r). it has been suggested to resuscitate antiviral immunity by interfering with il-10/il-10r pathway. an increased production of il-10 was observed in patients with chb [65] , which hints that blockade of il-10r might become a feasible therapeutic approach for chb. very recently, jiang et al. [54] successfully cloned woodchuck il-10r and generated antibodies against this molecule. the blockade of woodchuck il-10r enhanced the proliferation and degranulation of specific t cells from chronically whv-infected woodchucks in vitro. this work provides a basis for potential therapeutic approaches in chronic hbv infection. important woodchuck immune cell surface molecules which have been cloned so far can be divided into two categories based on their function: molecules involved in innate immunity and molecules involved in adaptive immunity. toll-like receptors (tlrs) are a class of molecules that play a key role in the innate immune system. recent progress in this field revealed that there are significant interactions between the tlr system and pathogens in chronic viral infections [66] . so far, tlr2, tlr3, tlr4, tlr7, tlr8 and tlr9 have been successfully cloned in woodchucks [67] . in a recent study, zhang et al. [66] showed that tlr2 ligands induced the activation of nf-κb, pi3k/akt and different arms of mapk signalling pathways and the production of pro-inflammatory cytokines in woodchuck hepatocytes. tlr2-mediated innate immune responses reduced replication and gene expression of hbv in hepg2.2.15 cells and whv in primary woodchuck hepatocytes (see also article from zhang and lu, in this issue). in chronic whv carriers woodchuck model, relatively low levels of tlr2 expression were found in pbmcs and in liver tissues. tlr2 expression in pbmcs was inversely correlated with whv dna titres in acute whv infection and in entecavir-treated chronic whv carriers. an effective immune response against viral infections depends on the activation of cd8 t cells that can clear infection by killing virus-infected cells. therefore, sequence information of woodchuck cd3, cd4 and cd8 has been used to determine the kinetic of the influx of t cells into the liver during incubation period and acute or chronic whv infection. in week two post-infection, an influx of cd3+ lymphocytes could be observed and reached higher levels prior and during the recovery phase. the peak level of cd4+ and cd8+ t cells coincided with recovery. during transient infection, t cells can accumulate in the liver and reach up to two-thirds of the total number of liver cells [35] . in the adaptive immune response, cd28 and ctla-4 are known to play important roles for the regulation of t cell activation by delivering costimulatory signals. the complete coding regions of woodchuck cd28 and cytotoxic t-lymphocyte-associated antigen 4 (ctla-4) have been cloned and sequenced [68] . woodchuck cd28 showed a similarity of 76 and 70 % to its human and mouse homologues, respectively, according to the deduced amino acid sequences. woodchuck ctla-4 has a higher similarity of 86 and 75 % to the corresponding human and mouse ctla-4 molecules, respectively. the strict conservation of critical amino acid residues like cysteine and asparagine residues in woodchuck cd28 and ctla-4 suggests that both molecules may structurally resemble their human or mouse homologues. a hexapeptide motif mypppy which has been supposed to be essential for the interaction with cd80 is present in both woodchuck cd28 and ctla-4 [68] . the advances in sequencing technology provide new tools to characterize genes of the woodchuck immune system in large scale. fletcher et al. [69] performed the sequencing, assembly and annotation of the woodchuck transcriptome, together with the generation of custom woodchuck microarrays. by using this new platform, they characterized the transcriptional response to persistent whv infection and whv-induced hcc. liu et al. have also performed de novo woodchuck transcriptome assembly by using deep sequencing technology (unpublished data). with the help of this advanced technology, sequence information of important immune genes such as apobec3 of woodchucks has been revealed. it has been shown that upregulation of apobec3 led to specific and non-hepatotoxic degradation of nuclear hbv cccdna [70] . therefore, future cloning and characterizing of apobec3 in the woodchuck model will evaluate the therapeutic potential for chb. in summary, these efforts on establishing the translational value of the woodchuck model can provide new insight into characterizing immune pathways which may play a role in the persistence of hbv infection. studies in patients underline the important role of hbvspecific t cell response as a leading factor of viral clearance. for many years, the lack of appropriate methods to evaluate antigen-specific t cell responses was the serious limitation of this model. the establishment of the assays for monitoring of cellular immune response in woodchucks is of great importance for a reliable evaluation of therapeutic and immunomodulatory strategies for treatment of chb in the woodchuck model. development of the 2[ 3 h]-adenine-based proliferation assay enabled to detect the t-helper lymphocyte responses after stimulation of woodchuck pbmcs [39, 41] . in addition, several t-helper epitopes within whcag [39, 41] were identified in pbmcs from acutely whv-infected animals. significant progress in studying the t cell response of woodchucks was achieved by introduction of the flow cytometric cd107a degranulation assay that enables the detection of whv-specific cytotoxic t cells (ctls) in woodchuck pbmcs and splenocytes [38] . several studies demonstrated that detection of cd107a, as a degranulation marker, is a suitable method for determination of antigenspecific cytotoxic t lymphocytes [71, 72] . introduction of the immunological tools for studying of the t cell response in woodchucks revealed a significant similarity between the pathogenesis of whv infection in woodchucks and hbv in humans. it was demonstrated that acute self-limiting and resolved whv infections correlate with robust multifunctional t-helper and cytotoxic t cell responses, while whv chronic carriers demonstrate weak or no virus-specific t cell responses against the viral proteins (table 2) [38, 39, 41]. moreover, these studies confirmed that the efficient cellular immune response to viral antigens results in liver injury and is necessary for viral clearance. recently described advancements in the characterization and monitoring of the woodchuck immune system during the whv infection made this animal model particularly useful for development of the immunomodulatory approaches in chb. the pioneer investigations with therapeutic vaccines based on whv core [73] or surface [76, 77] reporting that the t cell response to hbv was successfully restored in patients treated with lamivudine. in addition, the quantity of antigen particularly the whv surface antigen (whsag) to which the immune system is exposed can induce different degrees of functional impairment of antiviral t cells, up to physical t cell deletion [78, 79] . combination therapy using lamivudine and serumderived whsag vaccination showed no effect on induction of anti-whs antibodies or reduction of whv dna [80] . our group evaluated the efficacy of the combination therapy in the woodchuck model by combining lamivudine treatment, dna vaccination (three plasmids expressing whsag, whcag and woodchuck ifn-γ) and whsag/ anti-whs immunogenic complexes vaccination [81] . the triple combination led to a decrease in whv viral load up to 2.9 log, in serum whsag up to 92 % and in development of anti-whs antibodies. nevertheless, these effects were not sustained and all parameters reached the baseline levels shortly after withdrawal of lamivudine treatment. in addition, the vaccination did not induce whv-specific t cell responses in the majority of woodchucks, even in animals that exhibited virological responses. later, we modified this protocol by using the more potent antiviral drug entecavir (etv) and increasing the number of the immunizations (with plasmids expressing whsag and whcag from three to six) (lu et al., unpublished results). a significant delay of the rebound of viremia was observed in woodchucks which received additional vaccination, compared to controls treated only with etv. in another study, chronic whv carriers received a treatment of the potent antiviral drug clevudine in combination with an alumadsorbed whsag vaccine. combination treatment resulted in significant and sustained reduction of whv dna loads and whsag concentrations in most treated animals. compared to vaccination alone, combination treatment induced a more robust anti-whs response [82, 83] . the results of these studies clearly showed that combination of antiviral treatment and vaccination is more effective in inducing virus-specific t cell responses than therapeutic vaccination alone. nevertheless, the efficacy of these approaches was still too limited when applied for treatment of chb. the vaccination strategies used in some of these studies were even not able to boost a functional antiviral t cell response. a significantly better induction of whcagspecific t cells using more potent vaccines, such as recombinant viral vectors, may be required to achieve sustained antiviral response and viral clearance. recombinant adenoviral vectors (adv) proved to elicit a vigorous and sustained humoral and t cell responses to the transduced antigen [84, 85] . adenoviral vectors also act as a natural adjuvants causing dc maturation, enhanced antigen presentation and secretion of antiviral cytokines, such as ifn-α, tnf-α and il-6 [86] . however, even single immunization with recombinant adenoviruses may induce immunity, predominantly neutralizing antibodies, against the vector itself. this negative effect of the adenovirusinduced immunity against the vaccine may be overcome by heterologous prime-boost regimen. in particular, subsequent priming immunizations with plasmid dna vaccine followed by a booster vaccination with adv seem to be a very promising strategy. dna prime-adenovirus boost regimen proved to induce more robust and potent immune response in comparison with plasmid dna alone and provided protection against the pathogen challenge in several animal models of infectious diseases [87] [88] [89] (see also article from e. barnes in this issue). recently, our group has investigated whether the heterologous prime-boost immunization strategy using plasmid dna and recombinant adenoviral vectors may improve the efficacy of the therapeutic vaccination in chb in the woodchuck model. a new dna plasmid (pcgwhc) and an adenoviral serotype 5 vector (ad5whc) and a chimeric ad5 displaying ad35 fibre (ad35whc) showing high expression levels of whcag were constructed [90] . the increased antigen expression was achieved by insertion of an intron sequence in the expression cassette of the vaccines. preliminary results showed that the new vaccines are able to induce strong and sustained whcag-specific t cell response in mice and naïve woodchucks. interestingly, immunization with advs led to rapid and massive production of anti-whs antibodies and as a result resolution of infection after the whv challenge [90] . the dna prime-adv boost immunization strategy was further used as a therapeutic vaccine against chronic whv infection in combination with antiviral treatment with etv. six chronically whv-infected woodchucks were treated for 23 weeks with etv. starting from week eight, four of the six etv-treated animals received subsequently nine intramuscular immunizations with: dna plasmids expressing whcag (pcgwhc) and whsag (pwhsim), ad5whc and ad35whc. whsag-and whcag-specific t-helper and cytotoxic t cell responses were detected in all chronic carriers that received immunizations, but not in etv only treated animals. in addition, woodchucks receiving the combination therapy showed a prolonged suppression of whv replication and lower whsag levels compared to controls. excitingly, two of four immunized carriers remained whv dna negative after the end of etv treatment and developed anti-whs antibodies [91] . these data are encouraging and demonstrate that the combined antiviral and vaccination approach efficiently elicited sustained immunological control of chronic hepadnaviral infection in woodchucks. persistent hbv infection is associated with functional exhaustion of virus-specific cd8 t cells [92] . this defect in virus-specific t cells is one of the primary reasons for the inability of the host to eliminate the persisting pathogen. although it has been shown that nucleoside analogues treatment can induce the recovery of hbv-specific ctl activity in patients [76] , this effect is only transient [77] . those findings are consistent with our data obtained from the woodchuck model, in which etv treatment alone only induced either only transient ctl responses [91] or no responses at all [93] . therefore, additional strategies that can potently enhance t cell response need to be enroled for the treatment of chb infection. recent studies in chronic virus infection models indicate that the interaction between the inhibitory receptor programmed death-1 (pd-1) and its ligands plays a critical role in t cell exhaustion [94] [95] [96] [97] . in chronic hbv infections, upregulation of pd-1 on virus-specific t cells was observed, and restoration of the t cell function has been achieved by blocking the pd-1/pd-ligand 1 (pd-l1) interaction in vitro [98] . recently, the therapeutic effect of pd-1/pd-l1 blockade has also been investigated for chronic hcv infection in chimpanzees [99] and in patients [100] . however, limited effect on restoring t cell function was observed in these studies which used only pd-1/pd-l1 blockade. it has been recently clarified that the proportion of cd8 t cells expressing pd-1 and the levels of pd-1 on virus-specific t cells are strongly correlated with viral load in the plasma [101] [102] [103] . antiretroviral treatment resulted in the dramatic decline of plasma viral load, coincident with a decrease in the pd-1 expression level on virus-specific cd8 t cells [101, 103] . in line with this, a better restoration of t cell functions upon in vitro anti-pd-l1 treatment was observed in chronic hbv patients with lower viremia [104] . therefore, a combination therapy that includes direct antiviral drug and pd-l1 blockade is a reasonable strategy for the treatment of chronic hbv infection. in line with these findings, zhang et al. [105] and liu et al. [93] successfully cloned and characterized the woodchuck pd-1/pd-l system in the whv infection woodchuck model. a significant positive correlation between the viral load and the pd-1 expression on total cd8 t cells in chronic whv infection was observed. both the proportion of pd-1+ cd8 t cells and the levels of pd-1 expression on cd8 t cells were significantly higher in the woodchucks with chronic whv infection compared to naïve animals and resolvers. more importantly, during etv treatment of those chronic carriers, a reduction of serum viral load was correlated with a dramatic decrease in the level of pd-1 expression on cd8 t cells [93] . in vitro blockade of woodchuck pd-1/pd-l1 pathway by using a rabbit polyclonal pd-l1 blocking antibody could partially restore the t cell function in whv-infected woodchucks [105] . moreover, in vivo blockade of the pd-1/pd-l1 pathway on cd8 t cells, in combination with nucleoside analogue treatment and dna vaccination, synergistically enhanced the function of virus-specific t cells. the combination therapy potently suppressed whv replication, leading to sustained immunological control of viral infection, anti-whs antibody development and complete viral clearance in some woodchucks [93] . although similar approaches have been tried in other viruses in the past, such as lcmv, the data presented here may be an advance for the hbv field to new approaches for eliminating the virus itself rather than only suppressing its replication. the woodchuck is a valuable preclinical model for developing new therapeutic approaches in chronic hepadnaviral infections. even though several innovative approaches combining antiviral treatment with nucleoside analogues, dna vaccines and protein vaccines were tested in chronically infected woodchucks, the effectiveness of those strategies was very limited. strategies investigated so far were often hampered by weak t cell responses observed after immunization, suggesting a strong need for alternative strategies to enhance t cell functions during chronic hbv infection. recently, our group published two independent proof-of-concept studies, showing that using a very potent t cell vaccine and blockade of negative signalling in t cells may lead to the resolution of chronic hepatitis b in some woodchucks (table 3) . these data are encouraging and implicate the feasibility and usefulness of the immunotherapeutic strategies for the treatment of chronically hbv-infected patients. nevertheless, which factors influence the effect of therapeutic vaccination remains to be further investigated. it has been noticed that satisfactory therapeutic effects could not be documented in the studies using hbsagbased prophylactic vaccines. in the mean time, evidence has supported that hbcag-specific immunity is endowed with antiviral and liver-protecting capacities in chb patients and animal models. with the increasing number of available vaccine formulation, a more crucial question raised recently: what is the optimal combination of these vaccines. obviously, it is necessary to test the mutual influences of different types of vaccines to maximize their effects and avoid the negative interference between the vaccines. also, the question how hbv infection leads to defective immune responses to hbv proteins remains to be investigated. this issue is the key to a more rational design of new therapeutic approaches. figure 2 summarizes the ideas of a potential combination treatment for patients with chronic hepatitis b. the presence of viral components may be a main reason for t cell tolerance in chronic hbv infection. antiviral treatment with nucleoside analogues efficiently reduce hbv replication and release of new virions and may partly restore hbv-specific cd8 and cd4 t cell responses, thereby allowing successful therapeutic vaccination. however, hbv proteins are still produced as the transcription of mrnas for the s protein and the core protein on hbv cccdna is not affected by antiviral treatment. even when hbv dna disappears during antiviral treatment, hbsag and hbcag/hbeag are present in the liver or in blood at high levels. it is proposed to reduce hbv protein load by small interfering rnas (sirnas), which lead to the sequence-specific degradation of homologous mrna. using this rna interference (rnai) with chemically synthesized or vector-expressed sirnas, many clinically relevant viruses including the human immunodeficiency virus, hbv and hcv could be inhibited. in in vitro experiments showed that whv transcripts could be degraded by sirnas [106] . at the same time, the degradation of viral rnas resulted in the activation of multiple pathways of host innate immune responses [107] . however, future in vivo studies are required to demonstrate the usefulness of this technology. combining gene-silencing approach with nucleoside analogues may further facilitate the stimulation of the immune system by therapeutic vaccines. the epigenetic regulation provides an alternative to interfere with hbv gene expression. hbv minichromosome in hepatocytes is under the complex control of epigenetic mechanisms, and its transcriptional activity could be influenced by methylation, histone acetylation and other mechanisms [108] . therefore, exploring epigenetic drugs to modify, these regulatory processes may achieve an effective suppression of hbv gene expression and thereby replace antiviral treatment with nucleoside analogues. the stimulation of innate immune responses may contribute to the control of hbv infection. in this special issue, zhang and lu provided a review dedicating to the role of tlr system. interferons and interferon-stimulated genes (isgs) represent still an important part for anti-hbv treatment. a recent review about this aspect described the current progress (pei et al., in press) . recently, the antiviral functions of isgs are under studies. for example, interferon-induced protein with tetratricopeptide repeats 1 and 2 is a cellular factor that was shown to limit hepatitis b virus replication in hepatoma cells [109] . another recent report by lucifora et al. [70] about the role of apobecs in the degradation of cccdna was highly interesting, but remained to be controversial [110, 111] . future investigation is required to elucidate the functions of isgs and their relative contribution for control of hbv infection, before exploring these genes for antiviral treatment. global epidemiology of hepatitis b virus infection: new estimates of age-specific hbsag seroprevalence and endemicity pegylated interferon alfa-2b alone or in combination with lamivudine for hbeag-positive chronic hepatitis b: a randomised trial peginterferon alfa-2a, lamivudine, and the combination for hbeag-positive chronic hepatitis b cellular and virological mechanisms of hbv drug resistance hepatitis b virus resistance to nucleos(t)ide analogues long-lasting memory t cell responses following selflimited acute hepatitis b the hepatitis b virus persists for decades after patients' recovery from acute viral hepatitis despite active maintenance of a cytotoxic t-lymphocyte response cytotoxic t lymphocyte responsiveness after resolution of chronic hepatitis b virus infection specific vaccine therapy in chronic hepatitis b: induction of expression and purification of woodchuck tumour necrosis factor alpha molecular cloning of the woodchuck cytokines: tnf-alpha, ifn-gamma, and il-6 woodchuck gamma interferon upregulates major histocompatibility complex class i transcription but is unable to deplete woodchuck hepatitis virus replication intermediates and rnas in persistently infected woodchuck primary hepatocytes molecular characterization of woodchuck interleukin 15 (wil-15) and detection of its expression in liver samples of woodchucks infected with woodchuck hepatitis virus (whv) molecular cloning and expression of woodchuck granulocyte-macrophage colony stimulating factor expression of a new woodchuck ifn-alpha gene by a helper-dependent adenoviral vector in woodchuck hepatitis virus-infected primary hepatocytes molecular characterization of woodchuck type i interferons and their expression by woodchuck peripheral blood lymphocytes molecular characterization of the type i ifn receptor in two woodchuck species and detection of its expression in liver samples from woodchucks infected with woodchuck hepatitis virus (whv) hepatitis b virus-specific t-cell proliferation and cytokine secretion in chronic hepatitis b e antibody-positive patients treated with ribavirin and interferon alpha role of tolllike receptor 2 in the immune response against hepadnaviral infection lipopolysaccharide-induced innate immune responses in primary hepatocytes downregulates woodchuck hepatitis virus replication via interferon-independent pathways molecular characterization of cd28 and cytotoxic t-lymphocyte-associated antigen 4 (ctla-4) of woodchuck (marmota monax) transcriptomic analysis of the woodchuck model of chronic hepatitis b specific and nonhepatotoxic degradation of nuclear hepatitis b virus cccdna sensitive and viable identification of antigen-specific cd8+ t cells by a flow cytometric assay for degranulation ex vivo identification, isolation and analysis of tumor-cytolytic t cells the woodchuck: an animal model for hepatitis b virus infection in man therapeutic vaccination of woodchucks against chronic woodchuck hepatitis virus infection induction of antibodies to the pres region of surface antigens of woodchuck hepatitis virus (whv) in chronic carrier woodchucks by immunizations with whv surface antigens lamivudine treatment can overcome cytotoxic t-cell hyporesponsiveness in chronic hepatitis b: new perspectives for immune therapy transient restoration of anti-viral t cell responses induced by lamivudine therapy in chronic hepatitis b molecular signature of cd8+ t cell exhaustion during chronic viral infection reinvigorating exhausted hiv-specific t cells via pd-1-pd-1 ligand blockade t-helper cell response to woodchuck hepatitis virus antigens after therapeutic vaccination of chronically-infected animals treated with lamivudine combination of an antiviral drug and immunomodulation against hepadnaviral infection in the woodchuck model immunization with surface antigen vaccine alone and after treatment with 1-(2-fluoro-5-methyl-beta-larabinofuranosyl)-uracil (l-fmau) breaks humoral and cellmediated immune tolerance in chronic woodchuck hepatitis virus infection immunogenic effects of woodchuck hepatitis virus surface antigen vaccine in combination with antiviral therapy: breaking of humoral and cellular immune tolerance in chronic woodchuck hepatitis virus infection replication-incompetent adenoviral vaccine vector elicits effective anti-immunodeficiency-virus immunity augmentation of immune responses to sars coronavirus by a combination of dna and whole killed virus vaccines recombinant adenovirus induces maturation of dendritic cells via an nf-kappab-dependent pathway development of a preventive vaccine for ebola virus infection in primates attenuation of simian immunodeficiency virus siv-mac239 infection by prophylactic immunization with dna and recombinant adenoviral vaccine vectors expressing gag prime-boost vaccination with plasmid dna and a chimeric adenovirus type 5 vector with type 35 fiber induces protective immunity against hiv dna prime-adenovirus boost immunization induces a vigorous and multifunctional t-cell response against hepadnaviral proteins in the mouse and woodchuck model combination of dna prime-adenovirus boost immunization with entecavir elicits sustained control of chronic hepatitis b in the woodchuck model t cells and viral persistence: lessons from diverse infections enhancing virus-specific immunity in vivo by combining therapeutic vaccination and pd-l1 blockade in chronic hepadnaviral infection restoring function in exhausted cd8 t cells during chronic viral infection pd-1 blockade in rhesus macaques: impact on chronic infection and prophylactic vaccination pd-1:pd-l1 interactions contribute to the functional suppression of virus-specific cd8+ t lymphocytes in the liver enhancing siv-specific immunity in vivo by pd-1 blockade characterization of hepatitis b virus (hbv)-specific t-cell dysfunction in chronic hbv infection immunotherapy of chronic hepatitis c virus infection with antibodies against programmed cell death a randomized, double-blind, placebo-controlled assessment of bms-936558, a fully human monoclonal antibody to programmed death-1 (pd-1), in patients with chronic hepatitis c virus infection pd-1 expression on hiv-specific t cells is associated with t-cell exhaustion and disease progression dysfunction and functional restoration of hcv-specific cd8 responses in chronic hepatitis c virus infection pd-1 is a regulator of virus-specific cd8+ t cell survival in hiv infection antiviral intrahepatic t-cell responses can be restored by blocking programmed death-1 pathway in chronic hepatitis b the expression of pd-1 ligands and their involvement in regulation of t cell functions in acute and chronic woodchuck hepatitis virus infection inhibition of woodchuck hepatitis virus gene expression in primary hepatocytes by sirna enhances the cellular gene expression rnai induces innate immunity through multiple cellular signaling pathways regulation of hepatitis b virus replication by epigenetic mechanisms and micrornas interferon-induced proteins with tetratricopeptide repeats 1 and 2 are cellular factors that limit hepatitis b virus replication specific and nonhepatotoxic degradation of nuclear hepatitis b virus cccdna virology. response to comment on "specific and nonhepatotoxic degradation of nuclear hepatitis b virus cccdna a pilot study of the cy-1899 t-cell vaccine in subjects chronically infected with hepatitis b virus. the cy1899 t cell vaccine study group clinical and immunological efficacy of intradermal vaccine plus lamivudine with or without interleukin-2 in patients with chronic hepatitis b in vivo immunization by vaccine therapy following virus suppression by lamivudine: a novel approach for treating patients with chronic hepatitis b therapeutic vaccination of chronic hepatitis b patients with virus suppression by antiviral therapy: a randomized, controlled study of co-administration of hbsag/as02 candidate vaccine and lamivudine the authors thank dr. wolfram gerlich for his critical comments on this manuscript. a number of studies cited in this review were funded by deutsche forschungsgemeinschaft (gk 1045 and sfb/trr60). open access this article is distributed under the terms of the creative commons attribution license which permits any use, distribution, and reproduction in any medium, provided the original author(s) and the source are credited. key: cord-008821-rxzgfk1k authors: a. lucchiari, maria; modolell, manuel; eichmann, klaus; a. pereira, carlos title: in vivo depletion of interferon-gamma leads to susceptibility of a/j mice to mouse hepatitis virus 3 infection date: 2011-11-02 journal: immunobiology doi: 10.1016/s0171-2985(11)80089-6 sha: doc_id: 8821 cord_uid: rxzgfk1k the possible role of interferon-gamma (ifn-γ) in the resistance of a/j mice to mhv3 infection was investigated. monoclonal antibodies specific for ifn-γ, cd4 and cd8 molecules were administered in vivo to deplete selectively the ifn-y synthesized or the appropriate subset of t cells. the animals were then infected with mhv3 and the course of infection was followed by studying different parameters, such as, the mortality, the virus growth in the tissues and the ifn-γ synthesis in sera and peritoneal exudates. after mhv3 infection, a full resistance of control a/j mice was observed, in contrast to the high mortality rate observed among the depleted animals, where higher virus titers were found in different tissues. the ifn-γ synthesis in sera and peritoneal exudates of depleted mice, after mhv3 infection, drastically decreased when compared to that detected in control mice. the data presented are consistent with the hypothesis that ifn-γ plays an essential role in the resistance of a/j mice to mhv3 infection. the ai] mice have been reported to be resistant following mhv3 infection, since they develop a mild disease which disappears 4 to 6 days later (1, 2) . the mechanisms suggested to be involved in the resistance include the antiviral state induced by ifn, the virus replication in target cells and the expression of a mono kine that demonstrates pro coagulant activity (3) (4) (5) (6) (7) (8) . we have recently shown that the resistance of ai] mice this work was supported in part by grants from the funda"ao de amparo a pesquisa do estado de sao paulo (fapesp) and conselho nacional de pesquisa (cnpq). m.a.l was a recipient of the deutscher akademischer austauschdienst (daad) during part of this research. abbreviations; mhv3 = mouse hepatitis virus 3; ifn = interferon; fes = fetal calf serum; pfu = plaque-forming units; ip = intraperitoneally; ab = antibodies; pbs = phosphate buffer solution against our strain of mhv3 is acquired after immunization and the mechanism involved is dependent on the ifn -y synthesis and the macrophage sensitivity to ifn-y (9). cd4 and cds t lymphocytes are essential for the development of protective immunity to several infections (10) (11) (12) . one of the functions of these cells is the production of ifn-y, a major t celllymphokine, which can also be produced by nk cells. ifn-y activates macrophages, as assessed by the modulation of antiviral state (6), the increased expression of class ii major histocompatibility complex gene products (13) , the enhancement of phagocytosis (14) , the induction of production of reactive oxigen intermediates (15) and tumor necrosis factor (16) . in the experiments described here, with the aim to investigate the in vivo participation of ifn-y in the development of resistance to mhv3 infection, mice have been depleted in vivo of the ifn-y synthesized after infection or the cd4 and cds subsets of t cells, using monoclonal antibodies. the data show a direct evidence that the in vivo neutralization of synthesized ifn-y during the infection led to susceptibility to mhv3 and that mice depleted of cd4 or cds t lymphocytes were unable to synthesize the high levels of ifn-y that were found in the controls after mhv3 infection, and died of acute hepatitis. these results are further support to the already proposed crucial role of ifn -y in the expression of resistance of ai] mice to mhv3 (6, 7, 9) . six-week-old mice of the inbred ai] strain obtained from the institut pasteur, paris, france were bred in our mouse colony. the animals were periodically tested for the preexisting corona viruses or antibodies against mhv3 following procedures already described (9). mhv3 was cultivated and titrated by plaque assay on l929 cells at 37 dc as previously described (17) . aliquots containing 2 x 105 plaque-forming units per ml (pfu/ml) were stored at -80 dc and used in all experiments. for the determination of mhv3 titer in tissues, the animals were sacrificed, and the peritoneal exudates and livers were obtained. these were ground, resuspended in 2 ml of rpmi 1640 medium with 10 % fetal calf serum (fcs) (gibco ltd, paisley, scotland), penicillin (100 u/ml) plus streptomycin (100 [tg/ml) and the virus titrated on l 929 cells. the peritoneal exudates were collected by peritoneal lavage with 6 ml of medium, centrifuged at 750 x g for 10 min and the virus in the supernatants titrated on l 929 cells. the mhv3 titer obtained were expressed as pfu per milliliter of peritoneal exudate (pfulml), or pfu per gram of liver (pfu/g). cells producing rat monoclonal antibodies against mouse ifn-y (r4-6a2) was kindly donated by g. l. spitalny, trudeau institute, ny, usa (18) and cells producing rat monoclonal antibodies specific for cd4 and cd8 determinants (yts 191.1 and yts 169.4.2 respectively) were a gift of s. cobbold and h. waldmann, cambridge university, cambridge, u.k. (19) . the antibodies were purified from tissue culture supernatants by affinity chromatography on protein a-sepharose (pharmacia, uppsala, sweden). the immunoglobulin concentration was determined by spectrophotometric measurement at 280 nm. they were concentrated by dialysis and sterilized by filtration. the activity of the antibodies against ifn-y or cd4 and cd8 was tested, respectively, by neutralization of cytopathic effect or inhibition of t cell proliferation and cell binding by cytofluorometric assay. sera of 5 normal rats in a pool were used as a source of normal rat antibodies. groups of ai] mice were immunized once by intraperitoneal (ip) injection of 10 3 pfu of mhv3 inactivated by ultraviolet (uv) radiation. after a period of 30 days they were injected ip with normal rat antibodies (controls) or purified antibodies against ifn-y, cd4 or cd8 in pbs. groups of mice were injected ip with 200 ~g of purified antibodys at day -5, -1, 0,1,2, 3,4 and 500 pfu of mhv3 at day o. the mice were maintained under sterile conditions for the duration of the experiment and the t cell depletion, mortality, virus growth in the tissues and ifn synthesis determined. a cytopathic effect reduction test technique using monolayers of l 929 cells and encephalomyocarditis virus, described in detail in previous papers (17, 20) , was used as an ifn assay. for characterization of ifn-a/~ and ifn-y antibodies to mouse ifn-a/~, produced in rabbits, and monoclonal antibodies to recombinant mouse ifn-y (holland biotechnology, leiden, holland) with activity of 2 x 10 3 and 2 x 10 5 neutralizing units per mg, respectively, were always used. one unit was defined as the amount of antibodies sufficient for neutralizing one unit of ifn. these antibodies showed no cross-reactivity. as shown in table 1 , the treatment of ai] mice with antibodies against ifn-y or cd4 or cds t lymphocytes led to a high susceptibility to mhv3 infection. control a/j mice were fully resistant to the infection and the animals showed only a mild disease that disappeared 4 days later. in contrast, the treated animals had a period of disease characterized by ruffled fur, hunching, loss of weight, diarrhea and general lassitude, that started earlier and remained for a longer period. most of the animals died of acute hepatitis 7 to s days after the infection. it can be clearly seen in figure 1 previous work on mhv3 infection postulated that both resistance gene( s) controlling the degree of viral replication in target cells and the intact immune response are required for resistance (8, 21) . we have shown that a nutritionally induced hypercholesterolemia in resistant ai] mice caused susceptibility to mhv3 infection, and that the inhibition of the host resistance was a consequence of an impairment of kupffer cell functions, such as the sensitivity to the induction of an anti-mhv3 state by ifn (22) . recently, we have proposed that the mechanism involved in the ai] mice resistance to mhv3 is dependent on the t cells activity and rely on the ifn-y production and the macrophage sensitivity to ifn-y (6, 7, 9) . these mice have macrophages that are very sensitive to ifn -y in order to develop an anti-mhv3 state and are capable of producing reasonable amounts of ifn-y in the course of the immune response against mhv3. thus, soon after initiation of infection, the virus particles could be neutralized and the infection cleared in a few days. alternatively, susceptible balb/c mice have macrophages that are not sensitive to ifn -y in order to develop an anti-mhv3 state, and despite high concentrations of ifn -y produced during the first days of infection, the macrophages cannot display a restriction of virus multiplication and these animals die 5 to 6 days after infection (6) . in order to investigate the in vivo role of ifn-y during the mhv3 infection, we followed a direct approach by treating the ai] mice, previously immunized 30 days before with uv-inactivated mhv3, since the resistance is acquired after immunization (9), with monoclonal antibodies against ifn-y, which has been shown to neutralize different activities of the ifn-y (18) . since one of the features of cd4 and cd8 t lymphocytes is to produce and release ifn-y, it was of interest to investigate whether ai] mice treated with monoclonal antibodies against cd4 or cd8 molecules, in such a way that a depletion of cd4 or cd8 t lymphocytes occurred, could be rendered susceptible by an inhibition of ifn-y synthesized during the course of the infection by mhv3. the data obtained showed that ai] mice treated with anti-ifn-y, anti-cd4 or anti-cd8 antibodies became susceptible to mhv3, showing a longer period of disease (table 1) , and that the treatment induced a neutralization or inhibition of the ifn -y synthesis (fig. 3 ) that normally occurred in control mice during the mhv3 infection. the effect was due to the treatment with specific antibodies against ifn-y, cd4 or cd8, since mice treated with normal rat antibodies behave like those only infected with mhv3, showing that nonspecific effects of control antibodies were not involved. the finding that depletion of either cd4 or cd8 t cell subsets showed the same effect on the inhibition of ifn-y synthesis, mainly in the sera, after mhv3 infection is rather surprising and has been one of the subjects of our present investigations. although, in view of the crucial regulatory function of cd4 cells and the important role of cds cells as cytotoxic effector cells during a virusspecific immune response, the results cannot unequivocally exclude the interpretation that susceptibility of the immunosuppressed animals may be due to the absence of proper immune effector mechanisms, the lack of clearance of the virus in the peritoneum and liver of ai] treated and infected mice (figure 1) , which leads to high rates of mortality (table 1) , may be attributed to the lack of ifn-y in the serum or peritoneal exudate of them (fig. 3) , which has been shown in vitro to be effective to restrict the mhv3 growth in target cells such as macrophages (6, 7, 9) . furthermore, our previous observation that the lack of kupffer cells sensitivity to ifn correlated with the susceptibility of hypercholesterolemic ai] mice to mhv3 (22) , is in further support to the idea that ifn-y plays a crucial role in the resistance to mhv3. beside the direct evidence that ifn -y depleted mice became susceptible to mhv3 infection, our findings show the crucial intervention of cd4 and cds lymphocytes by one of their products, the ifn-y, in the development of ai] mice resistance to mhv3 infection. although the participation of other factors linked to the generation of the immune response can not be excluded, these results are in further support to the involvement of ifn-y in the mechanism of resistance against mhv3 infection. interactions between mouse hepatitis viruses and primary cultures of kupffer and endothelial liver cells from resistant and susceptible inbred mouse strain role of macrophages and interferon in natural resistance to mouse hepatitis virus infections adult mouse hepatocytes in primary monolayer culture express genetic resistance to mouse hepatitis virus type 3 susceptibilitylresistance to mouse hepatitis virus strain 3 and macrophage procoagulant activity are linked and controlled by two non-h-2 linked genes immunopathology of mouse hepatitis virus type 3 infection. 1. role of humoral and cell mediated immunity in resistance mechanisms a major role of macrophage activation by interferon gamma during mouse hepatitis virus type 3 infection. 1. genetically dependent resistance a major role of macrophage activation by interferon gamma during mouse hepatitis virus type 3 infection. ii role of interferon in the pathogenesis of viral diseases of mice as demonstrated by the use of anti-interferon serum. v. protective role in mouse hepatitis virus type 3 infection of susceptible and resistant strains of mice acquired immunity dependence of a/j mice resistance to mouse hepatitis virus 3 infection: dependence on interferon gamma synthesis and macrophage sensitivity to interferon gamma induction of maloney murine sarcoma virus tolerance in adult mice by anti-cd4 monoclonal antibody treatment roles of cd4-and cd8-bearing t lymphocytes in the immune response to erythrocytic stages of plasmodium chabaudi effective clearance of mouse hepatitis virus from the central nervous system requires both cd4+ and cd8+ t cells differential regulation of class ii mhc determinants on macrophages by ifn gamma and il-4 induction of crisis forms in the human malaria parasite plasmodium falciparum by gamma interferon activated, monocyte macrophages identification of interferon gamma as the lymphokine that activates human macrophage oxidative metabolism and antimicrobial activity activation of mouse peritoneal macrophages in vitro and in vivo by interferon gamma induction of natural killer cells and interferon during mouse hepatitis virus infection of resistant and susceptible inbred mouse strains monoclonal antibody to murine gamma interferon inhibits lymphokine-induced antiviral and macrophage tumoricidal activities therapy with monoclonal antibodies by elimination of t-cell subsets in vivo inhibition of mouse hepatitis virus type 3 multiplication in activated kupffer cells. braz genetically determined resistance to mouse hepatitis virus type 3 is expressed in hematopoietic donor cells in radiation chimeras increased susceptibility of mice to mhv3 infection induced by hypercholesterolemic diet: impairment of kupffer cell function laboratorio de imunologia viral, a v. dr. vital, brasil 1500 key: cord-005595-vzkcin1l authors: massa, p. t.; dörries, r.; ter meulen, v. title: viral particles induce ia antigen expression on astrocytes date: 1986 journal: nature doi: 10.1038/320543a0 sha: doc_id: 5595 cord_uid: vzkcin1l nan cally active but which do not bind antibody 259 have been prepared recently; cells transformed by such mutants were not altered morphologically, nor did they show greatly inhibited thymidine incorporation, after injection of antibody 259 26 • it is assumed that while viral oncogenes function without normal control, their mechanism of action is similar to that of related cellular genes. here we have presented evidence to support this idea in the case of growth factor receptor-like molecules and related oncogenes. it is therefore possible that these data might aid in understanding the way in which cellular genes interact in the control of normal proliferation. our results indicate that some receptor-like oncogenes depend on ras proteins while some cytoplasmic oncogenes do not. there are, of course, numerous oncogenes which we have not yet tested which might behave differently from those described here. with this limitation in mind and on the basis of our present data, we propose that an important class of proliferative signals are received at the cell surface by receptor molecules such as growth factor receptors, and the c-ras proteins are essential in the transfer of these signals to cytoplasmic effectors having serine kinase activity; the effectors then modify target molecules which are directly involved in initiating a proliferative cycle. accordingly, if the cytoplasmic effector were mutated such that it functioned without activation, proliferation would continue independently of c-ras proteins. receptor molecules, on the other hand, would always require c-ras to stimulate proliferation. while our data are consistent with the above scheme, they do not exclude many other possibilities involving multiple metabolic pathways and more complex interactions. for example, we have not reported results with nuclear oncogenes owing to their difficulty in transforming nih 3t3 cells. the proposed scheme is primarily attractive because of its similarity to the carefully studied mechanism of signal transduction involving cyclic amp. while it is unlikely that cyclic amp itself regulates proliferation 27 , g-regulatory proteins with enzymatic similarities to c-ras proteins are involved. these regulatory proteins control signal transduction from cell-surface receptors to cytoplasmic serine kinase effector molecules by regulating adenyl cyclase activity28. while the present study has examined only one aspect of what is likely to be a highly complex system for regulating proliferation, it does provide a means of functionally comparing separate viral oncogenes. injection of antibody has been used in other studies to characterize the types of molecules responsible for tumour cell proliferation. like nih 3t3 cells transformed by mos or raj genes, many tumour cells show no inhibition of proliferation when injected with anti-ras antibody. in this way their proliferation is distinct from that of the normal cell types studied, each of which was efficiently inhibited by the injected antibodyl6. this study relied on cell lines and plasmids prepared and characterized by several workers. in addition to those listed above, we thank h. temin, g. thornton, t. papas, s. goff and o. witte for supplying materials. we also thank t. curran and h.-f. kung for critical review of the manuscript and j. hansen for technical assistance. recent studies have shown that y-interferon (ifn-y) induces the expression of ia antigen on astrocytes l ,2. this observation is of immunological significance because such activated astrocytes can act as antigen-presenting cells, as demonstrated with myelin basic protein for antigen-specific encepbalitogenic t-cell lines 3 • however, the lack of lymphatic drainage in brain and the presence of the so-called blood-brain barrier restricting traffic of cells and macromolecules suggests that ifn-y may not be readily available, at least during the initial phases of viral infections. the question therefore arises as to whether astrocytes can be induced to express ia antigens by other signals directly related to viral infection and possibly independent of ifn-y. in the present report we demonstrate that a neurotropic murine hepatitis virus induces expression of ia antigen on astrocytes in tissue culture without infection, rendering these brain cells competent to participate directly in the immune response to a viral infection. the murine coronaviruses are a group of agents causing acute, subacute or chronic infections in mice or rats accompanied by different disease processes 4 , the jhm strain of this group is neurotropic and has been shown to induce acute or subacute encephalomyelitides which depend on virus as well as host factors 5 • one important factor in the case of subacute encephalomyelitis in lewis rats is the immune response, which is directed not only against the virus but also against brain tissue 6 • as various types of central nervous system (cns) disease are associated with this neurotropic murine coronavirus strain, we chose this virus to define its interaction with rat brain cells in culture with respect to the induction of ia antigen on astrocytes, as a baseline for our study, we analysed the response of lewis primary glial cell cultures consisting of macro phages carrying fc receptors (fc receptor+) and astrocytes expressing glial fibrillary acidic protein (gfap+) (fig,2a -c) to recombinant rat ifn-y (10 u ml-1 ; fig. la ). recombinant rat ifn-y induced the expression of ia on numerous cells in the primary cultures, fluorescence-activated cell sorting showed that 3-10% of the cells were induced to express ia compared with the control after 18 h treatment with 10 u ml-1 ifn-y, reaching a maximum at 48 h, when 20% of all cells were induced (fig. 1 a) . by immunofluorescence microscopy, ia+ cells also became apparent after 18 h of treatment whereas control cultures showed no ia + cells, double immunofluorescence microscopy revealed that (1 ,000 nu ml -' ) infectious jhm virus (10 3 pfu ml -i ) + ultraviolet-inactivated jhm virus (10 3 pfu ml -i ) + jhm virus (glial or dbt cell-derived) +anti-rat ifn-y + jhm virus+a non-neutralizing anti-jhm antibody + j h m virus + a neutralizing anti-jhm antibody + , detectable by immunofluorescence microscopy (induction of ia in at least 2,500-5,000 cells per cm 2 ); -, undetectable by immunofluorescence microscopy (induction of ia in 0-10 cells per cm 2 ). dmem, dulbecco's minimal essential medium; fcs, fetal calf serum. the stock preparation of recombinant rat ifn-y contained 1.2 x 10 5 u ml-i and 3 x 10 6 u per mg protein. polyclonal rabbit antiserum to rat ifn-y, given by dr van der meide, contained 1.0 x 10 5 neutralizing units (nu) ml -' . jhm virus was obtained from tissue culture supernatants of cells infected with wild-type jhm murine coronavirus. virus supernatants were produced from two different sources, primary glial cultures and a cell line permissive for jhm (designated dbt). the amount of virus in the supernatants was determined by titration (as pfu ml-i ) on dbt cells. stock virus from dbt cells numbered 2 x 10 5 pfu ml -' and from primary glial cultures, 2 x 10 4 pfu ml -i , when the cytopathic effect reached 90 %. virus preparations were completely inactivated with ultraviolet light (2,500 fl w cm-2 ) for 5 min. conditioned supernatants from uninfected cultures served as the control for the virus supernatant preparations. monoclonal antibody directed against the envelope glycoprotein e2 has been described elsewhere 9 . the neutralizing monoclonal antibody was used at a dilution sufficient to neutralize 10 5 pfu ml -i . cultures were treated as indicated for 4 days, stained for ia using ox6 monoclonal antibody, then examined by fluorescence microscopy as described in fig. 2 legend. the total number of cells in lo-day cultures was, on average, 10 5 cells em -2. macrophages as well as a small proportion of the type i astrocyte population were induced to express la ( fig. 2d-f ). treatment of primary glial cell cultures with either infectious or ultraviolet-inactivated jhm virus also induced la expression by astrocytes in a dose-dependent manner, peaking at 10 3 plaque-forming units (pfu) ml-1 (table 1) ; higher concentrations had a toxic effect on the cells. jhm virus at 10 3 pfu ml -1 gave the maximum response regardless of the source (table 1) . immunofluorescence microscopy of cultures treated with ultraviolet-inactivated jhm virus, using a polyclonal rabbit antiserum to jhm virus, confirmed the absence of infected cells throughout the cultures, indicating that there was no jhm virus replication. fluorescence-activated cell sorting showed that -10% of ali cells in the cultures became la-positive (fig. ib) . conditioned media from uninfected cultures, diluted correspondingly, had no effect on la expression. in contrast to rat ifn-y, jhm virus induced la primarily in the astrocytic cell population (fig.2g-i) , 90-100% of macrophages remaining negative, as determined by double immunofluorescence microscopy. in addition, the kinetics of induction was distinct from that seen with rat ifn-y in that noticeable induction required at least 3-4 days of treatment, reaching a peak at 4-7 days. double immunofluorescence analysis of gfap and la showed that between 90 and 100% of the induced cells were astrocytes (fig. 2g-i) . in cultures treated with infectious jhm virus, the numerous la-positive astrocytes induced were apparently uninmethods. viable lewis primary glial cell cultures were incubated for 1 h with a 1: 2 dilution of mouse monoclonal ox6 hybridoma supernatant (20 flg ml-' igg) in dmem with 20% normal horse serum (at 4°c). cultures were rinsed three times, then incubated with a 1 : 20 dilution of fitc-conjugated rabbit f(ab)2 anti-mouse immunoglobulin (dakopatts, denmark,) for 30 min. cultures were rinsed with 0.12 m phosphate buffer ph 7.2, containing 1 % bovine serum albumin (bsa), removed from the culture dishes and mechanically dissociated by pipette aspiration. mter addition of 0.2 flg ml-1 propidium iodide, cells were analysed immediately. gate window of forward angle light scatter (fals) lay between channels 10 and 255 . gate window for log integral red fluorescence was set for exclusion of non-viable cells stained bright red with propidium iodide. gate window for log integral green fitc fluorescence (ligfl) lay between channels 0 and 255 (abscissa). the number of la-positive cells was computed by integration from channel 10 to 255 for each sample containing 50,000 cells. macrophages without virus remained negative; this indicated that the ability of macrophages to express la was positively influenced by jhm virus, but that prostaglandins suppressed expression 7 • astrocytes appeared resistant to such suppression. to determine whether the jhm virus had a direct effect on astrocytes or whether the effect was due to a secondary signal released by macrophages 8 or astrocytes themselves, astrocytic cultures depleted of macrophages were tested for their responsiveness to jhm virus. macrophages were removed by panning of trypsinized primary cultures on hydrophobic plastic; the relatively non-adherent astrocytes remaining in suspension were removed and re-plated. after 4 days of treatment with 10 3 pfu ml-1 ultraviolet-inactivated virus, these astrocytes expressed la, just as in cultures containing macrophages. supernatants derived from either pure macrophage cultures or mixed primary cultures, after incubation with inactivated jhm virus, failed to induce la on naive astrocytes. this result indicated that secondary soluble factors were not involved in the induction of ia antigen on astrocytes. the possibility that ia induction was the result of virusinduced interferon synthesis in the cultures was also examined. primary cultures were treated with jhm virus (10 3 pfu ml-t, infectious or ultraviolet-inactivated) for 4 days, after which they na~t~u~r~e~v~o~l~.3~2~o~10~ap~r~il~19~86~ __________________ letterstonature---------------------------------~s~4s fig. 2 a15 ). c, phasecontrast photomicrograph. note the numerous lysosomal granules within the cytoplasm of fc+ macrophages. fc+ macrophages were found also to ingest large numbers of zymosan particles and to be nonspecific esterase-positive, both characteristic features of macro phages. d-j, double immunofluorescence and phase-contrast microscopy of one microscopic field of a 10-day primary glial cell culture treated for 18 h with recombinant rat ifn-y (10 u ml-i ). d, two macro phages (arrows) and one astrocyte (arrowhead) labelled for surface la. e, gfap+ astrocytes with characteristic gfap fibrillar staining pattern. not all gfap+ astrocytes are la-positive. the la + macrophages in d are clearly negative for g f ap staining and can be identified by their characteristic micros pikes (d)andlysosomalgranules (arrows in fl. f, phasecontrast photomicrograph. the astrocyte indi-i cated by an arrowhead in d-f is la+. g-i, double immunofluorescence and phase-contrast microscopy of one microscopic field of a lo-day primary glial cell culture treated during the previous 4 days with 10 3 pfu ml-1 ultraviolet-inactivated jhm virus. g, two astrocytes expressing cell-surface la (arrowheads). h, gfap+ astrocytes showing a fibrillar staining pattern. the arrowheads in hand i pinpoint the two la+ cells that are indicated by arrowheads in g, showing strong double immunofluorescence of ia and gfap for the same cells. note that the unlabelled gfap+ astrocytes in h are not la-positive in g. i, phase-contrast photomicrograph. the cell labelled with an arrow contains granules typical of macro phages and is negative for la (see g) and gfap (h). the other granule-containing macrophage indicated by the crossed arrow shows only weak expression of la in g at the lower pole of its cell body bearing microspikes. therefore, gfap+ astrocytes are selectively induced to express la while over 90% of macrophages remain ia-. parallel cultures stained for jhm antigen revealed no infected cells throughout the cultures. methods. primary glial cell cultures were established from newborn (1 day postnatal) lewis rat brain as described previousli 6 . six days after plating, at which stage the cultures were treated with ifn-y or jhm virus, three distinct cell populations were present: type i astrocytes, macrophages and a2b5+ precursors to both type ii astrocytes and galactocerebroside-positive oligodendrocytes 17. staining of fc receptors was achieved by incubating live cultures with a 1: 100 dilution of normal mouse serum, followed by goat anti-mouse igg conjugated to tritc (zymed, california), at 4°c (ref. 15 ). after fixation with 2% formaldehyde and permeabilization with 0.25% triton x-i00, gfap filaments were stained using a polyclonal rabbit igg directed against gfap (dakopatts, denmark) diluted 1: 250, followed by goat anti-rabbit igg conjugated to fitc (zymed). staining of rat la and gfap was as for fc receptors and gfap. a mouse monoclonal antibody directed against rat la (designated ox6; given by dr d. w. mason) was diluted to 20 f.lg ml-1 igg from hybridoma supernatant. as a control for the ox6 monoclonal antibody which is of the igg 1 subclass, two different mouse monoclonal igg 1 antibodies against unrelated antigens were tested and found to be negative. staining of jhm virus antigen was performed as for gfap, using a rabbit igg fraction directed against jhm, diluted to 20 f.lg ml-1 igg. were challenged with vesicular stomatitis virus (vsv; 100 pfu ml-i ). one day after infection, titrations of vsv released showed no reduction of pfu ml-i compared with the control. in addition, both untreated and jhm virus-treated cultures were totally destroyed by vsv, indicating an absence (or insufficient levels) of interferon(s). in addition, the application of a polyclonal rabbit antiserum to rat ifn-y in conjunction with jhm virus obtained from either infected dbt cells or primary glial cell cultures did not block or reduce ia induction. the concentration of rabbit anti-rat ifn-y used effectively eliminated the la-inducing capacity of 10 u ml-i recombinant rat ifn-y at 48 h post-treatment (table 1) . the ia antigen-inducing capacity of jhm appears to depend on direct interaction of jhm virus with the astrocytes. the envelope glycoprotein e2 would be the most appropriate candidate for such virus-cell interactions as it is responsible for binding of the virus to susceptible cells and for virus-induced cell fusion 9 • to test this notion, cultures were treated with jhm virus in conjunction with a neutralizing monoclonal antibody directed against the e2 glycoprotein. a non-neutralizing antibody to jhm virus was used as a control. after 4 days of treatment, cultures exposed to non-neutralizing antibody showed a typical pattern of ia induction, whereas the neutralizing antibody to e2 totally blocked jhm virus induction of la (table 1) ; this indicates that virus-cell binding mediated via the e2 glycoprotein of jhm has an important role in the induction of ia on astrocytes. here, we have described the ability of a neurotropic virus to induce la molecules on astrocytes, and we have presented evidence that jhm virus exerts its effects on astrocytes through direct interaction. this phenomenon is independent of viral replication in astrocytes as ultraviolet-inactivated virus is effective in inducing ia antigen. moreover, a monoclonal antibody to the e2 glycoprotein which blocks virus binding to susceptible cells 9 , also blocks the induction of ia antigen. these observations suggest that either binding of the virus to the surface of astrocytes through specific cell-surface receptors or phagocytosis of the virus particles initiates a series of events in astrocytes leading to ia expression. the mechanism by which jhm virus induces ia expression is unknown; it is conceivable that viral glycoproteins may act on particular target cells in the induction of la in a similar way to that described for bacterial endotoxin lo • the present results are particularly interesting as ifn-y released by t lymphocytes is thought to be indispensable in the induction of la antigen on certain antigen-presenting cells ll ,12, including astrocytes l -3 • astrocytes could be especially effective antigen-presenting cells in the brain owing to their ubiquity and their ability to phagocytose, process and present antigen 3 , in the case of virus invasion of the cns, this cell population may play an important part in mounting an immune response to effectively control the viral infection. on the other hand, high constitutive levels of la expression might carry the risk of inappropriate presentation of self antigens, as is thought to occur in autoimmune processes directed against the thyroid gland\3. this phenomenon may have special relevance to brain antigens and la-expressing astrocytes as the development of immune tolerance to self brain antigens, including myelin, may be hampered by the blood-brain barrier. the induction of la on astrocytes by jhm virus probably has a role in the jhm virusinduced chronic demyelinating disease of lewis rats, which involves induction of myelin basic protein-specific t lymphocytes 6 • the phenomenon reported here may represent a general feature of virus-astrocyte interactions and may have wider implications for human neurotropic viruses and the induction of immunologically mediated chronic demyelinating diseases l4 . we thank ines tschertner for technical assistance, helga kriesinger for preparing the manuscript, dr d. mason for the ox6 monoclonal antibody to rat la l8 , dr van der meide for the recombinant rat ifn-y, dr h. wege for monoclonal antibodies to jhm virus, and dr e. wecker for helpful discussion. this work was supported by hertie-foundation, deutsche forschungsgemeinschaft and phs-nrs a no.5 f32ns07293-02 to p.t.m. rna tumor viruses: molecular biology of tumor viroses 2nd end proc. natn. acad. sci. us.a. 78 proc. natn. acad. sc< us.a proc. natn. acad. sci. us.a. 77 progress in clinical & biological research. experimental allergic encephalomyelitis: a useful modelfor multiple sclerosis in mammals, the immunoglobulin heavy-chain variable region (v u) locus is organized in a linear fashion; individual v u, diversity (d u ), joining (j u ) and constant (c u ) region segments are linked in separate regions!. during somatic development, coding segments flanked by characteristic short recombination signal sequences, separated by intervening sequence regions that may exceed 2,000 kilobases (kb), are recombined. combinatorial joining of different segments as well as imprecision in this process contribute to the diversity of the primary antibody response; subsequent mutation further alters functionally rearranged genes. this basic somatic reorganization mechanism is shared by six major families of genes encoding antigen receptors2. previously, we have shown that multiple germline genes and mammalian-like recombination signal sequences are associated with the vu gene family of heterodontus franeisei (horned shark), a primitive elasmobranch 3 • studies presented here demonstrate that segmental reorganization involving mammalian-like du and j u segments occurs in the lymphoid tissues of this species. in marked contrast to the mammalian system, we find multiple instances of close linkage (-10 kb) between individual v u , d u , j u and c u segments. this unique organization may limit combinatorial joining and be a factor in the restricted antibody response of this lower vertebrate 4 ,5. a heterodontus spleen poly(a)+ messenger rna-complementary dna library was constructed in the vector agtll (ref. 6 ) and screened with a nick-translated probe that complements the entire coding region of hxia, a heterodontus germline v h gene 3 _ multiple positive hybridizing plaques were detected and the structure of one of these, hc-3, is illustrated in fig. 1 . regions exhibiting high degrees of nucleotide identity key: cord-003492-rodqdtfj authors: montaner-tarbes, sergio; del portillo, hernando a.; montoya, maría; fraile, lorenzo title: key gaps in the knowledge of the porcine respiratory reproductive syndrome virus (prrsv) date: 2019-02-20 journal: front vet sci doi: 10.3389/fvets.2019.00038 sha: doc_id: 3492 cord_uid: rodqdtfj the porcine reproductive and respiratory syndrome virus (prrsv) is one of the most important swine diseases in the world. it is causing an enormous economic burden due to reproductive failure in sows and a complex respiratory syndrome in pigs of all ages, with mortality varying from 2 to 100% in the most extreme cases of emergent highly pathogenic strains. prrsv displays complex interactions with the immune system and a high mutation rate, making the development, and implementation of control strategies a major challenge. in this review, the biology of the virus will be addressed focusing on newly discovered functions of non-structural proteins and novel dissemination mechanisms. secondly, the role of different cell types and viral proteins will be reviewed in natural and vaccine-induced immune response together with the role of different immune evasion mechanisms focusing on those gaps of knowledge that are critical to generate more efficacious vaccines. finally, novel strategies for antigen discovery and vaccine development will be discussed, in particular the use of exosomes (extracellular vesicles of endocytic origin). as nanocarriers of lipids, proteins and nucleic acids, exosomes have potential effects on cell activation, modulation of immune responses and antigen presentation. thus, representing a novel vaccination approach against this devastating disease. the porcine reproductive and respiratory syndrome virus (prrsv) is one of the most important swine diseases in the world. it is causing an enormous economic burden due to reproductive failure in sows and a complex respiratory syndrome in pigs of all ages, with mortality varying from 2 to 100% in the most extreme cases of emergent highly pathogenic strains. prrsv displays complex interactions with the immune system and a high mutation rate, making the development, and implementation of control strategies a major challenge. in this review, the biology of the virus will be addressed focusing on newly discovered functions of non-structural proteins and novel dissemination mechanisms. secondly, the role of different cell types and viral proteins will be reviewed in natural and vaccine-induced immune response together with the role of different immune evasion mechanisms focusing on those gaps of knowledge that are critical to generate more efficacious vaccines. finally, novel strategies for antigen discovery and vaccine development will be discussed, in particular the use of exosomes (extracellular vesicles of endocytic origin). as nanocarriers of lipids, proteins and nucleic acids, exosomes have potential effects on cell activation, modulation of immune responses and antigen presentation. thus, representing a novel vaccination approach against this devastating disease. keywords: porcine reproductive and respiratory syndrome virus, prrsv, virus biology, immunology, vaccinology, extracellular vesicles economic impact prrsv is responsible for respiratory disease in weaned and growing pigs, as well as reproductive failures in sows. it is considered one of the most important swine diseases worldwide, with an economic impact estimated at $664 million in losses every year to u.s. producers, representing an increase of 18.5% in the last 8 years (1, 2) . in europe, the situation is similar and economic disease models have been carried out to determine the economic burden in the best and worst case scenario combining reproductive failure and respiratory disease, estimating annual losses from a median of e75,724, if the farm was slightly affected during nursing and fattening, to a median of e650,090 if a farm of 1,000 sows is severely affected in all productive phases (3) . nevertheless, there is scarce of information about the economic impact of this disease as a consequence of multiple factors (vaccination, treatment, respiratory symptoms, reproductive failure, and other prrsv-related diseases) making a difficult task to quantify exactly this parameter under field conditions. thus, the exact economic impact of prrsv remains a key gap in the knowledge for this disease. the porcine reproductive and respiratory syndrome virus (prrsv) was first isolated in the early 1990s in europe and north america (4, 5) . it is an enveloped single-stranded positivesense rna virus of the family arteriviridae, genus porarterivirus according to the international committee of taxonomy of viruses (6) . presently, there are four distinct species included in this genus (porarterivirus), prrsv-1 and prrsv-2 (with 30-45% variation in nucleotide sequences), along with other two viruses that do not affect pigs (lactate dehydrogenaseelevating virus and rat arterivirus 1) (7) . the genome size of prrsv is about 15 kb with 10 open reading frames (orfs), with replicase genes located at the 5 ′ -end followed by the genes encoding structural proteins toward the 3 ′ -end (8) . the majority of the genome (∼60-70%) encodes non-structural figure 1 | genome structure and mature viral particle of prrsv virus. (a) non-structural proteins are located in the 5 ′ end of the genome, codifying for two different polyproteins pp1a and pp1ab that are cleaved into at least 14 nsps (nsp1 to nsp12 and nsp1α and nsp1β, and nsp7α, and nsp7β). structural proteins located near the 3 ′ end, are associated to the viral envelope and rna packaging. (b) prrsv mature viral particle, composed of a lipid bilayer envelop with viral receptor glycoproteins involved on infection and cell internalization. single stranded positive rna is associated with nucleocapsid protein in the internal layer of the virus. proteins involved in replication (orf1a and orf1ab), whereas orfs 2-7 encodes structural proteins (n, m, gp2-gp5, e) ( figures 1a,b) (9) . using orf5 in molecular epidemiological studies, an enormous genetic variability has been described (10) . yet, data on whole genome sequencing is scarce and constitute another important gap in the knowledge of this virus and its evolution (box 1). prrsv replicase genes consist of two orfs, orf1a and orf1b, which occupy the 5 ′ proximal three-quarters of the genome ( figure 1a) . both are expressed from the viral genome, with expression of orf1b depending on a conserved ribosomal frameshifting mechanism. subsequently, extensive proteolytic processing of the resulting pp1a and pp1ab polyproteins yields at least 14 functional non-structural proteins (nsps), specifically nsp1 to nsp12, with both the nsp1 and nsp7 parts being subject to internal cleavage (giving origin to nsp1α and nsp1β, and nsp7α, and nsp7β, respectively), most of which assemble into a membrane-associated replication and transcription complex (11) . recently, a programmed ribosomal frameshift encoding an alternative orf that generates two extra proteins, nsp2tf and nsp2n, was discovered in prrsv and other arteriviruses (12, 13). these nsps, described for prrsv, have proven to box 1 | gaps in knowledge in prrsv. be necessary and sufficient for the induction of membrane modifications resembling those found in infected cells (14) . most importantly, all positive rna viruses seem to induce one of two basic morphotypes of membrane modifications: invaginations or double-membrane vesicles. prrsv also has a set of 8 structural proteins, including a small non-glycosylated protein and a set of glycosylated ones: gp2ab, gp3, gp4, gp5, and gp5a, m and n proteins (15) . however, nsp2, traditionally classified as a non-structural protein, has been found to be incorporated in multiple isoforms within the viral envelope (ovarian tumor domain protease region, hypervariable region and c-terminal region) (16) , giving new insights into the structure of this virus ( figure 1b) . first, the nucleocapsid protein (n), as one of the most important parts of the mature viral particle, has been deeply characterized on prrsv, finding important features shared in most nonsegmented rna viruses. the n protein consists of 123 amino acids for genotype 2 and 128 amino acids for genotype 1. the viral envelope glycoproteins (gp2 to gp5) are the first interactors with host cell receptors to initiate infection and are exposed to the immune system when viral particles are in blood and lymphoid tissue circulation (figure 2 ). there is also another protein that contribute to virion structure, m protein, that is required during viral entry to interact with heparan sulfate cell receptor on macrophages. later, gp5 is thought to bind to sialoadhesin and virus internalization and uncoating is triggered by a formation of a viral heterotrimer (gp2a, gp3, and gp4) with scavenger receptor cd163 (figure 2 ) (17, 18) . gp5 is the most abundant glycoprotein. first, it interacts with two cell entry mediators, heparan sulfate glycosaminoglycans and sialoadhesin/cd169 (17, 18) to favor viral entry and then possibly with the n protein and its mhc-like domain to carry n-viral rna complex to the budding site (figure 2) . gp2, gp3, and gp4 are protected with glycan shields, like most prrsv membrane proteins, to avoid antibody recognition and neutralization. gp2 has two glycosylation sites, gp3 have seven and gp4 have four, figure 2 | interactions between viral proteins and cell receptors for virus attachment, entry, uncoating and release of genetic ssrna to cell cytoplasm. blocking cd163, cd151 tetraspanin or vimentin seems to inhibit viral replication or infection in the host cell, but reduced replication or no effect is seen when receptors such as heparan-sulfate or siglec-1 are blocked, demonstrating that some viral proteins and cell receptors are indispensable in terms of production of infectious viral progeny and dissemination in the host. three of which are directly related to virus survival, causing lethal damage in virus production when more than two of these sites are mutated (19) (figure 2 ). viral replication starts by interaction of viral glycoproteins with different cellular receptors (figure 2 ) (17) . cd163 and cd169 play a main role during infection, uncoating of the viral particle, activation of clathrin-mediated endocytosis and release of viral genome in the cytoplasm (20) . cd163 has been defined as the main receptor for viral infection by evaluating the effect of prrsv on cd163 knockout pigs, where there is complete resistance to infection (21) . cysteine-rich domain 5 in this receptor seems to be necessary to establish interactions with prrsv-1 species, since its deletion by crispr/cas9 system (exon 7 of the gene encoding this region) implies protection for a large panel of these viruses demonstrated by in vitro challenge of edited-pig macrophages and in vivo experiments with srcr5 animals (22) (23) (24) . more important, edited pigs show no side effects when kept under standard husbandry conditions and cd163 seems to maintain its biological function (hemoglobin-haptoglobin scavenger) regardless the lacking cysteine-rich 5 domain, nevertheless, other unknown functions could be impaired by this modification. in conclusion, gene-edited pigs lacking srcr5 region of cd163 could be an important asset to confront prrsv epidemics with the final goal of eradication. cd169 seems to be related only to co-interactions with sialic acid in the virion surface, however, knockout pigs for either exon 1, 2, or 3 of cd169 were not protected from infection and viral load as well as antibody responses were similar to heterozygous (cd169 +/− ) or wild type pigs (cd169 +/+ ) (25) . the former experiments suggested that other unknown mechanisms could be involved in prrsv infection such as other receptors, new unknown susceptible cell types different from macrophages or possible leaking of cd169 expression in the knockout model. other molecules are also involved in viral entry, such as cd151 (26) and vimentin (27) ; blocking of any of these four molecules (cd163, cd169, cd151, and vimentin) had an effect on viral infection, either on internalization or complete inhibition of viral replication (17) . after cell entry, prrsv causes a series of intracellular modifications to complete its replication cycle, which includes rearrangements of intracellular membrane organelles to generate the replication complex. these include the formation of perinuclear double membrane vesicles apparently derived from endoplasmic reticulum, synthesis of genomic rna (grna), transcription of segmented rna (sgrna) and expression of viral proteins (20, 28) . at late stages of replication, the mature virions accumulate in the intracellular membrane compartments and they are then released into the extracellular space through exocytosis (29) . a non-classical spread pathway has been detected in several viruses including prrsv where virus dissemination is mediated by cell to cell nanotubules (30) . it was reported that almost all prrsv proteins interact with myosin and actin (especially f-actin and myosin iia) where nanotubules connected cells allowing the movement of structural proteins and rna, infecting naïve cells in a non-classical way even in the presence of neutralizing antibodies in the cell media. in addition, this non-classical pathway demonstrated that prrsv cell entry receptors were not necessary to establish infection, as nonpermissive cells became infected when were contacted by infected cells via nanotubes. this spreading strategy has been proposed as a mechanism to facilitate infection either by surfing of viral particles between adjacent cell membranes or as a receptor-independent mechanism for infection (31) ; importantly, has been reported for other viruses such as hiv-1 where nanotube number on macrophages increases after infection (32) and herpesvirus transmission between bovine fibroblasts (33) . interestingly, although several viral proteins were detected in nanotubules (nsp1β, nsp2, nsp2tf, nsp4, nsp7, and nsp8, gp5 and n), gp4 was detected in only a few nanotubes. in particular, the role of gp4 in this nonclassical spread pathway is not fully understood and it will be interesting to further evaluate gp4 interaction with other cellular components to elucidate the reason why gp4 is not transported to new recipient naïve cells. altogether these data indicate that prrsv has evolved different pathways to spread even though, in vivo, the virus shows narrow cell tropism for monocytes and macrophages (34, 35) (box 1). the innate immune response is the first system any given pathogen encounters, specially to prevent viral replication and invasion into mucosal tissues (respiratory tract in the case of prrsv) and, importantly, to initiate the strong adaptive immune response to fight against intracellular infectious agents (7). type i interferons (ifn α/β) comprise one of the most potent mechanisms against invading viruses in the first stages of infection, triggering an array of ifn-stimulated genes (isg) (36) . generally speaking, all nucleated cells have the ability to produce ifn α/β, but plasmacytoid dc (pdc) are the most potent producers of this family of cytokines (37) . prrsv has evolved a set of mechanisms for suppressing ifn α/β in vivo, maintaining low expression levels of this cytokines on infected pigs (38) during almost all time-course of infection shortly after transient elevation in the lungs (39) . suppression of ifn α/β also takes place in vitro in prrsv infected marc-145 and porcine alveolar macrophages (38, 40, 41) . further studies have shown that ifn type i suppression is a major strategy of prrsv to modulate host antiviral defense. in fact, several viral proteins have been identified as ifn antagonists (nsp1α, nsp1β, nsp2, nsp4, nsp11, and n) (7, (42) (43) (44) . as an example for n protein, upon dsrna stimulation, ifn-β production was shown to decrease proportionally with increasing levels of n expression and additionally it was found to downregulate ifn-dependent gene production by dsrna interfering with dsrna-induced phosphorylation and nuclear translocation of irf3 (45) . among prrsv non-structural proteins with type i ifn modulation capacity, nsp1 has been considered as the strongest antagonist of ifn-β production by acting on interferon regulatory factor 3 (irf3) phosphorylation and nuclear translocation. almost all nsps, excepting nsp1, have been related to the perinuclear region, associated with intracellular membranes, supposedly derived from the endoplasmic reticulum (er), which are modified into vesicular double-membrane structures with which the viral replication and transcription complex (rtc) is thought to be associated with (14, 46, 47) . nsp1 translocates to the nucleus during the first hours of infection, where it is capable of inhibiting irf3 association with creb-binding protein (cbp), promoting cbp degradation by a proteasome-dependent mechanism, without which the transcription enhanceosome may not assemble the transcription machinery for the interferon expression (15, 46) . recently, post-transcription protein expression of ifn β was shown to be regulated by prrsv by means of upregulating cellular mirna in porcine alveolar macrophages (48) nsp2 is the largest (mature) prrsv protein and contains at least four distinct domains: the n-terminal cp/otu domain, a central hypervariable region, a putative transmembrane domain, and a c-terminal region of unknown function that is rich in conserved cysteine residues. this protein is unique in the context of prrsv due to its genetic heterogeneity, its participation in diverse roles supporting the viral replication cycle, and its packaging within the prrsv virion (16, 49) . previous studies suggest that nsp2 has different roles related to immune evasion mechanisms. it has been determined that nsp2 otu domain (thiol-dependent deubiquitinating domain) inhibits the nuclear factor kappa-light-chain-enhancer of activated b cells (nf-kb) by interfering with the polyubiquitination process of ikbα (nuclear factor of kappa light polypeptide gene enhancer in b-cells inhibitor) and, subsequently, preventing the degradation of the ikbα protein (50) . moreover, viable deletion mutants in nsp2, when infecting cells, caused a downregulation of cytokines (il-1β and tnf-α) mrna expression, in comparison with that of parental virus, suggesting that certain regions of nsp2 might contribute to the induction of a virus-specific host immune response and that deletion of such a region could produce a more virulent virus (51) . there are several isoforms of nsp2, sharing a consistent core set between viral strains, which are integrated into mature virion at the final stage of replication (figure 1b) , although some of them could be strain-specific. inclusion of nsp2 within the prrsv virion suggests that it may function in previously unknown roles related to extracellular function, entry, or immediate-early viral replication events (16) . truncated forms of nsp2 have also been identified, named nsp2tf and nsp2n, with apparent roles in modulation of immune evasion. when deletion mutants for those forms were used to infect cells, there was a significant change in gene expression, a strong activation of those involved in cytokine-cytokine receptor interaction, tnf signaling, toll-like receptor signaling, nodlike receptor signaling, nf-κb signaling, rig-i-like receptor signaling, chemokine signaling, jak-stat signaling, cytosolic dna-sensing, and nk cell mediated cytotoxicity (13) , suggesting that an active role (direct or indirect) is played by these truncated forms in modulating host cells innate immune response, making prrsv infectious cycle more complicated than it was initially thought. nsp11, is a nidovirus conserved endoribonuclease with an uridylate-specific endonuclease (nendou). it has been demonstrated in vitro that overexpression of nsp11 enhanced viral titter (52) . moreover, nsp11 antagonizes type i ifn, specifically ifnβ production, activated by the retinoic acid inducible gene 1 like receptor, showing substrate specificity toward mitochondrial antiviral signaling proteins (mavs) and rig-i (transcripts and proteins), and demonstrating that this activity was associated to the endoribonuclease activity of this protein in which transfection mutant viruses were unable to degrade mavs mrna and impair ifnβ production (53) . another mechanism whereby this protein limits antiviral response is related to inflammasome and synthesis of il-1β, due to its important role in both the innate and adaptive immune response and in pathological mechanisms. it has been shown that prrsv could activate nlrp3 inflammasome in early stages of infection but induce host's immunosuppression later as measured by determining the levels of pro-il-1β and procaspase-1 mrna and the mature il-1β protein in porcine alveolar macrophages (pam) (54) . it is not surprising that nsp11 also interacts with the rna-silencing complex (risc), as it has been demonstrated in vitro in a marc-145 cell line that this protein and nsp1α are responsible for inhibiting risc and downregulating argonaute-2 protein expression increasing viral titter significantly, which demonstrates a direct relationship between this silencing complex and viral replication at least in vitro (55) . other non-structural proteins have been studied but there is an important gap on information about in vivo and in vitro functions and interaction in signaling pathways. additionally, the enormous variation among strains makes it difficult to characterize all protein variants and interactions with cell systems (macrophages, dendritic cells "dcs, " monocytes and others) (box 1) . recently, a body of evidence associates host genetics with different outcomes following prrsv infection in the respiratory and reproductive form of the disease (56) (57) (58) (59) (60) . although pathways and mechanisms involved in specific disease-resistance traits have not yet been fully characterized, it is clear that the genetic variation in disease resilience is polygenic, regulating aspects of both innate resistance and acquired immunity (56) . in connection with innate response, the average daily gain (adg) after prrsv infection was associated with a single genomic region in chromosome 4 (ssc4) which is best represented by the snp tag marker wur, located in the 3 ′ non-coding region of the interferon-inducible guanylate-binding protein 1 (gbp1) gene (61) . the pig genetic resistance to prrsv infection has been historically overlooked in prrsv research probably generating a confounding factor in immune response studies. a key gap in the knowledge of prrsv is linked the pig genetic variability after prrsv infection with the enormous variability of the virus itself (box 1) . in pigs, prrsv replicates in cells belonging to the innate immune system. pams are the primary cells to be infected in the lungs as well as other cells of the monocyte/macrophage lineage, which later could disseminate the virus to other tissues or support replication to release viral particles into the bloodstream (17) (figure 2) . moreover, prrsv is thought to be able to infect professional antigen presenting cells such as dcs and monocyte derived dendritic cells, (modc) impairing their normal antigen presentation ability by inducing apoptosis, down-regulating the expression of ifn-α, mhc class i, mhc class ii, cd11b/c and cd14, upregulating the expression of il-10 and inducing minimal th1 cytokine secretion (62) (63) (64) (65) . nevertheless, new evidence suggest by in vivo and in vitro experiments that specifically lung cdc1, cdc2, and modcs are not infected by prrsv-1 viruses from subtypes 1 and 3 and one possible explanation is the lower expression of cd163 and cd169 in those 3 dc subtypes, associating previous results of infection in dcs to culture conditions of monocytes in vitro that could cause a sensibilization to infection by certain strains as lena (66) . in addition, these findings were also tested in tonsil cdc and tracheal cdc1 and cdc2 observing that those cell populations are not infected by prrsv virus (67, 68) . moreover, a new type of pam has been characterized and named porcine intravascular macrophages (pim) due to its association to endothelial lung capillaries and not to the alveoli, presenting strong capacity to phagocytised bloodrelated particles (69) . importantly, when infected pim cells gave similar results of viral load to those derived from infected pam, but significantly upregulates of tnfα and non-significantly il-6 and il-8 expression after infection when compared to normal alveolar macrophages, indicating that these cells have an important pro-inflammatory role during prrsv infection in the lungs (69) . new interactions between cells and the virus need to be further explored to unravel possible immunological features that leads to correlates of protection. recently, it has been shown that a domain within nsp1α is able to stimulate the secretion of cd83, which in turn inhibits modc function in vitro, impairing the ability of modc to stimulate t cell proliferation (70) . production of ifn α/β and the mechanisms for cell activation by pdc are severely suppressed during prrsv infection, although these cells are not permissive to prrsv infection (71, 72) . however, this phenomenon is strain dependent, as other prrsv strains are able to stimulate pdc for ifn α/β production in large quantities (73) . again, there is an enormous variability between prrsv strains in relation with their effect on antigen presenting cells which prevent scientists from finding common mechanisms. it might be of interest to link this key gap of knowledge for prrsv with host genetics (box 1) . moreover, in prrsvinfected cells, n is abundantly expressed benefiting from the discontinuous transcription mechanism (74) . this protein is also distributed in the nucleus, induced by two nuclear localization signals called cryptic nls or nls-1 and functional nls or nls-2 (positions 10-13 and 41-47, respectively) (75). the effect of n protein has been examined in pams and modcs using transfection, finding a significant upregulation of il-10 gene expression. natural killer (nk) cells constitute another powerful arm of the innate immune system against prrsv, particularly when considering the high percentage of circulating nk cells in pigs (76) . the cytotoxic function of nk cells is reduced in prrsv infected pigs from day 2 after infection up to 3-4 weeks (38, 77, 78) . initial studies using in vitro systems demonstrated that the stimulation of porcine nk cells with proinflammatory cytokines (il-2 and il-15) was capable of activating nk cells and inducing them to express high levels of ifn-γ and perforins to cause lysis of infected cells, but a different scenario appears if cells are evaluated post-infection, indicating that a virus such as prrsv is capable of impairing nk cell cytotoxicity (79) . in vitro, the nk cytotoxicity against prrsv-infected pams was decreased and degranulation of nk cells inhibited (80) . in vivo, the immune response is the same as that observed in vitro, with some studies reporting that approximately half of viremic pigs had a reduction >50% in nk cell-mediated cytotoxicity and enhanced secretion of il-4, il-12, and il-10 and reduced frequency of cytotoxic t-cells (cd4 − cd8 + t) and double positive t cells (cd4 + cd8 + t) and upregulated frequency regulatory t-cells (tregs) (81). innate immune responses against prrsv are obstructed by different mechanisms as are adaptive responses. the modest and delayed b cell mediated neutralizing antibody response is one of the main characteristics associated to prrsv acquired immune responses. even though prrsv specific antibodies appear early at 7-9 days post-infection, the efficacy of those antibodies remains unclear. neutralizing antibodies take longer, appearing nearly 1 month after infection (34) . however, passive transfer of these neutralizing antibodies conferred almost full protection in a prrsv reproductive model (95% of offspring alive after challenging pregnant sows with high neutralizing antibody titter). nevertheless, in another experiment using the reproductive model, when the presence of prrsv was examined after the transfer of neutralizing antibodies, lungs, tonsils, buffy coat cells, and peripheral lymph nodes contained replicating prrsv similar to infected controls, although pigs were apparently protected against infection. in summary, passive transfer of high neutralizing antibody titter conferred protection to gilts and offspring (not detectable viremia), but did not eliminate the presence of viral particles in peripheral tissues nor transmission to animals they were in contact with (82) (83) (84) . curiously, the role of neutralizing antibodies in the protection against the respiratory form of the disease is a key gap of knowledge for prrsv. this point is critical to define precisely targets for improved vaccines based on the humoral immune response against this virus (box 1). n protein is involved in several mechanisms for immune evasion and is also one of the most immunogenic structural proteins (75) . antibodies against n appear early during acute infection, together with those against m and gp5 proteins, but are non-neutralizing and could be involved in antibody dependent enhancement (85, 86) . there are other "antibody-related mechanisms" that do not necessarily involve neutralizing activity. antibody-dependent cell-mediated cytotoxicity (adcc), antibody-dependent complement-mediated cytotoxicity (cdc) and antibodydependent complement mediated virolysis (adcv) have been examined in the context of prrsv, although none of these mechanisms were evident during infection or have not been deeply investigated on in vitro and in vivo models of this virus (87) . it is important to note that neutralizing antibodies appear late in prrsv infection and other immune mechanisms (cellular or antibody mediated immune response) might be acting to suppress viral replication in blood, causing the virus to be isolated in lymphoid tissues and maintaining suboptimal replication that will finally end in viral clearance. for type prrsv-2 it has been demonstrated that immunization of pigs with ectodomain peptides from gp5/m complex did not induce neutralizing antibodies (88) although those ectodomain-specific antibodies generated were capable of binding virus. an important feature that makes difficult to validate the location of neutralizing epitopes is the number of glycosylations in or around it. for prrsv-1 strains, up to 3 glycosylations may be found in, or flanking the gp5 neutralizing epitope that is located between amino acids 37-45 (89) , whereas for prrsv-2 strains there are four potential glycosylation sites (90) . when tested, prrsv with mutations in gp5 glycosylation sites (either at n44 or in the hypervariable region, upstream the neutralizing epitope) enhanced immunogenicity with increased concentration of antibodies directed to this epitope 5-10 fold higher compared with those induced by the wild type strains (89) . same results were obtained when administering another deglycosylation mutant (double deglycosylation in the putative glycosylation moieties on gp5) twice, which conferred better protection against homologous challenge (91) . in addition, when this protein is expressed early during infection, it stimulates production of early neutralizing antibodies and ifn-β, two main antiviral mechanisms, demonstrating its role in induction of self-protection mechanisms from the host (92) . available data about neutralizing antibodies induced by this protein are controversial, which may be due to the high variation among prrsv strains (93) and, as previously commented, the host genetics. orf5 is also complemented by a small frameshift of the subgenomic mrna called orf5a, encoding a type i membrane protein consisting primarily of alpha helix with a membrane-spanning domain (called gp5a) that is incorporated into virions as a very minor component, playing a role in viral replication, as mutation in the initiation codon or premature termination related to expression for this protein leads to non-efficient viral replication and lower titter (94, 95) . this protein is capable of eliciting specific antibody immune response in natural infections and after immunizations, although those are not neutralizing neither protective in a challenge trial after infection, making difficult to define the role of this particular small protein in the whole immune response and viral clearance of prrsv infection (96) . in summary, the role of humoral immunity remains elusive in prrsv infection (neutralizing and non-neutralizing antibodies) and a better characterization will be required to overcome this relevant gap of knowledge (box 1) . treg typically increase in number in chronic viral diseases to prevent a persistent inflammatory response and pathological damage associated to viral infections. conversely, tregs are described as key contributors in modulating the host immune response to viral infection. this cell population is an important component in regulating the magnitude of the immune response to infection (in viruses such as hiv and hcv), thus preventing excessive inflammation and tissue damage. however, they can also be inappropriately induced by viruses to switch the balance of the immune response in favor of maintaining viral replication (97) . in prrsv, the role of tregs remains unclear and appears to be a consequence of il-10 induction of some strains as early as 2 days post infection (81) . in some experiments, in vitro infected dcs with prrsv-1 exhibited an unbalanced ability to stimulate t cell immune responses in a strain-dependent manner, but no tregs were detected, at least in vitro, as measured by expression of cd25 and foxp3 markers (98) . when using prrsv-2 strains, the case seems to be different, as the virus was capable of stimulating il-10 production with concomitant generation of tregs (99) which was associated to nucleocapsid protein expression in the in vitro system. this group also suggested that il-10 production and treg could be related to impaired gamma interferon (ifn-γ) production and altered development of protective t-cell response by inhibiting t-cell proliferation as seen in the early stage of infection with viruses such as hcv. vaccine strains currently in use in the united states do not provide adequate heterologous protection, one possible explanation could lay on their inability to induce an adequate ifn-γ response due to their ability to stimulate tregs, at least in vitro (100). structural conformation, but not nuclear localization, of the expressed n protein was suggested as essential for the ability to induce il-10 that, in consequence, causes induction of tregs as measured by markers cd4+cd25+foxp3+ (99) . it should be noted that when the role of the nuclear localization signal was evaluated using deletion mutants, results suggested that nls-2 was not essential for virus survival, although pigs developed a significantly shorter duration of viremia and higher neutralizing antibodies than those of wild-type prrsv-infected pigs (101) . the role of tregs cells in the immune response against prrsv is a key gap of knowledge in order to develop more efficacious prrsv vaccines (box 1) . moreover, reports have highlighted the impact of prrsv infection on thymic cellularity mainly as a loss of cd4 + /cd8 + cells in the thymus of prrsv-infected pigs. acute lymphopenia, thymic atrophy, and lymphadenopathy associated with the presence of prrsv antigen in the thymus are some of the mechanisms whereby prrsv suppresses the immune response. in addition, presence of prrsv antigens in the thymus could also induce tolerance and presents a mechanism that could explain the presence of tregs during prrsv infection (93) . nevertheless, the picture is not complete and basic knowledge about the effect of prrsv on cell development in the thymus would be of great interest to understand the effect of this viruses in the host. prrsv immunology thus remains an unsolved puzzle due to complex interactions between different viral strains and the host. similar immune responses could be the key feature of this virus, such as persistence viremia, a strong inhibition of innate cytokines (ifn-α/β, tnf-α, il-1β, ifn-γ), dysregulation of nk cell function (cytotoxicity and degranulation), rapid induction of non-neutralizing antibodies, delayed appearance of neutralizing antibody, late and low cd8+ t-cell response, and induction of regulatory t cells (tregs) (102) . as a whole, neutralizing antibodies and prrsv-specific ifn-γ secreting cells do not fully depict the immune effector functions related to protective immunity, as the viral targets related to them are unknown. as a consequence, correlates of protection remain elusive for this infection due to the laborious work in vitro and in vivo and the enormous genetic diversity that causes confusion and makes it difficult to predict how immune responses against one isolate or strain could be applied to another in a cross-protective immune prediction model (103, 104) . without any doubt, the most important gap of knowledge for prrsv is the lack of correlates of protection that makes extremely difficult to have robust models to check vaccines efficacy against this disease (box 1). since the beginning of prrsv outbreaks in europe and the usa, the development of efficacious prrsv vaccines has been a challenge. classical approaches are not working properly for several reasons: viral mutation can lead to more pathogenic strains, there is a lack of knowledge on how the porcine immune system interacts with all prrsv proteins, and most importantly, there is no robust parameter (surrogate marker) that can be unequivocally linked with viral clearance. thus, there is no relationship between complete homologous or heterologous protection and classic immunological parameters such as an increase/decrease in particular cell population (105) , ifnγ production, neutralizing antibodies (106), non-neutralizing antibodies and clinical outcome (107) . in addition, highly divergent strains make it more difficult to develop a universal vaccine for this virus (28) . several different vaccines against prrsv have reached the market and have been reviewed recently (108) . most of these vaccines rely upon modified live virus (porcilis prrs from merck, ingelvac prrsflex eu from boehringer ingelheim, amervac-prrs from hypra, pyrsvac-183 from syva) against prrsv-1, as well as some to control prrsv-2 (fostera prrs from zoetis, ingelvac prrs mlv/ingelvac prrsatp from boehringer ingelheim). there is also evidence that most mlv vaccines of both prrsv-1 and prrsv-2 species elicit specific humoral and cell-mediated immune (cmi) responses, as they confer protection to homologous parental strains and partial protection to heterologous strains. although it is possible to control some prrsv outbreaks by use of mlv in combination with good practices, there are major safety issues such as a high mutation rate leading to reversion to virulence and recombination among vaccine and wild type strains. cases have been reported in which new viruses have been introduced as a consequence of mlv vaccines. for example, nucleotide sequence identities of atypical danish isolates were between 99.2 and 99.5% with the vaccine virus respprrs and 99.0-99.3% with vr2332, which is the parental virus to the vaccine virus, supporting the conclusion that the introduction of prrsv-2 in denmark was due to the spread of vaccine virus (109) . in china a recombination event was reported in which a prrsv variant with nucleotide deletions and insertions in the non-structural protein 2 (nsp2) gene also showed a possible recombination event between a mlv strain and a prototype chinese field strain (110) . current inactivated vaccine approaches are not highly effective since elicited immune responses are not enough to prevent spreading of the virus. however, this type of vaccine can augment anamnestic virus neutralizing antibodies and virusspecific ifn-γ responses following a wild-type virus infection or prrsv-mlv vaccination which can contribute to viral clearance (111, 112) . thus, the combination of modified live vaccines with inactivated ones can be a reasonable approach to control the disease under field conditions (113) but unfortunately, there is no robust data comparing this approach with other options available on the market. on the other hand, most inactivated vaccines are not approved for use in the united states due to the poor efficacy showed in challenge trials (114) as measured by production of prrsv specific neutralizing and non-neutralizing antibodies and low cellular immune responses leading to their failure in the porcine market. according to the centre for food security and public health of iowa state university, only box 2 | exosomes and therapeutic applications in prrsv. frontiers in veterinary science | www.frontiersin.org "biosuis prrs inact eu+am" is approved to be used in the us. however, new strategies are being evaluated to overcome these problems (115) , including nanoparticle entrapped antigens (116) (117) (118) (119) , plant based approaches (120) or vectored vaccines (121) . several attempts have been made to use structural proteins to develop vaccines against prrsv because they are specific targets of neutralizing antibodies. for this reason, one may hypothesize that antibodies against those proteins could be the main key to inhibit viral replication and spread as it is common for many viruses. approaches such as vlps combining different structural proteins have been tested (122) (123) (124) , finding that anamnestic response is possible (boosted igg and ifn-γ producing cells) in previously vaccinated or infected pigs but not in the prechallenge period. these structural proteins are able to prime the immune system, but no reduction of viremia was observed after challenge (123) . those results suggest that other viral proteins may be targeted to induce a protective response in pigs. a plausible explanation for this finding may be based on the presence of few neutralizing epitopes in their sequences, most of which are located in variable regions of the proteins, to the phenomena of glycan shielding for epitopes and to the high variability observed between prrsv virus strains. again, a critical gap of knowledge for prrsv is to precisely characterize common epitopes that are present in all prrsv strains. epitopes responsible for generating an efficient immune response eliciting cross-protective immunity remained elusive. taken together, this evidence points to the need for new vaccination approaches that comply with a pathogen free strategy, capable of eliciting effective cellular and antibody responses with mid to long term protection against homologous strains and preferable to heterologous challenge as well. extracellular vesicles(evs) are gaining increased scientific attention as novel vaccines against infectious diseases, including animal diseases of veterinary importance by its capacity of self-antigen presentation, activation of host cell and antibody immune responses and more important, to induce protection in lethal challenge trials (125-131) (box 2). in the case of prrsv, artificial micrornas (amirna) were initially synthetized to try suppressing expression of sialoadhesin (sn) or cd163 by recombinant adenoviral vectors to be contained in exosomes, causing a subexpression of sn and cd163 at mrna and protein level, and reducing viral titter when porcine macrophages were pre-treated with amirna thus providing new evidence supporting the hypothesis that evs can also serve as an efficient small rna transfer vehicle for pig cells (132) . more recently, prrsv viral proteins associated to extracellular vesicles (evs) in the size range of exosomes, were reported (129) . moreover, a targeted-pig trial using evs from sera of infected pigs who had overcome the disease, demonstrated that evs are capable of inducing specific ifn-γ secreting cells after a prime-boost strategy, are safe, free-of-virus and can differentiate infected from vaccinated animals (133) , moreover, it was demonstrated that those evs contained antigenic viral proteins recognized by pig immune sera and not by the pre-immune one. of interest, however, a recent article indicated that prrsv derived evs are capable of transmitting the virus from one cell to another (134) . whether these discrepancies are due to in vivo vs. in vitro experimental work and methods applied to isolate evs from serum samples or culture supernatant, remains to be determined. evs have also been explored as novel control strategies in other viral diseases. for example, in respiratory syncytial virus infection, evs are released with a selected modified cargo when compared with uninfected epithelial cells. when analyzed in detail, several viral proteins and diverse species of rna were detected and capable of activating innate immune responses through induction of cytokine and chemokine release (135) . similar scenarios of viral proteins exported in evs have been observed and extensively reviewed for hiv/hcv/htlv-1 (136), ebv (137) , and other viral diseases. moreover, viral products of various origin and size including ebola virus vp24, vp40, and np, influenza virus np, crimean-congo haemorrhagic fever np, west nile virus ns3, and hepatitis c virus ns3, when fused with nef c-terminal domain through dna vectors, were directed to the evs membrane or packaged into them and remained stable after fusion. more importantly, when injected in mice, dna vectors expressing the diverse fusion products elicited a well detectable antigen-specific cd8+ t cell response associating with a cytotoxic activity potent enough to kill peptide-loaded and/or antigen-expressing syngeneic cells, proving its promising results as a cytotoxic t lymphocyte vaccine (138) . prrsv is a complex disease and several gaps in the knowledge of its economic impact, biology and evolution, genetic polymorphism, mechanism of viral infections, elicitation of protective immune responses and novel control strategies, have been reviewed here (box 1). since the late 1980's, different approaches have permitted to examine more closely this virus allowing the discovery of new features of the complex replication cycle, the identification of proteins and nucleic acids playing a role together with extracellular vesicles and nanotubules in facilitating spreading, and a better understanding of immune evasion (non-neutralizing antibodies, glycan shielding, mutation, recombination events, among others) to further vaccine development. presently available prrsv vaccines have many limitations in terms of heterologous protection, but some efforts have been made by combining new adjuvant formulations with modified live viruses, dna and peptide vaccines, as well as extracellular vesicles a new vaccination approach. advancing in all these gaps in knowledge, will eventually accelerate eliminating and eventually eradicating this devastating veterinary disease of such huge economic importance. sm-t: wrote the first draft of the manuscript. mm, hdp, and lf: wrote sections of the manuscript. all authors contributed to manuscript revision, read, and approved the submitted version. this review received support from the feder project (comrdi16-1-0035-03). assessment of the economic impact of porcine reproductive and respiratory syndrome virus on united states pork producers assessment of the economic impact of porcine reproductive and respiratory syndrome on swine production in the united states cost of porcine reproductive and respiratory syndrome virus at individual farm level -an economic disease model isolation of swine infertility and respiratory syndrome virus (isolate atcc vr-2332) in north america and experimental reproduction of the disease in gnotobiotic pigs mystery swine disease in the netherlands: the isolation of lelystad virus ratification vote on taxonomic proposals to the international committee on taxonomy of viruses porcine reproductive and respiratory syndrome virus (prrsv): pathogenesis and interaction with the immune system the molecular biology of arteriviruses the structural biology of prrsv a bayesian phylogeographical analysis of type 1 porcine reproductive and respiratory syndrome virus (prrsv) identification of porcine reproductive and respiratory syndrome virus orf1a-encoded non-structural proteins in virus-infected cells efficient−2 frameshifting by mammalian ribosomes to synthesize an additional arterivirus protein nonstructural proteins nsp2tf and nsp2n of porcine reproductive and respiratory syndrome virus (prrsv) play important roles in suppressing host innate immune responses biogenesis and architecture of arterivirus replication organelles regulation and evasion of antiviral immune responses by porcine reproductive and respiratory syndrome virus highly divergent strains of porcine reproductive and respiratory syndrome virus incorporate multiple isoforms of nonstructural protein 2 into virions prrsv receptors and their roles in virus infection membrane proteins of arterivirus particles: structure, topology, processing and function influence of n-linked glycosylation of minor proteins of porcine reproductive and respiratory syndrome virus on infectious virus recovery and receptor interaction overview: replication of porcine reproductive and respiratory syndrome virus cd163 knockout pigs are fully resistant to highly pathogenic porcine reproductive and respiratory syndrome virus precision engineering for prrsv resistance in pigs: macrophages from genome edited pigs lacking cd163 srcr5 domain are fully resistant to both prrsv genotypes while maintaining biological function pigs lacking the scavenger receptor cysteine-rich domain 5 of cd163 are resistant to porcine reproductive and respiratory syndrome virus 1 infection replacement of porcine cd163 scavenger receptor cysteine-rich domain 5 with a cd163-like homolog confers resistance of pigs to genotype 1 but not genotype 2 porcine reproductive and respiratory syndrome virus an intact sialoadhesin (sn/siglec1/cd169) is not required for attachment/internalization of the porcine reproductive and respiratory syndrome virus role of cd151, a tetraspanin, in porcine reproductive and respiratory syndrome virus infection prrsv structure, replication and recombination: origin of phenotype and genotype diversity effect of porcine reproductive and respiratory syndrome virus (prrsv) (isolate atcc vr-2385) infection on bactericidal activity of porcine pulmonary intravascular macrophages (pims): in vitro comparisons with pulmonary alveolar macrophages (pams) porcine reproductive and respiratory syndrome virus utilizes nanotubes for intercellular spread effect of malaria on hiv/aids transmission and progression tunneling nanotubes (tnt) are induced by hiv-infection of macrophages: a potential mechanism for intercellular hiv trafficking tunneling nanotubes as a novel route of cell-to-cell spread of herpesviruses innate and adaptive immunity against porcine reproductive and respiratory syndrome virus arterivirus molecular biology and pathogenesis interferon-stimulated genes: a complex web of host defenses production of type i interferons: plasmacytoid dendritic cells and beyond immune responses in pigs infected with porcine reproductive and respiratory syndrome virus (prrsv) differential production of proinflammatory cytokines in the pig lung during different respiratory virus infections: correlations with pathogenicity in vivo and in vitro interferon (ifn) studies with the porcine reproductive and respiratory syndrome virus (prrsv) interferon type i response in porcine reproductive and respiratory syndrome virus-infected marc-145 cells modulation of innate immune signaling by nonstructural protein 1 (nsp1) in the family arteriviridae interplay between interferonmediated innate immunity and porcine reproductive and respiratory syndrome virus modulation of host cell responses and evasion strategies for porcine reproductive and respiratory syndrome virus porcine reproductive and respiratory syndrome virus nucleocapsid protein modulates interferon-β production by inhibiting irf3 activation in immortalized porcine alveolar macrophages the prrsv replicase: exploring the multifunctionality of an intriguing set of nonstructural proteins open reading frame 1a-encoded subunits of the arterivirus replicase induce endoplasmic reticulumderived double-membrane vesicles which carry the viral replication complex porcine alveolar macrophage polarization is involved in inhibition of porcine reproductive and respiratory syndrome virus (prrsv) replication porcine reproductive and respiratory syndrome virus nonstructural protein 2 (nsp2) topology and selective isoform integration in artificial membranes the cysteine protease domain of porcine reproductive and respiratory syndrome virus nonstructural protein 2 possesses deubiquitinating and interferon antagonism functions immunodominant epitopes in nsp2 of porcine reproductive and respiratory syndrome virus are dispensable for replication, but play an important role in modulation of the host immune response nonstructural protein 11 (nsp11) of porcine reproductive and respiratory syndrome virus (prrsv) promotes prrsv infection in marc-145 cells nonstructural protein 11 of porcine reproductive and respiratory syndrome virus suppresses both mavs and rig-i expression as one of the mechanisms to antagonize type i interferon production the endoribonuclease activity essential for the nonstructural protein 11 of porcine reproductive and respiratory syndrome virus to inhibit nlrp3 inflammasome-mediated il-1β induction porcine reproductive and respiratory syndrome virus (prrsv) inhibits rnamediated gene silencing by targeting ago-2 novel insights into host responses and reproductive pathophysiology of porcine reproductive and respiratory syndrome caused by prrsv-2 comparison of host genetic factors influencing pig response to infection with two north american isolates of porcine reproductive and respiratory syndrome virus variation among sows in response to porcine reproductive and respiratory syndrome 1 genetic resistance -an alternative for controlling prrs? porc heal manag genetic analysis of reproductive traits and antibody response in a prrs outbreak herd 1 validation and further characterization of a major quantitative trait locus associated with host response to experimental infection with porcine reproductive and respiratory syndrome virus north american porcine reproductive and respiratory syndrome viruses inhibit type i interferon production by plasmacytoid dendritic cells porcine reproductive and respiratory syndrome virus infects mature porcine dendritic cells and up-regulates interleukin-10 production cytokine profiles and phenotype regulation of antigen presenting cells by genotype-i porcine reproductive and respiratory syndrome virus isolates differential type i interferon activation and susceptibility of dendritic cell populations to porcine arterivirus porcine reproductive and respiratory syndrome virus type 1.3 lena triggers conventional dendritic cells 1 activation and t helper 1 immune response without infecting dendritic cells tonsil conventional dendritic cells are not infected by porcine reproductive and respiratory syndrome virus response of the cdc1 and cdc2 subtypes of tracheal dendritic cells to porcine reproductive and respiratory syndrome virus porcine alveolar macrophage-like cells are pro-inflammatory pulmonary intravascular macrophages that produce large titers of porcine reproductive and respiratory syndrome virus nsp1α of porcine reproductive and respiratory syndrome virus strain bb0907 impairs the function of monocyte-derived dendritic cells via the release of soluble cd83 impact of genotype 1 and 2 of porcine reproductive and respiratory syndrome viruses on interferon-α responses by plasmacytoid dendritic cells characterization of the cytokine and maturation responses of pure populations of porcine plasmacytoid dendritic cells to porcine viruses and toll-like receptor agonists sensing of porcine reproductive and respiratory syndrome virus-infected macrophages by plasmacytoid dendritic cells the viral innate immune antagonism and an alternative vaccine design for prrs virus the role of porcine reproductive and respiratory syndrome (prrs) virus structural and non-structural proteins in virus pathogenesis perforin expression can define cd8 positive lymphocyte subsets in pigs allowing phenotypic and functional analysis of natural killer, cytotoxic t, natural killer t and mhc un-restricted cytotoxic t-cells cross-protective immunity to porcine reproductive and respiratory syndrome virus by intranasal delivery of a live virus vaccine with a potent adjuvant porcine reproductive and respiratory syndrome virus-induced immunosuppression exacerbates the inflammatory response to porcine respiratory coronavirus in pigs natural killer cells in host defense against veterinary pathogens suppression of nk cell-mediated cytotoxicity against prrsv-infected porcine alveolar macrophages in vitro evaluation of immune responses to porcine reproductive and respiratory syndrome virus in pigs during early stage of infection under farm conditions protection against porcine reproductive and respiratory syndrome virus (prrsv) infection through passive transfer of prrsv-neutralizing antibodies is dose dependent role of neutralizing antibodies in prrsv protective immunity passive transfer of virus-specific antibodies confers protection against reproductive failure induced by a virulent strain of porcine reproductive and respiratory syndrome virus and establishes sterilizing immunity the challenge of prrs immunology immunological responses of swine to porcine reproductive and respiratory syndrome virus infection mechanisms of adaptive immunity to porcine reproductive and respiratory syndrome virus dissociation of porcine reproductive and respiratory syndrome virus neutralization from antibodies specific to major envelope protein surface epitopes neutralizing antibody responses of pigs infected with natural gp5 n-glycan mutants of porcine reproductive and respiratory syndrome virus certainties, doubts and hypotheses in porcine reproductive and respiratory syndrome virus immunobiology protective humoral immune response induced by an inactivated porcine reproductive and respiratory syndrome virus expressing the hypo-glycosylated glycoprotein 5 gp5 expression in marc-145 cells inhibits porcine reproductive and respiratory syndrome virus infection by inducing beta interferon activity porcine reproductive and respiratory syndrome (prrs): an immune dysregulatory pandemic discovery of a small arterivirus gene that overlaps the gp5 coding sequence and is important for virus production novel structural protein in porcine reproductive and respiratory syndrome virus encoded by an alternative orf5 present in all arteriviruses immune response to orf5a protein immunization is not protective against porcine reproductive and respiratory syndrome virus infection regulatory t cells and infection: a dangerous necessity european genotype of porcine reproductive and respiratory syndrome (prrsv) infects monocyte-derived dendritic cells but does not induce treg cells role of porcine reproductive and respiratory syndrome virus nucleocapsid protein in induction of interleukin-10 and regulatory t-lymphocytes (treg) regulatory t cells in arterivirus and coronavirus infections: do they protect against disease or enhance it? mutations within the nuclear localization signal of the porcine reproductive and respiratory syndrome virus nucleocapsid protein attenuate virus replication immune control of prrs: lessons to be learned and possible ways forward immunological solutions for treatment and prevention of porcine reproductive and respiratory syndrome (prrs) antiviral regulation in porcine monocytic cells at different activation states porcine reproductive and respiratory syndrome virus isolates differ in their susceptibility to neutralization effector mechanisms of humoral immunity to porcine reproductive and respiratory syndrome virus improved vaccine against prrsv: current progress and future perspective. front microbiol sequence analysis of porcine reproductive and respiratory syndrome virus of the american type collected from danish swine herds complete genome sequence of a novel variant porcine reproductive and respiratory syndrome virus (prrsv) strain: evidence for recombination between vaccine and wild-type prrsv strains porcine reproductive and respiratory syndrome (prrs) virus-specific interferon-γ + t-cell responses after prrs virus infection or vaccination with an inactivated prrs vaccine failure of an inactivated vaccine against porcine reproductive and respiratory syndrome to protect gilts against a heterologous challenge with prrsv comparison of different vaccination schedules for sustaining the immune response against porcine reproductive and respiratory syndrome virus porcine reproductive and respiratory syndrome virus vaccines: immunogenicity, efficacy and safety aspects inactivated and subunit vaccines against porcine reproductive and respiratory syndrome: current status and future direction biodegradable nanoparticle-entrapped vaccine induces cross-protective immune response against a virulent heterologous respiratory viral infection in pigs plga nanoparticle entrapped killed porcine reproductive and respiratory syndrome virus vaccine helps in viral clearance in pigs evaluation of hydrophobic chitosan-based particulate formulations of porcine reproductive and respiratory syndrome virus vaccine candidate t cell antigens an innovative approach to induce cross-protective immunity against porcine reproductive and respiratory syndrome virus in the lungs of pigs through adjuvanted nanotechnology-based vaccination plant-based porcine reproductive and respiratory syndrome virus vlps induce an immune response in mice vectored vaccines to protect against prrsv development of a porcine reproductive and respiratory syndrome virus-like-particle-based vaccine and evaluation of its immunogenicity in pigs virus replicon particles expressing porcine reproductive and respiratory syndrome virus proteins elicit immune priming but do not confer protection from viremia in pigs generation of porcine reproductive and respiratory syndrome (prrs) viruslike-particles (vlps) with different protein composition exosomes as potent cell-free peptide-based vaccine. i dendritic cellderived exosomes transfer functional mhc class i/peptide complexes to dendritic cells induction of protective immunity against experimental eimeria tenella infection using serum exosomes extracellular vesicles in parasitic diseases exosomes from plasmodium yoelii-infected reticulocytes protect mice from lethal infections serumderived exosomes from non-viremic animals previously exposed to the porcine respiratory and reproductive virus contain antigenic viral proteins b lymphocytes secrete antigen-presenting vesicles biological properties of extracellular vesicles and their physiological functions inhibition of porcine reproductive and respiratory syndrome virus infection by recombinant adenovirusand/or exosome-delivered the artificial micrornas targeting sialoadhesin and cd163 receptors targeted-pig trial on safety and immunogenicity of serumderived extracellular vesicles enriched fractions obtained from porcine respiratory and reproductive virus infections exosomes mediate intercellular transmission of porcine reproductive and respiratory syndrome virus (prrsv) respiratory syncytial virus infection changes cargo composition of exosome released from airway epithelial cells exosomes and their role in the life cycle and pathogenesis of rna viruses pathogenic role of exosomes in epstein-barr virus (ebv)-associated cancers an exosome-based vaccine platform imparts cytotoxic t lymphocyte immunity against viral antigens we are particularly grateful to christa helwig for english editorial assistance. the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.the reviewer sg declared a past supervisory role with one of the authors mm to the handling editor.copyright © 2019 montaner-tarbes, del portillo, montoya and fraile. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord-002844-jv42o789 authors: marcos-villar, laura; díaz-colunga, juan; sandoval, juan; zamarreño, noelia; landeras-bueno, sara; esteller, manel; falcón, ana; nieto, amelia title: epigenetic control of influenza virus: role of h3k79 methylation in interferon-induced antiviral response date: 2018-01-19 journal: sci rep doi: 10.1038/s41598-018-19370-6 sha: doc_id: 2844 cord_uid: jv42o789 influenza virus stablishes a network of virus-host functional interactions, which depends on chromatin dynamic and therefore on epigenetic modifications. using an unbiased search, we analyzed the epigenetic changes at dna methylation and post-translational histone modification levels induced by the infection. dna methylation was unaltered, while we found a general decrease on histone acetylation, which correlates with transcriptional inactivation and may cooperate with the impairment of cellular transcription that causes influenza virus infection. a particular increase in h3k79 methylation was observed and the use of an inhibitor of the specific h3k79 methylase, dot1l enzyme, or its silencing, increased influenza virus replication. the antiviral response was reduced in conditions of dot1l downregulation, since decreased nuclear translocation of nf-kb complex, and ifn-β, mx1 and isg56 expression was detected. the data suggested a control of antiviral signaling by methylation of h3k79 and consequently, influenza virus replication was unaffected in ifn pathway-compromised, dot1l-inhibited cells. h3k79 methylation also controlled replication of another potent interferon-inducing virus such as vesicular stomatitis virus, but did not modify amplification of respiratory syncytial virus that poorly induces interferon signaling. epigenetic methylation of h3k79 might have an important role in controlling interferon-induced signaling against viral pathogens. chromatin is a dynamic structure that adapts itself to alter the spatial arrangement of genetic information and thus meet the changing demands of cell functions; it has a key role in the control of gene expression. epigenetic modifications of chromatin regulate heritable and reversible gene expression without altering the dna sequence, and can also modify chromatin accessibility. dna methylation and post-translational histone acetylation and methylation are major mechanisms responsible for epigenetic regulation of gene expression 1 . dna methylation typically represses gene transcription when takes place in the promoters of regulated genes 2 . histone acetylation opens the condensed chromatin structure by reducing dna affinity for histones; this enables the dna to uncoil from nucleosomes so that transcription factors and rna polymerase can access the dna and increase transcription 3 . histone methylation can increase or decrease gene transcription, depending on which amino acids in the histones are modified and how many methyl groups are added to specific residues 4 . influenza virus is an rna virus whose genome consists of eight single-stranded rna segments with negative polarity. the virus uses a very uncommon transcription mechanism. the heterotrimeric viral polymerase synthesizes capped, polyadenylated viral mrnas through an initiation process; it uses short-capped oligonucleotides as primers, which are scavenged by a viral endonuclease from newly synthesized host rna polymerase ii transcripts 5, 6 . the viral transcription strategy thus requires continuous cellular transcription for viral mrna infected cells. to identify epigenetic changes induced by influenza virus infection in the cell transcription machinery, we first analyzed dna methylation levels. human a549 lung epithelial cells were mock-infected or infected at high multiplicity of infection (m.o.i.; 3 pfu/ml) with a hypervirulent a/pr8/8/34 (pr8hv) strain [13] [14] [15] [16] (fig. 1a) . at 8 h post infection (hpi), dna was isolated and methylation profiles evaluated in triplicate using the 450 k infinium dna methylation beadchip. correlation analysis of the 444,751 valid probes showed an extremely strong relationship between samples (pearson correlation coefficient from r2 = 0.9945 to r2 = 0.9960; fig. 1b ). to identify specific differentially methylated candidates, we performed a parametric analysis to compare average beta values from infected with mock-infected cells, selecting those with differences in methylation levels >25 (−0.25>delta>0.25) and a standard deviation value <10% (desvest<0.10). we found no significant variations, even after analysis of selected cpg sites from infection-associated genes (not shown), which indicated that global methylation of dna does not change during influenza virus infection in this system. histone fractions were purified from the same samples used for the study of dna methylation and mass spectrometry (ms) analysis were performed. we used unbiased analysis with the nanolc-ms platform to identify and quantify influenza virus-induced epigenetic changes in histone post-translational modifications (ptm). three biological replicates were analyzed and quantitative analyses completed (see methods). we considered a mascot score threshold of 25 as high-confidence peptide identifications, and all peptides methylated/acetylated on lysine (above or below this threshold) were validated manually to assure correct tandem mass spectra matches. only ptms that were present in at least two replicas are presented (fig. 1c) and it has to be pointed out that all modified and unmodified peptides were identified reliably more than once. results in infected cells showed a reduction in lysine acetylation of histone 3 and 4. this decrease would impair host cell expression, in accordance with the role of acetylation in opening the condensed chromatin and activating transcription 17 . non-methylated h3k36 and non-acetylated h4k79 levels increased moderately during infection; since h3k36 methylation and h4k79 acetylation are hallmarks of transcription elongation 18, 19 , the increased levels of these unmodified residues would also contribute to transcriptional inactivation of host cells. histones have a large proportion of basic residues. this property impedes detection of specific residues, probably due to the presence of another lysine(s) that trypsin proteolysis would render as peptides too small to be identified reliably by ms-based techniques. lysines not detected by ms include h3k4 and h4k20, whose ptm are recognized as histone marks that control cell transcription. h3k4 trimethylation is a common modification in active chromatin 20 , whereas h4k20 methylation is linked to transcriptional repression 20, 21 . decreased h3k4me3 levels have been observed during influenza virus infection 16 ; here we evaluated possible changes in methylated h4k20 and in acetylated h4k16, as the latter is a conventional histone marker of active chromatin 22 . total extracts of a549 mock-infected or pr8hv-infected cells at high m.o.i. were collected at various times post-infection and levels of acetylated h4k16 and dimethylated h4k20 were tested by western blot. the results indicated that at late times after infection, influenza virus triggers a decrease in h4k16ac and an increase in h4k20me2 (fig. 1d ), which could contribute to a general decrease in host cell transcription activity. in contrast to this general effect on histone modifications, which seems to impair transcription, we detected a clear increase in methylation of lysine 79 of histone 3, especially monomethylation (fig. 1c ). this result was unanticipated, since studies in saccharomyces cerevisiae and in humans, h3k79 methylation levels correlated clearly with transcriptional activity 23 . as methylation of this lysine gave the highest score in ms analysis, we evaluated this increase by two additional methods. we purified histones from cells mock-or pr8hv-infected at high m.o.i.; at 8 hpi, h3k79me2 levels were evaluated by western blot analysis (fig. 1e ) and a quantitative colorimetric method based on anti-h3k79me2 antibody detection (epigentek) (fig. 1f ). both techniques verified the h3k79 methylation increase observed in proteomic analysis. these results indicate that epigenetic modifications induced by influenza virus infection mainly target the histone component of host cell chromatin, with h3k79 residue methylation the most frequently modified. that methylation of infected cell dna is unaffected indicates, that in these conditions, influenza virus does not permanently injure the host cell, but the epigenetic modifications may be transitory. methyltransferases modify different lysines, only one known methyltransferase, dot1l, mono-, di-and trimethylates h3k79 24 ; the inhibitor epz-5676 (epz) specifically inhibits dot1l. to confirm this ability to inhibit h3k79 methylation in our system, we incubated a549 cells with the inhibitor and used western blot to analyze h3k79me2 accumulation at various times after epz addition. treatment with 1 or 2 μm epz substantially decreased h3k79 methylation at 24-48 h post-epz addition, without affecting h3k4me3 levels (fig. s1a ). the effect of dot1l inhibition on cell viability was analyzed by mtt assay (see methods and 25 ); at these doses, epz did not notably affect cell viability, even at 72 h after treatment (fig. s1b) . to determine whether h3k79 methylation alters influenza virus infection, we inhibited dot1l and analyzed its effect on production of infectious particles. a549 cells were plated alone or with dot1l inhibitor (48 h), then infected at low m.o.i. with the pr8hv strain, with another laboratory-passaged influenza strain, a/wsn/33 (wsn) ( fig. 2a ) or with one of two natural 2009 pandemic isolates, a/california/04/2009 (cal04) and a/ california/07/2009 (cal07) (fig. 2b ) and analyzed production of infectious particles in each condition. dot1l inhibition caused an increase in viral replication, higher in cells infected with the natural isolates, which suggests a general role of h3k79 methylation in control of the influenza virus life cycle. to confirm the effect of dot1l activity on influenza virus replication, we tested the effect of dot1l knock-down on production of infective particles. for rnai-mediated dot1l silencing experiments, we used lentiviruses expressing shrna specific for dot1l (shdot1l 1, shdot1l 2) or a control that expresses irrelevant shrna (shtm) 7, 26 . treatment of a549 cells with the dot1l inhibitor or expression of the dot1l-shrnas reduced h3k79me2 levels compared with shtm, measured by western blot analysis (fig. 3a ) and quantitative colorimetric method (fig. 3b ). dot1l silencers decreased h3k79 methylation in infected cells in a lesser extent than epz treatment. to analyze the relevance of dot1l in virus replication, we transduced a549 cells with lentiviruses expressing the dot1l silencers or control shrna (5 days), followed by infection with the pr8hv ( role of h3k79 methylation in antiviral responses. the above results suggested that modification of lysine 79 of histone 3 may mediate the antiviral response; its demethylation could decrease the host response against the pathogen allowing higher production of infectious particles. in agreement with this proposal we did not find significant reduction of viral titer in cells infected at high m.o.i where ifn signaling does not play a major role, in the presence of the dot1l inhibitor (data not shown). these data suggested that h3k79 methylation would affect host response in conditions close to natural infections that take place in the epithelium of the respiratory tract at low multiplicity of infection and trigger an efficient ifn induction. nf-κb is a protein complex that controls dna transcription, cytokine production and cell survival. nf-κb is involved in cell responses to stimuli such as viral antigens and has a key role in regulating the innate immune response to infection 27 . nf-κb has been shown to be activated upon accumulation of influenza virus rna 28 . in response to specific stimuli, nf-κb translocates from cytoplasm to the nucleus, where it binds promoters of regulated genes, activates transcription of the interferon pathway 29 , and stimulates isg transcription. type i interferon, which includes the ifnα and β subtypes, is induced during influenza virus infection and has an essential role in host defense against the virus by activating expression of a large number of isg 30 . to analyze the effect of dot1l methylase in nf-κb activation and ifn signaling, we evaluated nf-κb subcellular distribution in cells treated with tumor necrosis factor (tnf)-α (10 ng/ml, 30 min), or infected with influenza virus (1 pfu/ml, 8 hpi), alone or with dot1l inhibitor (48 h). after tnf-α binding to the receptor, the inhibitory protein iκbα, which normally binds nf-κb and inhibits its translocation, is phosphorylated; this is followed by ubiquitination and degradation, releasing nf-κb, which is then translocated to the nucleus 29 . nf-κb distribution was monitored using antibodies to the p65 subunit of the nf-κb complex 31 and anti-lamin a/c antibodies to identify the nuclear envelope; influenza np detection was used to monitor infection. we analyzed >200 cells to quantitate nuclear and cytosolic nf-κb distribution (fig. 4) . tnf-α addition produced a significant increase in nuclear import of nf-κb, and dot1l inhibitor treatment significantly decreased its nuclear translocation (fig. 4a) . influenza virus infection similarly induced nf-κb nuclear import, and inhibition of h3k79 methylation reduced its nuclear translocation (fig. 4b) . subcellular distribution of nf-κb was additionally examined in cells previously transfected with lentiviruses that express the specific dot1l silencers or the control (5 days). using the conditions described above, the corresponding cells were treated with tnf-α, or infected with pr8hv influenza virus. in agreement with the results obtained using the dot1l inhibitor, the dot1l silencers decreased the nuclear translocation of nf-κb both, in tnf-α treated cells (fig. 5a ) or in influenza virus infected cells (fig. 5b) , whereas the expression of the control silencer did not change the subcellular distribution of nf-κb. together these results indicate that methylation of h3k79 modulates the pathway involved in the nuclear translocation of nk-κb. as dot1l inhibition impaired nf-κb activation, we examined its effect on steps that follow its nuclear translocation, that is, ifn and isg rna production. a549 cells were left untreated (control), treated with 100 u/ml ifnαβ (ifn) or were infected with pr8hv strain (1 pfu/cell) (pr8hv), alone or with dot1l inhibitor (48 h). after 8 h or 16 h of ifn treatment or virus infection, rna was extracted and used for qpcr detection. at 8 h, we found a weak increase on ifnβ, ifn-stimulated gene 56 (isg56) and interferon-induced protein mx1 (mx1) rna levels after ifnαβ addition or influenza virus infection, and dot1l inhibitor treatment did not significantly decreased their accumulation (fig. 6b,c) . at 16 h, we observed an increase in ifnβ, isg56 and mx1 rna levels and a reduction of these rnas when cells were pretreated with the h3k79 methylase inhibitor (fig. 6b,c) . the significant reduction in nf-κb nuclear translocation and decreased ifnβ, isg56 and mx1 rna expression after inhibition of dot1l both, support a role for h3k79 methylation in modulating the antiviral response. given the role of h3k79 methylation in the control of ifn signaling, we analyzed the effect of dot1l inhibitor on influenza virus replication in cells with normal or deficient ifn responses. we used pr8hv and cal07 strains at low m.o.i. to infect mdck, mdck v2 or mdck npro cells, untreated or treated with dot1l inhibitor. mdck v2 cells express v2 protein, which targets stat1 and 2 for degradation 32 , and mdck npro cells express npro protein, which targets irf-3 for polyubiquitination and subsequent degradation 33 ; ifn, but not isg, is thus induced in mdck v2 cells, and neither ifn nor isg are induced in mdck npro cells. we analyzed viral titers at different hpi and observed that the increased infectious particle production in mdck cells treated with the inhibitor and infected with pr8hv or cal07 (fig. 7a and b) was reduced or lost in 'ifn-compromised' cells treated with dot1l inhibitor. the effect of influenza virus infection on h3k79 methylation in mdck cells with normal and deficient response to ifn was checked by colorimetric assays. influenza virus infection increased h3k79 methylation levels in normal and interferon deficient cells, likewise in a549 cells (fig. s2a) . since h3k79 methylation does not affect influenza virus replication in cells with impaired ifn signaling, we analyzed the effect of dot1l inhibitor in subsequent stages of viral infection. we evaluated ifnβ, isg56 and mx1 rna amounts after influenza virus infection (1 pfu/cell) in ifn-competent or -deficient cells, untreated or treated with dot1l inhibitor. influenza virus infection increased ifnβ, isg56 and mx1 production in mdck control cells, and inhibitor treatment reduced their accumulation (fig. 7c, top) . infection of mdck v2 cells increased ifnβ production, which was reduced by dot1l inhibitor, whereas accumulation of isg was not observed, in accordance with the need for stat signaling for their induction (fig. 7c, center) . infection of mdck npro cells did not increase ifnβ, isg56 or mx1 rnas, independently of the presence of dot1l inhibitor (fig. 7c, bottom) , which coincided with the inability of these cells to produce ifn and isg. in addition, total extracts of mdck, mdck v2 and mdck npro cells infected with pr8hv at 3 m.o.i for 8 h were used for western blot analysis to detect irf-3, stat1, mx and isg56 to confirm the differential expression of these proteins in normal or ifn-deficient cells (fig. s2b) . these results indicate that the interferon antiviral response to influenza virus infection is at least partially regulated by h3k79 methylation via canonical type i ifn signaling mediated by the stat pathway at the level of ifn activation and response. these results indicated the ability of dot1l methylase to control interferon signaling pathways. to evaluate whether h3k79 methylase has a broader role in the control of virus multiplication, we tested the effect of dot1l inhibitor and the lentiviruses expressing the dot1l silencers on the replication of two distinct rna viruses, respiratory syncytial virus (rsv) and vesicular stomatitis virus (vsv). like influenza virus, rsv is a respiratory virus, and is the most common cause of viral lower respiratory tract infections in infants and children 34 . although ifnβ appears to restrict viral replication in lung epithelial cells, it is generally agreed that rsv is relatively resistant to ifnαβ, effects, and ifn has not been detected in natural infection 34 . vsv can infect insects, cattle, horses and pigs and is a potent ifn inducer both in cell culture and animal models 35 . human epithelial a549 cells, untreated or treated with dot1l inhibitor (48 h) (fig. 8a,c) , or transduced with lentiviruses expressing the dot1l silencers or control shrna (5 days) (fig. 8b,d) , were infected at low m.o.i. with rsv (1pfu/ml) (fig. 8a,b) or vsv (10 −3 pfu/ml) (fig. 8c,d) ; at various times post-infection, cell supernatants were obtained for viral titration (see methods). no changes were observed in rsv replication in the presence of the inhibitor or the dot1l silencers, whereas infectious particle production clearly increased in vsv-infected cells treated with dot1l inhibitor or when dot1l was down-regulated through the expression of the specific silencers. we also evaluated the expression of viral and isg proteins (isg56 and mxa) during infection with rsv (fig. 8b, right) , and vsv (fig. 8d, right) . the corresponding viral proteins were detected and we observed a lack of ifn-stimulated gene induction in rsv-infected cells, while infection with vsv efficiently induced ifn response in our system. these results support a role for h3k79 methylation in the control of ifn signaling, and a potential general dot1l methylase function in regulating pathogen infection controlled by the ifn pathway. viruses are obligate intracellular parasites that completely subordinate host cell metabolism. although influenza virus does not integrate into the genome of the infected cell, the virus efficiently switches off expression of host cell genes 36 , a result of a complex interplay of virus-induced activities closely coordinated to reduce the host response to eliminate the viral infection. consequently, during infection there is a network of viral-and host-induced modifications of cellular gene expression with a close dependence of chromatin-based functions and therefore of chromatin dynamic. accordingly, specific interactions between chromatin remodelers of the chd family and influenza virus proteins have been described such as the association of chd3 with the non-structural protein ns2 37 or chd6 and chd1 with the viral polymerase complex 16, 38, 39 . in spite of this association, little effort has been made to identify epigenetic changes in chromatin induced by the infection. few alterations in dna methylation have been reported during influenza virus infection [9] [10] [11] . among them changes in promoter methylation levels of some proinflammatory cytokines and interleukine genes when using the high virulence h5n1 strain have been described. infection of human respiratory epithelial cells with pr8hv strain did not show variations on dna methylation (fig. 1b) , however it is possible that changes in promoter methylation of specific genes and their subsequent inactivation, take place in response to particular virulent influenza strains and/or in different systems. previous studies showed that influenza virus infection modulates ptm of several isg. chromatin immunoprecipitation assays and antibodies that recognize canonical marks of transcription activation such as h3k4me3 or of inactivation such as h3k27me3 showed addition or removal of these ptm in several isg; modifications depended on strain virulence when h5n1 virus was compared with the h1n1 2009 pandemic strain 8 . this study emphasized the importance of epigenetic control in the antiviral response elicited by influenza virus infection; nonetheless, they focused on the search for specific canonical transcription activation or repression marks of histones that correlate with up-or down-transcriptional regulation of the corresponding genes and do not provide an overview of all possible changes to chromatin in response to the viral infection. for a broad overview of chromatin modifications in influenza virus-infected cells, we used an unbiased search for global changes in chromatin epigenetics at the dna and histone levels. we observed a general decrease on histone acetylation in h3 and h4 lysine residues after infection, as well as increased levels of unmodified h3k36, h4k79 and dimethylated h4k20 (fig. 1c and d) . in addition, previous studies showed reduction of h3k4me3; one of the hallmark of active chromatin 16 . histone acetylation has a key role opening the condensed chromatin, resulting in charge neutralization and a more relaxed, open, and transcriptionally active chromatin structure 17 . decreased acetylated histones, trimethylated h3k4 and increased non-methylated h3k36 and dimethylated h3k20, all associate with transcriptional inactivation [18] [19] [20] [21] . these epigenetic changes would impair host cell expression, in accordance with the transcription inactivation of the host cell that occurs during infection 40 and constitutes one of the major mechanisms triggered by the virus to inactivate host transcription machinery and consequently to decrease the antiviral response. unexpectedly, changes in methylation of lysine 79 of histone 3 were the most prominent. lysine 79 is located within the globular domain of histone h3 and is mono-, di-, and trimethylated by dot1l, which modifies this lysine exclusively 24 . h3k79 methylation is increased in actively transcribing genes and appears to have a role in cell cycle regulation and dna damage response 41 . dot1l has been studied particularly in the modulation of mixed-lineage leukemia (mll)-related leukemogenesis, as mll fusion target gene expression depends specifically on the functional dot1l gene 42 ; indeed, inhibition of dot1l activity is currently in clinical trials. the role of h3k79 methylation in viral infection, is poorly characterized. infection of human primary fibroblasts with human cytomegalovirus (hcmv), produces a marked increase in h3k79me2 levels and downregulation of dot1l expression results in a decreased viral growth 43 . the genome of hcmv is a double-stranded dna molecule that is maintained as episome during infection. the viral dna lacks histones when encapsidated in the virion however, upon infection the viral genome is transported to the nucleus, where it becomes associated with host cell histones. it has been speculated that dot1l directly mediates replication of the hcv genome, in agreement with the chromatinization of its dna genome 44 . the function of h3k79 methylation modulating influenza virus replication, suggest a very different mechanism, since influenza virus is an rna virus that does not integrate in the host chromatin and its genome lacks histones. in physiological conditions where antiviral signaling is induced, decreased h3k79 methylation clearly enhanced viral replication (figs 2 and 3) . moreover, nuclear translocation of nf-κb (figs 4 and 5) and accumulation of ifnβ and isgs (isg56 and mx1) decreased in influenza virus-infected cells with dot1l downregulated (fig. 6) . influenza virus and vesicular stomatitis virus induce a strong antiviral immune response characterized by robust production of antiviral type i interferons. during infection, double-stranded rna molecules are produced, which are recognized by the rig-i helicase 45 . after activation, rig-i recruits various tnf receptor-associated factors, which trigger phosphorylation and activation of the iκκ complex, leading to iκbα phosphorylation and degradation to allow nfκb nuclear translocation 46 . nf-κb and ifn regulatory factor 3 direct expression of type i ifn, which promote transcription of a variety of genes that further limit viral replication 45, 47 . we observed a marked increase on replication of influenza virus and vsv, both potent inducers of ifn, in cells with low levels of methylated h3k79 (figs 2, 3 and 8 ). this effect is lost when cells deficient on ifn signaling are infected with influenza virus (fig. 7) . together the data indicate that demethylation of h3k79 would decrease antiviral ifn signaling and therefore, viruses may develop different strategies attempting to control epigenetic modification of h3k79 that elicit the anti-pathogen response. methylation of lysine 79 of histone 3 might have a general role in controlling the host response of pathogens that are interferon inducers. epz5676 was purchased from novagen, human tumor necrosis factor α from sigma-aldrich, and recombinant interferon αβ (pbl 11200-2 universal type i ifn) was provided by s. guerra. lentiviral particle production and cell transduction. lentiviral particles were produced in hek293t cells by cotransfection of plasmids pspax2 and pmd2.g with each of the plko-based shrna vectors, as described 48, 49 . supernatants were collected 40 to 48 h post-transfection, filtered through a 0.45 μm filter, and used to transduce the corresponding cells. as the lentiviral vectors confer puromycin resistance, the minimum amount of supernatant necessary to confer 100% resistance to puromycin (5 μg/ml) was used. silencing was tested by western blotting or colorimetric assays, normally at 5 days post-transduction. western blot was performed as described 40 . to detect influenza virus proteins, the following antibodies were used: for pa, monoclonal antibodies (mab) 2 and 9 (1:250; 50 ; for pb2, mab 22 (1:100; 50 , and for pb1, rabbit polyclonal antibody (1:1000; 51 ; for β-tubulin, mouse anti-β-tubulin mab (1:1000; sigma). to analyze histones, we used h4k16ac ab109463 (1:1000; abcam) and h4k20me2 (1:1000; active motif). polyclonal antibodies to h3k79me2 d15e8, h3k4me3 c42d8, h4 #2592 and h3 d1h2 (1:1000) were from cell signaling. for vsv detection, a monoclonal antibody mix against g, n and m viral proteins provided by m. esteban was used. for rsv detection, a monoclonal antibody against np protein provided by j.a. melero was used. colorimetric determination of h3k79me2. the iquik global di-methyl histone h3k79 quantification kit (colorimetric) from epigentek was used for elisa-like measurement of total h3k79me2 amounts, following the supplier's protocol. proteomic analysis of histone modifications. enzymatic digestion and itraq-4plex labeling. the enriched histone fraction (30 μg) for each condition, prepared following the epigentek protocol, was precipitated by the methanol/chloroform method, reconstituted in 7 m urea/2 m thiourea/100 mm teab buffer (triethylammonium bicarbonate, ph 7.5), reduced with 50 mm tris(2-carboxyethyl) phosphine (tcep, sciex), and alkylated with 200 mm methyl methanethiosulfonate (mmts, pierce); this was followed by trypsin (sigma-aldrich) proteolysis at a 1:20 enzyme:protein ratio. the tryptic peptides were labeled using the itraq-4plex isobaric mass tagging kit (sciex) according to manufacturer's instructions (mock, tag-114; pr8lv, tag-115, pr8hv, tag-116). samples were pooled, dried and desalted on a sep-pak c18 cartridge (waters). peptide fractions were subjected to lc-ms/ms analysis using a nano liquid chromatography system (eksigent technologies nanolc ultra 1d plus) coupled to a high speed triple tof 5600 mass spectrometer (sciex) with a nanoelectrospray ion source. samples were injected on a c18 pepmap trap column (5 μm, 100 μm i.d. x 2 cm; thermo scientific) at 2 μl/min, in 0.1% formic acid in water, and the trap column was switched on-line to a c18 nanoacquity beh analytical column (1.7 μm, 100 å, 75 μm i.d. x 15 cm, waters), equilibrated in mobile phase a (0.1% formic acid in water), and peptide was eluted in a 120 min linear gradient from 5-40% b (0.1% formic acid in acetonitrile) at 250 nl/min. the mass spectrometer was operated in data-dependent acquisition mode. for tof scans, accumulation time was set to 250 ms, and up to 15 precursor ions were monitored per cycle. data analysis. ms and ms/ms data obtained were converted to mgf files, which were also searched against a homo sapiens protein database containing 138362 protein-coding genes (including reversed entries to calculate false discovery rate; fdr) using the mascot server v. 2.4 (matrix science). search parameters were set as follows: enzyme, trypsin; allowed missed cleavages, 3; fixed modifications, beta-methylthiolation of cysteine and itraq-4plex (n-term and k); variable modifications, oxidation of methionine, acetylation (k and n-term) and methylation and dimethylation (k). peptide mass tolerance was set to ±25 ppm for precursors and 0.05 da for fragment masses. the confidence interval for protein identification was set to ≥95% (p < 0.05) and only peptides with an individual ion score above the 1% fdr threshold were considered correctly identified. dna methylation. isolated dna was processed according to the manufacturer's protocols for illumina infinium assay, as described 52 . data filtering. from the initial 485,577 cpg, we excluded all probes with detection p-values > 0.001 (1135 cpg removed). we eliminated cpg containing single-nucleotide polymorphisms (snp) located within 10 bp of the target cpg (snp10) and internal controls (ch and rs), resulting in 39691 cpg removed. a total of 444,751 valid probes were included in the final analysis. methylation score was represented as β-values, average for each probe was calculated and β-value differences were used for analysis. confocal immunofluorescence microscopy. cells were fixed in 4% paraformaldehyde (20 min, room temperature) and stored in pbs. for immunofluorescence, cells were permeabilized (5 min) in pbs containing 1% triton x-100 and incubated with primary antibodies diluted in pbs/4% bsa (w/v) as follows: rat anti-np (1:5000 53 ;, anti-p65 ab16502 (1:500; abcam) and anti-lamin a/c (636)(1:200; sc-7292, santa cruz) to label nuclear envelope. confocal microscopy was performed with a leica tcs sp5 laser scanning system. images of 1024 × 1024 pixels and an eight-bit grayscale depth were acquired sequentially every 0.2-0.3 μm using las af version 2.2.1 software (leica) and analyzed using las af and metamorph premier version 7.5.2 image analysis software (molecular devices). p65 nuclear translocation was quantified by counting at least 200 cells/condition, and the ratio of relative p65 intensity in nucleus and cytoplasm of each cell was calculated. qrt-pcr analysis. for rna extraction, cell pellets were resuspended in 1 ml trizol reagent (invitrogen) and rna was purified as recommended by the manufacturer. rna was digested with rnase-free dnase (1 u/ mg; 1 h, 37 °c), extracted with phenol-chloroform-isoamyl alcohol and ethanol-precipitated. for reverse transcription, we used the high-capacity cdna rt kit (applied biosystems). pcr were performed in 96-well pcr plates using sybr green pcr master mix (applied biosystems). pcr were carried out in a prism 7000 sequence detection system (applied biosystems). the cycle threshold (ct) was determined with analytical software (sds; applied biosystems). serial dilutions of cdna were used to ensure amplification. chromatin remodeling, histone modifications, and dna methylation-how does it all fit together? cancer epigenomics: dna methylomes and histone-modification maps histone acetylation and chromatin remodeling histone modifications for human epigenome analysis the cap-snatching endonuclease of influenza virus polymerase resides in the pa subunit influenza virus transcription and replication influenza mrna translation revisited: is the eif4e cap-binding factor required for viral mrna translation? pathogenic influenza viruses and coronaviruses utilize similar and contrasting approaches to control interferon-stimulated gene responses changes in methylation of genomic dna from chicken immune organs in response to h5n1 influenza virus infection infection with influenza a viruses causes changes in promoter dna methylation of inflammatory genes interleukin-6 expression was regulated by epigenetic mechanisms in response to influenza virus infection or dsrna treatment influenza a virus-induced caspase-3 cleaves the histone deacetylase 6 in infected epithelial cells replication fitness determines high virulence of influenza a virus in mice carrying functional mx1 resistance gene attenuated strains of influenza a viruses do not induce degradation of rna polymerase ii specific residues of pb2 and pa influenza virus polymerase subunits confer the ability for rna polymerase ii degradation and virus pathogenicity in mice influenza virus and chromatin: role of the chd1 chromatin remodeler in the virus life cycle histone acetylation: a switch between repressive and permissive chromatin chromatin modifications by methylation and ubiquitination: implications in the regulation of gene expression hat4, a golgi apparatus-anchored b-type histone acetyltransferase, acetylates free histone h4 and facilitates chromatin assembly profile of histone lysine methylation across transcribed mammalian chromatin pr-set7-dependent methylation of histone h4 lys 20 functions in repression of gene expression and is essential for mitosis histone acetylation regulates chromatin accessibility: role of h4k16 in inter-nucleosome interaction the many faces of histone h3k79 methylation the diverse functions of dot1 and h3k79 methylation a rapid colorimetric assay of fungal viability with the tetrazolium salt mtt human staufen1 protein interacts with influenza virus ribonucleoproteins and is required for efficient virus multiplication the role of nuclear factor kappab in the interferon response influenza virus-induced nf-kappab-dependent gene expression is mediated by overexpression of viral proteins and involves oxidative radicals and activation of ikappab kinase the nf-kappab regulatory network signaling to nf-kappab x-ray crystal structure of an ikappabbeta x nf-kappab p65 homodimer complex in vitro and in vivo specificity of ubiquitination and degradation of stat1 and stat2 by the v proteins of the paramyxoviruses simian virus 5 and human parainfluenza virus type 2 the npro product of bovine viral diarrhea virus inhibits dna binding by interferon regulatory factor 3 and targets it for proteasomal degradation activity and regulation of alpha interferon in respiratory syncytial virus and human metapneumovirus experimental infections ability of the matrix protein of vesicular stomatitis virus to suppress beta interferon gene expression is genetically correlated with the inhibition of host rna and protein synthesis polypeptide synthesis in influenza-virus infected cells chd3 facilitates vrnp nuclear export by interacting with nes1 of influenza a virus ns2 pa subunit from influenza virus polymerase complex interacts with a cellular protein with homology to a family of transcriptional activators chd6 chromatin remodeler is a negative modulator of influenza virus replication that relocates to inactive chromatin upon infection influenza virus infection causes specific degradation of the largest subunit of cellular rna polymerase ii dot1l/kmt4 recruitment and h3k79 methylation are ubiquitously coupled with gene transcription in mammalian cells mll-rearranged leukemia is dependent on aberrant h3k79 methylation by dot1l quantitative proteomic discovery of dynamic epigenome changes that control human cytomegalovirus (hcmv) infection temporal dynamics of cytomegalovirus chromatin assembly in productively infected human cells activation and regulation of pathogen sensor rig-i mavs recruits multiple ubiquitin e3 ligases to activate antiviral signaling cascades. elife 2, e00785 emerging role of ubiquitination in antiviral rig-i signaling a third-generation lentivirus vector with a conditional packaging system lentivirus-delivered stable gene silencing by rnai in primary cells monoclonal antibodies against the influenza virus pb2 and np polypeptides interfere with the initiation step of viral mrna synthesis in vitro distinct regions of influenza virus pb1 polymerase subunit recognize vrna and crna templates molecular cloning. a laboratory manual genetic trans-complementation establishes a new model for influenza virus rna transcription and replication we are indebted to j. ortin, p. gastaminza and u. garaigorta for critique of the manuscript. the technical assistance of s. ciordia and r. navajas from the proteomics facility (cnb-csic, proteored isciii) is greatly appreciated. we thank c. mark for editorial assistance. lmv was supported by ciber de enfermedades respiratorias. this work was funded by the spanish ministry of economy and competitivness, plan nacional de investigacion científica, desarrollo e innovacion tecnologica (bfu2014-57797-r, (aei/feder)) and ciber de enfermedades respiratorias. js is a miguel servet researcher at isciii (ms13/00055). supplementary information accompanies this paper at https://doi.org/10.1038/s41598-018-19370-6.competing interests: the authors declare that they have no competing interests.publisher's note: springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.open access this article is licensed under a creative commons attribution 4.0 international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. key: cord-007696-83v9yfa6 authors: zisman, david a.; keane, michael p.; belperio, john a.; strieter, robert m.; lynch, joseph p. title: pulmonary fibrosis date: 2005 journal: fibrosis research doi: 10.1385/1-59259-940-0:003 sha: doc_id: 7696 cord_uid: 83v9yfa6 idiopathic pulmonary fibrosis (ipf) is a chronic fibrosing lung disease limited to the lungs and associated with the histologic appearance of usual interstitial pneumonia (uip) on surgical lung biopsy. the estimated prevalence in the united states is between 35,000 and 55,000 cases, and evidence suggests that the prevalence is increasing for ipf. risk factors associated with pulmonary fibrosis include smoking, environmental exposures, gastroesophageal reflux disease, commonly prescribed drugs, diabetes mellitus, infectious agents, and genetic factors. the diagnosis requires a careful history and physical examination, characteristic physiological and radiological studies, and, in some cases, a surgical lung biopsy. the natural history of ipf is not known, but evidence supports the concept of a continuum of idiopathic interstitial pneumonias that may overlap in time. most patients with ipf succumb to respiratory failure, cardiovascular disease, lung cancer, pulmonary embolism, infection, and other health problems. the median survival time for patients with ipf is less than 3 yr. factors that predict poor outcome include older age, male gender, severe dyspnea, history of cigarette smoking, severe loss of lung function, appearance and severity of fibrosis on radiological studies, lack of response to therapy, and prominent fibroblastic foci on histopathologic evaluation. conventional therapy (corticosteroids, azathioprine, cyclophosphamide) provides only marginal benefit. lung transplantation should be considered for patients with ipf refractory to medical therapy. in light of the poor prognosis and lack of response to available anti-inflammatory therapy, alternative approaches to therapy are being pursued. emerging strategies to treat patients with ipf include agents that inhibit epithelial injury or enhance repair, anticytokine approaches, agents that inhibit fibroblast proliferation or induce fibroblast apoptosis, and other novel approaches. the diagnosis and management of idiopathic interstitial pneumonias (iips) remains a challenge to the clinician. recently, there have been substantial changes in our understanding and approach to theses diseases. with greater comprehension of the clinical relevance of the different histopathological subgroups that make up the idiopathic interstitial pneumonias, the term idiopathic pulmonary fibrosis (ipf) is now reserved to patients with idiopathic usual interstitial pneumonia (uip) on surgical lung biopsy. the following review will provide an updated discussion of the epidemiology, risk factors, diagnosis, natural history, morbidity and mortality, prognosis, and conventional therapy of ipf, as well as emerging strategies for the treatment of patients with this disease. the true prevalence of ipf, also known as cryptogenic fibrosing alveolitis (cfa), is unknown. despite the poor quality of the data and the changes in diagnostic criteria and classification, there is evidence suggesting that ipf is increasing (1). there are approx 3 to 20.2 cases of ipf per 100,000 in the general population (1-5). the prevalence increases with older age, history of smoking, and male gender (3, 4, 6) . in a population-based study in bernalillo county, new mexico, the prevalence for adults from age 35 to 44 yr was 2.7 per 100,000, but surpassed 175 per 100,000 for individuals older than 75 yr (3). the prevalence of ipf is higher in men (20.2 cases per 100,000) than in women (13.2 cases per 100,000) (3). the estimated prevalence in the united states is between 35,000 and 55,000 cases (3). the incidence of ipf is estimated at 10.7 cases per 100,000 per year for males and 7.4 cases per 100,000 per year for females (3). vital statistics figures are limited and incomplete. in 1988, there were 30,000 hospitalizations and 4851 deaths in the united states owing to pulmonary fibrosis (compared with 665,000 hospitalizations owing to chronic obstructive pulmonary disease and asthma). in japan, the mortality rate for ipf per 100,000 population was estimated to be 3.3 in men and 2.5 in women, with an overall rate of 3.0 in both sexes (4). in the united kingdom, the annual number of deaths from ipf increased twofold between 1979 and 1988. the age-adjusted rate of pulmonary fibrosis among deceased in the united states increased from 48.6 per 100,000 in 1979 to 50.9 per 100,000 in 1991 in males, and from 21.4 per 100,000 in 1979 to 27.2 per 100,000 in 1991 in females (7). pulmonary fibrosis listed as a cause of death increased from 40% in 1979 to 56% in 1991. in the united states, the age-adjusted mortality rates are highest in the western and southwestern states and lowest in the midwest and northeast. in the united kingdom, highest mortality rates are found in industrialized areas of england and wales (7,8). in case-control studies, smoking has been identified as a possible risk factor for ipf with an odds ratio (or) of 1.6 (95% confidence interval [ci] 1.1-2.4) for ever-smoking, and 1.9 (95% ci 1.3-2.9) for former-smokers (4, 9, 10) . smokers of 21 to 40 pack-years have an or of 2.3 (95% ci 1.3-3.8) for pulmonary fibrosis (9). in a recent study in japan, the adjusted odds ratio for cigarette smoking was estimated at 5.40 (95% ci 2.30-12.66) (11). interestingly, three studies reported improved survival among current or former smokers with uip compared to never-smokers (12) (13) (14) . however, others found no such effect (15) (16) (17) . it is possible that the apparent protective effect of cigarette smoking may relate to the following: lead time bias; alterations in the balance of proteinases and antiproteinases that would influence net deposition of extracellular matrix in the lung; or inhibitory effects of cigarette smoke on lung fibroblast proliferation and chemotaxis (12,18). instillation of acid in several animal models results in aspiration-induced lung injury and pulmonary fibrosis (19) . clinical data suggest that a high percentage of patients with ipf have clinically silent gastroesophageal reflux disease (gerd) (19, 20) . small tracheobronchial aspirations of gastric acid may play a role in the pathogenesis of ipf; however, a causal relationship has not been established (19, 20) . investigators at the university of washington medical center are conducting an ongoing prospective study of 65 patients with ipf. interim results indicate that the prevalence of ipf patients with gerd is 95%, with only 40% of patients reporting symptoms (19) . however, the perpherial pattern of uip in the lung would be unusual if gerd was truly the cause, as compared with an association. in one case-control study, ipf was associated with exposure to antidepressants with an or of 1. 79 1.11-9.61] ). these associations were independent of smoking and occupational dust exposure. the authors concluded that exposure to antidepressants may be responsible for approx 10% of cases of ipf seen in their population. no significant association was noted between ipf and the other drug groups tested (anticonvulsants, β-blockers, antibiotics, and nonsteroidal anti-inflammatory drugs [nsaids]) (21). in a recent study, clinical and demographic data were extracted from medical records of 65 consecutive patients with ipf admitted to a japanese hospital. ipf was associated with diabetes mellitus (dm) with and or of 4.06 (95% ci 1. 80-9.15 ). the authors concluded that dm might be a risk factor for ipf (11). the etiology of ipf is unknown, but environmental factors may play a causative role. metal and wood dust environments may be important risk factors for pulmonary fibrosis. in one study, metal dust exposure was identified as a risk factor with an odds ratio (or) of 1.11 (95% ci 1.06-1. 16) , and wood dust exposure with an or of 1.12 (95% ci 1.02-1. 24 ). in that study, metal and wood dust exposure may have caused up to 13% and 10% of pulmonary fibrosis cases, respectively. dust containing brass, lead, cobalt, aluminum, zinc, cadmium, mercury, and pine dusts were associated with pulmonary fibrosis (10). certain occupations may predispose to pulmonary fibrosis. in one study, farming was identified as a potential risk factor with and or of 1.6 (95% ci 1.0-2.5) and exposure to livestock was associated with an or of 2.7 (95% ci 1.3-5.5). hairdressing was linked to pulmonary fibrosis with an or of 4.4 (95% ci 1.2-16.3), metal dust exposure with an or of 2.0 (95% ci 1.0-4.0), raising birds with an or of 4.5 (95% ci 1.6-14.1), stone cutting/polishing with an or of 3.9 (95% ci 1.2-12.7), and vegetable/animal dust exposure with an or of 4.7 (95% ci 2.1-10.4) (22). a number of viruses have been associated with pulmonary fibrosis, but true cause-effect relationships remain unproven. a serological survey found an association between active epstein-barr virus (ebv) infection and ipf (23). egan and colleagues reported immunohistochemical evidence of ebv-productive cycle antigens in type ii alveolar epithelial cells in ipf (24). subsequently, these investigators detected ebv dna by polymerase chain reaction (pcr) in the lung tissue of patients with ipf (25). further study demonstrated that productive ebv replication is common in ipf and it is not associated with immunosuppressive therapy (26). tang in a recent study, human t-lymphtropic virus type i (htvl-i) positive ipf patients had more affected lung parenchyma, demonstrated traction bronchiectasis with honeycomb change, and exhibited increased levels of specific cytokines that correlated with activated t-cells in the bronchoalveolar lavage fluid (balf). these findings suggested that htlv-i infection might contribute to the development of ipf via activation of t-cells (41). the latent nature together with episodic reactivation of many of these viruses may provide a scenario for the concept of "multiple hits" host defense followed by repair that these patients may experience during the course of their disease. the genetics of familial ipf have not been elucidated. an autosomal dominant trait with variable penetrance may account for approx 70% of cases; there is no clear mode of transmission in the remaining 30% (2,42). investigators have linked ipf to an increase in mz phenotype for α1-antitrypsin inhibition on chromosome 14 (43-45). using a candidate gene approach, researchers identified surfactant protein c gene mutations in large familial pulmonary fibrosis kindred, including adults with uip and children with nonspecific interstitial pneumonia (nsip) (46). in a separate study, selman and co-workers demonstrated that surfactant protein a and b genetic variants predispose to ipf (47). genetic polymorphisms for interleukin-1 receptor antagonist (il-1ra) and tumor necrosis factor (tfn)-α appear to be important in determining risk (48). in contrast, transforming growth factor (tgf)-β polymorphisms do not predispose to ipf, but these polymorphisms may affect the course of the disease (49). it is unknown what proportion of ipf is familial, but it is estimated that 0.5 to 2.2% of cases have a genetic basis (42). thirty-eight families affected by pulmonary fibrosis have been identified and are currently under active investigation. the familial aggregation in those families is consistent with a genetic basis in at least a subset of patients with ipf (50). ipf is a specific form of chronic fibrosing interstitial pneumonia limited to the lungs and associated with the histological pattern of uip on surgical lung biopsy (2). many earlier studies included various other idiopathic interstitial pneumonias under the term idiopathic pulmonary fibrosis, but the clinical term ipf is now reserved to patients with idiopathic uip. the diagnostic approach to any patient with diffuse lung disease must include a thorough history and physical examination with attention to symptoms or signs suggestive of a connective tissue disease, occupational or environmental exposures, use of fibrogenic drugs, and family history of pulmonary fibrosis. patient age at disease onset is generally between 50 and 70 yr of age and ipf is more common in males than females. ipf typically presents insidiously, with gradual onset of a nonproductive cough and dyspnea (2). patients are often treated for other conditions such as congestive heart failure, "walking pneumonia," bronchitis, or asthma before the diagnosis is made. the physical examination in most patients (>80%) reveals fine bibasilar inspiratory crackles ("velcro rales"), and clubbing is noted in up to 50% of patients (51,52). signs of right heart failure are evident in advanced cases (2). laboratory abnormalities are mild and nonspecific. one early study described "autoimmune factors" in blood of 8 patients (4 of whom had autoimmune-associated diseases) among 17 patients with diffuse fibrosing alveolitis (53). a positive rheumatoid factor occurred in 3 of 20 patients with unexplained pulmonary fibrosis and positive antinuclear factor in the serum (ana) (54). a later study examined serum specimens from 122 patients with ipf and compared them with specimens from age-and sex-matched controls; anas were present in 21% of patients with ipf and in 6% of the control subjects (55). positive circulating anas or rheumatoid factor occur in 10% to 20% of patients with ipf, but titers are rarely high (2,53,54,56 ). an elevated erythrocyte sedimentation rate, lactate dehydrogenase, or hypergammaglobulinemia may be found in patients with ipf, but are nondiagnostic (2). serological findings do not correlate with extent or severity of disease, and have no prognostic value (2,57). in the absence of symptoms of connective tissue disease, the presence of autoantibodies does not imply an underlying systemic disorder. recently, investigators reported the occurrence of low fasting triglyceride and high free fatty acid levels in patients with pulmonary fibrosis. because insulin-like growth factor (igf)-i is known to lower triglycerides and increase free fatty acids, the authors hypothesized that the reported increased production of igf-i in patients with ipf may explain such findings (58). classic chest radiographic findings in ipf include a basal predominant reticular, or reticulonodular, pattern associated with decreased lung volumes, and in later stages, cystic areas representing honeycomb (hc) lung (2,59-61). when a "confident" diagnosis of ipf is made on the basis of the chest radiograph, it is correct in 48 to 87% of cases (60,62,63). most patients with ipf will have an abnormal chest radiograph but, rarely, patients may present with a normal plain film (2). it is important to review all previous chest films to assess the rate of change in disease activity. in addition, radiographs are indicated if clinical deterioration occurs in order to identify superimposed infection or malignancy (2,64). pleural effusions, upper lobe predominant disease, airbronchograms, or prominent lymphadenopathy should suggest an alternative diagnosis. typical high-resolution chest computed tomography (hrct) features of ipf/uip include patchy, predominantly peripheral, subpleural, and symmetrical bibasilar honeycombing, reticular abnormalities, and limited "groundglass" opacities (ggo) ( [65] [66] [67] [68] . several studies have shown that experienced radiologists can make a "confident" diagnosis of uip with specificity greater than 95%, provided ct features are typical (65, [69] [70] [71] [72] . although a characteristic hrct is highly specific for ipf/uip, "typical" hrct identifies only 37 to 67% of patients with histological uip; therefore, a surgical lung biopsy (slb) is recommended when clinical and radiological information result in an uncertain diagnosis (14,65,70,73). one study evaluated the proficiency of physicians with expertise in interstitial lung diseases to identify accurately the hrct scans from patients with biopsy-proven ipf/uip. when these investigators made a "confident" diagnosis of ipf based on hrct scan and clinical data, they were right in more than 80% of the cases. however, more than half of the patients with ipf had an uncertain diagnosis on the basis of hrct and clinical assessment (70). in a subsequent analysis of these data, investigators identified hrct features associated with a pathological diagnosis of uip. on multivariate analysis, lower lobe honeycombing (or, 5.36), and upper-lung irregular lines (or, 6.28) were the only independent predictors of uip. when they combined those two factors, a diagnosis of uip was established with a sensitivity of 74%, a specificity of 81%, and a positive predictive value of 85% (65). interestingly, in that study, adenopathy was observed in 55% and 21% of patients with uip and without uip, respectively. this finding suggests that patients with ipf generate a marked lymphoproliferative response to an unknown antigen or antigens; we believe that this observation requires further investigation as it may provide important clues to further our understanding of the pathogenesis the disease. in a separate study, two radiologists independently assessed ct scans from a cohort of patients with either uip. ct features were "typical" for uip in only 37% patients, and all of them had histological uip on slb. typical ct features of uip are associated with advanced, latestage disease. among patients with earlier phases of uip, ct features may be atypical (74) or indeterminate (69). extensive ggo is not a major feature of uip, and suggests an alternative diagnosis such as desquamative interstitial pneumonia (dip), nsip, lymphocytic interstitial pneumonia (lip), cryptogenic organizing pneumonia (cop), hypersensitivity pneumonia (hp), or pulmonary alveolar proteinosis (pap) (75). in contrast, honeycomb change is a cardinal feature of uip, and is rare in other iips (71,76). pulmonary function tests (pfts) characteristically reveal a restrictive ventilatory defect with impaired gas exchange; however, smokers may have preserved lung volumes or airflow obstruction in the initial stages of the disease (2,77,78). impairments in gas exchange (i.e., carbon monoxide diffusing capasity [dl co ]) and oxygenation may be evident early in the course of the disease, even when spirometry and lung volumes are normal (79). the most appropriate and simple tests are vital capacity and dl co ; these are most useful for assessing the extent and monitoring the progression of the disease (80). cardiopulmonary exercise testing (cpet) demonstrates hypoxemia, widened a-a o 2 gradient, submaximal exercise endurance, reduced oxygen consumption (vo 2 ), high respiratory frequency, low tidal volume (v t ) breathing pattern, increased dead space (v d /v t ), increased minute ventilation for the level of vo 2 , and a low o 2 pulse (80-82). arterial desaturation and abnormal widening of a-a o 2 gradient with exercise may be elicited with relatively simple tests, such as the 6-min walk test (83,84). balf may play a role in the diagnosis of inorganic dust diseases, suspected malignancy, infections, some hematological disorders, drug-induced diseases, pulmonary alveolar proteinosis, langerhans' cell histiocytosis, and alveolar hemorrhage (85). balf is useful in research studies but of limited clinical application when evaluating iips (85). increases in polymorphonuclear leukocytes, eosinophils, mast cells, alveolar macrophages, and countless cytokines are noted in balf from patients with ipf/uip; lymphocyte numbers are usually normal (2). balf neutrophilia is present in 67 to 90% of patients with ipf/cfa (86,87), but does not predict prognosis or therapeutic responsiveness. elevations in balf eosinophils were associated with more severe clinical impairment (86,87), but balf eosinophil counts do not correlate consistently with prognosis (86,87). by contrast, the presence of balf lymphocytosis, found in fewer than 15% of cases, was associated with a greater responsiveness to corticosteroid therapy, a more cellular biopsy, and less honeycombing (86,87). data compiled from two studies documented favorable responses to corticosteroids in 12 of 13 patients exhibiting balf lymphocytosis, but in only 4 of 37 without lymphocytosis (86,87). because the studies citing balf lymphocytosis in steroid-responsive patients with ipf (87) or cfa (86) antedated the description of nsip, it is possible that balf lymphocytosis reflects disorders distinct from uip (such as cellular nsip). nevertheless, in a recent study, researchers hypothesized that balf findings may distinguish between uip and nsip; balf total and differential cell counts were not different between the two groups, and in neither group, were balf findings predictive of survival or changes in lung function (88). the american thoracic society (ats) and the european respiratory society (ers) in collaboration with american college of chest physicians (accp) published an international consensus statement on the diagnosis and treatment of ipf. this statement stated that the definite diagnosis of ipf requires an slb showing the uip pattern. however, an slb is not recommended in patients with suspected ipf in whom the clinical or radiographic information are stereotypical of ipf/uip. it has been suggested that, in the absence of an slb, the presence of all four major diagnostic criteria and at least three minor criteria increases the likelihood of an accurate diagnosis of ipf/uip (2). however, these criteria have not been prospectively validated. an slb, preferably by video-assisted thoracoscopy (vats), is recommended in patients with suspected ipf in whom the clinical or radiographic information are not typical of ipf/uip (2). given the patchy and heterogeneous nature of the uip lesion, a large piece of lung tissue is required and tbbx are used mainly to rule out other disorders that mimic ipf. however, emerging data suggest that tbbx findings may be more useful in diagnosing uip than previously recognized; characteristic histological features of uip (interstitial fibrosis with fibroblastic foci [ff] and/or hc change) can be appreciated even in a small sample obtained by tbbx; these findings in a patient with characteristic clinical and radiographic features may indicate a diagnosis of uip. however, these findings require prospective validation (89). significant advances have been made in our understanding of the idiopathic interstitial pneumonias (iips). the most important advancement has been the greater appreciation of the clinical relevance of the different histopathological subgroups that make up the iips. until recently, inflammatory or fibrotic lung disorders of unknown etiology were "lumped" under the term ipf (2,75,90). however, with the advent of vats lung biopsies and the greater availability and quality of surgical specimens, pathologists have recognized the heterogeneous nature of these disorders and described specific histopathological patterns that predict response to therapy and survival (74,91,92). ipf/uip is the most common of the iips, comprised of 47 to 71% of the cases (76,91,93,94). the uip lesion is characterized by temporal and geographic heterogeneity, with areas of old scar and hc change, admixed with granulation tissue and normal lung; the lesion has predilection for the subpleural and basilar regions of the lung, there is scant inflammation, and prominent aggregates of fibroblast and myofibroblasts, so-called "fibroblastic foci," which actively secreting extracellular matrix (75,92,95). additional features include smooth muscle hypertrophy, metaplasia and hyperplasia of type ii pneumocytes, destroyed and disrupted alveolar architecture, traction bronchiectasis and bronchioloectasis, and secondary pulmonary hypertension changes (75,92,95). other categories of iip that must be distinguished from ipf/uip include nsip, dip, respiratory bronchiolitis-associated interstitial lung disease (rbild), acute interstitial pneumonia (aip), cop, and lip (75). nsip is observed in approx 25% of patients with iip (75). this provisional category is used to describe a temporally homogeneous lesion with varying degrees of inflammation and fibrosis with favorable response to therapy and prognosis. nsip can be subdivided into nsip-cellular and nsip-fibrotic varieties depending on the degree of inflammation and fibrosis present in the surgical specimen (93). this subclassification provides important prognostic information; patients with idiopathic nsip, cellular pattern have a better 5-and 10-yr survival than those with idiopathic nsip, fibrosing pattern (100% vs 90% and 100% vs 35%, respectively) (93). in many patients, however, histological overlap between uip and nsip is evident. flaherty and co-workers reviewed slbs from 109 patients with iip who had multiple lobes biopsied and reported histopathological variability between lobes in 26% of patients. importantly, in that study, uip in at least one lobe defined prognosis (94). in a later study, the pathological findings in biopsy and subsequent explant specimens from 20 patients with uip were reviewed to refine histological criteria and to assess the relationship between uip and nsip. the important new finding was that nsip-like areas were present in the majority of uip patients (80%) in both biopsy and explants specimens, and in some, these areas were extensive, making accurate diagnosis of uip difficult (cases were misdiagnosed as nsip). the most useful feature for diagnosing uip in difficult cases is the presence of a distinct "patchwork" or variegated pattern of parenchymal involvement (95). rbild and dip comprise approx 15% of iips (75) . these entities are thought to be smoking-related diseases and, like nsip, tend to be responsive to anti-inflammatory therapy. this is not surprising, as pathologically, these lesions are characterized by varying degrees of intra-alveolar and/or peribronchial pigmented macrophage infiltration with scant or no fibrosis. aip is characterized by active fibrosis consisting of proliferating fibroblasts and myofibroblasts with minimal collagen deposition resembling the organizing stage of diffuse alveolar damage (dad) (92). finally within the iips, some include cop (previously termed bronchiolitis obliterans organizing pneumonia, or boop), and lip. both are relatively steroid-responsive lesions; pathologically, cop is characterized by the presence of intra-alveolar plugs of granulation tissue, and lip by lymphocytic infiltration of the alveolar walls. it should be noted, however, that many experts argue that lip should not be included within the iips because it is considered as lymphoproliferative disorder which, in turn, is rarely idiopathic and is mostly observed in association with infections (e.g., hiv) or collagen vascular diseases (cvds) (75). the natural history of the pathogenesis of ipf is not known. the description of temporal heterogeneity for the histopathological entity of uip would suggest that this process occurs over a significant period of time within the same low-power microscopic field of the lung. moreover, the description supports the notion that the lesions are not homogeneous for both the timing of the orginal injury and the subsequent response to the injury and repair. in addition, the histopathological entity of uip is not unique to ipf, and can be found in patients with connective tissue diseases, end-stage asbestosis, and end-stage hypersensitivity pneumonia. therefore, uip may represent an end-stage of a "process," not the beginning, intermediate, and end-stage of a disease. the only insight into the natural history of this process comes from two recent studies that suggest the potential concept of a continuum of iips that may overlap in time. in a study of 109 patients whom had multiple lobes biopsied, histological variability was evident in 26% of the patients. patients concordant for uip were older (63 ± 9 yr) than those discordant for uip (57 ± 12 yr) or with fibrotic nsip (56 ± 11 yr) or cellular nsip (50 ± 9 yr), suggesting that nsip may be an early lesion that progresses with time to uip (94). in a separate study, investigators found discordant histological diagnoses between lobes in 20% of the patients. nsip-like reactions were evident in 80% of patients with uip, suggesting that nsip may evolve into uip (95). in a genetic study, two different histopathological patterns of interstitial pneumonia were found to exist in members of a family who shared protein c gene mutations: adults with uip and children with nsip; this supports the notion that nsip may be a precursor lesion to uip (46). it has been suggested that most patients with ipf progress in a relentless and insidious manner with a median survival of less than 3 yr (91). this concept was challenged in a recent study of subcutaneous interferon (ifn)-γ1b (200 μg thrice weekly) in 330 patients with mild to moderate ipf (forced vital capacity [fvc] > 50% and dl co > 30%), where a trend toward lower mortality was seen in ifn-γ1b-treated patients compared with placebo-treated patients (96). interestingly, there were no significant differences in lung function or gas exchange between ifn-γ1b-treated and placebo-treated patients at 48 wk of follow-up. in both study groups, 70% of patients remained stable, 25% deteriorated, and the rest improved, suggesting that most patients with mild to moderate ipf remain stable for at least 1 yr on no specific therapy. the socalled "ipf exacerbations" may explain the trend toward lower mortality seen in this study, but this requires further study. ipf exacerbations may be defined as an accelerated deterioration of ipf in the absence of apparent infectious agents and heart failure (97,98). it is often a terminal event, with features of dad or organizing pneumonia on lung biopsy or autopsy (99,100). this syndrome is indistinguishable from idiopathic aip (101), and is similar to acute respiratory distress syndrome (ards). the factors responsible for this accelerated phase of ipf are unknown, but viral infections, high concentrations of oxygen, or drug reactions are plausible etiological factors (101). only one study has addressed mechanism of mortality of patients with ipf. panos and colleagues (102) found that most patients with ipf succumb to respiratory failure (39%), cardiovascular disease (27%), lung cancer (6-13%), pulmonary embolism (3%), infection (3%), or other health problems (18%) (102). although infection would appear unlikely as a cause of mortality in ipf patients, clinically we can only determine the micro-organism cause of community acquired pneumonia in 30% of all patients. therefore, we do not know the true incidence and prevalence of infection as a cause of mortality in patients with ipf, and perhaps a significant portion of the respiratory failure mortalities had infectious etiologies. with regard to cardiovascular disease, congestive heart failure and coronary artery disease (cad) account for 30% of deaths (102). patients with ipf appear to be at increased risk of developing cad. in a cross-sectional study of 630 patients referred for lung transplantation, fibrotic lung diseases were associated with an increased prevalence of cad compared with nonfibrotic diseases after adjustment for traditional risk factors (or 2.18; 95% ci, 1.17-4.06); the authors theorized that the fibroproliferative process may influence cells beyond the pulmonary compartment, and that mediator molecules produced in these disorders might promote atherogenesis (67). pulmonary arterial hypertension occurs in 70% of patients with advanced ipf and its presence correlates with a vital capacity (vc) below 50% of predicted or a dl co under 45% of predicted (102). left ventricular (lv) dysfunction occurs in less than 10% of patients and it is mostly (66%) a result of coexisting right heart failure. other causes of lv dysfunction include ischemic and hypertensive heart disease (5,102). six to 13% of patients with ipf develop bronchogenic carcinoma. lung cancer in patients with ipf typically presents as a peripheral squamous cell carcinoma in older male smokers (103). predisposing factors include squamous metaplasia, atypical epithelial cells, or occupational exposures (13,57,102) . hubbard and colleagues, in a population-based cohort in the united kingdom, studied 890 patients with ipf and 5884 controls. the risk ratio for ipf and lung cancer was 7.31 (95% ci 4.5-11.9). importantly, adjusting for previous smoking had little effect on this ratio, suggesting that ipf is an independent risk factor for lung cancer (104). pulmonary embolism occurs in approx 3 to 7% of patients; inactivity, heart failure, bronchogenic carcinoma, and possibly corticosteroid therapy predispose patients to thrombosis (102). pulmonary infection causes 2 to 4% of deaths in patients with ipf; immunosuppressive therapy, traction bronchiectasis, and possibly gerd are predisposing factors (102). pneumothorax occurs in up to 10% of patients with ipf and tends to be less responsive to tube thoracostomy, often necessitating surgical intervention (102). corticosteroids (cs) can cause a myriad of side effects including myopathy, peptic ulcer disease, cataracts, osteoporosis, compression fractures, fluid and electrolyte abnormalities, adrenal insufficiency, and infection (105,106) . in a cross-sectional study in patients with asthma, chronic obstructive pulmonary disease, or "alveolitis" taking oral corticosteroids (n = 367) vs controls (n = 734), the or for bone fractures was 1.8 (95% ci 1.3-2.6 [vertebral fracture or 10, hip fracture or 6, and ribs or sternum fracture or 3.2]). patients tak-ing corticosteroids experienced more cataracts, used more antacids, had more muscle weakness, back pain, bruising, and oral candidiasis, and had fewer teeth compared with controls (106). one prospective study included 41 patients with ipf; 24 received 100 mg/d of prednisone for 3 mo and 17 received 60 mg/d for 1 mo followed by 40 mg/d for 2 mo. patients were monitored monthly for steroid-related side effects. all patients experienced at least one side effect. common side effects included insomnia (76%), cushingoid change (73%), weight gain (71%), irritability (61%), infection (49%), blurred vision (41%), abdominal bloating (34%), glucose intolerance (24%), and fractures or avascular necrosis (10%) (105) . cytotoxic agents (cyclophosphamide [cp], azathioprine [aza]) can cause infections, bone marrow suppression, hepatitis, hemorrhagic cystitis (cp) and/ or malignancies (107,108) . in a prospective uncontrolled study, 19 patients with biopsy-proven uip who were unresponsive or intolerant to cs therapy were treated with oral cp (1-2 mg/kg/d) for 6 mo. nearly two-thirds of patients reported adverse effects, and 50% of patients discontinued therapy because of intolerable side effects. common side effects related to cp include nausea/ vomiting (26%), anorexia (26%), cytopenias (21%), weight loss (21%), alopecia (10%), infection (herpes zoster) (10%), and ovarian failure (5%) (108). several factors have been shown to predict poor outcome in ipf. these include older age at presentation, male gender, severe dyspnea at presentation, history of cigarette smoking, severe loss of lung function, severity of reticular opacities or honeycomb change on hrct, characteristic hrct appearance, lack of response to conventional therapy, and histopathological findings showing prominent ff (2,17,69,74,109) . investigators from the university of michigan evaluated the impact of histological diagnosis, baseline clinical, physiological, and radiographical factors on survival in 168 patients with suspected iip. the presence of histological uip was the most important risk factor for mortality (risk ratio [rr] of 28.46 [95% ci 5.5-148]), followed by the presence of honeycombing on hrct, a radiographic feature that was shown to be a good surrogate for histological uip (sensitivity of 90%, and a specificity of 86%) (14). the same group evaluated the impact of hrct appearance on survival in patients with iip. patients with histological uip and stereotypical hrct appearance of uip had a shorter survival (median survival 2.08 yr) when compared with patients with histological uip and indeterminate hrct scans (median survival 5.76 yr) (69). three studies have shown that a higher hrct-fibrosis score identify patients with worse prognosis. gay and colleagues did not find any measure of pulmo-nary function to be predictive of survival, but did find both the hrct-fibrosis score and the pathological fibrosis score to be useful in predicting survival (109) . similarly, investigators from the united kingdom found that baseline percent-predicted dl co and hrct-fibrosis score were independent predictors of mortality (110) . japanese investigators demonstrated that the baseline hc score and the rate of hc progression were both predictive of worse survival in patients with ipf (111). histopathological findings showing prominent ff identify patients with poor outcome. nicholson and associates retrospectively studied (53) patients with ipf/uip and analyzed the prognostic significance of four specific microscopic features of uip. multivariate analysis revealed that increasing ff and mononuclear cell infiltrate scores were associated with worsening lung function. higher profusion of ff and a lower dl co were independent predictors of mortality (112). these results supported the findings by king and co-workers, who also demonstrated that an increase in the number of ff correlated highly with mortality in patients with ipf/uip (13). in a separate study, expert pathologists reviewed slb from 108 patients with idiopathic or cvd-associated uip and assigned a score for ff. patients with idiopathic uip had more ff and worse survival compared with patients with cvd-associated uip (113). a number of composite scoring systems have been developed with which to predict survival in ipf. king and co-workers studied 238 patients with ipf/ uip and derived a clinical-radiological-physiological (crp) scoring system using clinical (age, smoking status, clubbing), radiographical (extent of interstitial opacities, presence of pulmonary hypertension on chest radiographs), and physiological parameters (reduced lung volume, abnormal gas exchange during maximal exercise). these investigators demonstrated that the crp score correlated with important histopathological findings and was helpful in predicting survival in patients with ipf. a second abbreviated crp scoring system that excluded pa o2 during maximal exercise was inferior in predicting survival (12,114) . similarly, investigators from the brompton hospital devised a composite physiological index (cpi) using radiographical and physiological information that predicted mortality more accurately than individual pft in patients with ipf (115). even though these composite scoring systems are accurate in their predictive ability, they are expensive and cumbersome to generate in clinical practice. with this in mind, the prognostic value of oxygen desaturation during a 6-min walking test was evaluated in patients with ipf. desaturation defined as a fall in oxygen saturation to 88% or less during the 6min walk test identified patients with higher mortality compared with patients who did not desaturate. the 4-yr survival rate of ipf patients who desaturated to that level was 34.5% compared with 69.1% in patients who did not desaturate (84). predicting survival in ipf has been centered on baseline radiographical, pathological, and/or physiological testing. recently, researchers have focused on the association of serial changes in pulmonary function or radiographical features and prognosis. one study determined that a decrease in fvc (>10% from baseline) during the initial 6 mo of follow-up was associated with increased mortality (hazard ratio 2.06; ci 1.09-3.89) (116). in a separate study, investigators concluded that at 6 and 12 mo of follow-up, serial pulmonary function trends (change in dl co , fvc, forced expiratory volume in 1 s [fev 1 ], and the cpi) provided important prognostic information in ipf (117). a third study showed that assessment of changes in clinical and physiological variables (dyspnea score, total lung capacity, thoracic gas volume, fvc, fev 1 , dl co , p o2 , oxygen saturation, and alveolar-arterial oxygen gradient) at 6 and 12 mo provide clinicians with more accurate prognostic information than baseline values alone (118). serum markers and nuclear medicine testing may have a predictive role in ipf. greene in a prospective study, investigators analyzed the usefulness of inhaled 99mlabeled diethylenetriamine penta-acetic acid ([99m] tc-dtpa) aerosol clearance and survival in a cohort of 106 patients with uip. multiple stepwise cox regression analysis identified fast clearance as an independent predictor of mortality (120). conventional therapy (cs, aza, or cp) for ipf provides only marginal benefit. unfortunately, in many studies, diagnoses were not based on the findings of lung biopsies or were not classified by current pathological criteria; thus, there is uncertainty as to the nature of the disease being treated. two recent meta-analyses searched two large databases for randomized controlled trials (rct) and controlled clinical trials (cct) using cs or non-cs agents in patients with histological uip or who fulfilled all ats criteria for ipf; the authors could not find rcts or ccts evaluating cs alone in ipf and concluded that there are scant good-quality data regarding the efficacy of non-cs agents in ipf (121,122) . the following is a brief discussion of anti-inflammatory (conventional) therapy in the treatment of ipf. cs were the mainstay of therapy for more than four decades, but are of unproven efficacy, and are associated with significant toxicities (105,123,124) . early studies of patients with ipf/cfa cited response rates of 10 to 30% with cs (alone or combined with immunosuppressive agents), but complete or sustained remissions were rare (105,125-127 ). more importantly, many responders likely had iips other than uip (e.g., nsip or rbild/dip). in recent studies, response rates to cs among patients with histological evidence for uip are low (0-17%) (16,57,74,76,123) . large retrospective studies of patients with ipf showed no survival benefit with cs (12; 15,123,128) . in one retrospective study from england, survival was worse among ipf patients treated with cs or cp, although this likely reflects a selection bias (15). given the potential severe toxicities associated with cs (105,124), recent international consensus statements argue that high-dose cs should not be used to treat ipf (2,61) . however, because anecdotal responses to cs are occasionally noted in patients with ipf/uip (14), these statements acknowledge that selected patients with clinical or physiological impairment or worsening pfts should be treated (2,61). both statements (2,61) advocate an individualized approach to treating ipf/uip. among patients requiring treatment, both statements recommend combining therapy with either oral aza or cp plus low-dose prednisone or prednisolone (0.5 mg/kg [lean body weight per d] for 4 wk, then 0.25 mg/kg for 8 wk, then 0.125 mg/kg). this represents a substantial departure from earlier regimens advocating high-dose prednisone (e.g., ≥1 mg/kg/d for ≥6-12 wk) (114,125,126) . combined therapy should be continued for 6 mo in the absence of adverse effects. treatment should be continued beyond 6 or 12 mo or later time points only if patients improve or remain stable. it should be emphasized that these recommendations (2,61) reflect expert opinion, but have not been validated in clinical trials. we believe cs should not be given to patients at high risk for adverse effects (e.g., age > 70 yr, osteoporosis, dm, extreme obesity, and so on). two prospective studies evaluated aza for ipf (125,126) . in both studies, aza was combined with prednisone. in the first study, 20 patients with progressive ipf were initially treated with prednisone alone for 3 mo (125). at that point, aza (3 mg/kg/d) was added and both agents were continued for an additional 9 mo or longer. twelve patients (60%) responded. the independent effect of aza was difficult to assess, because all patients received prednisone concomitantly. in a second, double-blind trial, raghu and associates compared the effect of aza plus prednisone on lung function with that of prednisone alone in previously untreated patients with ipf (the study population may have included patients with iip other than uip). forty-three percent of patients randomly assigned to aza plus prednisone died during the 9-yr follow-up period, compared with 77% of patients randomly assigned to prednisone alone. the difference became statistically significant only after adjustment for age (p = 0.02) (126). two randomized trials evaluated cp for ipf (127,129) . in one 6-mo trial, 28 patients with "mid-course" ipf were randomized to prednisone alone (n = 16); prednisone plus oral cp (1.5 mg/kg/d) (n = 9); or cp alone (n = 5) (129) . mean balf neutrophil counts declined in the cohort receiving cp, but pfts did not change in any group. johnson and colleagues compared the effect of prednisolone alone with that of prednisolone plus cp on breathlessness, radiographic appearance, and lung function in patients with ipf (the study population included patients with cvd and with iip other than uip). initial improvement occurred in 7 of the 22 patients in the prednisolone-only group and in 5 of the 21 patients in the cp plus prednisolone group. however, at 36 mo, only 2 of the 22 patients in the prednisolone-only group remained improved, and only 1 of the 21 patients in the cp-prednisolone group remained improved. life-table analysis suggested better survival in patients in the cp-prednisolone group, but this was not statistically significant (127). in a prospective uncontrolled study, zisman and associates studied the efficacy of cp in 19 patients with biopsyproven uip who were unresponsive or intolerant to cs therapy. only 1 patient improved; 7 remained stable, and 11 deteriorated. nearly two-thirds of the patients developed drug-related side effects and one half of the patients discontinued therapy due to intolerable side effects (108). intermittent, intravenous "pulse" cp, administered every 2 to 4 wk, has been tried for ipf refractory to cs in nonrandomized studies, but benefit was not convincing (130-132). lung transplantation should be considered for patients with ipf refractory to medical therapy (133,134) . two-year survival following single lung transplant (slt) ranges from 60 to 80%; 5-yr survival is 40 to 60% (134-136) . in one study, lung transplantation reduced the risk of death by 75% (135) . in addition, patients surviving lung transplantation appear to achieve considerable improvement in most dimensions of health-related quality of life (137) (138) (139) . unfortunately, owing to a shortage of donor organs, waiting time may be prolonged (up to 2-3 yr) and many patients with ipf die while awaiting transplantation (134,135) . one study evaluated baseline pft and hrct fibrosis scores and the relationship to 2-yr survival in patients with ipf younger than 65 yr of age; the optimal points on the receiving operator characteristics (roc) curves for discriminating between survivors and nonsurvivors corresponded to a combination of dl co of 39% predicted with hrct-fibrosis score of 2.25 (110) . in a separate study, investigators reviewed all transplant referrals for iip that were listed for lung transplantation at their center. the aim of the study was to determine a parameter that would discriminate between patients who survived and patients who died awaiting transplantation. the severity of hypoxemia at rest was the only significant difference between both groups (140). unless contraindications exist, patients with severe functional impairment (e.g., fvc <60% predicted, dl co <40% predicted), oxygen dependency, and a deteriorating course refractory to medical therapy should be listed promptly for transplantation (133,134). historically, the fibrotic process in ipf has been thought to be preceded by a chronic inflammatory process that injures the lung and modulates fibrogenesis (141). conventional management of ipf has been primarily based on the notion that suppressing inflammation may prevent progression to fibrosis. evidence against the notion that inflammation plays an important role in the pathogenesis of ipf comes from the lack of correlation of most markers of inflammation with disease stage or outcome, and the recognition that inflammation is not a prominent histopathological finding in uip (92,141) . additionally, emerging evidence suggests that inflammation is not required for the development of a fibrotic response (141). in light of the poor prognosis and lack of response to available anti-inflammatory therapy, alternative approaches to the treatment of ipf are being pursued. the following is a brief discussion of antifibrotic therapy and other promising agents in the treatment of ipf. colchicine is an alkaloid derivative of the plant colchicum autumnale, which has been used in acute attacks of gout. it is known to bind microtubular proteins necessary for intracellular trafficking and cellular mitosis, thus adversely affecting secretion of proteins from cells and cellular proliferation (142,143) . its antifibrotic activity was described following the discovery that colchicine inhibits secretion of collagen and other important growth factors necessary for fibroblast proliferation (144). however, further studies (145) with or without additional therapeutic agents, such as steroids, failed to document efficacy of colchicine in the treatment of human pulmonary fibrosis. it is thus not currently recommended for use in therapy of ipf. the d-isomer of penicillamine has been extensively studied in animal models of fibrosis, in which it has been shown to prevent accumulation of collagen in the lung by interrupting cross-linking of collagen molecules (146). this observation has led to its use in treating fibrotic lung disease associated with systemic sclerosis with good results (147). however, its efficacy in the treatment of ipf has been disappointing (148), and it is known to have toxic and significant adverse effects (2). thus it is currently not recommended as therapy for ipf. pirfenidone is a novel agent with broad-spectrum antifibrotic activity. numerous in vitro and animal model studies have demonstrated its effectiveness as an antifibrotic agent. in vitro studies have shown that pirfenidone significantly reduced mrna levels of type i and type iii collagen, and may act at the transcriptional or translational level of collagen synthesis (149). in vivo, it has been shown to inhibit tgf-β1-induced collagen synthesis, decrease extracellular matrix deposition, and suppress the overexpression of tgf-β in the bleomycin model of pulmonary fibrosis (150,151) . because ipf is becoming increasingly recognized as primarily a fibrotic process, the potential role of pirfenidone as a therapeutic agent is being explored. in a prospective open-label study, raghu and colleagues (152) treated 54 (42 biopsy-proven) consecutive patients with pirfenidone who were either unwilling to receive or unresponsive to conventional therapy. survival rates of 78% at 1 yr and 63% at 2 yr compared favorably with historical controls. in addition, 83% of patients discontinued prednisone therapy and the remaining 17% were able to reduce their daily dose. all patients treated with immunosuppressive therapy tolerated discontinuation of the drug. interestingly, patients whose lung function had deteriorated before enrollment appeared to stabilize after beginning pirfenidone. side effects were relatively common, with patients reporting nausea (44%), fatigue (44%), and photosensitivity (24%). despite these encouraging observations, the results of this study are difficult to interpret owing to the lack of appropriate controls, incomplete pre-entry and follow-up pulmonary function test data, a small study population, and a bias associated with survivorship effect (pulmonary function data of patients who died were not included in the analysis, and this could have biased the results). furthermore, the observed steroid-and immunosuppressive-sparing effects of pirfenidone may have simply reflected lack of efficacy of conventional therapy rather than a true effect of pirfenidone. in a second open-label trial, japanese investigators evaluated oral pirfenidone in eight patients with ipf and two with diffuse lung disease associated with systemic sclerosis; after 1 yr of therapy, there was no change in chest radiographic scores and arterial oxygen tension; the drug was well tolerated (153). early treatment with pirfenidone appears to slow the progression of pulmonary fibrosis in patients with hermansky-pudlak syndrome (154). a randomized-controlled trial focusing on early treatment is warranted to test the efficacy and safety of this agent in ipf. ifns play an integral role in the regulation of fibroblast proliferation and collagen synthesis, but the mechanism by which they exert their effect is not clearly understood. recent observations have shown that ifn-γ has antiproliferative, immunomodulatory, and antifibrotic effects (155), and thus may play a crucial role in the pathogenesis of ipf. ifn-γ decreases collagen content in the bleomycin model of lung fibrosis by inhibiting tgf-β transcription and subsequent procollagen mrna production (156). in addition, ifn-γ inhibits fibroblast proliferation in cultures derived from normal and fibrotic human lung, making an argument that ifn-γ may have therapeutic applications (157). lower levels of ifn-γ have been found in patients with ipf compared with patients with less fibrotic diseases such as pulmonary sarcoidosis (158). kuroki and associates (159) measured levels of type iii collagen in patients with progressive pulmonary fibrosis and found an inverse correlation with ifn-γ levels, particularly in patients with ipf. these studies suggest that patients with ipf may have a defect in ifn-γ production or function, which predisposes them to develop fibrosis following injury. however, the potential for developing fibrosis is not likely to be dependent on one factor; rather it is likely the result of a complex interplay of fibrotic mediators, differential gene expression, and feedback mechanisms. a study by shaw and colleagues (160) showed that alveolar macrophages from patients with interstitial lung disease had increased production of platelet-derived growth factor, which is a potent mitogen for fibroblasts. this increase in platelet-derived growth factor was upregulated following treatment with ifn-γ, suggesting that ifn-γ may act to potentiate fibrosis in certain cellular environments. clearly, there is a complex regulatory mechanism in place with regard to whether fibrosis occurs or not, and conflicting in vitro studies must be interpreted with caution. a randomized, prospectively controlled trial was conducted in 18 patients with ipf comparing ifn-γ1b and low-dose prednisolone with prednisolone alone for 12 mo (161) . the results were remarkable in that patients with progressive pulmonary fibrosis treated with ifn-γ1b plus lowdose prednisolone demonstrated improvement in pulmonary function, whereas those who received prednisolone alone experienced further decline in pulmo-nary function. the authors showed that all patients treated had almost undetectable levels of ifn-γ mrna, and increased levels of both tgf-β and connective tissue growth factor mrna in lung tissue. furthermore, after treatment with ifn-γ1b, transcription of tgf-β and connective tissue growth factor were both significantly decreased. several concerns have been raised regarding the findings reported by ziesche and co-workers (162), particularly the unexpectedly good results with ifn-γ1b. to address some of the issues raised, an outside panel of experts reanalyzed the study data by reviewing each patient's lung function studies, ct scans, and slbs to assess the clinical course and diagnosis of ipf according to the international consensus statement (2,163) . fifteen of 18 patients had either definite (n = 9) or probable (n = 6) ipf. the panel reanalyzed treatment response using published criteria and eliminated the patients who definitely did not have ipf. patients treated with ifn-γ1b plus low-dose prednisolone demonstrated either stability or improvement in pulmonary function and gas exchange after 1 yr of treatment, whereas treatment with prednisolone alone was associated with no improvement in all patients (163) . the observed benefit of ifn-γ1b on lung function has not been reproduced in subsequent studies. in one retrospective uncontrolled observation of 21 patients with ipf treated with ifn-γ1b, only one patient experienced objective improvement, 7 discontinued therapy (owing to lack of perceived benefit), and 11 died after 6 mo of therapy (164). in a separate study of five patients with ipf treated with ifn-γ1b, only one patient improved, two discontinued treatment owing to adverse effects and decline in lung function, and one died after 3 mo of therapy (165). in a recent prospective, randomized, placebo-controlled, double-blind, multicenter phase iii clinical trial of subcutaneous ifn-γ1b (200 μg thrice weekly) in 330 patients with mild to moderate idiopathic pulmonary fibrosis (fvc >50% and dl co >30%), a trend toward lower mortality was seen in ifn-γ1b-treated patients compared with placebo-treated patients. however, there were no significant differences in lung function or gas exchange between ifn-γ1-treated and placebo-treated patients after 48 wk of therapy (96). a prospective controlled multinational trial is planned to verify the possible survival benefit observed with ifn-γ1b therapy in ipf. ifn-β is used for the treatment of chronic hepatitis c and multiple sclerosis. in vitro ifn-β1a has been shown to reduce fibroblast proliferation (166), inhibit collagen production by fibroblasts (167), increase collagenase mrna (168), decrease pro-collagen mrna (169), and increase collagenase activity (167). a multicenter randomized, double-blind clinical trial examining the efficacy of ifnβ-1a was recently completed. patients were randomized into four groups: placebo or ifnβ-1a at 15, 30, or 60 μg intramuscularly twice per week for a minimum of 12 mo and up to 2.5 yr. preliminary results suggest that ifnβ-1a lacks significant efficacy (171). with a dismal response to existing therapy and its accompanied toxicity, the search for additional therapies has intensified in the past decade. the following is an overview of other therapeutic approaches, most of which are undergoing investigation and have not been adequately studied in humans. the prior discussion has centered on fibroblast proliferation and collagen deposition as two areas of therapeutic intervention. it is becoming more apparent that the fibrotic process has multiple pathogenetic mechanisms. one of the fundamental hypotheses of pulmonary fibrosis involves an imbalanced response to injury, in which the capacity of the alveolar epithelium to repair itself is compromised, ultimately leading to fibrosis. some investigators propose that the alveolar epithelium itself has antifibrotic properties, and that chronic loss of alveolar epithelium leads to an environment conducive to the development of fibrosis (172). support for this notion exists in the fact that induction of apoptosis of alveolar epithelium has been shown to occur following administration of bleomycin (173). therapies that either inhibit epithelial injury or enhance repair may limit the fibrotic response. in this regard, captopril, an angiotensin-converting enzyme inhibitor widely used in clinical practice, may have a role in the treatment of ipf. in vitro, captopril inhibits fibroblast proliferation, and in models of bleomycin-induced pulmonary fibrosis, it has been shown to reduce alveolar epithelial cell apoptosis and fibroproliferation. in addition, captopril abrogates fas-induced apoptosis in human alveolar epithelial cells (141,174) . there is currently an ongoing clinical trial at the national institute of respiratory diseases in mexico testing the efficacy of captopril in patients with ipf (141). another agent that may protect the alveolar epithelium is keratinocyte growth factor (kgf). this class of growth factor stimulates type ii cell proliferation with no direct effects on fibroblasts (175). keratinocyte growth factor increases surfactant protein gene expression and sodium/potassium adenosine triphosphatase, factors that may protect the alveolar epithelium (175). in vivo, kgf has been shown to protect animals from injury and subsequent development of fibrosis caused by a variety of insults (175). there is evidence that an exaggerated oxidant stress may play a role in the pathogenesis of pulmonary fibrosis by injuring the alveolar epithelial cells. this oxidant burden is thought to be a consequence of both increased levels of reactive oxygen species and a defective antioxidant response. a major protector of oxidant-induced injury of the alveolar epithelium is glutathione, which has been shown to be deficient in the balf of patients with ipf (176). moreover, in vitro studies have shown that n-acetylcysteine (nac), a precursor for glutathione synthesis, may augment the antioxidant defense system and protect the alveolar epithelium from free radical-induced injury (177). in vivo, hagiwara and associates (178) reported a significant inhibition of bleomycininduced lung fibrosis in mice following aerosolized nac during the early inflammatory phase of injury. whether this effect was secondary to nac inhibition of cellular inflammation, or its role as a scavenger of reactive oxygen species, is not clear. german investigators evaluated oral nac as a strategy to augment lung glutathione levels in 17 patients with biopsy-proven ipf. following therapy with nac, glutathione levels in balf were significantly increased compared with pretreatment levels (177). in a separate study, behr and colleagues (179) prospectively studied 18 patients with ipf and assessed the redox balance of the lung and changes in lung function following high-dose nac therapy for 12 wk. they reported an increase in the total and reduced form of glutathione concentration in the balf, and significant improvement in pulmonary function. the authors suggest that nac may be considered as an adjunct in the treatment of ipf. currently, there is a clinical trial in europe to evaluate the potential benefits of nac in ipf. as mechanisms of fibrosis at the cellular and molecular level become elucidated, their application to the development of novel therapeutic strategies appears promising. given the temporal heterogeneity of the uip lesion, early histopathological abnormalities may be present even in patients with advanced ipf. if early cytokine release is relevant to the initiation of this pathogenic response, then the targeting of early cytokines such as tnf-α should be considered. tnf-α appears to be upregulated soon after bleomycin-induced injury and has been implicated in a variety of inflammatory processes (180). sime and colleagues (181) showed that transient overexpression of tnf-α in rat lung led to fibrosis associated with concomitant tgf-β expression and proliferation of myofibroblasts (181). furthermore, upregulation of tnf-α expression has been shown to occur in inbred murine strains that are sensitive to bleomycin-induced lung fibrosis, with similar expression being absent in resistant strains (180). in addition, ortiz and colleagues (182) showed that tnf receptor-deficient mice did not develop pulmonary fibrosis following exposure to bleomycin despite increased tnf expression. studies of human lung biopsy specimens of patients with ipf have shown an upregulation of tnf-α mrna and protein (183). these observations along with other studies in animal models demonstrating abrogation of pulmonary fibrosis following treatment with soluble tnf receptors suggest that tnf-α may play an important role in the pathogenesis of pulmonary fibrosis (184). several agents that can block tnf-α are now available for human use (185). there is currently an ongoing clinical trial evaluating the safety and efficacy of etanercept, a tnf-α receptor antagonist, in patients with ipf. the expression of tnf-α is inhibited by certain cytokines such as il-10, which is produced by a variety of cells including t-helper (th) cells, monocytes, and alveolar macrophages (186, 187) . it is conceivable that il-10 may be useful in blunting the action of increased tnf-α observed following bleomycin-induced lung injury, therefore possibly inhibiting progression to fibrosis. arai and colleagues (188) investigated the possible inhibitory effects of il-10 by introducing the il-10 gene into mice exposed to bleomycin and found that bleomycin-induced pulmonary fibrosis was suppressed. these results suggest that treatment with il-10 during the early inflammatory phases of lung injury may be promising and requires further investigation. concerns regarding the role of this agent in treating pulmonary fibrosis exist in that il-10 is a type-2 th (th2) cytokine that could suppress ifn-γ expression and promote fibrogenesis (189). a clinical trial to study the effect of this cytokine is now underway in the united states. the realization that th cell subsets could be categorized on the basis of cytokine profiles has helped clarify our understanding of chronic cell-mediated immune responses. the type-1 (th1) cytokines include ifn-γ, il-2, il-12, il-18, and th2 cytokines include il-4, il-5, il-10, and il-13. analysis of subset populations of th cells within the interstitium of patients with ipf reveal a predominantly th2-type pattern of cytokine production, suggesting that alterations in t-cell subpopulations of th1 and th2 cells and their associated pattern of cytokine production may contribute to progression of ipf (190) . supporting evidence comes from studies demonstrating that ifn-γ (a th1 cytokine) has profound antifibrotic effects in ipf possibly because it shifts the balance away from a th2-dependent profibrotic environment. thus, it seems reasonable to target therapy to correct the th imbalance by either favoring a th1 phenotype or abrogating the predominant th2 response (e.g., administration of il-12 to promote ifn-γ expression, or inhibition of il-4, il-13, and so on). tgf-β is a critical cytokine for the promotion of fibrosis. in bleomycininduced pulmonary fibrosis, passive immunization with neutralizing antibod-ies against tgf-β reduces collagen deposition (191) . in addition, the overexpression of tgf-β results in a fibrogenic response resembling uip (i.e., abundant ff) (192) . in patients with ipf, increased expression of tgf-β is localized to bronchiolar epithelial cells, epithelial cells of hc cysts, and hyperplastic type ii pneumocytes (193) . it is possible that therapy with neutralizing antibodies against tgf-β1, or utilization of a tgf-β1 inhibitor such as decorin, may become useful in the treatment of ipf (194) . tgf-β signals through a receptor that activates transcription factors smad2 and smad3 promoting tgf-β gene transcription. interestingly, ifn-γ inhibits the activation of smad3 and induces the expression of smad7, an antagonistic molecule that inhibits tgf-β expression. smad7 can be produced in the laboratory and may become a useful molecule in the treatment of ipf (195) . monocyte chemoattractant protein (mcp)-1 is a member of the c-c subfamily of chemokines involved in monocyte/macrophage mediated inflammation (196, 197) . in addition, mcp-1 has been shown to stimulate pulmonary fibroblasts, tgf-β synthesis, and collagen production (198) . analysis of serum, balf, and lung biopsy specimens from patients with ipf reveal increased levels of this chemokine (196, 197, 199) . furthermore, serum mcp-1 levels correlate with clinical course in patients with interstitial lung disease (199) . further investigation into the clinical significance of mcp-1 and its contribution into the pathogenesis of ipf are necessary, and may provide the groundwork for novel therapies. another potential mediator of fibrosis produced in a variety of cells in the lung is the vasoactive peptide endothelin (et) -1 (200,201) . first thought to be primarily a vasoactive agent, et-1 has been shown to stimulate fibroblast proliferation, activate monocytes, induce collagen production, and regulate cytokine production (202). mutsaers and colleagues (203) revealed that et-1 levels are augmented following administration of bleomycin with increased localization of the agent in areas of fibrosis. with increases in et-1 synthesis following tnf-α and tgf-β stimulation, one may speculate that et-1 may play an important role in the cascade of events leading to pulmonary fibrosis (204, 205) . additional support for its role in pulmonary fibrosis comes from studies in animals in which fibrosis was attenuated following treatment with bosentan, an et receptor antagonist (206). et-1 has also been associated with pulmonary fibrosis in humans. in a study examining the expression of et-1 in patients with interstitial lung fibrosis, giaid and associates (200) found a striking expression of et-1 in lung tissue that correlated with parameters of disease activity in patients with ipf. there is currently an ongoing clinical trial evaluating the safety and efficacy of bosentan in patients with ipf. some have hypothesized that inducing fibroblast apoptosis may curb progression of fibrosis. lovastatin is a pharmacological agent widely used in the treatment of hypercholesterolemia that inhibits 3-hydroxy-3-methylglutarylcoenzyme a, therefore affecting many cellular functions essential for normal cell homeostasis including proliferation and cell survival. tan and associates showed that clinically achievable concentrations of lovastatin induced apoptosis of human lung fibroblasts in vitro, and in vivo reduced granulation tissue formation and induced fibroblast apoptosis in a guinea pig wound model of fibroproliferation (207) . with its known safety profile and potential antifibrotic effect, lovastatin is an attractive candidate in the treatment of ipf. suramin is a sulfonated napthylurea that has been used to treat onchocerciasis, acquired immunodeficiency virus, and prostate cancer. in vitro, suramin antagonizes the effects of a number of growth factors that promote fibrogenesis such as tgf-β, insulin-like growth factor-1, platelet-derived growth factor, epidermal-like growth factor, and fibroblast growth factor. in vivo, suramin has been shown to delay wound healing (175). relaxin is a protein secreted by the gravid uterus responsible for remodeling of the interpubic ligament and cervix during the later phases of pregnancy. relaxin inhibits the tgf-β-mediated overexpression of extracellular matrix, stimulates the expression of collagenases by lung fibroblasts in vitro, and has been shown to block bleomycin-induced fibrosis in mice (141). the eicosanoids are potential candidates for therapeutic intervention. the prostaglandin pge 2 is a potent inhibitor of fibroblast proliferation and extracellular matrix deposition and may ameliorate the fibrotic process in ipf (175). indomethacin, an inhibitor of cyclo-oxygenase, has been shown to decrease bleomycin-induced pulmonary fibrosis in an animal model but to our knowledge, it has not been evaluated in human ipf (175). the profibrotic leukotriene b4 has been shown to be increased in balf and lung tissue of patients with ipf (175). inhibition of leukotriene production may be an effective adjuvant therapy, and drugs are now available to block leukotriene synthesis. gene-specific antisense therapy against proteins known to be important in human lung fibroblast proliferation may become an effective approach in treating ipf patients. in vitro, chen and associates showed that gene-specific oligonucleotides (oligos) against c-ki-ras substantially inhibited the proliferation of human fibroblasts (141). beractan is a natural bovine lung extract containing phospholipids, neutral lipids, fatty acids, and surfactant-associated proteins. in vitro, beractan pro-voked fibroblast apoptosis, induced collagenase-1 expression, and decreased type i collagen (141). pulmonary fibrosis can be complicated by pulmonary hypertension limiting exercise tolerance and survival. german investigators performed a randomized-controlled, open-label trial in 16 individuals with pulmonary hypertension secondary to pulmonary fibrosis. they compared oral sildenafil with inhaled nitric oxide and infused epoprostenol. a single dose of sildenafil reduced pulmonary vascular resistance by nearly one-third and increased the mean arterial blood oxygen tension by 14 mmhg. the drug was well tolerated with no adverse effects on ventilation-perfusion matching (208). a clinical trial evaluating sildenafil in patients with ipf and pulmonary hypertension will be conducted soon. in recent years, significant advances have been made in our understanding and management of ipf. however, in order to further our knowledge and make significant progress in the care of these patients, it is critical that we improve our understanding of the natural history and pathogenesis of ipf. in addition, we need to pursue novel imaging and diagnostic technologies to improve earlier diagnosis and we must also educate primary care physicians and pulmonologists to refer patients early to an interstitial lung disease specialist or lung transplant center. therapeutic strategies must target specific aberrant pathways during the natural history of the pathogenesis of pulmonary fibrosis. only when these issues are in place will we be able to improve the prognosis of disorders associated with progressive pulmonary fibrosis. mortality rates from cryptogenic fibrosing alveolitis in seven countries idiopathic pulmonary fibrosis: diagnosis and treatment. international consensus statement the epidemiology of interstitial lung diseases idiopathic pulmonary fibrosis. epidemiologic approaches to occupational exposure idiopathic pulmonary fibrosis what causes cryptogenic fibrosing alveolitis? a case-control study of environmental exposure to dust pulmonary fibrosis deaths in the united states, 1979-1991. an analysis of multiple-cause mortality data rising mortality from cryptogenic fibrosing alveolitis cigarette smoking: a risk factor for idiopathic pulmonary fibrosis occupational exposure to metal or wood dust and aetiology of cryptogenic fibrosing alveolitis diabetes mellitus may increase risk for idiopathic pulmonary fibrosis predicting survival in idiopathic pulmonary fibrosis: scoring system and survival model idiopathic pulmonary fibrosis: relationship between histopathologic features and mortality clinical significance of histological classification of idiopathic interstitial pneumonia survival in patients with cryptogenic fibrosing alveolitis: a population-based cohort study the prognostic significance of the histologic pattern of interstitial pneumonia in patients presenting with the clinical entity of cryptogenic fibrosing alveolitis determinants of survival in idiopathic pulmonary fibrosis cigarette smoke inhibits lung fibroblast proliferation and chemotaxis the role of gastroesophageal reflux in idiopathic pulmonary fibrosis increased prevalence of gastroesophageal reflux in patients with idiopathic pulmonary fibrosis exposure to commonly prescribed drugs and the etiology of cryptogenic fibrosing alveolitis: a case-control study occupational and environmental risk factors for idiopathic pulmonary fibrosis: a multicenter casecontrol study. collaborating centers cryptogenic fibrosing alveolitis and epstein-barr virus: an association? epstein-barr virus replication within pulmonary epithelial cells in cryptogenic fibrosing alveolitis the detection of epstein-barr virus dna in lung tissue from patients with idiopathic pulmonary fibrosis a rearranged form of epstein-barr virus dna is associated with idiopathic pulmonary fibrosis herpesvirus dna is consistently detected in lungs of patients with idiopathic pulmonary fibrosis idiopathic pulmonary fibrosis and hepatitis c virus infection incidence of hepatitis c virus infection in italian patients with idiopathic pulmonary fibrosis idiopathic pulmonary fibrosis and high prevalence of serum antibodies to hepatitis c virus detection of adenovirus e1a dna in pulmonary fibrosis using nested polymerase chain reaction usual interstitial pneumonia following texas a2 influenza infection clinical course, management, and long-term sequelae of respiratory failure due to influenza viral pneumonia studies on influenza in the pandemic of 1957-1958. ii. pulmonary complications of influenza sequential virus infections, bacterial superinfections, and fibrogenesis giant cell pneumonia caused by parainfluenza type 3 in a patient with acute myelomonocytic leukemia three sensitive methods for the detection of cytomegalovirus in lung tissue of patients with interstitial pneumonitis computed tomography of the lungs in acquired immunodeficiency syndrome. an early indicator of interstitial pneumonia isolation of measles virus in primary rhesus monkey cells from a child with acute interstitial pneumonia who cytologically had giant-cell pneumonia without a rash surface and transmission ultrastructural characteristics of desquamative interstitial pneumonitis influence of human t lymphotrophic virus type i on cryptogenic fibrosing alveolitis-htlv-i associated fibrosing alveolitis: proposal of a new clinical entity adult familial cryptogenic fibrosing alveolitis in the united kingdom alpha 1-antitrypsin phenotypes in fibrosing alveolitis and rheumatoid arthritis genetic studies in familial fibrosing alveolitis. possible linkage with immunoglobulin allotypes (gm) al-pha1-antitrypsin phenotypes in patients with cryptogenic fibrosing alveolitis: a case-control study heterozygosity for a surfactant protein c gene mutation associated with usual interstitial pneumonitis and cellular nonspecific interstitial pneumonitis in one kindred surfactant protein a and b genetic variants predispose to idiopathic pulmonary fibrosis increased risk of fibrosing alveolitis associated with interleukin-1 receptor antagonist and tumor necrosis factor-alpha gene polymorphisms transforming growth factor-beta1 gene polymorphisms are associated with disease progression in idiopathic pulmonary fibrosis familial pulmonary fibrosis in the united states british thoracic society study of cryptogenic fibrosing alveolitis: current presentation and initial management. fibrosing alveolitis subcommittee of the research committee of the british thoracic society cryptogenic fibrosing alveolitis: clinical features and their influence on survival diffuse fibrosing alveolitis (diffuse interstitial fibrosis of the lungs) correlation of histology at biopsy with prognosis positive antinuclear factor in patients with unexplained pulmonary fibrosis definition and clinical relevance of antibodies to nuclear ribonucleoprotein and other nuclear antigens in patients with cryptogenic fibrosing alveolitis pathogenetic mechanisms of interstitial pulmonary fibrosis in patients with serum antinuclear factor. a histologic and clinical correlation usual interstitial pneumonia low fasting serum triglyceride and high free fatty acid levels in pulmonary fibrosis: a previously unreported finding idiopathic pulmonary fibrosis. clinical, histologic, radiographic, physiologic, scintigraphic, cytologic, and biochemical aspects chronic diffuse infiltrative lung disease: comparison of diagnostic accuracy of ct and chest radiography the diagnosis, assessment and treatment of diffuse parenchymal lung disease in adults chronic diffuse interstitial lung disease: diagnostic value of chest radiography and high-resolution ct accuracy of the typical computed tomographic appearances of fibrosing alveolitis a comparison of bronchiolitis obliterans with organizing pneumonia, usual interstitial pneumonia, and small airways disease radiologic findings are strongly associated with a pathologic diagnosis of usual interstitial pneumonia clinical usefulness of high resolution computed tomography in cryptogenic fibrosing alveolitis the association between pulmonary fibrosis and coronary artery disease idiopathic interstitial pneumonias: diagnostic accuracy of thin-section ct in 129 patients radiological versus histological diagnosis in uip and nsip: survival implications utility of a lung biopsy for the diagnosis of idiopathic pulmonary fibrosis idiopathic pulmonary fibrosis: role of high-resolution thin section computed tomographic scanning diffuse lung disease: diagnostic accuracy of ct in patients undergoing surgical biopsy of the lung the accuracy of the clinical diagnosis of new-onset idiopathic pulmonary fibrosis and other interstitial lung disease: a prospective study a histologic pattern of nonspecific interstitial pneumonia is associated with a better prognosis than usual interstitial pneumonia in patients with cryptogenic fibrosing alveolitis and by the ers executive committee idiopathic nonspecific interstitial pneumonia/fibrosis: comparison with idiopathic pulmonary fibrosis and boop the influence of cigarette smoking on lung function in patients with idiopathic pulmonary fibrosis lung and chest wall mechanics in ventilated patients with end stage idiopathic pulmonary fibrosis gas exchange at a given degree of volume restriction is different in sarcoidosis and idiopathic pulmonary fibrosis the role of pulmonary function testing in pulmonary fibrosis role of physiologic assessment in advanced lung disease cryptogenic fibrosing alveolitis with preserved lung volumes six-minute walk testing prognostic value of desaturation during a 6-minute walk test in idiopathic interstitial pneumonia bronchoalveolar lavage in interstitial lung disease the value of serial bronchoalveolar lavages in assessing the clinical progress of patients with cryptogenic fibrosing alveolitis idiopathic pulmonary fibrosis. pretreatment bronchoalveolar lavage cellular constituents and their relationships with lung histopathology and clinical response to therapy bal findings in idiopathic nonspecific interstitial pneumonia and usual interstitial pneumonia diagnostic value of transbronchial biopsy in usual interstitial pneumonia idiopathic pulmonary fibrosis: current concepts prognostic significance of histopathologic subsets in idiopathic pulmonary fibrosis idiopathic pulmonary fibrosis: clinical relevance of pathologic classification idiopathic nonspecific interstitial pneumonia: prognostic significance of cellular and fibrosing patterns: survival comparison with usual interstitial pneumonia and desquamative interstitial pneumonia histopathologic variability in usual and nonspecific interstitial pneumonias usual interstitial pneumonia: histologic study of biopsy and explant specimens a placebo-controlled trial of interferon gamma-1b in patients with idiopathic pulmonary fibrosis ct findings during phase of accelerated deterioration in patients with idiopathic pulmonary fibrosis circulating kl-6 predicts the outcome of rapidly progressive idiopathic pulmonary fibrosis acute exacerbation in idiopathic pulmonary fibrosis. analysis of clinical and pathologic findings in three cases outcome of patients with idiopathic pulmonary fibrosis admitted to the intensive care unit acute interstitial pneumonia clinical deterioration in patients with idiopathic pulmonary fibrosis: causes and assessment primary pulmonary carcinoma in patients with idiopathic pulmonary fibrosis lung cancer and cryptogenic fibrosing alveolitis. a population-based cohort study nonspecific interstitial pneumonia adverse effects of oral corticosteroids in relation to dose in patients with lung disease immunosuppressive and cytotoxic pharmacotherapy for pulmonary disorders cyclophosphamide in the treatment of idiopathic pulmonary fibrosis: a prospective study in patients who failed to respond to corticosteroids idiopathic pulmonary fibrosis: predicting response to therapy and survival pulmonary function in idiopathic pulmonary fibrosis and referral for lung transplantation serial evaluation of high-resolution computed tomography findings in patients with idiopathic pulmonary fibrosis in usual interstitial pneumonia the relationship between individual histologic features and disease progression in idiopathic pulmonary fibrosis fibroblastic foci in usual interstitial pneumonia: idiopathic versus collagen vascular disease a clinical, radiographic, and physiologic scoring system for the longitudinal assessment of patients with idiopathic pulmonary fibrosis idiopathic pulmonary fibrosis: a composite physiologic index derived from disease extent observed by computed tomography prognostic implications of physiologic and radiographic changes in idiopathic interstitial pneumonia fibrotic idiopathic interstitial pneumonia: the prognostic value of longitudinal functional trends changes in clinical and physiologic variables predict survival in idiopathic pulmonary fibrosis serum surfactant proteins-a and -d as biomarkers in idiopathic pulmonary fibrosis pulmonary (99m)tc-dtpa aerosol clearance and survival in usual interstitial pneumonia (uip) thorax 56 immunomodulatory agents for idiopathic pulmonary fibrosis corticosteroids for idiopathic pulmonary fibrosis idiopathic pulmonary fibrosis: impact of oxygen and colchicine, prednisone, or no therapy on survival colchicine versus prednisone as treatment of usual interstitial pneumonia diffuse interstitial pneumonitis. clinicopathologic correlations in 20 patients treated with prednisone/azathioprine azathioprine combined with prednisone in the treatment of idiopathic pulmonary fibrosis: a prospective double-blind, randomized, placebo-controlled clinical trial randomised controlled trial comparing prednisolone alone with cyclophosphamide and low dose prednisolone in combination in cryptogenic fibrosing alveolitis hospital-based historical cohort study of 234 histologically proven japanese patients with ipf pharmacologic suppression of the neutrophil component of the alveolitis in idiopathic pulmonary fibrosis outcome of subjects with idiopathic pulmonary fibrosis who fail corticosteroid therapy. implications for further studies use of intermittent, intravenous cyclophosphamide for idiopathic pulmonary fibrosis cyclophosphamide pulse therapy in idiopathic pulmonary fibrosis international guidelines for the selection of lung transplant candidates. the international society for heart and lung transplantation, the american thoracic society, the american society of transplant physicians, the european respiratory society lung transplantation survival benefit of lung transplantation for patients with idiopathic pulmonary fibrosis the registry of the international society for heart and lung transplantation: nineteenth official report-2002 single versus bilateral lung transplantation for idiopathic pulmonary fibrosis: a ten-year institutional experience health-related quality of life in lung transplant candidates and recipients research on the quality of life of lung transplant candidates and recipients: an integrative review predicting survival of lung transplantation candidates with idiopathic interstitial pneumonia: does pao(2) predict survival? idiopathic pulmonary fibrosis: prevailing and evolving hypotheses about its pathogenesis and implications for therapy colchicine: new uses of an old drug colchicine inhibition of plasma protein release from rat hepatocytes the effect of colchicine on the accumulation of hydroxyproline and on lung compliance after irradiation colchicine in the treatment of pulmonary fibrosis lung statics and connective tissues after penicillamine in bleomycin-treated hamsters a 15-year prospective study of treatment of rapidly progressive systemic sclerosis with d-penicillamine colchicine, d-penicillamine, and prednisone in the treatment of idiopathic pulmonary fibrosis: a controlled clinical trial pirfenidone: a novel pharmacological agent that inhibits leiomyoma cell proliferation and collagen production downregulation of procollagen genes at the transcriptional level in bleomycin hamster model of lung fibrosis effects of pirfenidone on transforming growth factor-beta gene expression at the transcriptional level in bleomycin hamster model of lung fibrosis treatment of idiopathic pulmonary fibrosis with a new antifibrotic agent, pirfenidone: results of a prospective, open-label phase ii study open-label compassionate use one year-treatment with pirfenidone to patients with chronic pulmonary fibrosis effect of pirfenidone on the pulmonary fibrosis of hermansky-pudlak syndrome interferon-gamma: biology and role in pathogenesis molecular mechanisms of antifibrotic effect of interferon gamma in bleomycin-mouse model of lung fibrosis: downregulation of tgf-beta and procollagen i and iii gene expression effect of gammainterferon on collagen synthesis by normal and fibrotic human lung fibroblasts in vivo levels and in vitro production of interferongamma in fibrosing interstitial lung diseases determination of various cytokines and type iii procollagen aminopeptide levels in bronchoalveolar lavage fluid of the patients with pulmonary fibrosis: inverse correlation between type iii procollagen aminopeptide and interferongamma in progressive patients pathogenesis of pulmonary fibrosis in interstitial lung disease. alveolar macrophage pdgf(b) gene activation and up-regulation by interferon gamma a preliminary study of long-term treatment with interferon gamma-1b and lowdose prednisolone in patients with idiopathic pulmonary fibrosis interferon gamma-1b for the treatment of idiopathic pulmonary fibrosis new approaches to managing idiopathic pulmonary fibrosis. continuing education monograph series interferon gamma-1b therapy for advanced idiopathic pulmonary fibrosis does interferon-gamma improve pulmonary function in idiopathic pulmonary fibrosis? interferon effects on the growth and division of human fibroblasts differential regulation of glycosaminoglycan, fibronectin, and collagenase production in cultured human dermal fibroblasts by interferon-alpha, -beta, and -gamma interferon-beta induces metalloproteinase mrna expression in human fibroblasts. role of activator protein-1 control of collagen synthesis in human chondrocyte cultures by immune interferon and interleukin-1 pulmonary changes induced by combined mouse beta-interferon (rmuifn-beta) and irradiation in normal mice-toxic versus protective effects feasibility of a trial of interferon beta-1a (ifn-beta-1a) in the treatment of idiopathic pulmonary fibrosis (ipf) relationship of alveolar epithelial injury and repair to the induction of pulmonary fibrosis induction of apoptosis and pulmonary fibrosis in mice in response to ligation of fas antigen abrogation of bleomycin-induced epithelial apoptosis and lung fibrosis by captopril or by a caspase inhibitor nhlbi workshop summary. pharmacological therapy for idiopathic pulmonary fibrosis. past, present, and future glutathione deficiency in the epithelial lining fluid of the lower respiratory tract in idiopathic pulmonary fibrosis the effect of oral nacetylcysteine on lung glutathione levels in idiopathic pulmonary fibrosis aerosolized administration of n-acetylcysteine attenuates lung fibrosis induced by bleomycin in mice antioxidative and clinical effects of high-dose n-acetylcysteine in fibrosing alveolitis. adjunctive therapy to maintenance immunosuppression lung cytokine production in bleomycininduced pulmonary fibrosis transfer of tumor necrosis factor-alpha to rat lung induces severe pulmonary inflammation and patchy interstitial fibrogenesis with induction of transforming growth factor-beta1 and myofibroblasts expression of tnf and the necessity of tnf receptors in bleomycin-induced lung injury in mice expression and localization of tumor necrosis factor-alpha and its mrna in idiopathic pulmonary fibrosis treatment by human recombinant soluble tnf receptor of pulmonary fibrosis induced by bleomycin or silica in mice treatment of rheumatoid arthritis with a recombinant human tumor necrosis factor receptor (p75)-fc fusion protein predominant role of tumor necrosis factor-alpha in human monocyte il-10 synthesis increased expression of the interleukin-10 gene by alveolar macrophages in interstitial lung disease introduction of the interleukin-10 gene into mice inhibited bleomycin-induced lung injury in vivo differential in vitro effects of il-4, il-10, and il-13 on proinflammatory cytokine production and fibroblast proliferation in rheumatoid synovium a type 2 (th2-like) pattern of immune response predominates in the pulmonary interstitium of patients with cryptogenic fibrosing alveolitis (cfa) effect of antibody to transforming growth factor beta on bleomycin induced accumulation of lung collagen in mice adenovector-mediated gene transfer of active transforming growth factor-beta1 induces prolonged severe fibrosis in rat lung tgf-beta 1, but not tgf-beta 2 or tgf-beta 3, is differentially present in epithelial cells of advanced pulmonary fibrosis: an immunohistochemical study transforming growth factorbeta in human glomerular injury inhibition of transforming growth factor-beta/smad signalling by the interferon-gamma/stat pathway elevated il-8 and mcp-1 in the bronchoalveolar lavage fluid of patients with idiopathic pulmonary fibrosis and pulmonary sarcoidosis expression of monocyte chemoattractant protein 1 mrna in human idiopathic pulmonary fibrosis costimulation of fibroblast collagen and transforming growth factor beta1 gene expression by monocyte chemoattractant protein-1 via specific receptors clinical significance of mcp-1 levels in balf and serum in patients with interstitial lung diseases expression of endothelin-1 in lungs of patients with cryptogenic fibrosing alveolitis a novel potent vasoconstrictor peptide produced by vascular endothelial cells a cytokine reborn? endothelin-1 in pulmonary inflammation and fibrosis effect of endothelin receptor antagonists (bq-485, ro 47-0203) on collagen deposition during the development of bleomycin-induced pulmonary fibrosis in rats elevated expression of endothelin-1 and endothelin-converting enzyme-1 in idiopathic pulmonary fibrosis: possible involvement of proinflammatory cytokines endothelins and pulmonary diseases increased endothelin-1 in bleomycin-induced pulmonary fibrosis and the effect of an endothelin receptor antagonist lovastatin induces fibroblast apoptosis in vitro and in vivo. a possible therapy for fibroproliferative disorders sildenafil for treatment of lung fibrosis and pulmonary hypertension: a randomised controlled trial key: cord-001848-idmj2d7p authors: onabajo, olusegun o.; porter-gill, patricia; paquin, ashley; rao, nina; liu, luyang; tang, wei; brand, nathan; prokunina-olsson, ludmila title: expression of interferon lambda 4 is associated with reduced proliferation and increased cell death in human hepatic cells date: 2015-11-01 journal: j interferon cytokine res doi: 10.1089/jir.2014.0161 sha: doc_id: 1848 cord_uid: idmj2d7p interferon lambda 4 (ifn-λ4) is a novel type-iii interferon that can be generated only in individuals carrying a δg frame-shift allele of an exonic genetic variant (rs368234815-δg/tt). the rs368234815-δg allele is strongly associated with decreased clearance of hepatitis c virus (hcv) infection. here, we further explored the biological function of ifn-λ4 expressed in human hepatic cells—a hepatoma cell line hepg2 and fresh primary human hepatocytes (phhs). we performed live confocal imaging, cell death and proliferation assays, mrna expression profiling, protein detection, and antibody blocking assays using transient and inducible stable in vitro systems. not only did we observe significant intracellular retention of ifn-λ4 but also detected secreted ifn-λ4 in the culture media of expressing cells. secreted ifn-λ4 induced strong activation of the interferon-stimulated genes (isgs) in ifn-λ4-expressing and surrounding cells in transwell assays. specifically, in phhs, secreted ifn-λ4 induced expression of the cxcl10 transcript and a corresponding pro-inflammatory chemokine, ip-10. in ifn-λ4-expressing hepg2 cells, we also observed decreased proliferation and increased cell death. all ifn-λ4-induced phenotypes—activation of isgs, decreased proliferation, and increased cell death—could be inhibited by an anti-ifn-λ4-specific antibody. our study offers new insights into biology of ifn-λ4 and its possible role in hcv clearance. w ith more than 170 million infected individuals, hepatitis c virus (hcv) infection represents a significant healthcare burden worldwide (mohd hanafiah and others 2013) . hcv infection is treated with interferon (ifn)-a-based regimens and recently approved ifn-a-free direct-acting antiviral agents (daa) (liang and ghany 2013) . genome-wide association studies identified a single nucleotide polymorphism rs12979860, located upstream of the ifnl3 (il28b) gene and thus initially referred to as the ''il28b marker,'' as one of several genetic variants strongly predictive of hcv clearance (ge and others 2009; thomas and others 2009) . further studies showed that rs12979860 is located in the intronic region of a novel gene, ifnl4, and is in high linkage disequilibrium (ld) with an ifnl4 exonic frameshift polymorphism rs368234815-tt/dg, initially designated as ss469415590 (prokunina-olsson and others 2013). the rs368234815-dg allele, which creates an open reading frame for a novel human interferon, interferon lambda 4 (ifn-l4), is associated with decreased hcv clearance (prokunina-olsson and others 2013) [reviewed in o'brien and others (2014) ]. the rs368234815-dg has allele frequency of *70% in individuals of african ancestry, *30% in europeans, while only 0%-5% in asians (prokunina-olsson and others 2013). in individuals of african ancestry, rs368234815 is more predictive of hcv clearance than rs12979860 (prokunina-olsson and others 2013; aka and others 2014); while in europeans and asians, these markers are in high ld and thus provide comparable predictive information (prokunina-olsson and others 2013). a genetic polymorphism rs117648444-c/t, which introduces an amino-acid substitution p70s in the ifn-l4 protein (prokunina-olsson and others 2013), is associated with reduced biological activity of ifn-l4 and increased hcv clearance (terczynska-dyla and others 2014), thereby supporting the critical role of ifn-l4 in this process. recent clinical trials showed that ifnl4 variants rs368234815 and rs12979860 are predictive of treatment efficacy even for daa therapies (fujino and others 2013; meissner and others 2014a; o'brien and pfeiffer 2015) and these markers, possibly together with p70s, could be used to optimize treatment regimens and duration in resource-limited settings. the functional importance of ifn-l4 is evidenced by the strong positive selection that favored elimination of ifn-l4 from human populations (key and others 2014) . although this selection cannot be explained by any known viral infection, it may reflect antiviral response to some extinct highly deadly infection. previously, we showed that transient transfection of an expression construct that generates ifn-l4 protein induced interferon signaling, with activation of interferon-stimulated genes (isgs) and generation of antiviral response in hepg2, a human hepatoma cell line (prokunina-olsson and others 2013). however, the function of ifn-l4 and its role in impaired hcv clearance remained unclear. here, we further explored this question by performing additional functional analyses of ifn-l4 transiently and stably overexpressed in human hepatic cells-fresh primary hepatocytes and hepg2 cells. the human hepatoma cell line hepg2 (atcc hb-8065) was purchased from the american tissue culture collection (atcc) and maintained in dulbecco's modified eagle's medium (dmem) supplemented with 10% heat-inactivated fetal bovine serum (fbs). the custom isre-luc-hepg2 cell line stably expressing a luciferase reporter under control of the interferon-stimulated response element (isre) was previously described (prokunina-olsson and others 2013); the cells were maintained in dmem supplemented with 10% fbs and 2 mg/ml puromycin. fresh primary human hepatocytes (phhs) were purchased from bioreclamation ivt. the cells were received in suspension within 6 h after isolation and were maintained in in-vitrogro hi culture media with torpedo antibiotic mix. the liver donor 1 was a 55-year-old woman who died of cardiac arrest, and the liver donor 2 was a 40-year-old man who died of anoxia. both were hcv-free caucasians. on arrival, phhs had 88% and 90% viability for donor 1 and 2, respectively. dna extracted from the phhs was genotyped with a custom taqman assay (prokunina-olsson and others 2013) for the exonic ifnl4 genetic variant rs368234815 (dg/tt). both donors were homozygous for the rs368234815-tt allele, which is associated with higher hcv clearance (prokunina-olsson and others 2013). the rs368234815-tt allele introduces a frame shift within the first exon of ifnl4 gene and eliminates the ifn-l4 protein. thus, no ifn-l4 protein could be endogenously produced in these phh samples. the expression constructs (ifnl4-halo, p131-halo, and control-halo) based on the pfc14a vector (promega) were previously described (prokunina-olsson and others 2013). the control-halo construct generates only a halo-tag protein (33 kda), the ifnl4-halo construct generates the ifn-l4-halo protein (53 kda) consisting of the ifn-l4 protein (20 kda) c-terminally fused with the halo-tag protein (33 kda), and the p131-halo generates a protein of 47 kda, consisting of the halo-tag c-terminally fused with a splicing protein isoform of ifn-l4 (131 aa, 14 kda, designated as p131), which lacks exon 3 and is nonfunctional in induction of interferon response (prokunina-olsson and others 2013). all constructs have been validated by western blotting with an a-halo antibody (promega) and/or an a-ifn-l4 antibody (mabf227; emd millipore). the ifnl3-halo construct generating the secreted ifn-l3-halo protein was created and validated the same way. hepg2 cells and phhs were transfected for 24 or 48 h with corresponding constructs; hepg2 cells were transfected using lipofectamine/ltx and opti-mem with standard protocols (life technologies), while phhs were transfected using a nucleofection protocol optimized for primary cells (lonza). briefly, for each transfection, 2 · 10 6 fresh phhs were resuspended in 100 ml of optimized nucleofection buffer l3 and transfected with 5 mg of corresponding constructs using 4d-nucleofector (lonza). immediately after transfection, dead cells were separated by centrifugation (4 min, 570 rpm) of the transfection mix overlaid with 900 ml of ficoll (ge healthcare) and 750 ml of supernatant with dead cells was removed. the remaining cells were resuspended in invitro-gro cp media with torpedo antibiotic mix (bioreclamation ivt), plated onto collagen-coated plates (bd biosciences), and incubated overnight. unattached and dead cells were removed 12 h post-transfection by replacing the transfection media with invitrogro hi culture media with torpedo antibiotic mix (bioreclamation ivt). tetracycline-inducible hepg2 cell lines for ifn-l4-gfp, p131-gfp, and control-gfp were generated using the tet-on 3g expression system (clontech). first, a stable hepg2 cell line expressing the tet-on 3g transactivator was generated by lentiviral transduction and blasticidin selection (5 mg/ml). corresponding cdnas were cloned into a ptreg3g plasmid (clontech) to generate tetracyclineinducible ifn-l4-gfp, p131-gfp, and control-gfp constructs, which were transfected into the stable tet-on 3g-hepg2 using lipofectamine/ltx. positive clones were identified by limiting dilution under neomycin selection (1 mg/ml) over 3 weeks. expression of corresponding proteins was induced by treatment with 1 mg/ml doxycycline for indicated experiment-specific time periods. hepg2 cells were transfected with corresponding constructs in 4-well coverslip chambers (4 · 10 4 cells/well; labtek). after 24 h, transfection media was replaced with live cell imaging solution (life technologies), supplemented with 20 mm glucose. cell-permeant halo-tag ligands (tmr red or oregon green; promega) were added to live cells (1:2,000 for 15 min), and live imaging was performed using a lsm700 confocal laser scanning microscope (carl zeiss) fitted with a temperature and co 2 -controlled chamber. when applicable, nuclei were visualized using fluorescent hoechst 33342 dye (life technologies). for cell death analysis, live cells were imaged using inverted oil lens at 40· magnification, with 6 s scanning per minute per field of view for 18 h, generating 1,080 images per field; 6 fields were imaged in sequence using stage-positioning software. each field of view had at least 10 transfected cells. signs of cell death were scored visually by counting membrane rupture events in the videos representing the 18-h imaging periods. cell death rates were determined as the percentages of ruptured cells of the total counts of fluorescent cells in each field of view. hepg2 cells were transfected with corresponding constructs and imaged live as described earlier using oregon green halo-tag ligand. the mobility of the ifn-l4-halo and control-halo proteins was evaluated with fluorescence recovery after photobleaching (frap) method (snapp and others 2003) . briefly, after 10 prebleach scans, a selected region was bleached at 100% of imaging power with 3 iterations and 2 ms per iteration, with 1.94 s per scan. imaging was continued for a total of 150 s. fluorescence intensity was plotted with curve fitting to evaluate fluorescence recovery in the bleached areas over time. half-time fluorescence recovery (t½) and the ratio of immobile fraction were averaged for 7 individually scanned cells for each construct (ifnl4-halo and control-halo). recovery data were normalized to unbleached regions inside the cells and adjusted for background fluorescence; data acquisition and analyses were performed using lsm700 frap software. the stable hepg2-isre-luc cells were seeded in 96-well plates that were either untransfected or transfected with corresponding constructs. after 24 h, the cells were treated for 2 h with the following antibodies: 20 mg/ml of a blocking a-il10r2 antibody (r&d systems), 40 mg/ml of a goat isotype igg control (abcam), a range (20, 10, 5, 2.5, or 1.25 mg/ml) of a custom rabbit monoclonal a-ifn-l4 antibody (prokunina-olsson and others 2013), or 40 mg/ml of a rabbit isotype igg control (cell signaling). recombinant human interferons-10 ng/ml of custom ifn-l3 (prokunina-olsson and others 2013), ifn-a and ifn-b (pbl assay science), or ifn-g (r&d systems) were added to the untransfected cells pretreated with the antibodies. negative controls included nontreated cells, phosphate-buffered saline (pbs) in media, and mock transfection with control-halo constructs. the cells were assayed for luciferase expression of the isre-luc reporter 48 h post-transfection. all transfections and treatments were done in 8 biological replicates. for other experiments, inducible stable hepg2 cells were seeded into 96-well plates and protein expression was induced for 12 h. antibodies (20 mg/ml of rabbit monoclonal a-ifn-l4 or 40 mg/ml of rabbit igg control) were added to shared culture media for 48 h. hepg2 and phhs were transfected with corresponding constructs and seeded onto 6-well plates. separately, transwell inserts with 0.4 mm pores (corning) were seeded with untransfected cells and placed into 6-well plates with culture media, with 1 insert per well. for phhs, both the plates and transwell inserts were collagen coated (corning). after 24 h, media was replaced and transwell inserts with growing cells were added to the plates containing transfected cells. the pore size of transwell inserts allowed free circulation of the media but not of the cells. for phhs from donor 2, the transwell experiment was expanded to include antibody treatment. phhs transfected with ifnl4-halo, ifnl3-halo, and control-halo constructs were incubated with transwell inserts seeded with untransfected phhs. antibodies (20 mg/ml of rabbit monoclonal a-ifn-l4 or 40 mg/ml of rabbit igg control) were added to shared culture media for 24 h. after 24 h of coincubation, cells from both chambers that shared culture media (transfected vs. untransfected transwell cells) were collected separately for rna extraction and stored in rlt buffer; shared culture media was collected for protein analysis and stored at -80°c until use. for transient transfections, hepg2 cells were transfected with corresponding constructs for 48 h and media was changed after overnight incubation. expression of halo-tag in live cells was detected using cell-permeant halo-tag tmr red ligand. for stable hepg2 cells, expression of proteins was induced for 72 h. in both systems, dead cells were identified using near-ir fixable live/dead marker (life technologies). for analysis of apoptosis, cells were additionally stained for annexin v (bd biosciences). for proliferation assays, the cells that were induced for 72 h were treated with 10 mm 5-bromo-2¢-deoxyuridine (brdu) for 3 h. dead cells were excluded by removing nonadherent cells, and the remaining cells were stained using the apc brdu flow kit (bd biosciences). multiparametric flow cytometry analysis was performed on facs aria iii (bd biosciences) with flowjo.10 software (tree star). for viability assays, the protein expression was induced for 5 days in 96-well plates and viability was evaluated with the multi tox-glo assay (promega). in some experiments, viability assays were performed after protein induction for 72 h, with similar results. quantitative reverse-transcriptase-polymerase chain reaction expression analysis total rna was isolated using an rneasy kit with oncolumn dnase i treatment (qiagen). rna quantity and quality were evaluated by nanodrop 8000 (thermo scientific). cdna was prepared from 40 to 100 ng of total rna with the rt 2 first-strand cdna kit and random hexamers with an additional dna-removal step (qiagen). quantitative reverse-transcriptase-polymerase chain reaction (qrt-pcr) mrna expression analysis was performed using sybr green antiviral response qrt-pcr plates (qiagen). the plates included 88 expression assays for target genes, as well as positive, negative, and endogenous normalization controls (supplementary tables s1-s3; supplementary data are available online at www.liebertpub.com/jir). a custom expression assay for ifnl3 (prokunina-olsson and others 2013) and predesigned taqman expression assays for ifnl1 (hs00601677_g1) and ifng (hs00989291_m1; life technologies) were not included on the predesigned plates and were used separately (supplementary table s4 ). the qrt-pcr reactions (5 or 10 ml) included expression master mixes (qiagen or life technologies), cdna, and corresponding expression assays; the quantification was performed in 2-4 technical replicates on the quantsudio 7 instrument (life technologies). expression was measured in c t values (pcr cycle at detection threshold), which are distributed on log 2 scale. expression was normalized by the geometric means of 5 endogenous controls (actb, b2m, gapdh, hprt1, rplp0) included on the qrt-pcr plates or of 2 endogenous controls used separately (assay 4326317e for gapdh, and assay 4326315e for actb; life technologies). differences in expression were calculated according to the relative quantification method, as dc t = c t (control) -c t (target). fold differences between expression of any 2 samples or groups of samples were calculated as 2 (dct1 -dct2) . heat-map analysis of auto-scaled expression data for a panel of selected isgs was performed with the genex software (multid). protein levels of ip-10 (encoded by cxcl10) in culture media from hepg2 and phhs transfected with corresponding constructs were measured with an elisa kit (r&d systems), which has a protein detection range of 7.8-500 pg/ml. culture media was collected 24 h after transfection media change (48 h post-transfection). media samples were diluted 1:10 or 1:20, and each sample was measured in technical triplicates. a standard curve was generated using the protein provided with the kit, with a correlation coefficient >0.98. the plates were analyzed using glomax luminometer (promega). ifn-l4 was detected with a custom-developed electrochemical elisa on the meso quickplex sq120 instrument [mesoscale discovery (msd)]. briefly, standard capacity multi-array 96-well plates (msd) were coated overnight at 4°c with 30 ml of 2 mg/ml custom monoclonal rabbit a-ifn-l4 antibody. blocking was performed with bovine serum albumin (msd blocker a) for 1 h at room temperature with rotation at 400 rpm, followed by 3 washes with pbs and 0.05% tween. standards, controls, and experimental samples were prepared using diluent 2 (msd). culture media samples were diluted 1:10 and incubated at room temperature for 2 h with rotation at 400 rpm. after additional wash steps, 25 ml of the 4 mg/ml detection antibody [mouse monoclonal a-ifn-l4 antibody (buffer exchanged into pbs, mabf227; emd millipore)], conjugated with a sulfo-tag (msd) was added to the wells and incubated for 1 h at room temperature with rotation at 400 rpm. after additional washing, the plates were scanned in a 2· read buffer (msd) on the meso quickplex sq120 and the results were analyzed with the msd discovery workbench software. purified recombinant ifn-l4 protein (prokunina-olsson and others 2013) was used as a standard at 9 concentrations in the range of 46 pg/ml to 300 ng/ml, and the detection range for the standards was determined as 150 pg/ ml to 300 ng/ml. hepg2 cells were transfected for 48 h with 50 ng of corresponding constructs with/without cotransfection with 1.0 ml of the endoplasmic reticulum stress response element (erse)-luc cignal reporter (qiagen). transfections were done in a 96-well plate, with 8 replicates per transfection. media was changed 24 h post-transfection, and the plate was assayed for luciferase and renilla using the glomax luminometer. data were presented as relative luciferase units, which correspond to luciferase/renilla ratio. the stable hepg2-isre-luc cells were transfected with corresponding constructs in 96-well plates; untransfected cells were treated with human recombinant interferons-ifna (2 ng/ml; pbl assay science) or custom ifn-l3 (10 ng/ ml). all experiments were represented by 8 biological replicates. the media was replaced 24 h post-transfection by 100 ml of full culture media with 1 or 10 mm jak inhibitor [active against jak1, jak2, and jak3, #420097; emd millipore, in 0.1% dimethyl sulfoxide (dmso)]. for il10r2 blocking experiment, the transfection culture media was replaced by 100 ml of media with 20 mg/ml of an a-il10r2 blocking antibody (mab874; r&d systems) or 20 mg/ml of a goat isotype igg control (abcam). for treatment experiments, cells were pretreated for 1 h with the jak inhibitor or for 2 h with the a-il10r2 or igg control antibodies and then treated with ifn-l3 (10 ng/ml) or ifn-a (2 ng/ml). negative controls included untreated cells, cells treated with 0.1% dmso in media, and cells transfected with control-halo construct. the cells were assayed for isre-luc reporter 48 h post-transfection, which corresponds to 24 h posttreatment with jak inhibitor and antibodies. sirna silencing of ifnlr1 in hepg2-isre-luc cells the stable hepg2-isre-luc cells were transfected with corresponding constructs in 2 identical 96-well plates. the cells were co-transfected with 1 pmol/well of a scrambled negative control sirna (am4615; ambion) or a set of sirnas against ifnlr1 (m-007981-00; thermo scientific). after 24 h, the cells were treated with culture media, ifn-l3 (10 ng/ml), or ifn-a (2 ng/ml). after 48 h, the first plate was assayed for the isre-luc reporter on the glomax luminometer. the second plate was used for rna extraction (zymo research) and mrna expression analysis. cdna was synthesized using super script iii reverse transcriptase (life technologies). specific taqman assays for ifnlr1 (hs00417120_m1) and endogenous control actb (assay 4326315e) from life technologies were used for qrt-pcr analysis on the quantstudio 7 instrument (life technologies). expression of ifnlr1 was normalized to expression of actb and then expression in samples treated with ifnlr1 sirna was compared with untreated samples (no sirna) or treated with scrambled sirna. all samples were represented by 6 biological replicates. compared with sirna-untreated samples (100%), the expression of ifnlr1 was decreased to 76% in si-scr samples and to 42% in si-ifnlr1 samples ( supplementary fig. s1d ). unless specified, data plotting and statistical analyses were performed with prism 6 (graphpad), p values are for 2-sided unpaired t-tests. shown are means and standard errors of the mean. previously, by performing western blot analysis, we were unable to detect ifn-l4 in culture media of hepg2 cells transiently transfected with an ifn-l4-producing construct, even though this transfection resulted in strong activation of interferon signaling (prokunina-olsson and others 2013). however, ifn-l4 was detectable at low levels in culture media of transfected cells by western blot analysis after acetone precipitation (hamming and others 2013) . now, we developed a mesoscale assay (an electrochemical elisa) and were able to detect ifn-l4 in culture media of transfected hepg2 cells (fig. 1a) . treatment of these cells with a custom a-ifn-l4 antibody decreased the interferon signaling by 70% (fig. 1b) , suggesting that ifn-l4 generated in this experimental system is a secreted biologically active interferon. simultaneously, the a-ifn-l4 antibody did not affect signaling of the main interferons (ifn-a, ifn-b, ifng, and ifn-l3, fig. 1c ). signaling induced by ifn-l4 was strongly attenuated by the jak inhibitor and by blocking of the ifn-l family receptors (ifnlr1 and il10r2, supplementary fig. s1a -c), confirming these canonical components of the jak/stat pathway as essential elements for the ifn-l4 signaling. we evaluated the biological activity of the secreted ifn-l4 by its ability to induce expression of a set of isgs (supplementary tables s1-s3). we analyzed mrna ex-pression in cells transiently transfected with ifnl4-halo or control-halo constructs and in bystander nontransfected cells exposed to media from the corresponding transfected cells in transwell assays (ifnl4-trans and halo-trans, fig. 2a ). in both hepg2 and phhs, cells transfected with ifnl4-halo or exposed to media from those cells (containing secreted ifn-l4) showed strong induction of isgs, such as ddx58 (rig-i), dhx58, ifih1 (mda5), isg15, mx1, oas2, and stat1 and chemokines cxcl10 and cxcl11 ( fig. 2a) . expression of cxcl10 and cxcl11 was much higher in phhs compared with hepg2, highlighting cell-specific differences ( fig. 2a) . ifn-l4 was detectable in culture media of ifnl4-transfected phhs and hepg2 cells, but not in corresponding halo-transfected cells (fig. 2b) . we did not detect expression of other interferons (ifna1, ifna2, ifnb, ifng, ifnl1, ifnl3) in any of the experimental conditions (supplementary table s4 ). cxcl10 encodes ifn-g inducible protein 10 (ip-10), which is a chemotactic factor for neutrophils. high levels of ip-10 have been associated with inflammation and pathogenesis of chronic hcv infection (harvey and others 2003; lagging and others 2006) . we measured the levels of ip-10 in culture media of phhs and hepg2 transfected with different constructs. in phhs, ip-10 was detectable in the media from samples transfected with ifnl4-halo and ifnl3-halo (fig. 3b) , while it was undetectable in hepg2 cells transfected with ifnl4-halo (data not shown), in accordance with a much lower cxcl10 mrna expression observed in hepg2 compared with phhs (supplementary tables s1-s3) . importantly, the a-ifn-l4 antibody added to the shared media strongly decreased mrna expression of isgs, including cxcl10, in nonexpressing transwell phhs (fig. 3a ) and the amount of ip-10 secreted to the shared media (fig. 3a, b) . even though some of ifn-l4 gets secreted, as described earlier, ifn-l4 was also detected as a cytoplasmic protein by confocal imaging in fixed cells-in hepg2 cells transiently expressing ifn-l4-halo, and in phhs induced to express endogenous ifn-l4 (prokunina-olsson and others 2013). we next performed live imaging of intracellular movements of ifn-l4-halo and control-halo proteins (supplementary video s1). we also monitored frap (fig. 4a ). this method does not detect secreted proteins such as ifn-l3, but it helps characterize intracellular proteins based on their mobility (fig. 4b) , which is proportional to the speed of fluorescence recovery (t½, in seconds). the control-halo protein showed very high mobility, with a t½ of 4.46-1.19 s (fig. 4c and supplementary video s2), while ifn-l4-halo showed a much lower mobility, with a t½ of 34.69 -6.94 s (fig. 4c and supplementary video s3). intracellular mobility of proteins could be limited by their attachment to structural elements, involvement in proteinprotein interactions, or confinement within vesicles (lippincott-schwartz and others 2001; snapp and others 2003). therefore, we also estimated the fraction of immobile expression of mrna transcripts was quantified using an antiviral response qrt-pcr plate. the cells were transfected with corresponding constructs for 48 h, and transwell cells shared culture media with the corresponding transfected cells for 24 h. all samples were represented by 2 biological replicates. expression of each target assay on the plate was measured using the same amount of cdna; expression was normalized to a geometric mean of 5 endogenous controls included on the plate. the results are presented as dc t values for targets normalized by endogenous controls, on a log 2 scale; less negative dc t values correspond to higher levels of expression. full expression data for hepg2 cells and phh are available as supplementary tables s1 and s2. protein, which is represented by unrecoverable fluorescence-nearly 30% of ifn-l4-halo (28.43% -4.22%) compared with *10% of control-halo protein (9.58% -2.43%) was estimated to be immobile (fig. 4d) . we conclude that in our experimental system the control-halo was mostly present as a mobile unattached cytoplasmic protein, in agreement with other studies that used halo-protein (huybrechts and others 2009) . in contrast, we detected ifn-l4-halo both as an intracellularly retained protein with limited mobility and as a mobile protein potentially available for secretion or release. while conducting live confocal imaging in transiently transfected hepg2 cells, we observed dying cells that underwent morphologic changes of swelling, membrane blebbing, and rupture, followed by release of cytoplasmic content (fig. 5a, b and supplementary videos s4-s6), suggesting necrotic-type cell death (fiers and others 1999; festjens and others 2006; galluzzi and others 2012) . cell rupture events over the course of 18 h of imaging were significantly more frequent in ifn-l4 + compared with control-halo + hepg2 cells (fig. 5c) . flow cytometry analysis showed that necrotic cells (defined as annexin v + and live/dead + ) were significantly more common in cells transfected with ifnl4-halo compared with p131-halo (a nonfunctional protein isoform of ifn-l4), ifnl3-halo, and control-halo constructs (fig. 5d , e). transfection efficiency was comparable for all these constructs (fig. 5g , but could not be determined for the secreted ifnl3-halo), and thus could not explain the observed differences in cell death. we also evaluated cell death in phhs transiently transfected with ifnl4-halo or control-halo constructs. in general, in phhs, cell death rates were very high even in untransfected cells and similar for both constructs (data not shown), possibly due to physiologically high baseline cell death rates in primary cells unrelated to the effect of ifn-l4. cell death can also be triggered as a result of an endoplasmic reticulum response to unfolded recombinant proteins generated in vitro. previously, we showed that transient expression of ifnl4-halo and its splicing forms-p131, p107, and p170-induced the erse-luc reporter in hepg2 cells (prokunina-olsson and others 2013). we next repeated this experiment for the erse-luc reporter cotransfected with ifnl4-halo or p131-halo. transfection with p131-halo did not induce cell death (fig. 5d , e), despite considerable induction of the esre-luc reporter (fig. 5f ). we conclude that potential activation of the unfolded protein response to in vitro generated ifn-l4 cannot explain the observed ifn-l4-induced cell death. in an effort to define the mechanism of ifn-l4-induced cell death, we assayed for ripk1-dependent necrosis (necroptosis) and caspase-1-dependent cell death (pyroptosis), using specific inhibitors (necrostatin and yvad). however, we saw no evidence of the activation of these pathways after transient transfection with the ifnl4-halo construct (data not shown). treatment with zvad, a pan-caspase inhibitor, caused a significant decrease in ifn-l4-induced cell death, which is suggestive of apoptosis, but this effect was also ) . graphs represent 1 of 3 independent experiments, n = 3. (c) cell viability was analyzed with multi tox-glo assay and presented as the percentage of induced to uninduced cells, n = 6. **p < 0.01, ***p < 0.001, based on t-tests. onabajo et al. observed in controls (data not shown), implying that transfection itself might be contributing to apoptosis in this experimental system. to further explore our observations done in transient expression system, we developed inducible stable hepg2 cell lines expressing ifn-l4 and p131 fused with c-terminal green fluorescent protein (gfp) and a control-gfp cell line, all of which were under control of a tetracycline-inducible promoter. stable clones showed 70%-90% intracellular expression ( fig 6a) . secreted ifn-l4-gfp was detectable in media after 24 h of induction (fig. 6b ) and was biologically active (fig. 6c ). to assess cell death, protein expression was induced for 72 h and cells were stained for live/dead + and annexin v + markers. the ifn-l4-gfp-expressing cells showed a significant increase in cell death compared with cells expressing p131-gfp or control-gfp (fig. 7a, b) . in addition, cell viability analysis showed that 5 days of induced ifn-l4 expression resulted in a 30% reduction in the counts of viable cells compared with uninduced cells, with no change observed for the controls (fig. 7c) . although we observed increased annexin v + staining in the induced ifn-l4-expressing cells (fig. 7a) suggesting apoptotic cell death, we did not detect any caspase-3 activation (data not shown), which is expected in apoptosis. we reasoned that cell death might be associated with decreased proliferation, and we evaluated cell proliferation after 72 h of induced ifn-l4 expression by measuring brdu incorporation. compared with p131-gfp and gfp control, induction of ifn-l4 was associated with a significant reduction of proliferation by 40% (fig. 8a, b) . we induced ifn-l4 expression in the presence or absence of the rabbit monoclonal a-ifn-l4 antibody, which we previously found to be able to block the ability of ifn-l4 to induce interferon signaling (fig. 1) . the a-ifn-l4 antibody treatment attenuated ifn-l4-induced cell death (fig. 9a) , while it increased proliferation (fig. 9b ) and improved cell viability (fig. 9c ). since the antibody is not expected to enter the cells and can only block the secreted protein, these results indicate that the observed cell death and decreased viability and proliferation are caused by the secreted ifn-l4. interestingly, this effect was localized to ifn-l4-expressing cells and was undetectable in nonexpressing bystander cells in the transwell assays (supplementary fig. s2) . thus, the effect of ifn-l4 may be concentration dependent and primarily affecting the ifn-l4-expressing cells. fig. 9. a-ifn-l4 antibody inhibits ifn-l4-induced cell death and proliferation defect. protein expression of ifn-l4-gfp, p131-gfp, or control-gfp in stable hepg2 cells was induced with 1 mg/ml doxycycline for 72 h. at 12 h, cells were treated with 20 mg/ ml of rabbit a-ifn-l4 antibody or 40 mg/ml of rabbit igg control (rigg). cells in different treatment conditions were assessed for cell death (a), cell proliferation (b), and cell viability (c). graphs represent 1 of 2 independent experiments, n = 3, *p < 0.05, **p < 0.01, ***p < 0.001 based on t-tests. we suggest that ifn-l4 may have at least 3 functions in human hepatic cells-activation of interferon signaling, inhibition of cell proliferation, and induction of cell death. these roles may be overlapping, synergistic, or independent, and some or all of them may be relevant for hcv clearance. activation of isgs by ifn-l4 was expected based on our previous observations in transiently transfected hepg2 cells (prokunina-olsson and others 2013). early activation of a panel of isgs has also been reported in both in vitro hcvinfected and adjacent uninfected phhs (sheahan and others 2014) . however, we now determined the following about ifn-l4-induced isg activation: it is specifically caused by the ifn-l4 secreted from cells at low but detectable amounts; it has an effect on both primary hepatocytes and hepatoma cells; it similarly affects the expressing and nonexpressing bystander cells; and it can be specifically blocked by the a-ifn-l4 antibody. anti-proliferative and cell-death-inducing anti-tumor effects are, in general, characteristic of interferons (wang and others 2011) , including in hepatoma cell lines (murata and others 2006) . however, we now present the first evidence of these effects caused by ifn-l4, the newest addition to the interferon family. while activation of isgs was observed in both the ifn-l4-expressing and nonexpressing bystander cells, the decrease of proliferation and induction of cell death were detectable only in the expressing cells. this could mean that these effects require different concentration thresholds. the dose-dependent anti-proliferative effect in hepatic cells has already been shown for ifn-a (lim and others 2006) , and the same might be true for ifn-l4. anti-proliferative activity of ifn-l4 is isg dependent as it was blocked by the same a-ifn-l4 antibody that blocks ifn-l4induced activation of isgs. some of the isgs induced by ifn-l4, including 2¢5¢oas and ip-10, are known for their contribution to the anti-proliferative and pro-apoptotic effects of interferons (rysiecki and others 1989; hassel and others 1993; aksoy and others 2006; liu and others 2011) . at this point, we are unable to determine the mechanism of ifn-l4-induced cell death. although we did not detect activation of caspase-3 by ifn-l4, therapeutic anti-tumor mechanisms of interferons have been related to cell growth arrest and induction of caspase-3-independent apoptosis via the p53 pathway (vogelstein and others 2000; takaoka and others 2003; , and this pathway should be tested for ifn-l4 as well. the effect of ifn-l4 on activation of interferon signaling and cell death might be complex and intertwined, and further studies are warranted. the genetic association between the ability to generate ifn-l4 (in carriers of the rs368234815-dg and rs12979860-t alleles) and impaired spontaneous and treatment-induced clearance of hcv is well established, but the molecular understanding of this association is still limited. recently, it has also been reported that the same individuals who are more likely to clear the hcv infection, in fact, are significantly more likely to develop liver fibro-proliferative disease (fibrosis), which is associated with increased mortality (eslam and others 2015) . our new findings regarding ifn-l4 function may help reconcile these counterintuitive connections. we suggest that the induction of interferon signaling by ifn-l4 may have both positive and negative implications. the ifn-l4-induced isgs might provide some antiviral protection, explaining the lower pretreatment hcv loads in individuals with the rs368234815-dg allele, compared with those who do not carry this allele (uccellini and others 2012) . on the other hand, the increased pretreatment expression levels of these isgs have been associated with refractoriness to ifn-a therapy (dill and others 2011), especially in carriers of the ifnl4-rs368234815-dg or rs12979860-t alleles (urban and others 2010; prokunina-olsson and others 2013) . this could be because ifn-a and ifn-l4 compete for induction of the same set of isgs, and, once activated by ifn-l4, these isgs do not respond to ifn-a. the fact that despite having lower pretreatment hcv levels, individuals with rs368234815-dg allele are more likely to develop chronic hcv and fail ifn-a-based treatment, suggests that either negative effects of ifn-l4 outweigh its positive effect on lowering hcv viral load or antiviral effects induced by ifn-l4 are insufficient for complete viral clearance. the ifn-l4 effect on proliferation and cell death might also be positive and negative. increased cell death has been correlated with inflammation during hcv infection and was suggested as a factor contributing to liver damage (bantel and schulze-osthoff 2003) . high rates of cell death in ifn-l4-expressing cells could amount to substantial cell loss and inflammation, contributing to the development of liver disease. simultaneously, the anti-proliferative effect of ifn-l4 might limit the tissue remodeling ability that leads to liver fibro-proliferative disease (fibrosis), as has been reported by the largest study on this subject to date (eslam and others 2015) . individuals who are genetically unable to produce ifn-l4 or in whom ifn-l4 is not sufficiently induced by specific stimuli may be more likely to clear the infection, spontaneously or after treatment, especially with ifn-abased therapies. however, they may not have the benefit of an anti-proliferative effect also contributed by ifn-l4, and will be more likely to develop fibrosis. since in our system the a-ifn-l4 antibody efficiently blocked activation of isgs (including ip-10), and ifn-l4induced cell death, blocking of ifn-l4 with therapeutic agents in carriers of the rs368234815-dg allele might be a plausible therapeutic option before or in conjunction with other treatments. blocking of ifn-l4 might boost mechanisms of endogenous host immunity, which are important for efficient clearance of hcv and prevention of posttreatment relapse (meissner and others 2014b) , and also decrease liver damage due to inflammation and cell death. although the antiviral role of ifn-l4 has been shown for a number of viruses (hamming and others 2013; lu and others 2015) , it remains to be found what other biologically relevant factors can trigger endogenous ifn-l4 expression. identification of these triggers in hepatic and other human cell types could help explore the role of ifn-l4 in other relevant clinical conditions. so far, expression of endogenous ifnl4 has only been detected in liver biopsies from patients infected with hcv, with significant correlations between hcv titers and expression levels of ifnl4 and isgs (amanzada and others 2013; konishi and others 2013; meissner and others 2014b; murakawa and others 2015). however, ifnl4 was undetectable in liver biopsies from patients infected with hepatitis b virus or affected by 898 onabajo et al. unrelated liver diseases (amanzada and others 2013) . in vitro, ifnl4 expression could be induced in phhs by hcv infection or treatment with polyi:c, but polyi:c treatments failed to induce ifnl4 expression in hepg2 cells. the ifnl4 region is absent in the genomes of mouse and rat, precluding the development of relevant rodent models. we acknowledge the limitations of our experimental system, which is based on expression of ifn-l4 in human hepatic cells-a hepatoma cell line hepg2 and phhs. the observed effects might differ from the physiologic conditions in the hcv-infected hepatic cells in the complex whole-organ environment by the magnitude, duration, and efficiency of ifn-l4 induction. we also did not address the mechanism of intracellular retention and the function of the nonsecreted ifn-l4. however, we provide a comprehensive functional analysis of ifn-l4, a novel human interferon, and suggest its role in activation of interferon signaling, inhibition of cell proliferation, and induction of cell death in human hepatic cells. association of the ifnl4-deltag allele with impaired spontaneous clearance of hepatitis c virus cxcr3 surface expression in human airway epithelial cells: cell cycle dependence and effect on cell proliferation interferon-lambda4 (ifnl4) transcript expression in human liver tissue samples apoptosis in hepatitis c virus infection interferon-induced gene expression is a stronger predictor of treatment response than il28b genotype in patients with hepatitis c interferon-lambda rs12979860 genotype and liver fibrosis in viral and non-viral chronic liver disease necrosis, a well-orchestrated form of cell demise: signalling cascades, important mediators and concomitant immune response more than one way to die: apoptosis, necrosis and reactive oxygen damage predictive value of the ifnl4 polymorphism on outcome of telaprevir, peginterferon, and ribavirin therapy for older patients with genotype 1b chronic hepatitis c molecular definitions of cell death subroutines: recommendations of the nomenclature committee on cell death genetic variation in il28b predicts hepatitis c treatment-induced viral clearance interferon lambda 4 signals via the ifnlambda receptor to regulate antiviral activity against hcv and coronaviruses expression of the chemokine ip-10 (cxcl10) by hepatocytes in chronic hepatitis c virus infection correlates with histological severity and lobular inflammation a dominant negative mutant of 2-5a-dependent rnase suppresses antiproliferative and antiviral effects of interferon peroxisome dynamics in cultured mammalian cells selection on a variant associated with improved viral clearance drives local, adaptive pseudogenization of interferon lambda 4 (ifnl4) interferon-lambda4 genetic polymorphism is associated with the therapy response for hepatitis c virus recurrence after a living donor liver transplant ip-10 predicts viral response and therapeutic outcome in difficult-to-treat patients with hcv genotype 1 infection current and future therapies for hepatitis c virus infection antiproliferative effects of interferon alpha on hepatic progenitor cells in vitro and in vivo studying protein dynamics in living cells the emerging role of cxcl10 in cancer (review) interferon-lambda4 is a cell-autonomous type iii interferon associated with pre-treatment hepatitis c virus burden ifnl4-deltag genotype is associated with slower viral clearance in hepatitis c, genotype-1 patients treated with sofosbuvir and ribavirin endogenous intrahepatic ifns and association with ifn-free hcv treatment outcome global epidemiology of hepatitis c virus infection: new estimates of age-specific antibody to hcv seroprevalence impaired induction of interleukin 28b and expression of interferon lambda 4 associated with nonresponse to interferon-based therapy in chronic hepatitis c a comparison of the antitumor effects of interferon-alpha and beta on human hepatocellular carcinoma cell lines reply: subgroup differences in response to 8 weeks of ledipasvir/sofosbuvir for chronic hepatitis c ifn-lambda4: the paradoxical new member of the interferon lambda family a variant upstream of ifnl3 (il28b) creating a new interferon gene ifnl4 is associated with impaired clearance of hepatitis c virus constitutive expression of a 2¢,5¢-oligoadenylate synthetase cdna results in increased antiviral activity and growth suppression interferon lambda alleles predict innate antiviral immune responses and hepatitis c virus permissiveness measuring protein mobility by photobleaching gfp chimeras in living cells integration of interferon-alpha/beta signalling to p53 responses in tumour suppression and antiviral defence new aspects of ifn-alpha/beta signalling in immunity, oncogenesis and bone metabolism reduced ifnlambda4 activity is associated with improved hcv clearance and reduced expression of interferon-stimulated genes genetic variation in il28b and spontaneous clearance of hepatitis c virus hcv rna levels in a multiethnic cohort of injection drug users: human genetic, viral and demographic associations il28b genotype is associated with differential expression of intrahepatic interferon-stimulated genes in patients with chronic hepatitis c surfing the p53 network interferon: current status and future prospects in cancer therapy the work has been supported by the intramural research program ( key: cord-012418-6ralcn8p authors: schwanke, hella; stempel, markus; brinkmann, melanie m. title: of keeping and tipping the balance: host regulation and viral modulation of irf3-dependent ifnb1 expression date: 2020-07-07 journal: viruses doi: 10.3390/v12070733 sha: doc_id: 12418 cord_uid: 6ralcn8p the type i interferon (ifn) response is a principal component of our immune system that allows to counter a viral attack immediately upon viral entry into host cells. upon engagement of aberrantly localised nucleic acids, germline-encoded pattern recognition receptors convey their find via a signalling cascade to prompt kinase-mediated activation of a specific set of five transcription factors. within the nucleus, the coordinated interaction of these dimeric transcription factors with coactivators and the basal rna transcription machinery is required to access the gene encoding the type i ifn ifnβ (ifnb1). virus-induced release of ifnβ then induces the antiviral state of the system and mediates further mechanisms for defence. due to its key role during the induction of the initial ifn response, the activity of the transcription factor interferon regulatory factor 3 (irf3) is tightly regulated by the host and fiercely targeted by viral proteins at all conceivable levels. in this review, we will revisit the steps enabling the trans-activating potential of irf3 after its activation and the subsequent assembly of the multi-protein complex at the ifnβ enhancer that controls gene expression. further, we will inspect the regulatory mechanisms of these steps imposed by the host cell and present the manifold strategies viruses have evolved to intervene with ifnβ transcription downstream of irf3 activation in order to secure establishment of a productive infection. the interferon (ifn) system provides mammalian cells with a potent framework to fend off intruding pathogens. interferons are signalling molecules first discovered more than 60 years ago, when virus-infected cells were found to release soluble compounds that could interfere with establishment of virus infection [1] . since this initial discovery, we have come to understand the pivotal role of interferon signalling for the immune response to invading pathogens, from conveying the very first notice of intrusion to eliciting a well-tailored immune reaction suited to thwart the infection. today, we differentiate three classes of interferons based on the receptor they employ for signal transduction. more than a dozen genes encoding ifnα subtypes and a single ifnb1 gene give rise to the majority of type i ifns in humans. they are the first messenger molecules released upon detection of a pathogen by infected cells and by bystanders to initiate the intrinsic defence mechanisms and to further involve dedicated cells of the immune system (recently reviewed in [2, 3] ). ifnγ, the only type ii ifn, presents in the latent state, a part of the linker and the most c-terminal portion form auto-inhibitory elements (aies). phosphorylation of two serine-rich clusters (c1 and c2) induces conformational rearrangements of the aies and frees the iad to participate in protein-protein interactions with coactivators and for dimerisation via the phosphorylated plxis motif (p: hydrophilic, x: any amino acid). the protein further contains a nuclear localization signal (nls) and a nuclear exit signal (nes) that enable constitutive shuttling between the cytosol and nucleus. irf3 and irf7 are the most closely related members of the irf family with especially high conservation of the aie, enabling formation of functional heterodimers and providing the basis for the shared activation mechanisms as well as the similar mode of action [24] [25] [26] . only in plasmacytoid dendritic cells and macrophages, irf7 is continuously expressed at high levels and crucial for the control of type i ifn induction following tlr7 and tlr9 engagement [27] . since these immune cells produce the major share of type i ifns in an infected host organism, irf7 was termed the "master regulator" of ifn expression (reviewed in [28, 29] ). in most other cell types, however, irf7 is expressed at very low levels in absence of stimulation [30] . in contrast to the ubiquitous irf3, participation of irf7 molecules seems less relevant during the very first response to virus detection at initial viral entry sites, which are usually composed of epithelial or endothelial cells or fibroblasts. nevertheless, irf7 is an important contributor of the type i ifn response. upon ifnα/β signalling via the interferon-alpha/beta receptor (ifnar), irf7 expression is highly induced as part of the positive feedback regulation of the antiviral response; in other terms, irf7 is the product of an ifn-stimulated gene (isg) [31] . newly synthesized irf7 undergoes an activation similar to irf3 and in concert with irf3 further amplifies transcription of ifnβ. this behaviour can interfere with studies focused on irf3 activity or modulation thereof in later stages. irf3 is degraded in the course of its activity, as discussed below (see section 4.3), while irf7, though quickly degraded, is constantly expressed during stimulation [28] . this shifts the control of gene expression in fibroblasts, epithelial and endothelial cells from irf3-mediated regulation upon initial sensing of an infection towards irf7-mediated in later phases [32] [33] [34] . knockout experiments in mice have shown that lack consists of an n-terminal dna-binding domain (dbd) linked by a flexible region to the c-terminal irf association domain (iad). in the latent state, a part of the linker and the most c-terminal portion form auto-inhibitory elements (aies). phosphorylation of two serine-rich clusters (c1 and c2) induces conformational rearrangements of the aies and frees the iad to participate in protein-protein interactions with coactivators and for dimerisation via the phosphorylated plxis motif (p: hydrophilic, x: any amino acid). the protein further contains a nuclear localization signal (nls) and a nuclear exit signal (nes) that enable constitutive shuttling between the cytosol and nucleus. irf3 and irf7 are the most closely related members of the irf family with especially high conservation of the aie, enabling formation of functional heterodimers and providing the basis for the shared activation mechanisms as well as the similar mode of action [24] [25] [26] . only in plasmacytoid dendritic cells and macrophages, irf7 is continuously expressed at high levels and crucial for the control of type i ifn induction following tlr7 and tlr9 engagement [27] . since these immune cells produce the major share of type i ifns in an infected host organism, irf7 was termed the "master regulator" of ifn expression (reviewed in [28, 29] ). in most other cell types, however, irf7 is expressed at very low levels in absence of stimulation [30] . in contrast to the ubiquitous irf3, participation of irf7 molecules seems less relevant during the very first response to virus detection at initial viral entry sites, which are usually composed of epithelial or endothelial cells or fibroblasts. nevertheless, irf7 is an important contributor of the type i ifn response. upon ifnα/β signalling via the interferon-alpha/beta receptor (ifnar), irf7 expression is highly induced as part of the positive feedback regulation of the antiviral response; in other terms, irf7 is the product of an ifn-stimulated gene (isg) [31] . newly synthesized irf7 undergoes an activation similar to irf3 and in concert with irf3 further amplifies transcription of ifnβ. this behaviour can interfere with studies focused on irf3 activity or modulation thereof in later stages. irf3 is degraded in the course of its activity, as discussed below (see section 4.3), while irf7, though quickly degraded, is constantly expressed during stimulation [28] . this shifts the control of gene expression in fibroblasts, epithelial and endothelial cells from irf3-mediated regulation upon initial sensing of an infection towards irf7-mediated in later phases [32] [33] [34] . knockout experiments in mice have shown that lack of irf3 delays the immune response, while cells lacking irf7 respond early but are unable to fend off the infection without the signal amplification [27] . accordingly, together with the hefty feed-forward amplification of ifnα/β signalling, this feature is essential for the induction of distinct and diverse cytokine subsets by irf7, especially of ifnα, that differentiate the reaction and prime cellular immunity [28, 35] . for this reason, viruses 2020, 12, 733 4 of 33 both irf3 and irf7 are crucial for the rapid induction and potent establishment of the antiviral response [27, 34] . the crucial role of irf3 and the posttranslational changes it undergoes upon viral infection were first reported more than 20 years ago: upon stimulation, irf3 gets phosphorylated and accumulates in the nucleus where it interacts with the coactivators creb-binding protein (cbp)/p300 to specifically bind to virus-inducible enhancer elements and exerts transcriptional activation of the ifnb1 gene [21, [36] [37] [38] ( figure 2 ). since these first observations, our knowledge of the mechanism of action of irf3 has been greatly refined. viruses 2020, 12, x for peer review 4 of 33 of irf3 delays the immune response, while cells lacking irf7 respond early but are unable to fend off the infection without the signal amplification [27] . accordingly, together with the hefty feed-forward amplification of ifnα/β signalling, this feature is essential for the induction of distinct and diverse cytokine subsets by irf7, especially of ifnα, that differentiate the reaction and prime cellular immunity [28, 35] . for this reason, both irf3 and irf7 are crucial for the rapid induction and potent establishment of the antiviral response [27, 34] . the crucial role of irf3 and the posttranslational changes it undergoes upon viral infection were first reported more than 20 years ago: upon stimulation, irf3 gets phosphorylated and accumulates in the nucleus where it interacts with the coactivators creb-binding protein (cbp)/p300 to specifically bind to virus-inducible enhancer elements and exerts transcriptional activation of the ifnb1 gene [21, [36] [37] [38] (figure 2 ). since these first observations, our knowledge of the mechanism of action of irf3 has been greatly refined. by rna or dna sensors in the cytosol. activation of retinoic acid-inducible gene i (rig-i)-like receptors (rlrs) like rig-i induces aggregation of the mitochondrial adaptor protein mitochondrial antiviral signalling protein (mavs). detection of dna by the dna sensor cyclic gmp-amp (cgamp) synthase (cgas) activates production of the second messenger cgamp, which in turn induces dimerisation of the adaptor protein stimulator of interferon genes (sting) and its translocation from the endoplasmic reticulum (er) to the golgi apparatus. higher-order structures of the adaptor molecules recruit the kinase tank-binding kinase 1 (tbk1) which leads to their tbk1-mediated phosphorylation. irf3 is recruited to this platform and gets phosphorylated by tbk1 at key residues in the aie, relieving the auto-inhibition. activated irf3 heterodimerises and associates with the coactivators cbp/p300 after translocation into the nucleus, yielding a holocomplex with trans-activation potential. in parallel, the heterodimeric transcription factors p50-p65 (nf-κb) and atf2-c-jun (ap-1) are activated and enter the nucleus. first, p50-p65 is recruited to the enhancer element upstream of the ifnb1 gene, followed by atf2-c-jun and the irf3-cbp/p300 holocomplex. the assembled ifnβ enhanceosome promotes recruitment of the basal transcription machinery for the expression of ifnb1. hyperphosphorylation is the first step in the activation of irf3 as a functional transcription factor. in unstimulated cells, the irf3 protein exists as two forms, an unphosphorylated (i) and a basally phosphorylated (ii) form [39] . after viral infection, iκb kinase-epsilon (ikkε) and tank-binding kinase 1 (tbk1), two homologs of the inhibitor of nf-κb kinase (ikk), are activated when prrs convey the sensing of virus infection to their adaptor proteins, inducing conformational changes and interactions that lead to the formation of a new interaction surface (reviewed in [13] ). the kinase binds to this signal-induced adaptor platform and phosphorylates the plxis motif (p: hydrophilic, x: any aa) on the surface of the adaptor proteins [40, 41] . irf3 is then recruited to the platform via its recognition site for the phosphorylated plxis motif and gets further phosphorylated, giving rise to two more protein forms (iii and iv) whose appearance correlates with cbp interaction and ifnβ induction [39, [42] [43] [44] . the serine-and threonine-rich region within the c-terminal aie of irf3 contains two clusters of potential phosphoacceptor residues: cluster 1 (s385/s386) and cluster 2 (s396/s398-s402/t404/s405) [19] . first, tbk1 phosphorylates cluster 2 residues of monomeric irf3, and the additional negative charge induces a reorientation of the aies n-and c-terminal of the iad [20, 45] . this unmasks a hydrophobic binding pocket required for further protein interactions and renders the c-terminal tail (ctt) accessible for interactions [45, 46] . additionally, the conformational change is relayed to the dbd to enhance the dna-binding affinity. the residues of cluster 2 are functionally largely redundant in terms of phosphorylation-mediated irf3 activation, though phosphorylation of s396 seems to predominate in vivo [37, 47] . the alternative use of phosphorylation sites could be the reason why some studies found that mutation of irf3 s396 to alanine sometimes retained biological function (for example in [48, 49] ). second, and induced by the first modification, irf3 gets further phosphorylated at s386 which is pivotal for dimerisation [45, 50] . consistently, 100% of irf3 dimers generated in vitro by incubation with tbk1 are phosphorylated at both clusters [45] . in contrast to cluster 2, added phosphate groups at cluster 1 residues have distinct effects: addition of a phosphate group at s386 promotes dimerisation of irf3 and strengthens interaction with the coactivator cbp, while phosphorylation of s385 negatively affects both interactions [19, 50, 51] . by relief of the auto-inhibition, phosphorylated irf3 can dimerise and associate with the coactivators cbp/p300 to form an active holocomplex. irf3 can also be rendered constitutively active by exchange of the five phosphoacceptor sites in cluster 2 to aspartic or glutamic acid residues (irf3-5d or -5e, respectively), and these mutants display a strong tendency to acquire the cluster 1 phosphorylation and dimerise [19, 45] . since the first description of irf3 dimerisation, it is generally noted as the second step of activation after phosphorylation [19] . formation of dimers requires homotypic interactions of the iad with a second phosphorylated irf3 molecule. structural studies revealed that phosphorylation of irf3 in fact modifies a plxis motif in the ctt, similar to the motif initiating recruitment of irf3 to the adaptor platform [42] . enabled by the negative charge at s386 after phosphorylation, the extended ctt can interact with the plxis-binding surface of a second phosphorylated irf3 monomer to form a domain-swapped dimer (indicated in figure 2 ). zhao and colleagues further proposed that irf3 dimerises at the adaptor platform to regain stability, but this was not yet confirmed. due to repulsion caused by the newly acquired negative charges, irf3 proteins could also dissociate from the adaptor complex before dimerisation and converge subsequently either (i) in the cytoplasm, (ii) after translocation into the nucleus or (iii) after engagement of coactivators during recruitment to the enhancer. further, the phosphorylation-induced rearrangement of the aie unmasks a hydrophobic binding site of irf3 and enables the interaction with transcriptional coactivators [21, 37, 52, 53] . association of irf3 with the histone-modifying lysine acetyltransferases (kats) creb-binding protein (cbp, or kat3a) and/or p300 (kat3b) in form of a holocomplex is pivotal for the ability of irf3 to trans-activate ifnb1 expression [22, 54, 55] . these coactivators are large proteins that contain several folded domains and additionally a big share of intrinsically disordered regions that allow for specific binding to numerous factors upon interaction [56] . their flexible structure enables the regulation of gene transcription by integrating the cues from several hundred transcription factors, yielding cbp/p300 the designation "master" coactivators of transcription. the closely related proteins cbp and p300 are usually regarded as functionally redundant-thus often referred to in combination as cbp/p300-and due to this assumption, studies characterising irf3 interactions assessed association of one or the other [57] . however, there is growing evidence that their activity is overlapping but can be distinctly involved in regulation of different pathways, as exemplified by brain development [58, 59] . considering that both cbp and p300 can participate in holocomplex formation with irf3 but do so with different shares [38, 52] , it would be interesting to determine whether engagement of cbp versus p300 is specific or coincidental. potentially, the contribution of the individual coactivators could change during the different phases of ifnb1 expression depending on integrated signals. to exercise its function as a transcription factor, it is pivotal that irf3 can reach the nucleus. in fact, the latent protein already shuttles between the cytoplasm and nucleus in unstimulated conditions driven by its nls and nes. as the influence of the nes seems to prevail the import, the main share of irf3 molecules localises to the cytoplasm [22] . the constitutive export of irf3 was shown to involve exportin 1 (crm1) [21, 22] , and the import through nuclear pore complexes involves the importins karyopherin (kpn) subunit α2 (kpna2), kpna3 and kpna4 (or qip1) [22, 60] . upon infection, phosphorylation of the aie and accompanying structural rearrangements enable irf3 to associate with cbp, and this interaction retains the activated holocomplex in the nucleus [22] . interestingly, in vitro experiments from several groups analysing the interaction between irf3 and the irf-binding domain (ibid) in the c-terminus of cbp have shown that independently of dimerisation, monomeric irf3 can interact with cbp once the auto-inhibition is relieved [22, 45, 46, 51] . moreover, in vitro analyses by chen and colleagues implied that the presence of cbp promotes oligomerisation of irf3 molecules with the required phosphomimetic mutations that enable dimerisation, while the mutant proteins stayed monomeric without cbp [51] . in a cellular scenario wherein irf3 dimerises directly after acquiring the required modifications in the vicinity of the adaptor platform in the cytoplasm, this interaction might not be relevant, though. characterisation of the holocomplex demonstrated that the homodimer of irf3 is more stable than the holocomplex of irf3 dimer plus p300 [53] , and the association of irf3 dimers with cbp is stronger than that of monomeric irf3 [51] , viruses 2020, 12, 733 7 of 33 favouring a model wherein irf3 first dimerises, and dimers subsequently interact with cbp. however, without determination of the precise temporal succession of interactions, this leaves the possibility that a phosphorylated irf3 monomer enters the nucleus and interacts with cbp/p300 before it encounters a second irf3 molecule to dimerise and swiftly induce a response, as suggested before [38] . in the end, the coactivator needs to associate with dimeric irf3 because only this can induce activation of the kat activity that is later required for assembly of the pre-initiation complex, as demonstrated for p300 [61] . several groups noted exceptions to the common model of irf3 activation followed above, drawing attention to the fact that assessment of the conventional signs like dimerisation that reflect the intermediate states is not always sufficient to infer on the downstream biological response [48, 62, 63] . due to these discrepancies we suggest to differentiate between phosphorylated irf3 that is relieved of the auto-inhibition and can "actively" participate in subsequent steps and irf3 in the holocomplex that can exercise its trans-activation potential as a transcription factor to "actively" induce ifnb1 expression. in fact, some of these studies assessed the activity of irf3 in terms of induction of isg56 [48, 62] , which intermingles the irf3-dependent regulation of type i ifns with directly irf3-regulated isg expression [64] . to our knowledge, it remains to be determined whether the form of irf3 induced for up-regulation of specific isg promoters is the same as the form of irf3 able to induce ifnb1 expression. moreover, reports clearly reflect that there are different extents of irf3 activity, and the protein might not yet be fully activated when initiating a first wave of ifnb1 expression. instead, irf3 might acquire modifications and engage in distinct interactions in different waves of the type i ifn response until it is fully activated. in line with this, we noticed several reports referring to "full" activity of irf3 in later loops of the response, for example enabled by phosphorylation at both clusters of the aie or determined only in the presence of ifnar signalling [55, 65] . this aligns with the kinetics of the ifnβ response we will briefly revisit below (see section 3.3), wherein only a part of the ifnb1 alleles are engaged in the early response. expression of ifnb1 is probably the best studied model of induced eukaryotic gene regulation. it exemplifies how factors activated by specific signals cooperate to prompt a defined expression programme, as reviewed before [17, [66] [67] [68] . upon viral stimulation, a multicomponent complex of transcription factors, coactivators and architectural proteins forms at the enhancer sequence upstream of the ifnb1 gene promoter. this higher-order nucleoprotein complex, termed the ifnβ enhanceosome, works as a molecular switch that turns on upon virus-induced activation of a specific set of transcription factors [69, 70] . when completed, the enhanceosome in turn recruits factors with chromatin-modifying activities as well as basal eukaryotic transcription factors to the nearby transcription start site (tss) to initiate gene expression. the assembly is regulated at several levels and highly specific due to an elaborate organisation of transcription factor binding motifs, the required set of activated transcription factors and architectural proteins and finely coordinated interactions of the individual participants as well as of the arising composite structures. from just slightly upstream of the tss of the human ifnb1 gene, a precisely arranged sequence of cis-acting dna constitutes the key enhancer element that defines ifnb1 expression. the ifnβ enhancer itself is accessible yet directly flanked by two nucleosomes, one of them covering the tss and a tata box [71] . this setup strategically restricts the access of the transcription machinery in latent conditions [72, 73] . the enhancer can be divided into four positive regulatory domains (prds) in the order iv-iii-i-ii that are discussed in detail in [68] . of note, however, is the overall architecture that is essential for the precise recognition and following interplay of the array of core transcription factors [74] . the prd farthest away from the promoter, prdiv, features motifs for binding an ap-1 heterodimer formed by activating transcription factor 2 (atf2) and c-jun of the basic-region leucine zipper (bzip) family [66, 75] . binding of the heterodimer over homodimers is favoured by the intrinsic architecture of the enhancer and important for subsequent interactions [76] . the last prd in the sequence of the enhancer, prdii, is recognised by a p50-p65 or p50-p50 nf-κb dimer [77] [78] [79] . prdiii and prdi in the middle section feature in total four overlapping irf-binding elements (irf-es) of the consensus sequence 5 -aanngaaa-3 that form two composite binding sites enabling binding of two irf dimers [34, 36, 80, 81] . this region was also termed p31 because these two prds act as a functional unit, an enhanson [38] . recent in-depth analysis of the binding requirements of different irf members suggests that, in addition to a slight variation of the core gaaa recognition motif, the flanking regions along with the spacing in between the irf-es contribute to specific binding of distinct irf family members, here favouring recruitment of irf3 and irf7 [81, 82] . the nucleotide sequence of the ifnβ enhancer causes a bent conformation of the dna helix in inactive conditions and in this way drives cooperative binding of the transcription factor heterodimers upon stimulation [68] . after stimulus-induced activation, the transcription factors translocate into the nucleus. first, the nf-κb dimer p50-p65 is recruited and nucleates assembly [71] . association of nf-κb is the most limiting factor of enhanceosome assembly, and to optimise recruitment, it is initially targeted to three loci at distinct chromatin regions via alu elements, also termed nf-κb reception centres (nrcs) [71, 83, 84] . the transcription factor thpok (t-helper-inducing poz/krüppel-like factor, also zbtb7b) binds cooperatively with nf-κb to these regions, and oligomerisation of thpok mediates interchromosomal interactions of the nrcs with the ifnβ enhancer to deliver nf-κb [83, 84] . alongside nf-κb, the architectural dna-binding high mobility group protein i(y) (hmg i(y), also hmga1) binds at two sites to the minor groove in prdii and straightens the dna conformation, thereby supporting interaction of nf-κb [69, [85] [86] [87] [88] . subsequently, the remaining virus-induced transcription factors are recruited. a second hmg i(y) molecule interacts with atf2-c-jun to promote binding of the heterodimer to prdv by straightening the dna helix [69, 88, 89] . additionally, nf-κb mediates capture and binding of the irf3-cbp/p300 holocomplex [38, 90] . in vitro, irf3 displays a surprisingly weak affinity for p31, which led to the former model in which irf1 is the critical irf member in ifnb1 induction because it freely associates with the enhancer sequence [91] [92] [93] . however, irf1 was then found to be dispensable for ifnb1 expression upon prr activation, which is congruent with its expression pattern as an isg [94] [95] [96] . instead, when phosphorylation releases the auto-inhibition of irf3 and enables interaction with cbp/p300, the coactivator strongly increases the affinity of irf3 for dna and enables association of the complex [18, 38, 54, 55] . further cooperativity is mediated by the p31 sequence that ensures concerted binding of atf2-c-jun and irf3 [97, 98] and by cbp/p300-mediated interactions within the enhanceosome complex [38, 90] . clustering of irf3 target regions with binding motifs for nf-κb or other transcription factors along with low chromatin accessibility in steady-state conditions was recently described as a common feature of irf3-dna interactions, implying an obligatory collaborative binding mode of irf3 that promotes access to chromatin [82] . at the ifnβ enhancer, a total of four irf3/7 molecules bound to four irf-es in tandem are required to induce gene expression of a luciferase-based ifnβ-reporter plasmid in the commonly used human embryonic kidney cell line hek293t [99] . structural analysis suggests that one irf dimer binds on one side of the duplex, occupying the first and the third irf-e, and the second dimer binds from the opposite side, binding to the second and fourth irf-e [81] . remarkably, the dbds of the eight transcription factors barely interact with each other despite the great overlap of their binding elements [81] . instead, the high level of cooperativity between binding events is achieved by the specific order of individual response elements that allows for binding-induced conformational changes of the dna to transmit allosteric effects [68, 74, 81, 100] . molecular dynamics simulations by pan and nussinov further showed that the combination of overlapping recognition viruses 2020, 12, 733 9 of 33 elements with an alternation between consensus and non-consensus motifs optimises the specific interactions by enhancing or restricting binding of neighbouring transcription factors. moreover, the signal integration by cbp/p300 that interacts with each transcription factor through different domains is essential for the functionality of the enhanceosome [90] . in addition to the pivotal architectural role of recruiting irf3 and mediating interactions with the transcription machinery as coactivator, cbp/p300 also participates in the subsequent chromatin remodelling in its vicinity [71, 101, 102] . as implied above, however, the precise number and identity of coactivator molecules participating in the formation of the active irf3-cbp/p300 holocomplex as well as in the enhanceosome is unknown. potentially, they present a module for further signal integration by participating in dynamic compositions. further, also the subtle modulation by the two hmg i(y) proteins is required for the maximal level of expression [86] , though their participation in the final assembly is controversial as previously discussed [17] . in line with the high synergy during assembly, the ifnβ enhanceosome is extraordinarily stable and enables multiple rounds of re-initiation in vitro [70, 88] . due to the binding-induced conformational changes of the dna foundation, the fully assembled structure encompasses a straight segment of dna covered by eight specifically bound transcription factors all of which interact with the essential coactivator cbp/p300. this final ifnβ enhanceosome presents a single new, continuous activating surface and provides the basis for the association of the basal transcription apparatus. before transcription of the ifnb1 gene can commence, the chromatin landscape needs to be modified so that the pre-initiation complex can be assembled and access the tss. first, the ifnβ enhanceosome mediates the transient recruitment of a complex of p300/cbp-associated factor (pcaf, or kat2a) and general control nonderepressible-5 (gcn5 or, kat2b) to the promoter and induces the remodelling, starting with gcn5-mediated acetylation of nucleosomes and hmg i(y) in its vicinity [70, 71, 103] . acetylation of hmg i(y) at k71 by gcn5 strengthens the association of nf-κb and thereby further stabilises the enhanceosome [70, 71] . next, the modification of the histone tails of nucleosomes at h3 and h4 promotes recruitment of the chromatin remodelling brg1-or brm-associated factor (baf) complex of the swi/sfn family [71, 87, 90, 102, 103] . in parallel, general eukaryotic transcription factors (tfs) tfiia, tfiib, tfiie, tfiih, tfiif and upstream stimulatory activity (usa) cofactors assemble at the promoter along with the rna polymerase ii (rnap ii) holoenzyme [71, 87, 101] . stimulated by the environment rich in acetylated histones, the atpase brahma-related gene 1 (brg1 or smarca4) of the baf complex induces a conformational change of the nucleosome at the tss [71, 104] . now, the tata box at the tss is available for binding by the tata box-binding protein (tbp) and allows association of the tfiid complex [71, 103] . notably, tfiid binds here after the rnap ii complex, as opposed to the classical sequence of events in assembly of the pre-initiation complex [105] . association of tfiid induces a bend of the dna helix so that the nucleosome covering the tata box slides 36 bp downstream of its latent position [73, 106] . with this, the arrangement of the pre-initiation complex can be completed, and ifnb1 transcription can begin [17, 107] . notably, while the rapid induction of the type i ifn response is crucial for the host defence, the first round of ifnb1 expression yields only low levels of ifnβ, as discussed by ford and thanos [17] . briefly, after stimulation of a cell population, ifnb1 is initially expressed only by a fraction of cells and from only one allele due to a stochastic phenomenon that indicates a limiting amount of required cellular factors [108] [109] [110] . the initial recruitment of nf-κb to other loci that collect the available transcription factor and transfer it to the required binding sequence highlights the p50-p65 heterodimer as the most limiting factor [83, 84] . the weak initial signal of secreted ifnβ induces high-level expression of irf7, and newly synthesized irf7 promotes enhanceosome assembly and ifnb1 transcription from the remaining allele and in more cells [83] . finally, the weak initial ifnβ signal is then amplified to eventually induce the cytokine storm of the antiviral response. the expression of ifnb1 is, at least in murine cells, tightly regulated by different forms of nf-κb dimers in the initial response to infection, as reviewed by balachandran and beg [111] . at resting conditions, p50 homodimers associate with the ifnβ promoter region and repress basal transcription in the absence of appropriate stimulation [66, 112, 113] . in addition, a binding site for nf-κb regulatory factor (nrf) overlaps the nf-κb binding element in prdii and negatively affects basal transcription [114, 115] . in line with a primary inhibitory effect, knockout of murine p50 does not affect induction of ifnb1 expression after virus infection [116] . however, the p50-imposed down-regulation in resting cells is competed with by p50-p65 dimers that are constitutively activated by ikkβ and cycle between the cytoplasm and nucleus [117] . this low level activity of p65 supports a basal level of ifnb1 expression that ensures rapid and robust responsiveness on demand, while the negative regulation by p50 ensures specificity [113] . after stimulation, p50-p65 is required to assist in the early recruitment of irf3 together with cbp/p300 to support ifnb1 expression [90, 113, 118] . only at high levels of irf3 activity, i.e., at the peak of activity later in infection or brought about by expression of the constitutively active irf3-s396d, p50-p65 is dispensable for ifnb1 transcription [118] . subsequent to the initial recruitment, the increasing levels of active irf3 and irf7 can fully assume the regulation of ifnβ production, and nf-κb proceeds to regulate expression of pro-inflammatory genes [118] . the p50-p65-mediated basal expression of ifnb1 raises the question how recruitment of the transcription machinery is achieved at resting conditions, considering that irf3 is missing to participate in a highly cooperative assembly as described above. moreover, since the type i ifn system is not 100% conserved between mice and humans, and all of these studies were carried out in murine systems, it remains to be confirmed whether the exact same mode applies for the participation of nf-κb in the human system. in addition to the proximal enhancer, dna elements further away from the promoter are involved in ifnb1 transcription, though again, most studies have thus far focused on the murine system. in non-infected mouse cells, a proximal region of the ifnb1 locus that contains binding sites for the transcription factor yin yang 1 (yy1) was shown to mediate association with pericentromeric heterochromatin (pch), a feature related to gene silencing [119] . after infection, the ifnb1 locus is repositioned away from pch, and this observation correlated with transcriptional activation of the promoter shortly thereafter. josse and colleagues suggested that this effect could be mediated by binding of yy1 to the proximal promoter. furthermore, yy1 interacts also in the absence of viral infection in mice with the signal transducer and activator of transcription 1 (stat1), one of the main factors mediating isg expression [35] , implying important roles of yy proteins at different stages during infection [120] . in the human cell, yy1 and yy2 regulate enhanceosome formation from yy-binding site-containing regions far upstream of the tss of the ifnb1 gene at −3 kb and −2 kb, termed dnase i hypersensitive sites 1 (hs1) and hs2, respectively [121] . yy1 and yy2 seem to modulate each other, with yy2 antagonising the negative effect of yy1. in analogy to the murine ifnβ promoter region, this long-range interaction was suggested as requirement for the recruitment of gcn5 to initiate chromatin remodelling at the ifnβ promoter [121] [122] [123] . a further dna element regulating ifnb1 transcription is the long-range enhancer l2, which was first identified in human cells but is conserved in mice [124] . l2 is activated upon viral stimulation and recruits irf3 and rnap ii dependent on the irf-e within its interferon-stimulated response element (isre). this induces expression of a non-coding rna from the enhancer, and this enhancer rna (erna) in turn supports ifnb1 expression. however, the mode of action of the erna was not yet determined. the rapid induction of the antiviral state is critical to overcome a nascent infection. while an insufficient response would be beneficial for the pathogen, overshooting activation of the immune system can cause severe damage to the organism. therefore, several mechanisms are implemented by the host organism to regulate irf3 at all levels, from moderating the potential to activate irf3 from its latent state, to promoting its participation in formation of the ifnβ enhanceosome, to supporting its activity during ongoing stimulation, through to terminating the response with deliberate timing. factors that contribute to the finely tuned ensemble of modulators can be continuously active, induced or inhibited upon stimulation. a wide array of molecular mechanisms that modulate the biological activity of irf3 are deployed, including protein-protein interactions, regulatory rnas and common as well as non-canonical posttranslational modifications. to illustrate the abundance of strategies, we will include also proteins expressed predominantly in immune cells. in particular, mechanisms aiming at dampening the activity of irf3 were usually identified in macrophages, highlighting the importance to keep the response primed but low until the full activation becomes necessary. since posttranslational modifications of irf3 and especially phosphorylation and ubiquitination were reviewed before [125, 126] , we will instead focus on how the host factors engage in the different steps to modulate the biologic activity of irf3 ( figure 3 ). viruses 2020, 12, x for peer review 11 of 33 system can cause severe damage to the organism. therefore, several mechanisms are implemented by the host organism to regulate irf3 at all levels, from moderating the potential to activate irf3 from its latent state, to promoting its participation in formation of the ifnβ enhanceosome, to supporting its activity during ongoing stimulation, through to terminating the response with deliberate timing. factors that contribute to the finely tuned ensemble of modulators can be continuously active, induced or inhibited upon stimulation. a wide array of molecular mechanisms that modulate the biological activity of irf3 are deployed, including protein-protein interactions, regulatory rnas and common as well as non-canonical posttranslational modifications. to illustrate the abundance of strategies, we will include also proteins expressed predominantly in immune cells. in particular, mechanisms aiming at dampening the activity of irf3 were usually identified in macrophages, highlighting the importance to keep the response primed but low until the full activation becomes necessary. since posttranslational modifications of irf3 and especially phosphorylation and ubiquitination were reviewed before [125, 126] , we will instead focus on how the host factors engage in the different steps to modulate the biologic activity of irf3 ( figure 3) . in addition to the essential steps of irf3 activation delineated above, various protein-protein interactions and additional posttranslational modifications were demonstrated to support the biological activity after induction. before stimulation, for example, protein phosphatase 1 (pp1) removes the phosphate groups at s385 and s396 in macrophages and consequently attenuates ifnβ production [127] . early after tlr-or rlr-mediated stimulation, however, activity of pp1 is down-regulated to increase the pool of active irf3 and augment the immune response [127] . also first identified in murine macrophages, the lysine methyltransferase nuclear receptor-binding set domain 3 (nsd3) targets nuclear irf3 after viral stimulation and modifies irf3 by adding a single methyl group at k366 [128] . this modification promotes the dissociation of phosphorylated irf3 from an isoform of pp1 catalytic subunit γ, thereby hindering the inhibitory effect of pp1 and maintaining the activated state of phosphorylated irf3. similarly, type i ifn up-regulates the expression of the long non-coding rna lnclrrc55-as, an antisense transcript to the gene of leucine-rich repeat-containing protein 55, which supports irf3 phosphorylation in macrophages by inhibiting an inhibitor [129] . in the cytosol, lnclrrc55-as associates with the protein phosphatase methylesterase 1 (pme-1), which in turn promotes the interaction of pme-1 and the protein phosphatase 2a (pp2a). pp2a is an inhibitor of irf3 signalling, but lnclrrc55-as mediates its demethylation and inactivation to maintain irf3 activity [129] [130] [131] . additionally, stability of the irf3 protein is promoted within the loop of the type i ifn response by covalent conjugation of newly synthesised isg15 to irf3 on k193, k360 or k366 mediated by herc5 (hect and rld domain-containing e3 ubiquitin protein ligase 5) [132, 133] . this modification, termed isgylation, disturbs the interaction between irf3 and peptidyl-prolyl isomerase 1 (pin1), delays the proteolytic degradation of irf3 and in this way prolongs the immune response [132] [133] [134] . the dual use of irf3 k366 for methylation by nsd3 versus isgylation by herc5, both of which contribute to protein stability, hints at a complex network that also modulates the modulators. while inactive irf3 already shuttles between the cytosol and nucleus in latent conditions, the translocation becomes crucial after its activation in order to exercise the trans-activation activity. just recently, cai and colleagues reported that the ubiquitin specific peptidase 22 (usp22) promotes the antiviral response from the cytoplasm by deubiquitinating importin kpna2 [60] . after viral infection, kpna2 associates with irf3 for nuclear import, and usp22 promotes this critical step by stabilizing kpna2. importin-mediated translocation is additionally regulated by the widely expressed transcription regulators yes associated protein 1 (yap1), yap2 and yap4: yap1/2/4 associate with latent as well as activated irf3 and block further interactions required for dimerisation or nuclear import [135] . after virus infection, however, activated ikkε phosphorylates a conserved motif of the yaps, which triggers their lysosomal degradation and reliefs the yap-mediated inhibition. further, an additional phosphate group within the dbd of irf3 at s97 is important for the nuclear translocation after viral stimulation [136] . this modification inhibits the nuclear import, and mass spectrometry revealed that about a fifth (18.2%) of irf3 molecules carry it at latent conditions when exogenously expressed in hek293. upon viral stimulation, the dual-specificity protein phosphatase and tensin homolog (pten) removes this phosphate group and consequently the negative regulation of irf3 [136] . finally, to keep irf3 in the nucleus, dna-pk is activated in response to virus infection and phosphorylates irf3 at t135 [137] . this phosphate moiety mediates the nuclear retention and delays proteolysis of the active transcription factor, thereby prolonging the irf3-driven response. the association of cbp and irf3 in the nucleus is supported by the constitutively active enzyme glutaredoxin-1 (glrx or grx1), which acts in the cytoplasm [138] . in resting cells, inactive irf3 is s-glutathionylated and this modification would impede the essential interaction of irf3 and cbp/p300. however, glrx removes this modification after infection and thus supports irf3 activity. while the deglutathionylation of irf3 is independent of its phosphorylation or dimerisation state, the accompanying structural changes could be involved in the recruitment of glrx to remove the modification after initial activation events [138] . within the nucleus, the interaction of irf3 and cbp is further promoted by a ubiquitously expressed subunit of the endosomal sorting complex required for viruses 2020, 12, 733 13 of 33 transport (escrt)-ii [139] . after virus infection, a fraction of the escrt-ii subunit ell-associated protein of 30 kda (eap30, also known as sfn8) localises to the nucleus where it interacts with activated irf3 and cbp and promotes binding of the holocomplex to target gene promoters. contrasting this, the protein argonaut 2 (ago2), a component of the rna-induced silencing complex, interacts with the iad of irf3 and inhibits its association with cbp in the nucleus in latent conditions [140] . upon viral infection, however, ago2 is exported from the nucleus, and its inhibition is revoked to promote ifnβ production during infection [140] . a further mechanism of signalling amplification is set into motion by type i ifn-stimulated production of the e74 like ets transcription factor 4 (elf4). elf4 is recruited to sting and activated by tbk1 similar to irf3 after viral stimulation, though without affecting irf3 activation itself [141] . activated elf4 enters the nucleus and binds to the ifnβ promoter region via ets/irf composite binding elements (eices). dna binding of elf4 synergises with binding of irf3, and thus promotes enhanceosome formation. the critical role of elf4 in the feed-forward amplification of the murine antiviral immune response was demonstrated in vivo, and notably, it is independent of the signalling in plasmacytoid dendritic cells [141] . in contrast, a mechanism that due to the predominating expression of both factors in tissues of the immune system applies exclusively to immune cells is the association of irf8 together with the transcription factor pu.1 (also spi1, spi-1 proto-oncogene) to the ifnβ promoter region mediated by eice [142] . li and colleagues proposed that irf8 and pu.1 support the rapid induction of transcription by forming a scaffold at the ifnβ enhancer to facilitate recruitment of activated irf3 [142] . a striking feature of irf3 modulation is reflected by mechanisms that are installed not to plainly heighten or terminate the biological activity of irf3 but to moderate its extent. some of the mechanisms delineated here affect the latent protein, others specifically target activated irf3 after viral stimulation and still others act on both. macrophages are a prime example in deploying numerous modulators to dampen fluctuations of irf3 activity until a certain threshold is passed and further to restrain the response once unleashed. for example, the ubiquitin-protein ligase e3c (ube3c) mediates k48-linked ubiquitination of irf3 and irf7, thereby targeting the master transcription regulators in dendritic cells for proteolysis, irrespective of their activation state [143] . in this way, ube3c helps to maintain low amounts of ifn production in resting conditions and additionally restrains the magnitude of the response after stimulation [125, 143] . another e3 ubiquitin ligase, tripartite motif-containing 21 (trim21 or ro52), was described by two groups to be important for regulation of irf3 but with opposing findings as already discussed by sin [125] : higgs and colleagues reported trim21-dependent mediation of irf3 degradation 48 hours after infection [144] , whereas yang and colleagues found that nine hours after infection, trim21 interferes with the interaction between irf3 and pin1 and in this way prevents pin1-mediated ubiquitination and degradation of irf3 to sustain the immune response [145] . sin suggested that, in addition to effects potentially imposed by different cell lines, the observation at different times of ongoing infection could have contributed to the contrasting findings [125] . stability of irf3 is further modified by addition or removal of small ubiquitin-like modifier (sumo) proteins. in hek293 cells, endogenous irf3 is sumoylated at k70 and k87 in its dbd, and viral infection slightly increases sumoylation [146] . the widely expressed human sentrin/sumo-specific protease 2 (senp2) removes these sumo moieties and in this way conditions irf3 for k48-linked ubiquitination at the same residues and subsequent degradation, thereby dampening the antiviral response [146] . senp2 targets wild-type irf3 as well as the constitutively active irf3-5d mutant, suggesting that the sumoylation initially masks the protein from degradation to prolong its activity but that this modification is constantly countered by senp2 activity. in addition, stimulus-induced sumoylation of murine irf3 at k152 was reported to attenuate the ifnβ response irrespective of the protein's phosphorylation status, though the mediating proteins are unknown [147] . regulation of auto-inhibition aside, phosphorylation of irf3 can contribute to the down-regulation of its activity also at subsequent steps. when screening the human kinome for phosphorylation events during nucleic acid sensing, meng and colleagues identified mammalian sterile 20-like kinase 1 (mst1, also stk4) as an inhibitor of ifnβ promoter induction and confirmed the effect of mst1 in knockout mice [148] . mst1 suppresses activation of tbk1 and ikkε and thereby inhibits the phosphorylation-dependent activation of irf3, but it also targets irf3 directly for deactivation by phosphorylating two sites of irf3 (t75, t253). demonstrating that this effect is independent of the inhibition of the upstream kinases by mst1, introduction of a single phosphomimetic mutation, t253d, is sufficient to impair the ability of the constitutively active irf3-5d to dimerise after stimulation [148] . a further level of negative regulation of ifnβ signalling is imposed by the ubiquitously expressed fas-associated factor 1 (faf1), which interacts with importin 5 (iop5, also kpnb3) in resting as well as stimulated cells [149] . after viral stimulation, irf3 increasingly associates with ipo5 for nuclear import, but this is dampened by the faf1-ipo5 interaction which consequently reduces the translocation of phosphorylated irf3 and the induction of ifnb1 expression. in the nucleus, formation of the irf3/p300 complex and p300-mediated acetylation of irf3 after virus infection is assisted by bromodomain-containing 3 (brd3) [150] . generally, interaction of brd3 with p300 promotes recruitment of the holocomplex to the ifnβ enhancer and facilitates ifnb1 transcription. this supporting mechanism seems to come with a time limit, however, as analysis in macrophages revealed that viral stimulation specifically down-regulates brd3 abundance and thereby terminates its support [150] . similarly, zhang and colleagues recently reported stimulation-induced non-canonical k6-linked ubiquitination at three positions in the dbd of irf3 (k39, k98, k105) and demonstrated that this modification is essential for the dna-binding capacity of irf3 in macrophages and in hek293t cells [151] . as a counter measure, virus-infected cells additionally up-regulate expression of the ovarian tumour domain-containing deubiquitinase (otud1), and otud1 then removes this ubiquitination from irf3, resulting in reduced irf3-binding to the ifnβ promoter region at later times [151] . to allow moderation of enhanceosome assembly after formation of the active irf3-cbp/p300 complex, other dna-binding proteins were reported to interact with motifs contained within the ifnβ enhancer sequence. the first irf-e overlaps with a consensus binding site for an activator of pro-inflammatory responses in macrophages, nfat5 (nuclear factor of activated t cells 5), which is conserved between human and mouse promoter sequences [152] . nfat5 constitutively forms dimers that can bind to the ifnβ enhancer region and competes with irf3 due to the overlapping recognition sequence, resulting in limited recruitment of irf3 to the enhancer and thus limited promoter induction. in a similar way, the transcription regulator maf bzip transcription factor b (mafb) binds in macrophages to the ifnβ enhancer sequence mediated by ap-1-like sites [153] . in resting conditions, mafb is a weak positive regulator of basal ifnb1 transcription. upon stimulation and activation of irf3, however, it impairs the interaction between the coactivators and antagonises enhanceosome formation. this dual role was suggested to allow the system to deal with fluctuations in irf3 activity [153] . during the course of the innate immune response, the signal that started it all has to be switched off again to allow other messengers to refine the defence line. the first mechanism to terminate the irf3-dependent response is set into action directly during the activation of irf3 by phosphorylation of the c-terminal aie [37] . in addition to releasing the auto-inhibition, the modification represents a signal for degradation of the protein, and so activated irf3 has a shorter half-life than the latent form [37, 154] . in this way, turnover of the activated protein by degradation rather than additional activation of repressors is an integral feature responsible for ending ifnb1 expression [155] . several molecules have been reported to mediate ubiquitination of irf3 in order to induce its depletion after initial stimulation [126] : pin1, foxo1 [156] , trim26, rbck1 and trim21 (see above, section 4.2) all mediate k48-linked ubiquitination and subsequent degradation of activated irf3. for example, the peptidyl-prolyl isomerase pin1 specifically mediates degradation of the activated transcription factor by recognition of a further phosphorylated motif (s339phos-p340) irf3 acquired during stimulation [134] . additionally, viral infection promotes the nuclear localisation of the e3 ubiquitin ligase trim26, allowing trim26 to bind phosphorylated irf3 in the nucleus and promote its k48-linked ubiquitination and degradation [157] . similarly, production of rbcc protein interacting with pkc1 (rbck1), another e3 ubiquitin ligase, is induced by viral stimulation and targets irf3 for ubiquitination [158] . furthermore, caspase-8 (casp8) is activated by cytosolic rig-i-dependent signalling and cleaves irf3 at a recognition motif between dbd and iad ( 118 sqpd 121 ), inducing ubiquitination and degradation of the fragments [159] . in parallel to the activation of irf3, viral stimulation also activates inhibitors of irf3. the transcriptional regulator krüppel-like factor 4 (klf4), which is widely expressed in human tissues, increasingly localises to the nucleus after viral infection where it binds to the ifnβ promoter region [160] . this reduces recruitment of irf3 and thus inhibits induction of ifnb1 expression. in immune cells, the lysine acetyltransferase kat8 was identified by an sirna-screen as a negative regulator of antiviral innate immunity [161] . viral infection promotes kat8-mediated acetylation of irf3 at k359, independent of the phosphorylation and dimerisation status of irf3, and this modification interferes with binding of irf3 to the ifnβ enhancer. a potential effect on assembly of the irf3-cbp/p300 holocomplex was not characterised, however, leaving the exact step of interference to be determined. a further line of down-regulation is introduced by the expression of isgs that negatively modulate irf3 activity. considering the multitude of induced isgs [162] , it is not surprising that they target various levels to terminate ifnb1 transcription. nuclear import is repressed by the increasing interaction of irf3 with the ifn-inducible dead-box rna helicase ddx56, as this interaction competes with the association of irf3 and ipo5 [163] . the irf3-dna interaction is inhibited by interaction of newly synthesized cell growth-regulating nucleolar protein lyar (ly1 antibody-reactive), which is usually low expressed in most cell tissues but induced by the ifnβ response [164] . lyar interacts specifically with the n-terminal domain of activated irf3 and in this way interferes with the dna-binding capacity of irf3 in the irf3-cbp/p300 holocomplex. additionally, a long non-coding rna directly targets the ifnβ promoter region and interferes with transcription factor binding in a unique way [165] : after rna deep sequencing revealed enhanced transcription of the long non-coding rna lnc-mxa from the mxa locus after viral infection, li and colleagues recently reported its mechanism of action. applying a chromatin isolation by rna purification assay and in vitro pulldown of an ifnβ promoter dsdna fragment with biotin-labelled lnc-mxa, they demonstrated that lnc-mxa forms an rna-dna triplex with the ifnβ promoter region. this changes the structure of the chromatin and interferes with binding of the transcription factors irf3 and nf-κb to their respective target sequence [165] . finally, the delayed generation of prdi-binding factor 1 (prdi-bf1, or pr/set domain 1) mediates recruitment of the histone h3 lysine methyltransferase g9a for epigenetic silencing of ifnb1 expression [166, 167] . the following host factors were additionally reported as negative modulators of irf3 activity, but it remains to be seen if they constantly inhibit irf3 or how they are regulated. for instance, calmodulin-like protein 6 (calml6 or caglp) was identified to negatively modulate irf3 and independently of the protein's ability to bind calcium ions [168] . calml6 interacts with the c-terminal aie of irf3, and this interaction is strengthened when irf3 is phosphorylated. after virus infection, calml6 thereby impairs dimerisation and nuclear import of irf3. the contribution of cellular flip long isoform protein (cflip l ) to regulation of the type i ifn response in several primary cancers was reported by several groups with contradicting outcomes, so gates and colleagues studied the underlying mechanism of action and concluded on an inhibitory function [169] . when ectopically expressed in hek293t cells, cflip l interacts with phosphorylated irf3 within the nucleus and hinders interaction with cbp and thus with the ifnβ enhancer. cflip l was then confirmed to be highly expressed in several human cancer cell lines and to interact with endogenous irf3, mediating reduction of isg expression. as opposed to the major isoform (variant 1) of irf3 that drives the type i ifn response as discussed so far, three of the five described splice-variants of human irf3 negatively modulate the biological activity of irf3, though their regulation remains to be determined. translation of variant 2 (irf3-cl) yields a slightly longer protein (452 aa) with a unique sequence of 125 aa in the c-terminus that lacks a part of the iad including the aie of irf3 [170] . irf3-cl constitutively forms homodimers that localize to the cytoplasm. after viral stimulation, ikk-mediated activation induces association of irf3-cl with phosphorylated irf3, but the heterodimers are retained in the cytoplasm and consequently, the association with this isoform keeps irf3 from the induction of ifnb1 expression. in contrast, variant 3 (irf3a) lacks the n-terminal part of the functional dbd of irf3 and is consequently unable to bind to classical irf-es [171, 172] . this isoform also heterodimerises with irf3 variant 1 after stimulation, but in line with the absent dbd, its presence selectively inhibits virus-induced ifnb1 transcription. interestingly, after virus stimulation, irf3a is degraded slower than phosphorylated irf3 and so the ratio of negative versus positive modulator increases with ongoing infection, potentially contributing to a downregulation of the type i ifn response [171] . the fourth variant, irf3e or irf3-nirs, was discovered when marozin and colleagues searched for the reason of the defect in ifnb1 expression in hepatocellular carcinoma (hcc) cells and discovered that a truncated variant of irf3 was constitutively expressed in primary cells of hcc or hcc cell lines but not in primary hepatocytes [173] . irf3-nirs is produced when an in-frame exon is aberrantly skipped during splicing, leading to the generation of a protein lacking 127 aa within the β-sandwich core of the iad. in line with this, this isoform is constitutively active and maintains the dna-binding ability. however, due to the compromised iad, irf3-nirs seems unable to exercise trans-activation activity and instead competes with irf3 for the limited irf binding sites in the ifnβ enhancer sequence [173] . surprisingly, given that association with the coactivator is normally essential for irf3 to bind to dna motifs, this implies that the interaction surface for cbp/p300 remains intact despite the missing adjacent structural module. in addition to the human isoforms, one variant of murine irf3, mirf-3a, was reported as a ubiquitously expressed negative modulator of ifnβ induction in mice [174] . during the generation of mirf-3a, an alternative donor splice site in exon 6 is used and leads to a frameshift with a premature termination codon, yielding a shorter variant (296 aa) which differs in the c-terminal region as compared to the major murine variant (419 aa). mirf-3a freely localises to the nucleus, binds to irf recognition sites and represses promoter activation. not long after the initial steps in the characterisation of the ifnβ enhanceosome were made, also the first viral proteins targeting its assembly were noticed [175, 176] . today, a long list of viral factors targeting every step from the stimulation of prrs by nucleic acids, to induction of transcription factor activation, to activity of ifn, through to the stimulation and activity of isgs are known, and the list still grows [177] [178] [179] [180] [181] [182] [183] [184] . the central role of irf3 in this pathway makes it an attractive target for viral evasion. strategies applied to interfere with the activation of irf3 in the cytoplasm include (i) the inhibition of irf3 expression; (ii) direct antagonism of essential phosphorylation events by targeting the essential kinases, their interaction with irf3 or dephosphorylating irf3 (recently joined by herpes simplex virus (hsv)-2 immediate early protein icp27 [185] , and the nucleocapsid protein of peste des petits ruminants virus [186] ); and (iii) mediating degradation of irf3. remarkably, even nuclear phosphorylated irf3 can specifically be targeted for ubiquitination and proteasome-mediated degradation after activation [187] . other viral factors aim to evade the type i ifn response more generally by limiting its induction, affecting production of ifn by transcriptional or translational shut-off or dysregulating the processing or trafficking of host mrnas [188, 189] . noting that strategies to inhibit irf3 activation and activity applied by viruses were frequently reviewed for specific virus families as well as in broad summaries [190] [191] [192] [193] [194] [195] , we want to emphasize here the molecular mechanisms viruses deploy to obstruct the function of irf3 after its trans-activation potential is enabled by phosphorylation (figure 4 ). obstruct the function of irf3 after its trans-activation potential is enabled by phosphorylation ( figure 4 ). as the activation of irf3 is a multistep process, in early studies the exact level of viral interference sometimes remained elusive. dimerisation of irf3 was among the first steps reported to be targeted in order to antagonize its trans-activation activity. the ml protein, a splice variant of the matrix protein of thogoto virus (thov) of the family orthomyxoviridae, blocks the formation of irf3 homodimers irrespective of the presence of the c-terminal aie and further inhibits association of irf3 with cbp [196, 197] . interestingly, this modulator does not affect nuclear translocation, suggesting that ml does not disable import of irf3 but renders otherwise activated irf3 monomers unable to participate in further interactions in the nucleus. the authors ruled out a lasting interaction as the activation of irf3 is a multistep process, in early studies the exact level of viral interference sometimes remained elusive. dimerisation of irf3 was among the first steps reported to be targeted in order to antagonize its trans-activation activity. the ml protein, a splice variant of the matrix protein of thogoto virus (thov) of the family orthomyxoviridae, blocks the formation of irf3 homodimers irrespective of the presence of the c-terminal aie and further inhibits association of irf3 with cbp [196, 197] . interestingly, this modulator does not affect nuclear translocation, suggesting that ml does not disable import of irf3 but renders otherwise activated irf3 monomers unable to participate in further interactions in the nucleus. the authors ruled out a lasting interaction between ml and irf3 that could for example directly compete with binding to cbp/p300 because they could not detect association of ml and irf3 by co-immunoprecipitation [197] . additionally, ml interferes with a later step in the induction of ifnb1 expression, namely, the cofactor function of tfiib at promoters that require de novo recruitment of rnap ii, which was indicated by the finding that ml also inhibited activity of the constitutively active irf3-5d mutant [198, 199] . the leader (l) protein of the cardioviruses of the picornaviridae also interferes with irf3 dimerisation. in fact, at first, the l protein of encephalomyocarditis virus (emcv) was reported for its ability to generally disturb trafficking of proteins between the cytoplasm and nucleus [200] . after infection with a related strain, mengovirus, however, irf3 still localized to the nucleus. to inhibit type i ifn transcription nonetheless, the mengovirus l protein antagonizes dimerisation of irf3 downstream of its phosphorylation [201] . due to the observation of irf3 translocation without dimerisation, the authors suggested that importin-mediated transport might be a requirement for dimerisation of irf3, which would take place within the nucleus, which is in line with nuclear translocation of monomeric irf3, as discussed above (see section 2.3). the l protein from a different cardiovirus species, theiler's murine encephalomyelitis virus (tmev), also interferes with the formation of irf3 dimers, though it was first identified because it additionally inhibits the nuclear translocation of irf3 [202, 203] . this function depends on the zinc finger motif of l [203] , but whether l affects preceding phosphorylation of irf3 as well remains undetermined. the strong impact of l on the antiviral innate immune response is additionally accounted for by the inhibition of mrna export from the nucleus which is achieved by l-mediated phosphorylation of nucleoporin 98 [203] . in contrast to the yet unclear molecular mechanism of ml and l, an interesting detail of dimerisation antagonism was described for the serine/threonine kinase us3 of hsv-1 [204] . us3 phosphorylates irf3 at s175, and this hyperphosphorylation interferes with both dimerisation and nuclear translocation of irf3. as also activity of irf3-5d could be inhibited by co-expressed us3 in a luciferase-based reporter assay, the effect of us3 seems independent of the activation status. this adds a further phosphate group to the posttranslational modifications that modulate irf3 activity and raises the question whether the cardiovirus l proteins might also interfere with the formation of irf3 dimers via small modifications, just as tmev l mediates inhibition of the nuclear pore protein by phosphorylation [203] . the v protein of simian virus 5 (sv5) was the first reported antagonist of irf3 translocation [205] , followed by the multifunctional ns1 protein of influenza b virus (ibv) [206] and the l protein of tmev [202] . again, these early studies did not yet dissect which form of irf3 is targeted because the role of the essential modifications was not known in detail. later reports of viral antagonists of irf3 translocation then included an assessment of the activation state of irf3, and some were found to specifically target irf3 after its activation. for example, hsv-1 initially stimulates activation of irf3 upon entry but subsequently inhibits irf3 activity by means of newly synthesized infected cell polypeptide 0 (icp0) [207] . during the viral replication cycle, a part of the multifunctional icp0 molecules localises to the cytoplasm and inhibits the translocation of activated irf3 into the nucleus in order to shorten the ongoing irf3-dependent innate immune response. curiously, while this cytosolic function of icp0 is independent of the activity of its e3 ubiquitin ligase domain, the functional host proteasome is required for the localisation of icp0 to the cytoplasm [207] . the inhibitory function on the innate immune response by the v protein of sendai virus (sev) was first accounted for by the interaction of v with the rna sensor melanoma differentiation-associated protein 5 (mda5) [208] . however, sev infection is predominantly detected by rig-i [209] [210] [211] , and sev infection still inhibits ifnβ production in mda5-knockout mice [212] . for this reason, the group of sakaguchi continued their studies and discovered that the sev v protein interacts with irf3 as well as irf3-5d in the cytosol and inhibits nuclear translocation. in line with this, also the v proteins of the related measles virus (mev) and newcastle disease virus (ndv) of the paramyxoviridae interact with irf3 and interfere with its trans-activation activity, though mev v seems to target irf3 after nuclear translocation [212] . in contrast, the non-structural protein ns5 of the flavivirus japanese encephalitis virus (jev) suppresses import of irf3 indirectly by interacting with the nuclear transport proteins kpna3 and kpna4 [213] . this interaction competitively blocks the interaction with the native cargo of the importins, including the transcription factors irf3 and p65. similarly, the ns3/4a protease of the related hepatitis c virus (hcv) triggers cleavage of importin β1 (ipob, also kpnb1) and in this way inhibits the transport of irf3 into the nucleus to interrupt ifnβ production [214] . once irf3 has reached the nucleus, it interacts with the coactivator cbp/p300 to obtain the potential to induce transcription. when studies concerned with the molecular requirements for the induction of type i ifns were still on their way, the critical role of the coactivators for irf3 activity was underlined by the observation that the antagonistic activity of adenovirus (adv) protein e1a on irf3-mediated stimulation was dependent on the interaction of e1a with cbp [176] . the inhibition of ifnb1 transcription by e1a could be competed with by overexpression of cbp/p300 but was not observed for a mutant of e1a that was defective in p300-binding. mechanistically, these observations implied that e1a competed with irf3 for binding to the essential coactivator [176] . to interfere with the productive association of irf3 and cbp/p300, the viral antagonists ns1 of human respiratory syncytial virus (rsv) [215] , e6 protein of human papillomavirus 16 (hpv-16) [175, 216] and the tegument protein vp16 of hsv-1 target both proteins simultaneously [217] . in contrast, the kinase orf36 of murine gammaherpesvirus 68 (mhv68) specifically interacts only with activated irf3 in the nucleus to interfere with the association of coactivators [218] . this protein is highly conserved, implying similar functions of its homologue in kaposi's sarcoma-associated herpesvirus (kshv), though the conserved kinase activity is not required for this function [218] . a striking example for the exploitation of structural homology by viruses are the viral homologues of irfs, termed virfs, which are encoded by some herpesviruses to interfere with the activity of host irfs. for details on the interplay of virfs and host irfs and their implications for the viral replication style, please refer to the recent review by myoung and colleagues [219] . shortly, virf1 of kshv binds to irf3 as well as p300 and in this way obstructs formation of the active holocomplex of irf3 and cbp/p300 [220] [221] [222] . additionally, kshv deploys virf2 to target irf3-driven gene expression [223] . similar to virf1, also the virion-associated virf r6 of the gammaherpesvirus rhesus macaque rhadinovirus (rrv) inhibits ifnb1 expression by targeting cbp and competing with phosphorylated irf3 for binding [224] . as tegument proteins, rrv r6 and also hsv-1 vp16 are released into the host cell alongside the entering virus and can directly take action to interfere with irf3 activity before a potent type i ifn response can be mounted. another strategy to hinder the participation of the essential coactivator cbp is applied by several rna viruses. the non-structural protein 1 (nsp1) subunits nsp1α of murine lactate dehydrogenase-elevating virus (ldv) [225] , nsp1γ of simian haemorrhagic fever virus (shfv) [225] and nsp1α of porcine reproductive and respiratory syndrome virus (prrsv) [225] [226] [227] of the arteriviridae family as well as nsp1 of porcine epidemic diarrhoea virus (pedv) [228] of the coronaviridae all suppress ifnb1 expression by specifically targeting cbp in the nucleus and mediating its proteasome-dependent degradation. this approach implies that these viruses do not require the activity of cbp to express their own genome, which is in line with their cytoplasmic replication. the final aim of irf3 activation is to enable its binding to recognition motifs within the ifnβ enhancer in order to allow assembly of the ifnβ enhanceosome and induce transcription. again, viral antagonists apply different mechanisms to interfere at this level. the np1 protein of human bocaparvovirus (bov) and us1 of hsv-2 prevent dna binding by interaction with the dbd of irf3 themselves [229, 230] . further, nss of sandfly fever sicilian virus (sfsv) of the bunyaviridae family was recently reported to apply this strategy to prevent association of irf3 with the ifnβ promoter [231] . wuerth and colleagues demonstrated that nss specifically interacts with irf3 via the dbd and by this masking competes for dna binding in a dose-dependent manner. several other strategies were identified in the herpesviridae family: (i) the nuclear share of the hsv-1 protein icp0 relocalises irf3 and cbp/p300 in the nucleus to special nuclear structures, thereby sequestering them away from their site of activity [232] . additionally, this mediates deactivation and promotes degradation of irf3. the icp0 variant of bovine herpesvirus 1 (bhv-1) also interacts with p300, but in contrast to hsv-1, this interaction hijacks the acetyltransferase activity of the coactivator and activates expression from viral promoters [233] . (ii) the kinase bglf4 of epstein-bar virus (ebv) directly interacts with irf3 and phosphorylates several residues between dbd and iad [234] . without affecting dimerisation or association with cbp, this modification prevents binding of irf3 to dna. (iii) kshv latency-associated nuclear antigen 1 (lana-1) competes with irf3 for binding to the prdiii-i region and in this way interferes with stable association of irf3 to the target dna [235] . the dna polymerase subunit ul44 of hcmv was recently reported to act in a similar way [236] : upon hcmv infection, exogenously expressed ul44 could be demonstrated to associate with the ifnβ promoter region in a chromatin immunoprecipitation assay and in parallel ul44 interfered with binding of the central transcription factors irf3 and p65. additionally, ul44 interacts with both transcription factors irrespective of their phosphorylation status. (iv) the kshv protein k-bzip binds to the ifnβ promoter region itself and prevents binding of irf3 [237] , but differently from lana-1 and ul44, k-bzip weakly induces gene expression while it prevents a high and detrimental activation of the type i ifn response. in contrast, the nss protein of rift valley fever virus (rvfv) applies a more indirect approach to inhibit binding of the irf3-cbp/p300 holocomplex [238] . nss interacts with the host factor sin3a-associated protein 30 (sap30), which belongs to the sin3a/ncor/hdacs repressor complexes. in turn, sap30 interacts with the transcription factor yy1, and this interaction enables recruitment of nss and sap30 to the murine ifnβ promoter region. finally, the presence of the nss-sap30-yy1 complex inhibits recruitment of cbp and thus transcriptional activation of ifnb1. since the beginning of the detailed characterisation of irf3 activation more than 20 years ago, we have gained profound insight into the mechanisms enabling the trans-activation activity of a transcription factor with low intrinsic binding affinity for its specific recognition motif and into the intricate network of interactions that allow its participation in the induction of ifnb1 expression. still, the fundamental principles were mostly characterised in vitro and sometimes biased from prior assumptions, rendering the sequence of events as it occurs in our cells incomplete. to reveal the full dynamics of the events summarised above, experiments with living cells will be required. the many studies reporting factors that affect irf3 dimerisation and/or translocation and/or trans-activation activity highlight the importance of pinpointing the modulatory mechanism as precisely as possible in future work. at the same time, the diversity of methods applied in the studies summarised here demonstrates that nowadays, dissection of the exact level of intervention with irf3 activity is within reach. as delineated in this review, the regulation of irf3 in the context of ifnb1 expression in itself presents us with a multi-layered circuit of promoting and dampening modulations. the ever-expanding catalogue of host and viral modulators of irf3 activity not only reflects the key role of this signalling pathway in the mammalian arsenal of antiviral defence mechanisms, but it further alludes to the possibility to use specific mechanisms for medical intervention. directed manipulation of host modulators could present a novel approach to stimulate the host organism to overcome an infection by its own resources or to reduce excessive ifn activity to healthy levels. more information on how to achieve this will surely emerge from the unceasing identification and characterisation of novel irf3 modulators deployed by host and virus. nevertheless, the multitude of additional genes targeted by irf3 signifies that we are still just beginning to understand the true complexity of irf3 fine regulation. in addition to the ongoing characterisation of the direct involvement in the expression of isgs, powerful in silico and sequencing approaches of recent years revealed a growing list of novel targets, including virus-inducible rnas [107, 239] . moreover, some of the identified host modulators hint at regulation of irf3 activity dependent on signalling of other pathways to enable the incorporation of further virus interference. i. the interferon global virus outbreaks: interferons as 1st responders type i interferons: distinct biological activities and current applications for viral infection the dual nature of type i and type ii interferons shared and distinct functions of type i and type iii interferons interferon-λ orchestrates innate and adaptive mucosal immune responses targeting interferons and their pathways in systemic lupus erythematosus specificity and function of irf family transcription factors: insights from genomics interferon response of chicken embryo fibroblasts to nucleic acids and related compounds inhibition of virus multiplication by foreign nucleic acid approaching the asymptote? evolution and revolution in immunology recognition of endogenous nucleic acids by the innate immune system detection of microbial infections through innate immune sensing of nucleic acids mechanisms controlling nucleic acid-sensing toll-like receptors molecular basis of nf-κb signaling the regulation of ap-1 activity by mitogen-activated protein kinases the transcriptional code of human ifn-beta gene expression identification of a member of the interferon regulatory factor family that binds to the interferon-stimulated response element and activates expression of interferon-induced genes structural and functional analysis of interferon regulatory factor 3: localization of the transactivation and autoinhibitory domains crystal structure of irf-3 reveals mechanism of autoinhibition and virus-induced phosphoactivation direct triggering of the type i interferon system by virus infection: activation of a transcription factor complex containing irf-3 and cbp/p300 regulated nuclear-cytoplasmic localization of interferon regulatory factor 3, a subunit of double-stranded rna-activated factor 1 bipartite nuclear localization signal controls nuclear import and dna-binding activity of ifn regulatory factor 3 analysis of functional domains of interferon regulatory factor 7 and its association with irf-3 gene induction pathways mediated by distinct irfs during viral infection structural insights into interferon regulatory factor activation irf-7 is the master regulator of type-i interferon-dependent immune responses irf7: activation, regulation, modification and function the multifaceted biology of plasmacytoid dendritic cells characterization of the interferon regulatory factor-7 and its potential role in the transcription activation of interferon a genes positive feedback regulation of type i ifn genes by the ifn-inducible transcription factor irf-7 differential viral induction of distinct interferon-alpha genes by positive feedback through interferon regulatory factor-7 selective dna binding and association with the creb binding protein coactivator contribute to differential activation of alpha/beta interferon genes by interferon regulatory factors 3 and 7 distinct and essential roles of transcription factors irf-3 and irf-7 in response to viruses for ifn-alpha/beta gene induction a positive feedback amplifier circuit that regulates interferon (ifn)-stimulated gene expression and controls type i and type ii ifn responses involvement of the irf family transcription factor irf-3 in virus-induced activation of the ifn-beta gene virus-dependent phosphorylation of the irf-3 transcription factor regulates nuclear translocation, transactivation potential, and proteasome-mediated degradation virus infection induces the assembly of coordinately activated transcription factors on the ifn-beta enhancer in vivo identification of distinct signaling pathways leading to the phosphorylation of interferon regulatory factor 3 sting specifies irf3 phosphorylation by tbk1 in the cytosolic dna signaling pathway phosphorylation of innate immune adaptor proteins mavs, sting, and trif induces irf3 activation structural basis for concerted recruitment and activation of irf-3 by innate immune adaptor proteins ikkepsilon and tbk1 are essential components of the irf3 signaling pathway triggering the interferon antiviral response through an ikk-related pathway interferon regulatory factor 3 is regulated by a dual phosphorylation-dependent switch crystal structure of irf-3 in complex with cbp identification of the minimal phosphoacceptor site required for in vivo activation of interferon regulatory factor 3 in response to virus and double-stranded rna phosphorylation of irf-3 on ser 339 generates a hyperactive form of irf-3 through regulation of dimerization and cbp association ser386 phosphorylation of transcription factor irf-3 induces dimerization and association with cbp/p300 without overall conformational change identification of ser-386 of interferon regulatory factor 3 as critical target for inducible phosphorylation that determines activation contribution of ser386 and ser396 to activation of interferon regulatory factor 3 interferon regulatory factor 3 and creb-binding protein/p300 are subunits of double-stranded rna-activated transcription factor draf1 analyses of virus-induced homomeric and heteromeric protein associations between irf-3 and coactivator cbp/p300 direct involvement of creb-binding protein/p300 in sequence-specific dna binding of virus-activated interferon regulatory factor-3 holocomplex transcriptional activity of interferon regulatory factor (irf)-3 depends on multiple protein-protein interactions role of intrinsic protein disorder in the function and interactions of the transcriptional coactivators creb-binding protein (cbp) and p300 target gene context influences the transcriptional requirement for the kat3 family of cbp and p300 histone acetyltransferases genome-wide assessment of differential roles for p300 and cbp in transcription regulation cbp/p300 in brain development and plasticity: disentangling the kat's cradle usp22 promotes irf3 nuclear translocation and antiviral responses by deubiquitinating the importin protein kpna2 transcription factor dimerization activates the p300 acetyltransferase differential modification of interferon regulatory factor 3 following virus particle entry inhibition of beta interferon induction by severe acute respiratory syndrome coronavirus suggests a two-step model for activation of interferon regulatory factor 3 transcriptional profiling of interferon regulatory factor 3 target genes: direct involvement in the regulation of interferon-stimulated genes complex regulation pattern of irf3 activation revealed by a novel dimerization reporter system structure and function of the interferon-beta enhanceosome the enhanceosome virus induction of human ifn beta gene expression requires the assembly of an enhanceosome coordination of a transcriptional switch by hmgi(y) acetylation ordered recruitment of chromatin modifying and general transcription factors to the ifn-beta promoter facilitated binding of tata-binding protein to nucleosomal dna modifying gene expression programs by altering core promoter chromatin architecture dna-binding landscape of irf3, irf5 and irf7 dimers: implications for dimer-specific gene regulation an atf/creb binding site is required for virus induction of the human interferon beta gene proc. natl. acad. sci. usa stability and dna-binding ability of the bzip dimers formed by the atf-2 and c-jun transcription factors induction of human interferon gene expression is associated with a nuclear factor that interacts with the nf-kappa b site of the human immunodeficiency virus enhancer the involvement of nf-kappa b in beta-interferon gene regulation reveals its role as widely inducible mediator of signal transduction double-stranded rna activates binding of nf-kappa b to an inducible element in the human beta-interferon promoter crystal structure of an irf-dna complex reveals novel dna recognition and cooperative binding to a tandem repeat of core sequences an atomic model of the interferon-beta enhanceosome specific enhancer selection by irf3, irf5 and irf9 is determined by isre half-sites, 5 and 3 flanking bases, collaborating transcription factors and the chromatin environment in a combinatorial fashion virus infection induces nf-kappab-dependent interchromosomal associations mediating monoallelic ifn-beta gene expression the transcription factor thpok orchestrates stochastic interchromosomal interactions required for ifnb1 virus-inducible gene expression the high mobility group protein hmg i(y) is required for nf-kappa b-dependent virus induction of the human ifn-beta gene reversal of intrinsic dna bends in the ifn beta gene enhancer by transcription factors and the architectural protein hmg i(y) the mechanism of transcriptional synergy of an in vitro assembled interferon-beta enhanceosome the role of hmg i(y) in the assembly and function of the ifn-beta enhanceosome mechanisms of transcriptional synergism between distinct virus-inducible enhancer elements recruitment of cbp/p300 by the ifn beta enhanceosome is required for synergistic activation of transcription induction of endogenous ifn-alpha and ifn-beta genes by a regulatory transcription factor, irf-1 evidence for a nuclear factor(s), irf-1, mediating induction and silencing properties to human ifn-beta gene regulatory elements critical role of a common transcription factor, irf-1, in the regulation of ifn-beta and ifn-inducible genes regulated expression of a gene encoding a nuclear factor, irf-1, that specifically binds to ifn-beta gene regulatory elements targeted disruption of irf-1 or irf-2 results in abnormal type i ifn gene induction and aberrant lymphocyte development mice devoid of interferon regulatory factor 1 (irf-1) show normal expression of type i interferon genes assembly of a functional beta interferon enhanceosome is dependent on atf-2-c-jun heterodimer orientation crystal structure of atf-2/c-jun and irf-3 bound to the interferon-beta enhancer structure of irf-3 bound to the prdiii-i regulatory element of the human interferon-beta enhancer the role of response elements organization in transcription factor selectivity: the ifn-beta enhanceosome example efficient recruitment of tfiib and cbp-rna polymerase ii holoenzyme by an interferon-beta enhanceosome in vitro virus infection leads to localized hyperacetylation of histones h3 and h4 at the ifn-beta promoter deciphering the transcriptional histone acetylation code for a human gene nucleosome sliding via tbp dna binding in vivo eukaryotic transcription turns 50 crystal structure of a yeast tbp/tata-box complex high-density nucleosome occupancy map of human chromosome 9p21-22 reveals chromatin organization of the type i interferon gene cluster identification of individual interferon-producing cells by in situ hybridization stochastic regulation in early immune response chromosome-specific and noisy ifnb1 transcription in individual virus-infected human primary dendritic cells defining emerging roles for nf-kappab in antivirus responses: revisiting the interferon-beta enhanceosome paradigm gene repression by coactivator repulsion the specificity of innate immune responses is enforced by repression of interferon response elements by nf-κb p50 the transcriptional silencer protein nrf: a repressor of nf-kappa b enhancers constitutive silencing of ifn-beta promoter is mediated by nrf (nf-kappab-repressing factor), a nuclear inhibitor of nf-kappab lack of essential role of nf-kappa b p50, rela, and crel subunits in virus-induced type 1 ifn expression distinct roles for the nf-kappa b rela subunit during antiviral innate immune responses nf-kappa b rela subunit is crucial for early ifn-beta expression and resistance to rna virus replication association of the interferon-β gene with pericentromeric heterochromatin is dynamically regulated during virus infection through a yy1-dependent mechanism yin yang 1 dynamically regulates antiviral innate immune responses during viral infection enhanceosome formation over the beta interferon promoter underlies a remote-control mechanism mediated by yy1 and yy2 transcription factor yy1 binds to the murine beta interferon promoter and regulates its transcriptional capacity with a dual activator/repressor role binding of yy1 to the proximal region of the murine beta interferon promoter is essential to allow cbp recruitment and k8h4/k14h3 acetylation on the promoter region after virus infection a novel virus-inducible enhancer of the interferon-β gene with tightly linked promoter and enhancer activities activation and regulation of interferon-β in immune responses post-translational regulation of antiviral innate signaling protein phosphatase pp1 negatively regulates the toll-like receptor-and rig-i-like receptor-triggered production of type i interferon by inhibiting irf3 phosphorylation at serines 396 and 385 in macrophage the methyltransferase nsd3 promotes antiviral innate immunity via direct lysine methylation of irf3 interferon-inducible cytoplasmic lnclrrc55-as promotes antiviral innate responses by strengthening irf3 phosphorylation recruitment of phosphatase pp2a by rack1 adaptor protein deactivates transcription factor irf3 and limits type i interferon signaling pp2a facilitates porcine reproductive and respiratory syndrome virus replication by deactivating irf3 and limiting type i interferon production isg15 enhances the innate antiviral response by inhibition of irf-3 degradation positive regulation of interferon regulatory factor 3 activation by herc5 via isg15 modification negative regulation of interferon-regulatory factor 3-dependent innate antiviral response by the prolyl isomerase pin1 yap antagonizes innate antiviral immunity and is targeted for lysosomal degradation through ikkε-mediated phosphorylation the tumor suppressor pten has a critical role in antiviral innate immunity interferon regulatory factor-3 is an in vivo target of dna-pk s-glutathionylation of irf3 regulates irf3-cbp interaction and activation of the ifn beta pathway pivotal role for the escrt-ii complex subunit eap30/snf8 in irf3-dependent innate antiviral defense ago2 negatively regulates type i interferon signaling pathway by competition binding irf3 with cbp/p300 elf4 is critical for induction of type i interferon and the host antiviral response irf8 and irf3 cooperatively regulate rapid interferon-beta induction in human blood monocytes the ubiquitin e3 ligase raul negatively regulates type i interferon through ubiquitination of the transcription factors irf7 and irf3 the e3 ubiquitin ligase ro52 negatively regulates ifn-beta production post-pathogen recognition by polyubiquitin-mediated degradation of irf3 trim21 is essential to sustain ifn regulatory factor 3 activation during antiviral response senp2 negatively regulates cellular antiviral response by desumoylating irf3 and conditioning it for ubiquitination and degradation virus infection triggers sumoylation of irf3 and irf7, leading to the negative regulation of type i interferon gene expression mst1 shuts off cytosolic antiviral defense through irf3 phosphorylation fas-associated factor 1 negatively regulates the antiviral immune response by inhibiting translocation of interferon regulatory factor 3 to the nucleus bromodomain protein brd3 promotes ifnb1 transcription via enhancing irf3/p300 complex formation and recruitment to ifnb1 promoter in macrophages otud1 negatively regulates type i ifn induction by disrupting noncanonical ubiquitination of irf3 the transcription factor nfat5 limits infection-induced type i interferon responses the transcription factor mafb antagonizes antiviral responses by blocking recruitment of coactivators to the transcription factor irf3 involvement of the ikappab kinase (ikk)-related kinases tank-binding kinase 1/ikki and cullin-based ubiquitin ligases in ifn regulatory factor-3 degradation negative regulation of interferon-β gene expression during acute and persistent virus infections foxo1 negatively regulates cellular antiviral response by promoting degradation of irf3 trim26 negatively regulates interferon-β production and antiviral response through polyubiquitination and degradation of nuclear irf3 negative feedback regulation of cellular antiviral signaling by rbck1-mediated degradation of irf3 caspase-8-mediated cleavage inhibits irf-3 protein by facilitating its proteasome-mediated degradation krüppel-like factor 4 negatively regulates cellular antiviral immune response kat8 selectively inhibits antiviral immunity by acetylating irf3 a diverse range of gene products are effectors of the type i interferon antiviral response ddx56 inhibits type i interferon by disrupting assembly of irf3-ipo5 to inhibit irf3 nucleus import lyar suppresses beta interferon induction by targeting phosphorylated interferon regulatory factor 3 long noncoding rna lnc-mxa inhibits beta interferon transcription by forming rna-dna triplexes at its promoter prdi-bf1 recruits the histone h3 methyltransferase g9a in transcriptional silencing identification and characterization of a novel repressor of beta-interferon gene expression the ef-hand protein calml6 suppresses antiviral innate immunity by impairing irf3 dimerization cflipl interrupts irf3-cbp-dna interactions to inhibit irf3-driven transcription interferon regulatory factor 3-cl, an isoform of irf3, antagonizes activity of irf3 functional characterization of interferon regulatory factor 3a (irf-3a), an alternative splice isoform of irf-3 dual utilization of an acceptor/donor splice site governs the alternative splicing of the irf-3 gene inhibition of the ifn-beta response in hepatocellular carcinoma by alternative spliced isoform of ifn regulatory factor-3 characterization of a novel isoform of murine interferon regulatory factor 3 human papillomavirus 16 e6 oncoprotein binds to interferon regulatory factor-3 and inhibits its transcriptional activity primary activation of interferon a and interferon b gene transcription by interferon regulatory factor 3 recent advances in understanding viral evasion of type i interferon cellular sensing of viral dna and viral evasion mechanisms early ifn type i response: learning from microbial evasion strategies viral evasion strategies in type i ifn signaling-a summary of recent developments viral dedication to vigorous destruction of interferon receptors on taking the sting out of immune activation cytosolic dna-sensing immune response and viral infection intracellular sensing of viral genomes and viral evasion herpes simplex virus type 2 immediate early protein icp27 inhibits ifn-β production in mucosal epithelial cells by antagonizing irf3 activation peste des petits ruminants virus nucleocapsid protein inhibits beta interferon production by interacting with irf3 to block its activation varicella-zoster virus immediate-early protein orf61 abrogates the irf3-mediated innate immune response through degradation of activated irf3 viral evasion and subversion of pattern-recognition receptor signalling strategies of highly pathogenic rna viruses to block dsrna detection by rig-i-like receptors: hide, mask, hit the interferon antiviral response: from viral invasion to evasion viral suppression of the interferon system interferons and viruses: an interplay between induction, signalling, antiviral responses and virus countermeasures ifn regulatory factors and antiviral innate immunity: how viruses can get better ten strategies of interferon evasion by viruses the molecular basis of viral inhibition of irf-and stat-dependent immune responses functional comparison of the two gene products of thogoto virus segment 6 thogoto virus ml protein suppresses irf3 function the interferon antagonist ml protein of thogoto virus targets general transcription factor iib viral targeting of tfiib impairs de novo polymerase ii recruitment and affects antiviral immunity nucleocytoplasmic traffic disorder induced by cardioviruses the mengovirus leader protein blocks interferon-alpha/beta gene transcription and inhibits activation of interferon regulatory factor 3 the leader protein of theiler's virus interferes with nucleocytoplasmic trafficking of cellular proteins inhibition of mrna export and dimerization of interferon regulatory factor 3 by theiler's virus leader protein herpes simplex virus 1 serine/threonine kinase us3 hyperphosphorylates irf3 and inhibits beta interferon production recovery of paramyxovirus simian virus 5 with a v protein lacking the conserved cysteine-rich domain: the multifunctional v protein blocks both interferon-beta induction and interferon signaling the n-and c-terminal domains of the ns1 protein of influenza b virus can independently inhibit irf-3 and beta interferon promoter activation cellular localization of the herpes simplex virus icp0 protein dictates its ability to block irf3-mediated innate immune responses analysis of interaction of sendai virus v protein and melanoma differentiation-associated gene 5 cell type-specific involvement of rig-i in antiviral response activation of innate defense against a paramyxovirus is mediated by rig-i and tlr7 and tlr8 in a cell-type-specific manner differential roles of mda5 and rig-i helicases in the recognition of rna viruses inhibition of interferon regulatory factor 3 activation by paramyxovirus v protein japanese encephalitis virus ns5 inhibits type i interferon (ifn) production by blocking the nuclear translocation of ifn regulatory factor 3 and nf-κb importin beta1 targeting by hepatitis c virus ns3/4a protein restricts irf3 and nf-kappab signaling of ifnb1 antiviral response a novel mechanism for the inhibition of interferon regulatory factor-3-dependent gene expression by human respiratory syncytial virus ns1 protein the e6 protein of human papillomavirus type 16 binds to and inhibits co-activation by cbp and p300 herpes simplex virus 1-encoded tegument protein vp16 abrogates the production of beta interferon (ifn) by inhibiting nf-κb activation and blocking ifn regulatory factor 3 to recruit its coactivator cbp conserved herpesviral kinase promotes viral persistence by inhibiting the irf-3-mediated type i interferon response beyond viral interferon regulatory factors: immune evasion strategies functional analysis of human herpesvirus 8-encoded viral interferon regulatory factor 1 and its association with cellular interferon regulatory factors and p300 viral interferon regulatory factor 1 of kaposi's sarcoma-associated herpesvirus (human herpesvirus 8) binds to, and inhibits transactivation of, creb-binding protein hhv-8 encoded virf-1 represses the interferon antiviral response by blocking irf-3 recruitment of the cbp/p300 coactivators inhibition of interferon signaling by the kaposi's sarcoma-associated herpesvirus full-length viral interferon regulatory factor 2 protein a rhesus rhadinovirus viral interferon (ifn) regulatory factor is virion associated and inhibits the early ifn antiviral response biogenesis of non-structural protein 1 (nsp1) and nsp1-mediated type i interferon modulation in arteriviruses degradation of creb-binding protein and modulation of type i interferon induction by the zinc finger motif of the porcine reproductive and respiratory syndrome virus nsp1alpha subunit modulation of type i interferon induction by porcine reproductive and respiratory syndrome virus and degradation of creb-binding protein by non-structural protein 1 in marc-145 and hela cells suppression of type i interferon production by porcine epidemic diarrhea virus and degradation of creb-binding protein by nsp1 human bocavirus np1 inhibits ifn-β production by blocking association of ifn regulatory factor 3 with ifnb promoter hsv-2 immediate-early protein us1 inhibits ifn-β production by suppressing association of irf-3 with ifn-β promoter nss protein of sandfly fever sicilian phlebovirus counteracts interferon (ifn) induction by masking the dna-binding domain of ifn regulatory factor 3 recruitment of activated irf-3 and cbp/p300 to herpes simplex virus icp0 nuclear foci: potential role in blocking ifn-beta induction bovine herpesvirus 1 immediate-early protein (bicp0) interacts with the histone acetyltransferase p300, which stimulates productive infection and gc promoter activity epstein-barr virus bglf4 kinase suppresses the interferon regulatory factor 3 signaling pathway kaposi sarcoma-associated herpesvirus latency-associated nuclear antigen inhibits interferon (ifn) beta expression by competing with ifn regulatory factor-3 for binding to ifnb promoter human cytomegalovirus dna polymerase subunit ul44 antagonizes antiviral immune responses by suppressing irf3-and nf-kappab-mediated transcription binding of kaposi's sarcoma-associated herpesvirus k-bzip to interferon-responsive factor 3 elements modulates antiviral gene expression a sap30 complex inhibits ifn-beta expression in rift valley fever virus infected cells we would like to thank friedemann weber for critically reading the manuscript. we would viruses 2020, 12 key: cord-000104-3b8b8p61 authors: mcwhirter, sarah m.; barbalat, roman; monroe, kathryn m.; fontana, mary f.; hyodo, mamoru; joncker, nathalie t.; ishii, ken j.; akira, shizuo; colonna, marco; chen, zhijian j.; fitzgerald, katherine a.; hayakawa, yoshihiro; vance, russell e. title: a host type i interferon response is induced by cytosolic sensing of the bacterial second messenger cyclic-di-gmp date: 2009-08-31 journal: j exp med doi: 10.1084/jem.20082874 sha: doc_id: 104 cord_uid: 3b8b8p61 the innate immune system responds to unique molecular signatures that are widely conserved among microbes but that are not normally present in host cells. compounds that stimulate innate immune pathways may be valuable in the design of novel adjuvants, vaccines, and other immunotherapeutics. the cyclic dinucleotide cyclic-di–guanosine monophosphate (c-di-gmp) is a recently appreciated second messenger that plays critical regulatory roles in many species of bacteria but is not produced by eukaryotic cells. in vivo and in vitro studies have previously suggested that c-di-gmp is a potent immunostimulatory compound recognized by mouse and human cells. we provide evidence that c-di-gmp is sensed in the cytosol of mammalian cells via a novel immunosurveillance pathway. the potency of cytosolic signaling induced by c-di-gmp is comparable to that induced by cytosolic delivery of dna, and both nucleic acids induce a similar transcriptional profile, including triggering of type i interferons and coregulated genes via induction of tbk1, irf3, nuclear factor κb, and map kinases. however, the cytosolic pathway that senses c-di-gmp appears to be distinct from all known nucleic acid–sensing pathways. our results suggest a novel mechanism by which host cells can induce an inflammatory response to a widely produced bacterial ligand. to sense infection, the innate immune system preferentially responds to conserved molecular signatures of microbes that are absent from normal host cells. there is currently great interest in determining what molecular features of pathogens are detected, and how these features are sensed by the innate immune system. a better understanding of the fundamental principles of how immune responses are stimulated is critical for the design of more effective vaccines, adjuvants, and immune therapeutics. one important class of ligands sensed by the innate immune system is nucleic acids. several distinct nucleic acid sensors have been described and have been found to be distributed to distinct subcellular locations and exhibit distinct specificities for different nucleic acid ligands. a common theme is that nucleic acid sensors tend to induce expression of type i ifn genes and, thus, appear to be particularly important for initiating immune responses to viruses. including listeria monocytogenes, mycobacterium tuberculosis, legionella pneumophila, francisella tularensis, group b streptococcus, and brucella abortus, it appears that induction of type i ifn is via a novel cytosolic pathway and is independent of tlrs. it has been suggested that these pathogens trigger the cytosolic dna-sensing pathway, but neither the host sensors nor the bacterial ligands that trigger cytosolic induction of type i ifns by these pathogens have been identified. intriguingly, evidence from l. monocytogenes suggests that bacterial multidrug efflux pumps are required for induction of type i ifn , and raises the possibility that a small molecule substrate of these pumps, rather than dna, is the bacterial trigger of type i ifn gene expression. nucleotide second messengers are critical transmitters of signaling in all living things. camp is used by bacteria and eukaryotes alike, whereas certain nucleotide second messengers are unique to bacteria. for example, guanosine tetraphosphate (ppgpp) is a key regulator of the stringent response in bacteria (srivatsan and wang, 2008) . another nucleotide second messenger, cyclic-di-gmp (c-di-gmp), is a relatively recently appreciated cyclic ribonucleotide (ross et al., 1987) synthesized by bacteria from two gtp precursors that are hydrolyzed and ultimately circularized via 5-to-3 monophosphate linkages. the bacterial diguanylate cyclases that synthesize c-di-gmp all contain a characteristic ggdef domain, whereas the phosphodiesterases that specifically degrade c-di-gmp to pgpg contain an eal domain. the ggdef domain, and presumably c-di-gmp, appears to be limited to bacteria (galperin et al., 2001) and is not found in archaea or eukarya. c-di-gmp appears to play complex roles as a second messenger in most bacterial species, and regulates diverse processes, such as motility, biofilm formation, and virulence gene expression (tamayo et al., 2007) . the molecular mechanisms by which c-di-gmp regulates these biological processes in bacteria are only beginning to be understood. it appears that c-di-gmp is capable of specific binding to regulatory proteins containing the pilz protein domain (cotter and stibitz, 2007) . the peld protein of p. aeruginosa is an additional non-pilz-containing c-di-gmp sensor protein (lee et al., 2007) . several recent reports have suggested that c-di-gmp can stimulate a variety of signaling pathways in mammalian cells in vivo. one early report demonstrated that 50 µm of exogenous c-di-gmp inhibited growth of human colon cancer cells (karaolis et al., 2005a) without toxicity against normal kidney cells. two additional reports have indicated that c-di-gmp can function as an adjuvant. one group demonstrated a highly significant (>200-fold; p < 0.001) induction of anti-clfa igg2a titers when mice were vaccinated with clfa and c-di-gmp as compared with vaccination with clfa alone (karaolis et al., 2007a) . another group found a similar induction of anti--gal titers when c-di-gmp was used as an adjuvant (ebensen et al., 2007) . both groups also found increased t cell responses in mice injected with c-di-gmp. karoalis et al. (2007a) also showed that c-di-gmp has immunostimulatory effects in vivo and in vitro on innate cell populations, toll-like receptor (tlr) 3, 7, 8, and 9 are transmembrane nucleic acid receptors that reside in intracellular compartments, where they detect endocytosed or autophagocytosed nucleic acids medzhitov, 2007) . tlr7, 8, and 9 all require the signaling adaptor myd88 to initiate downstream signaling, whereas tlr3 requires the signaling adaptor trif. thus, myd88 / trif / double-knockout cells are deficient in signaling through all known tlrs that sense nucleic acids (hoebe et al., 2003; yamamoto et al., 2003) . interestingly, recent work has established that myd88 / trif / cells are capable of responding to nucleic acid ligands via cytosolic immunosurveillance pathways. cytosolic rna appears to be sensed by two rna helicase-containing proteins, rig-i and mda5 (yoneyama et al., 2004) . interestingly, rig-i and mda5 do not perform redundant functions and appear to respond to distinct classes of viruses (gitlin et al., 2006; hornung et al., 2006; kato et al., 2006; pichlmair et al., 2006; . signaling by rig-i and mda5 requires a common adaptor molecule called mavs (also known as ips-1, cardif, or visa; kawai et al., 2005; meylan et al., 2005; seth et al., 2005; xu et al., 2005; sun et al., 2006) . mavs recruits the tbk1 kinase that phosphorylates and activates the irf3 transcription factor. other transcription factors such as atf2/c-jun and nk-b form a coordinated dna-bound complex with irf3, and are together required for robust activation of the ifn- promoter (maniatis et al., 1998; panne, 2008) . many cell types also appear to respond to the cytosolic presence of dna stetson and medzhitov, 2006a) . the cytosolic response to dna does not require mavs, but does require tbk1 and irf3 in most cell types stetson and medzhitov, 2006a; sun et al., 2006) . recently, a putative cytosolic sensor of dna was identified, and was named dna-dependent activator of ifn regulatory factors (dai; previously known as dlm-1 or zbp1; takaoka et al., 2007) . knockdown experiments indicated that dai was involved in sensing cytosolic dna in the l929 fibroblast-like and raw macrophage-like cell lines (takaoka et al., 2007; wang et al., 2008) . dai was expressed in other cell types, such as mouse embryonic fibroblasts (mefs), but was dispensable for the response to cytosolic dna in these cell types, suggesting that additional uncharacterized nucleic acid sensor proteins also exist . indeed, cells from dai knockouts appear to respond normally to cytosolic dna (ishii et al., 2008) , possibly because of redundancy with other cytosolic dna sensors. although type i ifns are primarily considered to be antiviral cytokines (stetson and medzhitov, 2006b) , there is growing appreciation for their complex role in bacterial infections as well. indeed, it is clear that type i ifns are induced by many, if not all, bacterial pathogens, and can contribute to diverse outcomes in vivo (coers et al., 2000; o'riordan et al., 2002; opitz et al., 2006; stetson and medzhitov, 2006a; henry et al., 2007; roux et al., 2007; stanley et al., 2007; charrel-dennis et al., 2008) . for several bacterial pathogens, (a) the c-di-gmp compound was chemically synthesized and >98% pure by hplc (not depicted), (b) phosphodiesterase treatment of c-di-gmp resulted in a product that is no longer stimulatory ( fig. 1 e) , and (c) macrophages deficient in all tlr signaling and unable to respond to lps still responded to c-di-gmp (see below). thus, these observations suggest that c-di-gmp is sensed in the cytosol, resulting in a potent induction of type i ifn in bone marrow macrophages. we performed microarray experiments to compare the global transcriptional response induced by cytosolic c-di-gmp with that induced by cytosolic dna (fig. 1 f and table s1 ). whole transcriptome spotted meebo microarrays (verdugo and medrano, 2006) were used in these experiments. the results indicated that the transcriptional profile of cells stimulated by cytosolic c-di-gmp was very similar to the transcriptional profile of cells stimulated with cytosolic dna. induction of type i ifns and known type i ifn-inducible genes dominated the transcriptional response, but other genes (e.g., il6, cd86, and cxcl9) were also strongly induced ( fig. 1 f and table s1 ). although the transcriptional profiles of cells stimulated with c-di-gmp and dna were highly similar, a small number of genes were reproducibly preferentially induced by c-di-gmp as compared with dna (e.g., htra serine peptidase 4 and claudin 23; table s1 ). collectively, these results suggested that similar but not identical downstream signaling pathways are triggered by dna and c-di-gmp. it was previously reported that hek293 cells stably transfected with various tlrs failed to respond to c-di-gmp (karaolis et al., 2007a) , but the reason for this failure was unclear. for example, hek293 cells might lack an essential accessory protein. to rule out a role for tlrs in sensing of c-di-gmp, we tested responses in myd88 / trif / double-knockout macrophages, which are deficient in all tlr signaling (yamamoto et al., 2003) . both wild-type and myd88 / trif / macrophages showed a robust induction of ifn- after c-di-gmp stimulation (fig. 2 a) . as expected, the myd88 / trif / macrophages did not respond to lps (fig. 2 a) . these results indicated that tlr signaling is not required for responsiveness to c-di-gmp, and are consistent with c-di-gmp signaling via a cytosolic surveillance pathway. robust transcription of the ifn- gene requires the coordinate activation of several transcription factors, including irf3 and nf-b (panne, 2008) . we therefore expected that the c-di-gmp-induced signaling pathway would also require these factors to induce type i ifn. irf3 activation requires phosphorylation by the tbk1 kinase, which leads to irf3 dimerization and nuclear translocation (fitzgerald et al., 2003; sharma et al., 2003; hemmi et al., 2004; mcwhirter et al., 2004; perry et al., 2004) . to address whether tbk1 is required for activation of type i ifn by c-di-gmp, we tested including monocytes, macrophages, and granulocytes. c-di-gmp induced dendritic cell maturation and led to the production of various cytokines and chemokines, including tnf, il-1, ip-10, rantes, and cxcr4 (karaolis et al., 2007a) . however, c-di-gmp did not stimulate induction of type i ifns by plasmacytoid dendritic cells. impressively, preinjection of 2.5 mg/kg c-di-gmp protected against subsequent lethal challenge with klebsiella pneumoniae or staphlyococcus aureus (karaolis et al., 2005b; karaolis et al., 2007b) . despite the impressive in vivo biological effects of c-di-gmp, the mechanism by which c-di-gmp stimulates host immunity remains unknown. in this study, we provide evidence that mammalian cells survey their cytosol for the presence of c-di-gmp. we find that c-di-gmp triggers a transcriptional response virtually indistinguishable from the response triggered by cytosolic dna. like the response to dna, the response to c-di-gmp requires the tbk1 kinase and irf3 transcription factor. however, the pathway for sensing c-di-gmp can be distinguished from that for sensing dna in certain cell types and, moreover, appears to be distinct from all other known cytosolic sensing pathways. our results suggest a novel mechanism by which host cells can induce an inflammatory response to a widely produced bacterial ligand. we hypothesized that c-di-gmp might be sensed by a cytosolic sensor leading to the induction of type i ifns. to test this hypothesis, we compared the ability of overlayed versus transfected c-di-gmp to induce type i ifn in bone marrow macrophages. at the concentrations used (up to 25 µg/ml), overlay of c-di-gmp did not elicit a significant response ( fig. 1 a) , although ifn- production could be detected at higher concentrations (50 or 100 µg/ml; not depicted). in contrast, delivery of c-di-gmp to the cytosol by transfection elicited robust activation of ifn- in a dose-dependent manner ( fig. 1 a) . a significant response was detected with a concentration of c-di-gmp as low as 2.5 µg/ml (equivalent to 3.6 µm). these results suggested that c-di-gmp signals via a cytosolic immunosurveillance pathway. we tested whether compounds related to c-di-gmp were capable of stimulating ifn- expression when delivered to the cytosol. we measured induction of endogenous type i ifn protein by bioassay ( fig. 1 b) , and induction of ifn- and ifn-5 mrna levels by quantitative rt-pcr ( fig. 1 , c and d). no detectable ifn- production was observed upon transfection with gtp, cgmp, ppgpp (a different bacterial nucleotide second messenger), or pgpg (hydrolyzed c-di-gmp), whereas the cytosolic response to 3.3 µg/ml of transfected c-di-gmp was of a similar magnitude to that of 3.3 µg/ml of transfected dna (pda:dt) and rna (pi:c), which are known cytosolic inducers of type i ifn. we do not believe that the cytosolic induction of type i ifns in response to c-di-gmp is caused by a contaminant (such as lps) because a role in ifn--mediated antiviral immunity (tenoever et al., 2007) , and found that ikbke / macrophages responded normally to c-di-gmp (fig. 2 c) . to examine the role of irf3 in c-di-gmp induction of ifn-, we tested the ability of c-di-gmp to induce signaling in macrophages lacking irf3. we observed that irf3 / macrophages failed to induce ifn- in response to c-di-gmp ( fig. 2 d) . we also tested irf7 / macrophages and observed a partial requirement for irf7 by the c-di-gmp pathway (fig. 2 d) . loss of irf7 is likely compensated for by irf3 in macrophages. the partial requirement for irf7 may reflect a c-di-gmp responses in tbk1 / macrophages (fig. 2 b) . we observed very little ifn- induction by c-di-gmp in cells lacking tbk1, suggesting that tbk1 is required for c-di-gmp signaling. we also observed that tbk1 is required for the induction of ifn- by pda:dt ( fig. 2 b) , in agreement with a recently published report (miyahira et al., 2009) . although the data indicate a key role for tbk1 in the response to c-di-gmp, it is formally possible, albeit unlikely, that tbk1 protein rather than tbk1 kinase activity is required for the response. we also tested the requirement for the tbk1related kinase ikbke (also called ikk or ikk-i), which plays (jiang et al., 2005) using recombinant ifn- as a standard. *, p < 0.01 as compared with transfected c-di-gmp (student's t test). (b) bone marrow macrophages were transfected with 3.3 µg/ml of the indicated molecules (or overlaid with 100 ng/ml lps), and type i ifn was assessed after 6 h by bioassay as in a. *, p < 0.001 as compared with unstimulated cells (student's t test). (c) bone marrow macrophages were transfected with 3.3 µg/ml of the indicated molecules (or overlaid with 100 ng/ml lps), and transcription of the ifn- gene (ifnb) was assessed by quantitative rt-pcr, with normalization to ribosomal protein rps17 message. n.d., not detectably induced above background (lipofectamine alone). *, p < 0.001 as compared with unstimulated cells (student's t test). (d) as in c, but transcription of the ifn-5 gene was assessed. n.d., not detectably induced above background (lipofectamine alone). *, p < 0.01 as compared with unstimulated cells (student's t test). (e) c-di-gmp, treated or untreated with snake venom phosphodiesterase, was transfected at 3.3 µg/ml into b6 bone marrow macrophages and analyzed after 6 h for ifn- production by bioassay, as in a. *, p < 0.02 as compared with c-di-gmp treatment alone (student's t test). results in a-e are representative of at least three independent experiments. data are means ± sd (n = 3). (f) bone marrow macrophages were transfected with poly da:dt (dna) or with c-di-gmp. total rna was isolated after 6 h of stimulation. probes were amplified and hybridized to whole-transcriptome spotted meebo microarrays. each dot represents a single gene, and its position is determined by its induction in response to dna (x axis) or c-di-gmp (y axis). most genes lie on the diagonal, indicative of similar induction ratios by both treatments. selected highly induced genes are labeled. two independent microarray experiments gave similar results. transcription factors, as well as phosphorylation of the p38, jnk, and erk map kinases, in response to c-di-gmp. activation of irf3 involves phosphorylation, dimerization, and translocation into the nucleus. we tested whether treatment with c-di-gmp resulted in significant nuclear accumulation of irf3. as expected, we found that c-di-gmp resulted in significant translocation of irf3 to the nucleus (fig. 3 a) . we also tested whether transfected c-di-gmp is capable of activating nf-b. this was particularly important to establish in light of a previous report that exogenous (untransfected) c-di-gmp failed to activate nf-b (karaolis et al., 2007a) . there have also been inconsistent reports in the literature as to whether cytosolic dna induces nf-b stetson and medzhitov, 2006a; sun et al., 2006) . we found that c-di-gmp treatment led to the activation of nf-b, as assessed by using an electromobility gel shift assay, with similar kinetics to that of transfected pi:c and pda:dt ( fig. 3 b) . the probe-bound complex was confirmed to contain nf-b p65 by supershifting with an anti-p65 antibody ( fig. 3 b) . we confirmed these results by using a raw role for irf 7 signaling as part of a positive feedback loop involving signaling through the type i ifn receptor (ifnar). indeed, ifnar / macrophages exhibited a diminished response to c-di-gmp (fig. 2 e) . irf5 and irf1 did not seem to be required for responsiveness to c-di-gmp (fig. 2 f) . in addition, macrophages deficient in rip2, nalp3, or doubly deficient in nod1/2 responded normally to c-di-gmp (unpublished data). we validated our results by examining production of a second secreted molecule known to be a target of the tbk1-irf3 signaling axis, the chemokine rantes (ccl5). rantes production was assessed by elisa (fig. s1 ). as expected, we found that rantes was induced by c-di-gmp in a manner dependent on tbk1, irf3, and ifnar, but largely or entirely independent of myd88/trif, irf1, irf5, irf7, or mavs (fig. s1 ). our genetic data suggested that the cytosolic responses to dna, rna, and c-di-gmp share a common downstream signaling pathway. if this were true, we would expect to be able to detect biochemical activation of the nf-b and irf3 myd88 / trif / mice were transfected with 3.3 µg/ml c-di-gmp or pda:dt (dna), or overlayed with 100 ng/ml lps as indicated. after 6 h of stimulation, induction of ifn- mrna was analyzed by quantitative rt-pcr, with normalization to ribosomal protein rps17 message. n.d., not detectable. (b) as in a, except tnfr1 / or tnfr1 / tbk1 / macrophages were analyzed. tbk1 deficiency is embryonic lethal, but viability can be rescued on the tnf receptor 1 (tnfr1)-deficient background. (c) as in a except ikbke (ikk/ikk-i)-deficient mice were analyzed. (d) as in a, except irf3 / and irf7 / macrophages were analyzed. (e) as in a, except macrophages deficient in the type i ifn receptor (ifnar / ) were analyzed. (f) as in a, except irf5 / and irf1 / macrophages were analyzed. results are representative of at least three independent experiments. data are means ± sd (n = 3). *, p < 0.05; and **, p < 0.001 as compared with wild-type bone marrow macrophages (student's t test) . discrepancy between our results and those of karaolis et al. (2007a) , it is more likely that we were able to detect map kinase activation because transfection of c-di-gmp triggers more robust responses than overlayed c-di-gmp, and because we examined later time points. cell type-specific responses to cytosolic dna and c-di-gmp collectively, these results suggest that there are strong similarities in the signaling pathways triggered by the cytosolic presence of c-di-gmp and other nucleic acids, such as dna and rna. however, c-di-gmp is not structurally similar to these nucleic acid ligands. the dna molecules capable of provoking cytosolic responses are double stranded and >25 deoxyribonucleotides in length stetson and medzhitov, 2006a) . in contrast, c-di-gmp is composed of two guanosine ribonucleotides (not deoxyribonucleotides) in an unusual cyclic conformation. c-di-gmp also lacks features consistent with recognition by rig-i or mda5, such as 5triphosphate, polyuridine, or double strandedness . therefore, we considered whether distinct sensors might be responsible for triggering responses to dna/rna and c-di-gmp, and if so, whether the responses could be distinguished in certain cell types. macrophage cell line stably transfected with an nf-b luciferase reporter. we observed activation of the nf-b luciferase reporter by transfected c-di-gmp (fig. 3 c) . both the gel shift and reporter assays showed that the activation of nf-b by transfected c-di-gmp was not as strong as observed with transfected pda:dt and pi:c. the subtle difference in nf-b induction between cytosolic dna and c-di-gmp may suggest that there are distinct signaling components upstream of nf-b in the two pathways. nonetheless, we concluded that transfected c-di-gmp is capable of activating nf-b. the relatively low levels and late time course of nf-b induction by c-di-gmp (as compared with induction by lps), as well as the fact that we transfected c-di-gmp into cells, may explain why nf-b induction was not previously observed in response to c-di-gmp (karaolis et al., 2007a) . we also tested whether map kinase pathways are activated by c-di-gmp. a previous report found that overlay of c-di-gmp induced erk but not p38 in human macrophages (karaolis et al., 2007a) . in contrast, we found that cytosolic c-di-gmp stimulated p38, erk1/2, and jnk phosphorylation in mouse bone marrow macrophages transfected with c-di-gmp (fig. 3 d) . although a difference between human and mouse macrophages may explain the data are means ± sd (n = 3). *, p < 0.05 compared with unstimulated cells (student's t test) . tion, and is downstream of two cytosolic viral rna sensors, rig-i and mda5 (sun et al., 2006) . to test if c-di-gmp signals through the cytosolic rna-sensing pathway, we tested c-di-gmp responses in mavs / and mda5 / macrophages. loss of mavs or mda5 did not affect the induction of ifn- by c-di-gmp (fig. 5, a and b; and fig. s1 c) , suggesting that c-di-gmp does not signal through the cytosolic rna signaling apparatus (gitlin et al., 2006; kato et al., 2008; . dai (encoded by the zpb1 gene) is a recently identified molecule that has been proposed to function as a sensor of cytosolic dna (takaoka et al., 2007; wang et al., 2008) . we found that bone marrow-derived macrophages from zbp1deficient mice (ishii et al., 2008) still responded to c-di-gmp (fig. 5 c) . however, as previously reported (ishii et al., 2008) , zbp1-deficient macrophages also responded normally to dna (fig. 5 c) . these results imply that macrophages express at least one cytosolic dna sensor that is distinct from in addition to bone marrow macrophages, we observed induction of ifn- by cytosolic c-di-gmp in a variety of other cell types, including peritoneal macrophages, conventional dendritic cells, l929 cells, and raw 264.7 macrophages (fig. 4, a-d) . these cells also responded to transfected dna and rna. interestingly, however, we were unable to detect significant induction of type i ifns by c-di-gmp in mefs or in 293t cells, even though the cytosolic pathways for detecting dna and rna are intact in these cells (fig. 4 , e and f). these results suggest that at least one component of the host signaling pathway responding to c-di-gmp is distinct from that used for responses to cytosolic rna or dna, and is differentially expressed in different cell types. known dna and rna sensing pathways are not required for responses to c-di-gmp mavs (also known as ips-1) is an essential adapter protein in the cytosolic rna pathway leading to type i ifn inducdata are means ± sd (n = 3). n.d., not detectable. *, p < 0.01; and **, p < 0.001 as compared with unstimulated cells (student's t test) . and measured their responsiveness to yac-1 target cells ex vivo. because nk cells respond to in vivo injection of poly i:c, a prototypical ifn inducer, we expected that c-di-gmp might also activate nk cells. indeed, nk cells obtained from b6 mice injected with c-di-gmp responded to yac-1 cells, as measured by intracellular cytokine staining for ifn- (fig. 6 b) . however, nk cells obtained from irf3/7 / mice stimulated with c-di-gmp were nonresponsive to yac-1 target cells. as a negative control, nk cells stimulated ex vivo with pbs did not produce ifn-. these observations indicate that c-di-gmp can stimulate cytokine and nk cell responses in vivo, and these responses require the irf3/7 transcription factors. to obtain a preliminary indication as to whether adaptive immune responses could be stimulated by c-di-gmp, we immunized mice with human serum albumin (hsa) and tested whether coinjected c-di-gmp could function as an adjuvant, as previously reported (ebensen et al., 2007; dai, and at least one of these uncharacterized dna sensors, or another independent sensor, may respond to c-di-gmp. to validate our findings in vivo, we injected mice intraperitoneally with c-di-gmp (200 nmol per mouse) and measured the production of type i ifns in the blood 18 h later. b6 mice injected with c-di-gmp produced an ifn response to c-di-gmp, and this response was abolished in irf3/7 double-deficient mice (fig. 6 a) . interestingly, single-deficient irf3 / mice responded to c-di-gmp in vivo (unpublished data), implying a role for irf7 in responses to c-di-gmp in vivo. to determine whether cytokine induction by c-di-gmp affected cellular responses in vivo, we collected splenic nk cells from mice injected with c-di-gmp figure 5 . c-di-gmp does not stimulate known cytosolic pathways for sensing nucleic acids. (a) mavs / and littermate wild-type macrophages were transfected with 3.3 µg/ml c-di-gmp, poly da:dt (pda:dt, dna), or poly i:c (pi:c, rna), and transcription of the ifn- gene (ifnb) was assessed by quantitative rt-pcr, with normalization to ribosomal protein rps17 message. n.d., not detectable. (b) mda5 / and wild-type control macrophages were stimulated and analyzed as in a. (c) zbp1 / (dai knockout) and wild-type control macrophages were stimulated and analyzed as in a. sev, sendai virus. results are representative of at least three independent experiments. data are means ± sd (n = 3). *, p < 0.01; and **, p < 0.001 as compared with wild-type bone marrow macrophages (student's t test) . (a and b) b6 and irf3/7 / mice were injected intraperitoneally with 200 nmol c-di-gmp, and (a) serum ifn was measured by bioassay 18h later, or (b) 36 h after injection, splenic nk responsiveness to yac-1 target cells (or pbs control) was measured ex vivo by intracellular staining for ifn-. (c) b6 and irf3/7 / mice were injected intraperitoneally with hsa ± 200 nmol c-di-gmp, and 2 wk later serum igg1 specific for hsa was determined by elisa. the experiments in a and b were repeated twice, and the experiment in c was performed once. horizontal bars represent means. **, p < 0.01; and *, p < 0.05 (student's t test) . respond well to c-di-gmp. given the marked chemical dissimilarities among dna, rna, and c-di-gmp, it is perhaps not surprising that these nucleic acids appear to be recognized by different sensors. indeed, we favor the idea that there are multiple cytosolic sensors for nucleic acids leading to induction of type i ifns. the specificities of these various sensors will require further dissection. for example, although dai knockout cells still responded to c-di-gmp, it remains formally possible that dai is just one of several redundant sensors for c-di-gmp. these possibilities can be addressed once additional cytosolic nucleic acid sensors are identified. our results suggest that cytosolic sensing of c-di-gmp is relatively specific. other related small nucleic acid compounds such as gtp, cgmp, ppgpp, and pgpg did not trigger transcriptional induction of type i ifns (fig. 1) . in addition, c-di-gmp that was hydrolyzed by snake venom phosphodiesterase did not induce type i ifn (fig. 1) . however, our results cannot eliminate the possibility that a metabolite of c-di-gmp, rather than c-di-gmp itself, is the true molecular moiety that is sensed in the cytosol. elimination of this possibility awaits identification of the c-di-gmp sensor and biophysical or crystallographic characterization of its binding to c-di-gmp. our results also cannot eliminate the possibility that c-di-gmp triggers type i ifn expression indirectly, for example, by stimulating the synthesis of a host ligand that functions as the "true" proximal trigger of type i ifn gene expression. it is also possible that c-di-gmp acts "pharmacologically" by disrupting host physiology in a way that results in type i ifn expression. moreover, because the signaling triggered by c-di-gmp and cytosolic dna are very similar, it is possible that the c-di-gmp pathway is a branch off of the dna-sensing pathway rather than a fully independent pathway. however, even if these alternative models are correct, our data indicate that the pathway downstream of c-di-gmp contains at least some novel components and is at least partially independent of known cytosolic or tlr signaling pathways. several bacterial pathogens, including l. monocytogenes, l. pneumophila, f. tularensis, m. tuberculosis, and group b streptococcus, have been reported to induce type i ifns (coers et al., 2000; o'riordan et al., 2002; opitz et al., 2006; stetson and medzhitov, 2006a; henry et al., 2007; roux et al., 2007; stanley et al., 2007; charrel-dennis et al., 2008) . the mechanism by which these pathogens induce type i ifn resembles that of c-di-gmp: in all cases, type i ifn induction is independent of myd88/trif and tlrs, requires tbk1 and irf3, and (with one exception; unpublished data; opitz et al., 2006) is independent of the cytosolic rna-sensing pathway. it is widely assumed that bacterial dna, perhaps released after bacterial cell lysis, is the relevant ligand that triggers type i ifns in response to pathogens. there are no data that eliminate or confirm this possibility, as sensors required for ifn induction in response to cytosolic bacteria have not yet been reported. however, it is noteworthy that in all cases, induction of type i ifns by cytosolic bacteria requires expression of an auxiliary secretion system, namely the esx-1 system of m. tuberculosis, the francisella pathogenicity island-encoded 2007a). serum was collected from immunized mice 2 wk after a single intraperitoneal injection. mice immunized with hsa alone did not mount a significant antibody response to hsa. in contrast, mice injected with hsa plus c-di-gmp produced variable but significant titers of hsa-specific igg1 antibodies, confirming that c-di-gmp can function as an adjuvant in vivo. importantly, our preliminary data indicated that the adjuvant effect of c-di-gmp depended on irf3/7, as antibody responses in irf3/7 / mice immunized with hsa and c-di-gmp were significantly (p < 0.05) reduced as compared with immunized b6 mice (fig. 6 c) . further studies will be required to establish the mechanism by which c-di-gmp functions as an adjuvant, but our results are consistent with a recent report indicating that the irf3/7 kinase, tbk1, is required for adaptive immune responses in a dna-vaccine model (ishii et al., 2008) . it has been well established that innate immune responses are initiated in response to certain microbial ligands that are evolutionarily conserved and that can be distinguished from selfligands. nucleic acids appear to be a favored target of immune recognition, and are sensed by a variety of endosomal and cytosolic sensor proteins. the cyclic dinucleotide c-di-gmp is a bacterial second messenger that exhibits several characteristics that are desirable in an immunostimulatory ligand: it is produced by numerous species of bacteria, it is a critical regulator of bacterial physiology, and it is not similar to host molecules. indeed, several previous reports have demonstrated that c-di-gmp can provoke potent immune responses when injected in vivo into mice (karaolis et al., 2005a; karaolis et al., 2005b; karaolis et al., 2007a; karaolis et al., 2007b) . however, the mechanism by which c-di-gmp stimulates immune responses remained unclear. our data provide evidence that c-di-gmp is sensed by a novel cytosolic immunosurveillance pathway. responsiveness to c-di-gmp is independent of tlrs that monitor the extracellular/endosomal compartments and is strongly potentiated by transfection of c-di-gmp into the host cell cytosol. moreover, c-di-gmp provokes a transcriptional response highly reminiscent of that triggered by the cytosolic presence of dna or rna, and involves activation of tbk-1, map kinases, and the irf-3 and nf-b transcription factors. thus, we propose that c-di-gmp is sensed in the cytosol. how many different cytosolic sensors exist for nucleic acids? our data suggest that sensing of c-di-gmp occurs via a novel cytosolic immunosurveillance pathway. known nucleic acid sensors include mda5 and rig-i, which sense viral rna and signal via mavs, as well as dai, a putative sensor of dna that signals independently of mavs. based on the finding that most cell types, including mefs, can still respond to dna in the absence of dai (ishii et al., 2008) , it has been proposed that an additional dna sensor must exist and that mefs express both of these sensors . our results now suggest that there is at least one additional nucleic acid sensor in the cytosol, because we find that mefs do not b. beutler (the scripps research institute, la jolla, ca), and raw-b cells were obtained from g. barton. animal protocols were approved by the university of california, berkeley animal care and use committee. reagents. c-di-gmp was synthesized as previously described (kawai et al., 2003) , poly i:c was obtained from ge biosciences, pda:dt (poly(da-dt): poly(da-dt)) was purchased from sigma-aldrich, ppgpp (guanosine-3,5bisdiphosphate) was obtained from trilink biotechnologies, pgpg (diguanosine) was purchased from iba gmbh, gtp and cgmp were obtained from sigma-aldrich, purified lps was purchased from invivogen, and sendai virus was obtained from charles river laboratories. theiler's virus was the gift of m. brahic (stanford university, stanford, ca). cell culture. l929, raw 264.7, and mef cell lines were cultured in dmem containing 10% fbs, glutamine, and penicillin-streptomycin. for bone marrow-derived macrophages, bone marrow cells from femurs and tibias were cultured for 7 d in rpmi 1640 media containing 10% fbs, glutamine, penicillin-streptomycin, and10% csf from 3t3 cells, with feeding on the fourth day of growth. for conventional dendritic cells (gm-csfdendritic cells), bone marrow cells were cultured for 5 d with rpmi 1640 containing 10% fbs, glutamine, penicillin-streptomycin, -mercaptoethanol, and gm-csf, with fresh media added on the second and fourth days of growth. peritoneal macrophages were elicited by injection of 2 ml 4% thioglycollate (fluid thioglycollate medium; bd), and were obtained 4 d later by lavage of the peritoneal cavity with rpmi 1640. cell stimulations (transfections). cells were transfected using lipofectamine 2000 (lf2000; invitrogen) according to the manufacturer's protocol. all nucleic acid stimulants were mixed with lf2000 at a ratio of 1 µl lf2000/1 µg nucleic acid, incubated at room temperature for 20-30 min, and added to cells at a final concentration of 3.3 µg/ml (96-well plates) or 4 µg/ml (6-well plates). for pi:c, 2 mg/ml of the stock solution was heated at 50°c for 10 min and cooled to room temperature before mixing with lf2000. transfection experiments were done for 6 h, unless otherwise stated in the figures. for phosphodiesterase treatment of c-di-gmp, 0.5 µg/µl c-di-gmp was incubated for 2 h at room temperature in optimem buffer (invitrogen), with 15 mm mgcl 2 and 1 u phosphodiesterase i (ge healthcare). quantitative pcr. stimulated cells were overlayed with rnalater (applied biosystems) and stored. rna was isolated using the rneasy kit (qiagen) according to the manufacturer's protocol, and was treated with rq1 rnase-free dnase (promega). 0.5 µg rna was reverse transcribed with superscript iii (invitrogen). platinum taq dna polymerase (invitrogen) and evagreen dye (biotium) were used for quantitative pcr assays and analyzed with a real-time pcr system (steponeplus; applied biosystems). all gene expression values were normalized to rps17 (mouse) or sp9 (human) levels for each sample. the following primer sequences were used: mouse ifnb, (forward) 5-ataagcagctccagctccaa-3 and (reverse) 5-ctgtctgctggtggagttca-3; mouse ifn5, (forward) 5-tgacctcaaagcctgtgtgatg-3 and (reverse) 5-aag-tatttcctcacagccagcag-3; mouse rps17, (forward) 5-cgcc-attatccccagcaag-3 and (reverse) 5-tgtcgggatccacc-t caatg-3; human ifn, (forward) 5-aaactcatgagcagtct-gca-3 and (reverse) 5-aggagatcttcagtttcggagg-3; and human sp9, (forward) 5-atccgccagcgccata-3 and (reverse) 5-tcaatgtgcttctgggaatcc-3. type i ifn bioassay and luciferase reporter assay. cell-culture supernatants from stimulated cells were overlayed on top of isre-l929 ifn reporter cells (jiang et al., 2005) and incubated for 4-6 h (96-well plate). the reporter cells were lysed in passive lysis buffer (promega) for 5 min at room temperature, mixed with firefly luciferin substrate (biosynth), and measured on a luminometer (lmaxii 384 ; mds analytical technologies). levels of type i ifn were calculated from a standard curve using recombinant mouse ifn- (r&d systems). secretion system of f. tularensis, the dot/icm system of l. pneumophila, or the mdrm multidrug efflux pump of l. monocytogenes. it is tempting to speculate that a small, bacterially derived molecule such as c-di-gmp could be transported or leak through these secretion systems. it has not yet been possible to test this idea directly because all of these bacterial species encode numerous c-di-gmp synthases, and a strain lacking all c-di-gmp synthesis has not been reported. in any case, interpretation of these experiments would be complicated by the fact that c-di-gmp plays important regulatory roles in bacterial physiology and pathogenesis. nevertheless, our results with synthetic purified c-di-gmp suggest that in addition to dna, c-di-gmp is a candidate for a conserved molecule unique to bacteria that is responsible for triggering transcription of type i ifn genes. in light of a recent report that bacteria appear to be able to synthesize c-di-amp (witte et al., 2008) , it is interesting to consider whether additional nucleic acids produced specifically by bacteria might also trigger host immunosurveillance pathways. non-nucleic acid small molecules, such as the drug dmxaa, also appear to be able to stimulate the tbk1-irf3 axis (roberts et al., 2007) . indeed, there may be multiple redundant pathways for cytosolic sensing of bacteria. collectively, the available evidence suggests that host cells may sense a wider array of bacterial ligands than was previously appreciated. our results may have important implications for the design of new adjuvants and vaccines. dna vaccines have attracted considerable enthusiasm as an approach for protecting against a variety of infectious diseases (wang et al., 2001; yang et al., 2004) , but there are safety concerns about the insertional mutagenic potential of dna vaccines and/or their potential to trigger pathogenic anti-dna autoimmune antibody responses (schalk et al., 2006) . the potency of dna vaccines appears to derive from their ability to stimulate the tbk1 and innate cytosolic dna-sensing pathways . thus, our demonstration that a synthetic nonself non-dna molecule such as c-di-gmp can stimulate an in vitro and in vivo innate and adaptive immune response (fig. 6 ) similar to that induced by dna, without similar autoimmune or mutagenic risks, suggests that c-di-gmp might have valuable application as a small-molecule adjuvant. understanding the molecular basis of c-di-gmp signaling in mammalian cells will be a crucial step toward achieving this aim. mice and cell lines. bone marrow macrophages were derived from various mouse strains, including mavs / (sun et al., 2006) , mda5 / (gitlin et al., 2006) , and zbp1 / (ishii et al., 2008) . wild-type c57bl/6 and tnfr1 / mice were from the jackson laboratory. myd88 / /trif / mice were obtained from g. barton (university of california, berkeley, berkeley, ca). irf1 / , irf3 / , irf5 / , and irf 7 / mice were obtained from t. taniguchi (university of tokyo, tokyo, japan). tbk1 / /tnfr1 / mice were generated by crossing tbk1 / mice (provided by w.-c. yeh, university of toronto, toronto, canada) with tnfr1 / mice. rip2 / mice were obtained from r. medzhitov (yale university, new haven, ct). nod1 / /nod2 / mice were obtained from d. portnoy (university of california, berkeley, berkeley, ca). nalp3 / mice were obtained from v. dixit (genentech, south san francisco, ca). isre-l929 ifn reporter cells were obtained from vance is a recipient of a cancer research institute investigator award, and is supported by the hellman family faculty fund and national institutes of health grants ai075039, ai082357, and ai080749. the authors have no conflicting financial interests tlr-independent type i interferon induction in response to an extracellular bacterial pathogen via intracellular recognition of its dna identification of icm protein complexes that play distinct roles in the biogenesis of an organelle permissive for legionella pneumophila intracellular growth c-di-gmp-mediated regulation of virulence and biofilm formation listeria monocytogenes multidrug resistance transporters activate a cytosolic surveillance pathway of innate immunity the bacterial second messenger cyclic digmp exhibits potent adjuvant properties ikkepsilon and tbk1 are essential components of the irf3 signaling pathway novel domains of the prokaryotic two-component signal transduction systems essential role of mda-5 in type i ifn responses to polyriboinosinic:polyribocytidylic acid and encephalomyocarditis picornavirus the roles of two ib kinase-related kinases in lipopolysaccharide and double stranded rna signaling and viral infection type i interferon signaling is required for activation of the inflammasome during francisella infection identification of lps2 as a key transducer of myd88-independent tir signalling 5-triphosphate rna is the ligand for rig-i a toll-like receptor-independent antiviral response induced by double-stranded b-form dna tank-binding kinase-1 delineates innate and adaptive immune responses to dna vaccines cd14 is required for myd88-independent lps signaling nk cell responsiveness is tuned commensurate with the number of inhibitory receptors for self-mhc class i: the rheostat model 5-cyclic diguanylic acid (c-di-gmp) inhibits basal and growth factor-stimulated human colon cancer cell proliferation the chemotherapeutic agent dmxaa potently and specifically activates the tbk1-irf-3 signaling axis regulation of cellulose synthesis in acetobacter xylinum by cyclic diguanylic acid brucella requires a functional type iv secretion system to elicit innate immune responses in mice differential recognition of double-stranded rna by rig-i-like receptors in antiviral immunity innate immunity induced by composition-dependent rig-i recognition of hepatitis c virus rna preclinical and clinical safety studies on dna vaccines identification and characterization of mavs, a mitochondrial antiviral signaling protein that activates nf-kappab and irf 3 triggering the interferon antiviral response through an ikk-related pathway control of bacterial transcription, translation and replication by (p) the type i ifn response to infection with mycobacterium tuberculosis requires esx-1-mediated secretion and contributes to pathogenesis recognition of cytosolic dna activates an irf3-dependent innate immune response type i interferons in host defense the specific and essential role of mavs in antiviral innate immune responses dai (dlm-1/zbp1) is a cytosolic dna sensor and an activator of innate immune response roles of cyclic diguanylate in the regulation of bacterial pathogenesis multiple functions of the ikk-related kinase ikkepsilon in interferon-mediated antiviral immunity comparison of gene coverage of mouse oligonucleotide microarray platforms induction of cd4(+) t cell-dependent cd8(+) type 1 responses in humans by a malaria dna vaccine regulation of innate immune responses by dai (dlm-1/zbp1) and other dna-sensing molecules structural biochemistry of a bacterial checkpoint protein reveals diadenylate cyclase activity regulated by dna recombination intermediates visa is an adapter protein required for virus-triggered ifn-beta signaling c-di-gmp (3-5-cyclic diguanylic acid) inhibits staphylococcus aureus cell-cell interactions and biofilm formation bacterial c-di-gmp is an immunostimulatory molecule cyclic di-gmp stimulates protective innate immunity in bacterial pneumonia differential roles of mda5 and rig-i helicases in the recognition of rna viruses length-dependent recognition of double-stranded ribonucleic acids by retinoic acid-inducible gene-i and melanoma differentiation-associated gene 5 a new synthetic approach to cyclic bis(3→5)diguanylic acid ips-1, an adaptor triggering rig-i-and mda5-mediated type i interferon induction distinct tlr-and nlr-mediated transcriptional responses to an intracellular pathogen a cyclic-di-gmp receptor required for bacterial exopolysaccharide production structure and function of the interferon-beta enhanceosome ifn-regulatory factor 3-dependent gene expression is defective in tbk1-deficient mouse embryonic fibroblasts recognition of microorganisms and activation of the immune response cardif is an adaptor protein in the rig-i antiviral pathway and is targeted by hepatitis c virus tank-binding kinase-1 plays an important role during in vitro and in vivo type i ifn responses to dna virus infections innate recognition of bacteria by a macrophage cytosolic surveillance pathway legionella pneumophila induces ifnbeta in lung epithelial cells via ips-1 and irf3, which also control bacterial replication the enhanceosome differential requirement for tank-binding kinase-1 in type i interferon responses to toll-like receptor activation and viral infection rig-i-mediated antiviral responses to single-stranded rna bearing 5-phosphates role of adaptor trif in the myd88-independent toll-like receptor signaling pathway a dna vaccine induces sars coronavirus neutralization and protective immunity in mice the rna helicase rig-i has an essential function in double-stranded rna-induced innate antiviral responses accepted: 10 july 2009 elisa. levels of rantes protein in supernatants from bone marrow macrophages were measured using the rantes duoset elisa kit (r&d systems) according to the manufacturer's protocol.microarray analysis. macrophage rna from 10 6 cells (6-well dishes) was isolated using the ambion rnaqueous kit (applied biosystems) and amplified with the ambion amino allyl messageamp ii arna amplification kit (applied biosystems) according to the manufacturer's protocol. microarrays were performed as previously described (leber et al., 2008) . in brief, spotted microarrays using the meebo 70-mer oligonucleotide set (illumina) were printed at the university of california, san francisco center for advanced technology. microarray probes were generated by coupling amplified rna to cy dyes. after hybridization, arrays were washed, scanned on a genepix 4000b scanner (mds analytical technologies), and gridded using spotreader software (niles scientific). analysis was performed using the genepix pro 6 and acuity 4 software packages (mds analytical technologies). two independent experiments were performed. microarray data have been deposited in the gene expression omnibus under accession no. gse16943.cell fractionation, immunoblotting, and gel shift assays. for analysis of map kinase activation, whole-cell extracts were prepared from 2 × 10 6 bone marrow macrophages stimulated for the times indicated in the figures and lysed in ripa buffer supplemented with 50 mm naf, 2 mm navo 3 , 25 mm -glycerol phosphate, 1 mm pmsf, and 1× complete protease inhibitor cocktail (roche). for analysis of irf3 nuclear translocation, nuclear extracts were prepared from 2 × 10 6 bone marrow macrophages stimulated for the times indicated in the figures using ne-per nuclear extraction reagent (pierce), supplemented with 1× complete protease inhibitor cocktail, using the manufacturer's protocol. protein levels were normalized using the micro-bca kit (thermo fisher scientific) and separated on 10% nupage bis-tris gels (invitrogen). proteins were transferred to polyvinylidene fluoride membranes and immunoblotted with various primary antibodies. whole-cell extracts were probed with anti-p38 map kinase, anti-phospho-p38 map kinase (thr180/tyr182), anti-erk1/2, anti-phospho-erk1/2 (thr202/ tyr204), anti-sapk/jnk, and anti-phospho-sapk/jnk(thr183/tyr185) antibodies from cell signaling technology. nuclear extracts were probed with anti-irf3 and anti-lamin b (fl-425 and m-20; santa cruz biotechnology, inc.). for emsa, nuclear extracts from stimulated bone marrow macrophages were incubated with a 32 p end-labeled nf-b probe (5-gat-cagttgaggggactttcccaggc-3). protein-dna complexes were resolved by native gel electorophoresis and visualized by autoradiography. anti-p65 (c-20) was obtained from santa cruz biotechnology, inc.in vivo experiments. c57bl/6 or irf3/7 / mice were injected intraperitoneally with 200 nmol of chemically synthesized c-di-gmp. for analysis of innate immune responses, mice were sacrificed and analyzed 18-36 h after injection, as indicated in the figures. serum was collected and tested for ifn as described above. splenic nk cells were assayed ex vivo for production of ifn- in response to yac-1 target cells, as described previously (joncker et al., 2009) . for antibody responses, mice were injected with 25 µg of endotoxin-free hsa (sigma-aldrich) mixed with 200 nmol c-di-gmp or saline. 2 wk after injection, mice were bled and serum igg1 specific for hsa was measured by elisa.online supplemental material. fig. s1 shows production of rantes in response to c-di-gmp in wild-type, myd88 / trif / , tnfr1 / , tbk1 / tnfr1 / , mavs / , irf1 / , irf3 / , irf5 / , irf 7 / , and ifnar / bone marrow-derived macrophages. table s1 contains whole-transcriptome microarray gene expression data for bone marrow-derived macrophages transfected with c-di-gmp and pda:dt. online supplemental material is available at http://www.jem.org/cgi/content/full/jem.20082874/dc1.we thank the vance, barton, and portnoy laboratories for discussions, j. leber for advice, and n. arpaia, l. lau, and t. kuo for advice and technical assistance. key: cord-007621-rapinodd authors: vidovic, maria; sparacio, shaun m.; elovitz, michal; benveniste, etty n. title: induction and regulation of class ii major histocompatibility complex mrna expression in astrocytes by interferon-γ and tumor necrosis factor-α date: 2002-11-13 journal: j neuroimmunol doi: 10.1016/0165-5728(90)90103-t sha: doc_id: 7621 cord_uid: rapinodd astrocytes can function as antigen-presenting cells (apc) upon expression of class ii major histocompatibility complex (mhc) antigens, which are induced by interferon-γ (ifn-γ). previous data from this laboratory had shown that the cytokine tumor necrosis factor-α (tnf-α) enhances ifn-γ-mediated class ii antigen expression on astrocytes. we have now investigated the effect of ifn-γ and tnf-α on class ii mhc mrna expression in astrocytes using northern blot analysis. astrocytes do not constitutively express mrna for class ii mhc. kinetic analysis of class ii mhc mrna expression in ifn-γ-treated cells demonstrated an 8 h time lag, which was followed by an increase over the next 16 h. optimal expression of class ii mrna was detected after a 24 h incubation with ifn-γ. this level of expression was further enhanced by the simultaneous addition of ifn-γ and tnf-α to the astrocytes, while tnf-α alone had no effect on class ii mrna expression. tnf-α does not act by increasing the stability of ifn-γ-induced class ii mrna, indicating its action is not at that specific level of post-transcriptional control. furthermore, astrocyte class ii mrna expression was inhibited when cycloheximide (chx) was added together with ifn-γ or ifn-γ/tnf-α, and when chx was added up to 4 h after treatment with ifn-γ or ifn-γ/tnf-α. these results indicate that astrocyte class ii mrna expression is mediated by newly synthesized proteins induced by ifn-γ and/or ifn-γ/tnf-α. the expression of class ii antigens on astrocytes, and cytokine modulation of their expression, may be important in the initiation and perpetuation of intracerebral immune responses. the non-neuronal cells of the central nervous system (cns) are made up of the macroglia (astrocytes, oligodendrocytes and ependymal cells) and the microglia. collectively, these glial cells perform a variety of active roles during development of the brain (rakic, 1971; silver and sapiro, 1981) and subsequently in the maintenance of normal cns physiology (hertz, 1981; janzer and raft, 1987) . recent work has suggested that glial cells such as astrocytes and microglia may be involved in immunological events occurring in the brain. the astrocyte can be stimulated to secrete a number of immunoregulatory molecules, including interleukin-1 (il-i) (fontana et al., 1982) , interleukin-3 (il-3) (frei et al., 1985) , interleukin-6 (il-6) (frei et al., 1989; benveniste eta[., 1990) , prostaglandins (fontana et al., 1982) , leukotriene 134 (llartung et al., 1988) , tumor necrosis factor-c~ (tnf-co (robbins et al., 1987; lieberman et al., 1989; chung and benveniste, 199(i) and ifn-c~/,8 (tedeschi et al., 1986) . the microglia can also be stimulated to secrete 1l-1 (giulian et al., 1986) , il-6 (frei et al., 1989) and tnf-a (frei et al., 1987) , thus providing the cns with numerous endogenous sources of cytokines necessary, for immunological response. more importantly, the astrocyte and microglia can function as antigen-presenting cells (apc) in the cns (fierz et al., 1985; frei et al., 1987) . these cells are able to internalize, process, express and present antigen to encephalitogenic t cells (fontana et al., 1984) . however, such a function is only possible upon expression of class i1 major histocompatibility complex (mhc) molecules. indeed, astrocytes can be induced to express class ii antigens both in the cns and in vitro, following exposure to interferon-7 (ifn-t) (wong et al.. 1984 : fierz et al., 1985 or virus (massa et al., 1986) . mhc-encoded class 11 molecules are heterodimerit glycoproteins which have a central role in the regulation of immune responses (benacerraf, 1981) . the expression of class ii antigens is primarily restricted to b cells, monocytes/macrophages and dendritic cells (hammerling et al., 1975) , although certain non-lymphoid cells can be induced to express class ii upon exposure to ifnq,, and function as apc. these include pancreatic beta cells (markmann et al.. 1988) , keratinocytes (gaspari et al., 1988) , brain endothelial cells (mc-carron et al., 1985) , and most pertinent to this study, astrocytes (fontana et al., 1984) . abnormal control in the level of expression of class i1 genes, and aberrant expression in cells normally class ii negative have been implicated in autoimmune phenomena. because of the importance of class ii mhc antigens, many studies have been directed toward understanding the regulatory mechanisms involved in class ii mhc gene expression. it is generally accepted that induction of class ii gene expression by ifn-y occurs at the transcriptional level (basta et al., 1987; blanar et al., 1988 : fertsch-ruggio et al., 1988 : rosa and fel-ious, 1988 : amaldi et al., 1989 , and that transacting factors interacting with cis-acting dna regulatory elements are involved in the transcriptional regulation of class ii mhc expression (accolla et al., 1985 " salter et al., 1985 sherman et al., 1987 sherman et al., , 1989 blanar et al., 1988 , ama[di et al., 1989 celada et al., 1989) . these trans-acting regulatory factors have been postulated to function positively or negatively, and to be expressed ubiquitously, or in a tissue-or stage-specific manner. although ifnq, is considered the primary inducer of class ii antigens, there is evidence for other cytokines contributing to class i1 expression. we have previously shown that tnf-~ enhances ifnq,-induced class ii antigen expression on astrocytes, and that this is a synergistic interaction as tnf-a alone has no effect on class i1 expression (benveniste et al., 1989) . the present study was undertaken to extend these previous findings, and to examine, at the molecular level, the effect of 1fn-y and tnf-a on astrocyte class ii gene expression. we report that astrocytes express class ii mrna 8 h after treatment with ifn-2¢ or 1fn-t/tni'-~,, indicating a long lag period between exposure to the cytokines and initiation of class ii gene expression. tnf-~ does not act to stabilize ifnq,-induced class ii mrna, suggesting it may act at other levels of post-transcriptional control or at the transcriptional level. furthermore, the expression of class ii mhc mrna was completely inhibited by cycloheximide (chx), suggesting a role for newly synthesized proteins in astrocyte class ii mhc expression. as astrocytes can be stimulated to secrete tnf-a (robbins et al., 1987; lieberman et al., 1989; chung and benveniste, 1990) , and express high affinity tnf-a receptors (benveniste et al., 1989) , tnf-a can act in an autocrine fashion to enhance class ii gene expression in astrocytes. by modulating class ii gene expression and thereby stimulating the apc function of astrocytes, ifn-y and tnf-a in concert may play a pivotal role in the regulation of intracerebral immune responses. rat recombinant ifn-y (specific activity: 4 x 106 u/mg) was purchased from amgen biologicais (thousand oaks, ca, u.s.a.), and human recombinant tnf-a (specific activity: 5.6 x 107 u/mg) was the generous gift of genentech (south san francisco, ca, u.s.a.). monoclonal antibody to glial fibrillary acidic protein (gfap) was obtained from boeringher mannheim (indianapolis, in, u.s.a.), and monoclonal antibody to rat class i1 mhc antigens (clone ox-6) was from accurate corporation (westbury, ny, u.s.a.). second antibody was affinity-purified goat anti-mouse lg conjugated to fluorescein-isothiocyanate (fitc) from southern biotechnology (birmingham, al, u.s.a.). cycloheximide and actinomycin-d were purchased from sigma chemical company (st. louis, mo, u.s.a.) . primary glial cell cultures were established from neonatal rat cerebra by a modification of the mccarthy and de vellis technique (1980) as previously described (benveniste and merrill, 1986) . meninges were removed from rat brains prior to glial cell dissociation and culture. culture medium (cm) was duibecco's modified essential medium (dmem), high glucose formula supplemented with glucose to a final concentration of 6 g/l, 2 mm glutamine, 0.1 mm non-essential amino acid mixture, 0.1% gentamicin, and 10% fetal bovine serum (hyclone, logan, ut, u.s.a.). after 10 days in primary culture, oligodendrocytes were separated from the glial cultures by mechanical dislodging, and the astrocytes were obtained by trypsinization (0.25% trypsin/0.02% edta) and replated at a density of 6-10 x 10 6 cells/100 mm 2 tissue culture plate and allowed to adhere for at least 24 h. the cells were counted using trypan blue; cell viability was 99-100%. the astrocytes were monitored for purity by immunofluorescence, and by non-specific esterase staining for contaminating microglia as previously described (benveniste and merrill, 1986) . the primary astrocytes were plated (5.0 x 104) on 12 mm glass coverslips, incubated in culture medium for 2 days, washed twice with phosphate-buffered saline (pbs), and fixed for 10 s in cold acetone. the cells were then stained for gfap, an intracellular antigen unique to astrocytes (bignami et al., 1972) , using a monoclonal antibody to gfap (1:4) for 30 min at room temperature, followed by a 30 min incubation with goat anti-mouse ig/fitc (1:20). the coverslips were then mounted in 30% glycerol, and visualized by fluorescent microscopy. astrocyte cultures were routinely > 97% positive for gfap, and less than 2% of the cells were microglia based on their positive staining for non-specific esterase. total cellular rna was isolated from confluent monolayers of astrocytes that were incubated for various intervals (0-48 h) without or with ifn-y and/or tnf-a. in some experiments, the protein synthesis inhibitor, chx (5 #g/ml) or the rna synthesis inhibitor, actinomycin d (5 #g/ml), were added to the cytokine-treated astrocytes for 0-24 h. rna isolation followed the procedure of chomczynski (1987) . the cells were collected, washed 2 times with cold pbs, and pelleted. rna was extracted with guanidinium isothiocyanate and phenol, and precipitated with ethanol. samples (15 ~g) of total cellular rna were denatured with formaldehyde for 15 rain at 55°c, and rna was size fractionated by electrophoresis through a 1.0% agarose gel" containing ethidium bromide for visualization of 28 s and 18 s ribosomal rna bands. the visualization of rna bands was useful for assessing the integrity of the rna and for varifying the amount of rna loaded. the rna was then transferred to nitrocellulose paper in 20 x standard saline citrate (ssc) (3 m naci and 0.3 m sodium citrate) at 4°c. after the transfer, the nitrocellulose paper was air-dried and the rna cross-linked in a uv stratalinker oven. prehybridization was performed at 42°(7 in a solution containing 50% (v/v) formamide, 5 x ssc, 1× denhardt's solution, 50 p,g/ml of denatured salmon sperm dna, and 0.1% sodium dodecyl sulfate (sds) for 8-24 h. hybridization was carried out at 420( ` for 48 h in prehybridization solution containing 10% dextran sulfate, 0.05 mm na phosphate buffer and denatured ?2p-labeled murine class i1 e-e~ cdna probe (2 x 1w' cpm/ml). the blots were then washed in 2 x ssc (twice for 20 min) at room temperature, followed by 1 x ssc containing 0.1% sds (twice for 30 min) at 42°c and finally in 0.1 x ssc for 30 rain at 42°c. the blots were dried between whatman filter paper and exposed to kodak x-omat ar film plus intensifying screens at -70°c. the autoradiographs were quantitated by scanning densitometry with a bio-rad model 620 video densitometer. filters were stripped to remove bound class 11 mhc probe, and rehybridized with a second control probe, cyclophilin. a edna probe (peacll) specific for mouse class ii e-a (mathis et al., 1983) was the generous gift of dr. jerold woodward, university of kentucky. the 1.08 kb ecori insert was isolated, and labeled with [et-s2p]deoxyctp using an amersham nick translation kit according to the manufacturer's instructions. a specific activity of 0.5-1 x 1() ~ cpm/~g dna was routinely attained. a edna probe for rat cyclophilin (plb15) (danielson et al., 1988) was the generous gift of dr. jim douglass, the oregon health sciences university. primary rat astrocytes were resuspended in dmem containing 10% fetal bovine serum (fbs), and plated at 4-5 x 10 ~ cells/well into 6-well (35 mm) plates (costar, cambridge, ma, u.s.a.). the plates were incubated overnight to allow recovery of the cells from trypsinization and to assure adherence of the astrocytes. after 24 h the original medium was aspirated off and fresh serum-free medium (1 ml) was added to the wells. triplicate wells of primary rat astrocytes were treated with 100 u/ml of recombinant rat ifn-y and/or 50 ng/ml of recombinant human tnf-a for various incubation periods (0 3 days). at each time point, the cells were trypsinized and stained for class it antigens, as previously described (benveniste et al., 1989) . briefly. astrocytes were incubated with 30 ~1 of ox-6 monoclonal antibody for 60 rain in the cold. washed 3 times with pbs containing 0.5% fbs and 0.02% azide (pbs-fbs-azide), and then incubated with 30 /tl of goat anti-mouse ig-fit(" (1:20) for another 30 rain in the cold. after washing 3 times with pbs-fbs-azide, the cells were fixed in a final volume of 100 ~1 of 1% paraformaldehydc and analyzed on the facstar (becton-dickinson, mountain view, ca, u.s.a.) for class ii antigen expression. negative controls were incubated with 30 /tl of pbs-fbs-azide in place of first antibody, or with an irrelevant monoclonal antibody of the same isotype. the gate window of forward-angle light scatter lay between channels 10 and 255: the gate window for log of green fifc fluorescence lay between channels 0 and 255. ten thousand cells were analyzed for each sample. the level of class i1 mhc mrna was examined in astrocytes following treatment for various times with ifn-y, tnf-a or a combination of the two cytokines. to determine the steady-state level of mrna for class ii, northern blot analysis was performed using a edna probe for murine class ii genes (e-a), with total rna isolated from cultured astrocytes. as seen in fig. 1 , a 1.3 kb class ii mhc mrna transcript was present in ifn-~, treated astrocytes (lanes 2 and 4) and absent in untreated cells (lanes 1 and 3) . class it m}tc mrna expression was more pronounced when the cells were cultured with ifn-y in serum-free medium (sfm) (fig. 1, lane 4) as opposed to serum-containing medium (fig. 1, lane 2) , thus, all the subsequent experiments were conin primary rat astrocytes. northern blot of rna from astrocytes that were incubated in serum containing media (lanes 1 and 2) or serum-free medium (sfm) (lanes 3 and 4) without (lanes 1 and 3) or with if'n-), (100 u/ml) (lanes 2 and 4) for 24 h. total rna was extracted and size fractionated by gel electrophoresis. hybridization was performed with a cdna probe (e-a) specific for a murine class 1i mhc gene. the blot was then exposed at -70°c for 24 h to kodak x-omat ar film plus two intensifying screens, kb, kilobases. ducted in sfm. optimal expression of class ii mrna was detected when cells were stimulated with 100-250 u/rnl of ifn-'r (data not shown). some variability in the concentration of ifn-t required for induction of class ii mrna was noted, and this variability was dependent on the lot of ifn-7 used. therefore, it was necessary to do a dose-response study for each lot of ifn-t used. for this study, 100 u/ml of ifn-t was sufficient for maximal expression of class ii mrna. the optimal time required for class ii mrna expression following treatment of astrocytes with ifn-t is illustrated in fig. 2 . astrocytes were incubated in sfm without or with ifn-~, for 12, 24 or 48 h prior to harvesting. a low level of class ii mhc mrna was detected at 12 h following treatment with ifn-~,, with maximal expression detected after a 24 h incubation with ifn-t. there was a 2.7-fold increase in class ii mhc mrna expression from 12 to 24 h, and a slight reduction at 48 h. we have previously shown that the level of class ii protein expression, based on fluorescenceactivated cell sorting (facs) analysis, was enhanced when the cells were treated with both ifn-t and tnf-a (benveniste et al., 1989) . similarly, in this present study, the incubation of as-2kb fig. 2 . kinetic analysis of ifn-y treatment on astrocyte class ii mhc mrna expression. astrocytes were cultured in sfm without (lanes 1, 3, and 5) or with ifn-'r (lanes 2, 4, and 6) for 12 h (lanes 1 and 2) , 24 h (lanes 3 and 4) or 48 h (lanes 5 and 6). total rna was extracted and analyzed for class 11 mrna by northern blot hybridization method. the blot was exposed to kodak x-omat ar film plus two intensifying ~reens at -70°c for24 h. trocytes with both ifn-y and tnf-a resulted in an enhanced expression of class ii mrna compared to ifn-7 alone (fig. 3) . optimal enhancement of class ii mrna was demonstrated using tnf-a at 50 ng/ml (fig. 3, lane 4) , which correlates with the concentration of tnf-a used for synergistic induction of class ii mhc protein (benveniste et al., 1989) . a 2.2-fold increase in class ii mrna expression in the presence of 50 ng/ml of tnf-a was detected, compared to ifn-y alone. as expected, tnf-a alone did not induce mrna for class ii antigens (data not shown). class ii mhc mrna expression induced by ifn-y/tnf-a was also enhanced when experi-2kb 1 2 3 4 5 , for 24 h. rna was isolated for analysis by northern blot hybridization method. the blot was probed with labeled e-a cdna, and exposed at -70°c for 24 h to kodak x-omat ar film plus two intensifying screens. lg4 ments were performed in sfm (data not shown), indicating that a serum component(s) has a slight inhibitory effect on class i1 mrna expression. in other cell types, a lag phase of approximately 6-8 h precedes the appearance of class ii mrna induced by ifn-7 (basta et al,, 1988; blanar et al., 1988, rosa and fellous, 1988; amaldi et al., 1989) . we performed a more indepth analysis of the kinetics of induction of class ii mrna by ifn-7 and ifn-7/tnf-a in astrocytes. analysis of mrna was performed at different times after induction with ifn-y (fig. 4 , lanes 2, 4, 6, and 8) and ifn-y/tnf-a (fig. 4, lanes 3 , 5, 7, and 9). no class ii mrna was detected until 8 h following treatment with ifn-7, with maximal exvression detected 24 h after exposure to ifn-y. similarly, class 11 mrna was not detected until 8 h in astrocytes that were stimulated with ifn-7/tnf-a; however, the intensity of the rna signal was increased in the presence of both cytokines, as expected. thus, there was an 8 h time lag before class ii mrna was detected in astrocytes. at early time points (8 and 12 h), mrna doublets are seen which ultimately merge into a diffuse, 6 and 7) . and 24 h (lanes 8 and 9). astrocytes in sfm alone (lane 1). the blot was exposed to kodak x-omat ar film plus two intensifying screens at -70 o c for 4 days. more intense 1.3 kb band at 24 h. this may be due to multiple transcription initiation sites described for the e-a gene (mathis et al., 1983) . in addition, a larger mrna species of 2.4 kb is seen at 24 h. the significance of this band is unknown at this time. results for the induction of class ii mhc antigen expression and mrna accumulation are summarized in fig. 5 . tnf-a increases ifn-v-induced class ii expression by increasing levels of mrna for the class ii molecule. however, it is not known whether tnf-a acts by increasing transcription or by stabilizing the mrna. experiments were conducted to assess class ii mrna stability in the presence of ifn-7 or ifn-y/tnf-a. class ii mrna was induced in astrocytes with either ifn-"/or ifn--//tnf-a for 24 h, then actinomycin d (a transcription inhibitor) was added for various times (1, 2, 4, 8, 16, and 24 h). total cellular rna was isolated and analyzed by northern blotting. preliminary results indicated that a decrease in class ii mrna was not detected until 8 h of actinomycin d treatment (data not shown). subsequent experiments were performed utilizing rna extracted after 8, 16 and 24 h of actinomycin d treatment. as shown in fig. 6 actinomycin d treatment, tnf-a did not appreciably affect the stability of e-a mrna compared to the stability of ifn-y-induced e-a mrna. in fact, it appears that tnf-a contributes to an accelerated destabilization of class ii mrna. the approximate half-life of e-a mrna in the presence of ifn-v/tnf-a was 19 h, compared to greater than 24 h in the presence of ifn-v alone. these same blots were reprobed for cyclophilin mrna to demonstrate that the integrity and quantity of rna loaded in each lane was similar (data not shown). these data indicate that tnf-a does not act by mrna stabilization to enhance if/q-v-induced class ii expression. the 8 h delay in class ii mrna expression after ifn-v or ifn-v/tnf-a stimulation of as-195 trocytes suggests that signal transmission initiated by these cytokines involves a number of intermediary steps, possibly the expression of newly synthesized gene products. to test this, we examined whether protein synthesis was required for induction of class 1i mrna by ifn-v and ifny/tnf-a. chx, an inhibitor of protein synthesis, was added to astrocytes at a concentration (5 /.tg/rnl) that inhibited protein synthesis by more than 92%, while still maintaining cell viability (data not shown). astrocytes were cultured for 24 h in the presence of ifn-y, ifn-y/tnf-a, chx alone, ifn-y plus chx, ifn-y/tnf-a plus chx, rna extracted, and then analyzed. fig. 7 demonstrates the effect of chx on the induction of class ii mrna by ifn-v and tnf-a. no mrna for class ii was detected in cells treated with chx alone, ifn-y plus chx, or ifny/tnf-a plus chx (fig. 7, lanes 4, 5, and 6 ). inhibition of protein synthesis completely abolished the induction of class ii mrna by ifn-y and ifn-y/tnf-ct. however, there was no inhibition of cyclophilin mrna expression (fig. 7b) , and no alteration in the pattern of ethidium bromide staining of rna in all the samples treated with chx alone or chx plus the cytokines (fig. 7c) , indicating that chx did not cause a generalized inhibition of mrna expression in astrocytes. cyclophilin was used as a control for these experiments as rna levels do not change upon treatment with ifn-v or ifn-v/tnf-a. that newly synthesized protein(s) is required for the induction of the class ii mhc gene in astrocytes treated with ifn-y or ifn-y/tnf-a was suggested by results in fig. 7 . the duration of protein synthesis required to allow expression of the class ii mhc gene in astrocytes was examined in cells that were pretreated with ifn-y or ifn-7/tnf-a for different lengths of time prior to the addition of chx. class ii mhc mrna was measured 24 h after the treatments were started. as shown in fig. 8a , when chx was added simultaneously with ifn-y/tnf-a or 1-2 h after ifn-y/tnf-a treatment, there was no detectable expression of class ii mhc mrna. however, when astrocytes were incubated with ifnhowever, in samples that were treated for 12 h with ifn-y/tnf-c~ before ctlx was added, there was still a 25% reduction in the expression of class ii mhc signal compared to the positive control of 1fn-y/tnf-a alone (fig. 8a, lane 2) , suggesting that continuous synthesis of protein is required for optimal expression of the class 11 mhc gene. chx treatment had no effect on the expression of cyclophilin rna (fig. 8b) . similar results were seen when astrocytes were incubated with ifn-t and chx, except that the expression of class ii mhc mrna was detected only after cells were incubated with ifn-7 for 8 h prior to the addition for 24 h. total rna was extracted, northern blot hybridization performed and the blot was exposed to kodak x-omat ar film plus two intensifying screens at -70°c for 3 days. autoradiograph of class ii mrna (a). autoradiograph for cyclophilin mrna was obtained by stripping class i1 probe and rehybridizing with a second probe to detect cyclophilin mrna expression (b). photograph of the original gel stained with ethidium bronude to show that there was no alteration in the quantity or the quality of rna in all the samples treated with chx (c). ~q 7/tnf-a for 4 h prior to addition of chx, and class ii rna measured 24 h later, a low level of class ii rna was detected. the increase in the level of class i1 mhc mrna detected parallels the increase in the amount of time the cells were treated with ifn-t/tnf-a before the addition of chx, i.e., the longer the treatment with ifn-7/tnf-a before the addition of chx, the stronger the mrna signal. these results, therefore, suggest that protein synthesis, initiated within 4 h of the cells encountering ifn-t/tnf-a, is critical for subsequent class ii mhc mrna expression. the blot was exposed at -70 ° c for 3 days to kodak x-omat ar film plus two intensifying screens (a). autoradiograph for cyclophilin mrna obtained by stripping class ii probe and rehybridizing with a second probe to detect cyclophilin mrna (b). photograph of the original gel stained with ethidium bromide (c). of chx (data not shown), indicating that 8 h of protein synthesis was critical for ifn-~-induced class ii mrna expression. in this study we have shown that primary neonatal rat astrocytes, upon stimulation with ifn-~,, express mrna transcripts for class ii mhc genes, and that tnf-a enhances the expression of ifn-~,-induced class ii mrna. these results support previous findings that ifn-~, and tnf-a synergize in the induction of class ii mhc protein expression in rat astrocytes (benveniste et al., 1989) . kinetic analysis demonstrated that class ii mrna was first detected after 8 h of treatment with ifn-y, followed by an increase in mrna expression over the next 16 h. when astrocytes were treated with ifn-~, and tnf-a simultaneously, the kinetics of class ii mrna expression did not change; however, the overall amount of steady-state class ii mrna was increased. optimal expression of class ii mrna was detected 24 h after incubation with ifn-3, alone or ifn-~,/tnf-a. although the predominant forms of gene regulation occur at the transcriptional level, a number of control mechanisms can act on rna once its transcription has been initiated. post-transcriptional regulatory mechanisms include (1) changes in mrna stability, (2) alternative rna splicing, (3) poly a addition, and (4) control of translational initiation. in our experiments, tnf-a did not increase the stability of ifn-3,-induced class ii mrna, indicating that tnf-a did not act at that level of post-transcriptional control. preliminary results from our laboratory suggest that the increase in class ii mrna occurs primarily by an increase in transcription of the e-a gene since nuclear run-on assays detected no transcription of the class ii genes without induction by ifn-y, and enhanced transcription in the presence of ifn-~, plus tnf-a. further experimentation is necessary to determine conclusively if tnf-a acts solely at the transcriptional level, or whether both transcriptional and post-transcriptional events result in increased class ii mhc mrna and protein. 197 the time required for the appearance of class ii mhc mrna following treatment with ifn-~, or ifn-~,/tnf-a (8 h) suggests that cytokine signal transmission is complex and may involve a number of intermediary steps. we examined whether protein synthesis was required for ifn-), or ifn-"t/tnf-a-induced expression of astrocyte class ii genes. the expression of class ii mrna was completely inhibited when chx was included with ifn-~, and ifn-'t/tnf-~ treatment, indicating that newly synthesized protein is required for astrocyte class ii mhc gene expression. a minimum of 4 h of active protein synthesis was required for subsequent ifn-t/tnf-a-induced class ii mrna expression, while 8 h was required for subsequent ifn-), expression. however, in experiments where chx was added 12 h after treatment with ifn-), or ifn-),/tnf-~, there was still a 45% and 25% reduction, respectively, in the expression of class ii mrna compared to astrocytes incubated with the cytokines alone. this indicates that the synthesis of novel proteins is required continuously for optimal class ii gene expression in astrocytes. other studies have shown that protein synthesis was required for up to 12 h after ifn-~, was added to murine p388d cells to detect an increase in the level of i-aa (boettger et al., 1988) , while in peritoneal mouse macrophages, a 30% decrease in i-at~ mrna levels was observed even when chx was added after 12 h of ifn-'rtreatment (fertsch et al., 1987) . in contrast, celada et al. (1989) demonstrated that protein synthesis was only required for 30 rain after murine macrophages were treated with ifn-), for an increase in i-aft mrna to be detected. thus, different cell types have varying requirements for active protein synthesis to express class ii mrna in response to ifn-),. other reports on ifn-t-induced expression of class i1 genes have indicated that protein synthesis is not required. induction of dra mrna in the human glioblastoma cell line u373-mg (basta et al., 1988) , dermal fibroblasts (collins et al., 1986) and i-aa in murine wehi-3 cells (woodward et al., 1989) , occurs in the absence of protein synthesis. this suggests that the expression of class ii mrna in these cells is mediated by pre-existing trans-acting factors that are triggered by ifn-'t (woodward et ai., 1989) . it is also important to note that primary astrocytes (this study) and glioblastoma cells (basta et al., 1988) differ in their requirements for protein synthesis for class 11 expression, illustrating fundamental differences between normal astrocytes and transformed glial cells. tnf-a may be an important enhancer of class i1 expression in the cns as it can function in an autocrine fashion on the astrocyte. in addition to responding to tnf-a and expressing specific high affinity receptors for this factor (benveniste et al., 1989) , astrocytes can also secrete tnf-a (robbins et al., 1987; sawada et al., 1989; chung and benveniste, 1990) . more importantly, ifn-t primes the astrocyte to produce tnf-a (chung and benveniste, 1990) , thus ifn-t can influence both tnf-a and class ii gene expression in the astrocyte. although class ii expression on astrocytes has been conclusively demonstrated in vitro, in vivo studies have generated conflicting results. direct injection of ifn-t into the brains of mice induced class ii antigens on astrocytes, indicating that astrocytes have the potential to express these antigens in vivo (wong et al., 1984) . many laboratories have examined whether astrocytes express class i1 antigens in a variety of immune-mediated disease states to better understand the possible role of the astrocyte as a local apc. traugott et al. (1985) demonstrated class ii expression on astrocytes in active chronic multiple sclerosis (ms) lesions, and then confirmed these studies by performing double-staining for both class ii and gfap (traugott and lebon, 1988) . a study by hofman et al. (1986) also identified class ll-posirive astrocytes in ms brain by double-staining. rodriguez et al. (1987) have studied class i1 expression on glial cells in an animal model of cns demyelination induced by theiler's virus. in susceptible strains of mice (bio.s and bio.asr2), the majority of class ii-positive glial cells had morphological characteristics of astrocytes, while uninfected mice or resistant strains (bio.s, (9r)) were class ii negative. in sjl mice with acute or chronic relapsing experimental allergic encephalomyelitis (eae), an animal model for ms, some class ii-positive ceils were identified as astrocytes (sakai et al., 1986) . however, other studies investigating the eae model in lewis rats failed to detect class ii-positive astrocytes in the brain (hickey et al., 1985 : matsumoto et al., 1986 vass et al., 1986) . thesc conflicting results may bc due solely to technical problems involved with antigen fixation and staining methodologies, or may indicate that the ability of astrocytes to function as apc in vivo may only bc relevant in certain diseases or specific stages of disease. another possibility may be the loss of class ii-positive astrocytes by class ii mhc-restricted t cell-mediated cytotoxicity as shown by sun and wekerle (1986) . the disease eae appears to be strain-specific as brown-norway rats and balb/c or c57bl/6 mice are resistant, whereas lewis rats and sjl mice are susceptible (linthicum and frelinger, 1982) . recent studies have demonstrated that astrocytes derived from susceptible strains express much higher levels of class ii antigen upon treatment with either ifn-~, or virus compared to astrocytes prepared from eae-resistant strains (massa et al., 1987a, b) . this hyperinduction of class ii in eae-susceptible animals was astrocyte specific as both peritoneal macrophages and microglial cells of susceptible and resistant strains showed identical patterns for class ii induction. this differential expression of class ii on astrocytes in response to ifn-t compared to microglia suggests that regulation of class i1 expression on astrocytes may correlate with antigen-presenting capacity and ultimately, disease development in the cns. we have begun, at the molecular level, to dissect the regulatory mechanisms utilized by primary rat astrocytes for class i1 mhc gene expression. future studies will focus on the regulation of gene expression at the transcriptional level, and ifn-t/tnf-a-induced trans-acting regulatory factors required for class ii gene expression. reactivation by a trans-acting factor of human mhc-ia gene expression in interspecies hybrids between an ia-negative human b cell variant and an la-positive mouse b cell lymphoma induction of hla class ii genes by ifn-y is transcriptional and requires a trans-acting protein identification of an interferon-y response region 5' of the human histocompatibility leukocyte antigen dret chain gene which is active in human glioblastoma multiform lines detailed delineation of an interferon-y-responsive element important in human hla-dra gene expression in a glioblastoma multiform line role of mhc gene products in immune regulation stimulation of oligodendroglial proliferation and maturation by interleukin-2 tumor necrosis factor-a enhances interferon-y mediated class i! antigen expression on astrocytes induction and regulation of interleukin-6 gene expression in rat astrocytes localization of the glial fibrillary acidic protein in astrocytes by immunofluorescence a (1988) transcriptional activation of hla-dra by interferon y requires a trans-acting protein cycloheximide, an inhibitor of protein synthesis, prevents y-interferon-induced expression of class ii mrna in a macrophage cell line lnterferon-y activates multiple pathways to regulate the expression of the genes for major histocompatibility class ii i-a#, tumor necrosis factor and complement component c3 in mouse macrophages single step method of rna isolation by acid guanidinium thiocyanate-phenolchloroform extraction tumor necrosis fac-199 tor-alpha production by astrocytes: induction by lipopolysaccharide, interferon-gamma and interleukin-1 recombinant human tumor necrosis factor increases mrna levels and surface expression of hla-a,b antigens in vascular endothelial cells and dermal fibroblasts in vitro plbl5: a cdna clone of the rat mrna encoding cyclophilin induction of macrophage la antigen expression by rlfn-y and down-regulation by ifn-a/fl and dexamethasone are mediated by changes in steady-state levels of la mrna induction of macrophage la antigen expression by rlfngamma and down regulation by ifn-alpha/beta and dexamethasone are regulated transcriptionally astrocytes as antigen presenting cells, t. induction of la antigen expression on astrocytes by t cells via immune interferon and its effect on antigen presentation production of prostaglandin e and an interleukin-l-like factor by cultured astrocytes and c6 glioma cells astrocytes present myelin basic protein to encephalitogenic t-cell lines astrocytes of the brain synthesize interleukin-3-1ike factors antigen presentation and tumor cytotoxicity by interferon-y-treated microglial cells on the cellular source and function of interleukin-6 produced in the central nervous system in viral diseases class ii mhc-bearing keratinocytes induce antigen-specific unresponsiveness in hapten-specific th1 clones interleukin-1 of the central nervous system is produced by ameboid microglia tissue distribution of la antigens: la on spermatozoa, macrophages and epidermal cells primary rat astroglial cultures can generate leukotriene b 4 functional interactions between astrocytes and neurons expression of la molecules by astrocytes during acute experimental allergic encephalomyelitis in the lewis rat immunoregulatory molecules and il-2 receptors identified in multiple sclerosis brain astrocytes induce bloodbrain barrier properties in endothelial cells production of tumor necrosis factor and other cytokines by astrocytes stimulated with lipopolysaccharide or a neurotropic virus acute autoimmune encephalomyelitis in mice. i1. susceptibility is controlled by the combination of h-2 and histamine sensitization genes antigen presenting function of class !i mhc expressing pancreatic beta cells viral particles induce la antigen expression on astrocytes inducibility of la antigen on astrocyte by murine coronavirus jhm is rat strain dependent hypersensitivity of la antigen on astrocytes correlates with strain-specific susceptibility to experimental autoimmune encephalomyelitis the murine e-a immune response gene lmmunohistochemical analysis of the rat central nervous system during experimental allergic encephalomyelitis, with special reference to la-positive cells with dendritic morphology presentation of myelin basic protein by murine cerebral vascular endothelial cells preparation of separate astroglial and oligodendroglial cell cultures from rat cerebral tissues neuron-glial relationship during granule cell migration in developing cerebellar cortex. a golgi and electromicroscopic study in maccacus rhesus production of cytotoxic factor for oligodendroeytcs by stimulated astrocytes immune response gene products (la antigens) on glial and endothelial cells in virus-induced demvelination regulation of hla-dr gene by ifn-~,. transcriptional and post-transcriptional control ) la expression in chronic relapsing experimental allergic encephalomyelitis induced by long-term cultured t cell lines in mice evidence for two trans-acting genes regulating hla class ii antigen expression pr(xluction of tumor necrosis factor-alpha by microgila and astrocytes in culture upstream dna sequences required for tissue-specific expression of the hla-dra gene class ii box consensus sequences in the hla-dra gene: transcriptional function and interaction with nuclear proteins axonal guidance during development of the optic nerve: the role of pigmented epithelia and other intrinsic factors ) la-restricted encephalitogenic t lymphocytes mediating eae lyse autoantigen-presenting astrocytes astrocytes produce interferon that enhances the expression of h-2 antigens on a subpopulation of brain cells interferon-'), and la antigen are present on astrocytes in active chronic multiple sclerosis lesions on the presence of la-positive endothelial cells and astrocytes in multiple sclerosis lesions and its relevance to antigen presentation the distribution of la antigen in the lesions of rat acute experimental allergic encephalomyelitis inducible expression of h-2 and la antigens on brain cells mhc class ii transcription in different mouse cell types: differential requirement for protein synthesis between b cells and macrophages this work was funded in part by grants rg 1954-a-2 and rg 2205-a-3 from the national multiple sclerosis society (e.n.b) and grant bns-8708233 from the national science foundation (e.n.b). we acknowledge the support of the university of alabama at birmingham flow cytometry core facility (am 20614).we thank mr. keith berry for facs analysis, and ii yup chung, j. gavin norris and john r. bethea for helpful discussions. key: cord-003300-994731g9 authors: wuerth, jennifer deborah; habjan, matthias; wulle, julia; superti-furga, giulio; pichlmair, andreas; weber, friedemann title: nss protein of sandfly fever sicilian phlebovirus counteracts interferon (ifn) induction by masking the dna-binding domain of ifn regulatory factor 3 date: 2018-11-12 journal: j virol doi: 10.1128/jvi.01202-18 sha: doc_id: 3300 cord_uid: 994731g9 sandfly fever sicilian virus (sfsv) is one of the most widespread and frequently identified members of the genus phlebovirus (order bunyavirales, family phenuiviridae) infecting humans. being transmitted by phlebotomus sandflies, sfsv causes a self-limiting, acute, often incapacitating febrile disease (“sandfly fever,” “pappataci fever,” or “dog disease”) that has been known since at least the beginning of the 20th century. we show that, similarly to other pathogenic phleboviruses, sfsv suppresses the induction of the antiviral type i interferon (ifn) system in an nss-dependent manner. sfsv nss interfered with the tbk1-interferon regulatory factor 3 (irf3) branch of the rig-i signaling pathway but not with nf-κb activation. consistently, we identified irf3 as a host interactor of sfsv nss. in contrast to irf3, neither the ifn master regulator irf7 nor any of the related transcription factors irf2, irf5, and irf9 were bound by sfsv nss. in spite of this specificity for irf3, nss did not inhibit its phosphorylation, dimerization, or nuclear accumulation, and the interaction was independent of the irf3 activation or multimerization state. in further studies, we identified the dna-binding domain of irf3 (amino acids 1 to 113) as sufficient for nss binding and found that sfsv nss prevented the association of activated irf3 with the ifn-β promoter. thus, unlike highly virulent phleboviruses, which either destroy antiviral host factors or sequester whole signaling chains into inactive aggregates, sfsv modulates type i ifn induction by directly masking the dna-binding domain of irf3. importance phleboviruses are receiving increased attention due to the constant discovery of new species and the ongoing spread of long-known members of the genus. outbreaks of sandfly fever were reported in the 19th century, during world war i, and during world war ii. currently, sfsv is recognized as one of the most widespread phleboviruses, exhibiting high seroprevalence rates in humans and domestic animals and causing a self-limiting but incapacitating disease predominantly in immunologically naive troops and travelers. we show how the nonstructural nss protein of sfsv counteracts the upregulation of the antiviral interferon (ifn) system. sfsv nss specifically inhibits promoter binding by ifn transcription factor 3 (irf3), a molecular strategy which is unique among phleboviruses and, to our knowledge, among human pathogenic rna viruses in general. this irf3-specific and stoichiometric mechanism, greatly distinct from the ones exhibited by the highly virulent phleboviruses, correlates with the intermediate level of pathogenicity of sfsv. keywords dna-binding domain, irf3, nss, sandfly fever sicilian virus, interferon beta promoter, interferon induction m embers of the genus phlebovirus (order bunyavirales, family phenuiviridae) are present worldwide and gain increasing attention as vector-borne agents of disease (1) . in addition to prominent, recently emerged phleboviruses such as severe fever with thrombocytopenia syndrome virus (sftsv) in asia and heartland virus (hrtv) in north america (2) , there are long-known members, such as rift valley fever virus (rvfv), punta toro virus (ptv), toscana virus (tosv), and sandfly fever sicilian virus (sfsv), that are often reemerging or spreading into new geographical areas (3) . in addition to these highly virulent (sftsv, hrtv, and rvfv) and intermediately virulent (tosv and sfsv) human pathogens, rapid progress in high-throughput sequencing enabled the identification of novel phleboviruses for which the disease potential is either recognized (e.g., sandfly fever turkey virus [4] and adria virus [5]) or not yet clarified (e.g., massilia virus [6] , aguacate virus [7] , and dashli virus [8] ). infection by sfsv and related sandfly fever viruses, all transmitted by phlebotomine sandflies, typically presents as an acute febrile disease with abrupt onset, often developing into incapacitating myalgia, headaches, malaise, leukocytopenia, or ocular or gastrointestinal symptoms (9, 10) . an outbreak of this so-called "sandfly fever," "pappataci fever," or "dog disease" during the sicilian campaign of world war ii in 1943 enabled albert sabin to isolate sfsv from infected soldiers (11) . sfsv later proved to be one of the most widespread phleboviruses; it is present across the entire mediterranean basin, in portugal, in the middle east inclusive of the arabian peninsula, in sudan, in ethiopia, and in somalia and in locations as distant as india and bangladesh (12) (13) (14) (15) (16) (17) (18) . in regions of endemicity, seroprevalence can reach levels of up to 50% in humans and close to 80% in dogs and other domestic animals, including cattle (12, 14, 19, 20) . hence, sandfly fever viruses are recognized as a significant public health threat, predominantly for immunologically naive groups such as soldiers or travelers (21) (22) (23) (24) . nonetheless, little is known about the molecular interplay of sfsv and sfsv-like viruses with the host organism. like all phleboviruses, sfsv contains a tripartite single-stranded rna genome (1, 3) . while the large (l) genome segment and the medium (m) genome segment encode the viral polymerase (pol) l and the glycoproteins, respectively, in a negative orientation, the small (s) segment codes for the nucleocapsid protein n and the nonstructural protein nss in an ambisense manner. the genomic rna segments are packaged into ribonucleoproteins (rnps) by the nucleocapsid n protein and the l polymerase and are transcribed and replicated in the cytoplasm (25) . due to complementarity of the 5= and 3= termini, the three rnp-packaged genome segments have the capacity to anneal to a so-called "panhandle." this rna structure, with its short double-stranded region and 5=-triphosphate moiety, is an activator of the cytoplasmic rna helicase rig-i, an important virus sensor of the antiviral type i interferon (ifn) system (26) . ligand-bound rig-i signals via the adaptor mitochondrial antiviral-signaling protein (mavs) and the kinases tbk1/ib kinase (ikk) to eventually activate the ubiquitously expressed transcription factor interferon regulatory factor 3 (irf3) (27) . the latter thereby becomes phosphorylated, dimerizes, and accumulates in the nucleus, where, together with nf-b and atf-2/c-jun, it transactivates the ifn-␤ promoter to kick off a first wave of ifn secretion (28) . autocrine and paracrine action of ifn-␤ then triggers the upregulation of irf7, which amplifies and diversifies the initial irf3-driven ifn response by inducing both the ifnb gene and multiple ifna genes (29) (30) (31) . simultaneously, it induces the transcription of ifn-stimulated genes (isgs), several of them with demonstrated antiphleboviral activity (3) . phleboviruses counteract the induction of the ifn response by means of their nss protein (3, 32) . the best-characterized nss, namely, that of rvfv, allows the full rig-i signaling cascade to reach the point of irf3 binding to the ifn-␤ promoter but then abrogates host gene expression by targeted sequestration and deletion of general transcription factors, as well as by the recruitment of corepressors and induction of an mrna export block (33) (34) (35) (36) (37) (38) . in the case of tosv, in contrast, the nss protein causes proteasomal degradation of rig-i (39) , and for sftsv, the nss sequesters multiple factors of the signaling cascade into cytoplasmic aggregates (40) (41) (42) (43) . for many phleboviruses, including the sandfly-borne sfsv, however, the mechanism of nss action is unclear. we and others previously found that the nss of sfsv, expressed by a recombinant rvfv, was able to block transcription of the ifnb gene (44, 45) . here, we investigated the molecular mechanism and identified irf3 as a functional target. sfsv nss inhibits ifn induction. sfsv nss expressed by recombinant rvfv was previously shown to inhibit the upregulation of the ifnb gene (44, 45) . accordingly, infection with parental sfsv strain sabin resulted in only limited upregulation of ifn-␤ mrna, as measured by reverse transcriptase quantitative pcr (rt-qpcr) (fig. 1a) . as controls, we used rvfv strain mp12 (expressing a functional rvfv nss) and clone 13 (expressing an internally deleted rvfv nss) in parallel (33) , which suppressed and activated ifn induction, respectively, in the expected manner. unlike rvfv, neither a natural nor a recombinant nss-deficient strain is available for sfsv. in order to abort nss function, we designed a pool of four small interfering rnas (sirnas) that specifically target the nss gene sequence. the efficiency of the sirnas was tested by cotransfection of an expression plasmid for 3ϫflag-tagged sfsv nss and either the nss-targeting sirna pool or a control sirna. the specific sirnas caused a significant reduction of sfsv nss rna levels in rt-pcr and a complete loss of the flag signal in immunoblot analysis, while the control sirna had no effect (fig. 1b) . in contrast, rna and protein levels of the 3ϫflag-tagged nss of ptv-a were not affected, confirming the specificity of the sirna pool for sfsv nss. we then combined transfection of the nss-specific sirna pool with infection by either sfsv or rvfv mp-12, followed by rt-qpcr analysis. of note, in infected cells the sirna pool as well as the pcr primers can target not only the nss transcript but also the entire s genome segment. therefore, we could not determine whether only the nss mrna was affected by the sirnas or whether the viral genome was also affected. however, due to encapsidation of the genome, we expect a certain level of protection, which in turn would result in an underestimation of sirna effects on nss transcripts. in any case, a substantial depletion of nss sequence-containing rna species (fold reduction, 3.3 ϯ 0.3) was observed (fig. 1c) . moreover, in the presence of the nss-specific sirnas, sfsv infection upregulated the amounts of ifn-␤ transcripts (fold increase, 5.1 ϯ 2.6) (fig. 1d) , despite the fact that virus replication (measured via analysis of l segment levels) was diminished (fold reduction, 2.0 ϯ 0.42) (fig. 1e) . for rvfv mp12, in contrast, the sfsv nss-specific sirnas affected neither the ifn-␤ mrna levels (fig. 1c ) nor the accumulation of its s segment (fig. 1f) . the same applied to clone 13, tosv, and the closely related sandfly fever turkey virus (data not shown), demonstrating both the specificity of the sirna pool and its effect on the induction of ifn-␤ by sfsv. furthermore, no intrinsic ifn-stimulatory activity of the sirna pool was observed in the mock samples (fig. 1c) . taking into consideration the opposing effects of the sirna on ifnb induction and on sfsv replication, a normalized fold induction of 9.8 ϯ 3.7 was calculated for ifnb, compared to 1.0 ϯ 0.4 for rvfv (fig. 1g, right column) . of note, the impairment of sfsv replication by the nss-specific sirna was far less pronounced in ifn-incompetent vero b4 cells (data now shown), indicating that it was largely mediated by the antiviral ifn system rather than by interference with the integrity of the genomic s segment. in summary, sirna knockdown of sfsv nss resulted in simultaneous upregulation of ifn induction and downregulation of sfsv replication in ifn-competent cells, reminiscent of the behavior of nss-deficient phleboviruses. together with the data from recombinant nss-expressing rvfv (44, 45) , this validates the identification of sfsv nss as an ifn induction antagonist. sfsv nss acts in a nondegradative manner. many pathogenic phleboviruses are known to counteract the ifn response by diminishing the levels of key host factors (3) . the nss of rvfv induces proteasomal degradation of cellular proteins such as tfiih-p62 (to block ifn induction) and protein kinase r (pkr) (to prevent the antiviral action of ifn) (35, 44, (46) (47) (48) . the nss of tosv was also shown to cause pkr degradation and to block ifn induction by decreasing rig-i levels (39, 49) . we investigated whether the nss protein of sfsv might execute a similar form of degradative activity on host proteins. as controls, we employed tosv nss and rvfv nss, and we also included the so far hpi, and analyzed by rt-qpcr analysis for ifnb (n ϭ 4; mean ϯ sd). (b) a549 cells were cotransfected with expression constructs for 3ϫflag-tagged sfsv or ptv-a nss and nontargeting control sirna or sfsv nss-specific sirna. samples were subjected to rt-pcr analysis (upper panels) and immunoblotting using anti-flag and anti-tubulin antibodies (lower panel) 24 h after transfection. to exclude amplification of nss sequences from plasmid dna, a duplicate set of reactions was performed without the reverse transcription step (no rt). (c to f) a549 cells were pretransfected with control or sfsv nss-targeting sirna and infected with sfsv or rvfv mp12 at an moi of 1. rna was isolated 12 hpi for rt-qpcr analysis for nss-containing rna (c), ifnb (d), the l segment of sfsv (e), and the s segment of rvfv mp12 (n ϭ 3; means ϯ sd) (f). (g) summary of the relative fold induction data depicted in panels c to f, normalized to the mock sample pretreated with control sirna as well as the fold induction of ifnb in sinss-treated cells over sictrl-treated cells that occurred in a manner independent of the viral burden (means ϯ sd). n.a., not applicable. little-investigated ptv nss, which is known to inhibit host cell transcription (45) . for ptv, there are two distinct strains, namely, adames (ptv-a) and balliett (ptv-b), which strongly and weakly suppress ifn induction, respectively (45, 50) . to directly compare the degradative capacities of the nss proteins of rvfv, tosv, sfsv, ptv-a, and ptv-b, we infected a549 cells with recombinant rvfv encoding the respective nss genes and monitored the intracellular levels of the known phleboviral targets tfiih-p62, pkr, and rig-i, as well as of the central rig-i signaling factors mavs, tbk1, and irf3. as shown in fig 2a, levels of tfiih-p62 were reduced only by rvfv nss. moreover, and in agreement with previous studies (44, 45, 49) , pkr levels were decreased upon expression of the nss of rvfv and tosv but not by those of sfsv and ptv. rig-i levels were left unchanged by the nss of rvfv or ptv-a, strongly decreased by the nss of tosv, and upregulated after infection with the recombinant rvfv expressing nss of sfsv (weakly) or ptv-b (strongly). in fact, in the presence of ptv-b nss the upregulation of rig-i was indistinguishable from the level seen with the nss-deficient control virus rzhδnss. the levels of mavs, tbk1, and irf3 were not affected by any of the nss proteins. these results were confirmed in cells infected with the parental sfsv strain sabin (fig. 2b) . thus, the nss proteins of sfsv and ptv do not degrade the host targets of other phleboviruses. sfsv nss inhibits the irf branch of ifn induction. for our further investigations, we focused on the nss of sfsv but also included those of rvfv (as a well-characterized control) and ptv. to interrogate their activity on ifn induction, we performed luciferase reporter assays. human hek293 cells were transfected with increasing amounts of expression plasmids encoding the respective nss proteins, along with a reporter construct harboring the firefly luciferase (ff-luc) gene under the control of the ifn-␤ promoter and a constitutively expressing renilla luciferase (r-luc) plasmid for normalization. activation of the ifn-␤ promoter was stimulated by cotransfection of a mavs cdna plasmid. as expected, overexpression of mavs strongly activated the ifn-␤ promoter, which was undisturbed by increasing doses of the n terminus of the human mxa protein (δmx [35] ) which was used as a negative control (fig. 3a) . expression of the nss proteins of rvfv, sfsv, and ptv-a, in contrast, suppressed the promoter in a dose-dependent manner. ptv-b nss showed only a partial effect in response to large plasmid amounts, in line with previous observations (50) . the ifn-␤ promoter contains several positive regulatory domains (prds), among which prdi binds transcription factors of the interferon regulatory factor (irf) family and prdii binds nf-b (51, 52) . reporter assays showed that the inhibitory effect of sfsv nss on the prdi promoter element was comparable to that seen with the full ifn-␤ promoter but that prdii activity was inhibited only weakly ( fig. 3b and c). this is in contrast to the nss of ptv-a, which, like the rvfv nss, inhibited the two prd reporters indiscriminately. as similar results were obtained when tbk1 was used for stimulation instead of mavs (data not shown), we concluded that sfsv nss specifically targets the irf branch of ifn induction at the level of tbk1 or further downstream, whereas ptv-a nss blocks ifn induction in a broad manner, as shown previously (45) . sfsv nss interacts with irf3 in a highly specific manner. previously, we took part in a large proteomics screen to identify host cell interactors of viral ifn antagonists that included sfsv nss (53) . the sfsv nss cdna, equipped with the sequence for a c-terminal tandem affinity purification (tap) tag, was inserted into recombinant rvfv to replace the rvfv nss gene (rrvfvδnss::nss sfsv -ctap). 293t cells were infected with this recombinant virus, tandem affinity purification was performed, and protein complexes were analyzed by liquid chromatography-mass spectrometry (lc-ms). strikingly, irf3 was among the host cell interactors of sfsv nss, which is compatible with the results of our reporter assays. in order to test the data obtained by mass spectrometry, , as well as stimulation-dependent firefly luciferase (ff-luc) and constitutively active renilla luciferase reporters. firefly luciferase was under the control of (a) the entire ifn-␤ promoter (n ϭ 3; means ϯ sd), (b) irf-driven prdi (n ϭ 3; means ϯsd), or (c) nf-b-driven prdii (n ϭ 3; means ϯ sd). cell lysates were harvested 24 h after transfection for dual-luciferase assays. firefly reporter activities were normalized to the renilla reporter activities, and the positive controls were set to 100% prior to calculating means and sd across biological replicates. we performed pulldown analyses. an enhanced green fluorescent protein (egfp)-irf3 fusion protein was coexpressed with the recombinant 3ϫflag-tagged nss of sfsv, rvfv, ptv-a, or ptv-b or with the negative-control δmx. cell lysates were then subjected to immunoprecipitation using a plate coated with a nanobody directed against gfp. the nss proteins of rvfv and ptv-a negatively affected the coexpression of egfp-irf3 ( fig. 4 and data not shown), but egfp-irf3 was enriched in all gfp precipitates nonetheless. sfsv nss clearly coprecipitated with egfp-irf3 but not with egfp alone. in contrast, neither of the other phleboviral nss proteins interacted with egfp-irf3. similar results were also observed in an inverse setting; i.e., sfsv nss was able to pull down egfp-irf3 (or hemagglutinin-irf3 [ha-irf3]), while ptv-a and δmx were not (data not shown). this confirms our earlier mass spectrometry data (53) and demonstrates that sfsv nss is unique among the tested phleboviral proteins in its interaction with irf3. we extended our assays to include other members of the irf family. irf7 is the family member most closely related to irf3 in both sequence and function (31) . however, sfsv nss did not coprecipitate with egfp-irf7 (fig. 5a) . likewise, egfp-irf2, egfp-irf5, and egfp-irf9 did not interact with sfsv nss (fig. 5b) . hence, we conclude that sfsv nss selectively targets the immediate early-acting ifn transcription factor irf3. sfsv nss does not inhibit irf3 activation. in uninfected cells, irf3 localizes predominantly to the cytoplasm. upon activation, irf3 becomes phosphorylated by tbk1/ikk, dimerizes, and accumulates in the nucleus, where it associates with the transcriptional cofactors cbp and p300 (52, (54) (55) (56) (57) . transiently expressed sfsv nss, on the other hand, localized diffusely to both the cytoplasm and the nucleoplasm (data not shown), suggesting that it could interfere with irf3 activation or function at any level. we thus simultaneously investigated the three classic hallmarks of irf3 activation in sfsv-infected cells. first, immunoblot analysis showed that irf3 phosphorylation was affected neither in sfsv-infected cells (fig. 6a ) nor in cells infected with a recombinant rvfv expressing sfsv nss (fig. 6b) . the latter experiment also demonstrated that ptv nss was acting downstream of irf3 phosphorylation. also, irf3 dimerization (fig. 6c ) and virus-triggered accumulation in the nucleus (fig. 6d) were not impaired by sfsv infection. thus, sfsv-like rvfv, which was used as a control (33)-was not preventing phosphorylation, dimerization, or nuclear localization of irf3. we tested the impact of sfsv nss on specific irf3 mutants. irf3(5d) is constitutively active and dimerized due to phosphomimetic aspartate residues that replace five serine and threonine phosphorylation sites in the region from amino acid (aa) 395 to aa 407 (54, 55) . sfsv nss was able to inhibit both ifn induction and prd i activation by irf3(5d) (fig. 7a and b) , just like the nss of ptv-a, which was used in parallel. sfsv nss, however, was additionally able to pull down irf3(5d) (fig. 7c ). sfsv nss also interacted with irf3 mutants that are deficient in dimerization, namely, irf3(s385a/s386a) (58) (fig. 7d ) as well as irf3(s385a/s386a-r211a/r213a) and irf3(s385a/s386a-r285a/ h288a/h290a), further derivatives with additional mutations of essential arginine and histidine residues within the dimerization interface (data not shown). in summary, these experiments demonstrated that sfsv nss inhibits a molecular step that takes place after the nuclear importation of activated irf3 but prior to irf3-driven transcription and that the interaction interface on irf3 is accessible in both the inactive and the active states. sfsv nss interacts with the dna-binding domain of irf3. irf3 possesses an n-terminal dna-binding domain (dbd; aa 1 to 113) (59) which also contains the bipartite nuclear localization signal (nls; k77/r78 and r86/k87) (60, 61) , followed by an activation domain comprising the nuclear export signal (nes; aa 139 to 150) (52, 60), a proline-rich domain (pro; aa 150 to 190), an irf association domain (iad; aa 190 to 384) (62) , and a serine-rich domain (sr; aa 384 to 427) that is phosphorylated upon activation (63) (fig. 8a ). crystal structures of the c-terminal portion of irf3 (aa 173/175 to 427) indicate that irf3 phosphorylation induces a marked conformational change in the iad, resulting in the exposure of residues that facilitate dimerization and the interaction with cbp/p300 (58, 64, 65) . we employed systematic deletion analysis to map the irf3 domain that is bound by sfsv nss. as a first step, we cut gfp-tagged irf3 into two halves at position 190. as shown in fig. 8b , only the n-terminal part, ranging from aa 1 to 190, was able to pull down nss. we then removed the remaining domains from this fragment one by one in the c-to n-terminal direction. in this way, we found that the n-terminal dbd alone (aa 1 to 113) was sufficient for binding sfsv nss (fig. 8c) . unfortunately, fine mapping by further c-terminal deletions was inconclusive, as were our attempts to map the corresponding irf3-interacting region within sfsv nss (data not shown). sfsv nss prevents irf3 from binding to the ifn promoter. we hypothesized that sfsv nss might interfere with the promoter-binding activity of irf3. to investigate this, we established an assay in which we used biotinylated ifn-␤ promoter oligonucleotides to pull down mavs-activated irf3 via the use of streptavidin-coated magnetic beads. egfp-irf3 and mavs were coexpressed in hek293 cells either on their own or together with increasing doses of 3ϫflag-tagged sfsv nss or the negative-control δmx. as observable in the input samples, overexpressed mavs induced the phosphorylation and dimerization of egfp-irf3, as expected (fig. 9 , left panels, and data not shown). the presence of sfsv nss did not affect irf3 activation, confirming our observations of sfsv-infected cells. analyzing the precipitated proteins (fig. 9 , right panels), we detected activated egfp-irf3 but not egfp, indicating specific binding to the ifn-␤ promoter oligonucleotide. furthermore, no protein precipitation was observed when empty beads without the biotinylated oligonucleotide were used (data not shown). the sequence specificity of egfp-irf3 binding was confirmed by the addition of an excess of nonbiotinylated ifn-␤ promoter oligonucleotide, which strongly diminished egfp-irf3 binding, whereas a scrambled control oligonucleotide had no such effect. importantly, coexpression of sfsv nss reduced the amount of promoter-bound egfp-irf3 in a dose-dependent manner, but the control protein δmx had no influence. of note, sfsv nss did not coprecipitate with the promoter oligonucleotide, indicating the absence of intrinsic or indirect dna-binding activity. thus, we conclude that sfsv nss stoichiometrically impairs the binding of irf3 to the ifn-␤ promoter by covering essential amino acid residues within the dbd. sfsv, first isolated in 1943 (11), is one of the geographically most widespread members of the genus phlebovirus, with high seroprevalence rates in regions of endemicity (12) (13) (14) (15) (16) (17) (18) (19) (20) . despite the long-standing association with an acute incapacitating disease, little is known about the interaction of sfsv with the host cell. also, there is no and total plasmid adjusted to equal levels with empty vector. firefly activities were normalized to those of renilla, and the stimulation control was set to 100% (n ϭ 3; means ϯ sd). (b) a dual-luciferase assay was performed in parallel with a prdi-responsive firefly luciferase reporter (n ϭ 3; means ϯ sd). (c and d) interaction with irf3 mutants. (c) 3ϫflag-tagged sfsv nss or ptv-a nss was coexpressed with irf3(5d) in hek293 cells. cell lysates were then subjected to immunoprecipitation using an antibody against flag that was covalently coupled to magnetic beads beforehand. (d) gfp-irf3(s385/386a), egfp-irf3, or egfp, as well as 3ϫflag-tagged sfsv nss, was obtained by transient transfection of hek293 cells. immunoprecipitation was performed via the use of gfp. established animal model (11, 24) , prompting earlier researchers to fall back on experiments with human volunteers (9) . we found that the induction of ifn-␤ in sfsv-infected cells is inhibited by nss, although sfsv does not destroy any of the key antiviral host factors that other dipteran-borne phleboviruses attack. rather, sfsv nss binds to the dbd of irf3, thus prohibiting ifn-␤ promoter activation. curiously, none of the other irf family members, including the master regulator irf7 (66), are targeted, indicating high specificity. although a significant role in the generation of a full ifn response has been attributed to the ifn-inducible irf7 (66), the constitutively expressed irf3 is indispensable for the induction of a first wave of ifn-␤ expression from virus-infected cells and the subsequent upregulation of irf7 expression (30, 31) . hence, irf3 knockout mice exhibit substantially increased susceptibility to viral infection (31, 67) . irf3 activation is the target of a number of virulence factors (28), e.g., human papillomavirus 16 (hpv16) e6 (68), the v protein of paramyxoviruses (69), herpes simplex virus 1 (hsv-1) vp16 (70), and rotavirus nsp1 (71) . however, in contrast to sfsv nss, these virulence factors affect phosphorylation, dimerization, nuclear accumulation, or the expression level of irf3. sfsv nss interacted with nonactivated and constitutively active (and dimerized) as well as dimerization-incompetent irf3, suggesting an ability to target irf3 both before and after it becomes activated. moreover, this interaction pattern pointed to a region of irf3 that is accessible independently of its activation and dimerization state. domain mapping consistently revealed that the n-terminal dbd alone was sufficient for binding of sfsv nss. taking the data together, including the interference with sfsv nss at a late stage in the signaling pathway on the one hand and the domain mapping on the other hand, a mechanism involving the sequestration of the dbd by sfsv nss from the ifn-␤ promoter was strongly implied, and its presence was confirmed by a promoter binding assay. the n-terminal dna-binding domain of interferon regulatory factors is about 120 amino acid residues long and displays a conserved architecture consisting of three ␣ helices, four ␤ sheets, and three loops (l1 to l3) in the order ␣1-␤1-␤2-l1-␣2-l2-␣3-␤3-l3-␤4 (51). as sfsv nss (i) interferes with the promoter binding activity of irf3 but (ii) does not interact with other irf family members, one could speculate that it targets amino acid residues that are involved in dna binding but that are not conserved within the irf family. irf3 residues l42, r78, and r86 are both nonconserved and involved in specific dna promoter binding (51) . however, r78 and r86 are also part of the bipartite irf3 nls. since sfsv nss does not interfere with the nuclear importation of irf3, these residues are less likely to mediate the interaction with sfsv nss. that leaves dna binding residue l42 (situated in loop l1) as well as less-conserved strands ␤3 and ␤4 and loops l2 and l3 as the most probable candidate binding sites for sfsv nss. among the other viral proteins known to target irf3, only us1 (also icp22) of herpes simplex virus 2 and np1 of human bocavirus have been described to employ a similar mechanism (72, 73) . like sfsv, both these viruses target the irf3 dbd and disrupt promoter binding, but whether this is restricted to irf3 or also true for any other member of the irf family was not addressed. kaposi's sarcoma-associated herpesvirus proteins k-bzip and lana-1 also prevent the binding of activated irf3 to its cognate promoter sites but do so by occupying the promoter sites themselves (74, 75) , which we did not observe for sfsv nss. in addition to these dna viruses, bovine viral diarrhea virus interferes with promoter binding and then induces the degradation of nuclear irf3 via its npro protein (76, 77) . a direct interaction between npro and irf3 could not be demonstrated, however. hence, to our knowledge sfsv nss seems to be the only virulence factor from an rna virus which acts by directly masking the dbd of irf3 to prevent promoter binding and ifn induction. given the remarkable diversity of the phleboviral nss proteins with respect to sequences, subcellular localizations, and molecular mechanisms, it is tempting to speculate on a correlation between the specific anti-ifn strategy of a given nss protein and the degree of virulence of the respective phlebovirus. the nss of highly virulent rvfv uses multiple strategies, mostly based on proteasomal degradation, to globally and rapidly blunt host gene expression at the transcriptional and posttranscriptional levels (3, 32) . the nss of the highly virulent sftsv abrogates ifn induction by sequestering several key signaling components, including rig-i and tbk1, into cytoplasmic aggregates (40) (41) (42) (43) . the intermediately pathogenic tosv acts by degrading rig-i itself (39) , but its nss seems to be degraded along with its host target, cutting down its inhibitory efficiency (78) . the nss of the apathogenic uukuniemi virus (uukv), in contrast, does not significantly inhibit ifn induction (79, 80) . how does the intermediately pathogenic sfsv fit into this picture? on the one hand, by masking the dbd to sterically hinder irf3 from binding the ifn-␤ promoter, sfsv nss blocks irf3 independently of its conformation or activation state. on the other hand, however, this stoichiometric mechanism requires nss to accumulate to levels that are sufficient for sequestering the cellular pool of irf3, which, during the early phase of infection, outnumbers nss. moreover, sfsv nss inhibition does not include irf7, the master regulator of innate immunity (66) . thus, sfsv nss fail to impair ifn induction in cells where upregulation of irf7 took place before infection or in cells with physiologically high basic levels of irf7, such as plasmacytoid dendritic cells (81) . in other words, the stoichiometric and irf3-specific nature of the anti-ifn induction strategy makes sfsv nss a modulator rather than a full antagonist of ifn induction. this places sfsv between tosv and uukv with regard to both anti-ifn strategy (rig-i degradation versus weak ifn antagonism) and virulence (fever and meningitis/encephalitis versus no disease). curiously, ptv-a does not seem to quite fit the picture; while its nss protein seems to act as a global host transcription inhibitor, infection of humans has so far been associated only with febrile symptoms. in rodent models, such as mouse and hamster, however, ptv-a and chimeric rvfvs that express ptv-a nss are also highly virulent (50, 82, 83) , suggesting that ptv may be an outlier with respect to humans but not other mammals. thus, the demonstration that the intermediately virulent sfsv specifically targets irf3 in a highly specific and stoichiometric (i.e. nondestructive) manner supports our hypothesis that the molecular strategy employed by the nss protein can correlate with the degree of virulence of the parental phlebovirus, although other factors, e.g., cell tropism, rna polymerase activity, species-specific host protein interactions, and escape from adaptive immunity, are of course equally important. cells, viruses, and plasmids. a549, bhk-21, hek293, hek293t, vero b4, and vero e6 cells were cultured in dulbecco's minimal essential medium (dmem) and ccm34 medium (dmem with addition of 17.8 mg/liter l-alanine, 0.7 g/liter glycine, 75 mg/liter l-glutamic acid, 25 mg/liter l-proline, 0.1 mg/liter biotin, 25 mg/liter hypoxanthine, and 3.7 g/liter sodium bicarbonate) supplemented with 10% fetal calf serum (fcs), 2 mm glutamine, 100 u/ml penicillin, and 100 g/ml streptomycin. the sabin strain of sfsv was obtained from the world reference center for emerging viruses and arboviruses (wrceva) and propagated in vero b4 cells. attenuated rvfv strains mp12 and clone 13 were propagated in bhk-21 cells. recombinant rvfv strains rzh548, rzh548δnss, rzh548δnss::nsssfsv, and rzh548δnss::nsstosv have been described previously (44, 84, 85) . rzh548δnss::nssptv-a, rzh548δnss:: ptv-b, and rzh548δnss::nsssfsv-ctap were generated using a polymerase i (pol i)/pol ii-based rescue system as described for the other recombinant rvfv strains (35, 53, 85) . in brief, nss coding sequences for ptv-a and ptv-b nss (genbank accession no. ef201835 and ef201834, respectively) were obtained by gene synthesis (mr. gene) and inserted into modified s-segment rescue plasmid phh21_rvfv_vn_tcs. the reading frame of sfsv nss was amplified from cdna of infected cells and inserted into rescue plasmid phh21_rvfv_vn_mcs_ctap, which contains a c-terminal tag for tandem affinity purification (tap). primer sequences are available on request. the resulting plasmids were transfected together with l-and m-segment rescue plasmids phh21_rvfv_vl and phh21_rvfv_vm, respectively, as well as helper plasmids pi.18_rvfv_l and pi.18_rvfv_n into cocultures of hek293t and bhk-21 cells. recombinant rvfv strains were harvested 5 days after transfection, propagated in vero e6 cells, and characterized by rt-pcr and sequencing of the n-and nss-coding regions. titers of all virus strains were determined on vero e6 cells via plaque assay. both the cell lines and the virus stocks were routinely tested for mycoplasma contamination. to generate constructs encoding 3ϫflag-tagged nss of sfsv (genbank accession no. ef201822.1), ptv-a, or ptv-b, the viral open reading frames were amplified from cdna (sfsv) or synthesized dna (ptv-a and ptv-b) and inserted into pi.18 by ligation-dependent cloning via the use of 5= bamhi and 3= xhoi restriction sites. primer sequences are available on request. pi.18-nssrvfv-3ϫflag and pi.18-3ϫflag-δmx were described before (35) . firefly luciferase reporter constructs p-125luc, p-55c1bluc, and p-55a2luc (52) were kindly donated by takashi fujita, and prl-sv40 was purchased from promega. expression plasmids for human tbk1 (86) and irf3(5d) (55) were kindly provided by john hiscott, for human mavs by shizuo akira (87) , and for full-length pegfp-c1-irf3 (88) dual-luciferase assay. hek293 cells seeded into 96-well plates (1.5 ϫ 10 4 per well) were transfected the following day with firefly and renilla luciferase reporter constructs (40 ng each), as well as expression constructs for mavs (10 ng) and nss proteins or the control protein δmx (0.1 ng, 1 ng, and 10 ng) via the use of transit-lt1 (mirus bio llc). the total plasmid dna amounts were adjusted to equal levels with empty vector pi.18. cells were processed 24 h after transfection, and luciferase activities were measured with a dual-luciferase reporter assay system (promega) according to the manufacturer's recommendations. firefly luciferase activities were normalized to those of renilla luciferase, and the stimulated control samples were set to 100% within each biological replicate. means and standard deviations (sd) were calculated across the indicated number of biological replicate data sets. proteomics. as described previously (35, 53) , approximately 2 ϫ 10 8 hek293t cells were infected with the recombinant rvfv strain expressing tap-tagged sfsv nss (rzh548δnss::nsssfsv-ctap) at an moi of 5. the cells were washed with and scraped off in prechilled pbs at 16 h postinfection (hpi). the cell pellet was snap-frozen in liquid nitrogen, lysed in tap buffer (50 mm tris-hcl [ph 7.5], 100 mm nacl, 0.2% np-40, 5% glycerol) supplemented with protease and phosphatase inhibitors, snap-frozen again, and stored at ϫ80°c until further processing. tap purification was performed by sequential pulldowns using streptavidin agarose and ha-agarose beads. bound protein complexes were eventually eluted in laemmli buffer and subjected to one-dimensional sds-page prior to trypsin digestion and peptide analysis by liquid chromatography-tandem mass spectrometry (lc-ms/ms), which was described in detail elsewhere (53) . coimmunoprecipitation. hek293 cells (2.5 ؋ 10 6 per 10-cm-diameter dish) were transfected with expression plasmids (4 g each) via the calcium phosphate method. cells were washed twice in pbs the following day and lysed in prechilled lysis buffer (50 mm tris-hcl [ph 7.0], 150 mm nacl, 1% igepal-630) freshly supplemented with protease (roche) (complete, edta-free) and phosphatase inhibitors (phosphatase inhibitor cocktail set ii; calbiochem). finally, cell debris was removed by centrifugation (10,000 ϫ g, 10 min, 4°c), and the supernatants were used for further processing. for immunoprecipitation via the use of gfp, supernatants were applied to prewashed wells of a gfp-multitrap (chromotek) and incubated at 4°c for 60 to 90 min under conditions of mild shaking. wells were washed extensively with lysis buffer and bound proteins eluted for 20 min with preheated laemmli buffer under conditions of strong agitation. for immunoprecipitation via the use of flag, magnetic beads (143-21d; invitrogen) were covalently coupled with flag m2 antibody (f3165; sigma) overnight and processed according to the manufacturer's recommendations. lysates were then added to the coupled beads followed by incubation under conditions of rotation at 4°c for 4 h. after extensive washing, bound proteins were eluted by boiling in laemmli buffer at 94°c for 5 min. to map the binding region within irf3, constructs comprising a t7 promoter, the open reading frames (orf) of the respective truncated irf3 mutants fused to egfp, a stop codon, and a poly(a) stretch were assembled via pcr (primer sequences available on request) and purified via gel extraction (omega bio-tek) and dna precipitation. the respective proteins were then produced by coupled in vitro transcription-translation using rabbit reticulocyte lysate (l4610; promega) and added to lysate of hek293 cells transiently expressing sfsv nss. immunoprecipitation via gfp was performed according to the aforementioned protocol. irf3 dimerization assay. a549 cells infected with sfsv were lysed as described above and then processed as described before (90) . in brief, 10% native polyacrylamide gels were prerun at 25 ma for 30 min in native running buffer (25 mm tris, 192 mm glycine, ph 8.3), with 1% deoxycholate added to the cathode buffer. samples were supplemented with native loading buffer (250 mm tris-hcl [ph 6.8], 50% glycerol, 1% deoxycholate, 0.5% bromophenol blue), run at 20 ma for the desired duration, and finally transferred to pvdf membranes via semidry blotting. immunofluorescence assay. a549 cells were seeded onto glass coverslips (1 ϫ 10 5 per 24 wells) 1 day prior to infection at an moi of 1. the cells were washed with pbs at 12 hpi and fixed overnight in pbs-4% paraformaldehyde (pfa) at 4°c. the coverslips were then washed with pbs, and the cells were permeabilized with pbs-0.1% triton x-100, washed again, and blocked in pbs-1% fcs. staining with primary antibodies diluted in blocking buffer (irf3 fl-425, 1:200; sfsv, 1:2,500) was performed for 1 h in a humid chamber. afterward, the coverslips were washed with pbs and incubated with secondary antibodies (alexa fluor 488 donkey anti-mouse [a21202] and alexa fluor 647 donkey anti-rabbit [a31573]; thermo fisher scientific) (both 1:500) and 4=,6-diamidino-2-phenylindole (dapi) (0.1 g/ml) for 45 min in a humid chamber. samples were washed again in pbs, rinsed in demineralized water, and mounted on microscopic slides using fluorsave reagent (calbiochem). confocal microscopy was performed using a leica sp5 microscope and the accompanying software. promoter binding assay. biotinylated dna covering the irf3-responsive positive regulatory domains within the human ifn-␤ promoter and the downstream sequence as a linker (genbank accession no. ef064725.1) was ordered as complementary single dna strands (sense, 5=-gac ata gga aaa ctg aaa ggg aga agt gaa agt ggg aaa ttc ctc tga ata gag aga gga cca tct cat ata aat agg cca tac cca tgg aga aag gac att-biotin-3=; antisense, 5=-aat gtc ctt tct cca tgg gta tgg cct att tat atg aga tgg tcc tct ctc tat tca gag gaa ttt ccc act ttc act tct ccc ttt cag ttt tcc tat gtc-3= ) and subsequently annealed by initial denaturation at 95°c for 5 min, followed by slow cooling (1°c/min) to room temperature (91) . the double-stranded biotinylated oligonucleotide (10 pmol per sample) was bound to streptavidin-coated magnetic beads (dynabeads m-280 streptavidin; invitrogen) (25 l per sample) according to the manufacturer's instructions. hek293 cells seeded into 6 wells (2.5 ϫ 10 5 per well) were transfected with plasmids coding for egfp-irf3 or for egfp (250 ng), mavs (500 ng), 3ϫflag-tagged sfsv nss, or δmx (25, 250, or 500 ng) and empty vector (to adjust plasmid amounts) via the use of transit-lt1 and lysed in the presence of protease and phosphatase inhibitors as we are grateful to besim berisha for excellent technical assistance. we are indebted to patricia aguilar and robert tesh from the world reference center for emerging viruses and arboviruses (wrceva) for providing the sabin strain of sfsv along with mouse immune ascites fluid and to alejandro brun for providing anti-rvfv antiserum. expression and luciferase reporter constructs were kind gifts from john hiscott, takashi fujita, luis martinez-sobrido, adolfo garcia-sastre, and shizuo akira. work in our laboratories was supported by the deutsche forschungsgemeinschaft (grant sfb 1021 to f.w. and grants pi1084/2, pi1084/3, trr179, and trr237 to a.p.), by an erc starting grant (erc-stg ivip no. 311339) to a.p., and by the bundesministerium für bildung und forschung (infect-era; grants "escential" and "erase" to f.w. and a.p., respectively). emerging phleboviruses a new phlebovirus associated with severe febrile illness in missouri phleboviruses and the type i interferon response characterization of a sandfly fever sicilian virus isolated during a sandfly fever epidemic in turkey novel phlebovirus in febrile child massilia virus, a novel phlebovirus (bunyaviridae) isolated from sandflies in the mediterranean aguacate virus, a new antigenic complex of the genus phlebovirus (family bunyaviridae) isolation and sequencing of dashli virus, a novel sicilian-like virus in sandflies from iran; genetic and phylogenetic evidence for the creation of one novel species within the phlebovirus genus in the bunyaviridae family clinical and serologic responses of volunteers infected with phlebotomus fever virus (sicilian type) phlebotomus or sandfly fever experimental studies on phlebotomus (pappataci, sandfly) fever during world war ii seroprevalence of sandfly-borne phleboviruses belonging to three serocomplexes (sandfly fever naples, sandfly fever sicilian and salehabad) in dogs from greece and cyprus using neutralization test neutralisation-based seroprevalence of toscana virus and sandfly fever sicilian virus in dogs and cats from portugal novel and emergent sandflyborne phleboviruses in asia minor: a systematic review sandfly fever in central isolation of phlebotomus (sandfly) fever virus from sandflies and humans during the same season in aurangabad district, maharashtra state presence of sandfly-borne phleboviruses of two antigenic complexes (sandfly fever naples virus and sandfly fever sicilian virus) in two different biogeographical regions of tunisia demonstrated by a microneutralisationbased seroprevalence study in dogs an outbreak of acute febrile illness caused by sandfly fever sicilian virus in the afar region of ethiopia high rates of neutralizing antibodies to toscana and sandfly fever sicilian viruses in livestock high prevalence rates of antibody to three sandfly fever viruses (sicilian, naples, and toscana) among cypriots sandfly fever among swedish tourists incidence of sand fly fever among swedish united nations soldiers on cyprus during 1985 seroconversion for infectious pathogens among uk military personnel deployed to afghanistan phlebotomus (pappataci or sandfly) fever a disease of military importance summary of existing knowledge and preliminary report of original investigations transcription and replication mechanisms of bunyaviridae and arenaviridae l proteins incoming rna virus nucleocapsids containing a 5'-triphosphorylated genome activate rig-i and antiviral signaling viral rna detection by rig-i-like receptors ifn regulatory factors and antiviral innate immunity: how viruses can get better type i interferon gene induction by the interferon regulatory factor family of transcription factors differential viral induction of distinct interferon-alpha genes by positive feedback through interferon regulatory factor-7 distinct and essential roles of transcription factors irf-3 and irf-7 in response to viruses for ifn-alpha/beta gene induction rift valley fever virus nss protein functions and the similarity to other bunyavirus nss proteins nss protein of rift valley fever virus blocks interferon production by inhibiting host gene transcription rift valley fever virus nss gene expression correlates with a defect in nuclear mrna export virulence factor nss of rift valley fever virus recruits the f-box protein fbxo3 to degrade subunit p62 of general transcription factor tfiih nss protein of rift valley fever virus promotes posttranslational downregulation of the tfiih subunit p62 tfiih transcription factor, a target for the rift valley hemorrhagic fever virus a sap30 complex inhibits ifn-beta expression in rift valley fever virus infected cells toscana virus nss protein inhibits the induction of type i interferon by interacting with rig-i suppression of type i and type iii ifn signalling by nss protein of severe fever with thrombocytopenia syndrome virus through inhibition of stat1 phosphorylation and activation viral suppression of innate immunity via spatial isolation of tbk1/ikkepsilon from mitochondrial antiviral platform hijacking of rig-i signaling proteins into virus-induced cytoplasmic structures correlates with the inhibition of type i interferon responses evasion of antiviral immunity through sequestering of tbk1/ikk epsilon/irf3 into viral inclusion bodies nss protein of rift valley fever virus induces the specific degradation of the double-stranded rnadependent protein kinase characterization of rift valley fever virus mp-12 strain encoding nss of punta toro virus or sandfly fever sicilian virus rift valley fever virus nss protein promotes post-transcriptional downregulation of protein kinase pkr and inhibits eif2alpha phosphorylation nss virulence factor of rift valley fever virus engages the f-box proteins fbxw11 and beta-trcp1 to degrade the antiviral protein kinase pkr protein kinase r degradation is essential for rift valley fever virus infection and is regulated by skp1-cul1-f-box (scf)(fbxw11-nss) e3 ligase toscana virus nss protein promotes degradation of double-stranded rna-dependent protein kinase the s segment of punta toro virus (bunyaviridae, phlebovirus) is a major determinant of lethality in the syrian hamster and codes for a type i interferon antagonist an atomic model of the interferon-beta enhanceosome direct triggering of the type i interferon system by virus infection: activation of a transcription factor complex containing irf-3 and cbp/ p300 viral immune modulators perturb the human molecular network by common and unique strategies structural and functional analysis of interferon regulatory factor 3: localization of the transactivation and autoinhibitory domains virus-dependent phosphorylation of the irf-3 transcription factor regulates nuclear translocation, transactivation potential, and proteasome-mediated degradation virus infection induces the assembly of coordinately activated transcription factors on the ifn-beta enhancer in vivo interferon regulatory factor 3 and creb-binding protein/p300 are subunits of double-stranded rnaactivated transcription factor draf1 x-ray crystal structure of irf-3 and its functional implications structure of irf-3 bound to the prdiii-i regulatory element of the human interferon-beta enhancer regulated nuclear-cytoplasmic localization of interferon regulatory factor 3, a subunit of double-stranded rna-activated factor 1 bipartite nuclear localization signal controls nuclear import and dna-binding activity of ifn regulatory factor 3 irf family of transcription factors as regulators of host defense roles of interferon-regulatory factors in t-helper-cell differentiation crystal structure of irf-3 reveals mechanism of autoinhibition and virus-induced phosphoactivation crystal structure of irf-3 in complex with cbp irf-7 is the master regulator of type-i interferon-dependent immune responses revisiting the role of irf3 in inflammation and immunity by conditional and specifically targeted gene ablation in mice human papillomavirus 16 e6 oncoprotein binds to interferon regulatory factor-3 and inhibits its transcriptional activity inhibition of interferon regulatory factor 3 activation by paramyxovirus v protein herpes simplex virus 1-encoded tegument protein vp16 abrogates the production of beta interferon (ifn) by inhibiting nf-kappa b activation and blocking ifn regulatory factor 3 to recruit its coactivator cbp rotavirus nonstructural protein 1 subverts innate immune response by inducing degradation of ifn regulatory factor 3 hsv-2 immediate-early protein us1 inhibits ifn-beta production by suppressing association of irf-3 with ifn-beta promoter human bocavirus np1 inhibits ifn-beta production by blocking association of ifn regulatory factor 3 with ifnb promoter kaposi sarcoma-associated herpesvirus latency-associated nuclear antigen inhibits interferon (ifn) beta expression by competing with ifn regulatory factor-3 for binding to ifnb promoter binding of kaposi's sarcoma-associated herpesvirus k-bzip to interferon-responsive factor 3 elements modulates antiviral gene expression inhibition of beta interferon transcription by noncytopathogenic bovine viral diarrhea virus is through an interferon regulatory factor 3-dependent mechanism the npro product of bovine viral diarrhea virus inhibits dna binding by interferon regulatory factor 3 and targets it for proteasomal degradation truncation of the c-terminal region of toscana virus nss protein is critical for interferon-beta antagonism and protein stability differential antagonism of human innate immune responses by tickborne phlebovirus nonstructural proteins generation of mutant uukuniemi viruses lacking the nonstructural protein nss by reverse genetics indicates that nss is a weak interferon antagonist plasmacytoid dendritic cells: recent progress and open questions pathogenesis of a phleboviral infection (punta toro virus) in golden syrian hamsters attenuation of pathogenic rift valley fever virus strain through the chimeric s-segment encoding sandfly fever phlebovirus nss or a dominant-negative pkr toscana virus induces interferon although its nss protein reveals antagonistic activity t7 rna polymerasedependent and -independent systems for cdna-based rescue of rift valley fever virus triggering the interferon antiviral response through an ikk-related pathway ips-1, an adaptor triggering rig-i-and mda5-mediated type i interferon induction the ebola virus vp35 protein inhibits activation of interferon regulatory factor 3 rapid detection of important human pathogenic phleboviruses interferon priming enables cells to partially overturn the sars coronavirus-induced block in innate immune activation high-throughput assay for determining specificity and affinity of protein-dna binding interactions key: cord-000409-lpf9lpky authors: chen, yongwen; wu, shengxi; guo, guoning; fei, lei; guo, sheng; yang, chengying; fu, xiaolan; wu, yuzhang title: programmed death (pd)-1-deficient mice are extremely sensitive to murine hepatitis virus strain-3 (mhv-3) infection date: 2011-07-07 journal: plos pathog doi: 10.1371/journal.ppat.1001347 sha: doc_id: 409 cord_uid: lpf9lpky the inhibitory receptor programmed death-1 (pd-1) has the capacity to maintain peripheral tolerance and limit immunopathological damage; however, its precise role in fulminant viral hepatitis (fh) has yet to be described. here, we investigated the functional mechanisms of pd-1 as related to fh pathogenesis induced by the murine hepatitis virus strain-3 (mhv-3). high levels of pd-1-positive cd4(+), cd8(+) t cells, nk cells and macrophages were observed in liver, spleen, lymph node and thymus tissues following mhv-3 infection. pd-1-deficient mice exhibited significantly higher expression of the effector molecule which initiates fibrinogen deposition, fibrinogen-like protein 2 (fgl2), than did their wild-type (wt) littermates. as a result, more severe tissue damage was produced and mortality rates were higher. fluorescence double-staining revealed that fgl2 and pd-1 were not co-expressed on the same cells, while quantitative rt-pcr demonstrated that higher levels of ifn-γ and tnf-α mrna transcription occurred in pd-1-deficient mice in response to mhv-3 infection. conversely, in vivo blockade of ifn-γ and tnf-α led to efficient inhibition of fgl2 expression, greatly attenuated the development of tissue lesions, and ultimately reduced mortality. thus, the up-regulation of fgl2 in pd-1-deficient mice was determined to be mediated by ifn-γ and tnf-α. taken together, our results suggest that pd-1 signaling plays an essential role in decreasing the immunopathological damage induced by mhv-3 and that manipulation of this signal might be a useful strategy for fh immunotherapy. although liver transplantation has emerged as an effective therapeutic approach for treating fulminant virus hepatitis (fh), mortality rates associated with fh remain very high worldwide [1] . the recent development of a mouse fh model, based upon infection with the murine hepatitis virus strain-3 (mhv-3), has provided insights into mechanisms underlying the disease pathogenesis and resulted in some novel treatment strategies [2] . mhv-3 is a single-stranded, positive-sense rna virus that belongs to the coronaviridae family. in inbred laboratory mice, the virus produces strain-dependent disease profiles that depend on the infection route, age, genetic background, and immune status of the host. for example, balb/c, c57bl/6 and dba/2 mice develop acute fulminant hepatitis, while c3h mice develop a mild chronic disease and mice of the a strain exhibit no evidence of hepatitis [3, 4] . in contrast to the resistant a strain mice, fh induced by mhv-3 in susceptible mice is characterized by the presence of sinusoidal thrombosis and hepatocellular necrosis [2, 3] . these pathological findings occur concomitantly with expression of fibrinogen-like protein 2 (fgl2), a virus-induced procoagulant molecule in the sinusoidal lining cells in the liver. fgl2 has the capacity to promote fibrinogen deposition and subsequently directly induce the coagulation cascades by the expression of procoagulant activity (pca) [5] . thus, up-regulation of fgl2 is an essential component of the lethal effects of mhv-3-induced fh. programmed death (pd)-1 is an inhibitory receptor expressed on activated t cells, b cells and myeloid cells. pd-1-deficient mice (pdcd1 2/2 ) develop various spontaneous autoimmune diseases, including glomerulonephritis and dilated cardiomyopathy, indicating that this receptor plays a critical role in maintenance of peripheral tolerance [6] . pd-l1 (b7-h1) and pd-l2 (b7-dc), two immunoregulatory molecules belonging to the b7 superfamily, were identified as ligands for pd-1, engagement of pd-1 with its ligands mediates negative signaling events via recruitment of phosphatases, such as shp-2, and dephosphorylation of some effector molecules involved in downstream t cell receptor (tcr) signaling [7, 8] . pd-1 signaling has also been shown to modulate the balance between antimicrobial immune defense and immune-mediated tissue damage. for example, pd-1-deficient mice develop more severe hepatocellular injury than their wild-type (wt) littermates in response to adenovirus infection [9] . in a herpes simplex virus (hsv) stromal keratitis mouse model, blockade of pd-1 signaling led to increased hsv-1-specific effector cd4 + t cell expansion, ifn-c production, and exacerbated keratitis [10] . a functionallysignificant high level of pd-1 expression has been found to be maintained by exhausted cd8 + t cells in mice chronically infected with lymphocytic choriomeningitis virus (lcmv), in primates exposed to simian immunodeficiency virus (siv), and in humans suffering from infection with human immunodeficiency virus (hiv), hepatitis b or c virus (hbv or hcv), or human tlymphotropic virus (htlv). however, blockade of the pd-1/pd-ls pathway efficiently restored the virus-specific effector functions of the exhausted t cells, and lead to substantially reduced in viral load [11, 12, 13, 14, 15] . the pd-1 signal is also known to play a key role in the chronicity of infections with bacteria (helicobacter pylori and schistosoma mansoni) [16, 17] , pathogenic fungus (histoplasma capsulatum) [18] , and parasitic worms (taenia crassiceps) [19] . it appears that a number of pathogenic microorganisms exploit the pd-1 signal in order to evade host immune responses and to establish persistent infection. although the influence of pd-1 signal activity has been studied in several infection models, there are no data available concerning the role of this pathway in fh. to this end, we used the mhv-3induced mouse fh model to demonstrate that pd-1 signaling acts to limit the immunopathological damage during disease progression. furthermore, our findings suggested that enhanced pd-1 signaling might represent a useful immunotherapeutic strategy for treating fh. pd-1 expression has been previously described as being induced on specific cell subsets in response to viral or bacterial infection [20] . thus, we first determined the status of pd-1 expression at 72 h after mhv-3 infection (10 pfu) by immunohistochemical techniques. pd-1-positive cells were observed in tissues from the thymus, spleen, lymph nodes and liver. cellular expression was localized to the cell membrane and in the cytoplasma while was completely absent from the nuclear compartment. pd-1-positive cells were distributed throughout the medulla and cortex of the thymus and lymph nodes. in the spleen, pd-1-positive cells were restricted to the germinal center under normal conditions, but extended to the red pulp after infection. in infected liver, more pd-1-positive cells were present in the portal and parenchymal areas, as opposed to the relatively low presence of pd-1-positive cells in only the portal area in phosphate-buffered saline (pbs) treated-mice (fig. 1a) . the amount of pd-1-positive cells in the different organs of infected and control mice were counted and compared, results showed that the number of positive cells was significantly higher in infected mice (fig. 1b) . furthermore, facs analysis revealed that pd-1 expression was enhanced on multiple subsets of immune cells, including the cd4 + and cd8 + t cells, nk1.1 + nk cells and cd68 + macrophages (fig. 1c) . pd-1positive cells were also observed in the lung, heart and kidney, however, the numbers of pd-1 positive cells in these tissues did not significantly increase in response to mhv-3 infection (fig. s1 ). to investigate the potential role of pd-1 signaling in regulating fh tissue pathology, organs from mhv-3 infected pd-1-deficient (pd-1 ko) and wt mice were assessed for morphological differences. small and discrete foci of necrosis with sparse polymorphonuclear leukocyte infiltration were observed in liver tissues from pd-1-deficient mice after 24 h of infection. in contrast, wt mice exhibited normal liver architecture at this time point. slight liver damage became apparent in wt mice after 48 h of infection, meanwhile, the damaged areas of pd-1-deficient mice had enlarged and confluent necrosis had become evident. by 72 h of infection, the damaged region in pd-1-deficient mice had extended throughout the entire liver, while wt mice suffered much less damage and up to 60% of their liver tissue remained normal at this time point ( fig. 2a) . likewise, higher levels of alanine aminotransferase (alt) and aspartate aminotransferase (ast) were observed in serum from pd-1-deficient mice after 72 h of infection (fig. 2b ). more interestingly, pd-1-deficient thymus, spleen and lymph node tissues infected with mhv-3 for 72 h exhibited severely disrupted architecture, loss of cellularity, and the presence of substantial amounts of karyorrhectic/apoptotic cell bodies. the histology of these organs from infected wt mice at 72 h was relatively normal (fig. 2c ). in conjunction with the apparent tissue necrosis, higher levels of cell apoptosis were also evidenced in the organs from pd-1-deficient mice by tunel staining (fig. 2d ). the architecture of other organs, including the heart, kidney and lung was relatively normal and only rare apoptosis events were observed in these tissues after infection (fig. s2 ). in all, these results demonstrated that pd-1 deficiency led to enhanced pathological damage by mhv-3 in the liver, spleen, lymph node and thymus, where higher levels of pd-1-positive cells were found after infection. the earlier and increased organ damage suffered by pd-1deficient mice infected with mhv-3 instigated our monitoring of the mortality rates of pd-1-deficient mice and their similarlyinfected (10 pfu) wt littermates. as shown in fig. 3 , all of the pd-1-deficient mice died within four days after infection, while the principal characteristic of fulminant viral hepatitis (fh) induced by the murine hepatitis virus strain-3 (mhv-3) is severe hepatocellular necrosis, which is mediated by the fibrinogen-like protein 2 (fgl2), a molecule that has the capacity to promote fibrinogen deposition and activate the coagulation cascades. here, we report that mhv-3 infection of program death-1 (pd-1)-deficient mice results in tissue damage throughout multiple organs, including the liver, spleen, thymus and lymph nodes. the liver damage, in particular, occurred earlier and was more severe in pd-1-deficient mice than in their wild type (wt) littermates. further investigation determined that mhv-3 infection was associated with high levels of ifn-c and tnf-a in the damaged organs of pd-1-deficient mice. conversely, intraperitoneal injection of a combination of anti-ifn-c and anti-tnf-a blocking mabs led to inhibition of fgl2 expression, greatly attenuated tissue lesions and reduced mortality. our results demonstrate that pd-1 signaling controls immunopathological damage following mhv-3 infection, indicating that manipulation of the pd-1 signal might represent a useful strategy for fh immunotherapy. 38% of the wt mice survived up to the end of the 15-day survey period (p = 0.007). these data indicated that pd-1 is likely a critical factor that controls mhv-3-mediated tissue damage and mortality. to understand the mechanisms of pd-1 deficiency-mediated tissue damage and mortality, we performed a comparative genome-wide microarray analysis (nimblegen) of genes expressed in liver tissues of pd-1-deficient and wt mice after 72 h of mhv-3 infection. the most notable finding was pronounced upregulation (3.75-fold) in the liver of pd-1-deficient mice of the fgl2 transcripts (fig. 4a) , the protein product of which has been demonstrated to induce lethality of mhv-3-induced fh [5] . in addition, the enhanced fgl2 expression was confirmed by quantitative (q)pcr, results revealed a 2.84-fold and 5.72-fold higher level was present in liver from wt and pd-1-deficient mice, respectively, after 72 h of mhv-3 infection, as compared to their uninfected controls. moreover, its level in pd-1-deficient liver was 2.01-fold higher than that in the wt group at this time point (fig. 4b) . immunohistochemistry was used to show that fgl2-positive cells were present in necrotic liver tissues in pd-1deficient mice at 24 h after mhv-3 injection. the protein expression was found to be enhanced rapidly upon infection, and the highest level occurred at 72 h post-infection. however, occasional fgl2-positive cells were detected in the livers of wt mice at 24 h post-mhv-3 infection and these cells were also present, and slightly enhanced in number, at both the 48 h and 72 h time point (fig. 4c ). western-blot was used to verify the higher fgl2 protein level in the livers of pd-1-deficient mice, as compared to wt littermates after 72 h of infection (fig. 4d ). fgl2 has the capacity to induce fibrinogen deposition, which then activates the coagulation cascades and finally induces procoagulant activity. therefore, the expression of fgl2 and fibrinogen deposition in damaged liver tissues was measured. dual fluorescent staining evidenced that substantial fibrinogen deposition occurred in the fgl2-positive damaged liver tissue (fig. 4e ). likewise, the level of fibrinogen deposition was more robust in livers from pd-1-deficient mice that in livers from wt littermates, at both the 48 h and 72 h time points (fig. 4f) . to determine whether fgl2-mediated pca activity was also involved in inducing damage in the other organs of pd-1-deficient mice, the expression of fgl2 was analyzed in the thymus, spleen and lymph nodes. immunohistochemistry evidenced that fgl2positive cells were also present in these organs. in thymus and lymph nodes, fgl2-positive cells were detected in both the medulla and cortex. in spleen, however, the positive cells were only found in the red pulp. again, the expression of fgl2 appeared to be restricted to the cell membrane and cytoplasma. the distribution of fgl2-positive cells in pd-1-deficient mice had not changed after 72 h of mhv-3 infection, but the number of positive cells in the examined organs was enhanced significantly and the levels of expression were much stronger (fig. 5a ). in addition, the transcription of fgl2 in the spleen of pd-1-deficient mice was also significantly increased in response to infection (fig. 5b) . meanwhile, higher levels of fibrinogen deposition were found in the spleen and lymph node tissues of pd-1-deficient mice (fig. 5c) . moreover, the level of fgl2 present in serum, as measured by elisa, was found to increase rapidly after infection, and the level in pd-1-deficient mice was significantly higher than that in wt littermates (fig. 5d ). to clarify the source of fgl2, fluorescent dual staining was performed on spleen tissues and results demonstrated that fgl2 was principally associated with cd11c-positive dendritic cells (dcs), cd68-positive macrophages and cd31-positive endothelial cells (fig. 5e ). all of these results indicated that the absence of pd-1 signaling can result in enhanced fgl2 expression, consequently inducing stronger fibrinogen deposition and more severe tissue necrosis in pd-1deficient mice following mhv-3 infection. we further examined whether fgl2 secretion was regulated by pd-1 directly or indirectly. fgl2/pd-1 dual fluorescent staining was performed and results indicated that fgl2 and pd-1 were not co-expressed on the same cells in the liver, thymus, spleen or lymph nodes (fig. 6a) . previous studies have shown that the secretion of fgl2 can be triggered by the pro-inflammatory factors ifn-c and tnf-a [21, 22] . on the other hand, the production of ifn-c and tnf-a by activated t cells, nk cells and macrophages can be inhibited by the pd-1 signal [6] . therefore, we compared the status of ifnc and tnf-a in pd-1-deficient and wt mice in response to mhv-3 infection. qpcr revealed that the transcription of the ifn-c gene in liver was significantly higher in pd-1-deficient mice than in wt mice at 72 h post-mhv-3 infection (fig. 6b) . in pd-1-deficient spleen, the transcription of both ifn-c and tnf-a was found to be rapidly enhanced upon mhv-3 exposure (fig. 6c ). facs analysis indicated that ifn-c secretion from nk cells, but not from cd3 + t cells, in the liver was much higher in pd-1deficient mice at 72 h after mhv-3 infection (fig. 6d ). the fact that ifn-c and tnf-a both have the capacity to initiate fgl2 expression may explain why higher fgl2 expression was observed in the pd-1-deficient mice. to further demonstrate that ifn-c and tnf-a were responsible for the observed fgl2 up-regulation in mhv-3 infected pd-1deficient mice, pd-1-deficient mice were infected with mhv-3 and simultaneously treated with a combination injection of anti-ifn-c and anti-tnf-a blocking mabs. the expression of fgl2 was measured by qpcr and protein detected by immunohistochemistry. the transcription of fgl2 mrna (fig. 7a ) and its protein levels (fig. 7b) were completely inhibited by 48 h after injection of anti-ifn-c and anti-tnf-a mabs, as compared to the control rat igg1 isotype antibodies-treated group. moreover, the tissue necrosis (fig. 7c ) and liver damage (as indicated by alt and ast levels) (fig. 7d ) in pd-1-deficient mice were also significantly reduced, thus the mhv-3-mediated mortality rates were decreased as well (fig. 7e ). the pd-1 signaling is best known for its ability to inhibit or dampen the immune response. most of the evidence for this function, however, comes from models of tolerance or chronic infections [11, 12, 13, 14 15] . although some studies have indicated that this signal might also participate in regulating acute infections [23, 24, 25, 26] , its functions in this disease condition are much less clear. here, we used a mouse fh model mediated by mhv-3 infection to describe the effects of pd-1 in this disease process. firstly, pd-1 was found to be significantly up-regulated on t cells, macrophages and nk cells within the thymus, spleen, lymph nodes and liver in response to mhv-3 infection. to determine the exact role of pd-1 in the pathogenesis of fh, pd-1deficient mice were used to establish an infection model. interestingly, mhv-3-induced liver damage in pd-1-deficient mice occurred rapidly and the lesion area was much larger than in their wt littermates. we then extended our investigation to the thymus, spleen and lymph nodes, where increased pd-1-positive cells were observed post-infection. surprisingly, severe tissue necrosis and substantial apoptosis was observed in these organs of pd-1-deficient mice at 72 h after mhv-3 infection. in contrast, these organs from wt mice exhibited relatively normal histology, a finding in agreement with previously reported results [27] . taken together, these results suggested that pd-1 deficiency promoted expansion of the pathological damage from the liver to the lymph organs, including the spleen, lymph node and thymus in this fh model, thereafter, the absence of pd-1 was associated with higher mortality rates in response to mhv-3 infection. murine fh induced by mhv-3 is a recognized and validated model for studying host resistance/susceptibility to human hepatitis virus, and several studies have shown that balb/c or c57bl/6 mice have an innate susceptibility to the infection [3, 4] . fgl2 has been proposed as a critical mediating factor of lethality in the mhv-3-induced fh mice due to the fact that it has the capacity to induce fibrinogen deposition, which in turn activates the coagulation cascades and induces procoagulant activity [5] . to clarify whether the tissue necrosis we observed in pd-1-deficient mice following infection was also mediated by fgl2, the expression of fgl2 was analyzed. results showed that the expression of fgl2 was principally associated with cd31-positive endothelial cells, cd68-positive macrophages and cd11c-positive dcs. surprisingly, significantly higher levels of fgl2 were observed after infection in all of pd-1-deficient organs, including the liver, thymus, spleen, lymph nodes, and serum than that in those from wt littermates. in addition, increased fibrinogen deposition was observed in the organs of pd-1-deficient mice. although we currently have no direct data to evidence that fgl2 directly mediates the mortality of our pd-1-deficient mice, data from other researchers have clearly shown that fgl2 promoted mouse mortality in response to mhv-3 infection [5, 28, 29, 30] . considering this, our results strongly indicate that the mortality of pd-1-deficient mice post-mhv-3 infection is due to the higher level of fgl2 secretion and increased fibrinogen deposition. indeed, it has been reported that both fgl2 and pd-1 are expressed on t cells, macrophages, and dcs, and that targeted deletion of fgl2 or pd-1 leads to impaired t cell activity, and these events are related to the development of autoimmune diseases [6, 31, 32] . we here also observed pd-1 expression as being enhanced on t cells (both cd4 + and cd8 + t cells). it was reasonable to propose that the expression of fgl2 may have been directly regulated by pd-1 signals. unexpectedly, our fgl2/pd-1 dual staining showed that pd-1-positive cells in the liver, thymus, spleen and lymph nodes did not co-express fgl2, indicating that the expression of fgl2 was not directly regulated by pd-1. on the other hand, the expression of fgl2 is believed to be induced by ifn-c and tnf-a [21, 22] , while pd-1 signaling has the capacity to inhibit ifn-c and tnf-a secretion from pd-1-positive immune cells [6] . therefore, we evaluated and compared the status of ifn-c and tnf-a in both pd-1-deficient and wt mice. definitively, the transcription of ifn-c and tnf-a genes was rapidly enhanced post-mhv-3 infection in pd-1-deficient mice, as compared to wt controls. in particular, a higher level of ifn-c was observed in nk cells but not in cd3 + t cells of pd-1deficient liver post-mhv-3 infection, indicating that the pd-1 signal can inhibit ifn-c secretion from nk cells under such condition. conversely, injection of a the combination of anti-ifnc and anti-tnf-a blocking mabs was able to successfully inhibit fgl2 mrna transcription and protein expression, resulting in reduced tissue damage and significantly protecting against mhv-3-mediated mortality in these mice. these results demonstrated that up-regulation of fgl2 in pd-1-deficient mice after mhv-3 infection was controlled, at least partially, by ifn-c and tnf-a. recently, the secretion of fgl2 from naturally occurring cd4 + foxp3 + regulatory t cells (tregs) was demonstrated and it was reported that deficiency of treg-produced fgl2 resulted in increased effector t cell proliferation [32] . more interestingly, levy and colleagues showed that the frequency of fgl2 + tregs was higher in lymphoid tissues of mhv-3 infected mice, and treatment with fgl2-specific antibodies reversed mhv-3induced liver injury and mortality in vivo. these findings demonstrated that fgl2 is an important effector cytokine of tregs that contributes to mhv-3-induced fh [30] . pd-1 signaling has also been described as participating in regulation of treg differentiation and function [33, 34] . in our study, we also analyzed the status of foxp3 + cells in both pd-1-deficient and wt controls. however, the number of foxp3-positive cells in the liver, spleen, lymph node or thymus was not significantly different between pd-1-deficient mice and their wt littermates after 72 h of mhv-3 infection (fig. s3) . therefore, foxp3 + cells are unlikely to be involved in the mortality of pd-1-deficient mice. however, the functional status of these tregs (for example, the level of fgl2 secretion) in pd-1-deficienct mice requires further investigation, and such studies are in progress in our lab. in conclusion, we have determined that pd-1 signaling can limit the immunopathological damage induced by mhv-3 infection in a mouse fh model. our results suggest that enhancing the pd-1 signal by an immunotherapeutic approach might be a useful treatment for fh. all experiments were approved by and conducted in accordance with the guidelines of the animal care and use committee of the third military medical university. all efforts were made to minimize animals' suffering. mice pd-1-ko-n10 (strain: balb/cj) mice were kindly provided by prof. t. honjo (department of immunology and genomic medicine, kyoto university, japan). the wt control mice were purchased from the animal center of beijing university school of medicine. all mice were maintained in micro-isolator cages and housed in the animal colony at the animal center, third military medical university, standard laboratory chow diet and water was supplied ad libitum. mice were used in experimental analysis at age of six weeks and at an average weight of 17 g (range: 16,18 g). mhv-3 was kindly provided by prof. q. ning (institute of infectious disease, tongji hospital of tongji medical college, wuhan, china). the virus was plaque-purified and then expanded in murine l2 cells. virus-containing supernatants were collected and stored at -80uc until use. mice were intraperitoneally (i.p.) injected with 10 pfu/mouse in a total volume of 200 ml. in some experiments, pd-1-deficient mice were infected with mhv-3 (10 pfu) and simultaneously treated with a infection was analyzed by immunohistochemistry. blue color indicates nuclear dapi staining. scale bar = 20 mm. magnification 6200. ns: not significant different. *p,0.05, ** p,0.01. doi:10.1371/journal.ppat.1001347.g004 combination injection of anti-ifn-c (200 mg/mouse per day, clone: r4-6a2, ebioscience, san diego, ca, usa) and anti-tnf-a (200 mg/mouse per day, clone: mp6-xt22, ebioscience) mabs, tissues were isolated for hematoxylin and eosin (h&e) staining to detect damage, and for fgl2 mrna transcription measured by qpcr (see below). serum alt and ast levels were measured by an au5400 automatic biochemistryanalyzer (olympus, japan). in order to monitor the mortality, anti-ifn-c and anti-tnf-a blocking mabs or rat igg1 control mabs were injected everyday for a total of 6 days. paraffin-embedded tissue blocks were cut into 5 mm slices which were mounted on polylysine-charged glass slides. endogenous peroxidase activity was blocked by exposure to 3.0% h 2 o 2 for 15 min. antigen retrieval was performed in a citrate buffer the expression of pd-1 on immune cells (cd4, cd8, nk and macrophages) from different organs was assessed by flow cytometry (facsaria cytometer; becton dickinson, germany). briefly, cell suspensions of liver, spleen, blood and thymus tissues were washed and resuspended in pbs. cells were then incubated for 30 min at room-temperature in the dark using primary antibodies (pe-pd-1, fitc-cd4, fitc-cd8, fitc-nk1.1 and fitc-cd68. ebioscience). to analyze the source of ifn-c in the liver, pd-1-deficient and wt mice were treated with mhv-3 (10 pfu). after 72 h, liver tissues were isolated and mechanically homogenized, lymphocytes were collected thereafter. cells were then treated with brefeldin a solution (bfa) for 4 h, and fitc-nk1.1, fitc-cd3 or pe-ifn-c mabs (ebioscience) were added and the solution incubated for an additional 1 h. for each analysis, 10000 cells were evaluated. flow cytometric data were analyzed with cellquest pro software. the microarray experiment was performed under contact by kangcheng co. ltd. (shanghai, china). briefly, total rna was isolated by trizol from liver tissue of pd-1-deficient and wt mice treated with 10 pfu mhv-3 for 72 h. rna concentration was measured on the nd-1000 spectrophotometer (nanodrop, wilmington, de, usa) and quality evaluated by denaturing gel electrophoresis. samples were then amplified and labeled using a nimblegen one-color dna labeling kit and hybridized using the nimblegen hybridization system (roche applied science, shanghai, china). after hybridization and washing, the processed slides were scanned by the axon genepix 4000b microarray scanner. three independent experiments were performed, and for each test and control sample, two hybridizations were carried out by a reverse fluorescent strategy. only genes whose alteration tendency was concordant between both microarray assays were selected as differentially expressed genes. total rna from the liver and spleen of wt and pd-1-deficient mice was isolated by trizol (invitrogen, carlsbad, ca, usa), according to the manufacturer's instructions. rna samples were quantitated by measurement of optical density at 260 nm. total mrna (2 mg) was reverse-transcribed to cdna using the revertaid h minus first strand cdna synthesis kit (fermentas china, shenzhen city, china), in accordance with the manufacturer's instructions. qpcr was performed to quantitatively analyze the gene transcription levels of fgl2, ifn-c and tnf-a genes. the primers for fgl2 were: sense 59-tggacaacaaagtgg-caaatct-39 and anti-sense 59-tggaacacttgccatc-caaa-39. the primers for ifn-c were: sense 59-tcaagtgg-catagatgtggaag-39, and anti-sense 59-cgcttatg-ttgttgctgatgg-39. the primers for tnf-a were: sense 59-cacgctcttctgtctactgaac-39 and anti-sense 59-atctgagtgtgagggtctgg-39. the primers for b-actin (internal control) were: sense 59-cactatcggcaatgag-cggttcc-39 and anti-sense 59-cagcactgtgttggca tagaggtc-39. the qpcr was performed at 95uc for 10 s followed by 40 cycles of 95uc for 5 s, 60uc for 15 s, and 72uc for 15 s. the specificity of pcr product was examined by a dissociation curve, and results were analyzed by the 2 2ddct method [35] . the expression of fgl2 in liver from mhv-3 infected (72 h) pd-1-deficient mice or their wt littermates was determined by western-blot; the protocol has been described previously [36] . the serum fgl2 level from mice infected with or without mhv-3 was detected by using the mouse fgl2 elisa kit (cat: e90512mu; uscn life science inc., wuhan, china) and following the manufacturer's instructions. all results shown are representative of at least three separate experiments. unpaired student's t-test (two-tailed) or the mann-whitney test was used for comparison of two groups where appropriate. kaplan meier curve with log-rank test (graphpad prism 4.03 software) was used to analyze the mortality rate. pvalue ,0.05 was considered as statistically significant. its protein expression in the liver, spleen and lymph node from pd-1-deficient mice after 72 h of mhv-3 infection in the presence of ifn-c and tnf-a mabs or rat igg1 isotype control antibodies was detected by qpcr and immunohistochemistry, respectively. (c) the ifn-c and tnf-a mabs treatment resulted in decreased damage to the liver, spleen, lymph node and thymus after 72 h of mhv-3 infection. (d) reduced fgl2 level by ifn-c and tnf-a mabs treatment resulted in reduced liver damage (indicated by ast and alt levels). (e) pd-1-deficient mice were infected with mhv-3 (10 pfu) and simultaneously treated with ifn-c and tnf-a blocking mabs (n = 4) or rat igg1 control (n = 6), the survival rate was monitored for a total of 15 days. p = 0.025,0.05 was considered significantly different. one representative of three experiments that yielded similar results is shown. magnification 6 400. ns: not significantly different. *p,0.05 and **p,0.01. n = 5/group. doi:10.1371/journal.ppat.1001347.g007 figure s3 the number of foxp3-positive cells was not changed significantly in pd-1-deficient mice after mhv-3 infection. foxp3-positive cells in the liver, thymus, spleen, and lymph nodes between pd-1-deficient and wt mice at 72 h after mhv-3 infection were detected by immunofluorescence staining (left). statistical analysis of the number of foxp3-positive cells in the indicated organs (right). blue color indicates nuclear dapi staining. scale bar = 20mm. ns: not significantly different. found at: doi:10.1371/journal.ppat.1001347.s003 (1.44 mb tif) viral hepatitis in the liver transplant recipient pattern of disease after murine hepatitis virus strain 3 infection correlates with macrophage activation and not viral replication induction of monocyte procoagulant activity by murine hepatitis virus type 3 parallels disease susceptibility in mice acquired immunity of a/j mice to mouse hepatitis virus 3 infection: dependence on interferon gamma synthesis and macrophage sensitivity to interferon gamma the fgl2/ fibroleukin prothrombinase contributes to immunologically mediated thrombosis in experimental and human viral hepatitis pd-1 and its ligands in tolerance and immunity pd-1 inhibits t-cell receptor induced phosphorylation of the zap70/cd3zeta signalosome and downstream signaling to pkctheta shp-1 and shp-2 associate with immunoreceptor tyrosine-based switch motif of programmed death 1 upon primary human t cell stimulation, but only receptor ligation prevents t cell activation pd-1 inhibits antiviral immunity at the effector phase in the liver cd274) inhibits the development of herpetic stromal keratitis (hsk restoring function in exhausted cd8 t cells during chronic viral infection enhancing sivspecific immunity in vivo by pd-1 blockade upregulation of pd-1 expression on hiv-specific cd8+ t cells leads to reversible immune dysfunction dysfunction and functional restoration of hcv-specific cd8 responses in chronic hepatitis c virus infection programmed death-1-induced interleukin-10 production by monocytes impairs cd4(+) t cell activation during hiv infection expression of b7-h1 on gastric epithelial cells: its potential role in regulating t cells during helicobacter pylori infection schistosoma mansoni worms induce anergy of t cells via selective up-regulation of programmed death ligand on macrophages the pd-1/pd-l costimulatory pathway critically affects host resistance to the pathogenic fungus histoplasma capsulatum role of the programmed death-1 pathway in the suppressive activity of alternatively activated macrophages in experimental cysticercosis novel strategies to eliminate persistent viral infections cytokineinduced hepatic apoptosis is dependent on fgl2/fibroleukin: the role of sp1/ sp3 and stat1/pu.1 composite cis elements intact type 1 immunity and immune-associated coagulative responses in mice lacking ifn gamma-inducible fibrinogen-like protein 2 role of pd-1 in regulating acute infections pd-1 expression by macrophages plays a pathologic role in altering microbial clearance and the innate inflammatory response to sepsis recall responses by helpless memory cd8+ t cells are restricted by the upregulation of pd-1 pd-1 on dendritic cells impedes innate immunity against bacterial infection a virus related to that causing hepatitis in mice (mhv) expression of the fgl2 and its protein product (prothrombinase) in tissues during murine hepatitis virus strain-3 (mhv-3) infection fulminant hepatic failure in murine hepatitis virus strain 3 infection: tissue-specific expression of a novel fgl2 prothrombinase the novel cd4+cd25+ regulatory t cell effector molecule fibrinogen-like protein 2 contributes to the outcome of murine fulminant viral hepatitis characterization of human fibroleukin, a fibrinogen-like protein secreted by t lymphocytes targeted deletion of fgl2 leads to impaired regulatory t cell activity and development of autoimmune glomerulonephritis pd-l1 negatively regulates cd4+cd25+foxp3+ tregs by limiting stat-5 phosphorylation in patients chronically infected with hcv pd-l1 regulates the development, maintenance, and function of induced regulatory t cells analysis of relative gene expression data using real-time quantitative pcr and the 2(-delta delta c(t)) method triptolide inhibits b7-h1 expression on proinflammatory factor activated renal tubular epithelial cells by decreasing nf-kappab transcription we thanks prof. t honjo kindly give us the pd-1 ko mice. mhv-3 virus was provided by prof. q ning and she also gave the research some invaluable suggestions. conceived and designed the experiments: yc cy. performed the experiments: yc sw gg lf sg xf. analyzed the data: yc cy yw. wrote the paper: yc yw. key: cord-023033-tgt69ir6 authors: nan title: poster session (pp. 78a–178a) date: 2006-02-10 journal: hepatology doi: 10.1002/hep.1840380503 sha: doc_id: 23033 cord_uid: tgt69ir6 nan , ala-60 (n=4), tyr-77 (n=2), gly-42 ( n = l ) and lys-89 ( n = l ) . the mean time from onset of symptoms to diagnosis was 3.4 years (0-10) and patients were listed for olt an average of 10.8 months (0-60) after diagnosis. the mean time from listing to olt was 8.2 months (1-18). five patients died after olt, during a mean follow-up of 3.5 years (4 months -6 years). one-year patient survival was 100% and threeyear patient survival was 92%. one patient required retransplantation for hepatic artery thrombosis. neurologic symptoms were the initial clinical manifestation in the majority of patients (7/12), followed by cardiopulmonary (4112) and gastrointestinal symptoms (1112). most patients experienced multiple symptoms. subjective evolution of symptoms as assessed by chart review demonstrated that symptoms referable to amyloidosis worsened after olt in seven patients, and improved or stabilized in five. following olt, neuropathy symptoms improved in four patients, worsened in seven patients and were unchanged in one patient. pre and post-olt nerve conduction velocities were available for 5 patients and the post-olt studies showed progression of disease in 3, improvement in 1 and were unchanged in 1 patient. prior to olt, all twelve patients had an increased ivst on echocardiogram. post olt cardiac symptoms improved in six patients (five of whom also had cardiac transplantation), worsened in three patients, and were unchanged in three patients (one of whom also had cardiac transplantation backgr0und :accurate delineation of the scope and magnitude of peri-operative donor risk is necessary to better allow for informed consent, to maximize the potential for donor safety, for comparative outcome analysis and ultimately to serve as a key determinant of the utility of adult-to-adult living donor liver transplantation (aaldlt). however, at present, there is lack of uniformity regarding what constitutes a complication in this setting. moreover, a system to stratify adverse events with respect to their life altering (quantity or quality) impact is lacking. aims: 1) to define a graded, inclusive classification schema for both early (e) and late (l) adverse operation-related events in live liver donors a n d 2) to apply this system to a retrospective review of events in individuals undergoing partial hepatectomy for live liver donation at our center. two patients (6.5%) suffered cut-surface bile leaks and one (3.2%) a right colon injury (grade 3e complications). one patient (3.2%) developed a transient bilateral ulnar neuropathy (grade 2e). two patients (6.5%) were readmitted within 30 days of operation for nausea and dyspnea respectively (grade 1e). one patient underwent repair of an incisional hernia 6 months post donation (grade 2l). one patient suffered positioning-related brachial plexopathy (grade 3l). conclusions: the definition and adoption of a graded, scale-based system of operation-related adverse events in live liver donors will allow for an inclusive, consistent and universally applicable method to collect, analyze and report donor complications. all aaldlt programs must be encouraged to fully review and report their donor related morbidity, ideally through the creation of a national donor registry initiation of antiviral therapy in the early post-transplant period, prior to overt evidence of hcv recurrence (i.e. preemptive therapy) may enhance rates of hcv eradication. aims: to determine the efficacy and tolerability of preemptive ifn versus ifnlrbv in anti-hcv positive lt patients. methods: consecutive and eligible lt recipients from a single center were enrolled. the goal was to initiate treatment within 6 wks of lt. patients were randomized to ifn or ifn plus rbv (400mg daily x 2wks, then 800mg daily x 2 wks, then 1.0-1.2g daily based upon body weight 75 kg). induction therapy with daily ifn was used for the first 8 wks (1.5mu daily x 2wks, then 3mu daily x 6 wks) followed by 3 mu tiw (n=39) or peg-ifn 1.5 uglkglwk (n=10) for 40 wks. key inclusion criteria were stable clinical status, cr45,ooo and wbc>3.0. dose reductions for side effects, especially cytopenias were standardized. growth factors were given for neutropenia (anc<1000) and anemia (hgb<9.0gldl) beginning 7l2001. virological (vr) and biochemical responses (br) were evaluated at end-of-treatment (et) and 6 mos post-treatment (sustained virological (svr) and biochemical (sbr) responses (table below) were associated with response. histological disease was mild in the majority of patients at treatment end (78% stage 0 fibrosis and 72% grade 1 or less necroinflammatory activity). conclusions: preemptive antiviral therapy was applicable to -60% of transplanted patients. biochemical responses were frequent but svrs uncommon. treatment discontinuation and lowering of rbv doses due to side effects likely reduced svrs and may have limited our ability to detect differences between treatment groups. the only predictor of svr was a negative qhcvrna pre-treatment, which implies that patients with low vl early post-lt may be the best candidates for preemptive therapy. compared to historical reports, the severity of histological disease was very mild at 1-year post-lt in the majority of treated patients, suggesting preemptive antiviral therapy may provide important histological benefits. the cadaveric organ shortage has led to the development of alternative strategies for organ transplantation. living donor liver transplant (ldlt) is one strategy that has offered many individuals transplantation before they die or become too sick for transplant.chronic hepatitis c (hcv) is the leading indication for liver transplantation. preliminary results from small single center studies suggest that recurrent hcv is more common after ldlt compared to cadaveric transplant with concern that graft survival may be lower after ldlt. &: to compare patient and graft survival in hcv recipients who undergo ldlt to cadaveric liver transplant recipients. methods: we analyzed the united network for organ sharing (unos) liver transplant database from january, 1999 -december, 2002. inclusion criteria included liver transplant recipients 218 years old transplanted from 1999-2002 with the transplant diagnosis of chronic hepatitis c. exclusion criteria included subjects who were hepatitis b surface antigen positive, history of prior organ transplant, concurrent kidney or heart transplant. the logrank test was used to compare survival between the two groups. results: from 1999-2002 6.6% of adult liver transplants were ldlt. 279 ldlt recipients and 3,955 cadaveric recipients transplanted for end stage liver disease from hcv were identified. the results are shown in the table: ldlt ( a greater proportion of ldlt recipients were female and ldlt recipients were less ill at the time of transplant.1 and 3 year patient and graft survival were not significantly different between the 2 groups, (p=o.11). if only the pre-meld era is considered (01l99 thru 2/02) 1 year graft survival for ldlt (n=240) and clt (n=3322) are 78% and 83%, respectively, p=o.lo. conclusions: our analysis demonstrates that short-term patient and graft survival are equivalent in ldlt and cadaveric recipients with hcv suggesting recurrent hepatitis c does not adversely affect survival over this time period. ldlt recipients are less ill at transplantation which did not confer a short-term survival advantage. continued follow-up should determine the impact of less advanced liver disease at the time of transplant and recurrent hepatitis c after transplant on long-term graft and patient survival in ldlt recipients. disclosures: introduction: histologic injury due to recurrent hcv has been reported in up to 90% of hcv infected patients who undergo liver transplantation with a cadaveric graft. however, the natural history of hcv following living donor liver transplantation (ldlt) is not as well described. anecdotal evidence suggests that hcv recurrence may be more severe in ldlt recipients. we hypothesize that post-operative liver regeneration in the partial graft procured from living donors may facilitate hcv replication, or increase the vulnerability of hepatocytes to infection. methods: we performed a retrospective analysis comparing outcomes, and the incidence, timing, and severity of histologic recurrence of hcv following transplantation in patients who underwent living donor liver transplant compared to recipients of cadaveric organs. between 12/98 and 3/02,68 hcv infected adult patients (age > 18 years old) underwent liver transplantation for hcv associated cirrhosis. 45 patients received a cadaveric graft (cad) and 23 received a graft from a living donor (ldlt). mean time of patient follow up was 25 months, with a range of 6 to 39 months. elevated serum transaminases, positive hcv rna, and liver biopsy consistent with histologic evidence of hcv defined recurrence. cholestatic hepatitis c (chc) was confirmed if all of the following criteria were met: serum total bilirubin greater than lomg/dl with no evidence of extra-hepatic biliary obstruction on ultrasound and cholangiography, and histologic features on liver biopsy consisting of portal expansion, ductular proliferation, and bile stasis with or without hepatocyte ballooning. immunosuppression, definition and treatment of rejection were standardized for both cad and ldlt. results: when comparing cad to ldlt, both the incidence of hcv recurrence and time to recurrence were not different. the overall incidence of "severe" sequelae of hcv recurrence, either cholestatic hepatitis, grade 111-iv inflammation, and/or hcv induced graft failure requiring re-transplantation was also not different when comparing cad to ldlt. however, when comparing cad versus ldlt, no cad patient developed cholestatic hepatitis c, compared to 17% of ldlt who developed this complication (p = 0.001). conclusions: in this patient population, the timing and overall incidence of hcv recurrence were not different when comparing cad versus ldlt, but the incidence of cholestatic hepatitis was significantly greater in patients with hcv who underwent ldlt. further multicenter studies are warranted to determine the incidence and risk factors for cholestatic hepatitis c following liver transplantation. ucsf, sun francisco, ca; fabien zoulim, inserm, lyon, france; carol l brosgart, michael wulfsohn, michael d miller, shelly xiong, gilead sciences, inc., foster city, ca background lamivudine (lam) resistance occurs in approximately 40% of chronic hepatitis b (chb) patients after 2 years of lam monotherapy. in contrast, resistance to adefovir dipivoxil (adv) occurs infrequently with 1.6% of chb patients developing the adefovir resistance mutation rtn236t after 2 years of adv therapy. pre-existing lam resistance mutations or concurrent use of immunosuppressive therapy by lt patients may increase the risk of resistance to adv. objective: to determine the incidence of adv resistance in a clinical trial of liver transplantation (pre and post) patients with lam-resistant hbv treated with adv for 96 weeks (lam therapy was maintained in most patients). methods: the hbv reverse transcriptase domain was sequenced for lt patients with detectable hbv dna by pcr (>lo00 copieslml) after 96 weeks of adv therapy (n=114). in vih-o drug susceptibility was determined following transfection of hepg2 cells with patient-derived hbv clones from baseline and week 96 serum samples. results: the rtn236t mutation wa!j observed in 21114 patients (1.8%) at week 96. lam had been discontinued at weeks 16 and 28 after initiation of adv in these two patients respectively. the baseline lam-resistant ymdd mutation reverted to wildtype prior to week 96 in both patients. emergence of rtn236t was associated with rebound in serum hbv dna and alt elevation in both patients. in vitro phenotypic analysis showed approximately 4-fold reduced susceptibility to adefovir with rtn236t. however, these adefovir-resistant hbv clones were fully susceptible to lam and entecavir in vitro. lam therapy was re-initiated, in addition to the ongoing adv therapy, after emergence of rtn236t in these patients resulting in a > 2.9 loglo copies1ml reduction in serum hbv dna in both patients. one patient also had a significant reduction (>80%) in alt within 5 months of lam treatment. no other mutations potentially associated with adefovir resistance were detected. conclusions: emergence of the adefovir resistance mutation rtn236t was observed infrequently after 2 years in liver transplantation patients (1.8%, 2/114) infected with lam-resistant hbv, similar to observations in treatment nayve non-liver transplantation patients. the adv-resistant hbv was sensitive to lam and addition of lam resulted in clinical stabiliation in both patients. local liver immune responses are thought to play a major role in chronic autoimmune diseases directed at biliary epithelium.using the apical sodium dependent bile acid transporter (asbt) promoter to drive biliary epithelial cell -specific expression of a membrane form of ovalbumin (ova), we have previously developed ova-bil transgenic mice. because these mice are tolerant to ova, we use ova-specific t cells from ot-1 and ot-i1 transgenic mice, restricted by mhc class i and class 11, respectively, with well defined peptide epitopes specific for ova to induce biliary damage in a dose dependent manner. aim: 1) to determine the liver mononuclear cell populations (mnc) involved in necroinflammatory disease, and 2) to determine where adoptively transferred cells home and proliferate. methods: 10 million ot-i and 2 million ot-i1 nayve t cells were adoptively transferred to ova-bil mice by intraperitoneal injection. at days 0, 5, and 7, liver mnc were isolated by collagenase digestion, purified by discontinuous percoll gradient centrifugation, and analyzed by flow cytometry. tail bleeds were performed at days 0, 3, 5, and 7 to follow serum alt. in a subset of experiments, ot-1 cells were labeled with carboxyfluorescein diacetate succinimidyl ester (cfse) and analyzed on day 3 and 5 by flow cytometry after adoptive transfer with unlabeled ot-i1 t cells into ova-bil mice. results: ova-bil mice develop normally without evidence of disease up to 2 years. after adoptive transfer of ova-specific t cells, there was a marked increase in serum alt. cd8+ ot-i t cells were required for liver damage and ova-specific cd4+ t cells markedly augmented this inflammation. adoptive transfer of ot-i1 cd4 t cells alone did not induce liver injury. there was extensive portal inflammation in every portal triad, centered around the bile ducts with infiltrating lymphocytes in the bile duct epithelia, apoptotic cells, loss of biliary epithelial cells as well as interface hepatitis. liver mnc were abundant in ova-bil mice and increased after adoptive transfer of ova-specific t cells. serum alt peaked at day 5 (mean 263 iulml), coincident with liver mnc peak. cfse labeling studies revealed robust homing of adoptively transferred ot-i cd8 cells to the liver, but not to the spleen, of ova-bil mice. the ova-specific cd8 cells, but not cd4, nk1.l, or cd19 cells, underwent cell division. conclu-sion: recognition of biliary epithelial antigen in ova-bil mice induces a necroinflammatory response in the liver as assessed by serum alt, liver mnc numbers and immunohistochemistry. the magnitude of this response correlates with influx of nahe ovaspecific cytotoxic t cells, which are activated and divide in the liver but not in the spleen. t cell recognition of antigen expressed on bile duct epithelium occurs rapidly, causes biliary specific inflammation with interface hepatitis, which may be a model of autoimmune bile duct injury or cholangiopathy. the etiology of autoimmune hepatitis (aih) is only poorly understood although the major autoantigens, such as cytochrome p450 2d6 (cyp2d6), could be identified and immunodominant epitopes have been mapped. one major reason for this lack of comprehension is the fact that there are currently only few valid animal models for aih and none of them involves the autoantigen targeted in humans. thus, we generated an animal model for human aih using a natural autoantigen (cyp2d6). self-tolerance in transgenic mice that express human cyp2d6 in the liver was challenged by infecting these cyp2d6-mice with an adenovirus-cyp2d6 vector (ad-2d6) in order to deliver large amounts of the critical antigen to the liver and to provide a inflammatory environment that would favour autoimmunity. infection with an empty adenovirus vector resulted in a transient form of focal necrosis 4 days after infection that subsequently disappeared after 2 weeks. in contrast, infection with ad-2d6 resulted in extended and persistent infiltration of cd4, cd8 lymphocytes as well as macrophages resulting in confluentlbridging necrosis at week 10 post-infection. in addition, the overall morphology of the liver was massively disturbed after ad-2d6 infection. at week 10 postinfection, the liver was approximately half the size of the control and its lobules were fused together and rounded up. first indications of fibrosis and hyperplasic nodules become apparent. the overall architecture of the liver was disrupted i disorganized and the liver parenchyma was partially collapsed. furthermore, theliver of ad-2d6 infected mice was surrounded by multiple layers of connective tissue as seen in some stages of liver cirrhosis. additional signs of cirrhosis started to become apparent in the form of nodules that are entrapped in fibrous tissue. in addition, accumulation of infiltrating cells are visible directly under the liver capsule. these observations indicate that the cyp2d6-mouse displays a persistent form of hepatitis after infection with ad-2d6 that may form the basis for a novel model system which would allow to study mechanisms involved in the initiation, propagation and precipitation of autoimmune processes involved in human autoimmune hepatitis. disclosures: urs christen -no relationships to disclose eric f johnson -no relationships to disclose michael p manns -no relationships to disclose antje rhode -no relationships to disclose matthias g von herrath -no relationships to disclose herkel, peter r galle, edgar schmitt, ansgar w lohse, johannes gutenberg university, mainz, germany background: in clinical hepatitis, hepatocytes express mhc class i1 molecules; we recently reported that mhc i1 expressing hepatocytes can function as antigen presenting cells that stimulate specific cd4 t cells (hepatology 2003; 37: 1079-85) . to understand the relevance of hepatocellular antigen presentation for hepatic immunity, we now examined whether hepatocytes may induce the differentiation of primary cd4 t cells. because inflammatory cd4 t cells in the liver are most likely primed by dendritic cells of the draining lymph nodes, we also examined whether hepatocytes may also re-differentiate dendritic cell-primed cd4 t cells. methods: primary cd4 t cells from ovalbumin-specific t cell receptor-transgenic mice were stimulated by antigen presenting hepatocytes from hepatocyte-specific ciita-transgenic mice or splenic dendritic cells.the response type was determined by measuring secreted interferon-gamma (ifn) and interleukin-4 (il-4). results: we found that antigen presenting hepatocytes by default induced differentiation of primary cd4 t cells to th2 cells (ifn: 200 ulml; 1l-4: 1800 ulml). in contrast, primary cd4 t cells stimulated by dendritic cells differentiated to thl effector cells (ifn: 2400 ulml; 1l-4 20 ulml). however, these dendritic cellprimed thl type cells, when re-stimulated by hepatocytes, had a decreased capacity to produce ifn (580 ulml vs. 2800 ulml after dentritic cell stimulation); and after repeated re-stimulation by hepatocytes, the dendritic-cell primed t cells even differentiated into th2 cells (ifn: 112 ulml; il-4: 1270 ulml). conclusions: these data show that antigen presenting hepatocytes induce th2 differentiation of undifferentiated cd4 t cells. most notably, even dendritic cell-primed cd4 t cells that are committed to thl differentiation could be reverted by hepatocytes to th2 type. thus, hepatocytes seem to have an extraordinary capacity to promote antiinflammatory hepatic immune responses. it is therefore conceivable that antigen presentation by hepatocytes associated with clinical hepatitis, in the absence of pro-inflammatory stimuli, seems to downregulate inflammatory infiltrating t cells. background nkt cells are a unique subset of regulatory lymphocytes with important immune modulatory effects. these cells recognize exogenous glycolipids anchored by a ceramide tail to the mhc-like cdld molecule, expressed by various antigen presenting cells. glycosylceramides, including the marine sponge-derived a-galactosylceramide can activate nkt cells, leading to exacerbation of hepatitis. concanavalin a induces immune mediated hepatitis in which nkt cells are key participants. glucocerebroside (gc) is a naturally occurring glycolipid. aims: to determine the immune modulatory effect of gc in a murine model of con a hepatitis. methods: five groups of balblc mice were studied group a and b mice were treated with gc (1.0 pg ip) two hours prior to and two hours following administration of 500 pg con a, respectively; group c mice were treated with con a alone; group d mice were treated with gc alone; group e mice did not receive any treatment. the degree of liver damage was evaluated by serum aspartate aminotransferase (ast) and alanine aminotransferase (alt) levels, and by liver histology. the immunmodulatory effect of gc was determined by facs analysis of intrahepatic and intrasplenic lymphocytes for nkt markers, and by elisa measurements of serum ifny, il2, il4, il10, and il12. the effect of gc on nkt lymphocyte proliferation was assessed in vitro. results: treatment with gc markedly reduced serum ast and alt levels in group a compared to group c (143 vs. 600 iu in group a and group c, respectively, for ast; 57 vs. 801 iu in group a and group c, respectively, for alt, p<0.05). administration of gc alone did not change ast or alt levels compared to naive controls. histological sections of livers from group a mice revealed markedly attenuated damage compared to sections from group c livers, in which massive hepatocyte necrosis was present. the beneficial effect of gc on immune mediated hepatitis was associated with a 20% decrease in the intrahepatic nkt lymphocyte number, and with significant lowering of serum ifny levels (3725 vs. 5620 pglml in groups a and c, respectively, p<0.05); administration of gc alone led to increased serum il-12 levels (573 pglml vs. 92 pglml in group d vs. group e, respectively, p<0.05). in vitro, administration of gc decreased nkt cell pro-liferation by 42% in the presence of dendritic cells, but not in their absence. conclusions: administration of gc led to significant amelioration of con a hepatitis that was associated with a dendritic cell-dependent decrease of nkt cell proliferation in vitro, and with decreased intrahepatic nkt lymphocytes and serum ifny levels in vivo. these results suggest that the effect of gc may be mediated by inhibition of intrahepatic nkt cells, resulting from competitive displacement of activating elements from the cdld molecule on antigen presenting cells. glucocerebroside, a naturally occurring glycolipid, holds promise as a new immunomodulatory agent, with a possible role in treatment of autoimmune hepatitis and other immune-mediated liver disorders. disclosures: background cd4+ cells constitutively expressing cd25 have a regulatory function, their experimental removal leading to spontaneous autoimmune disease in normal rodents. cd4+cd25+ regulatory t cells suppress both th1 and th2 responses, competing with effector cd4+ cells in recognizing the same peptide antigens. their ability of expansion is key to the maintenance of tolerance. autoimmune hepatitis type 2 (aih-2) is characterized by t cell immune responses against 6 well-defined regions on cytochrome p4502d6, the autoantigen of aih-2. aims: to investigate the ability of expansion of cd4+cd25+ after exposure to non-antigen-specific and cypzd6-specific stimuli in aih-2. methods: cd4+cd25+ t cells were analysed by triple colour flow-cytometry of freshlcryopreserved peripheral blood mononuclear cells (pbmcs) befare and after culture in the presence of a t cell expander capable of maintaining the original t cell function (cd3kd28 dynabeads t cell expander, dynal biotech, norway) and of 28 synthetic cyp2d6 peptides (10 pmol), spanning the 6 antigenic regions known to induce proliferative cd4+ responses in aih-2. patients and controls: nine patients with aih-2 (7 female, median age 11.4 yrs, range 2.5 to 21 yrs) were investigated, 3 at diagnosis and 6 while on sustained remission. nine healthy laboratory workers served as normal controls. results: before stimulation, the level of cd4+cd25+ t cells was significantly lower in aih-2 patients (3.59 2 1.08) than in normal controls (6.39 f 0.87; p=o.o2), a difference present both at diagnosis (2.24 ? 0.56, p<0.005) and during remission (4.25 2 0.35, p=o.ol). following exposure to the t cell expander, the level of cd4+cd25+ t cells increased 4.5 times (28.6 2 30.8) in normal controls, but only 1.85 times in am-2 patients (6.62 ? 1.85, p=o.ol). upon exposure to cymd6 peptides, cd4+cd25+ t cells remained numerically unchanged, in contrast to the pbmcs from the same patients giving a strong proliferative response (up to 6.9 times). summary & conclusion: our data show a numerical and functional impairment of regulatory t cells in am-2. this defect is likely to be key to the initiation and perpetuation of the autoaggressive process in this condition. orth, mark a mcniven, nicholas f larusso, mayo medical school, clinic and foundation, rochester, mn cryptosporidium parvum (cp) opportunistically infects intestinal and biliary epithelia causing worldwide morbidity, especially in patients with aids. epithelial invasion by cp involves host cell membrane alterations resulting in a parasitophorous vacuole that envelops the parasite and a dense-band within the host cell underlying the attachment site; both processes require host cell actin remodeling. since recent studies in other systems have demonstrated that cdc42, an actin-associated gtp-binding protein, plays a central role in microbial-induced actin remodeling, we tested the hypothesis that cp activates host cell cdc42 and its downstream effectors to induce actin rearrangement and microbial invasion. methods: we exposed cholangiocytes derived from normal human liver and immortalized by sv40 transformation to freshly excysted cp sporozoites and applied molecular and morphofogical approaches to test our hypothesis. results by immunofluorescent and immunoelectron microscopy, we found accumulation of cdc42 in cholangiocytes at the parasite-host cell interface during cp invasion. we confirmed activation of cdc42 in infected cholangiocytes by immunoprecipitation using an antibody to pak4, a downstream effector molecule that binds exclusively to the activated form of cdc42. phosphatidylinositol 3-kinase (pi-3k), a membrane-associated kinase associated with cdc42 activation, was also recruited to the site of attachment, as were n-wasp, pak4 and p34-arc, downstream effectors of cdc42. inhibition of pi-3k by wortmannin or ly294002 blocked cp-induced cdc42 accumulation. while overexpression in cholangiocytes of a constitutively active mutant of cdc42 promoted cp invasion (up to 50%), overexpression of function-deficient mutants of pi-3k, cdc42 or of the wa fragment of n-wasp inhibited cp invasion by 60 -80 %. moreover, inhibition of host cell cdc42 activation by dominant negative mutation inhibited cp-induced actin accumulation, and parasitophorous vacuole and dense-band formation at the attachment site. conclusions: these combined data suggest that cp invasion of cholangiocytes (and perhaps other target epithelia) results from the organism's ability to activate a complex host cell cdc42 signaling pathway that induces host cell actin remodeling at the attachment site. background: hepatitis b (hbv) and hepatitis c (hcv) infections are a world-wide health concern. studies of these infections have been hampered by a lack of a convenient animal model. we previously have shown that immunocompetent rats tolerized and transplanted with human hepatocytes can support hbv infection for at least 15 weeks. aim: to demonstrate that the immunocompetent rat which has been tolerized and transplanted with human hepatocytes can be used to sustain and study hcv infection. methods: sprague-dawley rats were injected in utero at 16-17 days gestation with one hundred thousand huh 7 cells (human hepatocyte cell line) to tolerized them to human hepatocytes. one week after birth, the tolerized newborns were intrasplenically transplanted with 5 million huh 7 cells. one week after transplantation, rodents were inoculated with one hundred thousand hcv rna copies, genotype l b human serum. animals were sacrificed at 6 and 12 weeks. the presence of human cells was determined by immunofluorescent staining of cryosectioned liver tissue using antibodies to human albumin. pcr and rt-pcr was used to confirm the presence of human albumin in liver tissue. the presence of hcv was assayed using an antibody to ns5a viral protein, and visualized using a rhodamine labeled secondary antibody. hcv infection in the liver was confirmed by the presence of negative strand rna using nested pcr. results: functional huh7 cells in the chimeric livers were confirmed by the presence of human albumin mrna and dna using rt-pcr and pcr. the presence of human albumin protein in the liver was further demonstrated by immunofluorescence of human albumin in frozen liver sections. eighty-one percent (n=57) of the tolerized, huh 7 transplanted rodents were positive for human albumin in the livers. seventy-two percent (n=36) of the chimeric animals infected with hcv were positive for the presence of ns5a hcv protein from immunofluorescence studies. livers of control rats that were only tolerized and control rats that were tolerized and transplanted with huh7 cells, but not inoculated with hcv were negative for ns5a (n=12). hcv viral replication could be demonstrated in livers of tolerized, transplanted and hcv infected rats by the presence of hcv rna bands of the correct size from nested pcr using negative strand specific primers. conclusion immunocompetent rats tolerized and transplanted with human hepatocytes can be a useful laboratory model to study hcv infection. ( we have previously observed that allogeneic hepatocellular transplants are highly immunogenic and are resistant to immunosuppressive therapy with a variety of agents. donor specific transfusion in combination with anti-cd40l mab is highly effective in inducing prolonged survival of skin and myoblasts and inducing indefinite and donor-specific tolerance to heart and islet allografts. the proposed mechanism of action for dst and anti-cd40l mab suggests peripheral deletion of alloreactive cd8+ t cells and induction of a regulatory cd4+ t cell population. resistance to induction of indefinite acceptance has been attributed to peripheral reappearance of alloreactive cd8+ t cells. in preliminary studies, we observed that dst and anti-cd40l mab prolonged fvbln hepatocyte (hc) survival in c57e3l/6 mice to median survival time (mst) of 88 days and induced indefinite acceptance (>90 days) in 43% of mice. hc rejection occurs by cd4-dependent and (cd4-independent) cd8-dependent pathways. this model permits evaluation on these two pathways separately. the current studies address the hypothesis that dst and anti-cd40l mab induces prolonged hc allograft acceptance by inducing immunoregulation of both alloreactive cd4+ and cd8+ t cells. methods: fvbln (halat-fvbin, h-24) hcs were transplanted into cd4 ko, cd8 ko, and c57bl/6 (all h-2b) mice. hc survival was monitored by detection of reporter halat protein in serum by elisa. for comparison, donor-matched islets were transplanted into streptozotocin-induced diabetic cd8 ko (h-2b) mice, and survival was monitored by blood glucose. recipient mice received peritransplant administration of dst ( 1 0~1 0~ fvbln splenocytes, day -7) and anti-cd4ol mab (1.0 mg, ip, d-7, -4, 0, 4) . some cd8 ko hosts were reconstituted cd8+ t cells (2x107 iv, d-10). results: when the cd8-dependent rejection was studied in isolation (cd4 ko mice), dst and anti-cd4ol mab prolonged hc survival to mst of 35 days (n=4) compared to 10 days in untreated cd4 ko (n=7); however, this was not significantly different from anti-cd40l mab alone. when cd4-dependent rejection was studied in isolation (cd8 ko mice), hcs were unexpectedly rejected with mst of 7 days (n=8) despite dst and anti-cd40l mab treatment. in contrast, islets of the same strain were accepted indefinitely in cd8 ko mice (n=5, mst>70 days). since this siraiegy effectively controlled hc rejection in c57bl16 mice which have both cd4-and cd8-dependent pathways available, the collective results suggested that perhaps host cd8+ t cells were necessary for the beneficial effects of the dst and anti-cd40l mab. to determine whether cd8+ t cells were required for induction of longterm hc survival with dst and anti-cd40l mab, cd8 ko hc recipients were reconstituted with cd8+ t cells prior to dst and anti-cd4ol mab treatment. cd8reconstituted cd8 kos accepted hcs for mst of 56 days. conclusion: the effects of dst and anti-cd40l mab on combined cd4-and cd8-initated hc rejection are more pronounced than on either pathway alone. cd8+ t cells appear to be required for induction of longterm hc survival under cover dst and anti-cd40l mab, which offers an apparently distinct mechanism compared to the existing paradigm for the mechanism of action of dst and anti-cd40l mab. engraftment of transplanted cells in the liver is affected by cellcell interactions involving hepatic endothelial cells and kupffer cells. the proximity of transplanted cells to hsc suggested that hsc could promote cell adhesion and extracellular matrix remodeling during cell engraftment. to investigate cell-cell interactions in the liver, we analyzed hsc activation in dppn-rats transplanted intrasplenically with f344 hepatocytes. analysis of tissues by immunostaining from animals 6h, 24h, 3d and 7d after cell transplantation showed appearance of desmin +ve hsc in periportal areas, reaching a peak on day 3 (4-10 fold increase in desmin f v e hsc in cell recipients, p<o.ool). using combined immunostaining and dppiv histochemistry, desmin +ve hsc were often observed in the immediate vicinity of transplanted cells. however, a-smooth muscle actin (sma) was not expressed in hsc perturbed by cell transplantation, whereas ccl&reatment in control animals induced sma expression in hsc, as shown by tissue immunostaining, as well as western blots of tissue extracts. after cell transplantation, perturbed hsc expressed desmin extensively, especially in areas with cell transplantation-induced ischemia and consequential ggt expression. to examine whether desmin +ve hsc were perturbed following activation of kupffer cells by cell transplantation, we administered carbon particles to hepatocyte recipients intrasplenically. these studies established that the majority (up to 72%) of desmin +ve hsc were located directly adjacent to carbon containing activated kupffer cells. as kupffer cells are activated within 6 hrs after cell transplantation, whereas hsc were perturbed maximally at a later time, we reasoned that these cell-cell interactions were likely mediated by soluble factors. to demonstrate whether tnf-alpha plays a role in the stellate cell activation process, we transplanted hepatocytes into animals treated with the tnf-alpha-blocker etanercept. however, etanercept did not prevent perturbation of hsc, suggesting that factors other than tnf-alpha must be involved. to eliminate the component of ischemia-mediated cell activations, we treated animals with intrasplenic nitroglycerine infusion before and during cell transplantation, which resulted in marked attenuation of hepatic ggt expression. interestingly, desmin +ve hsc were now distributed throughout the entire liver lobule, with 3-5 fold greater prevalence, compared to rats treated with cells alone without nitroglycerine, p<o.ool, suggesting that circulating soluble factors reached hsc more effectively. in contrast, treatment of animals with nitroglycerine alone did not perturb hsc. to determine effects on cell integration, bile canalicular reconstitution was analyzed with dppiv and atpase co-stainings. nitroglycerine pretreatment resulted in superior parenchymal integration of transplanted cells, in proximity with desmin +ve hsc. conclusions: transplantation of cells in liver sinusoids activates desmin, but not sma, expression in hsc. activated kupffer cells are likely mediators of hsc perturbation. improved cell engraftment following perturbation of hsc indicates that extracellular matrix remodeling could facilitate this process. these findings offer ways to further optimize liver repopulation with transplanted cells. knudsen, timothy b hummer, kim a buffers, kiyoshi ogata, cody a koch, jefiey l platt, mayo clinic, rochester, m n background hepatocyte transplantation has been of much interest as a biological liver support. how to treat hepatocytes to achieve optimum function has been uncertain. cultured, cryopreserved and hypothermically preserved hepatocytes have been employed for clinical hepatocyte transplantation, but the efficacy was rarely compared between those cell sources. using a xenogeneic hepatocyte transplantation model, the function of transplanted hepatocytes was quantified in vivo. methods: porcine hepatocytes were isolated by a two-step collagenase technique. the hepatocytes were: (1) immediately used; (2) kept in uw solution at 4°c; (3) cultured in supplemented williams e medium; or (4) cryopreserved in ljw solution with 10% dmso before use. the viability of hepatocytes was determined by trypan blue exclusion, and 1 x lo6 viable hepatocytes were injected into the spleens of rag-2-defficient mice. to evaluate the function of transplanted hepatocytes, the porcine albumin level in the recipient plasma was determined by a sandwich elisa. the number of surviving porcine cells in the recipients' spleen was estimated by a quantification of porcine genomic dna sequences. results: the viability of hepatocytes was as follows: freshly isolated hepatocytes, 92.2 2 1.9%; 24 hours, 48 hours, and 72 hours at 4"c, 85.8 t 3.1, 74.3 i 1.9, 71.0 i 3.2%; cryopreserved hepatocytes after thaw, 65.0 2 2.6%; cultured hepatocytes after dissociation, 78.4 t 6.3% (means t se). after transplanting comparable numbers of viable hepatocytes, the porcine albumin concentration was found to be lower in recipients of hypothermically-stored (group 2) and cultured hepatocytes (group 3) than recipients of fresh hepatocytes (group 1). cryopreserved hepatocytes (group 4) failed to secrete any albumin. because the half-life of porcine albumin in rag-2deficient mice was 13.5 hours, the porcine albumin concentration in the recipient plasma reflected real-time albumin secretion from the transplanted hepatocytes. the porcine genomic dna in the recipient spleen was quantified 4 weeks after transplantation. fewer cells were found in the hypothermically preserved hepatocyte-recipients (group 2). therefore, the lower level of the porcine albumin secretion in the group 2 was mainly attributable to the failure of the transplanted hepatocytes to survive. the amount of porcine genomic dna was comparable between cultured hepatocyte-recipients and freshly isolated hepatocyte-recipients, although the plasma of cultured hepatocyte-recipients included less porcine albumin. it suggested that the hepatocytes had dedifferentiated during culture and secreted less albumin thereafter in vivo. the cultured hepatocytes did not seem to differentiate again in murine spleen. conclusions: both the survival and differentiation of hepatocytes were important to maintenance of good function in vivo. the hypothermically preserved and cryopreserved hepatocytes did not survive long after transplantation contrary to their good viable look. cultured hepatocytes showed relatively good survival, although their dedifferentiation was not reversible resulting in poor outcome. freshly isolated hepatocytes are recommended for cell transplantation. medicine, ube-yamaguchi, japan; hiroshi nishina, university of tokyo, tokyo, japan; kiwamu okita, yamaguchi university school of medicine, ube-yamaguchi, japan obiective; we had developed an in vivo model to monitor the trans-differentiation and proliferation of bmcs into functional hepatocytes via hepatoblast phenotype (gfpicc14 model). in this model, transplanted gfp-positive bmcs via tail vein efficiently migrated to the peri-portal area of liver lobules under cc14induced chronic liver damage condition. transplanted bmcs gradually proliferated and spread from the peri-portal area (zone 1) into the central area (zone 2). under persistent liver damage condition, bmcs trans-differentiated into functional hepatocytes. previous analysis had shown that inflammatory signals had been important for the trans-differentiatin of bmcs. in this study, we focused on growth factors and analyzed which affected the transdifferentiation and proliferation of bmcs into hepatocytes in gfpl cc14 model. materials and methods; immunohistochemical staining and immunofluorescent double staining with antibody of gfp, several growth factors and their receptors were performed in gfpiccl4 model. hepatocyte growth factor (hgf), epidermal growth factor (egf), fibroblast growth factor (fgf), vascular endothelial growth factor (vegf), platelet-derived growth factor (pdgf), and transforming growth factor (tgf) were analyzed. the percentage of stained area was quantified in the sections at thirty random fields (n=6 in each group), and statistical analysis was performed using plsd test (anova). results; the most significant result was shown in fibroblast growth factors (fgfi, fgf2) and their receptors (flg, bek). in immunohistochemical staining, the stained region migrated and increased from zone1 into zone2 of liver lobules with the time after bmt. in immunofluorescent double staining, co-expressions both gfp and fgfs, fgf-receptors were detected at the same distribution. the percentage of stained area of fgfs and fgfreceptors gradually increased with significant difference (the data was shown in table 1 ). although expressions of other growth factors and their receptors were detected, the significant differences were not shown. conclusions; fgfs are the most important growth factor to transdifferentiate and proliferate bmcs into hepatocytes. cholangiocytes are the target cells of chronic cholestatic liver diseases (i.e., cholangiopathies), characterized by the dysregulation of the balance between apoptosis and proliferation that leads to changes in ductal bile secretion. in rats with bile duct ligation (bdl), there is increased cholangiocyte proliferation and enhanced basal and secretin-stimulated ductal secretion. we have shown that both cholinergic (by vagotomy) and adrenergic b y administration of a single intraportal injection of 6-hydroxydopamine (60hda)i denervation impairs the proliferative and secretory responses of intrahepatic bile ducts to bdl through an increase in cholangiocyte apoptosis. we have shown that bile acids and nerves co-ordinately regulate the balance between cholangiocyte apoptosislproliieration in hyperplastic rat livers. in bdl rats, while ursodeoxycholate and tauroursodeoxycholate feeding prevents the loss of bile ducts caused by vagotomy in a caz+/pkc-dependent manner, taurocholate feeding prevents vagotomy-induced duct damage in a pi3k dependent manner. no information exists regarding the interaction between bile acids and adrenergic innervation in the regulation of cholangiocyte function. thus, we tested the hypothesis that taurocholate feeding protects from bile duct damage induced by adrenergic denervation by 6-ohda. methods: immediately after bdl, rats received a single intraportal injection of 6-ohda (which induces degeneration of adrenergic terminal fibers, 50 mglkglbody weight) or vehicle, and subsequently were fed 1% taurocholate or control diet in the presence of daily injections of wortmannin (a pi3k inhibitor, 0.7 mgl kg body weight) or a control solution. we used 1 week bdl rats as control animals. seven days later, we evaluated cholangiocyte apoptosis by : (i) tunel assay in liver sections; and (ii) measurement of caspase 3 activity and protein expression for cleaved pro-caspase 3, and the number of purified cholangiocytes positive for annexin-v. cholangiocyte proliferation was evaluated by measurement of the number of pcna-and ck-19-positive cholangiocytes in liver sections, and pcna immunoblots in pure isolated cholangiocytes. the changes in ductal functional activity were evaluated by measurement of (i) secretinstimulated camp levels and cl-ihcos exchanger activity in pure cholangiocytes; and (ii) secretin-stimulated bile flow and bicarbonate secretion in bile fistula rats. results: taurocholate feeding prevented the increase in the number of apoptotic cholangiocytes and the enhancement of caspase 3 activity and protein expression for cleaved pro-caspase 3 induced by 6-ohda. taurocholate feeding prevented 6-ohda-induced decrease in the number of pcna-and ck-19 positive cholangiocytes, and the loss of pcna protein expression. at the functional level, taurocholate feeding prevented the decrease in secretin-stimulated camp levels, c1-l hco3 exchanger activity and bile and bicarbonate secretion. consistent with the concept that pi3k regulates the interaction of bile acids with adrenergic nerves in cholangiocytes, we found that administration of wortmannin blocks 6-ohda-induced activation of cholangiocyte apoptosis, and inhibition of cholangiocyte proliferation and ductal secretion. summarylconclusion: taurocholate feeding prevents the increase in cholangiocyte apoptosis and the loss of cholangiocyte proliferation and ductal functional activity induced by adrenergic denervation by 6-ohda. taurocholate effects are mediated by the pi3k pathway, since the simultaneous administration of wortmannin reverses such effects. these novel findings suggest that bile acids play a major role in sustaining the growth and functional activity of the intrahepatic biliary tree following liver transplantation. hepatocyte apoptosis by death receptors is emerging as a critical determinant in the initiation of hepatic inflammation and fibrosis during cholestatic liver diseases. however, the cellular sources of death ligands to activate the death receptors is unknown. we postulated that kupffer cells, which contribute to hepatic inflammation, may also contribute to hepatocyte apoptosis and liver fibrosis by generating death ligands. thus, our aim was to ascertain whether kupffer cells express death ligands and contribute to hepatocyte apoptosis and liver fibrosis in the bile duct ligated mouse, an animal model of cholestasis. methods: mice were subjected to bile duct ligation (bdl) or a sham operation and treated with gadolinium chloride, a kupffer cell intoxicant, or saline for three days. hepatocyte apoptosis in liver sections was assessed using the tunel assay. serum alt values were quantitated using commercially available kits. kupffer cells were isolated using arabinogalactan gradient centrifugation. real time pcr was used to measure expression of death ligands and fibrosis indicators. results: hepatocyte tunel positive cells, and serum alt values were 3.3-fold, and 5.2-fold, respectively, greater in 3 day-(bdl) saline-treated vs. gadolinium chloride-treated mice. kupffer cell expression of the death ligands tnf-alpha and fas ligand was 14 and 20-fold greater when kupffer cells were obtained from bdl vs. sham operated mice. the enhanced expression of fas ligand was verified by immunoblot analysis and tnf-alpha by an elisa. these data confirm that kupffer cells generate death ligands and contribute to hepatocyte apoptosis in cholestatic liver injury. consistent with a role for kupffer cellassociated hepatocyte apoptosis in liver inflammation, neutrophil infiltration into the bdl liver was abrogated by gadolinium chloride. hepatic mrna transcripts for a -smooth muscle actin, and collagen a 1(i), markers for stellate activation were all significantly attenuated gadolinium chloride vs. vehicle-treated animals. finally, the mechanisms for kupffer cell activation were explored in cell culture. isolated kupffer cells phagocytosed apoptotic bodies, which induced death ligand expression. in contrast, bile acids had no effect on kupffer cell gene expression. infection is associated with vigorous, durable cd8+ t cell responses against nonstructural hcv proteins. hcv persistence has been attributed to poor priming of cellular immune responses. one of the factors that contributes to poor priming of cellular immune responses may be the noncytopathic nature of hcv and to the subsequent absence or scarcity of virus-infected apoptotic or dead cells as source of exogenous antigens for crosspresentation. because crosspriming contributes to the induction of cd8+ t cells in vivo (nature 1999; 39877-80), we hypothesized that cross-priming with dendritic cells (dcs) containing self-replicating rna might be useful to induce strong, hcv-specific cellular immune responses. methods: choosing hcv ns3 as a model antigen, we generated a recombinant cytopathic pestivirus self-replicating rna to amplify hcv ns3 in dcs. the principal characteristic of this vector is its ability to replicate in transfected cells which in turn enhances production, processing and presentation of the encoded hcv ns3 antigen. moreover, the availability of cytopathic and noncytopathic forms of the same replicon enabled us to study the immunologic implications of dc apoptosis and antigen reprocessing, leading to crosspriming in vivo. results: the murine dendritic cell lines dc2.4 was transfected with cytopathic and noncytopathic recombinant replicon rna, respectively. hcv ns3 expression was detectable in more than 95% of the transfected cells by immunofluorescence. the time kinetics of apoptosis induction was monitored by flow cytometry using annexin v and propidium iodide staining. in contrast to the noncytopathic replicon, the cytopathic replicon led to dc apoptosis 12 hours after transfection. dc2.4 transfected with the cytopathic recombinant replicon rna were then used to immunize hla-a2 transgenic mice subcutaneously. ifn-positive, proliferative cd4+ t cells and ifn--y positive, cytotoxic cd8+ t cell responses were detectable after a single subcutaneous immunization with replicon-transfected dendritic cells and significantly stronger than after conventional, intramuscular dna immunization, the previous gold standard. crosspriming was demonstrated when t cells, primed by injection of h-2b+ dcs into the h-2b+ hla-a2+ mice, were tested with hla-a2+ antigen-presenting cells. transfer of cellular material from the vaccine dcs to the endogenous apcs, a phenomenon that underlies crosspriming, was directly visualized in vivo when fragments of csfe-labeled, injected h2b+ dcs were demonstrated in hla-a2+ host cells in lymph nodes and spleens by flow cytometry and if microscopy. finally, the protective effect and the in vivo function of the induced hcv-specific t cells were evaluated in a surrorgate challenge model with recombinant, hcv ns3 encoding vaccinia virus. whereas lo4 to lo7 pfu were detected in nonvaccinated control mice, no vaccinia virus was detected in mice vaccinated with replicon-transfected dcs, indicating complete protection. summary and conclusion: these findings demonstrate and explain the immunologic potential of the proposed vaccination strategy. moreover, they support the hypothesis that experimental forcing of exogenous antigens into the mhc class i pathway and subsequent promotion of cross-priming is particularly important to generate t cell responses against noncytopathic and tissue tropic viruses such as hcv. disclosures: introduction: hepatitis c virus (hcv) infection leads to chronic liver disease in about 85% of infected individuals while only a minority achieves spontaneous viral elimination in the initial phase of acute hepatitis c. anti viral mechanisms clearing the infection in these patients, however, could contribute to the genera-tion of therapeutic or prophylactic vaccines. the strong association of a broad, hla restricted, vigorous and long lived anti viral cd4+/ th1 t-cell response with spontaneously resolving acute hepatitis c has been demonstarted in the past. only few epitopes recognized by anti hcv specific cd4+ t cells that are associated with spontaneous viral clearance have been characterized so far. the aim of the present study was to identify and characterize hcv specific cd4+ t cell epitopes and their hla restriction during acute self limited hcv infection andlor viral control. methods: proliferative cd4+ t cell responses (3h-thymidin incorporation) against viral proteins (hcv-ns3, -ns4) and against overlapping 20-mer pep-tides covering the entire ns3 and ns4 (aa 1027-1972) region were performed in 25 patients with acute self limited hcv infection and in two patients during temporary viral control (hcv rna negative). ten patients with chronic hcv infection served as control group. cd4+ t cell clones were generated by limit-ing dilution of hcv protein specific t cell lines. hcv specific cd4+ t cell clones were further characterized fine specificity was assessed by proliferation assay after stimulation with overlapping peptides of the corresponding protein region, then the minimal epitope was defined with n-and c-terminal truncated peptides for each epitope. hla restriction was assessed by inhibition experi-ments (anti-dp, -dq, -dr) and cd25+ expression in the presence of a panel of ebv blasts by flowcytometry. results: we studied 25 patients with acute self limited hcv infection. all pa-tients displayed significant proliferative t cell responses against hcv-ns3 or -ns4 protein, that was strongly associated with self-limited disease and absence of hcv rna. proliferative responses against 20-mer peptides showed multiple responses within the ns3 and ns4 region, possibly reflecting multiple epitopes in this region. in contrast no virus specific t cell responses were detectable in a control group of patients with chronic hcv. for further characterization of t cell epitopes hcv specific t cell clones were generated from 6 patients against eight different hcv epitopes located within the ns3 (aa1027-1657, two epi-topes) and ns4alb (aa1658-1972, six epitopes) region. the minimal epitopes consist of 8 to 12 amino acids. all t cell clones were hla class i1 restricted and could be stimulated by protein or peptide pulsed ebv blasts expressing the cor-responding hla-dr or -dq. conclusion: the hcv ns3 and ns4 proteins contain several immunodominant cd4+ t cell epitopes which are associated with spontaneous viral clearance. the detailed characterization of minimal epitopes and hla restriction are a pre-requisite for the synthesis of hla class i1 tetramers and for the development of prophylactic and therapeutic t cell vaccines. background a brief period of tissue ischemia followed by reperfusion renders tissue resistant against subsequent prolonged ischemia and reperfusion, a phenomenon called ischemic preconditioning. hepatic ischemic preconditioning protects sinusoidal endothelial cells against subsequent cold storagelwarm reperfusion injury and improves graft survival after liver transplantation in rats. aim ischemic preconditioning also reduces liver injury after warm ischemia and reperfusion, in which hepatocyte damage and kupffer cell activation are dominant features. however, it is incompletely elucidated which cell type ischemic preconditioning affects and how it works. therefore, our aim was to investigate the mechanisms of ischemic preconditioning against warm ischemia and reperfusion injury. method male sprague-dawley rats were injected intravenously with 20 mglkg gadolinium chloride (gdcls), 10000 ulkg superoxide dismutase (sod) or 300 mglkg n-acethyl-l-cyctein (nac) to suppress kupffer cells, superoxide formation or oxygen radical formation, respectively. livers were then preconditioned by clamping the hepatic artery and portal vein to the median and left lobes for 10 min followed by 10 min reperfusion. subsequently, livers were subjected to warm ischemialreperfusion injury in in vivo experiments and in liver perfusion experiments as follows. in vivo experiments: the blood flow to the median and left lobes was occluded with a vascular clamp for 40 min. one hour after removal of the clamp, blood was collected for determination of alt activity and hyaluronic acid (ha) concentration. liver perfusion experiments: livers were perfused through the portal vein for 10 min with physiological buffer and stored at 37°c in the same buffer. after 40 min, livers were reperfused with the buffer containing 500 ng/ml ha for 60 min in a recirculating system for determination of alt activity in the perfusate and hepatic ha uptake as a marker of sinusoidal endothelial cell injury. livers of other non-treated rats were preconditioned by perfusion with the buffer containing h202 instead of ischemic preconditioning, and then subjected to warm ischemialreperfusion similarly. in other experiments, livers were subjected to ischemic preconditioning and perfused with the buffer containing 0.5 mglml nitro blue tetrazolium (nbt) for 10 min and fixed with formalin. since nbt reacts with superoxide to form insoluble blue formazan, kupffer cell superoxide formation was evaluated in histological sections. results ischemic preconditioning decreased serum alt activity compared to control (168 ? 34 [mean 5 semi vs 333 ? 52, p < 0.05), but did not change serum ha concentration. pretreatment with gdc13 reversed the effect of ischemic preconditioning (262 2 41), though gdc13 itself did not change alt activity after ischemialreperfusion (318 t_ 33). similar results were obtained in liver perfusion experiments, suggesting that ischemic preconditioning protected hepatocytes, but did not change sinusoidal endothelial cell injury. it is also suggested that kupffer cells mediate hepatocytes protection by ischemic preconditioning. in rats treated with sod or nac, decrease in alt activity in the perfusate by ischbackground: living donor liver transplantation (ldlt) and split livers have emerged as a solution to ease the shortage of liver transplants and have achieved remarkable success in pediatric populations. in adults, ldlt is limited by the size of the graft that can be safely harvested and transplanted. similar limitations apply when extensive liver resections are needed for the treatment of hepatic tumors. we have previously demonstrated that the anti-apoptotic gene a20 is part of the physiologic response of hepatocytes to injury, protecting them from apoptosis and limiting inflammation by inhibiting nf-kb activation. our objective was to check whether a20 expression would help decrease the minimal liver mass required for mice survival. methods and results: two-thirds partial hepatectomy is well tolerated and is an accepted model of liver regeneration in rodents. we performed an extended (78%) liver resection (n=12/group) in non-infected (ni) mice (10-12 week balb/c) and mice infected with recombinant a20 adenovirus (rad.a20) or a control adenovirus (rad.p-gal). surviving mice suffer a delay in regeneration as assessed by the number of proliferation cell nuclear antigen (pcna) positive nuclei 48 h following resection (5-10 positive cellslhpf in mice with 78% liver resection vs. 60-70 positive cells/ hpf in mice with a 2/3 liver resection). interestingly, such a delay was not noted in mice expressing a20 (70-80 pcna positive cells/ hpf were detected 48 h following 78% resection). a20 also prevented the development of coagulopathy as assessed by prothrombin time. we extended our resection to a radical hepatectomy comprising 87% of the liver (n=12lgroup). we observed 100% lethality in ni and rad.p-gal infected mice. expression of a20 resulted in the survival of 60% of the animals and correlated with increased pcna positive hepatocytes as an indicator of increased cell proliferation. a20 mediated increase in hepatocyte proliferation was not merely dependent upon its anti-apoptotic function. expression of a20 in hepatocyte cultures, in a nonapoptotic milieu, increased their proliferative response to serum and hgf as compared to ni and rad.p-gal infected cells. conclusion: these results demonstrate a novel pro-proliferatif function for a20 in hepatocytes and argue for its critical role in accelerating liver regeneration and promoting hepatocyte survival and function, even when facing extreme metabolic demands. gene therapy with a20 may allow for successful transplantation with smaller ldlt, split liver allografts and even hepatocellular grafts. additionally, a20 based therapy to the liver may allow for extensive liver resections in the setting of cancer therapy. employee graeme currie -gilead sciences, inc.: employee samuel didier -gilead sciences, inc.: investigator stefanos hadziyannis -gilead sciences, inc.: investigator craig james -gilead sciences, inc.: employee ching-lung lai -gilead sciences, inc.: investigator nicole lama -gilead sciences, inc.: employee pietro lampertico -gilead sciences, inc.: investigator peter neuhaus -gilead sciences, inc.: investigator eugene schiff -gilead sciences, inc.: investigator norah terrault -gilead sciences, inc.: investigator hans tillmann -gilead sciences, inc.: investigator 24 prolonged survival of porcine hepatocytes in cynomolgus monkeys. h nagata aliberti -no relationships to disclose sven erik behrens -no relationships to disclose vito racanelli -no relationships to disclose barbara rehermann -no relationships to disclose 35 characterization of novel cd4+ t cell epitopes in acute selflimited hcv infection previous studies from our lab show that interleukin-6 (il-6) is a complete biliary epithelial cell (bec) mitogen that is normally produced by bec at low levels and upregulated by mechanical, infectious, or immunologic biliary tract injury. bile duct ligation (bdl) in il-6-deficient (il-6-/-) mice results in a reproducible phenotype that includes rapidly decompensating biliary cirrhosis, which is at least partially attributable to impaired biliary tree integrity. methods and results: oligonucleotide array analysis of primary mouse il-64-and wild type il-6+/+ bec cultures was used to search for possible molecular mechanisms that might account for impaired biliary tree integrity in il-64-mice. the results showed upregulation of trefoil factor family (tff) messenger rna (mrna) in the il-6+/+ bec compared to the il-64-bec. tff are mucin-associated protease-resistant peptides that greatly contribute to barrier and epithelial repair in the gastrointestinal tract. various molecular analyses in vitro using bec cultures confirmed that il-6-mediated gp130 signaling through the signal transducer and activator of transcription 3 (stat3) importantly contributes to tff3 mrna and protein expression. in vivo, tff3 mrna and protein was expressed predominantly in the medium and large bile ducts and peribfiary glands in normal mouse liver in il-6+/+ mice, but weakly present or absent in il-64-mice. within several weeks after bdl, tff3 mrna and protein levels decreased transiently then increased to near baseline levels by twelve (12) weeks after bdl in the il-6+/+. in contrast, the il-64-mice failed to show the secondary recovery of tff3 by 12 weeks when mrna and protein levels were significantly lower than il-6+/+ (figure) . the retarded recovery of tff3 mrna and protein in the il-6-imice was reversed by daily treatment of recombinant il-6. similar results were obtained in analysis of normal human liver and various biliary tract diseases. conclusions: tff3 is the predominant trefoil factor expressed in the mouse and human biliary tree; its expression appears to be upregulated by stat3 signaling and suppressed by the mitogen-activated protein kinases (mapk) signaling in the bec. tff expression in the biliary tree importantly contributes to biliary tree integrity and repair reactions. blackard, carolynne zander, massachusetts general hospital, harvard medical school, boston, ma; gregory beck, university of massachusetts, boston, ma; emmett v schmidt, raymond t chung, massachusetts general hospital, harvard medical school, boston, ma backgroundlaims: the innate immune response (type i ifn system) is postulated to play a critical role in control of viral infection.the role of the ifn system in controlling hcv replication has been inferred but not proven. in addition, hcv protein expression has been shown to antagonize the ifn response in vitro. until recently, it has been impossible to assess the interaction between hcv and the type i ifn system in vivo without a model that successfully supports viral replication. we have developed a cell-based binary hcv replication model that successfully recapitulates the early stages of the hcv life cycle (pnas 98 9847,2001) . we therefore used this system (1) to assess the role of type i ifn in controlling hcv replication; and (2) to assess the effect of hcv expression or replication on ifn-induced intracellular signaling. methods: full-length hcv cdna corresponding to the ph77 strain previously shown to be infectious in the presence of ?7 was transfected into huh-t7 cells (a t7 stably-transfected huh7 be), as well as mutant m g h human fibroblast cell lines null for the ifn signaling proteins tyk2 (ul), irf9 (u2), statl (u3), jakl (u4), and stat2 (us) respectively. to test the effects of known ifn antagoflists on hcv replication, the ifn signaling antagonist sendai virus y2 protein was cotransfected into huh-t7 cells. in addition, neutralizing antibodies (nab) to type i ifn were added to selected huh-ti cells. to study the effect of hcv on ifn signaling, huh-ti cells were cotransfected with the reporter plasmids pisre-luc and prl-tk. ifna (1000 iu/ml) was added to selected cells after transfection. reporter gene expression was monitored by the dual-luciferase reporter assay system and measured as relative luciferase activity. hcv core protein was measured using the trak-c hcv core ag elisa. results ifna significantly reduced hcv core ag production from 203.5 pg/ml (ph77+/ifn-) to 41.3 pglml (ph77+/ifn t) (p=o.ool). in contrast, y2 protein enhanced hcv core ag production to 407.6 pglml in ph771y2 cotransfected cells (p=o.oool). control experiments confirmed that y2 protein inhibited ifna-induced isre-mediated signaling in huh-t7 cells; relative luciferase activity was reduced from 653 (ph771 ifnp nab increased hcv core ag replication by 42% and 23% compared to no treatment (p=o.o1). the combination of anti-1fna nab and anti-ifnp nab significantly enhanced hcv core ag production by 73% (p=o.004). the u3 cell line enhanced hcv core ag replication by over two-fold compared to the parental m g h and other mutant cell lines. hcv (ph77) transfection suppressed ifn-induced relative luciferase activity by 50% compared to ifn-treated pgem transfected cells (p= 0.w). transfection with a replication defective h77 mutant, subgenomic hcv core, or hcv structural protein mutant constructs had effects on suppression of isre-luc similar to h77.conclusions: using a binary, full length hcv replication model, we found that (i) hcv replication is enhanced in the presence of the ifn antagonist y2 as well as by ifn neutralizing antibodies, confirming the criticality of the type i ifn system in innate control of hcv replication.(2) statl null cell lines enhanced hcv replication, indicating that this protein is the critical signaling component in innate antiviral immunity to hcv. (3) hcv expression inhibits ifn signaling. hcv protein expression and specifically hcv core expression appears to be su€ficient to suppress ifn signaling. these findings demonstrate that the innate immune response exerts direct antiviral effects on hcv, and that hcv protein expression subverts type i ifn signaling. our replication model has the potential to provide new insights into the mechanisms of host control of hcv replication as well as the mechanism of hcv persistence. disclosures:gregory beck -no relationships to disclose jason blackard -no relationships to disclose raymond t chung -no relationships to disclose yoichi hiasa -no relationships to disclose yoshitaka kamegaya -no relationships to disclose wenyu lin -no relationships to disclose emmett v schmidt -no relationships to disclose carolynne zander -no relationships to disclose ifna) to 21.2 (ph77/ ifndy2) (p=o.o001). anti-ifna nab and anti-background cytotoxic t lymphocyte activator 4 downregulates t cell activation by ligating b7. single nucleotide polymorphisms (snps) at positions -318 in the promoter and +49 in exon 1 of the ctla4 gene have been associated with an increased risk for autoimmune diseases. the +49g allele has also been associated with a greater likelihood of response to interferon-alpha in patients treated for chronic hepatitis c. since some of the ctla4 snps either individually or linked together (haplotypes) may alter ctla4 expression or activity, we hypothesized that they may be associated with natural hepatitis c virus (hcv) clearance or persistence. methods: utilizing a nested case-control study design from an injection drug using cohort and a hemophiliac cohorts, we matched subjects with hcv clearance (anti-hcv positive and hcv rna negative on two occasions separated by a minimum of 6 months) to two persistently hcv-infected individuals (hcv rna positive on two occasions) based on h n status, race, and gender. five ctla4 snps, which define all common haplotypes, were genotyped using either sequence specific primers or a single base extension method. conditional logistic regression was used to determine odds ratios and p-values for these snps. haplotypes were constructed using the phase program and analyzed using the snpem program. results: included in this study were 190 and 370 subjects who either cleared or had a persistent infection with hcv, respectively. the study cohort was 47% black, 46% white, and 7% other. blacks who were homozygous for the mutant allele 49g were more likely to experience viral clearance (or 0.45, p=0.03). this association was not found in white subjects (or 0.92, p=0.81). since, hla-cw"o4 is associated with viral persistence in these cohorts (thio et a1 j virol2002), we stratified the 49g homozygous individuals by c w w . both blacks and whites who did not have cww4 were more likely to have viral clearance if they were homozygous for 49g (or 0.45, p=0.13 and or 0.49, p=o.11, respectively) . stratification by other hla alleles associated with hcv clearance or persistence did not reveal additional relationships. the 49g formed haplotypes with the wild type alleles at three of the four other snps examined, and those haplotypes did not have a stronger association than 49g alone. 49g was found in haplotypes with both the mutant and wild type alleles at the -1722 promoter position, but neither of these haplotypes had a stronger association with clearance. conclusions: homozygosity for the ctla4 49g snp is associated with clearance of hcv in blacks regardless of hla genotype, whereas in whites, homozygosity at this position trends toward an association with clearance in hla-cw*m-negative persons. since ctla4 49g has been reported to augment t ceil activation by decreasing ctla4 expression, these results point to the importance of t cell activation in hcv clearance. disclosures:jacquie astemborski -no relationships to disclose james g goedert -no relationships to disclose recurrence of hcv infection post liver transplant is universal. high levels of viraemia characterize the post transplant course. in this setting, acute rejection in association with hcv re-infection of the graft (ar (hcv)) and recurrent chronic hepatitis (chi) are recognized clinical entities. we aimed to 1) determine the intrahepatic gene profile in ar (hcv) and chi. 2) examine whether the gene profile in ar (hcv) is different to that of acute rejection in non-hcv infected allografts (ar non-hcv). 3) examine whether the gene profile of chi is different to chronic hepatitis c pre-transplant (ch). methods: genechip arrays were utilized to study pooled rna from 34 liver tissue; 6 patients with ar (hcv), 6 patients with ar (non-hcv), 8 patients with chi, 8 patients with ch and 6 normal livers. probe level data was analysed using the bioconductor affy library and genes with more than 2 fold changes were considered to be upregulated. results: ar (hcv) us ar (non-hcv): compared to normal tissue, both groups were similarly characterized by upregulation of several t cell dependent immune activation genes such as the lymphocyte antigen campath-1, mac-2 binding protein and ifn related genes. however, compared to ar (non-hcv), ar (hcv) was specifically characterized by upregulation of several ifn-alpha associated genes including 2-50as, and p27. chi vs c h compared to normal, both groups were characterized by upregulation of genes involved in antigen presentation (proteasome subunit 42, mdw), t cell activation (t cell receptor and ctl-17), ifn-gamma inducible genes (ifn-56kd, ip-10, humig and class i1 mhc) and ifn-alpha associated genes (p27 and 2-5 oas). however, the ifn-alpha and gamma genes were more upregulated in chi compared to ch. furthermore, compared to ch, the chi group displayed increased expression of genes involved in cellular proliferation (pcna, cyclin, beta integrin), apoptosis (cytochrome c, fas-l, caspase 4), inflammation (macrophage mannose receptor, complement factor h) and fibrosis (angiotensin i1 receptor, proteoglycan core protein). conclusions: at the transcriptome level, the pathological process in hcv infected allografts with acute rejection or recurrent chronic hepatitis is characterized by upregulation of several ifn-alpha and gamma related genes compared to other livers. moreover, chronic hcv post transplant is further characterized by an increased process of cellular proliferation, apoptosis and fibrosis compared to chronic hepatitis in the pretransplant setting. collectively the data suggest that the high levels of viraemia which characterize the post transplant setting drive the immune response to act at a higher level. in addition, the upregulation of several interferon related antiviral genes supports the notion that hcv is a strong inducer of intrahepatic interferons, but is relatively resistant to their antiviral activity. (hcv) infection is thought to be mediated by a multispecific immune response. hcv-specific t cells have been described in hcv-seronegative virus-exposed individuals such as i.v.-drug addicts, family members of chronic hcv-patients and lab-personnel. however, it is not known whether these individuals had recovered from previous asymptomatic acute hepatitis c. in this study, we prospectively followed 10 individuals who experienced an injury with an hcv-contaminated needle. methods: between january 2001 and march 2003 we collected pbmc from 10 individuals (medical health professionals at our institution; 3 females, 7 males; age 30-45 years) who experienced an injury with an hcv contaminated needle. blood samples were taken on the day or the day after the event and at different time points during follow-up for up to 12 months. low levels of hcv-rna were examined in serum by the highly sensitive transcription-mediated tma-assay (detection limit 5-50 iuiml) and in rna of pbmc. t cell-cytokine secretion (elispot-assays for ifn-gamma and il-10, flow-cytometry-based ifn-g capture assays), t cell-proliferation (3-thymidin incorporation, cfse-staining), and t cell-cytotoxicity (51-chromium release assays) were investigated directly ex vivo and in t cell lines. results: none of the individuals became positive for hcv-rna in serum (tma-assay) or pbmc and all of them remained anti-hcvnegative throughout follow-up. at the time of the needle stick injury, hcv-specific cd4+ t cell responses were already detectable in 2 of the individuals and became detectable thereafter in 2 additional persons. hcv-specific cd8+ t cell responses could be investigated in one hla-a2-positive individual in more detail at several time points. he was negative for hcv-specific interferon gamma-and il-10-producing cells on the day of the injury as determined by elispot assays. however, after 18 weeks, we detected for core-178-specific ifn-gamma producing cd8+ t cells. out of 7 mhc-class i-restricted hcv epitopes tested, one additional peptide (ns3-1406) became positive 8 weeks later. the presence of core-178 and ns3-1406-specific cd8+ t cells was confirmed by a second flow-cytometry-based assay. hcv-specific inf-gamma positive cells were cd8-dim and hla-dr-negative. the frequency of ns3-1406 and core-178-specific cd8+ t cells was about 114500 and 118500 pbmc, respectively, in the elispot assay and remained constant in this subject until month 11 of followup. the increase of cd8+ t cell responses was accompanied by the development of an hcv-specific cd4+ response targeting predominantly the hcv-core and hcv-helicase antigens. conclusions: we here demonstrate in a prospective study the development of hcv-specific t cells in hcv-exposed individuals after needle stick i n j q in the absence of viremia. surprisingly, these hcvspecific t cells became detectable rather late after the potential exposure. our data are in line with previous studies demonstrating hcvspecific t cell responses in chimpanzees inoculated with subinfectious doses of hcv. t cell immunity in medical health professionals against hcv may contribute to the low prevalence of hcv among doctors and nurses since hcv prevalence in medical health personnel has been shown to be equal or even lower as compared to the general population in most studies. aims: laparoscopic cholecystectomy is the treatment of choice for symptomatic gallstone disease at present. despite its wide spread application in the past decade, the incidence of iatrogenic bile duct injuries related to this procedure has remained unchanged around 0.3-0.6%. we review the investigation, management and outcomes of such injuries referred to a tertiary referral center in the united kingdom. methods: prospectively collected data on iatrogenic bile duct injuries following elective laparoscopic cholecystectomy referred to our center was analyzed. the strasberg classification was used for defining the types of injury. results: a series of 125 patients were referred for management of iatrogenic bile duct injuries between january 1991 and june 2003.median follow up period was 55 months. 87 patients had type e injuries (to%), followed by 20 with type a (16%) 14 with type d (11%) and 2 each of types b (1.6%) and c (1.6%). an uncomplicated primary operation was described in 74 patients (60%). despite the operation being converted to an open procedure in 27 patients only 18 injuries were recognized at the time of surgery.(l4.4%) although the median time to recognition of biliary tract injury was 2 days (1-21) the median time to referral increased to 10 days (0-99) for a type a injury, and 16 days (0-2200) for a type e injury. a total of 29 patients underwent surgical intervention prior to referral of which 20 had biliary reconstruction. nine of these patients required subsequent revision surgery. after referral 15(13%) patients were managed conservatively while 6(5%) had ercp and biliary stenting and 24(29%) had interventional radiology as the main procedure. surgical intervention was required in 79 patients (53%) at our center following referral. this included t-tube insertion (7), primary common bile duct repair (lo), biliary reconstruction (60) and liver resection (2) with three patients requiring subsequent revision surgery. major associated vascular injuries were present in eight patients. one patient suffers from recurrent cholangitis while five remain asymptomatic on follow up. two required liver resection due to severe ischaemic necrosis. bowel perforations were associated in three patients. both vascular and bowel injuries occurred in patients with type e bile duct injuries. nine patients (7%) had recurrent cholangitis following treatment. three patients developed end-stage liver disease due to secondary biliary cirrhosis with one undergoing orthotopic liver transplantation. there were five deaths related to bile duct injuries. two had associated vascular injuries and one had bowel perforation. a further two patients with type a injuries died following multiorgan failure due to sepsis. conclusions: iatrogenic bile duct injuries are associated with significant morbidity and mortality. delay in recognition and appropriate referral remain a continuing problem. a multi disciplinary approach in a specialized institution has a significantly better long term outcome. background radio-frequency ablation (rfa) is widely used as a treatment of inoperable tumor leasons in the liver. preliminary studies of a vx2 hepatoma model in rabbits showed tumor specific t lymphocytes after rfa treatment and a significantly prolonged survival of these treated animals compared to a control group. aim: the aim of the study was to assess whether the observed tumor specific t cell response after rfa treatment, found in the vx2 tumor model, is transferable to humans. methods: 12 patients were included into a pilot study. 6 patients had metastases of colonic cancer and 6 patients were suffering from hepatocellular carcinoma (hcc) with underlying cirrhosis. all patients were treated with rfa (elektrotom him 106, berchtold, germany).blood samples were taken before and 4 weeks after rfa. an interferon gamma cytokine secretion assay (miltenyi, germany) was carried out on whole blood and measured by a flowcytometer (facs calibur, bd science, germany). as test antigens served autologous liver tissue and tumor tissue received by biopsy. t cells were doublestained with cd 8 antibody to detect cytotoxic t cells. the basic ifn gamma secretion was also measured unstimulated. the activated/non activated cd8 positive t cell ratio (anr) was calculated and analysed statistically with spss. only rfa naive were included. results: before rfa patients had a mean stimulation of 0,022 anr (sd=0,001) against liver tissue and 0,027 anr (sd=0,002) against tumor tissue (see tab.1). 4 weeks after rfa a strong increase of anr was observed with 0,11 anr (hcc) and 0,15 anr (colorectal carcinoma) against tumor tissue. this increase was also detectable 8 weeks after rfa (0,14 anr (sd=0,003) (hcc) and 0,14 anr (0,003) (colorectal carcinoma)) (see fig. 1 and tab.1) . the anr levels were allways low against liver tissue with 0,03 anr (sd=0,002). discussion: rfa shows significant tumor specific t-cell stimulation in patients with primary and secondary tumors of the liver. rfa seems to overcome immune tolerance or presents alterated and/or cryptic tumor antigens, and causes a significant tumor specific cytotoxic tcell response. this effect could be exploided in multimodal antitumoral strategies. the strategy of primary hepatic resection followed by secondary liver transplantation (slt)was not evaluated in-term of operative risks and survival. between 1991-2001, among 88 cirrhotic patients transplanted for hcc with mazzafero's criteria on analysis of the specimen, 18 had liver resection prior to slt : for recurrence (n=11), deterioration of liver function (n=4) or high risk for recurrence (n=3) with a mean time between liver resection and listing for lt was 20 months .the comparison between slt group and patients who underwent primary liver transplantation (plt) showed similar median age (55 vs. 53 years), sex and etiology of liver disease (alcohol/viral biclother). overall time on lt waiting list of the two groups was similar (5 vs. 3 months). pathologic analysis of the specimen revealed similar number (1.7 vs. 1.6) and tumor size (2.3 vs. 2.2 cm). pen-and post-operative course were not different in terms of operative time (551 vs. 530 min), blood loss (1282 vs. 1191 ml), transfusion (3 vs. 2 units), icu (10 vs. 9 days) or hospital stay (32 vs. 31 days), morbidity (56% vs. 51%) or 30-day mortality (5.7% vs. 5.6%). during a median follow-up of 32 months (3 to 158 months), 3 patients recurred after plt and one after slt. after transplantation, 3 and 5-year overall survivals were not different between groups (82 vs. 82% and 59 vs. 61%). results of our study showed that liver resection for hcc prior to transplantation did not increased the morbidity or impair long-term survival following lt. therefore liver resection prior to transplantation can be integrated in the treatment strategy for hcc. aims: ct-1 is a member of the il-6 family of cytokines which protects myocardiocytes against thermal and ischemic insults. in this work we analyzed the expression of ct-1 by normal and diseased liver and whether this cytokine might protect the liver against ischemia-reperfusion injury. methods: rat ct-1 cdna was cloned, recombinat rat ct-1 was produced and used for generation of poly and monoclonal antibodies which were employed in immunochemistry, western blot and elisa. the recombinant cytokine was assayed for therapeutic use in rat models of ischemia-reperfusion injury. wistar rats were subjected to 60 min ischemia of 70% of the liver by clamping the corresponding branches of the hepatic and portal vein. after this period the clamp was released and serum and tissue samples were obtained at 6 hours. rats were divided into three groups: group 1 received 100 mcg ct-1 10 minutes prior to ischemia (n=8), group 2 received saline instead of ct-1 (n=8) and group 3 were sham operated (n=4). results: immunohistochemistry of normal livers demonstrated ct-1 expression selectively in hepatocytes of the centrolobular area. six hours after reperfusion of ischemic livers the values of serum alt were 10 times higher in group 2 than in group 1 (pco.01). severe focal hepatocellular necrosis was found in animals from group 2 while no apparent morpholgical changes were observed in rats from group 1. serum ct-1 levels were raised after reperfusion in untreated animals as compared with sham operated rats. western blot of liver samples showed an increase of ct-1 expression in the non-ischemic liver lobule as compared with the damaged part of the liver. conclusions: ct-1 is a hepatoprotective cytokine produced by hepatocytes in centrolobular areas. ct-1 synthesis increases after ischemic insults. administration of recombinant ct-1 protects very efficiently the liver against ischemia-reperfusion injury. ct-1 may be a valuable therapy to attenuate hepatic damage of the donor liver during orthotopic liver transplantation. disclosures: naiara beraza -no relationships to disclose matilde bustos -no relationships to disclose pun fortes -no relationships to disclose maria ifiiguez -no relationships to disclose eduardo martinez-ans6 -no relationships to disclose jesus prieto -no relationships to disclose cardiotrophin-1 (ct-1) is a potent background: in experimental and clinical acute liver failure, both a loss of cerebrovascular autoregulation and an increase in cerebral blood flow (cbf) have been implicated in the development of brain edema. it is unclear whether the pathogenesis of these two phenomena are linked. the accumulation of glutamine within astrocytes appears to be a critical first step in the development of cerebral hyperemia, as experimental inhibition of glutamine synthetase with methionine sulfoximine (mso) attenuates brain edema and corrects cerebral hyperemia. to study cerebrovascular autoregulation without the confounding variable of brain edema, we examined animals one day after portacaval anastomosis (pca), where a decreased cerebral blood flow has been noted in the setting of a hyperdynamic systemic circulation. material and methods: a pca or sham surgery was performed in male sld rats (300-400 gm). at 24 hrs, mean arterial pressure (map) was measured continuously in anesthetized rats. noradrenaline was infused at a dose of 1 pglkglmin for 60 minutes. mso or saline was given as an intraperitoneal injection (150 mglkg) three hours prior to na infusion. relative changes in cortical blood flow (cbf) were measured with a laser doppler probe placed over the parietal cortex. the animals were mechanically ventilated and arterial blood gases were monitored throughout the experiment. three groups of rats were studied (n= 18). results: the sham group had significantly higher map, in accordance with the systemic vasodilatation seen in pca rats. adding na to all groups significantly raised map and hr. the sham+na group did not exhibit an increase in cbf with a rise in map. the pca+na group had a significant increase in laser doppler flow (ldf) from baseline that coincided with an increase in map. pretreatment of the pca animals with mso prevented the rise in cbf despite a rise in map. there were no significant differences in pc02 among the three groups. as expected, arterial ammonia levels were higher in pca animals and increased further after inhibition of glutamine synthetase in the group that received mso. conclusions: a rise of map in rats after pca results in an increase of cbf, indicating a loss of cerebrovascular autoregulation. the restoration of autoregulation with mso, despite higher arterial ammonia levels, suggests that accumulation of glutamine within the brain is critical to the pathogenesis of altered cerebrovascular regulation in this model. as similar conclusions can be reached regarding the development of cerebral hyperemia in experimental acute liver failure, it appears that the mechanisms responsible for cerebral hyperemia and failed cerebrovascular autoregulation are linked. future studies should focus on the nature of the intracerebral signal that is responsible for the loss of cerebral autoregulation in this model. a decreased intra-hepatic production of nitric oxide (no) participates on the pathogenesis of portal hypertension in cirrhosis. although non-specific no donor substances, such as nitrates, are able to decrease the intra-hepatic vascular resistance to portal blood flow, the use of these vasodilators to treat portal hypertension in cirrhotic patients is limited by the occurrence of side effects (arterial hypotension, headaches etc). aim. to test the effect of a liver-specific no donor (ncx-1000), an ursodeoxycholic acid-derived compound, on both systemic and intra-hepatic hemodynamics in portal hypertensive cirrhotic rats. method. after the development of ascites, cirrhotic rats (cc4-induced) were treated with ncx-1000 (28 mglkg b.w.) or ursodeoxycholic acid (15 mglkg b.w., control group) by gavage once a day for 14 days and, then, used in 1 of 3 studies: 1) in vivo mean arterial pressure (map), portal pressure (pp), and superior mesenteric artery (sma) blood flow measurements before and during progressive blood volume expansion (blood infusion); 2) in situ liver perfusion to obtain doselresponse curves to methoxamine (alpha*-adrenergic agonist) or flowlpressure curves; and 3) cgmp concentration measurement in liver tissue. results. both map and heart rate were similar in both groups, indicating that ncx-1000 had no effect on systemic hemodynamics. pp was also similar in both groups. during blood infusion, ncx-1000 treated rats and control rats showed similar map (p=0.134) and sma blood flow (p=0.449) increases; however, the pp increase observed in control rats was blunted in ncx-1000 treated rats (p=0.015). flowlpressure curves obtained during liver perfusion were similar in both groups; however, livers from ncx-1000-treated rats showed a decreased response to methoxamine (p=0.016) than controls. the concentration of cgmp in ncx-1000-treated livers was significantly higher (p=0.015; ) than in ursodeoxycholic acid-treated livers (27.65 t 7.95 vs. 11.20 2 6.03 fmollmg of protein). conclusion. ncx-1000 improves the portal system adaptability to portal blood flow increase and decreases the intra-hepatic hyper-reactivity to methoxamine observed in cirrhotic rat livers without causing systemic side effects. these beneficial effects are probably due to increase in the intra-hepatic no bioavailability. the mechanisms behind the upregulation of endothelial nitric oxide synthase (enos) in portal hypertensive animals remain unknown. the aim of this study was to investigate the mechanism that initiates enos upregulation in the superior mesenteric artery (sma) in portal hypertension. it has been shown that the vasoconstriction of the sma with the consequent increase in the sma cyclic strain is the earliest event that occurs within hours after portal vein ligation (pvl). thus, we tested the hypothesis that this early vasoconstriction of the sma may initiate enos upregulation in pvl animals. methods: portal hypertension was induced by partial ligation of the portal vein (n=99). in a separate group of rats, mesenteric vasoconstriction was achieved also by renal artery ligation (ral, n=26). corresponding sham-operated rats were used as control groups (n=50). effects of vasoconstriction of the sma in pvl and ral rats were evaluated by measuring perfusion pressure changes in the isolated sma beds in response to methoxamine, nos activity and enos protein expression. data were compared to the corresponding sham group. hemodynamic features were also determined. mean arterial pressure (map), portal pressure ( i " ) and sma blood flow (qsma) were measured by catheterization and pulsed doppler flowmetry. sma vascular resistance was calculated from map, pp and qsma. results: there was a significant increase in the sma vascular resistance in pvl (2.33%0.13 vs 1.2220.03 rnmhgl %flow, k0.05) and ral rats (23220.18 vs 1.18%0.02 mmhgf%flow, p<0.05) compared to their corresponding sham-operated rats, demonstrating the presence of sma vasoconstriction in both pvl and ral groups. the mesenteric vasculature of pvl and ral animals showed hyporeactivity to methoxamine compared to their respective sham groups (p< 0.01). whiie pvl rats were portal hypertensive (p<o.ol), ral rats were not. the sma vascular hyporeactivity of pvl and ral were corrected by l-nmma and while there was not significant difference in enos protein expression nos enzyme activity was significantly higher in the pvl and ral rats compared to the corresponding sham-operated groups (see figure) . conclusions: mesenteric arterial vasoconstriction in itself leads to enos upregulation and therefore, plays a triggering role in the initial increase of enos catalyhc activity in sma of pvl rats. helen hendrickson, vijay shah, mayo clinic, rochester, m n background. diminished enos derived no production from the hepatic vascular endothelium contributes to hepatic vasoconstriction in cirrhotic portal hypertension. unfortunately, prior studies aimed at delivering a constitutively active form of enos (s1179denos) using an adenoviral vector (ads1179denos) were unsuccessful in correcting hepatic vasoconstriction in the bile duct ligated (bdl) rat liver. aim. to better understand the mechanism of s1179denos dysfunction in bdl liver by examining the influence of the enos inhibitory protein caveolin on recombinant s1179denos function in transduced hepatic stellate cells, which do not express enos endogenously but are a prominent cell target of systemically delivered adenoviral vectors. methods. cellular localization of caveolin was determined from fixed sham and bdl liver sections by immunogold electron microscopy. in vitro gene transfer was performed in the lx2 hsc line with ads1179denos and an adenoviral vector encoding caveolin (ad-cav). protein interactions were determined by immunoprecipita-tion and western blot analysis. nos activity was assessed by arginine to citrulline conversion assay. results. caveolin immunogold analysis in bdl liver demonstrated prominent expression not only in liver endothelial cells, but also in stellate cells. western blot analysis from lx2 cell lysates confirmed caveolin protein expression in vitro. in lx2 cells transduced with ads1179denos, immunoprecipitation of caveolin co-precipitated recombinant s1179denos and conversely, immunoprecipitation of s1179denos coprecipitated caveolin. to examine the influence of excess caveolin protein levels on s1179denos, lx2 cells were co-transduced with ads1179deno.s and adcav. co-transduction of adcav and ads1179d promotes the enhanced binding between s1179denos and caveolin as well as a decrease in the activity of recombinant s1179denos by 25% (p<0.05; ads1179denos vs ads1179denos +adcav; n=5). conclusion. these studies demonstrate a functional effect of enhanced caveolin expression and binding with s1179denos in stellate cells and indicate that the lack of function of recombinant s1179denos protein delivered in vivo to cirrhotic liver may be due to inhibition of s1179denos by caveolin-1 in stellate cells. background and aims: the presence of actin-like microfilaments in the neighborhood of sinusoidal endothelial fenestrae (sef) indicates that the cytoskeleton of sinusoidal endothelial cells (sec)s plays an important role in the modulation of sef. we have reported that a potent vasoconstrictor endothelin (et) causes contractions of sef as well as hepatic stellate cells via the eta and etb receptors, leading to the elevations of sinusoidal microvascular resistance supposed to be one the essential factors in the pathogenesis of portal hypertension. rho has emerged as an important regulator of the actin cytoskeleton, and consequently cell morphology. the present study aimed to examine how a rho stimulator; lysophophatidic acid (lpa), and a rho inhibitor; y-27632 rho-kinase inhibitor, affect the morphology of sef in vitro. methods: monolayers of cultured sec were obtained by collagenase infusion of a rat liver and then subjected to primary culture for 24 hours. the cells were separated into three groups; control, lpa-treated (lopm), and y-27632-treated (10pm) groups (treatment duration, 30 min). sef morphology was observed by scanning electron microscopy. formation of f-actin stress fibers was observed by confocal microscopy. actin cytoskeleton around sef was investigated by permealizing cells with streptolysin-0 (1.5 u) for 30 min and examined by transmission electron microscopy. rho a and phosphorylated myosin light chain kinase were analyzed by western blotting. rho activation was measured by rhotekin binding assay. results: following lpa treatment, f-actin stress fiber and active rho increased concomitant with a decrease in diameter of sef. however, following y-27632 treatment, f-actin stress fiber and active rho were reduced, concomitant with an increase in diameter of sef. actin cytoskeleton around sef increased in lpa-treated cell, but decreased in y-27632-treated cell. rho a levels were similar in control, lpa-treated, and y-27632treated secs. phosphorylated myosin light chain kinase levels were diminished gradually after 5 min in y-27632-treated secs and increased after 5 min in lpa-treated cells. lpa treatment increased the amount of active rho, compared to control and y-27632 treatment. conclusion: these results indicate that rho may play an essential role in the regulation of contraction and dilation of sef through the actomyosin system. disclosures: analysis of visual-evoked response patterns suggests that gabaergic tone is increased in brain in liver failure and that this is a major cause of hepatic encephalopathy (schafer et al., troenterolow 86:540-545,1984) . however, the precise mechanisms responsible have eluded researchers in this area for the last two decades. studies in brain tissue from both human and experimental animals with liver failure have consistently shown that gaba synthesis, gaba receptors and their associated benzodiazepine modulatory sites are intact and that expression of the subunit proteins of the gaba-a receptor complex are unchanged (zwingmann et al., hevatologv 37420-428,2003; ahboucha et al., neurochem. int., 2003) . on the other hand, there is emerging evidence to suggest that neurosteroids (ns), a novel class of compounds with potent gaba agonist properties are increased in brain in endstage chronic liver failure in humans (ahboucha et al., hepatola 36: 318a, 2002) . in order to further assess this possibility, ns concentrations were measured using a sensitive radioassay in samples of frontal cortex obtained at autopsy from 15 cirrhotic patients who died in hepatic coma compared to an equal number of controls matched for age, gender, and autopsy delay intervals. patient material contained up to 12-fold increases of ns and subsequent analysis by gas chromatography-mass spectrometry showed that the major component ns with positive allosteric properties at the gaba-a receptor was allopregnanolone. patient material did not contain significantly increased concentrations of either gaba or benzodiazepines. in a separate series of experiments, ns concentrations were found elevated up to 2-fold (p<0.02) in brains of rats following end-to-side portacaval anastomosis (pca) and 5.2-fold (p<o.ol) in brains of rats following hepatic devascularization (pca followed by hepatic artery ligation) compared to sham-operated controls. administration of inhibitors of the ns synthesis enzyme 3-hydroxysteroid dehydrogenase (trilostane or indomethacin) resulted in dose-dependent reductions in brain concentrations of allopregnanolone and a concomitant improvement in locomotor activity scores in pca rats. together, these findings offer unequivocal evidence that ns with positive allosteric modulatory properties are generated in brain in both human and experimental liver failure and that these substances contribute to the "increased gabaergic tone" characteristic of hepatic encephalopathy. pharmacological agents aimed at reduction of ns synthesis or modulation of the ns site on the gaba-a receptor complex could afford effective novel therapeutic strategies in the treatment of hepatic encephalopathy [funded by cihr canada] abstract primary hepatocellular carcinoma (hcc) is one of the most common malignancies, ranks fi&h in frequency among all cancers worldwide, and causes over 1 million deaths annually. although a number of oncogenes and alterations in signaling pathways have been identified in hcc, current treatment for this liver tumor is largely ineffective and has poor patient prognosis. one of the potentially curative approaches in hcc therapy is based on interfering with hcc progression by blocking cell division. in regenerating liver, expression of the proliferation-specific forkhead box m l b (foxmlb) transcription factor is reactivated prior to hepatocyte dna replication and its levels are sustained throughout the period of hepatocyte proliferation. deletion of the foxmlb fllfl (loxp targeted) allele using albumin promoterlenhancer driven-cre recombinase (alb-cre) transgene resulted in significant reduction in regenerating hepatocyte proliferation, which was associated with diminished expression of cell-cycle promoting cdc25a and cdc25b phosphatases and increased nuclear levels of cyclin dependent kinase (cdk) inhibitor p21cipl protein. in order to test the hypothesis that foxmlb is required for the development of hcc, we subjected alb-cre foxmlb -/-mice and foxmlb fllfl (control) mice to a diethylnitrosamine (den)lphenobarbital (w) liver tumor induction protocol. our data demonstrated that alb-cre foxmlb -imice are highly resistant to liver tumor formation as evidenced by their failure to develop either hepatic adenomas or hcc following 33 weeks of denlpb tumor induction protocol. in contrast, at 23 weeks following den/pb tumor induction protocol, foxmlb fllfl livers displayed a large number of adenomas that progressed to hcc by 33 weeks following den/pb treatment. the alb-cre foxmlb -iliver displayed enzyme-altered foci as evidenced by protein expression of the glutathione-s-transferase placental isoform. however, alb-cre foxmlb -/hepatocytes failed to develop hyper-proliferative foci, whereas abundant brdu labeling was found in hepatic adenomas and hcc of denlpb treated foxmlb fllfl mice. this reduced dna replication of foxmlb -/-hepatocytes was associated with increased nuclear levels of the cdk inhibitor p27kipl protein. furthermore, foxmlb fllfl tumors expressed high nuclear levels of m-phase promoting cdc25b protein, while undetectable nuclear levels of cdc25b were found in foxmlb -ihepatocytes after den/pb treatment. the cdc25b phosphatase is required for mitosis because it activates the m-phase promoting cyclin b-cdkl complex, and its increased expression is a prognostic marker for a variety of aggressive tumors. consistent with this finding, foxmlb -/-hepatocytes failed to enter mitosis following denlph tumor induction protocol and this was associated with accumulation of large polyploid hepatocytes. these studies demonstrate that foxmlb is required for the proliferative expansion of hepatic tumors, suggesting that foxmlb can be used as a potential target in treatment of patients with hcc. disclosures: robert h costa -no relationships to disclose vladimir v kalinichenko -no relationships to disclose joseph kuechle -no relationships to disclose brian shin -no relationships to disclose xinhe wang -no relationships to disclose yoder yoder -no relationships to disclose key: cord-004341-co9s26x1 authors: thukral, akanksha; ross, kathleen; hansen, chungyi; phanse, yashdeep; narasimhan, balaji; steinberg, howard; talaat, adel m. title: a single dose polyanhydride-based nanovaccine against paratuberculosis infection date: 2020-02-14 journal: npj vaccines doi: 10.1038/s41541-020-0164-y sha: doc_id: 4341 cord_uid: co9s26x1 mycobacterium avium subsp. paratuberculosis (m. paratuberculosis) causes johne’s disease in ruminants and is characterized by chronic gastroenteritis leading to heavy economic losses to the dairy industry worldwide. the currently available vaccine (inactivated bacterin in oil base) is not effective in preventing pathogen shedding and is rarely used to control johne’s disease in dairy herds. to develop a better vaccine that can prevent the spread of johne’s disease, we utilized polyanhydride nanoparticles (pan) to encapsulate mycobacterial antigens composed of whole cell lysate (pan-lysate) and culture filtrate (pan-cf) of m. paratuberculosis. these nanoparticle-based vaccines (i.e., nanovaccines) were well tolerated in mice causing no inflammatory lesions at the site of injection. immunological assays demonstrated a substantial increase in the levels of antigen-specific t cell responses post-vaccination in the pan-cf vaccinated group as indicated by high percentages of triple cytokine (ifn-γ, il-2, tnf-α) producing cd8(+) t cells. following challenge, animals vaccinated with pan-cf continued to produce significant levels of double (ifn-γ, tnf-α) and single cytokine (ifn-γ) secreting cd8(+) t cells compared with animals vaccinated with an inactivated vaccine. a significant reduction in bacterial load was observed in multiple organs of animals vaccinated with pan-cf, which is a clear indication of protection. overall, the use of polyanhydride nanovaccines resulted in development of protective and sustained immunity against johne’s disease, an approach that could be applied to counter other intracellular pathogens. m. paratuberculosis is the causative pathogen of johne's disease (jd) characterized by chronic gastroenteritis, diarrhea, weight loss and low milk yield in ruminants. 1 while jd is a worldwide problem, its prevalence in the united states is estimated to be >90% in dairy herds, 2 causing a combined loss of $200-250 million to the us dairy industry. 3, 4 the financial losses are incurred due to premature culling of infected animals, decreased milk production, and increased somatic cell infiltration in milk. 5, 6 jd is a slowly progressing disease and can infect 38-40% of the herd before becoming symptomatic in a single animal. 7, 8 currently there is no treatment for jd and controlling the disease progression by culling the infected animals is very expensive, whereas vaccination offers a reasonable alternative. 9 mycopar® (boehringer ingelheim) is an oil suspended, heat killed, whole cell vaccine licensed in the united states. however, mycopar® fails to completely protect against jd 10, 11 and can cause severe inflammatory lesions at the site of injection. 12 it also poses a health risk to vaccinators due to accidental inoculation, which leads to a chronic inflammatory reaction that potentially requires surgical intervention. 13 given the challenges to control jd with the current vaccine, we directed our efforts to develop a more effective and safe vaccine against jd using polyanhydride nanoparticles (pan). an ideal vaccine should elicit a robust immune response without causing untoward reactions in the vaccinee or risk to the vaccinator. another important aspect of vaccine development against mycobacterial infection is its capability to elicit a polyfunctional t cell response with simultaneous production of pro-inflammatory cytokines by t cells. 14, 15 to elicit robust immunity, antigens are often formulated with adjuvants to prolong their release and enhance their protective immunity. in this study, we used whole cell lysate and culture filtrate proteins encapsulated in biodegradable polyanhydride nanoparticles (adjuvant) that provide sustained release of m. paratuberculosis antigens by surface erosion. 16 pan-based vaccines (i.e., nanovaccines) have been shown to impart long lasting protective immunity against several infectious diseases including influenza, pneumonic plague, respiratory syncytial virus, and pneumonia, using pathogen-specific protein antigens. [16] [17] [18] [19] [20] [21] [22] the amphiphilicity of the pan chemistry provides antigen stability and the copolymer composition enables sustained release of the encapsulated immunogens. 21, [23] [24] [25] [26] [27] the small size (~200 nm) and large surface area of the nanoparticles allows them to carry antigens across cellular membranes and deliver them to their targets. [28] [29] [30] in addition, their molecular chemistry and size has pathogen-mimicking characteristics, allowing pan to be engulfed by, persist within, and subsequently stimulate antigen presenting cells (apcs). 31, 32 polyanhydride particles on their own exhibit adjuvant-like properties by activating apcs 31-33 and inducing both humoral and cell-mediated immune responses; 17, [33] [34] [35] formulating them with immune-stimulatory antigens results in protective immunity. 22, 35 finally, these particles have been shown to be safe and induce less inflammation at the administration site compared with traditional adjuvants such as alum and incomplete freund's adjuvant. 36, 37 m. paratuberculosis whole cell lysate and culture filtrate proteins have been shown to exhibit immunogenic properties and have previously been evaluated as a potential vaccine. 38, 39 therefore, we utilized m. paratuberculosis antigens together with pan to formulate nanovaccines that can elicit robust and sustainable protective immune responses. in this study, a single, subcutaneous dose of nanovaccine in c57bl/6 mice was evaluated for protection against m. paratuberculosis jtc-1285 challenge in comparison to both inactivated and live vaccine candidates. the live vaccine candidate lipn, developed previously by our group, was constructed by knockout of a fatty acid lipase/esterase gene lipn from m. paratuberculosis k10. this gene was significantly upregulated in m. paratuberculosis shed in the cow feces, as revealed by transcriptional profiling. 40 also, lipn mutant was analyzed and found to be attenuated in mice as indicated by reduced histopathological lesions and colonization of the liver. 41 its protective efficacy has been observed in goats challenged by virulent m. paratuberculosis strain. 42 the study was conducted in two phases, viz: trial i and trial ii. in the trial i studies, the focus was on the safety of the nanovaccine formulations while in the trial ii studies, the focus was on the efficacy of nanovaccine formulations (fig. 1) . scanning electron photomicrographs of m. paratuberculosis lysateencapsulated (pan-lysate) and culture-filtrate (pan-cf)-encapsulated polyanhydride nanoparticles showed similar spherical morphology and size as blank (i.e., empty) nanoparticles, indicating that antigen encapsulation did not change the average diameter, which was ca. 200 nm (fig. 2) . the encapsulation efficiency of the lysate into the nanoparticles was 40.0 ± 1.9% and that of the culture filtrate was 26.0 ± 0.4% and 2.5 wt% of the protein content of the lysate or culture filtrate (cf) was encapsulated into the particles. to evaluate nanovaccine safety, we monitored immunized mice on a daily basis. animals vaccinated with mycopar® gradually developed an abscess at the injection site which progressed and persisted throughout the study (supplemental fig. 7) . on the other hand, no lesions were observed in nanovaccine-immunized and live attenuated (lipn) vaccine immunized animals. at 6 weeks post-vaccination (wpv) and before any challenge, histopathology of vaccinated mice groups demonstrated lymphoid depletion in the spleens of animals immunized with the commercial vaccine, while minimal to moderate lymphocytic infiltration and granulomatous inflammation was observed in the livers of the rest of vaccinated animals regardless of formulation, which is indicative of induced immunity. no pathology was observed in the negative control group (pbsvaccinated mice). pre-challenge immune responses for trial i (i.e., safety study), the t cell response was evaluated at 6 weeks post-vaccination by performing ifn-γ elisa on spleen derived lymphocytes (described in methods) (supplemental fig. 1 ). the mycopar-vaccinated animals showed significantly higher ifn-γ levels than the rest of the groups (***p < 0.001) (supplemental fig. 2a ). for trial ii (i.e., efficacy study), spleen derived lymphocytes were stained with various antibody markers and analyzed using flow cytometry. we assessed antigen specific, polyfunctional t cell responses by multi-parametric flow-cytometry. in this analysis, lipn vaccinated mice showed significantly higher percentage of double cytokine (ifn-γ,tnf-α) and single cytokine (ifn-γ) producing cd4 + t cells as well cd8 + t cells in comparison with both pbs and mycopar® vaccinated animals ( fig. 3a, b) . interestingly, the percentage of triple cytokine producing (ifn-γ, il-2, tnf-α) cd8 + t cells was significantly higher in mice immunized with pan-cf when compared with pbs and mycopar®. also, the pan-cf vaccinated animals exhibited significantly higher double (ifn-γ, il-2) cytokine secreting cd8 + t cells in comparison with pbs vaccinated mice (fig. 3b) . of note was the breadth of the polyfunctional cd8 + t cell response observed from mice immunized with pan-cf. in contrast to all the other treatment groups where the majority of the cd8 + t cells was dominated by ifn-γ secreting single positive cells, pan-cf vaccinated mice showed a broader profile of triple, double and single cytokine secreting cd8 + t cells (fig. 3b) . post-challenge immune responses to evaluate t cell response in trial i/safety study ifn-γ elisa was performed and result of which depicted no significant differences in ifn-γ levels among the groups (supplemental fig. 2b ) while at 12 weeks post challenge mycopar and pan-lysate vaccinated animals showed significantly higher ifn-γ levels as compared with control animals given pbs (supplemental fig. 2c ). t cell responses for the trial ii/vaccine efficacy study were evaluated by flow cytometry for which spleens from vaccinated mice were collected at 12 and 18 weeks post-challenge (wpc). at 12 wpc, multiparametric flow cytometry analysis indicated that mice immunized with pan-cf elicited a significantly higher percent of antigen specific double cytokine (ifn-γ, tnf-α + ) and single cytokine (ifn-γ) producing cd8 + t cells compared with non-vaccinated and mycopar® vaccinated mice (fig. 4 ). in addition, pan-cf and mycopar®-vaccinated animals also displayed low levels of triple cytokine secreting cd8 + t cells. similar to the pre-challenge cd8 + t immune response, mice immunized with pan-cf showed a pre-challenge immune response specific to lysate of m. paratuberculosis. c57bl/6 mice (n = 5) were immunized with various vaccine groups and 6 wpv, five mice from each group were euthanized. spleens were harvested; lymphocytes were isolated and stimulated with the m. paratuberculosis lysate for 24 h. cells were then stained for cd4 + (a) and cd8 + (b) cell surface markers and intracellular cytokines. the total percentage of t cells secreting particular cytokines are indicated below each pie chart (denoted by t = number). the error bars show the standard error of the mean for five individually analyzed mice. * indicates p < 0.05; ** indicates p < 0.001. * denotes comparison with pbs while # denotes comparison with mycopar®. results were expressed as the increase in the percentage of the cells with positive staining relative to that of an unstimulated sample stained with the same antibody. early cellular responses in vaccine groups following challenge with a wild type strain of m. paratuberculosis. six to eight week-old c57bl/6 mice were immunized with various vaccine candidates. at 6 wpv they were challenged with m. paratuberculosis jtc-1285 and euthanized 12 weeks later (12 wpc). the lymphocytes were isolated from the spleens and stimulated with whole cell lysate of m. paratuberculosis for 24 h. cells were then stained for cd4 + (a) and cd8 + (b) cell surface markers and intracellular cytokines and were measured by flow cytometry. the total percentage of t cells secreting particular cytokines are indicated below each pie chart (denoted by t = number). the error bars show the standard error of the mean for five individually analyzed mice. * indicates p < 0.05; ** indicates p < 0.001. * denotes comparison with pbs while # denotes comparison with mycopar®. results were expressed as the increase in the percentage of the cells with positive staining relative to that of an unstimulated sample stained with the same antibody. broader profile of cytokine secreting cells at 12 wpc (see pie chart in fig. 3 ). the cumulative percentage of cd8 + t cells that were either triple, double or single cytokine secretors was also higher in animals vaccinated with pan-cf (total percentage of cells secreting cytokine; t = 6.23) in contrast to that in animals vaccinated with the other formulations, indicating the robustness of the induced cd8 + t cell response. also, animals receiving pan-cf + lysate showed significantly higher levels of double cytokine secreting (ifn-γ, il-2) cd8 + t cells in comparison with animals that received pbs and significantly higher levels of double cytokine secreting (ifn-γ, il-2) cd4 + t cells in comparison with animals receiving both pbs and mycopar®. at 18 wpc, the percentages of mycobacterial antigen-specific double positive cd8 + t and cd4 + t cells (ifn-γ + il-2 + ) were significantly higher in pan-cf vaccinated mice compared with pbs-vaccinated mice (supplemental fig. 3 ). protection against challenge with virulent strains of m. paratuberculosis in spite of the fact that safety was the main goal of the trial i studies, we were able to evaluate the protective efficacy of each vaccine candidate at 6wpc when bacterial colonization remained similar in organs of all vaccinated groups (supplemental fig. 4 ). at 12wpc, the bacterial load was significantly lower in the spleens and mesenteric lymph nodes of mycopar and pan-lysate and lysate vaccinated groups in comparison with non-vaccinated group. livers of mycopar and pan-lysate vaccine group showed significant reduction in comparison with pbs group (supplemental fig. 5 ). to better evaluate protection offered by each vaccine formulation in trial ii/efficacy study against recent isolates of m. paratuberculosis, we quantified the level of bacterial tissue colonization following a challenge with m. paratuberculosis jtc1285, a clinical isolate of the bovine origin. 43 as expected, mice that received pbs had high levels of bacterial load in all the tissues studied (liver, spleen, intestine and mesenteric lymph node) at 12 wpc (fig. 5 ). all the vaccinated mice showed significantly lower bacterial burden in the liver in comparison with pbs-treated mice (fig. 5a) . bacterial load was significantly lower in the spleens of all vaccine groups (including mycopar®, lipn, and pan-lysate) with a two-log reduction observed in the spleens of animals vaccinated with pan-cf in comparison with the load in the spleens of animals that received pbs. the pan-cf immunized mice also showed significant reduction in bacterial load burden compared with mycopar® vaccinated mice (fig. 5b) . interestingly, mycopar® did not provide any protection in terms of a reduced bacterial load in the small intestine (fig. 5c ) compared with the pbs-treated animals. in contrast, mice vaccinated with lipn mutant, pan-cf + lysate and pan-cf had significantly lower mycobacterial colonization levels in the small intestine. all mouse groups displayed a reduction in bacterial colonization of the mesenteric lymph nodes compared with the pbs control (fig. 5c, d) . at 18 wpc, no significant differences were observed in the bacterial colonization in the mouse tissues among any of the vaccine groups, including pbs (supplemental fig. 6 ). to analyze the level of tissue damage induced by challenge with the wild type m. paratuberculosis jtc1285 strain, we performed histopathology of the main body organs in all vaccine groups. at 12 wpc, all animals administered pbs had granulomatous inflammation in the liver while only 40% of the animals receiving the pan-lysate, pan-cf + lysate, or lipn vaccine exhibited minimal to mild pathology (table 1; fig. 6 ). the granulomatous lesions in livers involved variable size aggregates of lymphocytes with macrophages visible in some lesions. in the mycopar® vaccinated group, 60% of the animals had minimal to moderate granulomatous inflammation in the liver (table 1) . at 18 wpc, granulomatous and lymphocytic inflammation were larger in size and involved more sections of the liver in all groups with no significant differences among vaccine groups. despite many challenges and shortcomings, vaccination against map is still considered to be the most efective strategy to curb johne's disease. 44 the commercially available vaccines, such as mycopar®, gudair®, and silirum®, are comprised of whole inactivated map and provide moderate protection at best. 45, 46 the limited benefits provided by these vaccines are overshadowed by their drawbacks, which include granulomatous reaction at the injection site and poor protection against bacterial tissue colonization and shedding. 46, 47 in this report, we utilized the polyanhydride nanovaccine platform technology to design a safer and more efficacious vaccine aginst jd. previous studies have shown that pans can encapuslate a diverse array of biologics including subunit proteins, peptides, and antibiotics. 17, 35 here we demonstrate that polyanhydride nanoparticles can encapsulate complex payloads such as m. paratuberculosis proteins (from whole cell lysates and culture filtrate) and induce effective immune responses in mice indicating that the immunogenicity of the encapsulated cargo is intact. this novel delivery approach to inactivated vaccine improve their efficacy but maintain their safety profile. the strategy of using m. paratuberculosis proteins in both encapsulated and soluble form enables primed (provided by the soluble protein) and sustained (provided by the nanoparticleencapsulated protein) immune responses. 16, 33, 48 consistent with previous studies that examined the safety of inactivated vaccines (e.g., mycopar®), 47, 49 we observed abscess-like lesions at the inoculation site in several mice vaccinated with mycopar® (supplemental fig. 7) . in contrast, no adverse injection site reactions were observed in pan-vaccinated mice, clearly indicating their excellent safety profile. these results add to the body of literature on the minimal reacotogenicity of polyahydride nanovaccines, as demonstrated previously. 36, 50 although oral infection and mucosal immunizations against m. paratuberculosis could be more benificial in the target host (cattle), 51 we selected parental infection and subcutaenous injection of mice to test vaccine formulas using an entery level model for vaccine testing against paratuberculosis. 52 for both the murine and bovine models of paratuberculosis, cd4 + and cd8 + effector t cells play a crucial role in eliciting protective cell mediated immunity against mycobacterial infection. 53,54 effector t cells producing multiple pro-inflammatory cytokines such as ifnγ, 55 tnf-α, 56 and il-2 57 have been shown to be associated with protection against various intracellular pathogens including m. tuberculosis. 14,15 even though the exact mechanism(s) of polyfunctional t cell mediated protection are still not clear, it has been shown that two or more cytokines can work synergistically to control infection, as in the case of a closely related mycobacterium, m. tuberculosis 58 and leishmania spp. 59 indeed, pan-cf vaccinated (prechallenged) mice exhibited polyfunctional cd8 + t cells (ifn-γ+, il-2+, tnfα+) but showed superior protection (significantly lower bacterial burden) compared with both nonvaccinated (all organs) and mycopar (only spleen) vaccinated groups. we hypothesize that protection can be attributed to a robust polyfunctional cd8 + t cell response, especially for the nanovaccine formulas. recently, nano-peptide based adjuvant enhanced bcg primed immune response by induction of a robust polyfunctional cd8 + t cells. 60 overall, the presented analyses (figs 3, 4) clearly indicated robust induction of polyfunctional t cell responses in mice immunized by the nanovaccines as depicted by higher triple and double cytokine producing cd8 + t cells when compared with non-vaccinated (pbs) and mycopar®-vaccinated mice. it is noteworthy to mention here that for m. tuberculosis vaccines, polyfunctional t cells were mainly cd4 + unlike the predominantly cd8 + population observed in animals vaccinated with the pan-cf group. this is likely induced by the inclusion of polyanhydride nanoparticles in the pan-cf group, consistent with previous observations. 61, 62 the nanoparticle chemistry used in this study (i.e., 20:80 cpteg: cph) was rationally selected based on previous reports showing its potency as an adjuvant as exhibited by robust induction of cellular immune responses. 35 similarly, in this study pan-cf vaccinated mice at pre-challenge not only induced a polyfunctional cd8 + t cell response but also had more breadth as indicated by induction of more types of cytokine secreting cells. the robust polyfunctional t cell response observed in pan-cf vaccinated animals for 12 wpc may be attributed to sustained release of antigen from polyanhydride particles. 29 remarkably, pan-cf + lysate was able to produce double cytokine (ifn-γ + il-2 + ) cd4 + t cells significantly higher than pbs and mycopar® and exhibited lower bacterial burden in all the mice tissues as compared with non-vaccinated mice (fig. 6 ). most importantly, pan-cf vaccinated mice had the lowest bacterial burden in three out of the four tissues evaluated. moreover, despite this significant reduction was not maintained at 18 wpc, the level of polyfunctional t cells remained robust at this prolonged time. this indicates that several other paramters (beyond the scope of present study), such as th-17 induction, tissue homing properties of t cells, memory and effector phenotype could also play critical roles. 63, 64 these parameters deserve more attention, especially as we advance vaccine testing to ruminant models. as expected, histological lesions post-challenge correlated with protection based on bacterial burden in body organs. for example, granulomatous lesions that are typical of mycobacterial infection were seen in the liver of pbs and mycopar®-vaccinated animals in higher frequency than in the lipn or pan-vaccinated animals ( table 1) , consistent with the levels of mycobacterial colonization. similar to other vaccine candidates (lipn mutant), pan-based vaccines were able to significantly lower m. paratuberculosis levels in the liver, spleen and lymph nodes but did not prevent dissemination of infection. approaches focused on developing significant mucosal immunity at the main site of m. paratuberculosis entry (intestine) could definitely reduce organ dissemination of the infection. overall, the nanovaccines and lipn mutant were able to impart superior protective imunity against m. paratuberculosis challenge in mice compared with a commercial vaccine, mycopar®. further, in contrast to mycopar®, nanovaccines were also well tolerated and did not induce any adverse reactions at the site of injection. both these features makes pan nanovaccines ideal for further testing in larger animals such as goats and cattle following mucosal immunization and oral infections, to memic natural infection. although promising, nanovaccines have room for improvement before becoming suitable for field applications. for example, methods to improve the encapsulation efficiency of complex protein mixtures like whole cell lysate into pan are highly desirable and if developed, could improve vaccine production and facilitate immunization. such strategies could open the door for the development of more effective vaccines targeting other intracellular pathogens such as m. tuberculosis. for safety experiments, m. paratuberculosis -k10 was used to prepare lysate while for efficacy experiments, m. paratuberculosis strain jtc-1285 was used to derive lysate and culture-filtrate (cf) proteins for vaccine formulation. both isolates belong to c-type of m. paratuberculosis with almost identical genomes. 43 the same strains were used for the animal challenge studies, as described below. the strains were grown in middlebrooks 7h9 broth (difco, sparks, md) supplemented with 0.5% glycerol (v/v) and 10% (v/v) adc (albumin, dextrose, catalase) or tween 80 and 1 mg/ml mycobactin j (allied monitor, fayette, mo) in a shaking incubator at 37°c until it reached the log phase (od 600 = 0.5-1.0). 43 the m. paratuberculosis field isolate, jtc-1285, was sub-cultured in watson reid medium, 65 filter (nalgene). further, it was size fractionated by ultrafiltration (corning ultra spin columns, 5000 mwco) and the filtered volume retained on the membrane was dialyzed twice against 10 mm phosphate buffered saline (pbs) (ph 7.2). the concentrated culture filtrate proteins were quantified using bicinchoninic acid kit (pierce) and stored at −20°c. to obtain the lysate, the bacterial cell pellet was resuspended in protein lysis buffer (100 mm tris-cl, 100 mm nacl, 5 mm mgcl 2 , 1 mm pmsf, complete ultra protease inhibitor cocktail, ph 7.5) and placed in microcentrifuge tubes containing 0.1-mm zirconia/glass beads. tubes were shaken in the mini bead-beater cell disrupter for four 45 s pulses followed by 1-min incubation on ice. cellular debris and beads were pelleted by centrifugation at 3200 × g for 20 min. the supernatant was quantified for protein using bicinchoninic acid kit (thermo fisher scientific, rockford, il) and stored at −20°c. diacids of 1,8-bis(p-carboxyphenoxy)-3,6-dioxaoctane (cpteg) and 1,6-bis (p-carboxyphenoxy)hexane (cph) were synthesized as described in detail. 67, 68 next, melt polycondensation was used to synthesize 20:80 cpteg:cph copolymer. the purity and molecular weight of the copolymer were verified using 1 h nuclear magnetic resonance spectroscopy (vxr 300 mhz, varian, palo alto, ca) before proceeding to nanoparticle synthesis. nanoparticles were synthesized using solid-oil-oil double emulsion nanoprecipitation. 31 briefly, m. paratuberculosis lysate and culture filtrate proteins were dialyzed to nanopure water by using 5k mwco spin-x® uf concentrators (corning, corning, ny) and lyophilized overnight. the 20:80 cpteg:cph polymer (20 mg/ml) containing 2.5 wt% proteins was dissolved in methylene chloride. the solution was sonicated for 30 s to ensure uniform distribution of the protein throughout the solution. particles were precipitated by pouring the solution into chilled pentane (1:250 methylene chloride:pentane) and collected via vacuum filtration. nanoparticle size and morphology were characterized via scanning electron microscopy (fei quanta 250, fei, hillsboro, or). the encapsulation efficiency was determined by incubating the nanoparticles in pbs at 37°c. the released protein was quantified via a micro-bicinchoninic assay (pierce) and compared with the amount of protein theoretically encapsulated. the final nanovaccine formulation was a combination of free protein and nanoparticle-encapsulated protein. a total of 1 mg of nanoparticles encapsulating 25 µg protein was suspended in 100 μl pbs containing 75 µg free protein per mouse. the mixture was sonicated for 30 s on ice to disperse any nanoparticle aggregates prior to administration. female c57bl/6 mice (5-8 weeks of age) were obtained from taconic inc. and maintained in bio-safety level-2 containment. all procedures were in compliance with institutional animal care and use committee, university of wisconsin, madison. experiments were run in two trials ( table 2 , n = 15/ group) with trials i and ii comprising of four and six vaccine groups, respectively. animals were vaccinated subcutaneously as per the experimental design shown in table 2 . in all experiments, five mice from each group were sacrificed at 6 weeks post-vaccination. the remaining 10 animals in each group were challenged with 10 8 cfu of m. paratuberculosis jtc-1285 in 100 μl of pbs, injected intraperitoneally (ip). the subcutaneous vaccination regime and intraperitoneal challenge model has been successfully employed before in mouse models and the resulting data translated well to ruminant systems such as in goats. 42, 69, 70 the dose was confirmed by plating the serially diluted challenge inoculums on 7h10 plates. mice were monitored daily for adverse reaction(s) from vaccination and for the progression of infection. at 6 weeks (trial i), 12 weeks (trials i and ii) and 18 weeks (trial ii) post-challenge, mice (n = 5) were sacrificed and the liver, spleen, small intestine and mesenteric lymph nodes were harvested from each sacrificed mouse in order to quantify the bacterial burden. organs were homogenized in 1 ml pbs and undiluted and 10-fold serial diluted samples were plated onto antibiotic free and selective media (hygromycin 30 μg/ml) to differentiate between lipn and challenge strain. when the selective media were not used, organs were plated onto standard 7h10 plates supplemented with adc, mycobactin-j, and vancomycin (5 mg/ml), amphotericin b (30 mg/ml), and nalidixic acid (10 mg/ml) to reduce bacterial and fungal contamination. finally, tissue sections were collected for histopathology and stained with hematoxylin and eosin. 41 slides were scored by a trained pathologist blinded to the samples. the animal experimental design is shown in fig. 1 . spleens from five animals/group were aseptically harvested and placed in rpmi (corning, manassas, va) supplemented with 1% fbs (atlanta biological, lawrenceville, ga), 1% l-glutamine (gibco, grand island, ny), 1% penicillin-streptomycin (mediatech, inc. manassas, va) and 1% nonessential amino acids (gibco). spleens were pressed against the wire mesh screens to isolate splenocytes. the cells were washed with rpmi and resuspended in 1-2 ml of rbc lysis buffer (tris buffered ammonium chloride) for 1 min, washed, and resuspended in rpmi with 10% fbs. cells were counted using trypan blue dye to assess viability. a total of 10 6 cells/ well were seeded into 96-well round bottom plates and stimulated with 10 µg/ml of whole cell lysate of m. paratuberculosis k10 (trial i) or m. paratuberculosis jtc-1285 (trial ii) and 100 u/ml il-2 (bd biosciences, san jose, ca). a control of unstimulated cells from each sample was also plated and treated with 100 μl of media and 100 u/ml il-2. plates were incubated for 18 h at 37°c, 5% co 2 followed by addition of golgi-plug to each well and incubated for an additional 5 h. cells were harvested by centrifugation and stained with immune markers to be analyzed by flow cytometry and the supernatant was used to detect ifn-γ by elisa. supernatant from the stimulated splenocytes was collected and tested for ifn-γ levels using mouse ifn-γ elisa max tm deluxe kit (biolegend, san diego, ca) following the manufacturer's instructions. in brief, 96-well plates (maxisorp-immuno plates; nunc) were coated overnight with capture antibody (monoclonal capture antibody specific for mouse ifn-γ) at 1:200 dilutions in the coating buffer at 4°c. plates were washed with pbst (137 mm nacl, 2.7 mm kcl, 10.15 mm na 2 hpo 4 , 1.76 mm kh 2 po 4 , ph 7.4, 0.05% (v/v) tween 20), blocked with 200 μl of assay diluent, and incubated on a shaking plate for 1 h at room temperature. plates were washed five times with the pbst and 100 μl of sample and standards were added to the appropriate wells and incubated for 2 h on the shaking plate. plates were washed with pbst and 100 μl of detection antibody (biotinylated rat monoclonal anti-mouse antibody) was added and incubated for 1 h at room temperature. after three more washes, 100 μl of avidin-horse radish peroxidase conjugated solution was added to each well and incubated for 30 min on the shaking incubator. after a last few washes, 100 μl of freshly made 3', 3, 5, 5' -tetramethylbenzidine (tmb) substrate solution was added to the wells and incubated for 20 min in the dark. the reaction was stopped by adding 100 μl of stop solution (1 n h 2 so 4 ). the plates were read at a wavelength of 450 nm and analyzed with softmax pro software (molecular devices, sunnyvale, ca). splenocytes from five individual mice per group were counted and 1 × 10 6 cells/well were plated in 96-well plates. stimulated cells were harvested by centrifuging at 400 × g for 10 min at 4°c. supernatants were removed and a. thukral et al. cells were washed with pbs twice. fixable viability dye efluor 780 (ebioscience, san diego, ca) was diluted in pbs (1/10) and added to each well except unstained control, incubated for 30 min in dark at 2-8°c and cd16/cd32 receptors were blocked with fc block (bd pharmingen, san diego, ca). cell surfaces were stained with cocktail of buv496 conjugated anti-cd4 antibody, clone gk 1.5 (bd pharmingen); buv396 conjugated anti-cd8, clone 53-6.7 (bd pharmingen); bv711 conjugated anti-cd25, clone pc61 (biolegend) and incubated for 30 min in dark at 4°c. cells were washed with cold facs buffer twice and resuspended in 200 μl of fixation/permeabilization working solution (foxp3 staining buffer set, ebioscience, san diego, ca). after 1 h incubation, cells were washed with 1x permeabilization buffer and stained intracellularly with apc conjugated anti-ifn-γ, clone xmg1.2 (bd pharmingen); pe conjugated anti-il-2 (bd pharmingen); pecy7 conjugated anti-tnfα, clone mp6-xt22 (ebioscience); and alexa fluor 780 conjugated anti-foxp3, clone fjk16s (ebioscience). cells were analyzed using bd facscalibur and data were analyzed using flowjo software (flowjo, llc, ashland, or). results were expressed as the increase in the percentage of the cells with positive staining relative to that of an unstimulated sample stained with the same antibody. statistical analysis was performed using graphpad prism (la jolla, ca). data were analyzed using one-way anova followed by tukey's multiple comparison. results with p < 0.05 or better were considered significant. all research reported here was conducted in accordance with all relevant guidelines and procedures, and that the work was approved by the university of wisconsin-madison. further information on research design is available in the nature research reporting summary linked to this article. effect of paratuberculosis on culling, milk production, and milk quality in dairy herds herd-level prevalence of mycobacterium avium subsp. paratuberculosis infection in united states dairy herds in 2007 loss of income from cows shedding mycobacterium avium subspecies paratuberculosis prior to calving compared with cows not shedding the organism on two minnesota dairy farms herd-level economic losses associated with johne's disease on us dairy operations economic impact of paratuberculosis in dairy cattle herds: a review the effect of paratuberculosis on milk yield-a systematic review and meta-analysis evalution of the "iceberg phenomenon" in johne's disease through mathematical modelling ruminant paratuberculosis-a century of progress and frustration a rational framework for evaluating the next generation of vaccines against mycobacterium avium subspecies paratuberculosis immune reactions in cattle after immunization with a mycobacterium paratuberculosis vaccine and implications for the diagnosis of m. paratuberculosis and m. bovis infections efficacy of commercial and field-strain mycobacterium paratuberculosis vaccinations with recombinant il-12 in a bovine experimental infection model the effects of injection of bovine vaccine into a human digit: a case report accidental self-inoculation with mycobacterium paratuberculosis bacterin (johne's bacterin) by veterinarians in wisconsin multifunctional th1 cells define a correlate of vaccinemediated protection against leishmania major multifunctional, high-level cytokine-producing th1 cells in the lung, but not spleen, correlate with protection against mycobacterium tuberculosis aerosol challenge in mice rational design of pathogen-mimicking amphiphilic materials as nanoadjuvants polyanhydride nanovaccine platform enhances antigenspecific cytotoxic t cell responses hemagglutinin-based polyanhydride nanovaccines against h5n1 influenza elicit protective virus neutralizing titers and cell-mediated immunity a systems approach to designing next generation vaccines: combining α-galactose modified antigens with nanoparticle platforms design of a protective single-dose intranasal nanoparticlebased vaccine platform for respiratory infectious diseases retention of structure, antigenicity, and biological function of pneumococcal surface protein a (pspa) released from polyanhydride nanoparticles combination nanovaccine demonstrates synergistic enhancement in efficacy against influenza amphiphilic polyanhydrides for protein stabilization and release structural and antigenic stability of h5n1 hemagglutinin trimer upon release from polyanhydride nanoparticles carbohydrate-functionalized nanovaccines preserve hiv-1 antigen stability and activate antigen presenting cells encapsulation into amphiphilic polyanhydride microparticles stabilizes yersinia pestis antigens amphiphilic polyanhydride nanoparticles stabilize bacillus anthracis protective antigen pulmonary biodistribution and cellular uptake of intranasally administered monodisperse particles lung deposition and cellular uptake behavior of pathogenmimicking nanovaccines in the first 48 h effect of nanovaccine chemistry on humoral immune response kinetics and maturation polymer chemistry influences monocytic uptake of polyanhydride nanospheres the simultaneous effect of polymer chemistry and device geometry on the in vitro activation of murine dendritic cells single dose vaccine based on biodegradable polyanhydride microspheres can modulate immune response mechanism biodegradable particles as vaccine antigen delivery systems for stimulating cellular immune responses hemagglutinin-based polyanhydride nanovaccines against h5n1 influenza elicit protective virus neutralizing titers and cell-mediated immunity evaluation of biocompatibility and administration site reactogenicity of polyanhydride-particle-based platform for vaccine delivery safety and biocompatibility of carbohydratefunctionalized polyanhydride nanoparticles intranasal delivery of whole cell lysate of mycobacterium tuberculosis induces protective immune responses to a modified live porcine reproductive and respiratory syndrome virus vaccine in pigs immunogenic membrane-associated proteins of mycobacterium tuberculosis revealed by proteomics defining the stressome of mycobacterium avium subsp. paratuberculosis in vitro and in naturally infected cows superior protection elicited by liveattenuated vaccines in the murine model of paratuberculosis superior protection from live-attenuated vaccines directed against johne's disease genome-wide sequence variations among mycobacterium avium subspecies paratuberculosis isolates: a better understanding of johne's disease transmission dynamics cost-benefit analysis of vaccination against mycobacterium avium ssp. paratuberculosis in dairy cattle, given its cross-reactivity with tuberculosis tests a rational framework for evaluating the next generation of vaccines against mycobacterium avium subspecies paratuberculosis effect of subcutaneous administration of a killed mycobacterium avium subsp paratuberculosis vaccine on colonization of tissues following oral exposure to the organism in calves live mycobacterium avium subsp. paratuberculosis and a killed-bacterium vaccine induce distinct subcutaneous granulomas, with unique cellular and cytokine profiles nanoparticle chemistry and functionalization differentially regulates dendritic cell-nanoparticle interactions and triggers dendritic cell maturation paratuberculosis vaccination high throughput cell-based screening of biodegradable polyanhydride libraries early immune markers associated with mycobacterium avium subsp. paratuberculosis (map) infection in a neonatal calf model experimental challenge models for johne's disease: a review and proposed international guidelines immunoregulatory cytokines are associated with protection from immunopathology following mycobacterium avium subspecies paratuberculosis infection in red deer protection against mycobacterium tuberculosis infection offered by a new multistage subunit vaccine correlates with increased number of ifn-γ+ il-2+ cd4+ and ifn-γ+ cd8+ t cells an essential role for interferon gamma in resistance to mycobacterium tuberculosis infection structural deficiencies in granuloma formation in tnf genetargeted mice underlie the heightened susceptibility to aerosol mycobacterium tuberculosis infection, which is not compensated for by lymphotoxin rhull-2 adjunctive therapy in multidrug resistant tuberculosis: a comparison of two treatment regimens and placebo polyfunctional cd4(+) t cells as targets for tuberculosis vaccination tumor necrosis factor-alpha in combination with interferon-gamma, but not with interleukin 4 activates murine macrophages for elimination of leishmania major amastigotes nanoscale peptide self-assemblies boost bcg-primed cellular immunity against mycobacterium tuberculosis the effect of polyanhydride chemistry in particle-based cancer vaccines on the magnitude of the anti-tumor immune response polyanhydride nanovaccine induces robust pulmonary b and t cell immunity and confers protection against homologous and heterologous influenza a virus infections evaluation of a mycobacterium avium subsp paratuberculosis leud mutant as a vaccine candidate against challenge in a caprine model attenuated strains of mycobacterium avium subspecies paratuberculosis as vaccine candidates against johne's disease growth and metabolic characteristics of mycobacterium paratuberculosis tuberculin johnin and mallein derived from non-protein media. can synthesis and characterization of novel polyanhydrides with tailored erosion mechanisms poly[1,3-bis(p-carboxyphenoxy)-propane anhydride evaluation of novel oral vaccine candidates and validation of a caprine model of johne's disease. front evaluation of eight live attenuated vaccine candidates for protection against challenge with virulent mycobacterium avium subspecies paratuberculosis in mice all data presented in this paper are available through this report or the accompanied supplemental tables and figures.received: 14 february 2018; accepted: 27 january 2020; a.m.t. perceived the original idea and supervised the whole project. b.n. is the inventor of the p.a.n. delivery system and supervised the nanovaccine synthesis and charcaterization. a.t., k.r., c.h. and y.p. conducted all of the experiments while h.s. was responsible for the histology analysis. all authors contributed equally to the writing and editing of the paper. dr. adel m. talaat has an ownership interest in pan genome systems, inc, which is working in the area of animal vaccine development. also, dr. yashdeep phanse is currently employed by the same company. supplementary information is available for this paper at https://doi.org/10.1038/ s41541-020-0164-y.correspondence and requests for materials should be addressed to a.m.t. publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.open access this article is licensed under a creative commons attribution 4.0 international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons. org/licenses/by/4.0/. key: cord-002937-7xauocti authors: huang, chung-guei; lee, li-ang; wu, yi-cheng; hsiao, mei-jen; horng, jim-tong; kuo, rei-lin; huang, chih-heng; lin, ya-chu; tsao, kuo-chien; chen, min-chi; chen, tse-ching; shih, shin-ru title: a pilot study on primary cultures of human respiratory tract epithelial cells to predict patients’ responses to h7n9 infection date: 2018-02-20 journal: oncotarget doi: 10.18632/oncotarget.24537 sha: doc_id: 2937 cord_uid: 7xauocti avian influenza a(h7n9) virus infections frequently lead to acute respiratory distress syndrome and death in humans. we aimed to investigate whether primary cultures of human respiratory tract epithelial cells are helpful to understand h7n9 virus pathogenesis and tissue tropism, and to evaluate how patient-related characteristics can affect the host's response to infection. normal human bronchial epithelial cells (isolated from two different donors) and primary epithelial cells (harvested from 27 patients undergoing airway surgery) were experimentally infected with h7n9 and/or h1n1pdm for 72 h. after virus infection, the culture media were collected for viral rna quantitation and cytokine detection. both h7n9 and h1n1pdm viruses replicated and induced a cytokine response differently for each donor in the normal human bronchial epithelial model. h7n9 replicated equivalently in epithelial cells harvested from the inferior turbinate and paranasal sinus, and those from the larynx and bronchus, at 72 h post-infection. viral rna quantity at 72 h was significantly higher in patients aged 21–64 years than in patients aged ≥ 65 years; however, no effects of sex, medical comorbidities, and obesity were noted. h7n9-infected cultured cells released multiple cytokines within 72 h. levels of interleukin-1β, interleukin-6, interleukin-8, interferon-γ, and tumor necrosis factor-α were associated differently with patient-related characteristics (such as age, sex, obesity, and medical comorbidities). in the era of precision medicine, these findings illustrate the potential utility of this primary culture approach to predict a host's response to h7n9 infection or to future infection by newly emerging viral infections, and to dissect viral pathogenesis. avian influenza a(h7n9) virus infections are a serious public health threat. h7n9 infections were initially reported in china in march 2013 [1] . the clinical presentation of h7n9 infections varies with the individual; however, fever and cough generally represent the core symptoms [2] . serious complications include pneumonia, acute respiratory distress syndrome, and death. although the first outbreak has subsided, four subsequent seasonal epidemics were observed in china [3] . the severe illness of h7n9 infections and the presence of natural reservoirs represent significant concerns. in this context, the identification of vulnerable subjects is paramount to the prevention of spread. h7n9 transmission generally occurs from poultry to humans, and the closure of live poultry markets has been an effective control strategy [4] . however, evidence also indicates that: 1) h7n9 viruses are transmissible from ferrets in a direct contact setting [5] [6] [7] ; 2) they can bind to both human and avian receptors [8] ; 3) they can attach to the epithelium in both the upper and lower respiratory tracts of humans [9] ; and 4) they efficiently propagate in human alveolar tissue [10] . all of these features are important factors for human-to-human transmission. fortunately, h7n9 viruses do not appear to transmit easily from person to person. however, there are many more factors that are important in the adaptation to sustained inter-human transmission. in this scenario, primary cultures of human respiratory tract epithelial cells would be invaluable to understand h7n9 virus tissue tropism and pathogenesis, as well as to evaluate how patient-related characteristics can modulate the host's response to infection. notably, old age (≥ 65 years), male sex, medical comorbidities, and obesity have been previously identified as risk factors for the development of severe disease [2, [11] [12] [13] [14] [15] ]. an age-and sex-based analysis of 63 patients infected by the h7n9 virus showed that elderly men are most commonly affected, whereas no deaths were observed in elderly women. age-dependent changes in the airway epithelium mean that older patients may be more prone to respiratory tract infections [16] and the development of symptomatic h7n9 disease [11] . moreover, the presence of comorbidities is the only independent risk factor for acute respiratory distress syndrome in h7n9-infected individuals [2] . in the most recent epidemic episode in china (during september to december 2016 (n = 114)), distributions of age, sex, and severe illness remained unchanged compared with other epidemics [3] . nevertheless, the mechanisms by which age, sex, medical comorbidity, and obesity can influence h7n9 virus replication kinetics remain unclear. however, numerous studies have shown that increased levels of specific proinflammatory cytokines and chemokines are robust predictors of morbidity and mortality in h7n9infected patients [2, 8, [17] [18] [19] [20] . h1n1pdm virus is a swine-origin influenza virus that can transmit efficiently among humans. although the clinical severity of h1n1pdm was milder than that of avian h7n9 virus, there were many severe h1n1pdm patients because of the widespread transmission of the virus in the human population in 2009 [21] . compared with h7n9 virus infections, young age (≤ 29 years) and female sex were identified as risk factors for severe h1n1pdm infection [15] . respiratory epithelial cell culture systems have been used to investigate the interaction between influenza viruses, as well as other viruses, and their host, [8, [22] [23] [24] [25] . while these studies have recognized many important characteristics that can help to understand the pathogenesis of influenza viruses, few studies have investigated personto-person differences in virus replication and the influenzarelated cytokines and chemokines of respiratory epithelial cell cultures. recently, mindaye et al. [26] found different protein expression levels of pro-viral and antiviral factors among normal human bronchial epithelial (nhbe) cells isolated from three different donors, and suggested that this model may provide a way to identify individuals or population groups who are susceptible to severe influenza disease. in the present study, we addressed the hypothesis that respiratory epithelial cells from different human donors would vary in their response to influenza virus infections. to determine the impact of age on cellular response after influenza virus infection, commercial nhbe cells cultured from 24-year-old and 69-year-old donors were infected with both h1n1pdm virus (a/california/07/2009 [27] ) and avian h7n9 virus (a/taiwan/4-cgmh/2014 [28] ), and viral growth kinetics and the cytokine response were compared. we further explored how different donors' characteristics (i.e., age, sex, medical comorbidities, and obesity) could influence both virus replication kinetics and the cytokine response to experimental h7n9 infections. human respiratory tract primary epithelial cells were cultured from patients undergoing upper or lower airway surgery and experimentally infected with h7n9. the limited amount of primary epithelial cells available from each patient meant that only h7n9 virus infection was conducted. in consideration of biosafety issues, the viral rna quantity was determined to represent changes in viral titer at each time point. figure 1a demonstrates that the viral www.impactjournals.com/oncotarget figure 1 : changes in viral rna quantity (panel a) and core cytokine levels (panels b-g) in influenza virus infection can induce a cytokine storm, and tumor necrosis factor (tnf), interferon (ifn), interleukin (il)-1, il-6, and monocyte chemotactic protein-1 are key cytokine storm mediators [29] . an earlyonset cytokine storm is associated with h7n9-related mortality [18, 20] ; therefore, we specifically focused on the six cytokines that were found to most consistently predict death in previous studies (i.e., il-1β, il-6, il-8, ifn-γ, interferon gamma-induced protein 10 (ip-10), and tnf-α). such molecules are also produced by human airway epithelial cells [30] [31] [32] [33] [34] , and are termed "core cytokines" in the present study. in cases in which the cytokine concentration was undetectable, we assumed a value of 0.1 pg/ml for statistical purposes, in accordance with the recommendation of guo et al. [20] . figure 1b shows that the il-1β levels in the culture media of h7n9 virus-infected 24-year-old nhbe cells were significantly higher than those of the mock and h1n1pdm virusinfected 24-year-old nhbe cells at 48 and 72 h p.i. the il-1β levels of the h7n9 virus-infected 69-yearold nhbe cells were significantly higher than those of mock and h1n1pdm virus-infected 69-year-old nhbe cells at 48 h p.i. figure 1c reveals that the il-6 levels in the culture media were not related to virus type or the donor's age. in figure 1d , the il-8 level of the culture medium of h7n9-infected 24-year-old nhbe cells was significantly higher than those of the mock and h1n1pdm infected cells. figure 1e shows that the ifn-γ level of h7n9-infected nhbe cells (either 24-year-old or 69-yearold) was significantly higher than those of the mock and h1n1pdm infected cells. figure 1f demonstrates that levels of ip-10 in h7n9-infected 69-year-old nhbe cells were significantly higher than the others at 24, 48, and 72 h p.i., whereas those of h7n9 and h1n1pdm virus-infected 24-year-old nhbe culture media were significantly higher than that of mock at 72 h p.i. figure 1g demonstrates that the levels of tnf-α of h7n9 virus-infected nhbe cells obtained from 24-year-old and 69-year-old donors were significantly higher than the others at 72 h p.i. airway tissue specimens were collected from 27 patients ( table 2 ). there were 19 males (70%) and eight females (30%), with a mean age of 45.6 ± 15.7 years. patients were further dichotomized into "aged 21-64 years (n = 23 [85%])" and "aged ≥ 65 years (n = 4 [15%])" groups. seven (26%) patients had at least one medical comorbidity (hypertension [n = 6], diabetes mellitus [n = 2] and asthma [n = 2]); and nine (33%) were obese (body mass index ≥ 27 kg/m 2 [35] ). the anatomical sites from which the primary epithelial cell cultures were obtained were as follows: inferior turbinate (n = 14 [52%]), paranasal sinus (n = 4 [15%]), larynx (n = 1 [4%]), and bronchus (n = 8 [30%]). there were relatively fewer numbers of paranasal sinus-and larynxderived cultures; therefore, we combined the inferior turbinate and paranasal sinus-derived cultures as "upper anatomical location" (n = 18 [67%]) and larynx-and bronchus-derived cultures as "lower anatomical location" (n = 9 [33%]) for statistical evaluation. spearman's rank correlation coefficients revealed that a lower anatomical location was significantly inversely associated with male sex (r = -0.75, p < 0.001). age ≥ 65 years, medical comorbidity, and obesity were not significantly associated with the other patient characteristics (all p > 0.05). the epithelial origin (≥ 95%) of cultured cells was confirmed by immunofluorescence staining using an anti-cytokeratin 19 antibody. when all cultures were considered in combination, viral rna quantities increased significantly at both 24 and 48 h p.i. compared with the baseline, followed by a plateau between 48 and 72 h after infection (figure 2a ), as suggested by generalized estimating equations (gees), with or without adjustment for anatomical location, age, sex, medical comorbidities, and obesity. gees can account for possible correlations in repeated measures over time and are suitable to explore the differences among different time www.impactjournals.com/oncotarget points [36] . the percentage change in viral rna quantity between 24 and 72 h was also significant (table 3 ). with regard to virus tropism, viral rna quantities were significantly higher in epithelial cells obtained from the upper anatomical locations than from the lower anatomical locations, without adjustment (p = 0.030); however, the difference lost significance after adjustment for age, sex, medical comorbidities, and obesity (p = 0.490; figure 2b ). viral rna quantities in cells explanted from patients aged ≥ 65 years were significantly lower than those measured in patients aged 21-64 years, with or without adjustment for anatomical location, sex, medical comorbidities, and obesity (unadjusted p = 0.016, adjusted p = 0.019; figure 2c ). in contrast, no significant sexrelated differences in viral rna quantities were evident ( figure 2d ). the viral rna quantities in primary epithelial cells from patients with or without medical comorbidities did not differ significantly ( figure 2e) . similarly, the impact of obesity on viral rna quantities was not evident ( figure 2f ). the results of the cytokine analysis showed that the concentrations of all six cytokines in virus-infected culture supernatants increased significantly from 24 to 72 h p.i. ( table 3 ). the largest increase was observed for ip-10. ip-10 mediates both necrotic inflammation [37] and lung injury [38] ; therefore, our primary culture model supports the hypothesis that a cytokine storm could mediate airway tissue necrosis during the early stages of h7n9 infection. viral rna quantities were positively correlated with the concentrations of ip-10 and tnf-α at both 24 and 72 h p.i. (table 4) . notably, the concentrations of il-6, which were previously reported to be increased in the plasma of h7n9-infected patients during the first week p.i. [20] , were not significantly associated with viral rna quantities using this primary culture model. the associations between the observed increases in viral rna quantities over time (between 24 and 72 h p.i.) and core cytokine levels were then analyzed in relation to patient-related characteristics (table 5) . obesity was positively correlated with increases in viral rna finally, we graphically summarized the changes in core cytokine levels over time in relation to the anatomical sites of the explant and patient-related characteristics. at both 24 and 72 h p.i., the levels of il-8 and ifn-γ in virusinfected culture supernatants were significantly higher in the upper anatomical location group than in the lower anatomical location group, after adjustment for patientrelated characteristics ( figure 3a ). the levels of il-1β and il-8 at 24 and 72 h p.i. were significantly higher in patients aged 21-64 years, whereas the levels of il-6 were significantly higher in patients aged ≥ 65 years ( figure 3b ). male sex was significantly associated with higher levels of il-1β, il-6, il-8, and ifn-γ at 24 h p.i., as well as il-6, and ifn-γ at 72 h p.i. (figure 3c ). the presence of medical comorbidities was not significantly associated with cytokine levels at 24 and 72 h p.i.; however, changes in the il-8 level were significantly associated with medical comorbidities after adjustment for anatomical location and other patient-related characteristics ( figure 3d ). except for a change in il-8 level from 24 to 72 h p.i., obesity was not significantly associated with cytokine levels after adjustment, ( figure 3e ). in line with the observations obtained in mouse and ferret models [5] [6] [7] 39] , the h7n9 virus is not only able to penetrate the human respiratory epithelia [9] , but also can successfully replicate in ex vivo tissues. however, neither the mouse nor the ferret model can mimic the severity of h7n9 infection in humans. in the present study, our objectives were to discover whether viral replication and cytokine responses of nhbe cells from two donors of different ages are distinct after h7n9 and h1n1pdm virus infection, and to validate whether these changes are different after h7n9 virus infection using primary epithelial cells from the respiratory tracts of 27 donors with various patient-related characteristics. in the second model, we used a primary culture approach to possibly retain the effects of patient-related characteristics. a standard operation procedure was used to culture all primary epithelial cells to maintain comparable culture conditions. we found that a donor's age might have an effect on viral rna quantities (h7n9 and h1n1pdm) and cytokine levels (il-1β, il-8, ifn-γ, ip-10, and tnf-α) of the commercial nhbe culture supernatants, and certain patient-related characteristics could modulate viral replication and the cytokine response (e.g. age ≥ 65 years could decrease viral rna quantity and the levels of il-1β and il-8, and increase the il-6 level; male sex increased the levels of il-1β, il-6, il-8, and ifn-γ; medical comorbidity increased the il-8 level; and obesity abbreviations: fdr, false discovery rate; ifn, interferon; il, interleukin; ip-10, ifn-γ-induced protein; tnf, tumor necrosis factor. a indicates a statistically significant fdr when the benjamini and hochberg approach for multiple testing correction was applied. increased the il-8 level at 24 h p.i., and decreased the il-8 level at 72 h p.i., increased the changes in viral rna quantity, and decreased the changes in il-1β and ip-10; figure 4 ) for h7n9 infection in human respiratory tract primary epithelial cells. in addition, we also found that viral replication was equivalent in cells obtained from the upper and lower anatomical locations. specifically, our data suggested that the level of il-8 is markedly dependent on age, sex, medical comorbidities, and obesity. in general, the levels of several cytokines were found to increase significantly from 24 to 72 h p.i. in nhbe and primary epithelial cell models (table 1 & table 3 ). explant and patients' age, sex, medical comorbidities, and obesity (panels a-e) . a p < 0.05, b p < 0.01, and c p < 0.001, generalized estimating equations in which age, sex, medical comorbidity, obesity, and/or anatomical location were included as confounding variables. among them, il-1β and ifn-γ, widely recognized as early markers of h7n9 infection [8, 18, 20] , are involved in the modulation of the inflammatory response [40] and apoptosis [41] . high systemic levels of ifn-γ and il-8 are associated with neutrophil activation [42] and can predict the need for inpatient care in h7n9-infected patients [43] . one of the main findings of this study was that the h7n9 virus showed equivalent and efficient replication in epithelial cells derived from both the upper and lower anatomical locations. this finding indicates that avian influenza a(h7n9) virus a/taiwan/4/2014 [28] , similar to avian influenza a(h7n9) virus a/anhui/1/13 [9] , can bind to both α2, 6-linked and α2, 3-linked sialic acids of the upper and lower respiratory tract epithelial cells. intriguingly, infections of the upper airways were associated with a marked release of il-8, ifn-γ, and tnf-α within 72 h p.i. these observations suggested that the h7n9 virus infection induces a site-specific, amplified, local innate immune response [44] . notably, chan et al. [22] found that h7n9 virus infection did not enhance the mrna expression of il-8 in peripheral blood monocyte-derived macrophages. these findings suggested that il-8 released by h7n9-infected epithelial cells of the respiratory tract could be an important clue to the development of severe disease. we demonstrated that the h7n9 virus replicated more slowly and induced lower levels of il-1β and il-8, accompanied by a more pronounced increase in il-6 levels, in cell cultures obtained from patients aged > 65 years compared with those from patients aged 21-64 years. these observations may be attributable to "immunosenescence," i.e. an age-related decline in immune function accompanied by the dysfunction of chemokine signaling pathways [45] . in contrast, younger subjects are more likely to survive after h7n9 infection [2, [10] [11] [12] [13] [14] . it is possible that the high viral replication and 2) the individual cytokine response. the h7n9 virus can replicate equivalently in the epithelial cells obtained from the upper and lower anatomical locations, which may be negatively affected by age (≥ 65 years). after h7n9 infection, the anatomical location and patientrelated characteristics are significantly positively, negatively, or miscellaneously associated with elevated levels of certain core cytokines. occurring in their respiratory tract can act as a reservoir [46, 47] . by contrast, advanced age and increased il-6 levels have been shown to be significant predictors of mortality [2, 8, [10] [11] [12] [13] [14] 18] . male sex was significantly associated with higher levels of il-1β, il-6, il-8, and ifn-γ between 24 and 72 h p.i. these findings might explain why men are at a higher risk of more severe disease. in our study, cells from patients with medical comorbidities produced higher il-8 levels after experimental infection compared with those from otherwise healthy individuals, which might explain the absence of an association between medical comorbidities and the severity of h7n9 infections after adjustment for age and sex [14] . obesity was significantly associated with a higher increase in viral rna quantities; a higher level of il-8 at 24 h and a lower level at 72 h p.i.; and less pronounced increases in il-1β and ip-10. our data indicated that cells derived from obese individuals have subtle but widespread changes in viral replication and cytokine response following experimental h7n9 infection. furthermore, adiposity has been shown to be a critical factor in the age-associated lethal cytokine storm in an animal model [48] ; nevertheless, the corresponding clinical effect might be modest. some limitations of our study merit comment. first, we used a primary epithelial culture model. consequently, we cannot exclude the possibility that our findings related to h7n9 viral replication and its related cytokine response might be different in vivo. second, because of the limited amount of primary epithelial cells, we did not include control virus infections in the primary culture model of explanted cells to verify the viral response. however, we confirmed that the h7n9 virus could replicate and induce a cytokine response differently from h1n1pdm virus in the nhbe experiment. moreover, avian influenza a(h7n9) virus a/anhui/1/13 could attach moderately or abundantly to both the upper and lower respiratory tract, and could be replicated in primary cultures of the nasopharynx and bronchus [9] . nevertheless, the primary epithelial cells used in the present study seemed to reflect the properties of virus infection in patients, where the virus interacts with primary epithelial cells. finally, the relatively small sample size precluded the analysis of a greater number of host characteristics that could affect virus replication kinetics and cytokine response. further studies are needed to confirm and expand our findings. in conclusion, we describe primary epithelial cellular models that might be useful to clarify the influence of patient-related characteristics on h7n9 viral replication and the associated cytokine response. interestingly, il-1β, il-6, il-8, ifn-γ, and tnf-α were the most differentially influenced by grouped host-specific features. our results require independent replication in clinical studies before the widespread use of our primary cellular model can be recommended. however, our platform is less costly and labor-intensive than the use of animal models to mimic h7n9 virus infection. our preliminary results using a primary cellular model have revealed many interesting differences among patient responses, and might represent a potential tool to identify individuals more prone to developing severe h7n9 infections. ethical approval for human tissue explants and the collection of demographic and clinical data was granted by the institutional review board at the chang gung medical foundation, taoyuan, taiwan. (102-4819b). all the procedures described in the study complied with the principles of the declaration of helsinki and the standards of good clinical practice. written informed consent was obtained from all participants. all experimental procedures were conducted in biosafety level 2 facilities by personnel wearing biosafety level 3 personal protective equipment. between march 1, 2014 and april 30, 2016, we enrolled patients (n = 27) who were scheduled for upper or lower airway surgery at the linkou chang gung memorial hospital (taoyuan city, taiwan). inclusion criteria were as follows: 1) age > 20 years; 2) need for upper airway surgery (for chronic hypertrophic rhinitis, chronic rhinosinusitis, or laryngeal cancer) or lung surgery (for lung cancer; with no prior history of radiation or chemotherapy); and 3) the existence of clinical records detailing the presence of medical comorbidities. patients were excluded if they experienced respiratory tract viral infections within the two weeks preceding surgery, because some influenza viruses (such as h1n1) can trigger hypercytokinemia in the early stage and this effect can last for up to two weeks [20, 49] . patients were also excluded if they had a known history of chronic viral infections (e.g., human immunodeficiency virus, hepatitis b virus, or hepatitis c virus), and/or were unwilling to participate. (24yearold and 69-year-old female caucasians) were purchased from lonza (lonza walkersville, inc., walkersville, md, usa) and maintained in bronchial epithelial growth media (lonza walkersville, inc., walkersville, md, usa). cells at the third passage were grown into a confluent monolayer for subsequent infection experiments. airway tissue specimens were collected from the following anatomical sites: 1) inferior turbinate, 2) paranasal sinus, 3) larynx, and 4) bronchus. immediately after harvesting, tissues were submerged in appropriate volumes of ice-cold earl's balanced salt solution to preserve cell viability. epithelial cells were expanded in parallel using standard methods [23] [24] [25] . after rinsing and removal of excess tissue, specimens were minced into cubes (2-3-mm 3 ) and transferred onto a six-well transwell cell culture system. each transwell was coated with collagen (30 μg/ml), fibronectin (10 μg/ml), and fetal bovine serum (10 μg/ml) and allowed to adhere. culture medium (dulbecco's modified eagle's medium/ ham f12, 1% antibiotic/antimycotic, 1% fetal bovine serum) was added basolaterally to the co-culture dishes, and the plates were then incubated at 37 °c, with 5% co 2 in a humidified atmosphere. the culture solution was changed every two days. when the epithelial cells grew around the explant to cover a 1-2 cm 2 area (approximately 2-3 weeks), tissue explants were transferred onto new scratched and coated plates. when confluent monolayers were observed, epithelial cells in the transwells were infected with the h7n9 virus. for fixation, 4% paraformaldehyde was applied to the cell slides at room temperature for 15 min, followed by three washes with phosphate-buffered saline (pbs). cells were permeabilized with pbs containing 0.3% triton x-100 (sigma-aldrich co. llc, saint louis, mo, usa) twice for 15 min at 4 °c. the cells were then incubated with 2% bovine serum albumin (bionovas biotechnology co., ltd., toronto, ontario, canada) in pbs for 30 min. slides were then incubated at 37 °c for 1 h with anti-cytokeratin 19 antibodies (abcam plc., cambridge, uk) diluted 1:100 with blocking solution (final concentration 10 μg/ml), followed by three washes with pbs. fluorescein isothiocyanateconjugated affinipure goat anti-mouse igg + igm (h+l; jackson immunoresearch laboratories inc., west grove, pa, usa), diluted 1:100 to a final concentration of 15 μg/ ml, was applied as the secondary antibody and incubated at 37 °c for 30 min, followed by three washes with pbs. for nuclear staining, slides were incubated with 4′, 6-diamidino-2-phenylindole (life technologies, grand island, ny, usa) for 15 min at room temperature, followed by three washes with pbs. finally, the slide was dipped into pbs containing evans blue for background counterstaining to show the localization of the cells. we used influenza a/taiwan/4-cgmh/2014 (tw4/ h7n9) [28] and a/california/07/2009 (h1n1pdm) [27] in this study. all of the h7n9 infection-related procedures were conducted in biosafety level 3 facilities. a/taiwan/4-cgmh/2014 is genetically close to the a/anhui/1/2013 strain [28] . the viral rna quantities were determined by a plaque assay on madin-darby canine kidney epithelial cells. experimental infections of human respiratory tract primary epithelial cells (collected from different anatomical sites, as outlined above) were performed in triplicate. cells (1 × 10 5 cells/well) were initially exposed to serum-free bronchial epithelial cell basal medium (lonza walkersville, inc.) containing 0.25% trypsin, and subsequently infected with the h7n9 virus at a multiplicity of infection of 0.01 at 37 °c, 5% co 2 . mock-infected cells served as a negative control. after infection, cells were washed five times with hanks' balanced salt solution to remove unbound viruses. at each predetermined time-point (see "variables of interest"), virus-infected culture supernatants were collected and stored at −80°c until immediately before analysis. all procedures were conducted in biosafety level 2 facilities by personnel wearing biosafety level 3 personal protective equipment. quantitative real-time reverse transcription polymerase chain reaction (qrt-pcr) was used for h7n9 virus load quantitation. viral rna was extracted from culture supernatants using a qiaamp viral rna mini kit (qiagen inc., valencia, ca, usa) and subjected to qrt-pcr. we used the flua primers and probe originally developed by the world health organization collaborating centre in beijing, china (2013). the nucleotide sequences were as follows: forward primer, 5′-gaccratcctgtcacctctgac-3′; reverse primer, 5′-agggcattytggacaaakcgtcta-3′; probe, 5′-fam-tgcagtcctcgctcactgggcacg-bhq1-3′. in brief, 140 μl of supernatant was mixed with 560 μl of lysis buffer (350 μl for up to 5 × 10 6 cells). the volume of extracted rna was adjusted to 30 μl with rnase-free water. negative controls consisted of sterile water and were subjected to all the preparation steps in parallel with the extracted samples. a realtime ready rna virus master kit (roche diagnostics, gmbh mannheim, germany) was used for amplification. to set up the amplification reaction, 5 μl of template was added to each reaction tube; the working concentrations for the primers and probe were 10 and 5 μm, respectively. the final volume for the qrt-pcr was 25 μl. reverse transcription and amplification were carried out in a onestep reaction on a bio-rad pcr system (cfx96; bio-rad laboratories, hercules, ca, usa). the conditions for qrt-pcr were as follows: 45 °c for 10 min, 95 °c for 10 min; followed by 40 cycles at 95 °c for 15 s and 60 °c for 45 s. the qrt-pcr was repeated for samples with a ct value > 38. pcr products were cloned into a plasmid to generate positive controls. for quantification, plasmid dnas at six different concentrations, from 10 copies/μl to 10 6 copies/μl, were run in parallel with all the samples. the concentrations of six core cytokines in virusinfected culture supernatants were determined using the bio-plex pro human cytokine 27-plex panel (bio-rad laboratories). specifically, the following analytes were included for statistical analysis: il-1β, il-6, il-8, ifn-γ, ip-10, and tnf-α. samples were initially incubated on antibody-coupled beads for 60 min to allow binding, followed by incubation with detection antibodies for 30 min. conjugates were treated with streptavidin for 10 min, washed on a bio-plex pro ii wash station, resuspended, vortexed, and quantified by fluorescence. all data were analyzed using a bio-rad bio-plex luminex 200 instrument. standard curves (log (x) -linear (y)) were generated with the bio-rad bio-plex manager v6.0 software. triplicate measurements of all samples were performed. viral rna quantities and cytokine levels in the h7n9-infected culture supernatants at 24 h and 72 h p.i. served as the main variables of interest. the differences in viral rna quantities and cytokine levels at 24 h p.i. were expected to be less significant than those at 72 h p.i.; therefore, a more conservative power analysis was performed, based on the variables of interest at 24 h p.i. the priori sample size of the model of primary cultures of human respiratory tract epithelial cells was estimated using outcome effects (viral rna quantities and cytokine levels) based on a pilot study of the model of nhbe cells (mean 4.95 [standard deviation 0.94] and mean 3.61 [standard deviation 0.34] for "aged 24 years" and "aged 69 years", respectively), and a twotailed mann-whitney u test. to reach 90% power with an effect size of 1.9, a type i error of 0.05, and an allocation ratio 0.2, we obtained a minimum sample size of 26 (22 and four patients for "aged 21-64 years" and "aged ≥ 65 years", respectively) for comparison of ip-10 levels at 24 h p.i. viral rna quantities and cytokine levels were log-transformed (log 10 ) to improve the normality of the distribution. to avoid gaps in the assay results and inaccuracies in the estimates of concentrations, a value of 0.1 pg/ml was entered in the data set when an analyte was undetectable [20] . means and standard errors of the mean were used to summarize continuous variables. the viral rna quantities and cytokines were compared using the wilcoxon signed rank test, the kruskal-wallis test, or the mann-whitney u test, as appropriate. at selected time-points, viral rna quantities and cytokine levels were compared according to both the anatomical locations of the explant and patient-related characteristics of interest using gee models [36] , which were employed to determine whether changes in virus and cytokine levels were significantly different at specific time-points after adjusting for anatomical location and patient-related characteristics, or between different anatomical locations and patient-related characteristics after adjustment for other confounding variables. the use of gee allowed the monitoring of viral rna quantities and cytokine level as a function of time, anatomical location, age, sex, medical comorbidities, and obesity. the anatomical locations of the explants were dichotomized for the purpose of analysis in the upper (inferior turbinate and paranasal sinus) versus lower (larynx and bronchus) respiratory tract. spearman's rank correlation coefficients were used to analyze the associations between patient-related characteristics, viral rna quantities, and cytokine levels. we applied benjamini and hochberg's approach to control the false discovery rate. all statistical calculations were performed with the g * power 3.1.9.2 software (heinrich-heine university, dusseldorf, germany), the ibm spss (version 23; international business machines corp.), and the graphpad prism for windows (version 7.0; graphpad software, inc.) software packages. a p value < 0.05 (twotailed) was considered statistically significant. the study was approved by the institutional review board at the chang gung medical foundation, taoyuan, taiwan. (102-4819b, 104-9560c). human infection with a novel avian-origin influenza a (h7n9) virus clinical findings in 111 cases of influenza a (h7n9) virus infection sudden increase in human infection with avian influenza a(h7n9) virus in china effect of closure of live poultry markets on poultry-toperson transmission of avian influenza a h7n9 virus: an ecological study h7n9 influenza viruses are transmissible in ferrets by respiratory droplet the mouse and ferret models for studying the novel avian-origin human influenza a (h7n9) virus limited airborne transmission of h7n9 influenza a virus between ferrets biological features of novel avian influenza a (h7n9) virus novel avian-origin influenza a (h7n9) virus attaches to epithelium in both upper and lower respiratory tract of humans the novel human influenza a(h7n9) virus is naturally adapted to efficient growth in human lung tissue health organization outbreak response team. human infections with avian influenza a(h7n9) virus in china: preliminary assessments of the age and sex distribution clinical features and factors associated with outcomes of patients infected with a novel influenza a (h7n9) virus: a preliminary study risk factors for influenza a(h7n9) disease--china prognosis and survival of 128 patients with severe avian influenza a(h7n9) infection in zhejiang province age and gender adjusted comparison of clinical features between severe cases infected with h7n9 and h1n1pdm influenza a in jiangsu province mechanisms of dysfunction in senescent pulmonary endothelium host immunological response and factors associated with clinical outcome in patients with the novel influenza a h7n9 infection early hypercytokinemia is associated with interferon-induced transmembrane protein-3 dysfunction and predictive of fatal h7n9 infection clinical, virological and immunological features from patients infected with re-emergent avian-origin human h7n9 influenza disease of varying severity in guangdong province the serum profile of hypercytokinemia factors identified in h7n9-infected patients can predict fatal outcomes the infection attack rate and severity of 2009 pandemic h1n1 influenza in hong kong tropism and innate host responses of a novel avian influenza a h7n9 virus: an analysis of ex-vivo and in-vitro cultures of the human respiratory tract primary human bronchial epithelial cells grown from explants molecular modeling, organ culture and reverse genetics for a newly identified human rhinovirus c relative respiratory syncytial virus cytopathogenesis in upper and lower respiratory tract epithelium impact of influenza a virus infection on the proteomes of human bronchoepithelial cells from different donors phenotypic characteristics of novel swine-origin influenza a/california/07/2009 (h1n1) virus genomic signatures for avian h7n9 viruses adapting to humans into the eye of the cytokine storm effect of haemophilus influenzae endotoxin on the synthesis of il-6, il-8, tnf-alpha and expression of icam-1 in cultured human bronchial epithelial cells airway epithelial cells, cytokines, and pollutants macrophage inflammatory protein-1 human parainfluenza virus type 3 (hpiv3) induces production of ifngamma and rantes in human nasal epithelial cells (hnecs) human rhinovirus induced cytokine/ chemokine responses in human airway epithelial and immune cells obesity pandemic, correlated factors and guidelines to define, screen and manage obesity in taiwan longitudinal data analysis using generalized linear models cytokine and chemokine levels in patients infected with the novel avian influenza a (h7n9) virus in china characterization of cytokine/chemokine profiles of severe acute respiratory syndrome human h7n9 and h5n1 influenza viruses differ in induction of cytokines and tissue tropism circulating cytokines as mediators of fever role of apoptosis and cytokines in influenza virus morbidity a(h7n9) virus results in early induction of proinflammatory cytokine responses in both human lung epithelial and endothelial cells and shows increased human adaptation compared with avian h5n1 virus th1 and th17 hypercytokinemia as early host response signature in severe pandemic influenza influenza virus-induced lung injury: pathogenesis and implications for treatment immunosenescence-related transcriptomic and immunologic changes in older individuals following influenza vaccination how to interpret the transmissibility of novel influenza a(h7n9): an analysis of initial epidemiological data of human cases from china pathogenesis of influenza-induced acute respiratory distress syndrome adiposity induces lethal cytokine storm after systemic administration of stimulatory immunotherapy regimens in aged mice direct association between pharyngeal viral secretion and host cytokine response in severe pandemic influenza the authors thank mrs. hsin-ju wang and mr. jung-chin li for their expert technical assistance. www.impactjournals.com/oncotarget the authors declare no conflicts of interest. this study was financially supported by grants from the ministry of science and technology, taiwan, roc (103-2321-b-182-012 & 104-2321-b-182-004 to lal) and the chang gung medical foundation, taiwan, roc (cmrpg3g0341 to cgh; cmrpd1d0041, 1d0042, & 1d0043 to srs). key: cord-023372-ft8cp9op authors: rahman, q. k.; wikman, m.; vasconcelos, n.‐m.; berzins, k.; ståhl, s.; fernández, c. title: the immunomodulatory effect of heat shock protein 70: immunization with a dna construct based on the malarial antigen fused with a fragment of hsp 70 primes for a th‐1 type of response date: 2008-06-28 journal: scand j immunol doi: 10.1111/j.0300-9475.2004.01423aw.x sha: doc_id: 23372 cord_uid: ft8cp9op finding an appropriate adjuvant for human vaccination is crucial. heat shock proteins (hsps) act as adjuvants when coadministered with peptide antigens or given as fusion proteins. however, there is a potential risk of autoimmunity when using the complete molecules, because hsps are evolutionary conserved. to overcome this, we first evaluated the adjuvant effect against two different antigens of a less‐conserved fraction of plasmodium falciparum hsp70 (pf70c) and compared it to the whole hsp70 molecule from trypanosoma cruzi (tchsp70). we found that pf70c exhibited similar adjuvant properties as the whole molecule. we later evaluated the adjuvant potential of pf70c against the malarial antigen eb200 in a chimeric dna construct. no appreciable levels of eb200‐specific antibodies were detected in mice immunized only with the dna constructs. however, dna primed the immune system, because subsequent challenge with the corresponding recombinant fusion proteins elicited a strong th‐1 antibody response. in contrast, no priming effect was observed for ex vivo ifn‐γ production but stimulation with the hsp‐chimeric fusion protein induced a stronger secretion of ifn‐γin vitro than other proteins used. these results indicate that the use of hsps is promising in the design of new vaccines. the nuclear receptor heterodimers of liver x receptors (lxrs) are recently identified as key transcriptional regulators of genes involved in lipid homeostasis and inflammation. lxrs and their ligands are negative regulators of macrophage inflammatory gene expression. multiple sclerosis (ms), a demyelinating disease of the central nervous system of unknown cause, is characterized by recurrent inflammation involving macrophages and their inflammatory mediators. sweden belongs to the countries with a high ms incidence. in italy, incidence is lower, with an exception for sardinia where the incidence is even higher than that in sweden. subjects from sardinia are ethnically more homogeneous and differ from swedes, also regarding genetic background and environment. we studied lxrs and their related molecules of blood mononuclear cells (mncs) from female patients with untreated relapsing-remitting ms from sassari, sardinia and stockholm, sweden. sex-and age-matched healthy controls (hcs) were from both areas. mrna expression was evaluated by real-time pcr. lxr-a was lower (p < 0.05) in ms (mean ae sem: 3.1 ae 0.2; n ¼ 37) compared to hc (3.6 ae 0.1; n ¼ 37). lxr-a was lower in ms from stockholm (2.6 ae 0.2; n ¼ 22) compared to corresponding hc (3.4 ae 0.1; n ¼ 22; p < 0.01) and compared to ms (3.8 ae 0.2; n ¼ 15; p < 0.001) and hc (4 ae 0.2; n ¼ 15; p < 0.001) from sardinia. ms patients from stockholm, but not from sassari, also expressed lower (p < 0.05) lxr-b (à4.1 ae 0.4) compared to corresponding hc (à2.9 ae 0.3). ms from stockholm was associated with higher abca-1 (6.1 ae 0.4 versus 5.0 ae 0.3; p < 0.05) and higher estrogen receptor-b-cx (2.4 ae 0.4 versus 0.8 ae 0.4; p < 0.01) compared to corresponding hc. the hc from sassari had higher androgen receptor (2.9 ae 0.2) compared to ms from sassari (1.4 ae 0.3; p < 0.01), ms (1.3 ae 0.4; p < 0.01) and hc from stockholm (1.2 ae 0.3; p < 0.01). ms from sassari had lower cyclooxygenase-1 compared to corresponding hc (5.1 ae 0.4 versus 6.6 ae 0.3; p < 0.01) and lower prostaglandin-e (à0.03 ae 0.5) compared to the hc (1.4 ae 0.5; p < 0.05) and ms (2.7 ae 0.4; p < 0.05) and hc from stockholm (1.9 ae 0.4, p < 0.001). our findings identify lxrs and their related molecules as being involved in ms from stockholm but not from sassari, while sex hormone receptors seem to be involved in ms in sassari. multiple sclerosis: ifn-b induces cd123 + bdca2 -dendritic cells that produce il-6 and il-10 and have no enhanced type i interferon production y. m. huang, 1 s. adikari, 1 u. båve, 2 a. sanna 1,3 & g. alm 4 dc antigens (bdca) and investigate their ability to produce type i ifn in response to virus stimulation. we show that ifn-b induces development of cd123 þ dc from human blood monocytes, which coexpress bdca4 þ but are negative for bdca2 -, a specific marker for plasmacytoid dc. such ifn-b-modulated dc produce large amounts of il-6 and il-10, but no il-12p40 and have no enhanced ifn-b and ifn-b production. the findings indicate that ifn-bmodulated dc represent a myeloid dc subset with diminished cd11c, bdca-1 and cd1a expression, having potent th2-promoting function but lacking antiviral capacity. the association of psoriasis with throat infections by streptococcus pyogenes suggests a potential antigenic target for the t cells that are known to infiltrate dermis and epidermis of psoriatic skin. streptococcal m protein shares an extensive sequence homology with human epidermal keratins. keratins 16 (k16) and 17 (k17) are mostly absent from uninvolved skin but are upregulated in psoriatic lesions. there is increasing evidence that cd8 þ t cells play an important effector role in psoriasis and m proteinprimed t cells may recognize these shared epitopes in skin via molecular mimicry. to identify candidate epitopes, peptides with sequences from k17 were selected on the basis of predicted binding to hla-cw6 and sequence similarities with m6 protein. matched peptides from the sequence of m6 protein and a set of peptides with poor predicted binding were also selected. cw6 þ individuals with psoriasis and cw6 þ healthy controls, having a family history of psoriasis, were recruited. pbmcs were incubated with the peptide antigens. t-cell activation in the cd4 þ , cd8 þ and later the skin-homing cutaneous lymphocyteassociated antigen (cla)-expressing subset of cd8 þ t cells was evaluated by cd69 expression and intracellular ifn-g accumulation using flow cytometry. we demonstrate that cw6 þ psoriasis patients had significant cd8 þ t-cell ifn-g responses to peptides from k17 and m6 protein selected on the basis of sequence homology and predicted hla-cw*0602 binding. these responses were about 10 times more frequent in the skin-homing cutaneous lymphocyte-associated antigen-expressing (cla þ ) subset of cd8 þ t cells. cd4 þ t cells showed only borderline responses. cd8 þ t cells from cw6 þ nonpsoriatic individuals responded to some m6 peptides but very rarely to k17 peptides, and this also applied to the cla þ cd8 þ subset. these findings indicate that psoriatic individuals have cd8 þ t cells that recognize keratin self-antigens and that epitopes shared by streptococcal m protein and human keratin may be targets for the cd8 þ t cells that infiltrate psoriatic skin lesions. autoantibodies directed against citrulline-containing proteins have an impressive specificity of nearly 100% in ra patients and a suggestive involvement in the pathogenesis. the targeted epitopes are generated by a post-translational modification catalysed by the calcium-dependent enzyme peptidyl arginine deaminase that converts the positively charged arginine to polar but uncharged citrullin. the aim of this study was to analyse the presence of citrulline in the joints at different time points of collagen-induced arthritis in da rats by immunohistochemistry and to investigate how immunogenicity and arthritogenicity was affected by citrullination of rat serum albumin (rsa) and collagen type ii (cii). our results indicate that citrulline could be detected in joints of arthritic animals, first appearance at the onset of disease and increasing as disease progressed into a chronic state. unimmunized animals or time points before clinical signs of arthritis were negative. by morphology, we state that some infiltrating macrophages as well as the cartilage surface stain positive for citrulline, while the major source of citrullinated proteins appears to be fibrin depositions. a specific cit-rsa t-cell response was observed in animals challenged by citrullinated rsa, no response was recorded when rsa was used as a stimulus. the igg analysis reveals not only a response towards the modified protein but also cross-reactivity to native rsa. no t-cell or b-cell response was noted in animals injected with unmodified rsa. cit-cii induced a disease with higher incidence and earlier onset than did the native counterpart. we conclude that, in contrast to the human disease, citrulline does not seem to appear before clinical signs. as inflammation proceeds, citrulline is detected specifically in the joints. all other organs investigated were negative. we also conclude that citrullination of a protein can break tolerance and increase its arthritogenic properties. ectopic germinal centers (gcs) can be detected in the salivary glands of approximately 1/5 of patients with sjögren's syndrome (ss) and appear in both primary and secondary ss. previously, ectopic gc have been associated with increased local autoantibody production. the aim of this study was to determine whether gc in primary sjögren's syndrome (pss) defines a distinct seroimmunological phenotype. retrospectively, a material of 130 haematoxylin and eosin-stained paraffin-embedded tissue sections of minor salivary gland tissue from patients with pss was morphologically screened for the presence of ectopic gc. gc-like lesions were detected in 33/130 (25%) of the pss patients. seventy-two pss patients lacking these structures (gc-) were randomly selected for comparison. focus score was significantly increased in the gc þ patients compared to the gcpatients (p ¼ 0.035). in the gc þ group, 54.5% of the patients presented with anti-ro/ssa compared to 43.7% in the gcgroup. anti-la/ssb was detected in 31.3% of the gc þ patients compared to 25.7% of the gcpatients. sixty-one percentage of gc þ patients presented with increased levels of igg, a nonsignificant difference when compared to 39.4% in the gcpatients (p ¼ 0.089). levels of rf, ana, ena, igm and iga were similar in both patient groups, as were esr and crp. in conclusion, patients with ectopic gc have a higher focus score and more often present with autoantibodies and increased levels of igg compared to pss patients with regular focal infiltration (gc -). our findings may indicate a certain seroimmunological phenotype and warrant for further prospective studies. association between mannose-binding lectin and vascular complications in type 1 diabetes complement activation and inflammation have been suggested in the pathogenesis of diabetic vascular lesions. we investigated serum mannose-binding lectin (mbl) levels and polymorphisms in the mbl gene in type 1 diabetic (t1dm) patients with and without diabetic nephropathy and associated macrovascular complications. polymorphisms in the mbl gene and serum mbl levels were determined in 199 t1dm patients with overt nephropathy and 192 t1dm patients with persistent normoalbuminuria matched for age, sex and duration of diabetes as well as in 100 healthy control subjects. the frequencies of high and low expression mbl genotypes were similar in patients with t1dm and healthy controls. high mbl genotypes were significantly more frequent in diabetic patients with nephropathy than in the normoalbuminuric group, and the risk of having nephropathy, given a high mbl genotype, assessed by odds ratio was 1.52 (1.02-2.27), p ¼ 0.04. median serum mbl concentrations were significantly higher in patients with nephropathy than in patients with normoalbuminuria [2306 mg/l (iqr 753-4867 mg/l) versus 1491 mg/l (iqr 577-2944), p ¼ 0.0003], and even when comparing patients with identical genotypes, serum mbl levels were higher in the nephropathy group than in the normoalbuminuric group. patients with a history of cardiovascular disease had significantly elevated mbl levels independently of nephropathy status [3178 mg/l (iqr 636-5231 mg/l) versus 1741 mg/l (iqr 656-3149 mg/l), p ¼ 0.02]. the differences in mbl levels between patients with and without vascular complications were driven primarily by pronounced differences among carriers of high mbl genotypes (p < 0.0001). our findings suggest that mbl may be involved in the pathogenesis of microvascular and macrovascular complications in type 1 diabetes and that determination of mbl status might be used to identify patients at increased risk of developing these complications. neuroimmunology unit, center for molecular medicine, karolinska institutet, stockholm, sweden. e-mail: judit.wefer@cmm.ki.se dna vaccine coding for the encephalitogenic peptide mog 91-108 protects lew.1av1 from subsequent development of experimental autoimmune encephalomyelitis (eae). protection is associated with a type 1 immune response and is dependent on the presence of cpg dna motifs. the mechanisms underlying the observed reduction of eae development in protected rats have not been fully clarified. we investigated immunological characteristics of lymphocytes after dna vaccinaton and subsequent eae induction. we confirm that protection was not associated with suppression of t1 cells, as transcription of the novel molecule rat t-cell immunoglobulin-and mucindomain-containing molecule (tim-3), reported to be exclusively expressed on differentiated t1 cells, was not altered by dna vaccination. we did not note any clonal deletion upon tolerization, but detected an antigen-specific lymphocyte population upregulating ifng upon recall stimulation 3 weeks after protective dna vaccination. in protected rats, we observed (1) no alterations in antigenspecific th2 or th3 responses, (2) reduced mhc ii expression on splenocytes early after eae induction, (3) antigen-specific upregulation of ifnb upon recall stimulation and (4) reduced il-12rb2 on lymphocytes. we thus demonstrate an association of the protective effect of dna vaccination with expression of ifnb. we are currently investigating the cellular mechanisms behind this ifnb-mediated protection. multiple sclerosis (ms) is an autoimmune condition characterized by degeneration of nerve fibre myelin sheets. a candidate autoantigen, myelin basic protein (mbp), has especially attracted attention. the presence of anti-mbp antibodies is a predictor of definite ms, but their role in the pathogenesis remains obscure. t cells have long been known to play a pivotal role in the pathogenesis of ms. recently, an important role for b cells as autoantigen-presenting cells has been demonstrated in other autoimmune diseases, including rheumatoid arthritis and diabetes. the uptake of mbp by b cells and the presentation of mbp-derive peptides to t helper (th) cells by b cells may be promoted by the formation of complement (c) activating immune complexes (ics) between mbp and natural autoantibodies in healthy individuals and disease-associated anti-mbp antibodies in ms patients, respectively. we have investigated the formation of mbp-containing ic, the binding of mbp to b cells, the mbp-elicited induction of th-cell and b-cell proliferation and the cytokine production in peripheral blood mononuclear cells (pbmcs) from healthy donors grown in the presence of intact or c-inactivated serum from healthy donors or patients with ms. while mbp did not induce measurable proliferation of b cells nor cd4 þ t cells, we observed the production of tnf-a, ifn-g and il-10 by pbmc in response to incubation with mbp in the presence of sera from healthy controls as well as sera from ms patients. by contrast, no production of il-2, il-4 and il-5 was detected. we are currently investigating the capability of ms sera to promote the formation of mbp-containing ic and thereby enhance the cytokine responses, by virtue of elevated anti-mbp contents. the phagolysosomally localized acid sphingomyelinase (asmase) activated by proinflammatory cytokines such as tnf and ifn-g generates the signalling molecule ceramide which in turn results in the activation of proteases like cathepsin d. these characteristics of asmase suggest a possible role of this molecule in the phagocytotic uptake and phagosomal degradation processes of antigens or in antigen presentation. we show here that asmase -/mice fail to eliminate the noncytopathic lymphocytic choriomeningitis (lcm) virus as rapidly as littermate wildtype mice. investigation of the immune response revealed a reduced expansion of cd8 þ t cells. the secretion of ifn-g in response to contact with target cells as well as the cytolytic activity of virus-specific cd8 þ t cells was severely impaired. additionally, both phases of the lcm virus-specific dth response, mediated by cd8 þ and cd4 þ t cells consecutively, were diminished in asmase -/mice. however, the secondary memory response of virus-specific ctl was not altered, and the 614 abstracts ................................................................................................................................................................................................. virus was effectively controlled for at least 3 months by asmase -/mice. in conclusion, the results of this study suggest an involvement of the asmase in the activation, expansion or maturation of virus-specific cd8 þ t cells during the acute infection of mice with the lcm virus. novel markers for alternative activation of macrophages: macrophage galactose-type c-type lectins 1 and 2 in parallel with the th1/th2 dichotomy, macrophages are capable of developing into functionally and molecularly distinct subpopulations, due to differences in, for example cytokine environment and pathological conditions. while the best-studied, classically activated macrophage is induced by type i stimuli such as ifn-g, a type ii cytokine environment antagonizes the classical activation of macrophages and is capable of alternatively activating macrophages. however, molecular markers associated with these type ii cytokine-dependent, alternatively activated macrophages remain scarce. besides the earlier documented markers macrophage mannose receptor and arginase 1, we recently demonstrated that murine alternatively activated macrophages are characterized by increased expression of fizz1 and ym. we now report that expression of the two members of the mouse macrophage galactose-type c-type lectin gene family, termed mmgl1 and mmgl2, is induced in diverse populations of alternatively activated macrophages, including peritoneal macrophages elicited during infection with the protozoan trypanosoma brucei or the helminth taenia crassiceps, and alveolar macrophages elicited in a mouse model of allergic asthma. we also demonstrate that, in vitro, interleukin-4 and interleukin-13 upregulate mmgl1 and mmgl2 expression and that, in vivo, induction of mmgl1 and mmgl2 is dependent on interleukin-4 receptor signalling. moreover, we show that regulation of mgl expression is similar in human monocytes and monocyte-derived macrophages. hence, macrophage galactose-type c-type lectins represent novel markers for both murine and human alternatively activated macrophages; thus, paving the way for further characterization of the phenotype of macrophages occurring in th2 conditions. background: human parvovirus b19 (b19) is a ubiquitous pathogen, normally causing a mild self-limiting disease, but also capable of causing both significant pathology and long-term persistence. the small size and stability of the virus makes it suitable for mapping of the full breath and the kinetics of the cellular immune responses following acute viral infection. methods: five patients with acute primary b19 infection were included in the study and followed consecutively for up to 200 weeks. cellular immune responses were mapped by ifng enzyme-linked immunospot to overlapping peptides spanning the whole b19 genome. results: in all five acutely infected patients, we were able to monitor the kinetics of a strong specific cellular immune reaction. responses peaked at levels of 850-1850 sfc/ million pbmcs, roughly corresponding to 0.3-0.6% b19specific cd8 þ cells circulating in peripheral blood at 10-80 weeks post-infection. the responses in individual patients were directed to three or four different peptide pools, and the specificity was confined to the same cd8 epitopes present in the pools throughout the follow-up period. the majority of responses were directed to the virus nonstructural protein, only two patients showed any response to the capsid proteins, elicited by the same epitope in both cases. conclusion: the cellular immune responses to acute b19 infection are surprisingly narrow in distribution and remain at high levels for up to 80 weeks post-infection. the initial epitope specificity is maintained, and the majority of responses target the virus nonstructural protein, which is not included in vaccine preparations, evaluated against the infection. the relationship between malnutrition and malaria is controversial. on one hand, malaria may cause malnutrition, while on the other, malnutrition itself may modulate susceptibility to the disease. we investigated the association between plasmodium falciparum malaria and malnutrition in a cohort of children living on the coast of kenya. the study involved longitudinal follow-up for clinical malaria episodes and anthropometric measurements at four cross-sectional surveys. we used poisson regression analysis to investigate the association between malaria and nutritional status. compared to baseline (children with a waz or haz score of !à2), the crude incidence rate ratios (irrs) for malaria in children with low haz or waz scores (<à2) during the period prior to assessment were 1.17 (95% ci 0.91-1.50; 0 ¼ 0.21) and 0.94 (0.71-1.25; 0.67), respectively, suggesting no association between malaria and the subsequent development of pem. however, we found that age was acting as an effect modifier in the association between malaria and malnutrition. the irr for malaria in children 0-2 years old who were subsequently characterized as wasted was 1.65 (1.10-2.20; p ¼ 0.01), and a significant overall relationship between malaria and low-haz was found on regression analysis when adjusting for the interaction with age (irr 1.89; 1.01-3.53; p < 0.05). although children living on the coast of kenya continue to suffer clinical episodes of uncomplicated malaria throughout their first decade, the association between malaria and malnutrition appears to be limited to the first 2 years of life. a. astrinidou-vakaloudi, 1 s. xytsas, 1 i. diamanti, 1 h. ioannidis 2 & p. pangidis 2 1 microbiology department of general hospital of thessaloniki 'agios pavlos', thessaloniki, greece, and 2 nefrology, 2 nd ika hospital of thessaloniki, thessaloniki, greece. e-mail: stasa@hol.gr aim: renal dysfunction may influence the colonization of gastric mucosa by urea-splitting bacteria such as helicobacter pylori, by increasing urea concentrations in the gastric juice. our aim was to investigate the prevalence of h. pylori in patients with end-stage renal disease (esrd), receiving long-term haemodialysis treatment. methods: this study included 40 sera from patients with esrd (29 male and 11 female) undergoing periodic haemodialysis; mean time of treatment was 42.6 months. using elisa technique, we investigated the presence of igg and iga antibodies against h. pylori as well as igg caga (antibodies specific for caga(þ) strains of h. pylori). sera from 40 healthy blood donors were used as a control group. results: h. pylori igg antibodies were detected in 32 out of 40 (80%) patients in the dialysis group, while 31/40 (77.5%) tested positive for iga. igg caga antibodies were present in 13 out of 40 (32.5%). prevalence of h. pylori igg, iga and caga igg antibodies in the control group was 33, 7 and 15%, respectively. conclusions: although international data suggest that prevalence of h. pylori infection is the same in esrd patients as in healthy individuals, in our study that seems not to be the case. the higher blood and gastric juice urea levels may be a risk factor (among many others), but more studies are required in order to understand the relation of h. pylori infection in this group of patients. flanders interuniversity institute for biotechnology, department of molecular and cellular interactions, free university of brussels, brussels, and 2 pasteur institute of brussels, mycobacterial immunology, brussels, belgium. e-mail: tgartner@vub.ac.be immunity against tuberculosis (tb), caused by mycobacterium tuberculosis, depends largely on activation and maintenance of strong cell-mediated immune responses involving both cd4 þ and cd8 þ t cells and the ability to respond with th1-type cytokines, particularly ifn-g. recent studies suggested that bcg, the only licensed vaccine against m. tuberculosis, may fail to induce t-cell responses in the lung mucosa and may therefore not protect against pulmonary tb. a decrease in tb mortality may be achieved by enhancing immunity in the lung. the present study evaluated the induction of antigen-specific immunity in the lung by intranasal (i.n.) delivery of the lipoprotein i (opri) from pseudomonas aeruginosa. opri has shown to be a toll-like receptor 2/4 agonist that, when given subcutaneously, induces type-1 immune responses against heterologous antigens. here, a fusion of opri to ag85a of mtb (opri-ag85a) was used as a subunit vaccine in homologous prime-boost immunizations. in addition, opri-ag85a was combined with an ag85a-encoding dna vaccine (ag85a dna) or with bcg in heterologous prime-boost vaccinations. intranasal and parenteral delivery with opri-ag85a elicited comparable t-cell responses in the spleen; in addition, i.n. delivery elicited specific t-cell responses in the lung lymph nodes (llns). intramuscular delivery of ag85a dna induced significant systemic th1 immune responses. intranasal boosting with opri-ag85a enhanced this response and in addition induced an antigen-specific ifn-g response in lln. opri may therefore be an efficient adjuvant for mucosal boosting. we continue to evaluate the protection induced by opri-based prime-boost vaccinations against pulmonary tb. results on the immunogenicity and protection against intravenous mtb h37rv infection will be presented. toll-like receptors (tlrs) are pattern recognition receptors of the innate immune system, which recognize molecular structures on pathogens or cellular stress-associated molecules. tlr-ligand interactions trigger activation of inflammatory signal transduction and expression of genes involved in host defense. in this study, we have examined the requirement for different tlr adaptor molecules in virus-induced chemokine expression and are currently trying to identify the tlr involved. we have found that both a herpesvirus [herpes simplex virus (hsv)] and a paramyxovirus (sendai virus) require a functional genome to induce expression or proinflammatory chemokines in human and murine monocytic cell lines. for both viruses, this is independent of the tlr adaptor molecules trif and mal. however, overexpression of the vaccinia virus-encoded inhibitor of tlr-signalling a52r or dominant-negative myd88 totally inhibited hsv-induced rantes expression but only partially prevented sendai virus from inducing this chemokine. this suggests that hsv-induced rantes expression occurs via a tlr pathways, whereas sendai virus utilizes both tlr-dependent and -independent pathways to stimulate expression of rantes. we are currently trying to identify the tlrs involved. data from these studies will also be presented at the meeting. 2 0 -5 0 -oligoadenylate synthetases are interferon-induced, double-stranded rna-activated antiviral enzymes which are the only proteins known to catalyse 2 0 -specific nucleotidyl transfer. this first crystal structure of a 2 0 -5 0oligoadenylate synthetase reveals a structural conservation with the 3 0 -specific poly(a) polymerase that, coupled with structure-guided mutagenesis, supports a conserved catalytic mechanism for the 2 0 -and 3 0 -specific nucleotidyl transferases. comparison with structures of other superfamily members indicates that the donor substrates are bound by conserved active site features while the acceptor substrates are oriented by nonconserved regions. the 2 0 -5 0oligoadenylate synthetases are activated by viral doublestranded rna in infected cells and initiate a cellular response by synthesizing 2 0 -5 0 -oligoadenylates, that in turn activate rnase l. this crystal structure suggests that activation involves a domain-domain shift and identifies a putative dsrna activation site that is probed by mutagenesis. we demonstrated that this site is required both for the binding of dsrna and for the subsequent activation of oas. this rna-binding site is different from known rna-binding site; rather than forming a defined three-dimensional domain, it is located at the interface of the two major domains in oas. this novel architecture ensures that the dsrna helix can make simultaneously contact with both domains of oas and ensure the subsequent structural rearrangement leading to the activation of oas. our work provides structural insight into cellular recognition of double-stranded rna of viral origin and identifies a novel rna-binding motif. bacteria-specific iga antibodies are efficient opsonins for neutrophils and mononuclear phagocytes, provided that the phagocytes express the fca receptor (cd89). expression of cd89 can be stimulated by inflammatory cytokines, activated complement factors and certain microbial components. in one study, unstimulated phagocytes were able to ingest iga antibody-treated pneumococci, but only in the presence of complement, which was found to be activated by the iga antibodies along the alternative pathway. pneumococci produce iga1 protease that cleaves human iga1, but not iga2, molecules in the hinge region. this leaves iga1 as faba (monovalent) deprived of fca which contains the docking site for cd89. iga1 is the vastly predominant subclass of iga in the upper airways and circulation of humans. aims: to examine the effects of iga1 protease activity and complement on phagocytosis of iga antibody-coated pneumococci by an unstimulated human phagocytic cell line (hl60). materials and methods: iga1 and iga2 monoclonal antibodies to serotype 4 pneumococcal capsular polysaccharide (ps) were generated by heterohybridoma technique involving b cells from human vaccinees. isogenic serotype 4 pneumococci with and without iga1 protease activity, respectively, were obtained after inactivation of the iga gene of the tigr4 strain. opsonophagocytosis was quantitated using the assay described by romero-steiner et al. based on enumeration of surviving bacteria by culture. the integrity of iga molecules was examined by western blotting. results: both iga1 and iga2 antibody to type-4 polysaccharide-induced phagocytosis of iga1 protease-deficient type-4 pneumococci equally well in the absence as in the presence of complement. iga1 antibody to type-4 polysaccharide displayed a fourfold higher opsonophagocytosis titer against iga1 protease deficient compared to homologous wildtype target bacteria. a similar effect of iga1 protease activity of the target bacteria was not observed in a parallel experiment where iga2 antibody to type-4 polysaccharide served as opsonin. iga1 antibody extracted from iga1 protease-producing target bacteria was almost exclusively in the form of faba. conversely, iga1 from protease-deficient bacteria and iga2 from both types of bacteria were intact. conclusions: these results indicate that the iga1 protease activity of s. neumoniae may help the bacteria escape iga1 antibody-mediated opsonophagocytosis. besides, in these experiments, iga-mediated opsonophagocytosis was independent of complement. vitamins e and c have been found to increase the cellular and humeral immunity of pigs. vitamin e deficiency has also been found to predispose pigs to different diseases, e. coli infection is one among them. after weaning, the vitamin e status of pigs often decreases to a critical low level. in this experiment, we studied whether vitamin c supplementation would be a possible feeding strategy to optimize the immune status of weaners. the interaction between vitamin e and c is interesting due to the reported sparing action on vitamin e or synergism between these to vitamins. piglets were weaned at day 28 of age from sows fed increasing dietary vitamin e during lactation, and piglets were during the following 3 weeks fed either a control diet or this diet supplemented with 500 mg stay-c per kg. blood sampling was obtained weekly from day 28 and until day 49 of age. on the same days, one piglet per dietary treatment was killed and alveolar macrophages (am) were harvested. vitamin c supplementation increased the concentration of igm in serum of piglets throughout the weaning period. although the vitamin e concentration in am decreased with increasing age of the piglets, the concentration was numerically higher in piglets of sows fed the high dietary level of vitamin e. however, vitamin c supplementation tended to increase the total am concentration of vitamin e after weaning and increased the proportion of the biologically most active isomer of vitamin e [rrr-(a-tocopherol)] in the am. the eicosanoid synthesis by am was not influenced by the vitamin c supplementation, but the synthesis of leukotriene b4 was decreased 2 weeks after weaning compared to other days of am harvesting. in conclusion, dietary vitamin c supplementation improved the immune responses of piglets after weaning. a whole blood stimulation assay with escherichia coli (o111:b4) endotoxin was established to measure the capacity of dairy cows to produce the proinflammatory cytokine tumour necrosis factor-a (tnf-a) ex vivo. initially, a time-and dose-dependent study was carried out to find the optimal stimulation conditions for the tnf-a response. the tnf-a response peaked between 3 and 4 h at 38.5 c. a dose in the range of 5-10 g of e. coli lipopolysaccharide (lps)/ml whole blood was found to give the maximum tnf-a response. thirty-eight danish-holstein dairy cows were investigated for their tnf-a responsiveness ex vivo in the periparturient period. heparin-stabilized blood samples were collected seven times over a period of 4 months (weeks à3, à1, 2, 3, 5, 9 and 13 around calving) and stimulated with 5 g/ml of e. coli lps. indeed, fluctuations in the tnf-a responsiveness occurred over time. moreover, the mean tnf-a responsiveness of 38 cows was found to be significantly increased (p < 0.001) in the weeks close to calving. however, in the more stabile physiological periods, some cows had a consistently low tnf-a response, whereas others had high a tnf-a response. we are currently investigating whether high and low tnf-a responders to e. coli lps also exist in dairy cows in vivo. moreover, the importance of tnf-a responsiveness ex vivo to dairy cows' susceptibility and clinical response to experimental e. coli infections in the udder is being investigated. coelomic cytolytic factor (ccf) is a 42 kda invertebrate pattern recognition molecule isolated from the coelomic fluid of the earthworm eisenia foetida (oligochaeta, annelida). ccf displays a number of similarities with the mammalian cytokine tumour necrosis factor-a (tnfa) as a result of a shared n,n 0 -diacetylchitobiose lectin-like domain. however, these similarities are solely functional and are not based on any (dna or amino acid) sequence homology, thus suggesting a form of convergent evolution. in particular, the lectin-like domain of tnf-a has been shown to induce membrane depolarization in various mammalian cell types, through interactions with endogenous amiloride-sensitive ion channels. this nonreceptor-mediated activity of tnf-a has been reported to be involved in the resorption of oedema. likewise, the lectin-like domain of ccf also induces membrane depolarization in mammalian cells. here, we show that ccf appears to be able to induce oedema resorption in an alveolar epithelial cell line through its lectin-like domain. this lectin-like domain of ccf interacts (directly or indirectly) with endogenous sodium and/or chloride channels, and not potassium channels, on mammalian cells. additionally, we suggest that the jnk/sapk and erk1/2 pathways are involved in ccf-induced macrophage activation. these results further establish the functional analogy between an invertebrate pattern recognition molecule and a mammalian cytokine and, from a more applied point of view, suggest the possibility of utilizing ccf in the treatment of oedema. release of svegf and svegfr1 from white blood cells and platelets during surgery and stimulation with bacterial antigens introduction: the influence of surgery on release of soluble vascular endothelial growth factor (svegf) and the soluble vascular endothelial growth factor inhibitory receptor 1 (svegfr1) is unknown. we studied the effect of major and minor surgery on potential variations in svegf and svegfr1 concentrations in vivo and on bacterial antigen-induced release of svegf and svegfr1 from whole blood in vitro. methods: sixty-one patients with abdominal diseases undergoing five different surgical procedures were included. blood samples were drawn from anaesthetized patients before and after the operation. white blood cells and platelets were counted, and plasma svegf and svegfr1 was determined by an elisa method. whole blood from each blood sample was stimulated in vitro with bacteria-derived antigens (lps or protein-a) and svegf and svegfr1 levels were subsequently determined in the supernatants. stimulation with isotonic saline served as control assay. neither svegf or svegfr1 in plasma changed during surgery. in vitro stimulation of blood samples with bacteria-derived antigens resulted in a significant increase in svegf (p < 0.0001) and a less pronounced but still significant increase in svegfr1. release of svegf due to stimulation was significantly higher after the operation (nonsignificant), whereas svegfr1 release remained largely unchanged after surgery. correlation between bacterial antigen-induced release of svegf and neutrophile cell count was highly significant (p < 0.0001). there was no correlation between svegf and platelet cell count, and bacterial antigen-induced svegfr1 release did not correlate with counts of neutrophils and platelets. conclusions: plasma svegf and svegfr1 concentrations did not change during surgery. in vitro bacterial stimulation led to increased release of svegf and svegfr1, which was not significantly amplified during surgery and which may be related to number of circulating neutrophils. natural killer cell functions and subsets after in vitro stimulation with il-2 and il-12, with special emphasis on intracellular ifn-g and nk-cell cytotoxicity r. nyboe, 1,2 t. rix, 1,2 j. krog, 1,2 e. tønnesen 1 & m. hokland 2 1 department of anaesthesiology and intensive care, aarhus university hospital, and 2 institute of medical microbiology, and immunology, university of aarhus, aarhus, denmark. e-mail: rnsr@studmed.au.dk materials and methods: isolated cryopreserved human peripheral blood mononuclear cells (pbmcs) were stimulated with il-2 and il-12. this stimulation has previously been shown to activate nk cells. cell cytotoxicity was measured by flow cytometry after incubation with k562 cells. this method was compared to the current standard 51cr release assay. cells were treated with bfa to accumulate ifn-g, stained for surface markers, permeabilized and stained for intracellular ifn-g. flow cytometry was then performed to measure intracellular ifn-g production in pbmc, especially in nk cells. results: we have demonstrated that stimulation with il-2 and il-12 is effective in increasing the number of ifn-gpositive cells. there is a distinct difference between the cd3-cd56dim and the cd3-cd56bright subsets, with a much greater proportion of ifn-g-positive cells in the cd3-cd56bright subset. the effects of stimulation with il-2 and il-12 on cytotoxicity will be presented, as will the relation between ifn-g production and cytotoxicity. in addition, we will present results of these assays applied to porcine cells. discussion: in combination, these tests will address nk cell function by combining cytotoxicity with ifn-g production in nk cell subsets. the results will demonstrate whether this could serve as a useful tool in describing nkcell function, which could be of value in clinical and experimental settings. culture of regulatory t-cell lines from bronchial mucosa t lymphocytes play a major role in many immune responses. in the last decade, special focus has been on the function of th1 and th2 effector cells. now the importance of regulatory cd4 þ cd25 þ t cells in maintenance of the immunological homeostasis emerges. sarcoidosis is a multisystem granulomatous disorder often affecting the lungs. the typical sarcoid granulomas consists of epitheloid cells, macrophages and lymphocytes, mainly cd4 þ t cells of th1 phenotype. we have cultured t cells from bronchial biopsies of patients with sarcoidosis as well as from controls in high levels of interleukin 2 (il-2) and il-4 and demonstrate spontaneously arising cd4 þ cd25 þ populations and high concentrations of il-10 in these cultures. the main difference between cultures of sarcoid origin compared to controls is a very much higher concentration of the inflammatory cytokines il-6 and tnf-a in cultures of sarcoid origin. the effects of hyperbaric exposure on human peripheral blood mononuclear cells, with special emphasis on natural killer cell cytotoxicity and subsets materials and methods: as an experimental physiological stress model, we examined the effects of hyperbaric exposure on peripheral blood mononuclear cells (pbmcs) obtained from venous blood drawn from eight divers during a simulated heliox saturation dive. eight persons working in normobar atmosphere outside the pressurized chamber served as control donors. the spontaneous cytotoxicity of the pbmcs was estimated in a 4 h 51cr-release assay using k562 as nk-sensitive target cells. the pbmcs were characterized, using 4-colour flow cytometry, with special emphasis on the nk-cell subsets. the data were statistically analysed using a multivariate regression model (stata 8.2). p values <0.05 was considered statistically significant. results: the estimated cytotoxicity increased significantly in both the group of divers and control donors during the dive (pdivers < 0.01 and pcontrols < 0.01). although the cytotoxicity increased relatively more (p < 0.01) in the group of divers compared to the group of control donors between day 1 and 2. discussion: the increased cytotoxicity of pbmc estimated in the group of divers indicate that parts of the cellular immune system are affected during the extreme physiological conditions induced during the initial phase of the presented experimental hyperbaric setup. the increase in cytotoxicity observed in the group of control donors could hypothetically reflect the stress level in persons working outside the pressurized chamber during the dive. the interaction with adhesion molecules expressed by vascular endothelium is the first step in lymphocyte infiltration into tissues. at both cutaneous and mucosal sites interleukin-10 (il-10), il-12 and transforming growth factor (tgf)-b are important regulators of chronic inflammatory disease, where cutaneous lymphocyteassociated antigen (cla) and ae integrin (cd103) may be expressed. unlike cla, cd103 is not believed to play a role in tissue-specific homing but may help to retain t cells within epithelial layers. we have previously shown that il-12 alone can together with an unknown cofactor increase the expression of cla. stimulation with streptococcal pyrogenic exotoxin c (spec) increased the expression of cd103 by cd8 þ but not cd4 þ t cells. while il-12 increased superantigen-stimulated expression of cla, this cytokine strongly inhibited the cd103 expres-sion, and a combination of il-12 and tgf-b completely abrogated the induced cd103 expression. conversely, il-10 suppressed cla but increased cd103 expression. these findings indicate that, in addition to suppressing the development of th1-mediated inflammatory responses, il-10 may also inhibit the migration of cd8 þ t cells into the skin while il-12 promotes such migration. thus, the expression of cla and cd103 may be antagonistically regulated by il-10 and il-12, and the balance between these cytokines could influence the t-cell migration of inflammatory cells into epithelial tissues. during contact sensitivity reaction, immune cells proliferate. in order to study the histological picture of these proliferation phases, we used a mouse model of contact sensitivity in the oral mucosa and on skin. we also used bromodeoxyuridin (brdu, an analogue to thymidin) that is incorporated into the nucleus during cell replication. the hapten oxazolone (oxa) was used to sensitize and elicit the oral mucosa and/or the ear skin. mice were killed at various times after elicitation, and unsensitized animals were also exposed to the hapten as controls. brdu (25 mg/ kg animal) was injected i.p. 2 h before the kill. specimens from the oral mucosa, ear skin and submandibular and auricular lymph nodes were cut and fixed in 4% paraformaldehyde. they were then treated with acid and biotinylated anti-brdu antibody and developed using abc-kit and dab. the analyses were performed using a leica light microscope and the computer program analysis. in the oral mucosa, the frequency of proliferating cells were increasing during the observation period, 4-24 h after elicitation, regardless of site of sensitization. the proliferating cells were found mainly in the basal cell layer of the epithelium. similar patterns were found in ear skin. the regional lymph nodes demonstrated a few scattered proliferating cells 4 h after elicitation. after 24 h, these cells were found frequently in the whole lymph node. control animals exhibited considerable less proliferating cells at all times. we conclude that most proliferating cells were found 24 h after elicitation locally at the hapten-exposed sites (the oral mucosa or the ear skin) as well as in the regional lymph nodes. the endogenous nucleoside adenosine is released in excess during inflammation or other metabolic stress and is generally known to deliver tissue protective anti-inflammatory effects. adenosine acts via four adenosine receptors of which the a2a receptor is the predominant form in t cells. adenosine levels are elevated in asthmatic lung, and adenosine can directly induce mast cell degranulation and bronchoconstriction in these patients. instead, the role of anti-inflammatory mechanisms of adenosine on t cells in asthma is unclear. aim: to study the a2a receptor expression in peripheral blood cd4 þ t cells in asthmatic and healthy individuals using flow cytometric and quantitative real-time pcr methods. results: unstimulated cd4 þ cells of asthmatic patients expressed significantly lower levels (p < 0.001) of a2a receptor in protein level (mean percentage of cells positive ae sem: 76.8 ae 1.2, n ¼ 6) compared to healthy individuals (90.4% ae 1.9, n ¼ 4). double staining for cd69 expression showed that stimulation of cd4 þ cells decreased a2a expression in both groups but indicated that the detected lower levels of a2a in unstimulated cells of asthmatics was not due to preactivation in these patients. surprisingly, a2a mrna expression in unstimulated cd4 þ cells was significantly higher (p < 0.05) in asthmatics (n ¼ 28) compared to healthy controls (n ¼ 7). the expression did not correlate with serum total ige levels. conclusions: asthmatic individuals express less a2a adenosine receptor on their peripheral cd4 þ t cells. the higher mrna levels instead may point to a negative feedback regulation in the receptor expression. the role of possibly decreased adenosine-mediated anti-inflammatory effects in asthma pathogenesis require further studies on this t-cell mediated disease. the chronic inflammatory skin disease atopic eczema (ae) affects almost 15% of the population in many countries today. the pathogenesis of ae is not fully understood. a combination of genetic predisposition and environmental factors like microorganisms seems to contribute to the symptoms. the yeast malassezia sympodialis is part of our normal skin micro flora but can act as an allergen and elicit specific ige and t-cell reactivity in patients with ae. recently, we identified a novel major m. sympodialis allergen, designated mala s 11 (22.4 kda), with sequence similarity to the mitochondrial enzyme manganese superoxide dismutase (mnsod). interestingly, mala s 11 has a high degree of homology to human mnsod. the aim of this study was to examine the effects of recombinant mala s 11 on antigen-presenting dendritic cells. monocytederived dendritic cells (mddcs) from healthy blood donors were cultured with or without mala s 11 for different time periods. it was found that the maturation marker cd83 and the costimulatory molecules cd80 and cd86 were upregulated on the mddcs exposed to mala s 11 for 24 h, as demonstrated by flow cytometry. furthermore, coculture of mddcs with mala s 11 for 9 h induced an increased production of the inflammatory cytokines il-6 (200-fold), tnf-a (100-fold) and il-8 (sixfold), as detected by the cytometric bead array (cba) analysis. our results suggest that mala s 11 affects the immune response through dc maturation and production of inflammatory cytokines. the potential cross-reactivity with human mnsod needs to be explored and the exact role of mala s 11 in the pathogenesis of ae assessed in clinical studies involving skin prick and atopy patch tests. allergen-specific immunotherapy (sit) is commonly conducted with allergen extracts adsorbed to aluminium hydroxide (alum). drawbacks linked to the use of alum, such as the formation of granuloma at the site of injection, have led to suggestions of novel allergen carriers. an alternative carrier is 2 mm carbohydrate-based particles (cbps). in mouse, allergen-coupled cbps have been demonstrated to skew the allergen-specific immune response towards a th1-like activity (grönlund et al. immunology, 2002) . we here coupled the recombinant major cat allergen fel d 1 to cbps (cbp-fel d 1) by cyanogen-bromide activation, resulting in covalent binding. the effect of cbp-fel d 1 on monocyte-derived dendritic cells (mddcs) from healthy human blood donors was studied. we found that the majority of the cd1a þ mddcs were capable of taking up fitc-labelled cbp-fel d 1, as demonstrated by flow cytometry and confocal laser scanning microscopy. furthermore, incubation with cbp-fel d 1 resulted in an upregulation of the costimulatory molecule cd86 on the mddcs, which was not observed with fel d 1 or cbps alone. finally, cbp-fel d 1 induced a fivefold increase in the release of the pro-inflammatory cytokine tumour necrosis factor (tnf)-a and a fourfold increase in the release of the chemokine interleukin-8 from mddcs. taken together, the effects cbps possess make them interesting as novel allergen carriers for sit. the cysteine protease der p1 from dust mite of the genus dermatophagoides pteronyssinus is a major type i allergen. about 80% of house dust mite (hdm) allergic individuals are reactive to this protease in standard assays for detection of ige. a curative treatment for atopic allergy is immunotherapy (it) with hdm extracts which are complex mixtures occasionally resulting in anaphylactic reactions. novozymes focuses on developing a recombinant variant of der p1 which exhibit lowered risk of ige-mediated allergic reactions, while maintaining its ability to trigger proper th-cell responses. this may provide a safer alternative for specific it of hdm allergy. a secreted recombinant form of pro-der p 1 expressed by saccharamyces cerevisiae was obtained by fusion of the pro-enzyme to a fungal signal peptide. the n-glycosylation site of der p1 was mutated resulting in a deglycosylated pro-enzyme with a molecular mass of 35 kda. protein purification procedure was developed to obtain nearly pure der p1 protein followed by determination of concentration by active-site-titration with the cysteine protease inhibitor e64. the deglycosylated recombinant pro-der p 1 revealed immunologic similarity to the native der p 1 molecule when compared in basophile histamine release, ige-binding assays and t-cell proliferation assays. by in silico epitope mapping of a modelled 3-dimensional structure of der p1, five putative igg and ige epitopes were predicted. by protein engineering, the predicted epitopes were removed one by one in der p1 and screening for hypoallergenic variants was performed. combining inhaled long-acting b-2 agonist (laba) and inhaled corticosteroid (ics) seems to offer asthma control at a lower dose of ics than achieved by ics alone. fine mapping of t-cell surface markers by flow cytometry offers a detailed status of the individual's inflammatory response. the frequency of mt2 (cd4 þ cd45ra -cd62l þ cd11adim) and mt1 (cd4 þ cd45ra -cd62l -cd11abright) cells in peripheral blood, and their ratio, has been shown to differ predictably in atopics and patients with leprosy, where mt2 correlates with a th2 phenotype and mt1 with a th1 phenotype. stable asthmatics, requiring fluticasone propionate (fp) 750-1000 mg daily or equivalent, were randomized to receive, double-blinded, either seretide 1 [salmeterol and fluticasone propionate (sfc, n ¼ 16)] 50 mg/500 mg bd or fp 500 mg bd (n ¼ 17). if asthma was controlled based on lung function and symptoms at clinic visits every 6 weeks, ics dose was tapered until asthma exacerbated or 0 mg was reached. the frequency and ratio of mt2 and mt1 t cells of the patients was monitored at 6 week intervals. as treatment tapered, the frequency of mt2 cells decreased (p ¼ 0038 from first to final visit), whereas that of mt1 cells increased. the ratio of mt2/mt1 decreased (p ¼ 0049 from first to final visit). in patients receiving laba þ ics, the fall in mt2/mt1 ratio appeared to be more pronounced than in patients receiving ics alone. thus, the mt2 phenotype may be associated with stable asthma, whereas an imminent exacerbation may associate with an increase in the mt1 phenotype. laba may allow for a greater effect of fp on the mt ratio. activation of complement pathways, leading to production of c3a and c5a anaphylatoxins, has been postulated in the pathogenesis of asthma and allergic airway inflammation. the present study was undertaken to investigate the role of mannan-binding lectin (mbl), an initiator of the lectin pathway of complement, in asthma and allergic rhinitis. mbl levels and mbl-induced complement activity were determined in 19 patients of bronchial asthma with allergic rhinitis and 20 unrelated, age-matched controls of indian origin. mbl levels and activity were correlated with percent eosinophilia and percent predicted fev1 values of the patients. association of single nucleotide polymorphisms (snps) in exon 1 and intron 1 of the mbl with the disease, clinical markers, mbl levels and mbl-induced complement activity was analysed using standard statistical tools. significantly higher mbl levels and activity were observed in patients of bronchial asthma with allergic rhinitis as compared to the controls. we identified five snps, of which two, a816g in exon 1 and g1011a in intron 1 of the mbl, were novel. snp g1011a was significantly associated with the disease (p ¼ 0.0024, or ¼ 5.8696, 95% ci: 1.7316 < or < 19.8963). individuals with 'a' allele at position 1011 showed increased mbl levels, activity and disease severity. our results suggest that 'a' allele at position 1011 leading to high mbl levels and complement activity may be contributing to the severity of bronchial asthma and allergic airway inflammation. serum resistance of borrelia burgdorferi strains belonging to the b. afzelii and b. burgdorferi sensu stricto genospecies is dependent on binding of complement inhibitor factor h. we recently reported that factor h binding by b. burgdorferi is due to inducible expression of several approximately 20 kda plasmid-encoded, surface-exposed lipoproteins related to ospe (e.g. erpa, erpp and p21). in addition, a second class of factor h-binding proteins of approximately 27-35 kda has been described. the ospe-related lipoproteins are dramatically induced by b. burgdorferi during transmission from its tick vector into the mammalian host. the induction of ospe-related lipoproteins during mammalian infection may play a key a role in the borrelial evasion of the host's immune system. the goal of the present study was to define the factor h-binding regions of ospe-related proteins using mutagenesis, peptide mapping and surface plasmon resonance analysis (biacore). the combined studies revealed that the c-terminal regions of both human and mouse factor h (scrs 18-20) specifically bind to ospe-related lipoproteins. we also found fhr-1, whose c-terminal scrs 3-5 are homologous to scrs 18-20 of factor h, to bind to ospe. peptide mapping revealed five putative regions (designated i-v) in ospe that could directly interact with factor h. deleting the c-terminal 15 amino acid residues from region v of p21 abolished its ability to bind factor h. at the same time, however, synthetic peptides corresponding to the c-termini of ospe, p21 and erpp did not inhibit factor h binding to ospe. thus, the c-terminal-binding region v appears to be necessary but not sufficient for factor h binding. when a more specific mutation strategy was employed, where single amino acid residues in peptides spanning over the factor h-binding regions were mutated to alanines, we observed that lysines in the factor h-binding regions of ospe were required for factor h binding. the combined data have revealed that key lysine residues in ospe-related lipoproteins and ionic interactions are crucial for factor h interactions. furthermore, binding of ospe to the c-termini of both mouse and human factor h suggests that borrelia spirochetes utilize analogous complement resistance mechanisms in both rodents and man. in borrelia garinii strains, which in in vitro analyses have been found to be sensitive to complement killing, differences in the ospe sequences as well as in the expression of factor h-binding proteins may account for their susceptibility to serum lysis. role of yada, ail and lipopolysaccharide in serum resistance of yersinia enterocolitica serotype o:3 mannan-binding lectin (mbl), l-ficolin and h-ficolin are pattern recognition molecules of the innate immune system. we investigated the ability of these molecules to bind to different serotypes and noncapsulated variants of streptococcus pneumonia and staphylococcus aureus. we found that mbl binds to noncapsulated s. aureus strain (wood) but not any of the examined s. pneumoniae serotypes. l-ficolin binds to some capsulated s. pneumoniae serotypes (11a, 11d and 11f) as well as some capsulated s. aureus serotypes (type-1, -8, -9, -11 and -12). h-ficolin does not bind to any of the examined s. pneumoniae and s. aureus serotypes included in this study but did bind to a strain of aerococcus viridans. when bound to bacteria, mbl and h-ficolin initiated activation of complement factor c4, whereas l-ficolin did not. during this study, quantitative assays for the three proteins were developed and the concentration in 97 plasma samples were determined and the median values were estimated at 0.8 mg of mbl/ml, 3.3 mg of l-ficolin/ml and 18.4 mg of h-ficolin/ ml, respectively. the absence of early complement components (c1, c4 and c2 but not c3) is a predisposing factor for systemic lupus erythematosus (sle). recently, we demonstrated that, in c4-deficient (c4 def.) mice, igm-containing immune complexes (igm-ic) are filtered by the splenic barrier of marginal zone macrophages (mzm), resulting in an increased immune response against antigens within these igm-ic, but this could not be observed in wildtype or c3 def. mice. we hypothesized that splenic cd11b þ mzm play an important role in the induction of autoimmunity, and we therefore analysed their cytokine profile after isolation with the help of magnetic antibody cell sorting. mrna was isolated, and real-time pcr was performed with specific primers for murine ifn-g (ifn-g), interleukin-12 (il-12) and ifn-a (ifn-a). we observe a moderate increase of il-12 and ifn-g mrna in cd11b þ cells of c4 def. mice compared to wildtype cells. surprisingly, the concentration of ifn-a mrna is six times higher in c4 def. mice. preliminary results suggest that mrna in cd11b þ cells of c3 def. mice is even lower than that in wt. six hours following i.v. application of 20 mg of a abstracts 625 .................................................................................................................................................................................................. murine monoclonal igm anti-dsdna antibody, production of il-12, ifn-g and ifn-a mrna is increased in cd11b þ cells of both c4 def. and wt mice. several references described increased levels of inf-a in patients with sle. dendritic cells are discussed as a major source of ifn-a. our observation that c4-deficient, sle-susceptible mice demonstrate an increased spontaneous ifn-a production by splenic cd11b þ marginal zone macrophages could be an early sign and a trigger for the development of sle. this is supported by the fact that the absence of c3 is not a predisposing factor for sle and our observation that c3 def. animals display low levels of ifn-a mrna. 200-400 million people worldwide and represents one of the leading causes for liver cirrhosis and hepatocellular carcinoma. control over the hbv infection is achieved mainly by vaccination with hepatitis b surface antigen (hbsag). hbsag contains n-linked glycosylation side and is recognized by both mbl-a and mbl-c in a cadependent manner. hbsag-mbl complexes activate complement and may thus affect humoural immunity. to investigate the role of mbl in humoural responses to hbsag, we immununized mice that lack both mbl-a and mbl-c proteins with soluble hbsag. it has been shown that deficiencies in other complement components like c1q, c4 and c3 result in decreased antibody responses. however, mbl double ko animals mounted dramatically increased humoural responses. after priming, mbl double kos mounted hbsag-specific igm responses, which were threefold higher than wt controls. after boosting the hbsag, total igg was 10-fold higher in mbl ko than in wt control animals. similar to the response to hbsag, other glycosylated soluble antigens (e.g. invertase) induced better humoural responses in mbl double ko animals, suggesting that mbl plays an important role in a negative feedback regulation of adaptive immunity. reconstitution experiments with rmbl partially rescued the ko phenotype. we propose that the clearance of glycoprotein antigens in mbl ko is handled differently from the wt, resulting in better stimulation of humoural responses. alternatively, glycoprotein-ag-mbl-rich complexes inhibit b-cell responsiveness via putative mbl receptors. the complement system is an important part of the innate immune system. the activation of complement proceeds through three different pathways that converge in the generation of c3-activating enzyme complexes. complement activation via the lectin pathway is initiated when recognition molecules, mannan-binding lectin (mbl) or ficolin, bind to carbohydrate structures characteristic for microbial surfaces. in the circulation, mbl and ficolins are found in association with three structurally related mblassociated serine proteases (masp)-1, -2 and -3 and a small, nonenzymatic component, map19. masp-2 has been shown to elicit complement activation through the sequential proteolytic cleavage of c4 and c2 upon binding of mbl/masp-2 complexes to microbial surfaces. we have recently uncovered a polymorphism in the masp-2/map19 gene in a patient shown to be deficient in the lectin pathway of complement activation. the polymorphism results in a single amino acid substitution in the n-terminal part of the masp-2 protein. recombinant wildtype masp-2 and masp-2 containing the amino acid substitution in question was produced, and the ability to activate complement was studied. the mutation had a profound impact on masp-2 function, resulting in the lack of complement activation through the lectin pathway. elisa-based experiments showed that the mutation leads to the impairment of complement activation through influencing the binding of masp-2 to mbl or ficolins. deficiencies in the lectin pathway of complement activation have so far been accounted for only by lack of functional mbl. the mutation described above is the first defect described affecting both activation through mbl and the ficolins. .................................................................................................................................................................................................. th1, th2 and treg cell balance. dcs are present in the gut mucosa and may thus be target for modulation by gut microbes, including ingested probiotics. here, we tested the hypothesis that species of lactic acid bacteria, important members of the gut flora, differentially activate dc. a large panel of human gut-derived lactobacillus and bifidobacterium spp. was screened for dc-polarizing capacity by exposing bone marrow-derived murine dc to lethally irradiated bacteria. cytokines in culture supernatants and dc-surface maturation markers were analysed. substantial differences were found among strains in the capacity to induce interleukin-12 (il-12) and tumour necrosis factor (tnf)-a, while the differences for il-10 and il-6 were less pronounced. bifidobacteria tended to be weak il-12 and tnf-a inducers, while both strong and weak il-12 inducers were found among the strains of lactobacillus. remarkably, strains weak in il-12 induction inhibited il-12 and tnf-a production induced by an otherwise strong cytokine-inducing strain of lactobacillus casei, while il-10 production remained unaltered. selected strains were tested for induction of dc maturation markers. those lactobacilli with greatest capacity to induce il-12 were most effective in upregulating surface mhc class ii and cd86. moreover, l. casei-induced upregulation of cd86 was reduced in the presence of a weak il-12inducing l. reuteri. in conclusion, human lactobacillus and bifidobacterium spp. polarize differentially dc maturation. thus, the potential exists for th1/th2/treg-driving capacities of the gut dc to be modulated according to composition of gut flora including ingested probiotics. the intestinal micro flora is indispensable in developing and maintaining homeostasis of the gut-associated immune system. evidence indicates that lactic acid bacteria (lab), e.g. lactobacilli and bifidobacteria, have beneficial effects on the host. established health effects include increased gut maturation, antagonisms towards pathogens and immune modulation. the objective of this study is to evaluate the immunomodulating properties of a range of lab of human origin. as dendritic cells (dcs) play a pivotal role in the balance between tolerance and immunity to commensal microorganisms, in vitro-generated immature dcs serve as a suitable model for studying the immunomodulating effects of lab. human immature dcs were generated in vitro from monocytes and exposed to lethally uv-irradiated lab. the effect of various species of lab on dcs in direct contact was evaluated. furthermore, the maturation pattern of dcs separated from the bacteria by an epithelial cell layer (caco-2 cells), which should mimic the intestinal environment, was studied. cytokine secretion (il-12, il-10 and tnf-a) and upregulation of maturation surface markers on dcs (cd83 and cd86) was measured. different lab induced diverse cytokine responses. some strains were strong il-12 and tnf-a inducers and others weak. all strains induced il-10. different lab also differentially modulated expression of cd83 and cd86 on dcs. although some variation in the response to lab of dcs generated from different blood donors was observed, general differences in the effect of the various lab was revealed. experiments with the dc caco-2 coculture system are ongoing. different species of lab differentially affect dc maturation; this suggets that the gut flora plays a pivotal role in polarization of the immune response. natural killer (nk) cells are cells of the nonspecific immune system lysing altered self-cells. a noncytolytic subset of nk cells may serve a regulatory role by secreting cytokines. bacteria translocating across the gastrointestinal mucosa are presumed to gain access to nk cells, as consumption of certain lactic acid bacteria has been shown to increase in vivo nk cytotoxicity. here, we investigated how human gut flora-derived lactobacilli affect nk cells in vitro, by measuring proliferation and ifn-g production of human nk cells upon bacterial stimulation. cd3 -cd56 þ nk cells were isolated from buffy coats by negative isolation using non-nk lineage-specific antibodies and magnetic beads. nk cells were incubated with 10mg/ml uv-inactivated bacteria or 10mg/ml phytohemagglutinin (pha) for 4 days. proliferation was assessed by incorporation of radioactive thymidine into nk-cell dna. the ifn-g concentration was measured by elisa. incubation of nk cells with a lactobacillus acidophilus strain increased the proliferation of the nk cells and induced ifn-g production, both to levels comparable to pha stimulation. the proliferative response was further enhanced with autologous monocytes present, probably because cytokines, secreted by monocytes having engulfed bacteria, stimulated the nk cells. in contrast, a lactobacillus paracasei strain caused the nk cells to proliferate only in the presence of monocytes. these results demonstrate that various strains of lactobacilli have the capacity to activate nk cells in vitro, in a monocyte-dependent or -independent way. hence, the encounter of nk cells with lactic acid bacteria will affect nk-cell activation. such activation of nk cells may potentially skew an on-going or subsequent immune response towards a th1 response. lactobacilli are nonpathogenic gram-positive inhabitants of the normal human intestine known for their healthpromoting effects. in our earlier work, it is shown that human monoclonal antibody isolated from sera of a patient with waldenstrom macroglobulinaemia possess innate antibody characteristics and binds to lactic acid bacteria. according to the immune network model, immunization with this bacteria could induce the perturbations in immune system that might result in production of anti-lactobacillus antibodies, human monoclonal antibody like (ab1) and anti-idiotypic antibody (ab2). in this study, balb/c mice were immunized with two doses of bacteria lactobacillus acidophilus in complete and incomplete freund's adjuvant and phosphate-buffered saline (pbs), respectively. seven days after the last immunization, sera from immunized mice were collected and the presence of lactobacillus-specific ab1 and ab2 were determined by elisas. in the sera of immunized mice, antibodies specific to bacteria lactobacillus acidophilus were shown. the concentration of lactobacillus-specific antibodies was higher in the sera of hyperimmunized mice (mice immunized with 1 mg of igm dj) than in sera of mice immunized with 100 times lower doses of immunogen (0.01 mg per doses). moreover, ab1 and ab2 antibodies were detected in the sera of lactobacillus-hyperimmunized mice. in this study, we have shown the idiotypic network interactions in mice immunized with bacteria lactobacillus acidophilus. the normal gastrointestinal flora is crucial for the maturation of the acquired immunity via effects on antigenpresenting cells (apcs). here, we have investigated how two types of apcs, monocytes and dendritic cells (dcs), react to different bacterial strains typical of the commensal intestinal flora. purified monocytes and monocyte-derived dcs were stimulated with uv-inactivated gram-positive (lactobacillus plantarum and bifidobacterium adolescentis) and gram-negative (escherichia coli and veillonella parvula) bacterial strains. monocytes produced higher levels of il-12p70 and tnf, as detected by elisa, in response to l. plantarum than to e. coli and v. parvula. in contrast, dcs secreted high amounts of il-12p70, tnf, il-6 and il-10 in response to e. coli and v. parvula but were practically unresponsive to l. plantarum and b. adolescentis. the lack of response to the gram-positive strains correlated with a lower surface expression of toll-like reseptor 2 (tlr2) on dcs compared to monocytes. the surface expression of tlr4 on dcs was undetectable when analysed by flow cytometry, but blocking this receptor decreased the tnf production in response to v. parvula, indicating that low tlr4 expression on dcs is sufficient to mount an inflammatory response to gram-negative bacteria. ifn-g increased the expression of tlr4 on dcs and also potentiated the cytokine response to gram-negative bacteria. our results indicate that, when monocytes differentiate into dcs, their ability to respond to different commensal bacteria dramatically changes, thereby becoming unresponsive to probiotic gram-positive bacteria. these results may have important implications for the capacity of different groups of commensal bacteria to regulate mucosal and systemic immunity. probiotic bacteria, e.g. lactobacillus spp., may improve diseases such as chronic inflammatory bowel disease. we examined cytokine production and phenotypic change after in vitro stimulation of t cells from healthy volunteers using different probiotic strains. methods: t cells were cultured from colonic biopsies from eight healthy volunteers (agnholt and kaltoft, exp clin immunogenet 2001; 18:213-25) , and dendritic cells were matured from their peripheral blood mononuclear cells. t-cell cultures were stimulated with autologous bacterial sonicate or strains of lactobacillus spp., with and without the addition of dendritic cells. cytokine levels (tnf-a, ifn-g, il-10 and gm-csf) and phenotype (cd3, cd4, cd25 and cd69) were measured on day 4. results: lactobacillus spp. induced higher productions of tnf-a and il-10 than did autologous bacteria. in presence of dendritic cells, the production of all cytokines increased. however, the increases of ifn-g and tnf-a were more pronounced in wells with autologous bacteria than in wells with lactobacillus spp. the addition of dendritic cells upregulated cd25 expression without simultaneous upregulation of cd69. the upregulation was pronounced after stimulation with lactobacillus rhamnosus gg compared with autologous bacteria and other lactobacilli. discussion: in presence of dendritic cells, autologous bacteria induced inflammatory cytokines, while probiotics mainly induced regulatory cytokines. lactobacillus rhamnosus gg induced a regulatory phenotype (cd25 þ ), in part mediated by dendritic cells. future studies will address whether this shift to a cd25 þ phenotype represents a differentiation into competent regulatory t cells. in a clinical context, such cells might be used for treatment of inflammatory diseases. protein microarrays will play a key role in the postgenomic era and offer a unique possibility to perform highthroughput global proteome analysis. a chip can be printed with thousands of protein probes (e.g. antibodies), the biological sample added (e.g. a proteome) and any binding detected. we aim to develop protein microarrays based on human recombinant scfv antibody fragments for global proteome analysis. the concept of comparing proteomic maps of healthy versus diseased samples will allow diseasespecific proteins to be detected. in fact, antibody microarrays will allow us to perform comparative proteome analysis on any sample format in a species-independent manner, as long as a proteome can be isolated. however, the complexity of proteomes, containing several thousands of different proteins, is a problem. here, we have designed antibody microarrays targeting the water-soluble fraction of a proteome. to this end, an anticytokine antibody array was developed and human dendritic cells (aeactivation) was used as model system. the results showed that our antibody microarrays could be used to examine the cytokine profile in complex samples. furthermore, we have taken the first steps towards comparing our results with those of other technologies on both the protein and gene level. due to the complexity of the model proteome, we also examined the possibility to prefractionate the proteome in a simple one-step procedure (based on size) prior to the labelling step. in more detail, the sample proteome was fractionated into two fractions using membrane devices with different molecular weight cut-offs. the results showed that the fractionation considerably enhanced the assay sensitivity allowing cytokines in the pg/ml range to be readily detectable. the immunomodulatory effect of heat shock protein 70: immunization with a dna construct based on the malarial antigen fused with a fragment of hsp 70 primes for a th-1 type of response finding an appropriate adjuvant for human vaccination is crucial. heat shock proteins (hsps) act as adjuvants when coadministered with peptide antigens or given as fusion proteins. however, there is a potential risk of autoimmunity when using the complete molecules, because hsps are evolutionary conserved. to overcome this, we first evaluated the adjuvant effect against two different antigens of a less-conserved fraction of plasmodium falciparum hsp70 (pf70c) and compared it to the whole hsp70 molecule from trypanosoma cruzi (tchsp70). we found that pf70c exhibited similar adjuvant properties as the whole molecule. we later evaluated the adjuvant potential of pf70c against the malarial antigen eb200 in a chimeric dna construct. no appreciable levels of eb200-specific abstracts 629 .................................................................................................................................................................................................. antibodies were detected in mice immunized only with the dna constructs. however, dna primed the immune system, because subsequent challenge with the corresponding recombinant fusion proteins elicited a strong th-1 antibody response. in contrast, no priming effect was observed for ex vivo ifn-g production but stimulation with the hsp-chimeric fusion protein induced a stronger secretion of ifn-g in vitro than other proteins used. these results indicate that the use of hsps is promising in the design of new vaccines. high-throughput proteomics on antibody-based microarrays: the importance of probe and surface design in analogy to dna microarrays, protein microarrays offer a new distinct possibility to perform sensitive highthroughput global proteome analysis. however, the development of the protein microarray technology will place high demands upon the design of both probes and solid supports. the analysis of thousands of heterogeneous proteins on a single microarray requires the use of uniform probes, such as antibodies, directly designed for protein microarray applications. we have recently generated a human recombinant single-chain fv antibody library, genetically constructed around one framework, the ncoder-library, containing 2 â 1010 clones. single framework antibody fragments (sinfabs) selected from this library were successfully applied as probes for microarrays providing sensitive detection in the 600 attomol (mass spectrometry) and the 300 zeptomole range (fluorescence). however, the choice of framework is critical. we have shown that the selected ncoder framework displayed excellent functional on-chip stability and arrayed dehydrated probes retained their activity for several months. furthermore, we have addressed the issues of biocompatibility of the solid support and immobilization strategies for our microarray setup. an in-house-designed substrate, macroporous silicon coated with nitrocellulose (map3-nc7), displayed properties equal to, or better than, those of five commercially available supports used as reference surfaces. we have also evaluated different coupling strategies, such as adsorption, covalent coupling, diffusion and affinity coupling. using a novel affinity tag, the double-(his)6-tag, we increased the binding efficiency of sinfab-molecules to ni2 þ -coated solid supports, thereby allowing nonpurified probes to be directly applied. the mannan-binding lectin (mbl) pathway is part of the innate immune system providing a first line of defence against infections. mbl and ficolins circulate in complexes with mbl-associated serine proteases (masp-1, -2 and -3). after recognition of a microorganism by mbl, activation of the complement system occurs. masp-1 and masp-3 share five domains (making up the so-called a-chain), whereas they have unique protease domains (b-chains). before the identification of masp-3, an assay for masp was presented, based on antibodies against the a-chain of masp-1. with the new knowledge of the three masps, and the sharing of domains by masp-1 and masp-3, assays specific for the protease domains have to be constructed, if one wishes to measure the proteins individually. we present an assay for quantifying total masp-3 in plasma and serum samples. the assay is a sandwich-type assay using as catching antibody a monoclonal antibody against the common a-chain of masp-1/3 and a developing secondary antibody against the c-terminal part of the protease domain of masp-3. we have used this assay for estimating the normal concentration of the protein as well as the concentration in patients and also for characterizing by gel permeation chromatography the masp-3 protein in serum. inducible costimulator ligand (icosl) is a costimulatory molecule related to b7.1 (cd80) and b7.2 (cd86). b cells, monocytes, dendritic cells and endothelial cells express icosl. inducible costimulator (icos) interacts with icosl, and this interaction leads to signals involved in isotype switching and the development of immunological memory. hitherto, no polymorphisms of this gene have been described. the aim of this study was to reveal variation of the icosl gene in normal individuals. all eight exons, except exon 1, were sequenced with flanking introns in 10 healthy blood donors. eight single nucleotide polymorphisms (snps) and two length polymorphisms were found. one of the snps was found in the coding regions of the gene. the base involved was located in exon 3 and caused a conservative amino acid change from valine (gtt) to isoleucine (att). three individuals were heterozygous g/a for the exon polymorphism, while the remaining seven individuals were homozygous for the wildtype g/g. exon 3 encodes the immunoglobulin variable (igv)-like domain of the molecule which is situated outside the cell. this means that the amino acid could be critical for the stability of the molecule or could constitute part of the binding site for icos. the results form the basis for further experiments to find possible associations of the alleles to diseases caused by immune dysregulation. especially, the exon 3 variant is interesting and could play a role for the development of immunological diseases. besides, it would be interesting to see whether both exon 3 alleles are expressed or only the wildtype allele is functional. interferon-a/b (ifn-a/b) is increasingly used as antiviral and immunomodulatory therapies. unfortunately, bioavailability varies with ifn species and mode of administration, and all ifn species are potentially immunogenic. assays for antiviral activity (ifn) and antiviral neutralization (antibodies, nab) have been used for some time to monitor patients on ifn biologicals. these assays require laborious titrations making them unsuitable for large-scale clinical use. myxovirus a (mxa) is a resistance gtpbinding protein that is specifically induced by treatment with type 1 ifns. for example, ifn-b-induced mxa in blood leucocytes has been used as a biomarker in ifn-btreated patients with multiple sclerosis. however, the degree of specificity of mxa in this regard is unclear, and measurements of mxa protein and/or mrna are not yet suitable for routine clinical use. in an attempt to find new and better reporter genes (and, hopefully, genes and gene products with proven specificity for ifn-a and -b), microarray screenings with u133a genechips (affymetrix) were carried using human blood leucocytes and the human lung carcinoma cell line a549. we studied the simultaneous expression of 22,000 transcripts before and after exposure to human recombinant ifn-a and ifn-b and other antiviral and immunomodulatory cytokines. the results will be presented at the conference. interferon-a/b (ifn-a/b) is increasingly used as antiviral and immunomodulatory therapies. unfortunately, bioavailability varies with ifn species and mode of administration, and all ifn species are potentially immunogenic. assays for antiviral activity (ifn) and antiviral neutralization (antibodies, nab) have been used for some time to monitor patients on ifn biologicals. these assays require laborious titrations making them unsuitable for large-scale clinical use. our laboratories have therefore modified the antiviral assays for ifn bioactivity and nab, so that they are suitable for large-scale screening in specialized laboratories. the read-out is survival of a subcloned a549 cell line in the presence of an otherwise lethal amount of virus. thus, survival increases in the presence of type 1 ifn and decreases in the presence of nab against the ifn added to the cells. mxa is induced by type 1 ifn and can be used for measuring the nab activity. in another assay, the mxa level in the a549 cell line is measured. in an attempt to find a new and better reporter gene for type 1 ifn than mxa and genes specific for either ifn-a or -b, a micro array screen was carried using the u133a chip from affymetrix. the expression of 22,000 genes can be studied simultaneous with this technology. the results will be presented at the conference. in our laboratory, we have developed a database system, which we believe is of immediate interest to the general scientific community. the database represents a computerbased replacement for the laboratory notebooks used in the majority of research laboratories worldwide. in addition, the database provides an effective tool for organizing and managing laboratory information at all levels, spanning from managing and revising standard operating procedures and producing documentation of research activities to keeping track of data and conclusions. using the commercially available database toolkit software filemaker pro, we have developed a relational database solution for management of laboratory information. the system consists of a hierarchy of five interrelated databases, each pertaining to a separate type of information, namely, overall project information, information relating to individual experiment setups, documentation of daily research activity, generated data and descriptions of standard operating procedures. like other databases, each individual database consists of a number of records, each comprised of a set of fields in which information is entered. in each record, a certain field is reserved to specify the relation of the record to a record in another database at a higher level. thus, the database is essentially five databases linked by a hierarchy of one-to-many relations, organizing information in a folder-like structure. importantly, the database system allows multiple users to access and edit records simultaneously, and the data entered in one database immediately becomes accessible through the other databases. the limitations of laboratory notebooks are apparent when looking for information, which is dispersed throughout one or more notebooks, or possibly on loose sheets of paper or printouts 'somewhere'. the often complicated process of gathering laboratory data or results when writing grant applications or research papers is made considerably easier with the database system. thus, the database solution presented should be broadly attractive to researchers, irrespective of their scientific discipline. an effective sars vaccine is likely to include components that can induce specific cytotoxic t-cell (ctl) responses. the specificities of such responses are governed by hlarestricted presentation of sars-derived peptide epitopes. exact knowledge of how the immune system handles protein antigens would allow for the identification of such linear sequences directly from genomic/proteomic sequence information. the latter was recently established when a causative coronavirus (sars cov) was isolated and full-length sequenced. here, we have combined advanced bioinformatics and high-throughput immunology to perform an hla supertype, genome-wide scan for sars-specific cytotoxic t cell epitopes. the scan includes all nine human hla supertypes in total covering >99% of all major human populations. for each hla supertype, we have selected the 15 top candidates for test in biochemical-binding assays. at this time (approximately 6 months after the genome was established), we have tested the majority of the hla supertypes and identified almost 100 potential vaccine candidates. these should be further validated in sars survivors and used for vaccine formulation. we suggest that immunobioinformatics may become a fast and valuable tool in rational vaccine design. rationale: major histocompatibility complex class i (mhc i) molecules monitor the protein content of the cell by binding small derived peptides and presenting them to cytotoxic cd8 þ t cells. the goal of the human mhc project is to predict the binding strength of any given peptide/mhc complex. this prediction allows the design of peptide-based vaccines. the prediction requires representative binding data from mhc alleles from all the nine hla supertypes. here, we describe the genetic construction, protein production and purification as well as the establishment-binding assays for two recombinant mhc supertype alleles, hla-b*1501 and hla-b*5801. methods: using the quikchange multisite directed mutagenesis kit (stratagene), codon-optimized genes encoding hla-b*1501 and hla-b*5801 are created. the two mhc i molecules are fermented and purified by ion exchange chromatography, hydrophobic interaction chromatography and size exclusion chromatography. the binding (kd) of natural t-cell epitopes, as well as predicted peptide ligands, is described by radioactive immunoassays (rias) and enzyme-linked immunosorbent assays (elisas). the mhc molecules are biotinylated during expression. results: the expression of mhc i resulted in multiple disulfide bond isomers, which are separated by hydrophobic interaction chromatography and used in subsequent binding studies resulting in the determination of kd for various peptide ligands ranging from strong binders we have previously demonstrated that bioinformatics tools such as artificial neural networks (anns) are capable of performing pathogen-, genome-and hlawide predictions of peptide-hla interactions. these tools may therefore enable a fast and rational approach to epitope identification and thereby assist in the development of vaccines and immunotherapy. a crucial step in the generation of such bioinformatics tools is the selection of data representing the event in question (in casu peptide-hla interaction). this is particularly important when it is difficult and expensive to obtain data. herein, we demonstrate the importance in selecting information-rich data and we develop a computational method, query-bycommittee, which can perform a global identification of such information-rich data in an unbiased and automated manner. furthermore, we demonstrate how this method can be applied to an efficient iterative development strategy for these bioinformatics tools. methods: a large panel of binding affinities of peptides binding to hla a*0204 was measured by a radioimmunoassay (ria). this data was used to develop multiple first generation anns, which formed a virtual committee. this committee was used to screen (or 'queried') for peptides, where the anns agreed ('low-qbc'), or disagreed ('high-qbc'), on their hla-binding potential. seventeen low-qbc peptides and 17 high-qbc peptides were synthesized and tested. the high-or low-qbc data were added to the original data, and new high-or low-qbc second generation anns were developed, respectively. this procedure was repeated 40 times. the high-qbc-enriched ann performed significantly better than the low-qbc-enriched ann in 37 of the 40 tests. conclusion: these results demonstrate that high-qbcenriched networks perform better than low-qbc-enriched networks in selecting informative data for developing peptide-mhc-binding predictors. this improvement in selecting data is not due to differences in network training performance but due to the difference in information content in the high-qbc experiment and in the low-qbc experiment. finally, it should be noted that this strategy could be used in many contexts where generation of data is difficult and costly. interleukin-18 (il-18), a pro-inflammatory cytokine that is produced by both lymphoid and nonlymphoid cells, has a critical role in modulation of innate and adaptive immunity. its primary function in stimulation of ifn-g production and stimulation of nk-cell-cytotoxic activities makes this cytokine a candidate for cancer immunotherapy. in oral cavity, this cytokine is produced by oral epithelia and carcinoma cells and is related to tumour regression in nude mice bearing salivary adenocarcinoma. however, direct effects of this cytokine on oral cancer cells have not been elucidated. in this project, we investigated il-18 effect on an oral carcinoma (kb) cell line. with rt-pcr technique, kb-cell line was found to express il-18 receptors (il-18ra and il-18rb), indicating that this oral carcinoma line is a target for il-18 study. we showed that recombinant human il-18 inhibited kb-cell proliferation by 17% at concentration of 100 ng/ml (p < 0.05), whereas ldh release by these cells in treatment group and control groups was comparable, indicating that il-18 suppression of cell proliferation was not mediated by the induction of cell death. to further address this hypothesis, we found that il-18 treatment did not induce apoptotic cell death, as studied by dna laddering and tunel assays. in addition, expression pattern of cell death-controlling genes (bcl-2 and bax) was not altered by this cytokine. findings in these studies indicated that suppression of kb-cell proliferation may be attributed to control of cell cycle, growth arrest or induction of cell differentiation. the data presented in this project could provide an insight of how cancer cell directly responds to il-18, as this cytokine is an important regulator of anticancer mechanisms. aloe emodin (ae) is a naturally occurring compound with wide spectrum of biological properties, including antimicrobial, vasorelaxant, immunosuppressive and anticancer actions. this anthraquinone induces apoptosis in several tumour cell lines with special affinity to tumours of neuroectodermal origin. high amounts of nitric oxide (no) released by activated macrophages induce tumour cell death. therefore, we explored the capacity of ae to modulate no-mediated antitumour response in vitro. interestingly, while ae markedly suppressed no release from macrophages alone, it significantly potentiated no production in cocultures of macrophages and c6 cells, after 48 h of cultivation. accordingly, the viability of c6 cells cocultivated with macrophages was reduced in the presence of ae. moreover, the observed ae-imposed potentiation of no production in macrophages was closely related to macrophage culture cell density. according to these data, we proposed that no modulator capacity of ae strongly depended on intercellular contact, indicating that macrophage antitumour response was not compromised but even potentiated by ae. immunotherapy represents an attractive fourth-modality therapeutic approach, especially in the light of the shortcomings of conventional surgery, radiation and chemotherapies in the management of metastatic cancer. to this end, a large number of peptide antigens derived from taa have been applied in immunotherapeutic trials for the treatment of various malignancies, e.g. cancers of the breast, prostate and kidney, in addition to haematological cancers. in some cases the response rates have been impressive and no adverse autoimmunity have been observed. a major strategic difficulty associated with these trials relates to the choice of best-suited peptide antigens. the vast majority of the antigens described thus far is not vital for survival and growth of the tumour cells, and immunoselection of antigen-loss variants may therefore prove to be an additional obstacle for the clinical applicability of most of the known peptide epitopes. in this respect, the development of acquired antigen loss during immunotherapy has been demonstrated in several cases. obviously, the development of loss-variant tumour cells implies that these cells acquire a pronounced growth advantage and are left unaffected by further treatment. ideally, target antigens should be derived from proteins required for survival and growth of tumour cells, as antigens with these characteristics would not be inflicted by the development of loss-variant tumour cells. in this respect, several inhibitors of apoptosis proteins (iaps) are universally expressed among tumours and play an important role in tumour cell escape from apoptosis. we have characterized spontaneous t-cell reactivity against iapderived peptides in cancer patients. from the iap survivin, we have characterized peptides restricted to the class i molecules hla-a1, a2, a3, a11, b7 and b35. furthermore, we have demonstrated that survivin-specific t cells infiltrate metastatic lesions and that isolated survivinspecific ctls are capable of killing hla-matched tumour cells. survivin-derived peptides are now in clinical trial, and continued work in our lab has demonstrated that other iaps are targets for spontaneous t-cell reactivity in cancer patients. we previously reported that in mice with large progressing t-cell lymphoma tumours, dysfunctions in the antitumour ctl activity occur, associated with an accumulation of splenic arginase-producing myeloid suppressor cells (mscs). in this study, we first demonstrate that both the presence and the activation state of these msc depends on tumour evolution. while in tumour regressors hardly any arginase-producing msc can be found, both the amount and the arginase activity of this population expands from early over late progressors. this gradual induction of mscs is paralleled by an increasing suppression of ctl activity and th1, but not th2, cytokine production. upon analysing the molecular repertoire of msc in vitro, we found, besides arginase1, a well-established marker for alternatively activated myeloid cells or m2, a strong upregulation of fizz1 and ym, two additional recently identified markers for m2. further evaluation of molecular markers by microarray analysis in msc yielded genes involved in wound healing (e.g. coagulation factor xiiia), anti-inflammation (e.g. selenoprotein p), immunomodulation (e.g. pd-l2) and fat and sugar metabolism (e.g. leptin receptor). of note, many of these genes are regulated by type 2 cytokines (il-4, il-13 and il-10) and are therefore rather m2 associated. overall, our data provide new markers for msc in cancer and further establish their m2 activation state. study. only sp-a showed a significant expression in normal mucosa which was downregulated in crc. as the absolute signal level was below the noise threshold, these results have to be interpreted with caution and require confirmation by direct measurenment of the proteins. our results suggest that there is no major role for the human collectins in colorectal cancer. tetramerization is visualized by sds-page. conclusion: an effective method for the production of highly pure mhc i molecules has been applied to hla-b*1501 and hla-b*5801, and ria and elisa binding assays for those alleles have been established background: proliferation, differentiation and apoptosis are essential processes in the normal functions of the mammary epithelium. the hypothesis examined in this study is that the transcription factor bcl-6 is critically important not only for regulating b-cell growth and development but also for mammary epithelial apoptosis. methodology: twenty breast cancer cases and 31 healthy controls were used to investigate whether bcl-6 protein in involved in breast cancer (grade iii). full length bcl-6 cdna was retrovirally transduced into eph-4 cell line. we then used flow cytometry of brdurd-stained cells to investigate the cell-cycle duration of the control and transduced cell lines. tunel was used as a marker of apoptosis to find out differences in the frequencies of apoptotic cells in the control and transduced cell lines. finally, immunohistochemistry staining was performed to detect bcl-6 in breast cancer (iii). results: restoration of bcl-6 into eph-4 cells not only inhibits apoptosis but also prolongs the cell cycle and results in increased cell size and protein content. the results also indicated that the cell-cycle time of bcl-6-transduced eph-4 cells is prolonged by about 3 h, presumably as a result of the action of bcl-6 at the bcl-6 at the g1/s transition. we found differences in the frequencies of viable and apoptotic cells in cultures of the parent eph-4 cells, control-transduced eph-4 cells and bcl-6-transduced eph-4 cells. consistently, we demonstrated that bcl-6 is expressed in 90% of high grade of breast carcinoma, which is considered as the most aggressive of tumours. conclusion: together, these results suggest that bcl-6 is likely to be involved in mammary gland development and carcinogenesis. inflammatory cytokines have a critical role in modulation of both innate and adaptive immunity in response to foreign antigen. they also play an important role in anticancer immunity. for example, they can promote cell-mediated immunity against cancer cells. with their immunostimulatory effects, these cytokines are being tested for cancer treatment in the form of dna vaccine or adjuvant or therapeutic cytokines. direct effect of these cytokines on cancer cell, however, is still unclear. in this project, we investigated whether il-1( and il-18 can modulate cancer cell proliferation. we employed a simple nonradioactive proliferation (mtt) assay and detection of lactate dehydrogenase (ldh) to test the effect of these recombinant human cytokines on various cancer cell lines, including breast cancer cell line (mcf-7), oral carcinoma cell line (kb), colon cancer cell line (caco-2) and choriocarcinoma cell line (jar). cytokines used in this study had both inhibitory and stimulatory effect on cell proliferation. findings in this project could provide an insight of cancer cell response to these cytokines and this could lead to a consideration on using cytokine as immunotherapy for cancer treatment.capacity of ae to modulate nitric oxide production depended on intercellular contact donor t cells are involved in the antitumour effects observed after bmt. thus, patients receiving t-celldepleted bmt have a higher risk of leukaemia relapse compared to patients receiving nonmanipulated bmt, and patients experiencing graft-versus-host disease (gvhd) have a lower risk of disease relapse than patients who do not experience gvhd. although the importance of donor t cells for the curative action of bmt has been established, the exact mechanisms and molecules involved in this graft-versus-tumour effect remain largely unknown. in a recently initiated project, we have conducted a longitudinal study of t-cell clonotypes in patients who received peripheral blood stem cell grafts after nonmyeloablative conditioning. peripheral blood samples were obtained sequentially after transplant, and the mononuclear cells (mncs) were isolated and cryopreserved. cd8 þ t cells were isolated from the mncs by use of immunomagnetic beads or facs and analysed for the presence of clonally expanded cells by t-cell receptor clonotype mapping based on rt-pcr and denaturing gradient gel electrophoresis (dgge). using this gel-based methodology, clonally expanded t cells were monitored after transplant and compared to the clinical data of the patients. the preliminary results demonstrates the presence of clonally expanded cd8 þ t cells at all time points analysed. furthermore, a number of clonotypes persisted for more than 6 months, and other clonotypes emerged during this period. the appearance of newly emerged clonotypes which coincided with clinical gvhd could indicate a role for these t cells in the pathogenesis of gvhd. background: deficiency of the mannan-binding lectin (mbl) pathway of innate immunity leads to increased susceptibility to infections. in patients with colorectal cancer, postoperative infection is associated with poor prognosis. the aim of the present study was to evaluate (1) the relation between the mbl pathway and postoperative infectious complications and survival of patients resected for colorectal cancer and (2) the role of mbl as acute phase reactant compared to crp. methods: preoperative mbl concentration, mbl/mblassociated serine protease (masp) activity and crp were determined in serum from 611 patients and 150 healthy controls. the patients were observed for 8 years. postoperative infections, recurrence and survival were recorded. results: the mbl pathway components were increased in the patients (p < 0.0001) compared to healthy controls. low mbl levels were predictive of pneumonia (p ¼ 0.01), and pneumonia (n ¼ 87) was associated with poor survival (p ¼ 0.003, hr ¼ 1.5, 95% ci 1.1-1.9). mbl and mbl/ masp activity could not predict postoperative overall infections. mbl showed no correlation (spearman's r ¼ 0.02, 95% ci à0.06-0.10) with crp. conclusions: low preoperative mbl levels are predictive of pneumonia, which is associated with poorer survival. mbl concentration and mbl/masp activity was not predictive of other postoperative infections or long-term prognosis. mbl apparently is not a surrogate measure of crp. department of surgery, university hospital of erlangen, erlangen, germany. e-mail: michael.siassi@rzmail.uni-erlangen.de introduction: the human collectins, mannan-binding lectin (mbl), surfactant protein-a (sp-a) and surfactantprotein-d (sp-d) play a central role in the innate immune system. immunological responses to malignant transformation of epithelial cells gained increasing interest recently. a former study could demonstrate binding of mbl to certain colorectal carcinoma (crc) cell lines in vitro. we therefore examined the expression of human collectins in normal colon mucosa and in colorectal carcinomas. materials and methods: colon samples from 20 crc patients and 10 normal mucosa samples were collected immediately after surgery. the tissue was microdissected and rna isolated (qiagen, rneasy-kit). gene expression profiles were analysed using gene-chips (affymetrix, hg-u133). we analysed the data for the expression of mbl, its associated serine proteases mannan-binding lectinassociated serine protease 1/2 (masp 1/2), sp-a and sp-d. the signal intensity of the genes of interest was compared using the mann-whitney u-test. results: the expression of human collectins in normal human colon mucosa was generally low. only the expression of sp-a and masp-2 reached the noise threshold of 250 signals. these genes were significantly downregulated in crc specimens. the expression of the other proteins showed no difference in normal mucosa and crc. conclusion: as demonstrated before, the expression of human collectins in normal colon was low in this being the first lymph node to receive drainage from the tumour area, the sentinel node offers a unique possibility to obtain tumour-reactive lymphocytes. we investigated antitumour immune responses in sentinel nodes from patients with bladder cancer, by assaying tumour-specific proliferation and tcr vb repertoires. during tumour surgery, sentinel lymph nodes were identified by peritumoural injection of blue dye. fresh specimens of tumour, sentinel and nonsentinel lymph nodes were obtained, and single-cell suspensions were prepared. cells were assayed for reactivity against autologous tumour extract in [ 3 h]-thymidine incorporation assays and characterized by flow cytometry. parallel analyses of the expression of vb gene families were performed with padlock probes, linear oligonucleotides which upon target recognition can be converted to circular molecules by a ligase. probes were reacted with cdna prepared from magnetically separated cd4 þ cells, and the tcr repertoire was determined by hybridizing the products to oligonucleotide microarrays. dose-dependent proliferation in response to tumour extract could be detected in sentinel lymph nodes. common clonal expansions were detected among tumourinfiltrating lymphocytes and in sentinel lymph nodes. nonsentinel lymph nodes displayed a divergent tcr vb repertoire. these results indicate an ongoing immune response against tumour antigens in sentinel nodes, draining urinary bladder cancer. identification of sentinel lymph nodes makes it possible to obtain tumour-reactive lymphocytes for use in adoptive immunotherapy. key: cord-023387-tyeh14wz authors: hvas, c. l.; kelsen, j.; agnholt, j.; höllsberg, p.; dahlerup, j. f. title: probiotic bacteria induce regulatory cytokine production via dendritic cells date: 2008-06-28 journal: scand j immunol doi: 10.1111/j.0300-9475.2004.01423au.x sha: doc_id: 23387 cord_uid: tyeh14wz probiotic bacteria, e.g. lactobacillus spp., may improve diseases such as chronic inflammatory bowel disease. we examined cytokine production and phenotypic change after in vitro stimulation of t cells from healthy volunteers using different probiotic strains. methods: t cells were cultured from colonic biopsies from eight healthy volunteers (agnholt and kaltoft, exp clin immunogenet 2001;18:213–25), and dendritic cells were matured from their peripheral blood mononuclear cells. t‐cell cultures were stimulated with autologous bacterial sonicate or strains of lactobacillus spp., with and without the addition of dendritic cells. cytokine levels (tnf‐α, ifn‐γ, il‐10 and gm‐csf) and phenotype (cd3, cd4, cd25 and cd69) were measured on day 4. results: lactobacillus spp. induced higher productions of tnf‐α and il‐10 than did autologous bacteria. in presence of dendritic cells, the production of all cytokines increased. however, the increases of ifn‐γ and tnf‐α were more pronounced in wells with autologous bacteria than in wells with lactobacillus spp. the addition of dendritic cells upregulated cd25 expression without simultaneous upregulation of cd69. the upregulation was pronounced after stimulation with lactobacillus rhamnosus gg compared with autologous bacteria and other lactobacilli. discussion: in presence of dendritic cells, autologous bacteria induced inflammatory cytokines, while probiotics mainly induced regulatory cytokines. lactobacillus rhamnosus gg induced a regulatory phenotype (cd25(+)), in part mediated by dendritic cells. future studies will address whether this shift to a cd25(+) phenotype represents a differentiation into competent regulatory t cells. in a clinical context, such cells might be used for treatment of inflammatory diseases. the nuclear receptor heterodimers of liver x receptors (lxrs) are recently identified as key transcriptional regulators of genes involved in lipid homeostasis and inflammation. lxrs and their ligands are negative regulators of macrophage inflammatory gene expression. multiple sclerosis (ms), a demyelinating disease of the central nervous system of unknown cause, is characterized by recurrent inflammation involving macrophages and their inflammatory mediators. sweden belongs to the countries with a high ms incidence. in italy, incidence is lower, with an exception for sardinia where the incidence is even higher than that in sweden. subjects from sardinia are ethnically more homogeneous and differ from swedes, also regarding genetic background and environment. we studied lxrs and their related molecules of blood mononuclear cells (mncs) from female patients with untreated relapsing-remitting ms from sassari, sardinia and stockholm, sweden. sex-and age-matched healthy controls (hcs) were from both areas. mrna expression was evaluated by real-time pcr. lxr-a was lower (p < 0.05) in ms (mean ae sem: 3.1 ae 0.2; n ¼ 37) compared to hc (3.6 ae 0.1; n ¼ 37). lxr-a was lower in ms from stockholm (2.6 ae 0.2; n ¼ 22) compared to corresponding hc (3.4 ae 0.1; n ¼ 22; p < 0.01) and compared to ms (3.8 ae 0.2; n ¼ 15; p < 0.001) and hc (4 ae 0.2; n ¼ 15; p < 0.001) from sardinia. ms patients from stockholm, but not from sassari, also expressed lower (p < 0.05) lxr-b (à4.1 ae 0.4) compared to corresponding hc (à2.9 ae 0.3). ms from stockholm was associated with higher abca-1 (6.1 ae 0.4 versus 5.0 ae 0.3; p < 0.05) and higher estrogen receptor-b-cx (2.4 ae 0.4 versus 0.8 ae 0.4; p < 0.01) compared to corresponding hc. the hc from sassari had higher androgen receptor (2.9 ae 0.2) compared to ms from sassari (1.4 ae 0.3; p < 0.01), ms (1.3 ae 0.4; p < 0.01) and hc from stockholm (1.2 ae 0.3; p < 0.01). ms from sassari had lower cyclooxygenase-1 compared to corresponding hc (5.1 ae 0.4 versus 6.6 ae 0.3; p < 0.01) and lower prostaglandin-e (à0.03 ae 0.5) compared to the hc (1.4 ae 0.5; p < 0.05) and ms (2.7 ae 0.4; p < 0.05) and hc from stockholm (1.9 ae 0.4, p < 0.001). our findings identify lxrs and their related molecules as being involved in ms from stockholm but not from sassari, while sex hormone receptors seem to be involved in ms in sassari. multiple sclerosis: ifn-b induces cd123 + bdca2 -dendritic cells that produce il-6 and il-10 and have no enhanced type i interferon production y. m. huang, 1 s. adikari, 1 u. båve, 2 a. sanna 1,3 & g. alm 4 dc antigens (bdca) and investigate their ability to produce type i ifn in response to virus stimulation. we show that ifn-b induces development of cd123 þ dc from human blood monocytes, which coexpress bdca4 þ but are negative for bdca2 -, a specific marker for plasmacytoid dc. such ifn-b-modulated dc produce large amounts of il-6 and il-10, but no il-12p40 and have no enhanced ifn-b and ifn-b production. the findings indicate that ifn-bmodulated dc represent a myeloid dc subset with diminished cd11c, bdca-1 and cd1a expression, having potent th2-promoting function but lacking antiviral capacity. the association of psoriasis with throat infections by streptococcus pyogenes suggests a potential antigenic target for the t cells that are known to infiltrate dermis and epidermis of psoriatic skin. streptococcal m protein shares an extensive sequence homology with human epidermal keratins. keratins 16 (k16) and 17 (k17) are mostly absent from uninvolved skin but are upregulated in psoriatic lesions. there is increasing evidence that cd8 þ t cells play an important effector role in psoriasis and m proteinprimed t cells may recognize these shared epitopes in skin via molecular mimicry. to identify candidate epitopes, peptides with sequences from k17 were selected on the basis of predicted binding to hla-cw6 and sequence similarities with m6 protein. matched peptides from the sequence of m6 protein and a set of peptides with poor predicted binding were also selected. cw6 þ individuals with psoriasis and cw6 þ healthy controls, having a family history of psoriasis, were recruited. pbmcs were incubated with the peptide antigens. t-cell activation in the cd4 þ , cd8 þ and later the skin-homing cutaneous lymphocyteassociated antigen (cla)-expressing subset of cd8 þ t cells was evaluated by cd69 expression and intracellular ifn-g accumulation using flow cytometry. we demonstrate that cw6 þ psoriasis patients had significant cd8 þ t-cell ifn-g responses to peptides from k17 and m6 protein selected on the basis of sequence homology and predicted hla-cw*0602 binding. these responses were about 10 times more frequent in the skin-homing cutaneous lymphocyte-associated antigen-expressing (cla þ ) subset of cd8 þ t cells. cd4 þ t cells showed only borderline responses. cd8 þ t cells from cw6 þ nonpsoriatic individuals responded to some m6 peptides but very rarely to k17 peptides, and this also applied to the cla þ cd8 þ subset. these findings indicate that psoriatic individuals have cd8 þ t cells that recognize keratin self-antigens and that epitopes shared by streptococcal m protein and human keratin may be targets for the cd8 þ t cells that infiltrate psoriatic skin lesions. autoantibodies directed against citrulline-containing proteins have an impressive specificity of nearly 100% in ra patients and a suggestive involvement in the pathogenesis. the targeted epitopes are generated by a post-translational modification catalysed by the calcium-dependent enzyme peptidyl arginine deaminase that converts the positively charged arginine to polar but uncharged citrullin. the aim of this study was to analyse the presence of citrulline in the joints at different time points of collagen-induced arthritis in da rats by immunohistochemistry and to investigate how immunogenicity and arthritogenicity was affected by citrullination of rat serum albumin (rsa) and collagen type ii (cii). our results indicate that citrulline could be detected in joints of arthritic animals, first appearance at the onset of disease and increasing as disease progressed into a chronic state. unimmunized animals or time points before clinical signs of arthritis were negative. by morphology, we state that some infiltrating macrophages as well as the cartilage surface stain positive for citrulline, while the major source of citrullinated proteins appears to be fibrin depositions. a specific cit-rsa t-cell response was observed in animals challenged by citrullinated rsa, no response was recorded when rsa was used as a stimulus. the igg analysis reveals not only a response towards the modified protein but also cross-reactivity to native rsa. no t-cell or b-cell response was noted in animals injected with unmodified rsa. cit-cii induced a disease with higher incidence and earlier onset than did the native counterpart. we conclude that, in contrast to the human disease, citrulline does not seem to appear before clinical signs. as inflammation proceeds, citrulline is detected specifically in the joints. all other organs investigated were negative. we also conclude that citrullination of a protein can break tolerance and increase its arthritogenic properties. ectopic germinal centers (gcs) can be detected in the salivary glands of approximately 1/5 of patients with sjögren's syndrome (ss) and appear in both primary and secondary ss. previously, ectopic gc have been associated with increased local autoantibody production. the aim of this study was to determine whether gc in primary sjögren's syndrome (pss) defines a distinct seroimmunological phenotype. retrospectively, a material of 130 haematoxylin and eosin-stained paraffin-embedded tissue sections of minor salivary gland tissue from patients with pss was morphologically screened for the presence of ectopic gc. gc-like lesions were detected in 33/130 (25%) of the pss patients. seventy-two pss patients lacking these structures (gc-) were randomly selected for comparison. focus score was significantly increased in the gc þ patients compared to the gcpatients (p ¼ 0.035). in the gc þ group, 54.5% of the patients presented with anti-ro/ssa compared to 43.7% in the gcgroup. anti-la/ssb was detected in 31.3% of the gc þ patients compared to 25.7% of the gcpatients. sixty-one percentage of gc þ patients presented with increased levels of igg, a nonsignificant difference when compared to 39.4% in the gcpatients (p ¼ 0.089). levels of rf, ana, ena, igm and iga were similar in both patient groups, as were esr and crp. in conclusion, patients with ectopic gc have a higher focus score and more often present with autoantibodies and increased levels of igg compared to pss patients with regular focal infiltration (gc -). our findings may indicate a certain seroimmunological phenotype and warrant for further prospective studies. association between mannose-binding lectin and vascular complications in type 1 diabetes complement activation and inflammation have been suggested in the pathogenesis of diabetic vascular lesions. we investigated serum mannose-binding lectin (mbl) levels and polymorphisms in the mbl gene in type 1 diabetic (t1dm) patients with and without diabetic nephropathy and associated macrovascular complications. polymorphisms in the mbl gene and serum mbl levels were determined in 199 t1dm patients with overt nephropathy and 192 t1dm patients with persistent normoalbuminuria matched for age, sex and duration of diabetes as well as in 100 healthy control subjects. the frequencies of high and low expression mbl genotypes were similar in patients with t1dm and healthy controls. high mbl genotypes were significantly more frequent in diabetic patients with nephropathy than in the normoalbuminuric group, and the risk of having nephropathy, given a high mbl genotype, assessed by odds ratio was 1.52 (1.02-2.27), p ¼ 0.04. median serum mbl concentrations were significantly higher in patients with nephropathy than in patients with normoalbuminuria [2306 mg/l (iqr 753-4867 mg/l) versus 1491 mg/l (iqr 577-2944), p ¼ 0.0003], and even when comparing patients with identical genotypes, serum mbl levels were higher in the nephropathy group than in the normoalbuminuric group. patients with a history of cardiovascular disease had significantly elevated mbl levels independently of nephropathy status [3178 mg/l (iqr 636-5231 mg/l) versus 1741 mg/l (iqr 656-3149 mg/l), p ¼ 0.02]. the differences in mbl levels between patients with and without vascular complications were driven primarily by pronounced differences among carriers of high mbl genotypes (p < 0.0001). our findings suggest that mbl may be involved in the pathogenesis of microvascular and macrovascular complications in type 1 diabetes and that determination of mbl status might be used to identify patients at increased risk of developing these complications. neuroimmunology unit, center for molecular medicine, karolinska institutet, stockholm, sweden. e-mail: judit.wefer@cmm.ki.se dna vaccine coding for the encephalitogenic peptide mog 91-108 protects lew.1av1 from subsequent development of experimental autoimmune encephalomyelitis (eae). protection is associated with a type 1 immune response and is dependent on the presence of cpg dna motifs. the mechanisms underlying the observed reduction of eae development in protected rats have not been fully clarified. we investigated immunological characteristics of lymphocytes after dna vaccinaton and subsequent eae induction. we confirm that protection was not associated with suppression of t1 cells, as transcription of the novel molecule rat t-cell immunoglobulin-and mucindomain-containing molecule (tim-3), reported to be exclusively expressed on differentiated t1 cells, was not altered by dna vaccination. we did not note any clonal deletion upon tolerization, but detected an antigen-specific lymphocyte population upregulating ifng upon recall stimulation 3 weeks after protective dna vaccination. in protected rats, we observed (1) no alterations in antigenspecific th2 or th3 responses, (2) reduced mhc ii expression on splenocytes early after eae induction, (3) antigen-specific upregulation of ifnb upon recall stimulation and (4) reduced il-12rb2 on lymphocytes. we thus demonstrate an association of the protective effect of dna vaccination with expression of ifnb. we are currently investigating the cellular mechanisms behind this ifnb-mediated protection. multiple sclerosis (ms) is an autoimmune condition characterized by degeneration of nerve fibre myelin sheets. a candidate autoantigen, myelin basic protein (mbp), has especially attracted attention. the presence of anti-mbp antibodies is a predictor of definite ms, but their role in the pathogenesis remains obscure. t cells have long been known to play a pivotal role in the pathogenesis of ms. recently, an important role for b cells as autoantigen-presenting cells has been demonstrated in other autoimmune diseases, including rheumatoid arthritis and diabetes. the uptake of mbp by b cells and the presentation of mbp-derive peptides to t helper (th) cells by b cells may be promoted by the formation of complement (c) activating immune complexes (ics) between mbp and natural autoantibodies in healthy individuals and disease-associated anti-mbp antibodies in ms patients, respectively. we have investigated the formation of mbp-containing ic, the binding of mbp to b cells, the mbp-elicited induction of th-cell and b-cell proliferation and the cytokine production in peripheral blood mononuclear cells (pbmcs) from healthy donors grown in the presence of intact or c-inactivated serum from healthy donors or patients with ms. while mbp did not induce measurable proliferation of b cells nor cd4 þ t cells, we observed the production of tnf-a, ifn-g and il-10 by pbmc in response to incubation with mbp in the presence of sera from healthy controls as well as sera from ms patients. by contrast, no production of il-2, il-4 and il-5 was detected. we are currently investigating the capability of ms sera to promote the formation of mbp-containing ic and thereby enhance the cytokine responses, by virtue of elevated anti-mbp contents. the phagolysosomally localized acid sphingomyelinase (asmase) activated by proinflammatory cytokines such as tnf and ifn-g generates the signalling molecule ceramide which in turn results in the activation of proteases like cathepsin d. these characteristics of asmase suggest a possible role of this molecule in the phagocytotic uptake and phagosomal degradation processes of antigens or in antigen presentation. we show here that asmase -/mice fail to eliminate the noncytopathic lymphocytic choriomeningitis (lcm) virus as rapidly as littermate wildtype mice. investigation of the immune response revealed a reduced expansion of cd8 þ t cells. the secretion of ifn-g in response to contact with target cells as well as the cytolytic activity of virus-specific cd8 þ t cells was severely impaired. additionally, both phases of the lcm virus-specific dth response, mediated by cd8 þ and cd4 þ t cells consecutively, were diminished in asmase -/mice. however, the secondary memory response of virus-specific ctl was not altered, and the 614 abstracts ................................................................................................................................................................................................. virus was effectively controlled for at least 3 months by asmase -/mice. in conclusion, the results of this study suggest an involvement of the asmase in the activation, expansion or maturation of virus-specific cd8 þ t cells during the acute infection of mice with the lcm virus. novel markers for alternative activation of macrophages: macrophage galactose-type c-type lectins 1 and 2 in parallel with the th1/th2 dichotomy, macrophages are capable of developing into functionally and molecularly distinct subpopulations, due to differences in, for example cytokine environment and pathological conditions. while the best-studied, classically activated macrophage is induced by type i stimuli such as ifn-g, a type ii cytokine environment antagonizes the classical activation of macrophages and is capable of alternatively activating macrophages. however, molecular markers associated with these type ii cytokine-dependent, alternatively activated macrophages remain scarce. besides the earlier documented markers macrophage mannose receptor and arginase 1, we recently demonstrated that murine alternatively activated macrophages are characterized by increased expression of fizz1 and ym. we now report that expression of the two members of the mouse macrophage galactose-type c-type lectin gene family, termed mmgl1 and mmgl2, is induced in diverse populations of alternatively activated macrophages, including peritoneal macrophages elicited during infection with the protozoan trypanosoma brucei or the helminth taenia crassiceps, and alveolar macrophages elicited in a mouse model of allergic asthma. we also demonstrate that, in vitro, interleukin-4 and interleukin-13 upregulate mmgl1 and mmgl2 expression and that, in vivo, induction of mmgl1 and mmgl2 is dependent on interleukin-4 receptor signalling. moreover, we show that regulation of mgl expression is similar in human monocytes and monocyte-derived macrophages. hence, macrophage galactose-type c-type lectins represent novel markers for both murine and human alternatively activated macrophages; thus, paving the way for further characterization of the phenotype of macrophages occurring in th2 conditions. background: human parvovirus b19 (b19) is a ubiquitous pathogen, normally causing a mild self-limiting disease, but also capable of causing both significant pathology and long-term persistence. the small size and stability of the virus makes it suitable for mapping of the full breath and the kinetics of the cellular immune responses following acute viral infection. methods: five patients with acute primary b19 infection were included in the study and followed consecutively for up to 200 weeks. cellular immune responses were mapped by ifng enzyme-linked immunospot to overlapping peptides spanning the whole b19 genome. results: in all five acutely infected patients, we were able to monitor the kinetics of a strong specific cellular immune reaction. responses peaked at levels of 850-1850 sfc/ million pbmcs, roughly corresponding to 0.3-0.6% b19specific cd8 þ cells circulating in peripheral blood at 10-80 weeks post-infection. the responses in individual patients were directed to three or four different peptide pools, and the specificity was confined to the same cd8 epitopes present in the pools throughout the follow-up period. the majority of responses were directed to the virus nonstructural protein, only two patients showed any response to the capsid proteins, elicited by the same epitope in both cases. conclusion: the cellular immune responses to acute b19 infection are surprisingly narrow in distribution and remain at high levels for up to 80 weeks post-infection. the initial epitope specificity is maintained, and the majority of responses target the virus nonstructural protein, which is not included in vaccine preparations, evaluated against the infection. the relationship between malnutrition and malaria is controversial. on one hand, malaria may cause malnutrition, while on the other, malnutrition itself may modulate susceptibility to the disease. we investigated the association between plasmodium falciparum malaria and malnutrition in a cohort of children living on the coast of kenya. the study involved longitudinal follow-up for clinical malaria episodes and anthropometric measurements at four cross-sectional surveys. we used poisson regression analysis to investigate the association between malaria and nutritional status. compared to baseline (children with a waz or haz score of !à2), the crude incidence rate ratios (irrs) for malaria in children with low haz or waz scores (<à2) during the period prior to assessment were 1.17 (95% ci 0.91-1.50; 0 ¼ 0.21) and 0.94 (0.71-1.25; 0.67), respectively, suggesting no association between malaria and the subsequent development of pem. however, we found that age was acting as an effect modifier in the association between malaria and malnutrition. the irr for malaria in children 0-2 years old who were subsequently characterized as wasted was 1.65 (1.10-2.20; p ¼ 0.01), and a significant overall relationship between malaria and low-haz was found on regression analysis when adjusting for the interaction with age (irr 1.89; 1.01-3.53; p < 0.05). although children living on the coast of kenya continue to suffer clinical episodes of uncomplicated malaria throughout their first decade, the association between malaria and malnutrition appears to be limited to the first 2 years of life. a. astrinidou-vakaloudi, 1 s. xytsas, 1 i. diamanti, 1 h. ioannidis 2 & p. pangidis 2 1 microbiology department of general hospital of thessaloniki 'agios pavlos', thessaloniki, greece, and 2 nefrology, 2 nd ika hospital of thessaloniki, thessaloniki, greece. e-mail: stasa@hol.gr aim: renal dysfunction may influence the colonization of gastric mucosa by urea-splitting bacteria such as helicobacter pylori, by increasing urea concentrations in the gastric juice. our aim was to investigate the prevalence of h. pylori in patients with end-stage renal disease (esrd), receiving long-term haemodialysis treatment. methods: this study included 40 sera from patients with esrd (29 male and 11 female) undergoing periodic haemodialysis; mean time of treatment was 42.6 months. using elisa technique, we investigated the presence of igg and iga antibodies against h. pylori as well as igg caga (antibodies specific for caga(þ) strains of h. pylori). sera from 40 healthy blood donors were used as a control group. results: h. pylori igg antibodies were detected in 32 out of 40 (80%) patients in the dialysis group, while 31/40 (77.5%) tested positive for iga. igg caga antibodies were present in 13 out of 40 (32.5%). prevalence of h. pylori igg, iga and caga igg antibodies in the control group was 33, 7 and 15%, respectively. conclusions: although international data suggest that prevalence of h. pylori infection is the same in esrd patients as in healthy individuals, in our study that seems not to be the case. the higher blood and gastric juice urea levels may be a risk factor (among many others), but more studies are required in order to understand the relation of h. pylori infection in this group of patients. flanders interuniversity institute for biotechnology, department of molecular and cellular interactions, free university of brussels, brussels, and 2 pasteur institute of brussels, mycobacterial immunology, brussels, belgium. e-mail: tgartner@vub.ac.be immunity against tuberculosis (tb), caused by mycobacterium tuberculosis, depends largely on activation and maintenance of strong cell-mediated immune responses involving both cd4 þ and cd8 þ t cells and the ability to respond with th1-type cytokines, particularly ifn-g. recent studies suggested that bcg, the only licensed vaccine against m. tuberculosis, may fail to induce t-cell responses in the lung mucosa and may therefore not protect against pulmonary tb. a decrease in tb mortality may be achieved by enhancing immunity in the lung. the present study evaluated the induction of antigen-specific immunity in the lung by intranasal (i.n.) delivery of the lipoprotein i (opri) from pseudomonas aeruginosa. opri has shown to be a toll-like receptor 2/4 agonist that, when given subcutaneously, induces type-1 immune responses against heterologous antigens. here, a fusion of opri to ag85a of mtb (opri-ag85a) was used as a subunit vaccine in homologous prime-boost immunizations. in addition, opri-ag85a was combined with an ag85a-encoding dna vaccine (ag85a dna) or with bcg in heterologous prime-boost vaccinations. intranasal and parenteral delivery with opri-ag85a elicited comparable t-cell responses in the spleen; in addition, i.n. delivery elicited specific t-cell responses in the lung lymph nodes (llns). intramuscular delivery of ag85a dna induced significant systemic th1 immune responses. intranasal boosting with opri-ag85a enhanced this response and in addition induced an antigen-specific ifn-g response in lln. opri may therefore be an efficient adjuvant for mucosal boosting. we continue to evaluate the protection induced by opri-based prime-boost vaccinations against pulmonary tb. results on the immunogenicity and protection against intravenous mtb h37rv infection will be presented. toll-like receptors (tlrs) are pattern recognition receptors of the innate immune system, which recognize molecular structures on pathogens or cellular stress-associated molecules. tlr-ligand interactions trigger activation of inflammatory signal transduction and expression of genes involved in host defense. in this study, we have examined the requirement for different tlr adaptor molecules in virus-induced chemokine expression and are currently trying to identify the tlr involved. we have found that both a herpesvirus [herpes simplex virus (hsv)] and a paramyxovirus (sendai virus) require a functional genome to induce expression or proinflammatory chemokines in human and murine monocytic cell lines. for both viruses, this is independent of the tlr adaptor molecules trif and mal. however, overexpression of the vaccinia virus-encoded inhibitor of tlr-signalling a52r or dominant-negative myd88 totally inhibited hsv-induced rantes expression but only partially prevented sendai virus from inducing this chemokine. this suggests that hsv-induced rantes expression occurs via a tlr pathways, whereas sendai virus utilizes both tlr-dependent and -independent pathways to stimulate expression of rantes. we are currently trying to identify the tlrs involved. data from these studies will also be presented at the meeting. 2 0 -5 0 -oligoadenylate synthetases are interferon-induced, double-stranded rna-activated antiviral enzymes which are the only proteins known to catalyse 2 0 -specific nucleotidyl transfer. this first crystal structure of a 2 0 -5 0oligoadenylate synthetase reveals a structural conservation with the 3 0 -specific poly(a) polymerase that, coupled with structure-guided mutagenesis, supports a conserved catalytic mechanism for the 2 0 -and 3 0 -specific nucleotidyl transferases. comparison with structures of other superfamily members indicates that the donor substrates are bound by conserved active site features while the acceptor substrates are oriented by nonconserved regions. the 2 0 -5 0oligoadenylate synthetases are activated by viral doublestranded rna in infected cells and initiate a cellular response by synthesizing 2 0 -5 0 -oligoadenylates, that in turn activate rnase l. this crystal structure suggests that activation involves a domain-domain shift and identifies a putative dsrna activation site that is probed by mutagenesis. we demonstrated that this site is required both for the binding of dsrna and for the subsequent activation of oas. this rna-binding site is different from known rna-binding site; rather than forming a defined three-dimensional domain, it is located at the interface of the two major domains in oas. this novel architecture ensures that the dsrna helix can make simultaneously contact with both domains of oas and ensure the subsequent structural rearrangement leading to the activation of oas. our work provides structural insight into cellular recognition of double-stranded rna of viral origin and identifies a novel rna-binding motif. bacteria-specific iga antibodies are efficient opsonins for neutrophils and mononuclear phagocytes, provided that the phagocytes express the fca receptor (cd89). expression of cd89 can be stimulated by inflammatory cytokines, activated complement factors and certain microbial components. in one study, unstimulated phagocytes were able to ingest iga antibody-treated pneumococci, but only in the presence of complement, which was found to be activated by the iga antibodies along the alternative pathway. pneumococci produce iga1 protease that cleaves human iga1, but not iga2, molecules in the hinge region. this leaves iga1 as faba (monovalent) deprived of fca which contains the docking site for cd89. iga1 is the vastly predominant subclass of iga in the upper airways and circulation of humans. aims: to examine the effects of iga1 protease activity and complement on phagocytosis of iga antibody-coated pneumococci by an unstimulated human phagocytic cell line (hl60). materials and methods: iga1 and iga2 monoclonal antibodies to serotype 4 pneumococcal capsular polysaccharide (ps) were generated by heterohybridoma technique involving b cells from human vaccinees. isogenic serotype 4 pneumococci with and without iga1 protease activity, respectively, were obtained after inactivation of the iga gene of the tigr4 strain. opsonophagocytosis was quantitated using the assay described by romero-steiner et al. based on enumeration of surviving bacteria by culture. the integrity of iga molecules was examined by western blotting. results: both iga1 and iga2 antibody to type-4 polysaccharide-induced phagocytosis of iga1 protease-deficient type-4 pneumococci equally well in the absence as in the presence of complement. iga1 antibody to type-4 polysaccharide displayed a fourfold higher opsonophagocytosis titer against iga1 protease deficient compared to homologous wildtype target bacteria. a similar effect of iga1 protease activity of the target bacteria was not observed in a parallel experiment where iga2 antibody to type-4 polysaccharide served as opsonin. iga1 antibody extracted from iga1 protease-producing target bacteria was almost exclusively in the form of faba. conversely, iga1 from protease-deficient bacteria and iga2 from both types of bacteria were intact. conclusions: these results indicate that the iga1 protease activity of s. neumoniae may help the bacteria escape iga1 antibody-mediated opsonophagocytosis. besides, in these experiments, iga-mediated opsonophagocytosis was independent of complement. vitamins e and c have been found to increase the cellular and humeral immunity of pigs. vitamin e deficiency has also been found to predispose pigs to different diseases, e. coli infection is one among them. after weaning, the vitamin e status of pigs often decreases to a critical low level. in this experiment, we studied whether vitamin c supplementation would be a possible feeding strategy to optimize the immune status of weaners. the interaction between vitamin e and c is interesting due to the reported sparing action on vitamin e or synergism between these to vitamins. piglets were weaned at day 28 of age from sows fed increasing dietary vitamin e during lactation, and piglets were during the following 3 weeks fed either a control diet or this diet supplemented with 500 mg stay-c per kg. blood sampling was obtained weekly from day 28 and until day 49 of age. on the same days, one piglet per dietary treatment was killed and alveolar macrophages (am) were harvested. vitamin c supplementation increased the concentration of igm in serum of piglets throughout the weaning period. although the vitamin e concentration in am decreased with increasing age of the piglets, the concentration was numerically higher in piglets of sows fed the high dietary level of vitamin e. however, vitamin c supplementation tended to increase the total am concentration of vitamin e after weaning and increased the proportion of the biologically most active isomer of vitamin e [rrr-(a-tocopherol)] in the am. the eicosanoid synthesis by am was not influenced by the vitamin c supplementation, but the synthesis of leukotriene b4 was decreased 2 weeks after weaning compared to other days of am harvesting. in conclusion, dietary vitamin c supplementation improved the immune responses of piglets after weaning. a whole blood stimulation assay with escherichia coli (o111:b4) endotoxin was established to measure the capacity of dairy cows to produce the proinflammatory cytokine tumour necrosis factor-a (tnf-a) ex vivo. initially, a time-and dose-dependent study was carried out to find the optimal stimulation conditions for the tnf-a response. the tnf-a response peaked between 3 and 4 h at 38.5 c. a dose in the range of 5-10 g of e. coli lipopolysaccharide (lps)/ml whole blood was found to give the maximum tnf-a response. thirty-eight danish-holstein dairy cows were investigated for their tnf-a responsiveness ex vivo in the periparturient period. heparin-stabilized blood samples were collected seven times over a period of 4 months (weeks à3, à1, 2, 3, 5, 9 and 13 around calving) and stimulated with 5 g/ml of e. coli lps. indeed, fluctuations in the tnf-a responsiveness occurred over time. moreover, the mean tnf-a responsiveness of 38 cows was found to be significantly increased (p < 0.001) in the weeks close to calving. however, in the more stabile physiological periods, some cows had a consistently low tnf-a response, whereas others had high a tnf-a response. we are currently investigating whether high and low tnf-a responders to e. coli lps also exist in dairy cows in vivo. moreover, the importance of tnf-a responsiveness ex vivo to dairy cows' susceptibility and clinical response to experimental e. coli infections in the udder is being investigated. coelomic cytolytic factor (ccf) is a 42 kda invertebrate pattern recognition molecule isolated from the coelomic fluid of the earthworm eisenia foetida (oligochaeta, annelida). ccf displays a number of similarities with the mammalian cytokine tumour necrosis factor-a (tnfa) as a result of a shared n,n 0 -diacetylchitobiose lectin-like domain. however, these similarities are solely functional and are not based on any (dna or amino acid) sequence homology, thus suggesting a form of convergent evolution. in particular, the lectin-like domain of tnf-a has been shown to induce membrane depolarization in various mammalian cell types, through interactions with endogenous amiloride-sensitive ion channels. this nonreceptor-mediated activity of tnf-a has been reported to be involved in the resorption of oedema. likewise, the lectin-like domain of ccf also induces membrane depolarization in mammalian cells. here, we show that ccf appears to be able to induce oedema resorption in an alveolar epithelial cell line through its lectin-like domain. this lectin-like domain of ccf interacts (directly or indirectly) with endogenous sodium and/or chloride channels, and not potassium channels, on mammalian cells. additionally, we suggest that the jnk/sapk and erk1/2 pathways are involved in ccf-induced macrophage activation. these results further establish the functional analogy between an invertebrate pattern recognition molecule and a mammalian cytokine and, from a more applied point of view, suggest the possibility of utilizing ccf in the treatment of oedema. release of svegf and svegfr1 from white blood cells and platelets during surgery and stimulation with bacterial antigens introduction: the influence of surgery on release of soluble vascular endothelial growth factor (svegf) and the soluble vascular endothelial growth factor inhibitory receptor 1 (svegfr1) is unknown. we studied the effect of major and minor surgery on potential variations in svegf and svegfr1 concentrations in vivo and on bacterial antigen-induced release of svegf and svegfr1 from whole blood in vitro. methods: sixty-one patients with abdominal diseases undergoing five different surgical procedures were included. blood samples were drawn from anaesthetized patients before and after the operation. white blood cells and platelets were counted, and plasma svegf and svegfr1 was determined by an elisa method. whole blood from each blood sample was stimulated in vitro with bacteria-derived antigens (lps or protein-a) and svegf and svegfr1 levels were subsequently determined in the supernatants. stimulation with isotonic saline served as control assay. neither svegf or svegfr1 in plasma changed during surgery. in vitro stimulation of blood samples with bacteria-derived antigens resulted in a significant increase in svegf (p < 0.0001) and a less pronounced but still significant increase in svegfr1. release of svegf due to stimulation was significantly higher after the operation (nonsignificant), whereas svegfr1 release remained largely unchanged after surgery. correlation between bacterial antigen-induced release of svegf and neutrophile cell count was highly significant (p < 0.0001). there was no correlation between svegf and platelet cell count, and bacterial antigen-induced svegfr1 release did not correlate with counts of neutrophils and platelets. conclusions: plasma svegf and svegfr1 concentrations did not change during surgery. in vitro bacterial stimulation led to increased release of svegf and svegfr1, which was not significantly amplified during surgery and which may be related to number of circulating neutrophils. natural killer cell functions and subsets after in vitro stimulation with il-2 and il-12, with special emphasis on intracellular ifn-g and nk-cell cytotoxicity r. nyboe, 1,2 t. rix, 1,2 j. krog, 1,2 e. tønnesen 1 & m. hokland 2 1 department of anaesthesiology and intensive care, aarhus university hospital, and 2 institute of medical microbiology, and immunology, university of aarhus, aarhus, denmark. e-mail: rnsr@studmed.au.dk materials and methods: isolated cryopreserved human peripheral blood mononuclear cells (pbmcs) were stimulated with il-2 and il-12. this stimulation has previously been shown to activate nk cells. cell cytotoxicity was measured by flow cytometry after incubation with k562 cells. this method was compared to the current standard 51cr release assay. cells were treated with bfa to accumulate ifn-g, stained for surface markers, permeabilized and stained for intracellular ifn-g. flow cytometry was then performed to measure intracellular ifn-g production in pbmc, especially in nk cells. results: we have demonstrated that stimulation with il-2 and il-12 is effective in increasing the number of ifn-gpositive cells. there is a distinct difference between the cd3-cd56dim and the cd3-cd56bright subsets, with a much greater proportion of ifn-g-positive cells in the cd3-cd56bright subset. the effects of stimulation with il-2 and il-12 on cytotoxicity will be presented, as will the relation between ifn-g production and cytotoxicity. in addition, we will present results of these assays applied to porcine cells. discussion: in combination, these tests will address nk cell function by combining cytotoxicity with ifn-g production in nk cell subsets. the results will demonstrate whether this could serve as a useful tool in describing nkcell function, which could be of value in clinical and experimental settings. culture of regulatory t-cell lines from bronchial mucosa t lymphocytes play a major role in many immune responses. in the last decade, special focus has been on the function of th1 and th2 effector cells. now the importance of regulatory cd4 þ cd25 þ t cells in maintenance of the immunological homeostasis emerges. sarcoidosis is a multisystem granulomatous disorder often affecting the lungs. the typical sarcoid granulomas consists of epitheloid cells, macrophages and lymphocytes, mainly cd4 þ t cells of th1 phenotype. we have cultured t cells from bronchial biopsies of patients with sarcoidosis as well as from controls in high levels of interleukin 2 (il-2) and il-4 and demonstrate spontaneously arising cd4 þ cd25 þ populations and high concentrations of il-10 in these cultures. the main difference between cultures of sarcoid origin compared to controls is a very much higher concentration of the inflammatory cytokines il-6 and tnf-a in cultures of sarcoid origin. the effects of hyperbaric exposure on human peripheral blood mononuclear cells, with special emphasis on natural killer cell cytotoxicity and subsets materials and methods: as an experimental physiological stress model, we examined the effects of hyperbaric exposure on peripheral blood mononuclear cells (pbmcs) obtained from venous blood drawn from eight divers during a simulated heliox saturation dive. eight persons working in normobar atmosphere outside the pressurized chamber served as control donors. the spontaneous cytotoxicity of the pbmcs was estimated in a 4 h 51cr-release assay using k562 as nk-sensitive target cells. the pbmcs were characterized, using 4-colour flow cytometry, with special emphasis on the nk-cell subsets. the data were statistically analysed using a multivariate regression model (stata 8.2). p values <0.05 was considered statistically significant. results: the estimated cytotoxicity increased significantly in both the group of divers and control donors during the dive (pdivers < 0.01 and pcontrols < 0.01). although the cytotoxicity increased relatively more (p < 0.01) in the group of divers compared to the group of control donors between day 1 and 2. discussion: the increased cytotoxicity of pbmc estimated in the group of divers indicate that parts of the cellular immune system are affected during the extreme physiological conditions induced during the initial phase of the presented experimental hyperbaric setup. the increase in cytotoxicity observed in the group of control donors could hypothetically reflect the stress level in persons working outside the pressurized chamber during the dive. the interaction with adhesion molecules expressed by vascular endothelium is the first step in lymphocyte infiltration into tissues. at both cutaneous and mucosal sites interleukin-10 (il-10), il-12 and transforming growth factor (tgf)-b are important regulators of chronic inflammatory disease, where cutaneous lymphocyteassociated antigen (cla) and ae integrin (cd103) may be expressed. unlike cla, cd103 is not believed to play a role in tissue-specific homing but may help to retain t cells within epithelial layers. we have previously shown that il-12 alone can together with an unknown cofactor increase the expression of cla. stimulation with streptococcal pyrogenic exotoxin c (spec) increased the expression of cd103 by cd8 þ but not cd4 þ t cells. while il-12 increased superantigen-stimulated expression of cla, this cytokine strongly inhibited the cd103 expres-sion, and a combination of il-12 and tgf-b completely abrogated the induced cd103 expression. conversely, il-10 suppressed cla but increased cd103 expression. these findings indicate that, in addition to suppressing the development of th1-mediated inflammatory responses, il-10 may also inhibit the migration of cd8 þ t cells into the skin while il-12 promotes such migration. thus, the expression of cla and cd103 may be antagonistically regulated by il-10 and il-12, and the balance between these cytokines could influence the t-cell migration of inflammatory cells into epithelial tissues. during contact sensitivity reaction, immune cells proliferate. in order to study the histological picture of these proliferation phases, we used a mouse model of contact sensitivity in the oral mucosa and on skin. we also used bromodeoxyuridin (brdu, an analogue to thymidin) that is incorporated into the nucleus during cell replication. the hapten oxazolone (oxa) was used to sensitize and elicit the oral mucosa and/or the ear skin. mice were killed at various times after elicitation, and unsensitized animals were also exposed to the hapten as controls. brdu (25 mg/ kg animal) was injected i.p. 2 h before the kill. specimens from the oral mucosa, ear skin and submandibular and auricular lymph nodes were cut and fixed in 4% paraformaldehyde. they were then treated with acid and biotinylated anti-brdu antibody and developed using abc-kit and dab. the analyses were performed using a leica light microscope and the computer program analysis. in the oral mucosa, the frequency of proliferating cells were increasing during the observation period, 4-24 h after elicitation, regardless of site of sensitization. the proliferating cells were found mainly in the basal cell layer of the epithelium. similar patterns were found in ear skin. the regional lymph nodes demonstrated a few scattered proliferating cells 4 h after elicitation. after 24 h, these cells were found frequently in the whole lymph node. control animals exhibited considerable less proliferating cells at all times. we conclude that most proliferating cells were found 24 h after elicitation locally at the hapten-exposed sites (the oral mucosa or the ear skin) as well as in the regional lymph nodes. the endogenous nucleoside adenosine is released in excess during inflammation or other metabolic stress and is generally known to deliver tissue protective anti-inflammatory effects. adenosine acts via four adenosine receptors of which the a2a receptor is the predominant form in t cells. adenosine levels are elevated in asthmatic lung, and adenosine can directly induce mast cell degranulation and bronchoconstriction in these patients. instead, the role of anti-inflammatory mechanisms of adenosine on t cells in asthma is unclear. aim: to study the a2a receptor expression in peripheral blood cd4 þ t cells in asthmatic and healthy individuals using flow cytometric and quantitative real-time pcr methods. results: unstimulated cd4 þ cells of asthmatic patients expressed significantly lower levels (p < 0.001) of a2a receptor in protein level (mean percentage of cells positive ae sem: 76.8 ae 1.2, n ¼ 6) compared to healthy individuals (90.4% ae 1.9, n ¼ 4). double staining for cd69 expression showed that stimulation of cd4 þ cells decreased a2a expression in both groups but indicated that the detected lower levels of a2a in unstimulated cells of asthmatics was not due to preactivation in these patients. surprisingly, a2a mrna expression in unstimulated cd4 þ cells was significantly higher (p < 0.05) in asthmatics (n ¼ 28) compared to healthy controls (n ¼ 7). the expression did not correlate with serum total ige levels. conclusions: asthmatic individuals express less a2a adenosine receptor on their peripheral cd4 þ t cells. the higher mrna levels instead may point to a negative feedback regulation in the receptor expression. the role of possibly decreased adenosine-mediated anti-inflammatory effects in asthma pathogenesis require further studies on this t-cell mediated disease. the chronic inflammatory skin disease atopic eczema (ae) affects almost 15% of the population in many countries today. the pathogenesis of ae is not fully understood. a combination of genetic predisposition and environmental factors like microorganisms seems to contribute to the symptoms. the yeast malassezia sympodialis is part of our normal skin micro flora but can act as an allergen and elicit specific ige and t-cell reactivity in patients with ae. recently, we identified a novel major m. sympodialis allergen, designated mala s 11 (22.4 kda), with sequence similarity to the mitochondrial enzyme manganese superoxide dismutase (mnsod). interestingly, mala s 11 has a high degree of homology to human mnsod. the aim of this study was to examine the effects of recombinant mala s 11 on antigen-presenting dendritic cells. monocytederived dendritic cells (mddcs) from healthy blood donors were cultured with or without mala s 11 for different time periods. it was found that the maturation marker cd83 and the costimulatory molecules cd80 and cd86 were upregulated on the mddcs exposed to mala s 11 for 24 h, as demonstrated by flow cytometry. furthermore, coculture of mddcs with mala s 11 for 9 h induced an increased production of the inflammatory cytokines il-6 (200-fold), tnf-a (100-fold) and il-8 (sixfold), as detected by the cytometric bead array (cba) analysis. our results suggest that mala s 11 affects the immune response through dc maturation and production of inflammatory cytokines. the potential cross-reactivity with human mnsod needs to be explored and the exact role of mala s 11 in the pathogenesis of ae assessed in clinical studies involving skin prick and atopy patch tests. allergen-specific immunotherapy (sit) is commonly conducted with allergen extracts adsorbed to aluminium hydroxide (alum). drawbacks linked to the use of alum, such as the formation of granuloma at the site of injection, have led to suggestions of novel allergen carriers. an alternative carrier is 2 mm carbohydrate-based particles (cbps). in mouse, allergen-coupled cbps have been demonstrated to skew the allergen-specific immune response towards a th1-like activity (grönlund et al. immunology, 2002) . we here coupled the recombinant major cat allergen fel d 1 to cbps (cbp-fel d 1) by cyanogen-bromide activation, resulting in covalent binding. the effect of cbp-fel d 1 on monocyte-derived dendritic cells (mddcs) from healthy human blood donors was studied. we found that the majority of the cd1a þ mddcs were capable of taking up fitc-labelled cbp-fel d 1, as demonstrated by flow cytometry and confocal laser scanning microscopy. furthermore, incubation with cbp-fel d 1 resulted in an upregulation of the costimulatory molecule cd86 on the mddcs, which was not observed with fel d 1 or cbps alone. finally, cbp-fel d 1 induced a fivefold increase in the release of the pro-inflammatory cytokine tumour necrosis factor (tnf)-a and a fourfold increase in the release of the chemokine interleukin-8 from mddcs. taken together, the effects cbps possess make them interesting as novel allergen carriers for sit. the cysteine protease der p1 from dust mite of the genus dermatophagoides pteronyssinus is a major type i allergen. about 80% of house dust mite (hdm) allergic individuals are reactive to this protease in standard assays for detection of ige. a curative treatment for atopic allergy is immunotherapy (it) with hdm extracts which are complex mixtures occasionally resulting in anaphylactic reactions. novozymes focuses on developing a recombinant variant of der p1 which exhibit lowered risk of ige-mediated allergic reactions, while maintaining its ability to trigger proper th-cell responses. this may provide a safer alternative for specific it of hdm allergy. a secreted recombinant form of pro-der p 1 expressed by saccharamyces cerevisiae was obtained by fusion of the pro-enzyme to a fungal signal peptide. the n-glycosylation site of der p1 was mutated resulting in a deglycosylated pro-enzyme with a molecular mass of 35 kda. protein purification procedure was developed to obtain nearly pure der p1 protein followed by determination of concentration by active-site-titration with the cysteine protease inhibitor e64. the deglycosylated recombinant pro-der p 1 revealed immunologic similarity to the native der p 1 molecule when compared in basophile histamine release, ige-binding assays and t-cell proliferation assays. by in silico epitope mapping of a modelled 3-dimensional structure of der p1, five putative igg and ige epitopes were predicted. by protein engineering, the predicted epitopes were removed one by one in der p1 and screening for hypoallergenic variants was performed. combining inhaled long-acting b-2 agonist (laba) and inhaled corticosteroid (ics) seems to offer asthma control at a lower dose of ics than achieved by ics alone. fine mapping of t-cell surface markers by flow cytometry offers a detailed status of the individual's inflammatory response. the frequency of mt2 (cd4 þ cd45ra -cd62l þ cd11adim) and mt1 (cd4 þ cd45ra -cd62l -cd11abright) cells in peripheral blood, and their ratio, has been shown to differ predictably in atopics and patients with leprosy, where mt2 correlates with a th2 phenotype and mt1 with a th1 phenotype. stable asthmatics, requiring fluticasone propionate (fp) 750-1000 mg daily or equivalent, were randomized to receive, double-blinded, either seretide 1 [salmeterol and fluticasone propionate (sfc, n ¼ 16)] 50 mg/500 mg bd or fp 500 mg bd (n ¼ 17). if asthma was controlled based on lung function and symptoms at clinic visits every 6 weeks, ics dose was tapered until asthma exacerbated or 0 mg was reached. the frequency and ratio of mt2 and mt1 t cells of the patients was monitored at 6 week intervals. as treatment tapered, the frequency of mt2 cells decreased (p ¼ 0038 from first to final visit), whereas that of mt1 cells increased. the ratio of mt2/mt1 decreased (p ¼ 0049 from first to final visit). in patients receiving laba þ ics, the fall in mt2/mt1 ratio appeared to be more pronounced than in patients receiving ics alone. thus, the mt2 phenotype may be associated with stable asthma, whereas an imminent exacerbation may associate with an increase in the mt1 phenotype. laba may allow for a greater effect of fp on the mt ratio. activation of complement pathways, leading to production of c3a and c5a anaphylatoxins, has been postulated in the pathogenesis of asthma and allergic airway inflammation. the present study was undertaken to investigate the role of mannan-binding lectin (mbl), an initiator of the lectin pathway of complement, in asthma and allergic rhinitis. mbl levels and mbl-induced complement activity were determined in 19 patients of bronchial asthma with allergic rhinitis and 20 unrelated, age-matched controls of indian origin. mbl levels and activity were correlated with percent eosinophilia and percent predicted fev1 values of the patients. association of single nucleotide polymorphisms (snps) in exon 1 and intron 1 of the mbl with the disease, clinical markers, mbl levels and mbl-induced complement activity was analysed using standard statistical tools. significantly higher mbl levels and activity were observed in patients of bronchial asthma with allergic rhinitis as compared to the controls. we identified five snps, of which two, a816g in exon 1 and g1011a in intron 1 of the mbl, were novel. snp g1011a was significantly associated with the disease (p ¼ 0.0024, or ¼ 5.8696, 95% ci: 1.7316 < or < 19.8963). individuals with 'a' allele at position 1011 showed increased mbl levels, activity and disease severity. our results suggest that 'a' allele at position 1011 leading to high mbl levels and complement activity may be contributing to the severity of bronchial asthma and allergic airway inflammation. serum resistance of borrelia burgdorferi strains belonging to the b. afzelii and b. burgdorferi sensu stricto genospecies is dependent on binding of complement inhibitor factor h. we recently reported that factor h binding by b. burgdorferi is due to inducible expression of several approximately 20 kda plasmid-encoded, surface-exposed lipoproteins related to ospe (e.g. erpa, erpp and p21). in addition, a second class of factor h-binding proteins of approximately 27-35 kda has been described. the ospe-related lipoproteins are dramatically induced by b. burgdorferi during transmission from its tick vector into the mammalian host. the induction of ospe-related lipoproteins during mammalian infection may play a key a role in the borrelial evasion of the host's immune system. the goal of the present study was to define the factor h-binding regions of ospe-related proteins using mutagenesis, peptide mapping and surface plasmon resonance analysis (biacore). the combined studies revealed that the c-terminal regions of both human and mouse factor h (scrs 18-20) specifically bind to ospe-related lipoproteins. we also found fhr-1, whose c-terminal scrs 3-5 are homologous to scrs 18-20 of factor h, to bind to ospe. peptide mapping revealed five putative regions (designated i-v) in ospe that could directly interact with factor h. deleting the c-terminal 15 amino acid residues from region v of p21 abolished its ability to bind factor h. at the same time, however, synthetic peptides corresponding to the c-termini of ospe, p21 and erpp did not inhibit factor h binding to ospe. thus, the c-terminal-binding region v appears to be necessary but not sufficient for factor h binding. when a more specific mutation strategy was employed, where single amino acid residues in peptides spanning over the factor h-binding regions were mutated to alanines, we observed that lysines in the factor h-binding regions of ospe were required for factor h binding. the combined data have revealed that key lysine residues in ospe-related lipoproteins and ionic interactions are crucial for factor h interactions. furthermore, binding of ospe to the c-termini of both mouse and human factor h suggests that borrelia spirochetes utilize analogous complement resistance mechanisms in both rodents and man. in borrelia garinii strains, which in in vitro analyses have been found to be sensitive to complement killing, differences in the ospe sequences as well as in the expression of factor h-binding proteins may account for their susceptibility to serum lysis. role of yada, ail and lipopolysaccharide in serum resistance of yersinia enterocolitica serotype o:3 mannan-binding lectin (mbl), l-ficolin and h-ficolin are pattern recognition molecules of the innate immune system. we investigated the ability of these molecules to bind to different serotypes and noncapsulated variants of streptococcus pneumonia and staphylococcus aureus. we found that mbl binds to noncapsulated s. aureus strain (wood) but not any of the examined s. pneumoniae serotypes. l-ficolin binds to some capsulated s. pneumoniae serotypes (11a, 11d and 11f) as well as some capsulated s. aureus serotypes (type-1, -8, -9, -11 and -12). h-ficolin does not bind to any of the examined s. pneumoniae and s. aureus serotypes included in this study but did bind to a strain of aerococcus viridans. when bound to bacteria, mbl and h-ficolin initiated activation of complement factor c4, whereas l-ficolin did not. during this study, quantitative assays for the three proteins were developed and the concentration in 97 plasma samples were determined and the median values were estimated at 0.8 mg of mbl/ml, 3.3 mg of l-ficolin/ml and 18.4 mg of h-ficolin/ ml, respectively. the absence of early complement components (c1, c4 and c2 but not c3) is a predisposing factor for systemic lupus erythematosus (sle). recently, we demonstrated that, in c4-deficient (c4 def.) mice, igm-containing immune complexes (igm-ic) are filtered by the splenic barrier of marginal zone macrophages (mzm), resulting in an increased immune response against antigens within these igm-ic, but this could not be observed in wildtype or c3 def. mice. we hypothesized that splenic cd11b þ mzm play an important role in the induction of autoimmunity, and we therefore analysed their cytokine profile after isolation with the help of magnetic antibody cell sorting. mrna was isolated, and real-time pcr was performed with specific primers for murine ifn-g (ifn-g), interleukin-12 (il-12) and ifn-a (ifn-a). we observe a moderate increase of il-12 and ifn-g mrna in cd11b þ cells of c4 def. mice compared to wildtype cells. surprisingly, the concentration of ifn-a mrna is six times higher in c4 def. mice. preliminary results suggest that mrna in cd11b þ cells of c3 def. mice is even lower than that in wt. six hours following i.v. application of 20 mg of a abstracts 625 .................................................................................................................................................................................................. murine monoclonal igm anti-dsdna antibody, production of il-12, ifn-g and ifn-a mrna is increased in cd11b þ cells of both c4 def. and wt mice. several references described increased levels of inf-a in patients with sle. dendritic cells are discussed as a major source of ifn-a. our observation that c4-deficient, sle-susceptible mice demonstrate an increased spontaneous ifn-a production by splenic cd11b þ marginal zone macrophages could be an early sign and a trigger for the development of sle. this is supported by the fact that the absence of c3 is not a predisposing factor for sle and our observation that c3 def. animals display low levels of ifn-a mrna. 200-400 million people worldwide and represents one of the leading causes for liver cirrhosis and hepatocellular carcinoma. control over the hbv infection is achieved mainly by vaccination with hepatitis b surface antigen (hbsag). hbsag contains n-linked glycosylation side and is recognized by both mbl-a and mbl-c in a cadependent manner. hbsag-mbl complexes activate complement and may thus affect humoural immunity. to investigate the role of mbl in humoural responses to hbsag, we immununized mice that lack both mbl-a and mbl-c proteins with soluble hbsag. it has been shown that deficiencies in other complement components like c1q, c4 and c3 result in decreased antibody responses. however, mbl double ko animals mounted dramatically increased humoural responses. after priming, mbl double kos mounted hbsag-specific igm responses, which were threefold higher than wt controls. after boosting the hbsag, total igg was 10-fold higher in mbl ko than in wt control animals. similar to the response to hbsag, other glycosylated soluble antigens (e.g. invertase) induced better humoural responses in mbl double ko animals, suggesting that mbl plays an important role in a negative feedback regulation of adaptive immunity. reconstitution experiments with rmbl partially rescued the ko phenotype. we propose that the clearance of glycoprotein antigens in mbl ko is handled differently from the wt, resulting in better stimulation of humoural responses. alternatively, glycoprotein-ag-mbl-rich complexes inhibit b-cell responsiveness via putative mbl receptors. the complement system is an important part of the innate immune system. the activation of complement proceeds through three different pathways that converge in the generation of c3-activating enzyme complexes. complement activation via the lectin pathway is initiated when recognition molecules, mannan-binding lectin (mbl) or ficolin, bind to carbohydrate structures characteristic for microbial surfaces. in the circulation, mbl and ficolins are found in association with three structurally related mblassociated serine proteases (masp)-1, -2 and -3 and a small, nonenzymatic component, map19. masp-2 has been shown to elicit complement activation through the sequential proteolytic cleavage of c4 and c2 upon binding of mbl/masp-2 complexes to microbial surfaces. we have recently uncovered a polymorphism in the masp-2/map19 gene in a patient shown to be deficient in the lectin pathway of complement activation. the polymorphism results in a single amino acid substitution in the n-terminal part of the masp-2 protein. recombinant wildtype masp-2 and masp-2 containing the amino acid substitution in question was produced, and the ability to activate complement was studied. the mutation had a profound impact on masp-2 function, resulting in the lack of complement activation through the lectin pathway. elisa-based experiments showed that the mutation leads to the impairment of complement activation through influencing the binding of masp-2 to mbl or ficolins. deficiencies in the lectin pathway of complement activation have so far been accounted for only by lack of functional mbl. the mutation described above is the first defect described affecting both activation through mbl and the ficolins. .................................................................................................................................................................................................. th1, th2 and treg cell balance. dcs are present in the gut mucosa and may thus be target for modulation by gut microbes, including ingested probiotics. here, we tested the hypothesis that species of lactic acid bacteria, important members of the gut flora, differentially activate dc. a large panel of human gut-derived lactobacillus and bifidobacterium spp. was screened for dc-polarizing capacity by exposing bone marrow-derived murine dc to lethally irradiated bacteria. cytokines in culture supernatants and dc-surface maturation markers were analysed. substantial differences were found among strains in the capacity to induce interleukin-12 (il-12) and tumour necrosis factor (tnf)-a, while the differences for il-10 and il-6 were less pronounced. bifidobacteria tended to be weak il-12 and tnf-a inducers, while both strong and weak il-12 inducers were found among the strains of lactobacillus. remarkably, strains weak in il-12 induction inhibited il-12 and tnf-a production induced by an otherwise strong cytokine-inducing strain of lactobacillus casei, while il-10 production remained unaltered. selected strains were tested for induction of dc maturation markers. those lactobacilli with greatest capacity to induce il-12 were most effective in upregulating surface mhc class ii and cd86. moreover, l. casei-induced upregulation of cd86 was reduced in the presence of a weak il-12inducing l. reuteri. in conclusion, human lactobacillus and bifidobacterium spp. polarize differentially dc maturation. thus, the potential exists for th1/th2/treg-driving capacities of the gut dc to be modulated according to composition of gut flora including ingested probiotics. the intestinal micro flora is indispensable in developing and maintaining homeostasis of the gut-associated immune system. evidence indicates that lactic acid bacteria (lab), e.g. lactobacilli and bifidobacteria, have beneficial effects on the host. established health effects include increased gut maturation, antagonisms towards pathogens and immune modulation. the objective of this study is to evaluate the immunomodulating properties of a range of lab of human origin. as dendritic cells (dcs) play a pivotal role in the balance between tolerance and immunity to commensal microorganisms, in vitro-generated immature dcs serve as a suitable model for studying the immunomodulating effects of lab. human immature dcs were generated in vitro from monocytes and exposed to lethally uv-irradiated lab. the effect of various species of lab on dcs in direct contact was evaluated. furthermore, the maturation pattern of dcs separated from the bacteria by an epithelial cell layer (caco-2 cells), which should mimic the intestinal environment, was studied. cytokine secretion (il-12, il-10 and tnf-a) and upregulation of maturation surface markers on dcs (cd83 and cd86) was measured. different lab induced diverse cytokine responses. some strains were strong il-12 and tnf-a inducers and others weak. all strains induced il-10. different lab also differentially modulated expression of cd83 and cd86 on dcs. although some variation in the response to lab of dcs generated from different blood donors was observed, general differences in the effect of the various lab was revealed. experiments with the dc caco-2 coculture system are ongoing. different species of lab differentially affect dc maturation; this suggets that the gut flora plays a pivotal role in polarization of the immune response. natural killer (nk) cells are cells of the nonspecific immune system lysing altered self-cells. a noncytolytic subset of nk cells may serve a regulatory role by secreting cytokines. bacteria translocating across the gastrointestinal mucosa are presumed to gain access to nk cells, as consumption of certain lactic acid bacteria has been shown to increase in vivo nk cytotoxicity. here, we investigated how human gut flora-derived lactobacilli affect nk cells in vitro, by measuring proliferation and ifn-g production of human nk cells upon bacterial stimulation. cd3 -cd56 þ nk cells were isolated from buffy coats by negative isolation using non-nk lineage-specific antibodies and magnetic beads. nk cells were incubated with 10mg/ml uv-inactivated bacteria or 10mg/ml phytohemagglutinin (pha) for 4 days. proliferation was assessed by incorporation of radioactive thymidine into nk-cell dna. the ifn-g concentration was measured by elisa. incubation of nk cells with a lactobacillus acidophilus strain increased the proliferation of the nk cells and induced ifn-g production, both to levels comparable to pha stimulation. the proliferative response was further enhanced with autologous monocytes present, probably because cytokines, secreted by monocytes having engulfed bacteria, stimulated the nk cells. in contrast, a lactobacillus paracasei strain caused the nk cells to proliferate only in the presence of monocytes. these results demonstrate that various strains of lactobacilli have the capacity to activate nk cells in vitro, in a monocyte-dependent or -independent way. hence, the encounter of nk cells with lactic acid bacteria will affect nk-cell activation. such activation of nk cells may potentially skew an on-going or subsequent immune response towards a th1 response. lactobacilli are nonpathogenic gram-positive inhabitants of the normal human intestine known for their healthpromoting effects. in our earlier work, it is shown that human monoclonal antibody isolated from sera of a patient with waldenstrom macroglobulinaemia possess innate antibody characteristics and binds to lactic acid bacteria. according to the immune network model, immunization with this bacteria could induce the perturbations in immune system that might result in production of anti-lactobacillus antibodies, human monoclonal antibody like (ab1) and anti-idiotypic antibody (ab2). in this study, balb/c mice were immunized with two doses of bacteria lactobacillus acidophilus in complete and incomplete freund's adjuvant and phosphate-buffered saline (pbs), respectively. seven days after the last immunization, sera from immunized mice were collected and the presence of lactobacillus-specific ab1 and ab2 were determined by elisas. in the sera of immunized mice, antibodies specific to bacteria lactobacillus acidophilus were shown. the concentration of lactobacillus-specific antibodies was higher in the sera of hyperimmunized mice (mice immunized with 1 mg of igm dj) than in sera of mice immunized with 100 times lower doses of immunogen (0.01 mg per doses). moreover, ab1 and ab2 antibodies were detected in the sera of lactobacillus-hyperimmunized mice. in this study, we have shown the idiotypic network interactions in mice immunized with bacteria lactobacillus acidophilus. the normal gastrointestinal flora is crucial for the maturation of the acquired immunity via effects on antigenpresenting cells (apcs). here, we have investigated how two types of apcs, monocytes and dendritic cells (dcs), react to different bacterial strains typical of the commensal intestinal flora. purified monocytes and monocyte-derived dcs were stimulated with uv-inactivated gram-positive (lactobacillus plantarum and bifidobacterium adolescentis) and gram-negative (escherichia coli and veillonella parvula) bacterial strains. monocytes produced higher levels of il-12p70 and tnf, as detected by elisa, in response to l. plantarum than to e. coli and v. parvula. in contrast, dcs secreted high amounts of il-12p70, tnf, il-6 and il-10 in response to e. coli and v. parvula but were practically unresponsive to l. plantarum and b. adolescentis. the lack of response to the gram-positive strains correlated with a lower surface expression of toll-like reseptor 2 (tlr2) on dcs compared to monocytes. the surface expression of tlr4 on dcs was undetectable when analysed by flow cytometry, but blocking this receptor decreased the tnf production in response to v. parvula, indicating that low tlr4 expression on dcs is sufficient to mount an inflammatory response to gram-negative bacteria. ifn-g increased the expression of tlr4 on dcs and also potentiated the cytokine response to gram-negative bacteria. our results indicate that, when monocytes differentiate into dcs, their ability to respond to different commensal bacteria dramatically changes, thereby becoming unresponsive to probiotic gram-positive bacteria. these results may have important implications for the capacity of different groups of commensal bacteria to regulate mucosal and systemic immunity. probiotic bacteria, e.g. lactobacillus spp., may improve diseases such as chronic inflammatory bowel disease. we examined cytokine production and phenotypic change after in vitro stimulation of t cells from healthy volunteers using different probiotic strains. methods: t cells were cultured from colonic biopsies from eight healthy volunteers (agnholt and kaltoft, exp clin immunogenet 2001; 18:213-25) , and dendritic cells were matured from their peripheral blood mononuclear cells. t-cell cultures were stimulated with autologous bacterial sonicate or strains of lactobacillus spp., with and without the addition of dendritic cells. cytokine levels (tnf-a, ifn-g, il-10 and gm-csf) and phenotype (cd3, cd4, cd25 and cd69) were measured on day 4. results: lactobacillus spp. induced higher productions of tnf-a and il-10 than did autologous bacteria. in presence of dendritic cells, the production of all cytokines increased. however, the increases of ifn-g and tnf-a were more pronounced in wells with autologous bacteria than in wells with lactobacillus spp. the addition of dendritic cells upregulated cd25 expression without simultaneous upregulation of cd69. the upregulation was pronounced after stimulation with lactobacillus rhamnosus gg compared with autologous bacteria and other lactobacilli. discussion: in presence of dendritic cells, autologous bacteria induced inflammatory cytokines, while probiotics mainly induced regulatory cytokines. lactobacillus rhamnosus gg induced a regulatory phenotype (cd25 þ ), in part mediated by dendritic cells. future studies will address whether this shift to a cd25 þ phenotype represents a differentiation into competent regulatory t cells. in a clinical context, such cells might be used for treatment of inflammatory diseases. protein microarrays will play a key role in the postgenomic era and offer a unique possibility to perform highthroughput global proteome analysis. a chip can be printed with thousands of protein probes (e.g. antibodies), the biological sample added (e.g. a proteome) and any binding detected. we aim to develop protein microarrays based on human recombinant scfv antibody fragments for global proteome analysis. the concept of comparing proteomic maps of healthy versus diseased samples will allow diseasespecific proteins to be detected. in fact, antibody microarrays will allow us to perform comparative proteome analysis on any sample format in a species-independent manner, as long as a proteome can be isolated. however, the complexity of proteomes, containing several thousands of different proteins, is a problem. here, we have designed antibody microarrays targeting the water-soluble fraction of a proteome. to this end, an anticytokine antibody array was developed and human dendritic cells (aeactivation) was used as model system. the results showed that our antibody microarrays could be used to examine the cytokine profile in complex samples. furthermore, we have taken the first steps towards comparing our results with those of other technologies on both the protein and gene level. due to the complexity of the model proteome, we also examined the possibility to prefractionate the proteome in a simple one-step procedure (based on size) prior to the labelling step. in more detail, the sample proteome was fractionated into two fractions using membrane devices with different molecular weight cut-offs. the results showed that the fractionation considerably enhanced the assay sensitivity allowing cytokines in the pg/ml range to be readily detectable. the immunomodulatory effect of heat shock protein 70: immunization with a dna construct based on the malarial antigen fused with a fragment of hsp 70 primes for a th-1 type of response finding an appropriate adjuvant for human vaccination is crucial. heat shock proteins (hsps) act as adjuvants when coadministered with peptide antigens or given as fusion proteins. however, there is a potential risk of autoimmunity when using the complete molecules, because hsps are evolutionary conserved. to overcome this, we first evaluated the adjuvant effect against two different antigens of a less-conserved fraction of plasmodium falciparum hsp70 (pf70c) and compared it to the whole hsp70 molecule from trypanosoma cruzi (tchsp70). we found that pf70c exhibited similar adjuvant properties as the whole molecule. we later evaluated the adjuvant potential of pf70c against the malarial antigen eb200 in a chimeric dna construct. no appreciable levels of eb200-specific abstracts 629 .................................................................................................................................................................................................. antibodies were detected in mice immunized only with the dna constructs. however, dna primed the immune system, because subsequent challenge with the corresponding recombinant fusion proteins elicited a strong th-1 antibody response. in contrast, no priming effect was observed for ex vivo ifn-g production but stimulation with the hsp-chimeric fusion protein induced a stronger secretion of ifn-g in vitro than other proteins used. these results indicate that the use of hsps is promising in the design of new vaccines. high-throughput proteomics on antibody-based microarrays: the importance of probe and surface design in analogy to dna microarrays, protein microarrays offer a new distinct possibility to perform sensitive highthroughput global proteome analysis. however, the development of the protein microarray technology will place high demands upon the design of both probes and solid supports. the analysis of thousands of heterogeneous proteins on a single microarray requires the use of uniform probes, such as antibodies, directly designed for protein microarray applications. we have recently generated a human recombinant single-chain fv antibody library, genetically constructed around one framework, the ncoder-library, containing 2 â 1010 clones. single framework antibody fragments (sinfabs) selected from this library were successfully applied as probes for microarrays providing sensitive detection in the 600 attomol (mass spectrometry) and the 300 zeptomole range (fluorescence). however, the choice of framework is critical. we have shown that the selected ncoder framework displayed excellent functional on-chip stability and arrayed dehydrated probes retained their activity for several months. furthermore, we have addressed the issues of biocompatibility of the solid support and immobilization strategies for our microarray setup. an in-house-designed substrate, macroporous silicon coated with nitrocellulose (map3-nc7), displayed properties equal to, or better than, those of five commercially available supports used as reference surfaces. we have also evaluated different coupling strategies, such as adsorption, covalent coupling, diffusion and affinity coupling. using a novel affinity tag, the double-(his)6-tag, we increased the binding efficiency of sinfab-molecules to ni2 þ -coated solid supports, thereby allowing nonpurified probes to be directly applied. the mannan-binding lectin (mbl) pathway is part of the innate immune system providing a first line of defence against infections. mbl and ficolins circulate in complexes with mbl-associated serine proteases (masp-1, -2 and -3). after recognition of a microorganism by mbl, activation of the complement system occurs. masp-1 and masp-3 share five domains (making up the so-called a-chain), whereas they have unique protease domains (b-chains). before the identification of masp-3, an assay for masp was presented, based on antibodies against the a-chain of masp-1. with the new knowledge of the three masps, and the sharing of domains by masp-1 and masp-3, assays specific for the protease domains have to be constructed, if one wishes to measure the proteins individually. we present an assay for quantifying total masp-3 in plasma and serum samples. the assay is a sandwich-type assay using as catching antibody a monoclonal antibody against the common a-chain of masp-1/3 and a developing secondary antibody against the c-terminal part of the protease domain of masp-3. we have used this assay for estimating the normal concentration of the protein as well as the concentration in patients and also for characterizing by gel permeation chromatography the masp-3 protein in serum. inducible costimulator ligand (icosl) is a costimulatory molecule related to b7.1 (cd80) and b7.2 (cd86). b cells, monocytes, dendritic cells and endothelial cells express icosl. inducible costimulator (icos) interacts with icosl, and this interaction leads to signals involved in isotype switching and the development of immunological memory. hitherto, no polymorphisms of this gene have been described. the aim of this study was to reveal variation of the icosl gene in normal individuals. all eight exons, except exon 1, were sequenced with flanking introns in 10 healthy blood donors. eight single nucleotide polymorphisms (snps) and two length polymorphisms were found. one of the snps was found in the coding regions of the gene. the base involved was located in exon 3 and caused a conservative amino acid change from valine (gtt) to isoleucine (att). three individuals were heterozygous g/a for the exon polymorphism, while the remaining seven individuals were homozygous for the wildtype g/g. exon 3 encodes the immunoglobulin variable (igv)-like domain of the molecule which is situated outside the cell. this means that the amino acid could be critical for the stability of the molecule or could constitute part of the binding site for icos. the results form the basis for further experiments to find possible associations of the alleles to diseases caused by immune dysregulation. especially, the exon 3 variant is interesting and could play a role for the development of immunological diseases. besides, it would be interesting to see whether both exon 3 alleles are expressed or only the wildtype allele is functional. interferon-a/b (ifn-a/b) is increasingly used as antiviral and immunomodulatory therapies. unfortunately, bioavailability varies with ifn species and mode of administration, and all ifn species are potentially immunogenic. assays for antiviral activity (ifn) and antiviral neutralization (antibodies, nab) have been used for some time to monitor patients on ifn biologicals. these assays require laborious titrations making them unsuitable for large-scale clinical use. myxovirus a (mxa) is a resistance gtpbinding protein that is specifically induced by treatment with type 1 ifns. for example, ifn-b-induced mxa in blood leucocytes has been used as a biomarker in ifn-btreated patients with multiple sclerosis. however, the degree of specificity of mxa in this regard is unclear, and measurements of mxa protein and/or mrna are not yet suitable for routine clinical use. in an attempt to find new and better reporter genes (and, hopefully, genes and gene products with proven specificity for ifn-a and -b), microarray screenings with u133a genechips (affymetrix) were carried using human blood leucocytes and the human lung carcinoma cell line a549. we studied the simultaneous expression of 22,000 transcripts before and after exposure to human recombinant ifn-a and ifn-b and other antiviral and immunomodulatory cytokines. the results will be presented at the conference. interferon-a/b (ifn-a/b) is increasingly used as antiviral and immunomodulatory therapies. unfortunately, bioavailability varies with ifn species and mode of administration, and all ifn species are potentially immunogenic. assays for antiviral activity (ifn) and antiviral neutralization (antibodies, nab) have been used for some time to monitor patients on ifn biologicals. these assays require laborious titrations making them unsuitable for large-scale clinical use. our laboratories have therefore modified the antiviral assays for ifn bioactivity and nab, so that they are suitable for large-scale screening in specialized laboratories. the read-out is survival of a subcloned a549 cell line in the presence of an otherwise lethal amount of virus. thus, survival increases in the presence of type 1 ifn and decreases in the presence of nab against the ifn added to the cells. mxa is induced by type 1 ifn and can be used for measuring the nab activity. in another assay, the mxa level in the a549 cell line is measured. in an attempt to find a new and better reporter gene for type 1 ifn than mxa and genes specific for either ifn-a or -b, a micro array screen was carried using the u133a chip from affymetrix. the expression of 22,000 genes can be studied simultaneous with this technology. the results will be presented at the conference. in our laboratory, we have developed a database system, which we believe is of immediate interest to the general scientific community. the database represents a computerbased replacement for the laboratory notebooks used in the majority of research laboratories worldwide. in addition, the database provides an effective tool for organizing and managing laboratory information at all levels, spanning from managing and revising standard operating procedures and producing documentation of research activities to keeping track of data and conclusions. using the commercially available database toolkit software filemaker pro, we have developed a relational database solution for management of laboratory information. the system consists of a hierarchy of five interrelated databases, each pertaining to a separate type of information, namely, overall project information, information relating to individual experiment setups, documentation of daily research activity, generated data and descriptions of standard operating procedures. like other databases, each individual database consists of a number of records, each comprised of a set of fields in which information is entered. in each record, a certain field is reserved to specify the relation of the record to a record in another database at a higher level. thus, the database is essentially five databases linked by a hierarchy of one-to-many relations, organizing information in a folder-like structure. importantly, the database system allows multiple users to access and edit records simultaneously, and the data entered in one database immediately becomes accessible through the other databases. the limitations of laboratory notebooks are apparent when looking for information, which is dispersed throughout one or more notebooks, or possibly on loose sheets of paper or printouts 'somewhere'. the often complicated process of gathering laboratory data or results when writing grant applications or research papers is made considerably easier with the database system. thus, the database solution presented should be broadly attractive to researchers, irrespective of their scientific discipline. an effective sars vaccine is likely to include components that can induce specific cytotoxic t-cell (ctl) responses. the specificities of such responses are governed by hlarestricted presentation of sars-derived peptide epitopes. exact knowledge of how the immune system handles protein antigens would allow for the identification of such linear sequences directly from genomic/proteomic sequence information. the latter was recently established when a causative coronavirus (sars cov) was isolated and full-length sequenced. here, we have combined advanced bioinformatics and high-throughput immunology to perform an hla supertype, genome-wide scan for sars-specific cytotoxic t cell epitopes. the scan includes all nine human hla supertypes in total covering >99% of all major human populations. for each hla supertype, we have selected the 15 top candidates for test in biochemical-binding assays. at this time (approximately 6 months after the genome was established), we have tested the majority of the hla supertypes and identified almost 100 potential vaccine candidates. these should be further validated in sars survivors and used for vaccine formulation. we suggest that immunobioinformatics may become a fast and valuable tool in rational vaccine design. rationale: major histocompatibility complex class i (mhc i) molecules monitor the protein content of the cell by binding small derived peptides and presenting them to cytotoxic cd8 þ t cells. the goal of the human mhc project is to predict the binding strength of any given peptide/mhc complex. this prediction allows the design of peptide-based vaccines. the prediction requires representative binding data from mhc alleles from all the nine hla supertypes. here, we describe the genetic construction, protein production and purification as well as the establishment-binding assays for two recombinant mhc supertype alleles, hla-b*1501 and hla-b*5801. methods: using the quikchange multisite directed mutagenesis kit (stratagene), codon-optimized genes encoding hla-b*1501 and hla-b*5801 are created. the two mhc i molecules are fermented and purified by ion exchange chromatography, hydrophobic interaction chromatography and size exclusion chromatography. the binding (kd) of natural t-cell epitopes, as well as predicted peptide ligands, is described by radioactive immunoassays (rias) and enzyme-linked immunosorbent assays (elisas). the mhc molecules are biotinylated during expression. results: the expression of mhc i resulted in multiple disulfide bond isomers, which are separated by hydrophobic interaction chromatography and used in subsequent binding studies resulting in the determination of kd for various peptide ligands ranging from strong binders we have previously demonstrated that bioinformatics tools such as artificial neural networks (anns) are capable of performing pathogen-, genome-and hlawide predictions of peptide-hla interactions. these tools may therefore enable a fast and rational approach to epitope identification and thereby assist in the development of vaccines and immunotherapy. a crucial step in the generation of such bioinformatics tools is the selection of data representing the event in question (in casu peptide-hla interaction). this is particularly important when it is difficult and expensive to obtain data. herein, we demonstrate the importance in selecting information-rich data and we develop a computational method, query-bycommittee, which can perform a global identification of such information-rich data in an unbiased and automated manner. furthermore, we demonstrate how this method can be applied to an efficient iterative development strategy for these bioinformatics tools. methods: a large panel of binding affinities of peptides binding to hla a*0204 was measured by a radioimmunoassay (ria). this data was used to develop multiple first generation anns, which formed a virtual committee. this committee was used to screen (or 'queried') for peptides, where the anns agreed ('low-qbc'), or disagreed ('high-qbc'), on their hla-binding potential. seventeen low-qbc peptides and 17 high-qbc peptides were synthesized and tested. the high-or low-qbc data were added to the original data, and new high-or low-qbc second generation anns were developed, respectively. this procedure was repeated 40 times. the high-qbc-enriched ann performed significantly better than the low-qbc-enriched ann in 37 of the 40 tests. conclusion: these results demonstrate that high-qbcenriched networks perform better than low-qbc-enriched networks in selecting informative data for developing peptide-mhc-binding predictors. this improvement in selecting data is not due to differences in network training performance but due to the difference in information content in the high-qbc experiment and in the low-qbc experiment. finally, it should be noted that this strategy could be used in many contexts where generation of data is difficult and costly. interleukin-18 (il-18), a pro-inflammatory cytokine that is produced by both lymphoid and nonlymphoid cells, has a critical role in modulation of innate and adaptive immunity. its primary function in stimulation of ifn-g production and stimulation of nk-cell-cytotoxic activities makes this cytokine a candidate for cancer immunotherapy. in oral cavity, this cytokine is produced by oral epithelia and carcinoma cells and is related to tumour regression in nude mice bearing salivary adenocarcinoma. however, direct effects of this cytokine on oral cancer cells have not been elucidated. in this project, we investigated il-18 effect on an oral carcinoma (kb) cell line. with rt-pcr technique, kb-cell line was found to express il-18 receptors (il-18ra and il-18rb), indicating that this oral carcinoma line is a target for il-18 study. we showed that recombinant human il-18 inhibited kb-cell proliferation by 17% at concentration of 100 ng/ml (p < 0.05), whereas ldh release by these cells in treatment group and control groups was comparable, indicating that il-18 suppression of cell proliferation was not mediated by the induction of cell death. to further address this hypothesis, we found that il-18 treatment did not induce apoptotic cell death, as studied by dna laddering and tunel assays. in addition, expression pattern of cell death-controlling genes (bcl-2 and bax) was not altered by this cytokine. findings in these studies indicated that suppression of kb-cell proliferation may be attributed to control of cell cycle, growth arrest or induction of cell differentiation. the data presented in this project could provide an insight of how cancer cell directly responds to il-18, as this cytokine is an important regulator of anticancer mechanisms. aloe emodin (ae) is a naturally occurring compound with wide spectrum of biological properties, including antimicrobial, vasorelaxant, immunosuppressive and anticancer actions. this anthraquinone induces apoptosis in several tumour cell lines with special affinity to tumours of neuroectodermal origin. high amounts of nitric oxide (no) released by activated macrophages induce tumour cell death. therefore, we explored the capacity of ae to modulate no-mediated antitumour response in vitro. interestingly, while ae markedly suppressed no release from macrophages alone, it significantly potentiated no production in cocultures of macrophages and c6 cells, after 48 h of cultivation. accordingly, the viability of c6 cells cocultivated with macrophages was reduced in the presence of ae. moreover, the observed ae-imposed potentiation of no production in macrophages was closely related to macrophage culture cell density. according to these data, we proposed that no modulator capacity of ae strongly depended on intercellular contact, indicating that macrophage antitumour response was not compromised but even potentiated by ae. immunotherapy represents an attractive fourth-modality therapeutic approach, especially in the light of the shortcomings of conventional surgery, radiation and chemotherapies in the management of metastatic cancer. to this end, a large number of peptide antigens derived from taa have been applied in immunotherapeutic trials for the treatment of various malignancies, e.g. cancers of the breast, prostate and kidney, in addition to haematological cancers. in some cases the response rates have been impressive and no adverse autoimmunity have been observed. a major strategic difficulty associated with these trials relates to the choice of best-suited peptide antigens. the vast majority of the antigens described thus far is not vital for survival and growth of the tumour cells, and immunoselection of antigen-loss variants may therefore prove to be an additional obstacle for the clinical applicability of most of the known peptide epitopes. in this respect, the development of acquired antigen loss during immunotherapy has been demonstrated in several cases. obviously, the development of loss-variant tumour cells implies that these cells acquire a pronounced growth advantage and are left unaffected by further treatment. ideally, target antigens should be derived from proteins required for survival and growth of tumour cells, as antigens with these characteristics would not be inflicted by the development of loss-variant tumour cells. in this respect, several inhibitors of apoptosis proteins (iaps) are universally expressed among tumours and play an important role in tumour cell escape from apoptosis. we have characterized spontaneous t-cell reactivity against iapderived peptides in cancer patients. from the iap survivin, we have characterized peptides restricted to the class i molecules hla-a1, a2, a3, a11, b7 and b35. furthermore, we have demonstrated that survivin-specific t cells infiltrate metastatic lesions and that isolated survivinspecific ctls are capable of killing hla-matched tumour cells. survivin-derived peptides are now in clinical trial, and continued work in our lab has demonstrated that other iaps are targets for spontaneous t-cell reactivity in cancer patients. we previously reported that in mice with large progressing t-cell lymphoma tumours, dysfunctions in the antitumour ctl activity occur, associated with an accumulation of splenic arginase-producing myeloid suppressor cells (mscs). in this study, we first demonstrate that both the presence and the activation state of these msc depends on tumour evolution. while in tumour regressors hardly any arginase-producing msc can be found, both the amount and the arginase activity of this population expands from early over late progressors. this gradual induction of mscs is paralleled by an increasing suppression of ctl activity and th1, but not th2, cytokine production. upon analysing the molecular repertoire of msc in vitro, we found, besides arginase1, a well-established marker for alternatively activated myeloid cells or m2, a strong upregulation of fizz1 and ym, two additional recently identified markers for m2. further evaluation of molecular markers by microarray analysis in msc yielded genes involved in wound healing (e.g. coagulation factor xiiia), anti-inflammation (e.g. selenoprotein p), immunomodulation (e.g. pd-l2) and fat and sugar metabolism (e.g. leptin receptor). of note, many of these genes are regulated by type 2 cytokines (il-4, il-13 and il-10) and are therefore rather m2 associated. overall, our data provide new markers for msc in cancer and further establish their m2 activation state. study. only sp-a showed a significant expression in normal mucosa which was downregulated in crc. as the absolute signal level was below the noise threshold, these results have to be interpreted with caution and require confirmation by direct measurenment of the proteins. our results suggest that there is no major role for the human collectins in colorectal cancer. tetramerization is visualized by sds-page. conclusion: an effective method for the production of highly pure mhc i molecules has been applied to hla-b*1501 and hla-b*5801, and ria and elisa binding assays for those alleles have been established background: proliferation, differentiation and apoptosis are essential processes in the normal functions of the mammary epithelium. the hypothesis examined in this study is that the transcription factor bcl-6 is critically important not only for regulating b-cell growth and development but also for mammary epithelial apoptosis. methodology: twenty breast cancer cases and 31 healthy controls were used to investigate whether bcl-6 protein in involved in breast cancer (grade iii). full length bcl-6 cdna was retrovirally transduced into eph-4 cell line. we then used flow cytometry of brdurd-stained cells to investigate the cell-cycle duration of the control and transduced cell lines. tunel was used as a marker of apoptosis to find out differences in the frequencies of apoptotic cells in the control and transduced cell lines. finally, immunohistochemistry staining was performed to detect bcl-6 in breast cancer (iii). results: restoration of bcl-6 into eph-4 cells not only inhibits apoptosis but also prolongs the cell cycle and results in increased cell size and protein content. the results also indicated that the cell-cycle time of bcl-6-transduced eph-4 cells is prolonged by about 3 h, presumably as a result of the action of bcl-6 at the bcl-6 at the g1/s transition. we found differences in the frequencies of viable and apoptotic cells in cultures of the parent eph-4 cells, control-transduced eph-4 cells and bcl-6-transduced eph-4 cells. consistently, we demonstrated that bcl-6 is expressed in 90% of high grade of breast carcinoma, which is considered as the most aggressive of tumours. conclusion: together, these results suggest that bcl-6 is likely to be involved in mammary gland development and carcinogenesis. inflammatory cytokines have a critical role in modulation of both innate and adaptive immunity in response to foreign antigen. they also play an important role in anticancer immunity. for example, they can promote cell-mediated immunity against cancer cells. with their immunostimulatory effects, these cytokines are being tested for cancer treatment in the form of dna vaccine or adjuvant or therapeutic cytokines. direct effect of these cytokines on cancer cell, however, is still unclear. in this project, we investigated whether il-1( and il-18 can modulate cancer cell proliferation. we employed a simple nonradioactive proliferation (mtt) assay and detection of lactate dehydrogenase (ldh) to test the effect of these recombinant human cytokines on various cancer cell lines, including breast cancer cell line (mcf-7), oral carcinoma cell line (kb), colon cancer cell line (caco-2) and choriocarcinoma cell line (jar). cytokines used in this study had both inhibitory and stimulatory effect on cell proliferation. findings in this project could provide an insight of cancer cell response to these cytokines and this could lead to a consideration on using cytokine as immunotherapy for cancer treatment.capacity of ae to modulate nitric oxide production depended on intercellular contact donor t cells are involved in the antitumour effects observed after bmt. thus, patients receiving t-celldepleted bmt have a higher risk of leukaemia relapse compared to patients receiving nonmanipulated bmt, and patients experiencing graft-versus-host disease (gvhd) have a lower risk of disease relapse than patients who do not experience gvhd. although the importance of donor t cells for the curative action of bmt has been established, the exact mechanisms and molecules involved in this graft-versus-tumour effect remain largely unknown. in a recently initiated project, we have conducted a longitudinal study of t-cell clonotypes in patients who received peripheral blood stem cell grafts after nonmyeloablative conditioning. peripheral blood samples were obtained sequentially after transplant, and the mononuclear cells (mncs) were isolated and cryopreserved. cd8 þ t cells were isolated from the mncs by use of immunomagnetic beads or facs and analysed for the presence of clonally expanded cells by t-cell receptor clonotype mapping based on rt-pcr and denaturing gradient gel electrophoresis (dgge). using this gel-based methodology, clonally expanded t cells were monitored after transplant and compared to the clinical data of the patients. the preliminary results demonstrates the presence of clonally expanded cd8 þ t cells at all time points analysed. furthermore, a number of clonotypes persisted for more than 6 months, and other clonotypes emerged during this period. the appearance of newly emerged clonotypes which coincided with clinical gvhd could indicate a role for these t cells in the pathogenesis of gvhd. background: deficiency of the mannan-binding lectin (mbl) pathway of innate immunity leads to increased susceptibility to infections. in patients with colorectal cancer, postoperative infection is associated with poor prognosis. the aim of the present study was to evaluate (1) the relation between the mbl pathway and postoperative infectious complications and survival of patients resected for colorectal cancer and (2) the role of mbl as acute phase reactant compared to crp. methods: preoperative mbl concentration, mbl/mblassociated serine protease (masp) activity and crp were determined in serum from 611 patients and 150 healthy controls. the patients were observed for 8 years. postoperative infections, recurrence and survival were recorded. results: the mbl pathway components were increased in the patients (p < 0.0001) compared to healthy controls. low mbl levels were predictive of pneumonia (p ¼ 0.01), and pneumonia (n ¼ 87) was associated with poor survival (p ¼ 0.003, hr ¼ 1.5, 95% ci 1.1-1.9). mbl and mbl/ masp activity could not predict postoperative overall infections. mbl showed no correlation (spearman's r ¼ 0.02, 95% ci à0.06-0.10) with crp. conclusions: low preoperative mbl levels are predictive of pneumonia, which is associated with poorer survival. mbl concentration and mbl/masp activity was not predictive of other postoperative infections or long-term prognosis. mbl apparently is not a surrogate measure of crp. department of surgery, university hospital of erlangen, erlangen, germany. e-mail: michael.siassi@rzmail.uni-erlangen.de introduction: the human collectins, mannan-binding lectin (mbl), surfactant protein-a (sp-a) and surfactantprotein-d (sp-d) play a central role in the innate immune system. immunological responses to malignant transformation of epithelial cells gained increasing interest recently. a former study could demonstrate binding of mbl to certain colorectal carcinoma (crc) cell lines in vitro. we therefore examined the expression of human collectins in normal colon mucosa and in colorectal carcinomas. materials and methods: colon samples from 20 crc patients and 10 normal mucosa samples were collected immediately after surgery. the tissue was microdissected and rna isolated (qiagen, rneasy-kit). gene expression profiles were analysed using gene-chips (affymetrix, hg-u133). we analysed the data for the expression of mbl, its associated serine proteases mannan-binding lectinassociated serine protease 1/2 (masp 1/2), sp-a and sp-d. the signal intensity of the genes of interest was compared using the mann-whitney u-test. results: the expression of human collectins in normal human colon mucosa was generally low. only the expression of sp-a and masp-2 reached the noise threshold of 250 signals. these genes were significantly downregulated in crc specimens. the expression of the other proteins showed no difference in normal mucosa and crc. conclusion: as demonstrated before, the expression of human collectins in normal colon was low in this being the first lymph node to receive drainage from the tumour area, the sentinel node offers a unique possibility to obtain tumour-reactive lymphocytes. we investigated antitumour immune responses in sentinel nodes from patients with bladder cancer, by assaying tumour-specific proliferation and tcr vb repertoires. during tumour surgery, sentinel lymph nodes were identified by peritumoural injection of blue dye. fresh specimens of tumour, sentinel and nonsentinel lymph nodes were obtained, and single-cell suspensions were prepared. cells were assayed for reactivity against autologous tumour extract in [ 3 h]-thymidine incorporation assays and characterized by flow cytometry. parallel analyses of the expression of vb gene families were performed with padlock probes, linear oligonucleotides which upon target recognition can be converted to circular molecules by a ligase. probes were reacted with cdna prepared from magnetically separated cd4 þ cells, and the tcr repertoire was determined by hybridizing the products to oligonucleotide microarrays. dose-dependent proliferation in response to tumour extract could be detected in sentinel lymph nodes. common clonal expansions were detected among tumourinfiltrating lymphocytes and in sentinel lymph nodes. nonsentinel lymph nodes displayed a divergent tcr vb repertoire. these results indicate an ongoing immune response against tumour antigens in sentinel nodes, draining urinary bladder cancer. identification of sentinel lymph nodes makes it possible to obtain tumour-reactive lymphocytes for use in adoptive immunotherapy. key: cord-023388-btbf6wkg authors: hoffmann, h. j.; nielsen, l. p.; blumberga, g.; dahl, r. title: decrease in fine t‐cell subset ratio mt2/mt1 during steroid reduction of asthmatic patients date: 2008-06-28 journal: scand j immunol doi: 10.1111/j.0300-9475.2004.01423ah.x sha: doc_id: 23388 cord_uid: btbf6wkg combining inhaled long‐acting β‐2 agonist (laba) and inhaled corticosteroid (ics) seems to offer asthma control at a lower dose of ics than achieved by ics alone. fine mapping of t‐cell surface markers by flow cytometry offers a detailed status of the individual's inflammatory response. the frequency of mt2 (cd4(+)cd45ra(–)cd62l(+)cd11adim) and mt1 (cd4(+)cd45ra(–)cd62l(–)cd11abright) cells in peripheral blood, and their ratio, has been shown to differ predictably in atopics and patients with leprosy, where mt2 correlates with a th2 phenotype and mt1 with a th1 phenotype. stable asthmatics, requiring fluticasone propionate (fp) 750–1000 µg daily or equivalent, were randomized to receive, double‐blinded, either seretide(®)[salmeterol and fluticasone propionate (sfc, n = 16)] 50 µg/500 µg bd or fp 500 µg bd (n = 17). if asthma was controlled based on lung function and symptoms at clinic visits every 6 weeks, ics dose was tapered until asthma exacerbated or 0 µg was reached. the frequency and ratio of mt2 and mt1 t cells of the patients was monitored at 6 week intervals. as treatment tapered, the frequency of mt2 cells decreased (p = 0038 from first to final visit), whereas that of mt1 cells increased. the ratio of mt2/mt1 decreased (p = 0049 from first to final visit). in patients receiving laba + ics, the fall in mt2/mt1 ratio appeared to be more pronounced than in patients receiving ics alone. thus, the mt2 phenotype may be associated with stable asthma, whereas an imminent exacerbation may associate with an increase in the mt1 phenotype. laba may allow for a greater effect of fp on the mt ratio. the nuclear receptor heterodimers of liver x receptors (lxrs) are recently identified as key transcriptional regulators of genes involved in lipid homeostasis and inflammation. lxrs and their ligands are negative regulators of macrophage inflammatory gene expression. multiple sclerosis (ms), a demyelinating disease of the central nervous system of unknown cause, is characterized by recurrent inflammation involving macrophages and their inflammatory mediators. sweden belongs to the countries with a high ms incidence. in italy, incidence is lower, with an exception for sardinia where the incidence is even higher than that in sweden. subjects from sardinia are ethnically more homogeneous and differ from swedes, also regarding genetic background and environment. we studied lxrs and their related molecules of blood mononuclear cells (mncs) from female patients with untreated relapsing-remitting ms from sassari, sardinia and stockholm, sweden. sex-and age-matched healthy controls (hcs) were from both areas. mrna expression was evaluated by real-time pcr. lxr-a was lower (p < 0.05) in ms (mean ae sem: 3.1 ae 0.2; n ¼ 37) compared to hc (3.6 ae 0.1; n ¼ 37). lxr-a was lower in ms from stockholm (2.6 ae 0.2; n ¼ 22) compared to corresponding hc (3.4 ae 0.1; n ¼ 22; p < 0.01) and compared to ms (3.8 ae 0.2; n ¼ 15; p < 0.001) and hc (4 ae 0.2; n ¼ 15; p < 0.001) from sardinia. ms patients from stockholm, but not from sassari, also expressed lower (p < 0.05) lxr-b (à4.1 ae 0.4) compared to corresponding hc (à2.9 ae 0.3). ms from stockholm was associated with higher abca-1 (6.1 ae 0.4 versus 5.0 ae 0.3; p < 0.05) and higher estrogen receptor-b-cx (2.4 ae 0.4 versus 0.8 ae 0.4; p < 0.01) compared to corresponding hc. the hc from sassari had higher androgen receptor (2.9 ae 0.2) compared to ms from sassari (1.4 ae 0.3; p < 0.01), ms (1.3 ae 0.4; p < 0.01) and hc from stockholm (1.2 ae 0.3; p < 0.01). ms from sassari had lower cyclooxygenase-1 compared to corresponding hc (5.1 ae 0.4 versus 6.6 ae 0.3; p < 0.01) and lower prostaglandin-e (à0.03 ae 0.5) compared to the hc (1.4 ae 0.5; p < 0.05) and ms (2.7 ae 0.4; p < 0.05) and hc from stockholm (1.9 ae 0.4, p < 0.001). our findings identify lxrs and their related molecules as being involved in ms from stockholm but not from sassari, while sex hormone receptors seem to be involved in ms in sassari. multiple sclerosis: ifn-b induces cd123 + bdca2 -dendritic cells that produce il-6 and il-10 and have no enhanced type i interferon production y. m. huang, 1 s. adikari, 1 u. båve, 2 a. sanna 1,3 & g. alm 4 dc antigens (bdca) and investigate their ability to produce type i ifn in response to virus stimulation. we show that ifn-b induces development of cd123 þ dc from human blood monocytes, which coexpress bdca4 þ but are negative for bdca2 -, a specific marker for plasmacytoid dc. such ifn-b-modulated dc produce large amounts of il-6 and il-10, but no il-12p40 and have no enhanced ifn-b and ifn-b production. the findings indicate that ifn-bmodulated dc represent a myeloid dc subset with diminished cd11c, bdca-1 and cd1a expression, having potent th2-promoting function but lacking antiviral capacity. the association of psoriasis with throat infections by streptococcus pyogenes suggests a potential antigenic target for the t cells that are known to infiltrate dermis and epidermis of psoriatic skin. streptococcal m protein shares an extensive sequence homology with human epidermal keratins. keratins 16 (k16) and 17 (k17) are mostly absent from uninvolved skin but are upregulated in psoriatic lesions. there is increasing evidence that cd8 þ t cells play an important effector role in psoriasis and m proteinprimed t cells may recognize these shared epitopes in skin via molecular mimicry. to identify candidate epitopes, peptides with sequences from k17 were selected on the basis of predicted binding to hla-cw6 and sequence similarities with m6 protein. matched peptides from the sequence of m6 protein and a set of peptides with poor predicted binding were also selected. cw6 þ individuals with psoriasis and cw6 þ healthy controls, having a family history of psoriasis, were recruited. pbmcs were incubated with the peptide antigens. t-cell activation in the cd4 þ , cd8 þ and later the skin-homing cutaneous lymphocyteassociated antigen (cla)-expressing subset of cd8 þ t cells was evaluated by cd69 expression and intracellular ifn-g accumulation using flow cytometry. we demonstrate that cw6 þ psoriasis patients had significant cd8 þ t-cell ifn-g responses to peptides from k17 and m6 protein selected on the basis of sequence homology and predicted hla-cw*0602 binding. these responses were about 10 times more frequent in the skin-homing cutaneous lymphocyte-associated antigen-expressing (cla þ ) subset of cd8 þ t cells. cd4 þ t cells showed only borderline responses. cd8 þ t cells from cw6 þ nonpsoriatic individuals responded to some m6 peptides but very rarely to k17 peptides, and this also applied to the cla þ cd8 þ subset. these findings indicate that psoriatic individuals have cd8 þ t cells that recognize keratin self-antigens and that epitopes shared by streptococcal m protein and human keratin may be targets for the cd8 þ t cells that infiltrate psoriatic skin lesions. autoantibodies directed against citrulline-containing proteins have an impressive specificity of nearly 100% in ra patients and a suggestive involvement in the pathogenesis. the targeted epitopes are generated by a post-translational modification catalysed by the calcium-dependent enzyme peptidyl arginine deaminase that converts the positively charged arginine to polar but uncharged citrullin. the aim of this study was to analyse the presence of citrulline in the joints at different time points of collagen-induced arthritis in da rats by immunohistochemistry and to investigate how immunogenicity and arthritogenicity was affected by citrullination of rat serum albumin (rsa) and collagen type ii (cii). our results indicate that citrulline could be detected in joints of arthritic animals, first appearance at the onset of disease and increasing as disease progressed into a chronic state. unimmunized animals or time points before clinical signs of arthritis were negative. by morphology, we state that some infiltrating macrophages as well as the cartilage surface stain positive for citrulline, while the major source of citrullinated proteins appears to be fibrin depositions. a specific cit-rsa t-cell response was observed in animals challenged by citrullinated rsa, no response was recorded when rsa was used as a stimulus. the igg analysis reveals not only a response towards the modified protein but also cross-reactivity to native rsa. no t-cell or b-cell response was noted in animals injected with unmodified rsa. cit-cii induced a disease with higher incidence and earlier onset than did the native counterpart. we conclude that, in contrast to the human disease, citrulline does not seem to appear before clinical signs. as inflammation proceeds, citrulline is detected specifically in the joints. all other organs investigated were negative. we also conclude that citrullination of a protein can break tolerance and increase its arthritogenic properties. ectopic germinal centers (gcs) can be detected in the salivary glands of approximately 1/5 of patients with sjögren's syndrome (ss) and appear in both primary and secondary ss. previously, ectopic gc have been associated with increased local autoantibody production. the aim of this study was to determine whether gc in primary sjögren's syndrome (pss) defines a distinct seroimmunological phenotype. retrospectively, a material of 130 haematoxylin and eosin-stained paraffin-embedded tissue sections of minor salivary gland tissue from patients with pss was morphologically screened for the presence of ectopic gc. gc-like lesions were detected in 33/130 (25%) of the pss patients. seventy-two pss patients lacking these structures (gc-) were randomly selected for comparison. focus score was significantly increased in the gc þ patients compared to the gcpatients (p ¼ 0.035). in the gc þ group, 54.5% of the patients presented with anti-ro/ssa compared to 43.7% in the gcgroup. anti-la/ssb was detected in 31.3% of the gc þ patients compared to 25.7% of the gcpatients. sixty-one percentage of gc þ patients presented with increased levels of igg, a nonsignificant difference when compared to 39.4% in the gcpatients (p ¼ 0.089). levels of rf, ana, ena, igm and iga were similar in both patient groups, as were esr and crp. in conclusion, patients with ectopic gc have a higher focus score and more often present with autoantibodies and increased levels of igg compared to pss patients with regular focal infiltration (gc -). our findings may indicate a certain seroimmunological phenotype and warrant for further prospective studies. association between mannose-binding lectin and vascular complications in type 1 diabetes complement activation and inflammation have been suggested in the pathogenesis of diabetic vascular lesions. we investigated serum mannose-binding lectin (mbl) levels and polymorphisms in the mbl gene in type 1 diabetic (t1dm) patients with and without diabetic nephropathy and associated macrovascular complications. polymorphisms in the mbl gene and serum mbl levels were determined in 199 t1dm patients with overt nephropathy and 192 t1dm patients with persistent normoalbuminuria matched for age, sex and duration of diabetes as well as in 100 healthy control subjects. the frequencies of high and low expression mbl genotypes were similar in patients with t1dm and healthy controls. high mbl genotypes were significantly more frequent in diabetic patients with nephropathy than in the normoalbuminuric group, and the risk of having nephropathy, given a high mbl genotype, assessed by odds ratio was 1.52 (1.02-2.27), p ¼ 0.04. median serum mbl concentrations were significantly higher in patients with nephropathy than in patients with normoalbuminuria [2306 mg/l (iqr 753-4867 mg/l) versus 1491 mg/l (iqr 577-2944), p ¼ 0.0003], and even when comparing patients with identical genotypes, serum mbl levels were higher in the nephropathy group than in the normoalbuminuric group. patients with a history of cardiovascular disease had significantly elevated mbl levels independently of nephropathy status [3178 mg/l (iqr 636-5231 mg/l) versus 1741 mg/l (iqr 656-3149 mg/l), p ¼ 0.02]. the differences in mbl levels between patients with and without vascular complications were driven primarily by pronounced differences among carriers of high mbl genotypes (p < 0.0001). our findings suggest that mbl may be involved in the pathogenesis of microvascular and macrovascular complications in type 1 diabetes and that determination of mbl status might be used to identify patients at increased risk of developing these complications. neuroimmunology unit, center for molecular medicine, karolinska institutet, stockholm, sweden. e-mail: judit.wefer@cmm.ki.se dna vaccine coding for the encephalitogenic peptide mog 91-108 protects lew.1av1 from subsequent development of experimental autoimmune encephalomyelitis (eae). protection is associated with a type 1 immune response and is dependent on the presence of cpg dna motifs. the mechanisms underlying the observed reduction of eae development in protected rats have not been fully clarified. we investigated immunological characteristics of lymphocytes after dna vaccinaton and subsequent eae induction. we confirm that protection was not associated with suppression of t1 cells, as transcription of the novel molecule rat t-cell immunoglobulin-and mucindomain-containing molecule (tim-3), reported to be exclusively expressed on differentiated t1 cells, was not altered by dna vaccination. we did not note any clonal deletion upon tolerization, but detected an antigen-specific lymphocyte population upregulating ifng upon recall stimulation 3 weeks after protective dna vaccination. in protected rats, we observed (1) no alterations in antigenspecific th2 or th3 responses, (2) reduced mhc ii expression on splenocytes early after eae induction, (3) antigen-specific upregulation of ifnb upon recall stimulation and (4) reduced il-12rb2 on lymphocytes. we thus demonstrate an association of the protective effect of dna vaccination with expression of ifnb. we are currently investigating the cellular mechanisms behind this ifnb-mediated protection. multiple sclerosis (ms) is an autoimmune condition characterized by degeneration of nerve fibre myelin sheets. a candidate autoantigen, myelin basic protein (mbp), has especially attracted attention. the presence of anti-mbp antibodies is a predictor of definite ms, but their role in the pathogenesis remains obscure. t cells have long been known to play a pivotal role in the pathogenesis of ms. recently, an important role for b cells as autoantigen-presenting cells has been demonstrated in other autoimmune diseases, including rheumatoid arthritis and diabetes. the uptake of mbp by b cells and the presentation of mbp-derive peptides to t helper (th) cells by b cells may be promoted by the formation of complement (c) activating immune complexes (ics) between mbp and natural autoantibodies in healthy individuals and disease-associated anti-mbp antibodies in ms patients, respectively. we have investigated the formation of mbp-containing ic, the binding of mbp to b cells, the mbp-elicited induction of th-cell and b-cell proliferation and the cytokine production in peripheral blood mononuclear cells (pbmcs) from healthy donors grown in the presence of intact or c-inactivated serum from healthy donors or patients with ms. while mbp did not induce measurable proliferation of b cells nor cd4 þ t cells, we observed the production of tnf-a, ifn-g and il-10 by pbmc in response to incubation with mbp in the presence of sera from healthy controls as well as sera from ms patients. by contrast, no production of il-2, il-4 and il-5 was detected. we are currently investigating the capability of ms sera to promote the formation of mbp-containing ic and thereby enhance the cytokine responses, by virtue of elevated anti-mbp contents. the phagolysosomally localized acid sphingomyelinase (asmase) activated by proinflammatory cytokines such as tnf and ifn-g generates the signalling molecule ceramide which in turn results in the activation of proteases like cathepsin d. these characteristics of asmase suggest a possible role of this molecule in the phagocytotic uptake and phagosomal degradation processes of antigens or in antigen presentation. we show here that asmase -/mice fail to eliminate the noncytopathic lymphocytic choriomeningitis (lcm) virus as rapidly as littermate wildtype mice. investigation of the immune response revealed a reduced expansion of cd8 þ t cells. the secretion of ifn-g in response to contact with target cells as well as the cytolytic activity of virus-specific cd8 þ t cells was severely impaired. additionally, both phases of the lcm virus-specific dth response, mediated by cd8 þ and cd4 þ t cells consecutively, were diminished in asmase -/mice. however, the secondary memory response of virus-specific ctl was not altered, and the 614 abstracts ................................................................................................................................................................................................. virus was effectively controlled for at least 3 months by asmase -/mice. in conclusion, the results of this study suggest an involvement of the asmase in the activation, expansion or maturation of virus-specific cd8 þ t cells during the acute infection of mice with the lcm virus. novel markers for alternative activation of macrophages: macrophage galactose-type c-type lectins 1 and 2 in parallel with the th1/th2 dichotomy, macrophages are capable of developing into functionally and molecularly distinct subpopulations, due to differences in, for example cytokine environment and pathological conditions. while the best-studied, classically activated macrophage is induced by type i stimuli such as ifn-g, a type ii cytokine environment antagonizes the classical activation of macrophages and is capable of alternatively activating macrophages. however, molecular markers associated with these type ii cytokine-dependent, alternatively activated macrophages remain scarce. besides the earlier documented markers macrophage mannose receptor and arginase 1, we recently demonstrated that murine alternatively activated macrophages are characterized by increased expression of fizz1 and ym. we now report that expression of the two members of the mouse macrophage galactose-type c-type lectin gene family, termed mmgl1 and mmgl2, is induced in diverse populations of alternatively activated macrophages, including peritoneal macrophages elicited during infection with the protozoan trypanosoma brucei or the helminth taenia crassiceps, and alveolar macrophages elicited in a mouse model of allergic asthma. we also demonstrate that, in vitro, interleukin-4 and interleukin-13 upregulate mmgl1 and mmgl2 expression and that, in vivo, induction of mmgl1 and mmgl2 is dependent on interleukin-4 receptor signalling. moreover, we show that regulation of mgl expression is similar in human monocytes and monocyte-derived macrophages. hence, macrophage galactose-type c-type lectins represent novel markers for both murine and human alternatively activated macrophages; thus, paving the way for further characterization of the phenotype of macrophages occurring in th2 conditions. background: human parvovirus b19 (b19) is a ubiquitous pathogen, normally causing a mild self-limiting disease, but also capable of causing both significant pathology and long-term persistence. the small size and stability of the virus makes it suitable for mapping of the full breath and the kinetics of the cellular immune responses following acute viral infection. methods: five patients with acute primary b19 infection were included in the study and followed consecutively for up to 200 weeks. cellular immune responses were mapped by ifng enzyme-linked immunospot to overlapping peptides spanning the whole b19 genome. results: in all five acutely infected patients, we were able to monitor the kinetics of a strong specific cellular immune reaction. responses peaked at levels of 850-1850 sfc/ million pbmcs, roughly corresponding to 0.3-0.6% b19specific cd8 þ cells circulating in peripheral blood at 10-80 weeks post-infection. the responses in individual patients were directed to three or four different peptide pools, and the specificity was confined to the same cd8 epitopes present in the pools throughout the follow-up period. the majority of responses were directed to the virus nonstructural protein, only two patients showed any response to the capsid proteins, elicited by the same epitope in both cases. conclusion: the cellular immune responses to acute b19 infection are surprisingly narrow in distribution and remain at high levels for up to 80 weeks post-infection. the initial epitope specificity is maintained, and the majority of responses target the virus nonstructural protein, which is not included in vaccine preparations, evaluated against the infection. the relationship between malnutrition and malaria is controversial. on one hand, malaria may cause malnutrition, while on the other, malnutrition itself may modulate susceptibility to the disease. we investigated the association between plasmodium falciparum malaria and malnutrition in a cohort of children living on the coast of kenya. the study involved longitudinal follow-up for clinical malaria episodes and anthropometric measurements at four cross-sectional surveys. we used poisson regression analysis to investigate the association between malaria and nutritional status. compared to baseline (children with a waz or haz score of !à2), the crude incidence rate ratios (irrs) for malaria in children with low haz or waz scores (<à2) during the period prior to assessment were 1.17 (95% ci 0.91-1.50; 0 ¼ 0.21) and 0.94 (0.71-1.25; 0.67), respectively, suggesting no association between malaria and the subsequent development of pem. however, we found that age was acting as an effect modifier in the association between malaria and malnutrition. the irr for malaria in children 0-2 years old who were subsequently characterized as wasted was 1.65 (1.10-2.20; p ¼ 0.01), and a significant overall relationship between malaria and low-haz was found on regression analysis when adjusting for the interaction with age (irr 1.89; 1.01-3.53; p < 0.05). although children living on the coast of kenya continue to suffer clinical episodes of uncomplicated malaria throughout their first decade, the association between malaria and malnutrition appears to be limited to the first 2 years of life. a. astrinidou-vakaloudi, 1 s. xytsas, 1 i. diamanti, 1 h. ioannidis 2 & p. pangidis 2 1 microbiology department of general hospital of thessaloniki 'agios pavlos', thessaloniki, greece, and 2 nefrology, 2 nd ika hospital of thessaloniki, thessaloniki, greece. e-mail: stasa@hol.gr aim: renal dysfunction may influence the colonization of gastric mucosa by urea-splitting bacteria such as helicobacter pylori, by increasing urea concentrations in the gastric juice. our aim was to investigate the prevalence of h. pylori in patients with end-stage renal disease (esrd), receiving long-term haemodialysis treatment. methods: this study included 40 sera from patients with esrd (29 male and 11 female) undergoing periodic haemodialysis; mean time of treatment was 42.6 months. using elisa technique, we investigated the presence of igg and iga antibodies against h. pylori as well as igg caga (antibodies specific for caga(þ) strains of h. pylori). sera from 40 healthy blood donors were used as a control group. results: h. pylori igg antibodies were detected in 32 out of 40 (80%) patients in the dialysis group, while 31/40 (77.5%) tested positive for iga. igg caga antibodies were present in 13 out of 40 (32.5%). prevalence of h. pylori igg, iga and caga igg antibodies in the control group was 33, 7 and 15%, respectively. conclusions: although international data suggest that prevalence of h. pylori infection is the same in esrd patients as in healthy individuals, in our study that seems not to be the case. the higher blood and gastric juice urea levels may be a risk factor (among many others), but more studies are required in order to understand the relation of h. pylori infection in this group of patients. flanders interuniversity institute for biotechnology, department of molecular and cellular interactions, free university of brussels, brussels, and 2 pasteur institute of brussels, mycobacterial immunology, brussels, belgium. e-mail: tgartner@vub.ac.be immunity against tuberculosis (tb), caused by mycobacterium tuberculosis, depends largely on activation and maintenance of strong cell-mediated immune responses involving both cd4 þ and cd8 þ t cells and the ability to respond with th1-type cytokines, particularly ifn-g. recent studies suggested that bcg, the only licensed vaccine against m. tuberculosis, may fail to induce t-cell responses in the lung mucosa and may therefore not protect against pulmonary tb. a decrease in tb mortality may be achieved by enhancing immunity in the lung. the present study evaluated the induction of antigen-specific immunity in the lung by intranasal (i.n.) delivery of the lipoprotein i (opri) from pseudomonas aeruginosa. opri has shown to be a toll-like receptor 2/4 agonist that, when given subcutaneously, induces type-1 immune responses against heterologous antigens. here, a fusion of opri to ag85a of mtb (opri-ag85a) was used as a subunit vaccine in homologous prime-boost immunizations. in addition, opri-ag85a was combined with an ag85a-encoding dna vaccine (ag85a dna) or with bcg in heterologous prime-boost vaccinations. intranasal and parenteral delivery with opri-ag85a elicited comparable t-cell responses in the spleen; in addition, i.n. delivery elicited specific t-cell responses in the lung lymph nodes (llns). intramuscular delivery of ag85a dna induced significant systemic th1 immune responses. intranasal boosting with opri-ag85a enhanced this response and in addition induced an antigen-specific ifn-g response in lln. opri may therefore be an efficient adjuvant for mucosal boosting. we continue to evaluate the protection induced by opri-based prime-boost vaccinations against pulmonary tb. results on the immunogenicity and protection against intravenous mtb h37rv infection will be presented. toll-like receptors (tlrs) are pattern recognition receptors of the innate immune system, which recognize molecular structures on pathogens or cellular stress-associated molecules. tlr-ligand interactions trigger activation of inflammatory signal transduction and expression of genes involved in host defense. in this study, we have examined the requirement for different tlr adaptor molecules in virus-induced chemokine expression and are currently trying to identify the tlr involved. we have found that both a herpesvirus [herpes simplex virus (hsv)] and a paramyxovirus (sendai virus) require a functional genome to induce expression or proinflammatory chemokines in human and murine monocytic cell lines. for both viruses, this is independent of the tlr adaptor molecules trif and mal. however, overexpression of the vaccinia virus-encoded inhibitor of tlr-signalling a52r or dominant-negative myd88 totally inhibited hsv-induced rantes expression but only partially prevented sendai virus from inducing this chemokine. this suggests that hsv-induced rantes expression occurs via a tlr pathways, whereas sendai virus utilizes both tlr-dependent and -independent pathways to stimulate expression of rantes. we are currently trying to identify the tlrs involved. data from these studies will also be presented at the meeting. 2 0 -5 0 -oligoadenylate synthetases are interferon-induced, double-stranded rna-activated antiviral enzymes which are the only proteins known to catalyse 2 0 -specific nucleotidyl transfer. this first crystal structure of a 2 0 -5 0oligoadenylate synthetase reveals a structural conservation with the 3 0 -specific poly(a) polymerase that, coupled with structure-guided mutagenesis, supports a conserved catalytic mechanism for the 2 0 -and 3 0 -specific nucleotidyl transferases. comparison with structures of other superfamily members indicates that the donor substrates are bound by conserved active site features while the acceptor substrates are oriented by nonconserved regions. the 2 0 -5 0oligoadenylate synthetases are activated by viral doublestranded rna in infected cells and initiate a cellular response by synthesizing 2 0 -5 0 -oligoadenylates, that in turn activate rnase l. this crystal structure suggests that activation involves a domain-domain shift and identifies a putative dsrna activation site that is probed by mutagenesis. we demonstrated that this site is required both for the binding of dsrna and for the subsequent activation of oas. this rna-binding site is different from known rna-binding site; rather than forming a defined three-dimensional domain, it is located at the interface of the two major domains in oas. this novel architecture ensures that the dsrna helix can make simultaneously contact with both domains of oas and ensure the subsequent structural rearrangement leading to the activation of oas. our work provides structural insight into cellular recognition of double-stranded rna of viral origin and identifies a novel rna-binding motif. bacteria-specific iga antibodies are efficient opsonins for neutrophils and mononuclear phagocytes, provided that the phagocytes express the fca receptor (cd89). expression of cd89 can be stimulated by inflammatory cytokines, activated complement factors and certain microbial components. in one study, unstimulated phagocytes were able to ingest iga antibody-treated pneumococci, but only in the presence of complement, which was found to be activated by the iga antibodies along the alternative pathway. pneumococci produce iga1 protease that cleaves human iga1, but not iga2, molecules in the hinge region. this leaves iga1 as faba (monovalent) deprived of fca which contains the docking site for cd89. iga1 is the vastly predominant subclass of iga in the upper airways and circulation of humans. aims: to examine the effects of iga1 protease activity and complement on phagocytosis of iga antibody-coated pneumococci by an unstimulated human phagocytic cell line (hl60). materials and methods: iga1 and iga2 monoclonal antibodies to serotype 4 pneumococcal capsular polysaccharide (ps) were generated by heterohybridoma technique involving b cells from human vaccinees. isogenic serotype 4 pneumococci with and without iga1 protease activity, respectively, were obtained after inactivation of the iga gene of the tigr4 strain. opsonophagocytosis was quantitated using the assay described by romero-steiner et al. based on enumeration of surviving bacteria by culture. the integrity of iga molecules was examined by western blotting. results: both iga1 and iga2 antibody to type-4 polysaccharide-induced phagocytosis of iga1 protease-deficient type-4 pneumococci equally well in the absence as in the presence of complement. iga1 antibody to type-4 polysaccharide displayed a fourfold higher opsonophagocytosis titer against iga1 protease deficient compared to homologous wildtype target bacteria. a similar effect of iga1 protease activity of the target bacteria was not observed in a parallel experiment where iga2 antibody to type-4 polysaccharide served as opsonin. iga1 antibody extracted from iga1 protease-producing target bacteria was almost exclusively in the form of faba. conversely, iga1 from protease-deficient bacteria and iga2 from both types of bacteria were intact. conclusions: these results indicate that the iga1 protease activity of s. neumoniae may help the bacteria escape iga1 antibody-mediated opsonophagocytosis. besides, in these experiments, iga-mediated opsonophagocytosis was independent of complement. vitamins e and c have been found to increase the cellular and humeral immunity of pigs. vitamin e deficiency has also been found to predispose pigs to different diseases, e. coli infection is one among them. after weaning, the vitamin e status of pigs often decreases to a critical low level. in this experiment, we studied whether vitamin c supplementation would be a possible feeding strategy to optimize the immune status of weaners. the interaction between vitamin e and c is interesting due to the reported sparing action on vitamin e or synergism between these to vitamins. piglets were weaned at day 28 of age from sows fed increasing dietary vitamin e during lactation, and piglets were during the following 3 weeks fed either a control diet or this diet supplemented with 500 mg stay-c per kg. blood sampling was obtained weekly from day 28 and until day 49 of age. on the same days, one piglet per dietary treatment was killed and alveolar macrophages (am) were harvested. vitamin c supplementation increased the concentration of igm in serum of piglets throughout the weaning period. although the vitamin e concentration in am decreased with increasing age of the piglets, the concentration was numerically higher in piglets of sows fed the high dietary level of vitamin e. however, vitamin c supplementation tended to increase the total am concentration of vitamin e after weaning and increased the proportion of the biologically most active isomer of vitamin e [rrr-(a-tocopherol)] in the am. the eicosanoid synthesis by am was not influenced by the vitamin c supplementation, but the synthesis of leukotriene b4 was decreased 2 weeks after weaning compared to other days of am harvesting. in conclusion, dietary vitamin c supplementation improved the immune responses of piglets after weaning. a whole blood stimulation assay with escherichia coli (o111:b4) endotoxin was established to measure the capacity of dairy cows to produce the proinflammatory cytokine tumour necrosis factor-a (tnf-a) ex vivo. initially, a time-and dose-dependent study was carried out to find the optimal stimulation conditions for the tnf-a response. the tnf-a response peaked between 3 and 4 h at 38.5 c. a dose in the range of 5-10 g of e. coli lipopolysaccharide (lps)/ml whole blood was found to give the maximum tnf-a response. thirty-eight danish-holstein dairy cows were investigated for their tnf-a responsiveness ex vivo in the periparturient period. heparin-stabilized blood samples were collected seven times over a period of 4 months (weeks à3, à1, 2, 3, 5, 9 and 13 around calving) and stimulated with 5 g/ml of e. coli lps. indeed, fluctuations in the tnf-a responsiveness occurred over time. moreover, the mean tnf-a responsiveness of 38 cows was found to be significantly increased (p < 0.001) in the weeks close to calving. however, in the more stabile physiological periods, some cows had a consistently low tnf-a response, whereas others had high a tnf-a response. we are currently investigating whether high and low tnf-a responders to e. coli lps also exist in dairy cows in vivo. moreover, the importance of tnf-a responsiveness ex vivo to dairy cows' susceptibility and clinical response to experimental e. coli infections in the udder is being investigated. coelomic cytolytic factor (ccf) is a 42 kda invertebrate pattern recognition molecule isolated from the coelomic fluid of the earthworm eisenia foetida (oligochaeta, annelida). ccf displays a number of similarities with the mammalian cytokine tumour necrosis factor-a (tnfa) as a result of a shared n,n 0 -diacetylchitobiose lectin-like domain. however, these similarities are solely functional and are not based on any (dna or amino acid) sequence homology, thus suggesting a form of convergent evolution. in particular, the lectin-like domain of tnf-a has been shown to induce membrane depolarization in various mammalian cell types, through interactions with endogenous amiloride-sensitive ion channels. this nonreceptor-mediated activity of tnf-a has been reported to be involved in the resorption of oedema. likewise, the lectin-like domain of ccf also induces membrane depolarization in mammalian cells. here, we show that ccf appears to be able to induce oedema resorption in an alveolar epithelial cell line through its lectin-like domain. this lectin-like domain of ccf interacts (directly or indirectly) with endogenous sodium and/or chloride channels, and not potassium channels, on mammalian cells. additionally, we suggest that the jnk/sapk and erk1/2 pathways are involved in ccf-induced macrophage activation. these results further establish the functional analogy between an invertebrate pattern recognition molecule and a mammalian cytokine and, from a more applied point of view, suggest the possibility of utilizing ccf in the treatment of oedema. release of svegf and svegfr1 from white blood cells and platelets during surgery and stimulation with bacterial antigens introduction: the influence of surgery on release of soluble vascular endothelial growth factor (svegf) and the soluble vascular endothelial growth factor inhibitory receptor 1 (svegfr1) is unknown. we studied the effect of major and minor surgery on potential variations in svegf and svegfr1 concentrations in vivo and on bacterial antigen-induced release of svegf and svegfr1 from whole blood in vitro. methods: sixty-one patients with abdominal diseases undergoing five different surgical procedures were included. blood samples were drawn from anaesthetized patients before and after the operation. white blood cells and platelets were counted, and plasma svegf and svegfr1 was determined by an elisa method. whole blood from each blood sample was stimulated in vitro with bacteria-derived antigens (lps or protein-a) and svegf and svegfr1 levels were subsequently determined in the supernatants. stimulation with isotonic saline served as control assay. neither svegf or svegfr1 in plasma changed during surgery. in vitro stimulation of blood samples with bacteria-derived antigens resulted in a significant increase in svegf (p < 0.0001) and a less pronounced but still significant increase in svegfr1. release of svegf due to stimulation was significantly higher after the operation (nonsignificant), whereas svegfr1 release remained largely unchanged after surgery. correlation between bacterial antigen-induced release of svegf and neutrophile cell count was highly significant (p < 0.0001). there was no correlation between svegf and platelet cell count, and bacterial antigen-induced svegfr1 release did not correlate with counts of neutrophils and platelets. conclusions: plasma svegf and svegfr1 concentrations did not change during surgery. in vitro bacterial stimulation led to increased release of svegf and svegfr1, which was not significantly amplified during surgery and which may be related to number of circulating neutrophils. natural killer cell functions and subsets after in vitro stimulation with il-2 and il-12, with special emphasis on intracellular ifn-g and nk-cell cytotoxicity r. nyboe, 1,2 t. rix, 1,2 j. krog, 1,2 e. tønnesen 1 & m. hokland 2 1 department of anaesthesiology and intensive care, aarhus university hospital, and 2 institute of medical microbiology, and immunology, university of aarhus, aarhus, denmark. e-mail: rnsr@studmed.au.dk materials and methods: isolated cryopreserved human peripheral blood mononuclear cells (pbmcs) were stimulated with il-2 and il-12. this stimulation has previously been shown to activate nk cells. cell cytotoxicity was measured by flow cytometry after incubation with k562 cells. this method was compared to the current standard 51cr release assay. cells were treated with bfa to accumulate ifn-g, stained for surface markers, permeabilized and stained for intracellular ifn-g. flow cytometry was then performed to measure intracellular ifn-g production in pbmc, especially in nk cells. results: we have demonstrated that stimulation with il-2 and il-12 is effective in increasing the number of ifn-gpositive cells. there is a distinct difference between the cd3-cd56dim and the cd3-cd56bright subsets, with a much greater proportion of ifn-g-positive cells in the cd3-cd56bright subset. the effects of stimulation with il-2 and il-12 on cytotoxicity will be presented, as will the relation between ifn-g production and cytotoxicity. in addition, we will present results of these assays applied to porcine cells. discussion: in combination, these tests will address nk cell function by combining cytotoxicity with ifn-g production in nk cell subsets. the results will demonstrate whether this could serve as a useful tool in describing nkcell function, which could be of value in clinical and experimental settings. culture of regulatory t-cell lines from bronchial mucosa t lymphocytes play a major role in many immune responses. in the last decade, special focus has been on the function of th1 and th2 effector cells. now the importance of regulatory cd4 þ cd25 þ t cells in maintenance of the immunological homeostasis emerges. sarcoidosis is a multisystem granulomatous disorder often affecting the lungs. the typical sarcoid granulomas consists of epitheloid cells, macrophages and lymphocytes, mainly cd4 þ t cells of th1 phenotype. we have cultured t cells from bronchial biopsies of patients with sarcoidosis as well as from controls in high levels of interleukin 2 (il-2) and il-4 and demonstrate spontaneously arising cd4 þ cd25 þ populations and high concentrations of il-10 in these cultures. the main difference between cultures of sarcoid origin compared to controls is a very much higher concentration of the inflammatory cytokines il-6 and tnf-a in cultures of sarcoid origin. the effects of hyperbaric exposure on human peripheral blood mononuclear cells, with special emphasis on natural killer cell cytotoxicity and subsets materials and methods: as an experimental physiological stress model, we examined the effects of hyperbaric exposure on peripheral blood mononuclear cells (pbmcs) obtained from venous blood drawn from eight divers during a simulated heliox saturation dive. eight persons working in normobar atmosphere outside the pressurized chamber served as control donors. the spontaneous cytotoxicity of the pbmcs was estimated in a 4 h 51cr-release assay using k562 as nk-sensitive target cells. the pbmcs were characterized, using 4-colour flow cytometry, with special emphasis on the nk-cell subsets. the data were statistically analysed using a multivariate regression model (stata 8.2). p values <0.05 was considered statistically significant. results: the estimated cytotoxicity increased significantly in both the group of divers and control donors during the dive (pdivers < 0.01 and pcontrols < 0.01). although the cytotoxicity increased relatively more (p < 0.01) in the group of divers compared to the group of control donors between day 1 and 2. discussion: the increased cytotoxicity of pbmc estimated in the group of divers indicate that parts of the cellular immune system are affected during the extreme physiological conditions induced during the initial phase of the presented experimental hyperbaric setup. the increase in cytotoxicity observed in the group of control donors could hypothetically reflect the stress level in persons working outside the pressurized chamber during the dive. the interaction with adhesion molecules expressed by vascular endothelium is the first step in lymphocyte infiltration into tissues. at both cutaneous and mucosal sites interleukin-10 (il-10), il-12 and transforming growth factor (tgf)-b are important regulators of chronic inflammatory disease, where cutaneous lymphocyteassociated antigen (cla) and ae integrin (cd103) may be expressed. unlike cla, cd103 is not believed to play a role in tissue-specific homing but may help to retain t cells within epithelial layers. we have previously shown that il-12 alone can together with an unknown cofactor increase the expression of cla. stimulation with streptococcal pyrogenic exotoxin c (spec) increased the expression of cd103 by cd8 þ but not cd4 þ t cells. while il-12 increased superantigen-stimulated expression of cla, this cytokine strongly inhibited the cd103 expres-sion, and a combination of il-12 and tgf-b completely abrogated the induced cd103 expression. conversely, il-10 suppressed cla but increased cd103 expression. these findings indicate that, in addition to suppressing the development of th1-mediated inflammatory responses, il-10 may also inhibit the migration of cd8 þ t cells into the skin while il-12 promotes such migration. thus, the expression of cla and cd103 may be antagonistically regulated by il-10 and il-12, and the balance between these cytokines could influence the t-cell migration of inflammatory cells into epithelial tissues. during contact sensitivity reaction, immune cells proliferate. in order to study the histological picture of these proliferation phases, we used a mouse model of contact sensitivity in the oral mucosa and on skin. we also used bromodeoxyuridin (brdu, an analogue to thymidin) that is incorporated into the nucleus during cell replication. the hapten oxazolone (oxa) was used to sensitize and elicit the oral mucosa and/or the ear skin. mice were killed at various times after elicitation, and unsensitized animals were also exposed to the hapten as controls. brdu (25 mg/ kg animal) was injected i.p. 2 h before the kill. specimens from the oral mucosa, ear skin and submandibular and auricular lymph nodes were cut and fixed in 4% paraformaldehyde. they were then treated with acid and biotinylated anti-brdu antibody and developed using abc-kit and dab. the analyses were performed using a leica light microscope and the computer program analysis. in the oral mucosa, the frequency of proliferating cells were increasing during the observation period, 4-24 h after elicitation, regardless of site of sensitization. the proliferating cells were found mainly in the basal cell layer of the epithelium. similar patterns were found in ear skin. the regional lymph nodes demonstrated a few scattered proliferating cells 4 h after elicitation. after 24 h, these cells were found frequently in the whole lymph node. control animals exhibited considerable less proliferating cells at all times. we conclude that most proliferating cells were found 24 h after elicitation locally at the hapten-exposed sites (the oral mucosa or the ear skin) as well as in the regional lymph nodes. the endogenous nucleoside adenosine is released in excess during inflammation or other metabolic stress and is generally known to deliver tissue protective anti-inflammatory effects. adenosine acts via four adenosine receptors of which the a2a receptor is the predominant form in t cells. adenosine levels are elevated in asthmatic lung, and adenosine can directly induce mast cell degranulation and bronchoconstriction in these patients. instead, the role of anti-inflammatory mechanisms of adenosine on t cells in asthma is unclear. aim: to study the a2a receptor expression in peripheral blood cd4 þ t cells in asthmatic and healthy individuals using flow cytometric and quantitative real-time pcr methods. results: unstimulated cd4 þ cells of asthmatic patients expressed significantly lower levels (p < 0.001) of a2a receptor in protein level (mean percentage of cells positive ae sem: 76.8 ae 1.2, n ¼ 6) compared to healthy individuals (90.4% ae 1.9, n ¼ 4). double staining for cd69 expression showed that stimulation of cd4 þ cells decreased a2a expression in both groups but indicated that the detected lower levels of a2a in unstimulated cells of asthmatics was not due to preactivation in these patients. surprisingly, a2a mrna expression in unstimulated cd4 þ cells was significantly higher (p < 0.05) in asthmatics (n ¼ 28) compared to healthy controls (n ¼ 7). the expression did not correlate with serum total ige levels. conclusions: asthmatic individuals express less a2a adenosine receptor on their peripheral cd4 þ t cells. the higher mrna levels instead may point to a negative feedback regulation in the receptor expression. the role of possibly decreased adenosine-mediated anti-inflammatory effects in asthma pathogenesis require further studies on this t-cell mediated disease. the chronic inflammatory skin disease atopic eczema (ae) affects almost 15% of the population in many countries today. the pathogenesis of ae is not fully understood. a combination of genetic predisposition and environmental factors like microorganisms seems to contribute to the symptoms. the yeast malassezia sympodialis is part of our normal skin micro flora but can act as an allergen and elicit specific ige and t-cell reactivity in patients with ae. recently, we identified a novel major m. sympodialis allergen, designated mala s 11 (22.4 kda), with sequence similarity to the mitochondrial enzyme manganese superoxide dismutase (mnsod). interestingly, mala s 11 has a high degree of homology to human mnsod. the aim of this study was to examine the effects of recombinant mala s 11 on antigen-presenting dendritic cells. monocytederived dendritic cells (mddcs) from healthy blood donors were cultured with or without mala s 11 for different time periods. it was found that the maturation marker cd83 and the costimulatory molecules cd80 and cd86 were upregulated on the mddcs exposed to mala s 11 for 24 h, as demonstrated by flow cytometry. furthermore, coculture of mddcs with mala s 11 for 9 h induced an increased production of the inflammatory cytokines il-6 (200-fold), tnf-a (100-fold) and il-8 (sixfold), as detected by the cytometric bead array (cba) analysis. our results suggest that mala s 11 affects the immune response through dc maturation and production of inflammatory cytokines. the potential cross-reactivity with human mnsod needs to be explored and the exact role of mala s 11 in the pathogenesis of ae assessed in clinical studies involving skin prick and atopy patch tests. allergen-specific immunotherapy (sit) is commonly conducted with allergen extracts adsorbed to aluminium hydroxide (alum). drawbacks linked to the use of alum, such as the formation of granuloma at the site of injection, have led to suggestions of novel allergen carriers. an alternative carrier is 2 mm carbohydrate-based particles (cbps). in mouse, allergen-coupled cbps have been demonstrated to skew the allergen-specific immune response towards a th1-like activity (grönlund et al. immunology, 2002) . we here coupled the recombinant major cat allergen fel d 1 to cbps (cbp-fel d 1) by cyanogen-bromide activation, resulting in covalent binding. the effect of cbp-fel d 1 on monocyte-derived dendritic cells (mddcs) from healthy human blood donors was studied. we found that the majority of the cd1a þ mddcs were capable of taking up fitc-labelled cbp-fel d 1, as demonstrated by flow cytometry and confocal laser scanning microscopy. furthermore, incubation with cbp-fel d 1 resulted in an upregulation of the costimulatory molecule cd86 on the mddcs, which was not observed with fel d 1 or cbps alone. finally, cbp-fel d 1 induced a fivefold increase in the release of the pro-inflammatory cytokine tumour necrosis factor (tnf)-a and a fourfold increase in the release of the chemokine interleukin-8 from mddcs. taken together, the effects cbps possess make them interesting as novel allergen carriers for sit. the cysteine protease der p1 from dust mite of the genus dermatophagoides pteronyssinus is a major type i allergen. about 80% of house dust mite (hdm) allergic individuals are reactive to this protease in standard assays for detection of ige. a curative treatment for atopic allergy is immunotherapy (it) with hdm extracts which are complex mixtures occasionally resulting in anaphylactic reactions. novozymes focuses on developing a recombinant variant of der p1 which exhibit lowered risk of ige-mediated allergic reactions, while maintaining its ability to trigger proper th-cell responses. this may provide a safer alternative for specific it of hdm allergy. a secreted recombinant form of pro-der p 1 expressed by saccharamyces cerevisiae was obtained by fusion of the pro-enzyme to a fungal signal peptide. the n-glycosylation site of der p1 was mutated resulting in a deglycosylated pro-enzyme with a molecular mass of 35 kda. protein purification procedure was developed to obtain nearly pure der p1 protein followed by determination of concentration by active-site-titration with the cysteine protease inhibitor e64. the deglycosylated recombinant pro-der p 1 revealed immunologic similarity to the native der p 1 molecule when compared in basophile histamine release, ige-binding assays and t-cell proliferation assays. by in silico epitope mapping of a modelled 3-dimensional structure of der p1, five putative igg and ige epitopes were predicted. by protein engineering, the predicted epitopes were removed one by one in der p1 and screening for hypoallergenic variants was performed. combining inhaled long-acting b-2 agonist (laba) and inhaled corticosteroid (ics) seems to offer asthma control at a lower dose of ics than achieved by ics alone. fine mapping of t-cell surface markers by flow cytometry offers a detailed status of the individual's inflammatory response. the frequency of mt2 (cd4 þ cd45ra -cd62l þ cd11adim) and mt1 (cd4 þ cd45ra -cd62l -cd11abright) cells in peripheral blood, and their ratio, has been shown to differ predictably in atopics and patients with leprosy, where mt2 correlates with a th2 phenotype and mt1 with a th1 phenotype. stable asthmatics, requiring fluticasone propionate (fp) 750-1000 mg daily or equivalent, were randomized to receive, double-blinded, either seretide 1 [salmeterol and fluticasone propionate (sfc, n ¼ 16)] 50 mg/500 mg bd or fp 500 mg bd (n ¼ 17). if asthma was controlled based on lung function and symptoms at clinic visits every 6 weeks, ics dose was tapered until asthma exacerbated or 0 mg was reached. the frequency and ratio of mt2 and mt1 t cells of the patients was monitored at 6 week intervals. as treatment tapered, the frequency of mt2 cells decreased (p ¼ 0038 from first to final visit), whereas that of mt1 cells increased. the ratio of mt2/mt1 decreased (p ¼ 0049 from first to final visit). in patients receiving laba þ ics, the fall in mt2/mt1 ratio appeared to be more pronounced than in patients receiving ics alone. thus, the mt2 phenotype may be associated with stable asthma, whereas an imminent exacerbation may associate with an increase in the mt1 phenotype. laba may allow for a greater effect of fp on the mt ratio. activation of complement pathways, leading to production of c3a and c5a anaphylatoxins, has been postulated in the pathogenesis of asthma and allergic airway inflammation. the present study was undertaken to investigate the role of mannan-binding lectin (mbl), an initiator of the lectin pathway of complement, in asthma and allergic rhinitis. mbl levels and mbl-induced complement activity were determined in 19 patients of bronchial asthma with allergic rhinitis and 20 unrelated, age-matched controls of indian origin. mbl levels and activity were correlated with percent eosinophilia and percent predicted fev1 values of the patients. association of single nucleotide polymorphisms (snps) in exon 1 and intron 1 of the mbl with the disease, clinical markers, mbl levels and mbl-induced complement activity was analysed using standard statistical tools. significantly higher mbl levels and activity were observed in patients of bronchial asthma with allergic rhinitis as compared to the controls. we identified five snps, of which two, a816g in exon 1 and g1011a in intron 1 of the mbl, were novel. snp g1011a was significantly associated with the disease (p ¼ 0.0024, or ¼ 5.8696, 95% ci: 1.7316 < or < 19.8963). individuals with 'a' allele at position 1011 showed increased mbl levels, activity and disease severity. our results suggest that 'a' allele at position 1011 leading to high mbl levels and complement activity may be contributing to the severity of bronchial asthma and allergic airway inflammation. serum resistance of borrelia burgdorferi strains belonging to the b. afzelii and b. burgdorferi sensu stricto genospecies is dependent on binding of complement inhibitor factor h. we recently reported that factor h binding by b. burgdorferi is due to inducible expression of several approximately 20 kda plasmid-encoded, surface-exposed lipoproteins related to ospe (e.g. erpa, erpp and p21). in addition, a second class of factor h-binding proteins of approximately 27-35 kda has been described. the ospe-related lipoproteins are dramatically induced by b. burgdorferi during transmission from its tick vector into the mammalian host. the induction of ospe-related lipoproteins during mammalian infection may play a key a role in the borrelial evasion of the host's immune system. the goal of the present study was to define the factor h-binding regions of ospe-related proteins using mutagenesis, peptide mapping and surface plasmon resonance analysis (biacore). the combined studies revealed that the c-terminal regions of both human and mouse factor h (scrs 18-20) specifically bind to ospe-related lipoproteins. we also found fhr-1, whose c-terminal scrs 3-5 are homologous to scrs 18-20 of factor h, to bind to ospe. peptide mapping revealed five putative regions (designated i-v) in ospe that could directly interact with factor h. deleting the c-terminal 15 amino acid residues from region v of p21 abolished its ability to bind factor h. at the same time, however, synthetic peptides corresponding to the c-termini of ospe, p21 and erpp did not inhibit factor h binding to ospe. thus, the c-terminal-binding region v appears to be necessary but not sufficient for factor h binding. when a more specific mutation strategy was employed, where single amino acid residues in peptides spanning over the factor h-binding regions were mutated to alanines, we observed that lysines in the factor h-binding regions of ospe were required for factor h binding. the combined data have revealed that key lysine residues in ospe-related lipoproteins and ionic interactions are crucial for factor h interactions. furthermore, binding of ospe to the c-termini of both mouse and human factor h suggests that borrelia spirochetes utilize analogous complement resistance mechanisms in both rodents and man. in borrelia garinii strains, which in in vitro analyses have been found to be sensitive to complement killing, differences in the ospe sequences as well as in the expression of factor h-binding proteins may account for their susceptibility to serum lysis. role of yada, ail and lipopolysaccharide in serum resistance of yersinia enterocolitica serotype o:3 mannan-binding lectin (mbl), l-ficolin and h-ficolin are pattern recognition molecules of the innate immune system. we investigated the ability of these molecules to bind to different serotypes and noncapsulated variants of streptococcus pneumonia and staphylococcus aureus. we found that mbl binds to noncapsulated s. aureus strain (wood) but not any of the examined s. pneumoniae serotypes. l-ficolin binds to some capsulated s. pneumoniae serotypes (11a, 11d and 11f) as well as some capsulated s. aureus serotypes (type-1, -8, -9, -11 and -12). h-ficolin does not bind to any of the examined s. pneumoniae and s. aureus serotypes included in this study but did bind to a strain of aerococcus viridans. when bound to bacteria, mbl and h-ficolin initiated activation of complement factor c4, whereas l-ficolin did not. during this study, quantitative assays for the three proteins were developed and the concentration in 97 plasma samples were determined and the median values were estimated at 0.8 mg of mbl/ml, 3.3 mg of l-ficolin/ml and 18.4 mg of h-ficolin/ ml, respectively. the absence of early complement components (c1, c4 and c2 but not c3) is a predisposing factor for systemic lupus erythematosus (sle). recently, we demonstrated that, in c4-deficient (c4 def.) mice, igm-containing immune complexes (igm-ic) are filtered by the splenic barrier of marginal zone macrophages (mzm), resulting in an increased immune response against antigens within these igm-ic, but this could not be observed in wildtype or c3 def. mice. we hypothesized that splenic cd11b þ mzm play an important role in the induction of autoimmunity, and we therefore analysed their cytokine profile after isolation with the help of magnetic antibody cell sorting. mrna was isolated, and real-time pcr was performed with specific primers for murine ifn-g (ifn-g), interleukin-12 (il-12) and ifn-a (ifn-a). we observe a moderate increase of il-12 and ifn-g mrna in cd11b þ cells of c4 def. mice compared to wildtype cells. surprisingly, the concentration of ifn-a mrna is six times higher in c4 def. mice. preliminary results suggest that mrna in cd11b þ cells of c3 def. mice is even lower than that in wt. six hours following i.v. application of 20 mg of a abstracts 625 .................................................................................................................................................................................................. murine monoclonal igm anti-dsdna antibody, production of il-12, ifn-g and ifn-a mrna is increased in cd11b þ cells of both c4 def. and wt mice. several references described increased levels of inf-a in patients with sle. dendritic cells are discussed as a major source of ifn-a. our observation that c4-deficient, sle-susceptible mice demonstrate an increased spontaneous ifn-a production by splenic cd11b þ marginal zone macrophages could be an early sign and a trigger for the development of sle. this is supported by the fact that the absence of c3 is not a predisposing factor for sle and our observation that c3 def. animals display low levels of ifn-a mrna. 200-400 million people worldwide and represents one of the leading causes for liver cirrhosis and hepatocellular carcinoma. control over the hbv infection is achieved mainly by vaccination with hepatitis b surface antigen (hbsag). hbsag contains n-linked glycosylation side and is recognized by both mbl-a and mbl-c in a cadependent manner. hbsag-mbl complexes activate complement and may thus affect humoural immunity. to investigate the role of mbl in humoural responses to hbsag, we immununized mice that lack both mbl-a and mbl-c proteins with soluble hbsag. it has been shown that deficiencies in other complement components like c1q, c4 and c3 result in decreased antibody responses. however, mbl double ko animals mounted dramatically increased humoural responses. after priming, mbl double kos mounted hbsag-specific igm responses, which were threefold higher than wt controls. after boosting the hbsag, total igg was 10-fold higher in mbl ko than in wt control animals. similar to the response to hbsag, other glycosylated soluble antigens (e.g. invertase) induced better humoural responses in mbl double ko animals, suggesting that mbl plays an important role in a negative feedback regulation of adaptive immunity. reconstitution experiments with rmbl partially rescued the ko phenotype. we propose that the clearance of glycoprotein antigens in mbl ko is handled differently from the wt, resulting in better stimulation of humoural responses. alternatively, glycoprotein-ag-mbl-rich complexes inhibit b-cell responsiveness via putative mbl receptors. the complement system is an important part of the innate immune system. the activation of complement proceeds through three different pathways that converge in the generation of c3-activating enzyme complexes. complement activation via the lectin pathway is initiated when recognition molecules, mannan-binding lectin (mbl) or ficolin, bind to carbohydrate structures characteristic for microbial surfaces. in the circulation, mbl and ficolins are found in association with three structurally related mblassociated serine proteases (masp)-1, -2 and -3 and a small, nonenzymatic component, map19. masp-2 has been shown to elicit complement activation through the sequential proteolytic cleavage of c4 and c2 upon binding of mbl/masp-2 complexes to microbial surfaces. we have recently uncovered a polymorphism in the masp-2/map19 gene in a patient shown to be deficient in the lectin pathway of complement activation. the polymorphism results in a single amino acid substitution in the n-terminal part of the masp-2 protein. recombinant wildtype masp-2 and masp-2 containing the amino acid substitution in question was produced, and the ability to activate complement was studied. the mutation had a profound impact on masp-2 function, resulting in the lack of complement activation through the lectin pathway. elisa-based experiments showed that the mutation leads to the impairment of complement activation through influencing the binding of masp-2 to mbl or ficolins. deficiencies in the lectin pathway of complement activation have so far been accounted for only by lack of functional mbl. the mutation described above is the first defect described affecting both activation through mbl and the ficolins. .................................................................................................................................................................................................. th1, th2 and treg cell balance. dcs are present in the gut mucosa and may thus be target for modulation by gut microbes, including ingested probiotics. here, we tested the hypothesis that species of lactic acid bacteria, important members of the gut flora, differentially activate dc. a large panel of human gut-derived lactobacillus and bifidobacterium spp. was screened for dc-polarizing capacity by exposing bone marrow-derived murine dc to lethally irradiated bacteria. cytokines in culture supernatants and dc-surface maturation markers were analysed. substantial differences were found among strains in the capacity to induce interleukin-12 (il-12) and tumour necrosis factor (tnf)-a, while the differences for il-10 and il-6 were less pronounced. bifidobacteria tended to be weak il-12 and tnf-a inducers, while both strong and weak il-12 inducers were found among the strains of lactobacillus. remarkably, strains weak in il-12 induction inhibited il-12 and tnf-a production induced by an otherwise strong cytokine-inducing strain of lactobacillus casei, while il-10 production remained unaltered. selected strains were tested for induction of dc maturation markers. those lactobacilli with greatest capacity to induce il-12 were most effective in upregulating surface mhc class ii and cd86. moreover, l. casei-induced upregulation of cd86 was reduced in the presence of a weak il-12inducing l. reuteri. in conclusion, human lactobacillus and bifidobacterium spp. polarize differentially dc maturation. thus, the potential exists for th1/th2/treg-driving capacities of the gut dc to be modulated according to composition of gut flora including ingested probiotics. the intestinal micro flora is indispensable in developing and maintaining homeostasis of the gut-associated immune system. evidence indicates that lactic acid bacteria (lab), e.g. lactobacilli and bifidobacteria, have beneficial effects on the host. established health effects include increased gut maturation, antagonisms towards pathogens and immune modulation. the objective of this study is to evaluate the immunomodulating properties of a range of lab of human origin. as dendritic cells (dcs) play a pivotal role in the balance between tolerance and immunity to commensal microorganisms, in vitro-generated immature dcs serve as a suitable model for studying the immunomodulating effects of lab. human immature dcs were generated in vitro from monocytes and exposed to lethally uv-irradiated lab. the effect of various species of lab on dcs in direct contact was evaluated. furthermore, the maturation pattern of dcs separated from the bacteria by an epithelial cell layer (caco-2 cells), which should mimic the intestinal environment, was studied. cytokine secretion (il-12, il-10 and tnf-a) and upregulation of maturation surface markers on dcs (cd83 and cd86) was measured. different lab induced diverse cytokine responses. some strains were strong il-12 and tnf-a inducers and others weak. all strains induced il-10. different lab also differentially modulated expression of cd83 and cd86 on dcs. although some variation in the response to lab of dcs generated from different blood donors was observed, general differences in the effect of the various lab was revealed. experiments with the dc caco-2 coculture system are ongoing. different species of lab differentially affect dc maturation; this suggets that the gut flora plays a pivotal role in polarization of the immune response. natural killer (nk) cells are cells of the nonspecific immune system lysing altered self-cells. a noncytolytic subset of nk cells may serve a regulatory role by secreting cytokines. bacteria translocating across the gastrointestinal mucosa are presumed to gain access to nk cells, as consumption of certain lactic acid bacteria has been shown to increase in vivo nk cytotoxicity. here, we investigated how human gut flora-derived lactobacilli affect nk cells in vitro, by measuring proliferation and ifn-g production of human nk cells upon bacterial stimulation. cd3 -cd56 þ nk cells were isolated from buffy coats by negative isolation using non-nk lineage-specific antibodies and magnetic beads. nk cells were incubated with 10mg/ml uv-inactivated bacteria or 10mg/ml phytohemagglutinin (pha) for 4 days. proliferation was assessed by incorporation of radioactive thymidine into nk-cell dna. the ifn-g concentration was measured by elisa. incubation of nk cells with a lactobacillus acidophilus strain increased the proliferation of the nk cells and induced ifn-g production, both to levels comparable to pha stimulation. the proliferative response was further enhanced with autologous monocytes present, probably because cytokines, secreted by monocytes having engulfed bacteria, stimulated the nk cells. in contrast, a lactobacillus paracasei strain caused the nk cells to proliferate only in the presence of monocytes. these results demonstrate that various strains of lactobacilli have the capacity to activate nk cells in vitro, in a monocyte-dependent or -independent way. hence, the encounter of nk cells with lactic acid bacteria will affect nk-cell activation. such activation of nk cells may potentially skew an on-going or subsequent immune response towards a th1 response. lactobacilli are nonpathogenic gram-positive inhabitants of the normal human intestine known for their healthpromoting effects. in our earlier work, it is shown that human monoclonal antibody isolated from sera of a patient with waldenstrom macroglobulinaemia possess innate antibody characteristics and binds to lactic acid bacteria. according to the immune network model, immunization with this bacteria could induce the perturbations in immune system that might result in production of anti-lactobacillus antibodies, human monoclonal antibody like (ab1) and anti-idiotypic antibody (ab2). in this study, balb/c mice were immunized with two doses of bacteria lactobacillus acidophilus in complete and incomplete freund's adjuvant and phosphate-buffered saline (pbs), respectively. seven days after the last immunization, sera from immunized mice were collected and the presence of lactobacillus-specific ab1 and ab2 were determined by elisas. in the sera of immunized mice, antibodies specific to bacteria lactobacillus acidophilus were shown. the concentration of lactobacillus-specific antibodies was higher in the sera of hyperimmunized mice (mice immunized with 1 mg of igm dj) than in sera of mice immunized with 100 times lower doses of immunogen (0.01 mg per doses). moreover, ab1 and ab2 antibodies were detected in the sera of lactobacillus-hyperimmunized mice. in this study, we have shown the idiotypic network interactions in mice immunized with bacteria lactobacillus acidophilus. the normal gastrointestinal flora is crucial for the maturation of the acquired immunity via effects on antigenpresenting cells (apcs). here, we have investigated how two types of apcs, monocytes and dendritic cells (dcs), react to different bacterial strains typical of the commensal intestinal flora. purified monocytes and monocyte-derived dcs were stimulated with uv-inactivated gram-positive (lactobacillus plantarum and bifidobacterium adolescentis) and gram-negative (escherichia coli and veillonella parvula) bacterial strains. monocytes produced higher levels of il-12p70 and tnf, as detected by elisa, in response to l. plantarum than to e. coli and v. parvula. in contrast, dcs secreted high amounts of il-12p70, tnf, il-6 and il-10 in response to e. coli and v. parvula but were practically unresponsive to l. plantarum and b. adolescentis. the lack of response to the gram-positive strains correlated with a lower surface expression of toll-like reseptor 2 (tlr2) on dcs compared to monocytes. the surface expression of tlr4 on dcs was undetectable when analysed by flow cytometry, but blocking this receptor decreased the tnf production in response to v. parvula, indicating that low tlr4 expression on dcs is sufficient to mount an inflammatory response to gram-negative bacteria. ifn-g increased the expression of tlr4 on dcs and also potentiated the cytokine response to gram-negative bacteria. our results indicate that, when monocytes differentiate into dcs, their ability to respond to different commensal bacteria dramatically changes, thereby becoming unresponsive to probiotic gram-positive bacteria. these results may have important implications for the capacity of different groups of commensal bacteria to regulate mucosal and systemic immunity. probiotic bacteria, e.g. lactobacillus spp., may improve diseases such as chronic inflammatory bowel disease. we examined cytokine production and phenotypic change after in vitro stimulation of t cells from healthy volunteers using different probiotic strains. methods: t cells were cultured from colonic biopsies from eight healthy volunteers (agnholt and kaltoft, exp clin immunogenet 2001; 18:213-25) , and dendritic cells were matured from their peripheral blood mononuclear cells. t-cell cultures were stimulated with autologous bacterial sonicate or strains of lactobacillus spp., with and without the addition of dendritic cells. cytokine levels (tnf-a, ifn-g, il-10 and gm-csf) and phenotype (cd3, cd4, cd25 and cd69) were measured on day 4. results: lactobacillus spp. induced higher productions of tnf-a and il-10 than did autologous bacteria. in presence of dendritic cells, the production of all cytokines increased. however, the increases of ifn-g and tnf-a were more pronounced in wells with autologous bacteria than in wells with lactobacillus spp. the addition of dendritic cells upregulated cd25 expression without simultaneous upregulation of cd69. the upregulation was pronounced after stimulation with lactobacillus rhamnosus gg compared with autologous bacteria and other lactobacilli. discussion: in presence of dendritic cells, autologous bacteria induced inflammatory cytokines, while probiotics mainly induced regulatory cytokines. lactobacillus rhamnosus gg induced a regulatory phenotype (cd25 þ ), in part mediated by dendritic cells. future studies will address whether this shift to a cd25 þ phenotype represents a differentiation into competent regulatory t cells. in a clinical context, such cells might be used for treatment of inflammatory diseases. protein microarrays will play a key role in the postgenomic era and offer a unique possibility to perform highthroughput global proteome analysis. a chip can be printed with thousands of protein probes (e.g. antibodies), the biological sample added (e.g. a proteome) and any binding detected. we aim to develop protein microarrays based on human recombinant scfv antibody fragments for global proteome analysis. the concept of comparing proteomic maps of healthy versus diseased samples will allow diseasespecific proteins to be detected. in fact, antibody microarrays will allow us to perform comparative proteome analysis on any sample format in a species-independent manner, as long as a proteome can be isolated. however, the complexity of proteomes, containing several thousands of different proteins, is a problem. here, we have designed antibody microarrays targeting the water-soluble fraction of a proteome. to this end, an anticytokine antibody array was developed and human dendritic cells (aeactivation) was used as model system. the results showed that our antibody microarrays could be used to examine the cytokine profile in complex samples. furthermore, we have taken the first steps towards comparing our results with those of other technologies on both the protein and gene level. due to the complexity of the model proteome, we also examined the possibility to prefractionate the proteome in a simple one-step procedure (based on size) prior to the labelling step. in more detail, the sample proteome was fractionated into two fractions using membrane devices with different molecular weight cut-offs. the results showed that the fractionation considerably enhanced the assay sensitivity allowing cytokines in the pg/ml range to be readily detectable. the immunomodulatory effect of heat shock protein 70: immunization with a dna construct based on the malarial antigen fused with a fragment of hsp 70 primes for a th-1 type of response finding an appropriate adjuvant for human vaccination is crucial. heat shock proteins (hsps) act as adjuvants when coadministered with peptide antigens or given as fusion proteins. however, there is a potential risk of autoimmunity when using the complete molecules, because hsps are evolutionary conserved. to overcome this, we first evaluated the adjuvant effect against two different antigens of a less-conserved fraction of plasmodium falciparum hsp70 (pf70c) and compared it to the whole hsp70 molecule from trypanosoma cruzi (tchsp70). we found that pf70c exhibited similar adjuvant properties as the whole molecule. we later evaluated the adjuvant potential of pf70c against the malarial antigen eb200 in a chimeric dna construct. no appreciable levels of eb200-specific abstracts 629 .................................................................................................................................................................................................. antibodies were detected in mice immunized only with the dna constructs. however, dna primed the immune system, because subsequent challenge with the corresponding recombinant fusion proteins elicited a strong th-1 antibody response. in contrast, no priming effect was observed for ex vivo ifn-g production but stimulation with the hsp-chimeric fusion protein induced a stronger secretion of ifn-g in vitro than other proteins used. these results indicate that the use of hsps is promising in the design of new vaccines. high-throughput proteomics on antibody-based microarrays: the importance of probe and surface design in analogy to dna microarrays, protein microarrays offer a new distinct possibility to perform sensitive highthroughput global proteome analysis. however, the development of the protein microarray technology will place high demands upon the design of both probes and solid supports. the analysis of thousands of heterogeneous proteins on a single microarray requires the use of uniform probes, such as antibodies, directly designed for protein microarray applications. we have recently generated a human recombinant single-chain fv antibody library, genetically constructed around one framework, the ncoder-library, containing 2 â 1010 clones. single framework antibody fragments (sinfabs) selected from this library were successfully applied as probes for microarrays providing sensitive detection in the 600 attomol (mass spectrometry) and the 300 zeptomole range (fluorescence). however, the choice of framework is critical. we have shown that the selected ncoder framework displayed excellent functional on-chip stability and arrayed dehydrated probes retained their activity for several months. furthermore, we have addressed the issues of biocompatibility of the solid support and immobilization strategies for our microarray setup. an in-house-designed substrate, macroporous silicon coated with nitrocellulose (map3-nc7), displayed properties equal to, or better than, those of five commercially available supports used as reference surfaces. we have also evaluated different coupling strategies, such as adsorption, covalent coupling, diffusion and affinity coupling. using a novel affinity tag, the double-(his)6-tag, we increased the binding efficiency of sinfab-molecules to ni2 þ -coated solid supports, thereby allowing nonpurified probes to be directly applied. the mannan-binding lectin (mbl) pathway is part of the innate immune system providing a first line of defence against infections. mbl and ficolins circulate in complexes with mbl-associated serine proteases (masp-1, -2 and -3). after recognition of a microorganism by mbl, activation of the complement system occurs. masp-1 and masp-3 share five domains (making up the so-called a-chain), whereas they have unique protease domains (b-chains). before the identification of masp-3, an assay for masp was presented, based on antibodies against the a-chain of masp-1. with the new knowledge of the three masps, and the sharing of domains by masp-1 and masp-3, assays specific for the protease domains have to be constructed, if one wishes to measure the proteins individually. we present an assay for quantifying total masp-3 in plasma and serum samples. the assay is a sandwich-type assay using as catching antibody a monoclonal antibody against the common a-chain of masp-1/3 and a developing secondary antibody against the c-terminal part of the protease domain of masp-3. we have used this assay for estimating the normal concentration of the protein as well as the concentration in patients and also for characterizing by gel permeation chromatography the masp-3 protein in serum. inducible costimulator ligand (icosl) is a costimulatory molecule related to b7.1 (cd80) and b7.2 (cd86). b cells, monocytes, dendritic cells and endothelial cells express icosl. inducible costimulator (icos) interacts with icosl, and this interaction leads to signals involved in isotype switching and the development of immunological memory. hitherto, no polymorphisms of this gene have been described. the aim of this study was to reveal variation of the icosl gene in normal individuals. all eight exons, except exon 1, were sequenced with flanking introns in 10 healthy blood donors. eight single nucleotide polymorphisms (snps) and two length polymorphisms were found. one of the snps was found in the coding regions of the gene. the base involved was located in exon 3 and caused a conservative amino acid change from valine (gtt) to isoleucine (att). three individuals were heterozygous g/a for the exon polymorphism, while the remaining seven individuals were homozygous for the wildtype g/g. exon 3 encodes the immunoglobulin variable (igv)-like domain of the molecule which is situated outside the cell. this means that the amino acid could be critical for the stability of the molecule or could constitute part of the binding site for icos. the results form the basis for further experiments to find possible associations of the alleles to diseases caused by immune dysregulation. especially, the exon 3 variant is interesting and could play a role for the development of immunological diseases. besides, it would be interesting to see whether both exon 3 alleles are expressed or only the wildtype allele is functional. interferon-a/b (ifn-a/b) is increasingly used as antiviral and immunomodulatory therapies. unfortunately, bioavailability varies with ifn species and mode of administration, and all ifn species are potentially immunogenic. assays for antiviral activity (ifn) and antiviral neutralization (antibodies, nab) have been used for some time to monitor patients on ifn biologicals. these assays require laborious titrations making them unsuitable for large-scale clinical use. myxovirus a (mxa) is a resistance gtpbinding protein that is specifically induced by treatment with type 1 ifns. for example, ifn-b-induced mxa in blood leucocytes has been used as a biomarker in ifn-btreated patients with multiple sclerosis. however, the degree of specificity of mxa in this regard is unclear, and measurements of mxa protein and/or mrna are not yet suitable for routine clinical use. in an attempt to find new and better reporter genes (and, hopefully, genes and gene products with proven specificity for ifn-a and -b), microarray screenings with u133a genechips (affymetrix) were carried using human blood leucocytes and the human lung carcinoma cell line a549. we studied the simultaneous expression of 22,000 transcripts before and after exposure to human recombinant ifn-a and ifn-b and other antiviral and immunomodulatory cytokines. the results will be presented at the conference. interferon-a/b (ifn-a/b) is increasingly used as antiviral and immunomodulatory therapies. unfortunately, bioavailability varies with ifn species and mode of administration, and all ifn species are potentially immunogenic. assays for antiviral activity (ifn) and antiviral neutralization (antibodies, nab) have been used for some time to monitor patients on ifn biologicals. these assays require laborious titrations making them unsuitable for large-scale clinical use. our laboratories have therefore modified the antiviral assays for ifn bioactivity and nab, so that they are suitable for large-scale screening in specialized laboratories. the read-out is survival of a subcloned a549 cell line in the presence of an otherwise lethal amount of virus. thus, survival increases in the presence of type 1 ifn and decreases in the presence of nab against the ifn added to the cells. mxa is induced by type 1 ifn and can be used for measuring the nab activity. in another assay, the mxa level in the a549 cell line is measured. in an attempt to find a new and better reporter gene for type 1 ifn than mxa and genes specific for either ifn-a or -b, a micro array screen was carried using the u133a chip from affymetrix. the expression of 22,000 genes can be studied simultaneous with this technology. the results will be presented at the conference. in our laboratory, we have developed a database system, which we believe is of immediate interest to the general scientific community. the database represents a computerbased replacement for the laboratory notebooks used in the majority of research laboratories worldwide. in addition, the database provides an effective tool for organizing and managing laboratory information at all levels, spanning from managing and revising standard operating procedures and producing documentation of research activities to keeping track of data and conclusions. using the commercially available database toolkit software filemaker pro, we have developed a relational database solution for management of laboratory information. the system consists of a hierarchy of five interrelated databases, each pertaining to a separate type of information, namely, overall project information, information relating to individual experiment setups, documentation of daily research activity, generated data and descriptions of standard operating procedures. like other databases, each individual database consists of a number of records, each comprised of a set of fields in which information is entered. in each record, a certain field is reserved to specify the relation of the record to a record in another database at a higher level. thus, the database is essentially five databases linked by a hierarchy of one-to-many relations, organizing information in a folder-like structure. importantly, the database system allows multiple users to access and edit records simultaneously, and the data entered in one database immediately becomes accessible through the other databases. the limitations of laboratory notebooks are apparent when looking for information, which is dispersed throughout one or more notebooks, or possibly on loose sheets of paper or printouts 'somewhere'. the often complicated process of gathering laboratory data or results when writing grant applications or research papers is made considerably easier with the database system. thus, the database solution presented should be broadly attractive to researchers, irrespective of their scientific discipline. an effective sars vaccine is likely to include components that can induce specific cytotoxic t-cell (ctl) responses. the specificities of such responses are governed by hlarestricted presentation of sars-derived peptide epitopes. exact knowledge of how the immune system handles protein antigens would allow for the identification of such linear sequences directly from genomic/proteomic sequence information. the latter was recently established when a causative coronavirus (sars cov) was isolated and full-length sequenced. here, we have combined advanced bioinformatics and high-throughput immunology to perform an hla supertype, genome-wide scan for sars-specific cytotoxic t cell epitopes. the scan includes all nine human hla supertypes in total covering >99% of all major human populations. for each hla supertype, we have selected the 15 top candidates for test in biochemical-binding assays. at this time (approximately 6 months after the genome was established), we have tested the majority of the hla supertypes and identified almost 100 potential vaccine candidates. these should be further validated in sars survivors and used for vaccine formulation. we suggest that immunobioinformatics may become a fast and valuable tool in rational vaccine design. rationale: major histocompatibility complex class i (mhc i) molecules monitor the protein content of the cell by binding small derived peptides and presenting them to cytotoxic cd8 þ t cells. the goal of the human mhc project is to predict the binding strength of any given peptide/mhc complex. this prediction allows the design of peptide-based vaccines. the prediction requires representative binding data from mhc alleles from all the nine hla supertypes. here, we describe the genetic construction, protein production and purification as well as the establishment-binding assays for two recombinant mhc supertype alleles, hla-b*1501 and hla-b*5801. methods: using the quikchange multisite directed mutagenesis kit (stratagene), codon-optimized genes encoding hla-b*1501 and hla-b*5801 are created. the two mhc i molecules are fermented and purified by ion exchange chromatography, hydrophobic interaction chromatography and size exclusion chromatography. the binding (kd) of natural t-cell epitopes, as well as predicted peptide ligands, is described by radioactive immunoassays (rias) and enzyme-linked immunosorbent assays (elisas). the mhc molecules are biotinylated during expression. results: the expression of mhc i resulted in multiple disulfide bond isomers, which are separated by hydrophobic interaction chromatography and used in subsequent binding studies resulting in the determination of kd for various peptide ligands ranging from strong binders we have previously demonstrated that bioinformatics tools such as artificial neural networks (anns) are capable of performing pathogen-, genome-and hlawide predictions of peptide-hla interactions. these tools may therefore enable a fast and rational approach to epitope identification and thereby assist in the development of vaccines and immunotherapy. a crucial step in the generation of such bioinformatics tools is the selection of data representing the event in question (in casu peptide-hla interaction). this is particularly important when it is difficult and expensive to obtain data. herein, we demonstrate the importance in selecting information-rich data and we develop a computational method, query-bycommittee, which can perform a global identification of such information-rich data in an unbiased and automated manner. furthermore, we demonstrate how this method can be applied to an efficient iterative development strategy for these bioinformatics tools. methods: a large panel of binding affinities of peptides binding to hla a*0204 was measured by a radioimmunoassay (ria). this data was used to develop multiple first generation anns, which formed a virtual committee. this committee was used to screen (or 'queried') for peptides, where the anns agreed ('low-qbc'), or disagreed ('high-qbc'), on their hla-binding potential. seventeen low-qbc peptides and 17 high-qbc peptides were synthesized and tested. the high-or low-qbc data were added to the original data, and new high-or low-qbc second generation anns were developed, respectively. this procedure was repeated 40 times. the high-qbc-enriched ann performed significantly better than the low-qbc-enriched ann in 37 of the 40 tests. conclusion: these results demonstrate that high-qbcenriched networks perform better than low-qbc-enriched networks in selecting informative data for developing peptide-mhc-binding predictors. this improvement in selecting data is not due to differences in network training performance but due to the difference in information content in the high-qbc experiment and in the low-qbc experiment. finally, it should be noted that this strategy could be used in many contexts where generation of data is difficult and costly. interleukin-18 (il-18), a pro-inflammatory cytokine that is produced by both lymphoid and nonlymphoid cells, has a critical role in modulation of innate and adaptive immunity. its primary function in stimulation of ifn-g production and stimulation of nk-cell-cytotoxic activities makes this cytokine a candidate for cancer immunotherapy. in oral cavity, this cytokine is produced by oral epithelia and carcinoma cells and is related to tumour regression in nude mice bearing salivary adenocarcinoma. however, direct effects of this cytokine on oral cancer cells have not been elucidated. in this project, we investigated il-18 effect on an oral carcinoma (kb) cell line. with rt-pcr technique, kb-cell line was found to express il-18 receptors (il-18ra and il-18rb), indicating that this oral carcinoma line is a target for il-18 study. we showed that recombinant human il-18 inhibited kb-cell proliferation by 17% at concentration of 100 ng/ml (p < 0.05), whereas ldh release by these cells in treatment group and control groups was comparable, indicating that il-18 suppression of cell proliferation was not mediated by the induction of cell death. to further address this hypothesis, we found that il-18 treatment did not induce apoptotic cell death, as studied by dna laddering and tunel assays. in addition, expression pattern of cell death-controlling genes (bcl-2 and bax) was not altered by this cytokine. findings in these studies indicated that suppression of kb-cell proliferation may be attributed to control of cell cycle, growth arrest or induction of cell differentiation. the data presented in this project could provide an insight of how cancer cell directly responds to il-18, as this cytokine is an important regulator of anticancer mechanisms. aloe emodin (ae) is a naturally occurring compound with wide spectrum of biological properties, including antimicrobial, vasorelaxant, immunosuppressive and anticancer actions. this anthraquinone induces apoptosis in several tumour cell lines with special affinity to tumours of neuroectodermal origin. high amounts of nitric oxide (no) released by activated macrophages induce tumour cell death. therefore, we explored the capacity of ae to modulate no-mediated antitumour response in vitro. interestingly, while ae markedly suppressed no release from macrophages alone, it significantly potentiated no production in cocultures of macrophages and c6 cells, after 48 h of cultivation. accordingly, the viability of c6 cells cocultivated with macrophages was reduced in the presence of ae. moreover, the observed ae-imposed potentiation of no production in macrophages was closely related to macrophage culture cell density. according to these data, we proposed that no modulator capacity of ae strongly depended on intercellular contact, indicating that macrophage antitumour response was not compromised but even potentiated by ae. immunotherapy represents an attractive fourth-modality therapeutic approach, especially in the light of the shortcomings of conventional surgery, radiation and chemotherapies in the management of metastatic cancer. to this end, a large number of peptide antigens derived from taa have been applied in immunotherapeutic trials for the treatment of various malignancies, e.g. cancers of the breast, prostate and kidney, in addition to haematological cancers. in some cases the response rates have been impressive and no adverse autoimmunity have been observed. a major strategic difficulty associated with these trials relates to the choice of best-suited peptide antigens. the vast majority of the antigens described thus far is not vital for survival and growth of the tumour cells, and immunoselection of antigen-loss variants may therefore prove to be an additional obstacle for the clinical applicability of most of the known peptide epitopes. in this respect, the development of acquired antigen loss during immunotherapy has been demonstrated in several cases. obviously, the development of loss-variant tumour cells implies that these cells acquire a pronounced growth advantage and are left unaffected by further treatment. ideally, target antigens should be derived from proteins required for survival and growth of tumour cells, as antigens with these characteristics would not be inflicted by the development of loss-variant tumour cells. in this respect, several inhibitors of apoptosis proteins (iaps) are universally expressed among tumours and play an important role in tumour cell escape from apoptosis. we have characterized spontaneous t-cell reactivity against iapderived peptides in cancer patients. from the iap survivin, we have characterized peptides restricted to the class i molecules hla-a1, a2, a3, a11, b7 and b35. furthermore, we have demonstrated that survivin-specific t cells infiltrate metastatic lesions and that isolated survivinspecific ctls are capable of killing hla-matched tumour cells. survivin-derived peptides are now in clinical trial, and continued work in our lab has demonstrated that other iaps are targets for spontaneous t-cell reactivity in cancer patients. we previously reported that in mice with large progressing t-cell lymphoma tumours, dysfunctions in the antitumour ctl activity occur, associated with an accumulation of splenic arginase-producing myeloid suppressor cells (mscs). in this study, we first demonstrate that both the presence and the activation state of these msc depends on tumour evolution. while in tumour regressors hardly any arginase-producing msc can be found, both the amount and the arginase activity of this population expands from early over late progressors. this gradual induction of mscs is paralleled by an increasing suppression of ctl activity and th1, but not th2, cytokine production. upon analysing the molecular repertoire of msc in vitro, we found, besides arginase1, a well-established marker for alternatively activated myeloid cells or m2, a strong upregulation of fizz1 and ym, two additional recently identified markers for m2. further evaluation of molecular markers by microarray analysis in msc yielded genes involved in wound healing (e.g. coagulation factor xiiia), anti-inflammation (e.g. selenoprotein p), immunomodulation (e.g. pd-l2) and fat and sugar metabolism (e.g. leptin receptor). of note, many of these genes are regulated by type 2 cytokines (il-4, il-13 and il-10) and are therefore rather m2 associated. overall, our data provide new markers for msc in cancer and further establish their m2 activation state. study. only sp-a showed a significant expression in normal mucosa which was downregulated in crc. as the absolute signal level was below the noise threshold, these results have to be interpreted with caution and require confirmation by direct measurenment of the proteins. our results suggest that there is no major role for the human collectins in colorectal cancer. tetramerization is visualized by sds-page. conclusion: an effective method for the production of highly pure mhc i molecules has been applied to hla-b*1501 and hla-b*5801, and ria and elisa binding assays for those alleles have been established background: proliferation, differentiation and apoptosis are essential processes in the normal functions of the mammary epithelium. the hypothesis examined in this study is that the transcription factor bcl-6 is critically important not only for regulating b-cell growth and development but also for mammary epithelial apoptosis. methodology: twenty breast cancer cases and 31 healthy controls were used to investigate whether bcl-6 protein in involved in breast cancer (grade iii). full length bcl-6 cdna was retrovirally transduced into eph-4 cell line. we then used flow cytometry of brdurd-stained cells to investigate the cell-cycle duration of the control and transduced cell lines. tunel was used as a marker of apoptosis to find out differences in the frequencies of apoptotic cells in the control and transduced cell lines. finally, immunohistochemistry staining was performed to detect bcl-6 in breast cancer (iii). results: restoration of bcl-6 into eph-4 cells not only inhibits apoptosis but also prolongs the cell cycle and results in increased cell size and protein content. the results also indicated that the cell-cycle time of bcl-6-transduced eph-4 cells is prolonged by about 3 h, presumably as a result of the action of bcl-6 at the bcl-6 at the g1/s transition. we found differences in the frequencies of viable and apoptotic cells in cultures of the parent eph-4 cells, control-transduced eph-4 cells and bcl-6-transduced eph-4 cells. consistently, we demonstrated that bcl-6 is expressed in 90% of high grade of breast carcinoma, which is considered as the most aggressive of tumours. conclusion: together, these results suggest that bcl-6 is likely to be involved in mammary gland development and carcinogenesis. inflammatory cytokines have a critical role in modulation of both innate and adaptive immunity in response to foreign antigen. they also play an important role in anticancer immunity. for example, they can promote cell-mediated immunity against cancer cells. with their immunostimulatory effects, these cytokines are being tested for cancer treatment in the form of dna vaccine or adjuvant or therapeutic cytokines. direct effect of these cytokines on cancer cell, however, is still unclear. in this project, we investigated whether il-1( and il-18 can modulate cancer cell proliferation. we employed a simple nonradioactive proliferation (mtt) assay and detection of lactate dehydrogenase (ldh) to test the effect of these recombinant human cytokines on various cancer cell lines, including breast cancer cell line (mcf-7), oral carcinoma cell line (kb), colon cancer cell line (caco-2) and choriocarcinoma cell line (jar). cytokines used in this study had both inhibitory and stimulatory effect on cell proliferation. findings in this project could provide an insight of cancer cell response to these cytokines and this could lead to a consideration on using cytokine as immunotherapy for cancer treatment.capacity of ae to modulate nitric oxide production depended on intercellular contact donor t cells are involved in the antitumour effects observed after bmt. thus, patients receiving t-celldepleted bmt have a higher risk of leukaemia relapse compared to patients receiving nonmanipulated bmt, and patients experiencing graft-versus-host disease (gvhd) have a lower risk of disease relapse than patients who do not experience gvhd. although the importance of donor t cells for the curative action of bmt has been established, the exact mechanisms and molecules involved in this graft-versus-tumour effect remain largely unknown. in a recently initiated project, we have conducted a longitudinal study of t-cell clonotypes in patients who received peripheral blood stem cell grafts after nonmyeloablative conditioning. peripheral blood samples were obtained sequentially after transplant, and the mononuclear cells (mncs) were isolated and cryopreserved. cd8 þ t cells were isolated from the mncs by use of immunomagnetic beads or facs and analysed for the presence of clonally expanded cells by t-cell receptor clonotype mapping based on rt-pcr and denaturing gradient gel electrophoresis (dgge). using this gel-based methodology, clonally expanded t cells were monitored after transplant and compared to the clinical data of the patients. the preliminary results demonstrates the presence of clonally expanded cd8 þ t cells at all time points analysed. furthermore, a number of clonotypes persisted for more than 6 months, and other clonotypes emerged during this period. the appearance of newly emerged clonotypes which coincided with clinical gvhd could indicate a role for these t cells in the pathogenesis of gvhd. background: deficiency of the mannan-binding lectin (mbl) pathway of innate immunity leads to increased susceptibility to infections. in patients with colorectal cancer, postoperative infection is associated with poor prognosis. the aim of the present study was to evaluate (1) the relation between the mbl pathway and postoperative infectious complications and survival of patients resected for colorectal cancer and (2) the role of mbl as acute phase reactant compared to crp. methods: preoperative mbl concentration, mbl/mblassociated serine protease (masp) activity and crp were determined in serum from 611 patients and 150 healthy controls. the patients were observed for 8 years. postoperative infections, recurrence and survival were recorded. results: the mbl pathway components were increased in the patients (p < 0.0001) compared to healthy controls. low mbl levels were predictive of pneumonia (p ¼ 0.01), and pneumonia (n ¼ 87) was associated with poor survival (p ¼ 0.003, hr ¼ 1.5, 95% ci 1.1-1.9). mbl and mbl/ masp activity could not predict postoperative overall infections. mbl showed no correlation (spearman's r ¼ 0.02, 95% ci à0.06-0.10) with crp. conclusions: low preoperative mbl levels are predictive of pneumonia, which is associated with poorer survival. mbl concentration and mbl/masp activity was not predictive of other postoperative infections or long-term prognosis. mbl apparently is not a surrogate measure of crp. department of surgery, university hospital of erlangen, erlangen, germany. e-mail: michael.siassi@rzmail.uni-erlangen.de introduction: the human collectins, mannan-binding lectin (mbl), surfactant protein-a (sp-a) and surfactantprotein-d (sp-d) play a central role in the innate immune system. immunological responses to malignant transformation of epithelial cells gained increasing interest recently. a former study could demonstrate binding of mbl to certain colorectal carcinoma (crc) cell lines in vitro. we therefore examined the expression of human collectins in normal colon mucosa and in colorectal carcinomas. materials and methods: colon samples from 20 crc patients and 10 normal mucosa samples were collected immediately after surgery. the tissue was microdissected and rna isolated (qiagen, rneasy-kit). gene expression profiles were analysed using gene-chips (affymetrix, hg-u133). we analysed the data for the expression of mbl, its associated serine proteases mannan-binding lectinassociated serine protease 1/2 (masp 1/2), sp-a and sp-d. the signal intensity of the genes of interest was compared using the mann-whitney u-test. results: the expression of human collectins in normal human colon mucosa was generally low. only the expression of sp-a and masp-2 reached the noise threshold of 250 signals. these genes were significantly downregulated in crc specimens. the expression of the other proteins showed no difference in normal mucosa and crc. conclusion: as demonstrated before, the expression of human collectins in normal colon was low in this being the first lymph node to receive drainage from the tumour area, the sentinel node offers a unique possibility to obtain tumour-reactive lymphocytes. we investigated antitumour immune responses in sentinel nodes from patients with bladder cancer, by assaying tumour-specific proliferation and tcr vb repertoires. during tumour surgery, sentinel lymph nodes were identified by peritumoural injection of blue dye. fresh specimens of tumour, sentinel and nonsentinel lymph nodes were obtained, and single-cell suspensions were prepared. cells were assayed for reactivity against autologous tumour extract in [ 3 h]-thymidine incorporation assays and characterized by flow cytometry. parallel analyses of the expression of vb gene families were performed with padlock probes, linear oligonucleotides which upon target recognition can be converted to circular molecules by a ligase. probes were reacted with cdna prepared from magnetically separated cd4 þ cells, and the tcr repertoire was determined by hybridizing the products to oligonucleotide microarrays. dose-dependent proliferation in response to tumour extract could be detected in sentinel lymph nodes. common clonal expansions were detected among tumourinfiltrating lymphocytes and in sentinel lymph nodes. nonsentinel lymph nodes displayed a divergent tcr vb repertoire. these results indicate an ongoing immune response against tumour antigens in sentinel nodes, draining urinary bladder cancer. identification of sentinel lymph nodes makes it possible to obtain tumour-reactive lymphocytes for use in adoptive immunotherapy. key: cord-023414-xxw5kptr authors: chistensen, h. r.; frøkiær, h. title: characterization of a large panel of lactic acid bacteria derived from the human gut for their capacity to polarize dendritic cell date: 2008-06-28 journal: scand j immunol doi: 10.1111/j.0300-9475.2004.01423ap.x sha: doc_id: 23414 cord_uid: xxw5kptr dendritic cells (dcs) are the principal stimulators of naïve t helper (th) cells and play a pivotal regulatory role in the th1, th2 and treg cell balance. dcs are present in the gut mucosa and may thus be target for modulation by gut microbes, including ingested probiotics. here, we tested the hypothesis that species of lactic acid bacteria, important members of the gut flora, differentially activate dc. a large panel of human gut‐derived lactobacillus and bifidobacterium spp. was screened for dc‐polarizing capacity by exposing bone marrow‐derived murine dc to lethally irradiated bacteria. cytokines in culture supernatants and dc‐surface maturation markers were analysed. substantial differences were found among strains in the capacity to induce interleukin‐12 (il‐12) and tumour necrosis factor (tnf)‐α, while the differences for il‐10 and il‐6 were less pronounced. bifidobacteria tended to be weak il‐12 and tnf‐α inducers, while both strong and weak il‐12 inducers were found among the strains of lactobacillus. remarkably, strains weak in il‐12 induction inhibited il‐12 and tnf‐α production induced by an otherwise strong cytokine‐inducing strain of lactobacillus casei, while il‐10 production remained unaltered. selected strains were tested for induction of dc maturation markers. those lactobacilli with greatest capacity to induce il‐12 were most effective in upregulating surface mhc class ii and cd86. moreover, l. casei‐induced upregulation of cd86 was reduced in the presence of a weak il‐12‐inducing l. reuteri. in conclusion, human lactobacillus and bifidobacterium spp. polarize differentially dc maturation. thus, the potential exists for th1/th2/treg‐driving capacities of the gut dc to be modulated according to composition of gut flora including ingested probiotics. the nuclear receptor heterodimers of liver x receptors (lxrs) are recently identified as key transcriptional regulators of genes involved in lipid homeostasis and inflammation. lxrs and their ligands are negative regulators of macrophage inflammatory gene expression. multiple sclerosis (ms), a demyelinating disease of the central nervous system of unknown cause, is characterized by recurrent inflammation involving macrophages and their inflammatory mediators. sweden belongs to the countries with a high ms incidence. in italy, incidence is lower, with an exception for sardinia where the incidence is even higher than that in sweden. subjects from sardinia are ethnically more homogeneous and differ from swedes, also regarding genetic background and environment. we studied lxrs and their related molecules of blood mononuclear cells (mncs) from female patients with untreated relapsing-remitting ms from sassari, sardinia and stockholm, sweden. sex-and age-matched healthy controls (hcs) were from both areas. mrna expression was evaluated by real-time pcr. lxr-a was lower (p < 0.05) in ms (mean ae sem: 3.1 ae 0.2; n ¼ 37) compared to hc (3.6 ae 0.1; n ¼ 37). lxr-a was lower in ms from stockholm (2.6 ae 0.2; n ¼ 22) compared to corresponding hc (3.4 ae 0.1; n ¼ 22; p < 0.01) and compared to ms (3.8 ae 0.2; n ¼ 15; p < 0.001) and hc (4 ae 0.2; n ¼ 15; p < 0.001) from sardinia. ms patients from stockholm, but not from sassari, also expressed lower (p < 0.05) lxr-b (à4.1 ae 0.4) compared to corresponding hc (à2.9 ae 0.3). ms from stockholm was associated with higher abca-1 (6.1 ae 0.4 versus 5.0 ae 0.3; p < 0.05) and higher estrogen receptor-b-cx (2.4 ae 0.4 versus 0.8 ae 0.4; p < 0.01) compared to corresponding hc. the hc from sassari had higher androgen receptor (2.9 ae 0.2) compared to ms from sassari (1.4 ae 0.3; p < 0.01), ms (1.3 ae 0.4; p < 0.01) and hc from stockholm (1.2 ae 0.3; p < 0.01). ms from sassari had lower cyclooxygenase-1 compared to corresponding hc (5.1 ae 0.4 versus 6.6 ae 0.3; p < 0.01) and lower prostaglandin-e (à0.03 ae 0.5) compared to the hc (1.4 ae 0.5; p < 0.05) and ms (2.7 ae 0.4; p < 0.05) and hc from stockholm (1.9 ae 0.4, p < 0.001). our findings identify lxrs and their related molecules as being involved in ms from stockholm but not from sassari, while sex hormone receptors seem to be involved in ms in sassari. multiple sclerosis: ifn-b induces cd123 + bdca2 -dendritic cells that produce il-6 and il-10 and have no enhanced type i interferon production y. m. huang, 1 s. adikari, 1 u. båve, 2 a. sanna 1,3 & g. alm 4 dc antigens (bdca) and investigate their ability to produce type i ifn in response to virus stimulation. we show that ifn-b induces development of cd123 þ dc from human blood monocytes, which coexpress bdca4 þ but are negative for bdca2 -, a specific marker for plasmacytoid dc. such ifn-b-modulated dc produce large amounts of il-6 and il-10, but no il-12p40 and have no enhanced ifn-b and ifn-b production. the findings indicate that ifn-bmodulated dc represent a myeloid dc subset with diminished cd11c, bdca-1 and cd1a expression, having potent th2-promoting function but lacking antiviral capacity. the association of psoriasis with throat infections by streptococcus pyogenes suggests a potential antigenic target for the t cells that are known to infiltrate dermis and epidermis of psoriatic skin. streptococcal m protein shares an extensive sequence homology with human epidermal keratins. keratins 16 (k16) and 17 (k17) are mostly absent from uninvolved skin but are upregulated in psoriatic lesions. there is increasing evidence that cd8 þ t cells play an important effector role in psoriasis and m proteinprimed t cells may recognize these shared epitopes in skin via molecular mimicry. to identify candidate epitopes, peptides with sequences from k17 were selected on the basis of predicted binding to hla-cw6 and sequence similarities with m6 protein. matched peptides from the sequence of m6 protein and a set of peptides with poor predicted binding were also selected. cw6 þ individuals with psoriasis and cw6 þ healthy controls, having a family history of psoriasis, were recruited. pbmcs were incubated with the peptide antigens. t-cell activation in the cd4 þ , cd8 þ and later the skin-homing cutaneous lymphocyteassociated antigen (cla)-expressing subset of cd8 þ t cells was evaluated by cd69 expression and intracellular ifn-g accumulation using flow cytometry. we demonstrate that cw6 þ psoriasis patients had significant cd8 þ t-cell ifn-g responses to peptides from k17 and m6 protein selected on the basis of sequence homology and predicted hla-cw*0602 binding. these responses were about 10 times more frequent in the skin-homing cutaneous lymphocyte-associated antigen-expressing (cla þ ) subset of cd8 þ t cells. cd4 þ t cells showed only borderline responses. cd8 þ t cells from cw6 þ nonpsoriatic individuals responded to some m6 peptides but very rarely to k17 peptides, and this also applied to the cla þ cd8 þ subset. these findings indicate that psoriatic individuals have cd8 þ t cells that recognize keratin self-antigens and that epitopes shared by streptococcal m protein and human keratin may be targets for the cd8 þ t cells that infiltrate psoriatic skin lesions. autoantibodies directed against citrulline-containing proteins have an impressive specificity of nearly 100% in ra patients and a suggestive involvement in the pathogenesis. the targeted epitopes are generated by a post-translational modification catalysed by the calcium-dependent enzyme peptidyl arginine deaminase that converts the positively charged arginine to polar but uncharged citrullin. the aim of this study was to analyse the presence of citrulline in the joints at different time points of collagen-induced arthritis in da rats by immunohistochemistry and to investigate how immunogenicity and arthritogenicity was affected by citrullination of rat serum albumin (rsa) and collagen type ii (cii). our results indicate that citrulline could be detected in joints of arthritic animals, first appearance at the onset of disease and increasing as disease progressed into a chronic state. unimmunized animals or time points before clinical signs of arthritis were negative. by morphology, we state that some infiltrating macrophages as well as the cartilage surface stain positive for citrulline, while the major source of citrullinated proteins appears to be fibrin depositions. a specific cit-rsa t-cell response was observed in animals challenged by citrullinated rsa, no response was recorded when rsa was used as a stimulus. the igg analysis reveals not only a response towards the modified protein but also cross-reactivity to native rsa. no t-cell or b-cell response was noted in animals injected with unmodified rsa. cit-cii induced a disease with higher incidence and earlier onset than did the native counterpart. we conclude that, in contrast to the human disease, citrulline does not seem to appear before clinical signs. as inflammation proceeds, citrulline is detected specifically in the joints. all other organs investigated were negative. we also conclude that citrullination of a protein can break tolerance and increase its arthritogenic properties. ectopic germinal centers (gcs) can be detected in the salivary glands of approximately 1/5 of patients with sjögren's syndrome (ss) and appear in both primary and secondary ss. previously, ectopic gc have been associated with increased local autoantibody production. the aim of this study was to determine whether gc in primary sjögren's syndrome (pss) defines a distinct seroimmunological phenotype. retrospectively, a material of 130 haematoxylin and eosin-stained paraffin-embedded tissue sections of minor salivary gland tissue from patients with pss was morphologically screened for the presence of ectopic gc. gc-like lesions were detected in 33/130 (25%) of the pss patients. seventy-two pss patients lacking these structures (gc-) were randomly selected for comparison. focus score was significantly increased in the gc þ patients compared to the gcpatients (p ¼ 0.035). in the gc þ group, 54.5% of the patients presented with anti-ro/ssa compared to 43.7% in the gcgroup. anti-la/ssb was detected in 31.3% of the gc þ patients compared to 25.7% of the gcpatients. sixty-one percentage of gc þ patients presented with increased levels of igg, a nonsignificant difference when compared to 39.4% in the gcpatients (p ¼ 0.089). levels of rf, ana, ena, igm and iga were similar in both patient groups, as were esr and crp. in conclusion, patients with ectopic gc have a higher focus score and more often present with autoantibodies and increased levels of igg compared to pss patients with regular focal infiltration (gc -). our findings may indicate a certain seroimmunological phenotype and warrant for further prospective studies. association between mannose-binding lectin and vascular complications in type 1 diabetes complement activation and inflammation have been suggested in the pathogenesis of diabetic vascular lesions. we investigated serum mannose-binding lectin (mbl) levels and polymorphisms in the mbl gene in type 1 diabetic (t1dm) patients with and without diabetic nephropathy and associated macrovascular complications. polymorphisms in the mbl gene and serum mbl levels were determined in 199 t1dm patients with overt nephropathy and 192 t1dm patients with persistent normoalbuminuria matched for age, sex and duration of diabetes as well as in 100 healthy control subjects. the frequencies of high and low expression mbl genotypes were similar in patients with t1dm and healthy controls. high mbl genotypes were significantly more frequent in diabetic patients with nephropathy than in the normoalbuminuric group, and the risk of having nephropathy, given a high mbl genotype, assessed by odds ratio was 1.52 (1.02-2.27), p ¼ 0.04. median serum mbl concentrations were significantly higher in patients with nephropathy than in patients with normoalbuminuria [2306 mg/l (iqr 753-4867 mg/l) versus 1491 mg/l (iqr 577-2944), p ¼ 0.0003], and even when comparing patients with identical genotypes, serum mbl levels were higher in the nephropathy group than in the normoalbuminuric group. patients with a history of cardiovascular disease had significantly elevated mbl levels independently of nephropathy status [3178 mg/l (iqr 636-5231 mg/l) versus 1741 mg/l (iqr 656-3149 mg/l), p ¼ 0.02]. the differences in mbl levels between patients with and without vascular complications were driven primarily by pronounced differences among carriers of high mbl genotypes (p < 0.0001). our findings suggest that mbl may be involved in the pathogenesis of microvascular and macrovascular complications in type 1 diabetes and that determination of mbl status might be used to identify patients at increased risk of developing these complications. neuroimmunology unit, center for molecular medicine, karolinska institutet, stockholm, sweden. e-mail: judit.wefer@cmm.ki.se dna vaccine coding for the encephalitogenic peptide mog 91-108 protects lew.1av1 from subsequent development of experimental autoimmune encephalomyelitis (eae). protection is associated with a type 1 immune response and is dependent on the presence of cpg dna motifs. the mechanisms underlying the observed reduction of eae development in protected rats have not been fully clarified. we investigated immunological characteristics of lymphocytes after dna vaccinaton and subsequent eae induction. we confirm that protection was not associated with suppression of t1 cells, as transcription of the novel molecule rat t-cell immunoglobulin-and mucindomain-containing molecule (tim-3), reported to be exclusively expressed on differentiated t1 cells, was not altered by dna vaccination. we did not note any clonal deletion upon tolerization, but detected an antigen-specific lymphocyte population upregulating ifng upon recall stimulation 3 weeks after protective dna vaccination. in protected rats, we observed (1) no alterations in antigenspecific th2 or th3 responses, (2) reduced mhc ii expression on splenocytes early after eae induction, (3) antigen-specific upregulation of ifnb upon recall stimulation and (4) reduced il-12rb2 on lymphocytes. we thus demonstrate an association of the protective effect of dna vaccination with expression of ifnb. we are currently investigating the cellular mechanisms behind this ifnb-mediated protection. multiple sclerosis (ms) is an autoimmune condition characterized by degeneration of nerve fibre myelin sheets. a candidate autoantigen, myelin basic protein (mbp), has especially attracted attention. the presence of anti-mbp antibodies is a predictor of definite ms, but their role in the pathogenesis remains obscure. t cells have long been known to play a pivotal role in the pathogenesis of ms. recently, an important role for b cells as autoantigen-presenting cells has been demonstrated in other autoimmune diseases, including rheumatoid arthritis and diabetes. the uptake of mbp by b cells and the presentation of mbp-derive peptides to t helper (th) cells by b cells may be promoted by the formation of complement (c) activating immune complexes (ics) between mbp and natural autoantibodies in healthy individuals and disease-associated anti-mbp antibodies in ms patients, respectively. we have investigated the formation of mbp-containing ic, the binding of mbp to b cells, the mbp-elicited induction of th-cell and b-cell proliferation and the cytokine production in peripheral blood mononuclear cells (pbmcs) from healthy donors grown in the presence of intact or c-inactivated serum from healthy donors or patients with ms. while mbp did not induce measurable proliferation of b cells nor cd4 þ t cells, we observed the production of tnf-a, ifn-g and il-10 by pbmc in response to incubation with mbp in the presence of sera from healthy controls as well as sera from ms patients. by contrast, no production of il-2, il-4 and il-5 was detected. we are currently investigating the capability of ms sera to promote the formation of mbp-containing ic and thereby enhance the cytokine responses, by virtue of elevated anti-mbp contents. the phagolysosomally localized acid sphingomyelinase (asmase) activated by proinflammatory cytokines such as tnf and ifn-g generates the signalling molecule ceramide which in turn results in the activation of proteases like cathepsin d. these characteristics of asmase suggest a possible role of this molecule in the phagocytotic uptake and phagosomal degradation processes of antigens or in antigen presentation. we show here that asmase -/mice fail to eliminate the noncytopathic lymphocytic choriomeningitis (lcm) virus as rapidly as littermate wildtype mice. investigation of the immune response revealed a reduced expansion of cd8 þ t cells. the secretion of ifn-g in response to contact with target cells as well as the cytolytic activity of virus-specific cd8 þ t cells was severely impaired. additionally, both phases of the lcm virus-specific dth response, mediated by cd8 þ and cd4 þ t cells consecutively, were diminished in asmase -/mice. however, the secondary memory response of virus-specific ctl was not altered, and the 614 abstracts ................................................................................................................................................................................................. virus was effectively controlled for at least 3 months by asmase -/mice. in conclusion, the results of this study suggest an involvement of the asmase in the activation, expansion or maturation of virus-specific cd8 þ t cells during the acute infection of mice with the lcm virus. novel markers for alternative activation of macrophages: macrophage galactose-type c-type lectins 1 and 2 in parallel with the th1/th2 dichotomy, macrophages are capable of developing into functionally and molecularly distinct subpopulations, due to differences in, for example cytokine environment and pathological conditions. while the best-studied, classically activated macrophage is induced by type i stimuli such as ifn-g, a type ii cytokine environment antagonizes the classical activation of macrophages and is capable of alternatively activating macrophages. however, molecular markers associated with these type ii cytokine-dependent, alternatively activated macrophages remain scarce. besides the earlier documented markers macrophage mannose receptor and arginase 1, we recently demonstrated that murine alternatively activated macrophages are characterized by increased expression of fizz1 and ym. we now report that expression of the two members of the mouse macrophage galactose-type c-type lectin gene family, termed mmgl1 and mmgl2, is induced in diverse populations of alternatively activated macrophages, including peritoneal macrophages elicited during infection with the protozoan trypanosoma brucei or the helminth taenia crassiceps, and alveolar macrophages elicited in a mouse model of allergic asthma. we also demonstrate that, in vitro, interleukin-4 and interleukin-13 upregulate mmgl1 and mmgl2 expression and that, in vivo, induction of mmgl1 and mmgl2 is dependent on interleukin-4 receptor signalling. moreover, we show that regulation of mgl expression is similar in human monocytes and monocyte-derived macrophages. hence, macrophage galactose-type c-type lectins represent novel markers for both murine and human alternatively activated macrophages; thus, paving the way for further characterization of the phenotype of macrophages occurring in th2 conditions. background: human parvovirus b19 (b19) is a ubiquitous pathogen, normally causing a mild self-limiting disease, but also capable of causing both significant pathology and long-term persistence. the small size and stability of the virus makes it suitable for mapping of the full breath and the kinetics of the cellular immune responses following acute viral infection. methods: five patients with acute primary b19 infection were included in the study and followed consecutively for up to 200 weeks. cellular immune responses were mapped by ifng enzyme-linked immunospot to overlapping peptides spanning the whole b19 genome. results: in all five acutely infected patients, we were able to monitor the kinetics of a strong specific cellular immune reaction. responses peaked at levels of 850-1850 sfc/ million pbmcs, roughly corresponding to 0.3-0.6% b19specific cd8 þ cells circulating in peripheral blood at 10-80 weeks post-infection. the responses in individual patients were directed to three or four different peptide pools, and the specificity was confined to the same cd8 epitopes present in the pools throughout the follow-up period. the majority of responses were directed to the virus nonstructural protein, only two patients showed any response to the capsid proteins, elicited by the same epitope in both cases. conclusion: the cellular immune responses to acute b19 infection are surprisingly narrow in distribution and remain at high levels for up to 80 weeks post-infection. the initial epitope specificity is maintained, and the majority of responses target the virus nonstructural protein, which is not included in vaccine preparations, evaluated against the infection. the relationship between malnutrition and malaria is controversial. on one hand, malaria may cause malnutrition, while on the other, malnutrition itself may modulate susceptibility to the disease. we investigated the association between plasmodium falciparum malaria and malnutrition in a cohort of children living on the coast of kenya. the study involved longitudinal follow-up for clinical malaria episodes and anthropometric measurements at four cross-sectional surveys. we used poisson regression analysis to investigate the association between malaria and nutritional status. compared to baseline (children with a waz or haz score of !à2), the crude incidence rate ratios (irrs) for malaria in children with low haz or waz scores (<à2) during the period prior to assessment were 1.17 (95% ci 0.91-1.50; 0 ¼ 0.21) and 0.94 (0.71-1.25; 0.67), respectively, suggesting no association between malaria and the subsequent development of pem. however, we found that age was acting as an effect modifier in the association between malaria and malnutrition. the irr for malaria in children 0-2 years old who were subsequently characterized as wasted was 1.65 (1.10-2.20; p ¼ 0.01), and a significant overall relationship between malaria and low-haz was found on regression analysis when adjusting for the interaction with age (irr 1.89; 1.01-3.53; p < 0.05). although children living on the coast of kenya continue to suffer clinical episodes of uncomplicated malaria throughout their first decade, the association between malaria and malnutrition appears to be limited to the first 2 years of life. a. astrinidou-vakaloudi, 1 s. xytsas, 1 i. diamanti, 1 h. ioannidis 2 & p. pangidis 2 1 microbiology department of general hospital of thessaloniki 'agios pavlos', thessaloniki, greece, and 2 nefrology, 2 nd ika hospital of thessaloniki, thessaloniki, greece. e-mail: stasa@hol.gr aim: renal dysfunction may influence the colonization of gastric mucosa by urea-splitting bacteria such as helicobacter pylori, by increasing urea concentrations in the gastric juice. our aim was to investigate the prevalence of h. pylori in patients with end-stage renal disease (esrd), receiving long-term haemodialysis treatment. methods: this study included 40 sera from patients with esrd (29 male and 11 female) undergoing periodic haemodialysis; mean time of treatment was 42.6 months. using elisa technique, we investigated the presence of igg and iga antibodies against h. pylori as well as igg caga (antibodies specific for caga(þ) strains of h. pylori). sera from 40 healthy blood donors were used as a control group. results: h. pylori igg antibodies were detected in 32 out of 40 (80%) patients in the dialysis group, while 31/40 (77.5%) tested positive for iga. igg caga antibodies were present in 13 out of 40 (32.5%). prevalence of h. pylori igg, iga and caga igg antibodies in the control group was 33, 7 and 15%, respectively. conclusions: although international data suggest that prevalence of h. pylori infection is the same in esrd patients as in healthy individuals, in our study that seems not to be the case. the higher blood and gastric juice urea levels may be a risk factor (among many others), but more studies are required in order to understand the relation of h. pylori infection in this group of patients. flanders interuniversity institute for biotechnology, department of molecular and cellular interactions, free university of brussels, brussels, and 2 pasteur institute of brussels, mycobacterial immunology, brussels, belgium. e-mail: tgartner@vub.ac.be immunity against tuberculosis (tb), caused by mycobacterium tuberculosis, depends largely on activation and maintenance of strong cell-mediated immune responses involving both cd4 þ and cd8 þ t cells and the ability to respond with th1-type cytokines, particularly ifn-g. recent studies suggested that bcg, the only licensed vaccine against m. tuberculosis, may fail to induce t-cell responses in the lung mucosa and may therefore not protect against pulmonary tb. a decrease in tb mortality may be achieved by enhancing immunity in the lung. the present study evaluated the induction of antigen-specific immunity in the lung by intranasal (i.n.) delivery of the lipoprotein i (opri) from pseudomonas aeruginosa. opri has shown to be a toll-like receptor 2/4 agonist that, when given subcutaneously, induces type-1 immune responses against heterologous antigens. here, a fusion of opri to ag85a of mtb (opri-ag85a) was used as a subunit vaccine in homologous prime-boost immunizations. in addition, opri-ag85a was combined with an ag85a-encoding dna vaccine (ag85a dna) or with bcg in heterologous prime-boost vaccinations. intranasal and parenteral delivery with opri-ag85a elicited comparable t-cell responses in the spleen; in addition, i.n. delivery elicited specific t-cell responses in the lung lymph nodes (llns). intramuscular delivery of ag85a dna induced significant systemic th1 immune responses. intranasal boosting with opri-ag85a enhanced this response and in addition induced an antigen-specific ifn-g response in lln. opri may therefore be an efficient adjuvant for mucosal boosting. we continue to evaluate the protection induced by opri-based prime-boost vaccinations against pulmonary tb. results on the immunogenicity and protection against intravenous mtb h37rv infection will be presented. toll-like receptors (tlrs) are pattern recognition receptors of the innate immune system, which recognize molecular structures on pathogens or cellular stress-associated molecules. tlr-ligand interactions trigger activation of inflammatory signal transduction and expression of genes involved in host defense. in this study, we have examined the requirement for different tlr adaptor molecules in virus-induced chemokine expression and are currently trying to identify the tlr involved. we have found that both a herpesvirus [herpes simplex virus (hsv)] and a paramyxovirus (sendai virus) require a functional genome to induce expression or proinflammatory chemokines in human and murine monocytic cell lines. for both viruses, this is independent of the tlr adaptor molecules trif and mal. however, overexpression of the vaccinia virus-encoded inhibitor of tlr-signalling a52r or dominant-negative myd88 totally inhibited hsv-induced rantes expression but only partially prevented sendai virus from inducing this chemokine. this suggests that hsv-induced rantes expression occurs via a tlr pathways, whereas sendai virus utilizes both tlr-dependent and -independent pathways to stimulate expression of rantes. we are currently trying to identify the tlrs involved. data from these studies will also be presented at the meeting. 2 0 -5 0 -oligoadenylate synthetases are interferon-induced, double-stranded rna-activated antiviral enzymes which are the only proteins known to catalyse 2 0 -specific nucleotidyl transfer. this first crystal structure of a 2 0 -5 0oligoadenylate synthetase reveals a structural conservation with the 3 0 -specific poly(a) polymerase that, coupled with structure-guided mutagenesis, supports a conserved catalytic mechanism for the 2 0 -and 3 0 -specific nucleotidyl transferases. comparison with structures of other superfamily members indicates that the donor substrates are bound by conserved active site features while the acceptor substrates are oriented by nonconserved regions. the 2 0 -5 0oligoadenylate synthetases are activated by viral doublestranded rna in infected cells and initiate a cellular response by synthesizing 2 0 -5 0 -oligoadenylates, that in turn activate rnase l. this crystal structure suggests that activation involves a domain-domain shift and identifies a putative dsrna activation site that is probed by mutagenesis. we demonstrated that this site is required both for the binding of dsrna and for the subsequent activation of oas. this rna-binding site is different from known rna-binding site; rather than forming a defined three-dimensional domain, it is located at the interface of the two major domains in oas. this novel architecture ensures that the dsrna helix can make simultaneously contact with both domains of oas and ensure the subsequent structural rearrangement leading to the activation of oas. our work provides structural insight into cellular recognition of double-stranded rna of viral origin and identifies a novel rna-binding motif. bacteria-specific iga antibodies are efficient opsonins for neutrophils and mononuclear phagocytes, provided that the phagocytes express the fca receptor (cd89). expression of cd89 can be stimulated by inflammatory cytokines, activated complement factors and certain microbial components. in one study, unstimulated phagocytes were able to ingest iga antibody-treated pneumococci, but only in the presence of complement, which was found to be activated by the iga antibodies along the alternative pathway. pneumococci produce iga1 protease that cleaves human iga1, but not iga2, molecules in the hinge region. this leaves iga1 as faba (monovalent) deprived of fca which contains the docking site for cd89. iga1 is the vastly predominant subclass of iga in the upper airways and circulation of humans. aims: to examine the effects of iga1 protease activity and complement on phagocytosis of iga antibody-coated pneumococci by an unstimulated human phagocytic cell line (hl60). materials and methods: iga1 and iga2 monoclonal antibodies to serotype 4 pneumococcal capsular polysaccharide (ps) were generated by heterohybridoma technique involving b cells from human vaccinees. isogenic serotype 4 pneumococci with and without iga1 protease activity, respectively, were obtained after inactivation of the iga gene of the tigr4 strain. opsonophagocytosis was quantitated using the assay described by romero-steiner et al. based on enumeration of surviving bacteria by culture. the integrity of iga molecules was examined by western blotting. results: both iga1 and iga2 antibody to type-4 polysaccharide-induced phagocytosis of iga1 protease-deficient type-4 pneumococci equally well in the absence as in the presence of complement. iga1 antibody to type-4 polysaccharide displayed a fourfold higher opsonophagocytosis titer against iga1 protease deficient compared to homologous wildtype target bacteria. a similar effect of iga1 protease activity of the target bacteria was not observed in a parallel experiment where iga2 antibody to type-4 polysaccharide served as opsonin. iga1 antibody extracted from iga1 protease-producing target bacteria was almost exclusively in the form of faba. conversely, iga1 from protease-deficient bacteria and iga2 from both types of bacteria were intact. conclusions: these results indicate that the iga1 protease activity of s. neumoniae may help the bacteria escape iga1 antibody-mediated opsonophagocytosis. besides, in these experiments, iga-mediated opsonophagocytosis was independent of complement. vitamins e and c have been found to increase the cellular and humeral immunity of pigs. vitamin e deficiency has also been found to predispose pigs to different diseases, e. coli infection is one among them. after weaning, the vitamin e status of pigs often decreases to a critical low level. in this experiment, we studied whether vitamin c supplementation would be a possible feeding strategy to optimize the immune status of weaners. the interaction between vitamin e and c is interesting due to the reported sparing action on vitamin e or synergism between these to vitamins. piglets were weaned at day 28 of age from sows fed increasing dietary vitamin e during lactation, and piglets were during the following 3 weeks fed either a control diet or this diet supplemented with 500 mg stay-c per kg. blood sampling was obtained weekly from day 28 and until day 49 of age. on the same days, one piglet per dietary treatment was killed and alveolar macrophages (am) were harvested. vitamin c supplementation increased the concentration of igm in serum of piglets throughout the weaning period. although the vitamin e concentration in am decreased with increasing age of the piglets, the concentration was numerically higher in piglets of sows fed the high dietary level of vitamin e. however, vitamin c supplementation tended to increase the total am concentration of vitamin e after weaning and increased the proportion of the biologically most active isomer of vitamin e [rrr-(a-tocopherol)] in the am. the eicosanoid synthesis by am was not influenced by the vitamin c supplementation, but the synthesis of leukotriene b4 was decreased 2 weeks after weaning compared to other days of am harvesting. in conclusion, dietary vitamin c supplementation improved the immune responses of piglets after weaning. a whole blood stimulation assay with escherichia coli (o111:b4) endotoxin was established to measure the capacity of dairy cows to produce the proinflammatory cytokine tumour necrosis factor-a (tnf-a) ex vivo. initially, a time-and dose-dependent study was carried out to find the optimal stimulation conditions for the tnf-a response. the tnf-a response peaked between 3 and 4 h at 38.5 c. a dose in the range of 5-10 g of e. coli lipopolysaccharide (lps)/ml whole blood was found to give the maximum tnf-a response. thirty-eight danish-holstein dairy cows were investigated for their tnf-a responsiveness ex vivo in the periparturient period. heparin-stabilized blood samples were collected seven times over a period of 4 months (weeks à3, à1, 2, 3, 5, 9 and 13 around calving) and stimulated with 5 g/ml of e. coli lps. indeed, fluctuations in the tnf-a responsiveness occurred over time. moreover, the mean tnf-a responsiveness of 38 cows was found to be significantly increased (p < 0.001) in the weeks close to calving. however, in the more stabile physiological periods, some cows had a consistently low tnf-a response, whereas others had high a tnf-a response. we are currently investigating whether high and low tnf-a responders to e. coli lps also exist in dairy cows in vivo. moreover, the importance of tnf-a responsiveness ex vivo to dairy cows' susceptibility and clinical response to experimental e. coli infections in the udder is being investigated. coelomic cytolytic factor (ccf) is a 42 kda invertebrate pattern recognition molecule isolated from the coelomic fluid of the earthworm eisenia foetida (oligochaeta, annelida). ccf displays a number of similarities with the mammalian cytokine tumour necrosis factor-a (tnfa) as a result of a shared n,n 0 -diacetylchitobiose lectin-like domain. however, these similarities are solely functional and are not based on any (dna or amino acid) sequence homology, thus suggesting a form of convergent evolution. in particular, the lectin-like domain of tnf-a has been shown to induce membrane depolarization in various mammalian cell types, through interactions with endogenous amiloride-sensitive ion channels. this nonreceptor-mediated activity of tnf-a has been reported to be involved in the resorption of oedema. likewise, the lectin-like domain of ccf also induces membrane depolarization in mammalian cells. here, we show that ccf appears to be able to induce oedema resorption in an alveolar epithelial cell line through its lectin-like domain. this lectin-like domain of ccf interacts (directly or indirectly) with endogenous sodium and/or chloride channels, and not potassium channels, on mammalian cells. additionally, we suggest that the jnk/sapk and erk1/2 pathways are involved in ccf-induced macrophage activation. these results further establish the functional analogy between an invertebrate pattern recognition molecule and a mammalian cytokine and, from a more applied point of view, suggest the possibility of utilizing ccf in the treatment of oedema. release of svegf and svegfr1 from white blood cells and platelets during surgery and stimulation with bacterial antigens introduction: the influence of surgery on release of soluble vascular endothelial growth factor (svegf) and the soluble vascular endothelial growth factor inhibitory receptor 1 (svegfr1) is unknown. we studied the effect of major and minor surgery on potential variations in svegf and svegfr1 concentrations in vivo and on bacterial antigen-induced release of svegf and svegfr1 from whole blood in vitro. methods: sixty-one patients with abdominal diseases undergoing five different surgical procedures were included. blood samples were drawn from anaesthetized patients before and after the operation. white blood cells and platelets were counted, and plasma svegf and svegfr1 was determined by an elisa method. whole blood from each blood sample was stimulated in vitro with bacteria-derived antigens (lps or protein-a) and svegf and svegfr1 levels were subsequently determined in the supernatants. stimulation with isotonic saline served as control assay. neither svegf or svegfr1 in plasma changed during surgery. in vitro stimulation of blood samples with bacteria-derived antigens resulted in a significant increase in svegf (p < 0.0001) and a less pronounced but still significant increase in svegfr1. release of svegf due to stimulation was significantly higher after the operation (nonsignificant), whereas svegfr1 release remained largely unchanged after surgery. correlation between bacterial antigen-induced release of svegf and neutrophile cell count was highly significant (p < 0.0001). there was no correlation between svegf and platelet cell count, and bacterial antigen-induced svegfr1 release did not correlate with counts of neutrophils and platelets. conclusions: plasma svegf and svegfr1 concentrations did not change during surgery. in vitro bacterial stimulation led to increased release of svegf and svegfr1, which was not significantly amplified during surgery and which may be related to number of circulating neutrophils. natural killer cell functions and subsets after in vitro stimulation with il-2 and il-12, with special emphasis on intracellular ifn-g and nk-cell cytotoxicity r. nyboe, 1,2 t. rix, 1,2 j. krog, 1,2 e. tønnesen 1 & m. hokland 2 1 department of anaesthesiology and intensive care, aarhus university hospital, and 2 institute of medical microbiology, and immunology, university of aarhus, aarhus, denmark. e-mail: rnsr@studmed.au.dk materials and methods: isolated cryopreserved human peripheral blood mononuclear cells (pbmcs) were stimulated with il-2 and il-12. this stimulation has previously been shown to activate nk cells. cell cytotoxicity was measured by flow cytometry after incubation with k562 cells. this method was compared to the current standard 51cr release assay. cells were treated with bfa to accumulate ifn-g, stained for surface markers, permeabilized and stained for intracellular ifn-g. flow cytometry was then performed to measure intracellular ifn-g production in pbmc, especially in nk cells. results: we have demonstrated that stimulation with il-2 and il-12 is effective in increasing the number of ifn-gpositive cells. there is a distinct difference between the cd3-cd56dim and the cd3-cd56bright subsets, with a much greater proportion of ifn-g-positive cells in the cd3-cd56bright subset. the effects of stimulation with il-2 and il-12 on cytotoxicity will be presented, as will the relation between ifn-g production and cytotoxicity. in addition, we will present results of these assays applied to porcine cells. discussion: in combination, these tests will address nk cell function by combining cytotoxicity with ifn-g production in nk cell subsets. the results will demonstrate whether this could serve as a useful tool in describing nkcell function, which could be of value in clinical and experimental settings. culture of regulatory t-cell lines from bronchial mucosa t lymphocytes play a major role in many immune responses. in the last decade, special focus has been on the function of th1 and th2 effector cells. now the importance of regulatory cd4 þ cd25 þ t cells in maintenance of the immunological homeostasis emerges. sarcoidosis is a multisystem granulomatous disorder often affecting the lungs. the typical sarcoid granulomas consists of epitheloid cells, macrophages and lymphocytes, mainly cd4 þ t cells of th1 phenotype. we have cultured t cells from bronchial biopsies of patients with sarcoidosis as well as from controls in high levels of interleukin 2 (il-2) and il-4 and demonstrate spontaneously arising cd4 þ cd25 þ populations and high concentrations of il-10 in these cultures. the main difference between cultures of sarcoid origin compared to controls is a very much higher concentration of the inflammatory cytokines il-6 and tnf-a in cultures of sarcoid origin. the effects of hyperbaric exposure on human peripheral blood mononuclear cells, with special emphasis on natural killer cell cytotoxicity and subsets materials and methods: as an experimental physiological stress model, we examined the effects of hyperbaric exposure on peripheral blood mononuclear cells (pbmcs) obtained from venous blood drawn from eight divers during a simulated heliox saturation dive. eight persons working in normobar atmosphere outside the pressurized chamber served as control donors. the spontaneous cytotoxicity of the pbmcs was estimated in a 4 h 51cr-release assay using k562 as nk-sensitive target cells. the pbmcs were characterized, using 4-colour flow cytometry, with special emphasis on the nk-cell subsets. the data were statistically analysed using a multivariate regression model (stata 8.2). p values <0.05 was considered statistically significant. results: the estimated cytotoxicity increased significantly in both the group of divers and control donors during the dive (pdivers < 0.01 and pcontrols < 0.01). although the cytotoxicity increased relatively more (p < 0.01) in the group of divers compared to the group of control donors between day 1 and 2. discussion: the increased cytotoxicity of pbmc estimated in the group of divers indicate that parts of the cellular immune system are affected during the extreme physiological conditions induced during the initial phase of the presented experimental hyperbaric setup. the increase in cytotoxicity observed in the group of control donors could hypothetically reflect the stress level in persons working outside the pressurized chamber during the dive. the interaction with adhesion molecules expressed by vascular endothelium is the first step in lymphocyte infiltration into tissues. at both cutaneous and mucosal sites interleukin-10 (il-10), il-12 and transforming growth factor (tgf)-b are important regulators of chronic inflammatory disease, where cutaneous lymphocyteassociated antigen (cla) and ae integrin (cd103) may be expressed. unlike cla, cd103 is not believed to play a role in tissue-specific homing but may help to retain t cells within epithelial layers. we have previously shown that il-12 alone can together with an unknown cofactor increase the expression of cla. stimulation with streptococcal pyrogenic exotoxin c (spec) increased the expression of cd103 by cd8 þ but not cd4 þ t cells. while il-12 increased superantigen-stimulated expression of cla, this cytokine strongly inhibited the cd103 expres-sion, and a combination of il-12 and tgf-b completely abrogated the induced cd103 expression. conversely, il-10 suppressed cla but increased cd103 expression. these findings indicate that, in addition to suppressing the development of th1-mediated inflammatory responses, il-10 may also inhibit the migration of cd8 þ t cells into the skin while il-12 promotes such migration. thus, the expression of cla and cd103 may be antagonistically regulated by il-10 and il-12, and the balance between these cytokines could influence the t-cell migration of inflammatory cells into epithelial tissues. during contact sensitivity reaction, immune cells proliferate. in order to study the histological picture of these proliferation phases, we used a mouse model of contact sensitivity in the oral mucosa and on skin. we also used bromodeoxyuridin (brdu, an analogue to thymidin) that is incorporated into the nucleus during cell replication. the hapten oxazolone (oxa) was used to sensitize and elicit the oral mucosa and/or the ear skin. mice were killed at various times after elicitation, and unsensitized animals were also exposed to the hapten as controls. brdu (25 mg/ kg animal) was injected i.p. 2 h before the kill. specimens from the oral mucosa, ear skin and submandibular and auricular lymph nodes were cut and fixed in 4% paraformaldehyde. they were then treated with acid and biotinylated anti-brdu antibody and developed using abc-kit and dab. the analyses were performed using a leica light microscope and the computer program analysis. in the oral mucosa, the frequency of proliferating cells were increasing during the observation period, 4-24 h after elicitation, regardless of site of sensitization. the proliferating cells were found mainly in the basal cell layer of the epithelium. similar patterns were found in ear skin. the regional lymph nodes demonstrated a few scattered proliferating cells 4 h after elicitation. after 24 h, these cells were found frequently in the whole lymph node. control animals exhibited considerable less proliferating cells at all times. we conclude that most proliferating cells were found 24 h after elicitation locally at the hapten-exposed sites (the oral mucosa or the ear skin) as well as in the regional lymph nodes. the endogenous nucleoside adenosine is released in excess during inflammation or other metabolic stress and is generally known to deliver tissue protective anti-inflammatory effects. adenosine acts via four adenosine receptors of which the a2a receptor is the predominant form in t cells. adenosine levels are elevated in asthmatic lung, and adenosine can directly induce mast cell degranulation and bronchoconstriction in these patients. instead, the role of anti-inflammatory mechanisms of adenosine on t cells in asthma is unclear. aim: to study the a2a receptor expression in peripheral blood cd4 þ t cells in asthmatic and healthy individuals using flow cytometric and quantitative real-time pcr methods. results: unstimulated cd4 þ cells of asthmatic patients expressed significantly lower levels (p < 0.001) of a2a receptor in protein level (mean percentage of cells positive ae sem: 76.8 ae 1.2, n ¼ 6) compared to healthy individuals (90.4% ae 1.9, n ¼ 4). double staining for cd69 expression showed that stimulation of cd4 þ cells decreased a2a expression in both groups but indicated that the detected lower levels of a2a in unstimulated cells of asthmatics was not due to preactivation in these patients. surprisingly, a2a mrna expression in unstimulated cd4 þ cells was significantly higher (p < 0.05) in asthmatics (n ¼ 28) compared to healthy controls (n ¼ 7). the expression did not correlate with serum total ige levels. conclusions: asthmatic individuals express less a2a adenosine receptor on their peripheral cd4 þ t cells. the higher mrna levels instead may point to a negative feedback regulation in the receptor expression. the role of possibly decreased adenosine-mediated anti-inflammatory effects in asthma pathogenesis require further studies on this t-cell mediated disease. the chronic inflammatory skin disease atopic eczema (ae) affects almost 15% of the population in many countries today. the pathogenesis of ae is not fully understood. a combination of genetic predisposition and environmental factors like microorganisms seems to contribute to the symptoms. the yeast malassezia sympodialis is part of our normal skin micro flora but can act as an allergen and elicit specific ige and t-cell reactivity in patients with ae. recently, we identified a novel major m. sympodialis allergen, designated mala s 11 (22.4 kda), with sequence similarity to the mitochondrial enzyme manganese superoxide dismutase (mnsod). interestingly, mala s 11 has a high degree of homology to human mnsod. the aim of this study was to examine the effects of recombinant mala s 11 on antigen-presenting dendritic cells. monocytederived dendritic cells (mddcs) from healthy blood donors were cultured with or without mala s 11 for different time periods. it was found that the maturation marker cd83 and the costimulatory molecules cd80 and cd86 were upregulated on the mddcs exposed to mala s 11 for 24 h, as demonstrated by flow cytometry. furthermore, coculture of mddcs with mala s 11 for 9 h induced an increased production of the inflammatory cytokines il-6 (200-fold), tnf-a (100-fold) and il-8 (sixfold), as detected by the cytometric bead array (cba) analysis. our results suggest that mala s 11 affects the immune response through dc maturation and production of inflammatory cytokines. the potential cross-reactivity with human mnsod needs to be explored and the exact role of mala s 11 in the pathogenesis of ae assessed in clinical studies involving skin prick and atopy patch tests. allergen-specific immunotherapy (sit) is commonly conducted with allergen extracts adsorbed to aluminium hydroxide (alum). drawbacks linked to the use of alum, such as the formation of granuloma at the site of injection, have led to suggestions of novel allergen carriers. an alternative carrier is 2 mm carbohydrate-based particles (cbps). in mouse, allergen-coupled cbps have been demonstrated to skew the allergen-specific immune response towards a th1-like activity (grönlund et al. immunology, 2002) . we here coupled the recombinant major cat allergen fel d 1 to cbps (cbp-fel d 1) by cyanogen-bromide activation, resulting in covalent binding. the effect of cbp-fel d 1 on monocyte-derived dendritic cells (mddcs) from healthy human blood donors was studied. we found that the majority of the cd1a þ mddcs were capable of taking up fitc-labelled cbp-fel d 1, as demonstrated by flow cytometry and confocal laser scanning microscopy. furthermore, incubation with cbp-fel d 1 resulted in an upregulation of the costimulatory molecule cd86 on the mddcs, which was not observed with fel d 1 or cbps alone. finally, cbp-fel d 1 induced a fivefold increase in the release of the pro-inflammatory cytokine tumour necrosis factor (tnf)-a and a fourfold increase in the release of the chemokine interleukin-8 from mddcs. taken together, the effects cbps possess make them interesting as novel allergen carriers for sit. the cysteine protease der p1 from dust mite of the genus dermatophagoides pteronyssinus is a major type i allergen. about 80% of house dust mite (hdm) allergic individuals are reactive to this protease in standard assays for detection of ige. a curative treatment for atopic allergy is immunotherapy (it) with hdm extracts which are complex mixtures occasionally resulting in anaphylactic reactions. novozymes focuses on developing a recombinant variant of der p1 which exhibit lowered risk of ige-mediated allergic reactions, while maintaining its ability to trigger proper th-cell responses. this may provide a safer alternative for specific it of hdm allergy. a secreted recombinant form of pro-der p 1 expressed by saccharamyces cerevisiae was obtained by fusion of the pro-enzyme to a fungal signal peptide. the n-glycosylation site of der p1 was mutated resulting in a deglycosylated pro-enzyme with a molecular mass of 35 kda. protein purification procedure was developed to obtain nearly pure der p1 protein followed by determination of concentration by active-site-titration with the cysteine protease inhibitor e64. the deglycosylated recombinant pro-der p 1 revealed immunologic similarity to the native der p 1 molecule when compared in basophile histamine release, ige-binding assays and t-cell proliferation assays. by in silico epitope mapping of a modelled 3-dimensional structure of der p1, five putative igg and ige epitopes were predicted. by protein engineering, the predicted epitopes were removed one by one in der p1 and screening for hypoallergenic variants was performed. combining inhaled long-acting b-2 agonist (laba) and inhaled corticosteroid (ics) seems to offer asthma control at a lower dose of ics than achieved by ics alone. fine mapping of t-cell surface markers by flow cytometry offers a detailed status of the individual's inflammatory response. the frequency of mt2 (cd4 þ cd45ra -cd62l þ cd11adim) and mt1 (cd4 þ cd45ra -cd62l -cd11abright) cells in peripheral blood, and their ratio, has been shown to differ predictably in atopics and patients with leprosy, where mt2 correlates with a th2 phenotype and mt1 with a th1 phenotype. stable asthmatics, requiring fluticasone propionate (fp) 750-1000 mg daily or equivalent, were randomized to receive, double-blinded, either seretide 1 [salmeterol and fluticasone propionate (sfc, n ¼ 16)] 50 mg/500 mg bd or fp 500 mg bd (n ¼ 17). if asthma was controlled based on lung function and symptoms at clinic visits every 6 weeks, ics dose was tapered until asthma exacerbated or 0 mg was reached. the frequency and ratio of mt2 and mt1 t cells of the patients was monitored at 6 week intervals. as treatment tapered, the frequency of mt2 cells decreased (p ¼ 0038 from first to final visit), whereas that of mt1 cells increased. the ratio of mt2/mt1 decreased (p ¼ 0049 from first to final visit). in patients receiving laba þ ics, the fall in mt2/mt1 ratio appeared to be more pronounced than in patients receiving ics alone. thus, the mt2 phenotype may be associated with stable asthma, whereas an imminent exacerbation may associate with an increase in the mt1 phenotype. laba may allow for a greater effect of fp on the mt ratio. activation of complement pathways, leading to production of c3a and c5a anaphylatoxins, has been postulated in the pathogenesis of asthma and allergic airway inflammation. the present study was undertaken to investigate the role of mannan-binding lectin (mbl), an initiator of the lectin pathway of complement, in asthma and allergic rhinitis. mbl levels and mbl-induced complement activity were determined in 19 patients of bronchial asthma with allergic rhinitis and 20 unrelated, age-matched controls of indian origin. mbl levels and activity were correlated with percent eosinophilia and percent predicted fev1 values of the patients. association of single nucleotide polymorphisms (snps) in exon 1 and intron 1 of the mbl with the disease, clinical markers, mbl levels and mbl-induced complement activity was analysed using standard statistical tools. significantly higher mbl levels and activity were observed in patients of bronchial asthma with allergic rhinitis as compared to the controls. we identified five snps, of which two, a816g in exon 1 and g1011a in intron 1 of the mbl, were novel. snp g1011a was significantly associated with the disease (p ¼ 0.0024, or ¼ 5.8696, 95% ci: 1.7316 < or < 19.8963). individuals with 'a' allele at position 1011 showed increased mbl levels, activity and disease severity. our results suggest that 'a' allele at position 1011 leading to high mbl levels and complement activity may be contributing to the severity of bronchial asthma and allergic airway inflammation. serum resistance of borrelia burgdorferi strains belonging to the b. afzelii and b. burgdorferi sensu stricto genospecies is dependent on binding of complement inhibitor factor h. we recently reported that factor h binding by b. burgdorferi is due to inducible expression of several approximately 20 kda plasmid-encoded, surface-exposed lipoproteins related to ospe (e.g. erpa, erpp and p21). in addition, a second class of factor h-binding proteins of approximately 27-35 kda has been described. the ospe-related lipoproteins are dramatically induced by b. burgdorferi during transmission from its tick vector into the mammalian host. the induction of ospe-related lipoproteins during mammalian infection may play a key a role in the borrelial evasion of the host's immune system. the goal of the present study was to define the factor h-binding regions of ospe-related proteins using mutagenesis, peptide mapping and surface plasmon resonance analysis (biacore). the combined studies revealed that the c-terminal regions of both human and mouse factor h (scrs 18-20) specifically bind to ospe-related lipoproteins. we also found fhr-1, whose c-terminal scrs 3-5 are homologous to scrs 18-20 of factor h, to bind to ospe. peptide mapping revealed five putative regions (designated i-v) in ospe that could directly interact with factor h. deleting the c-terminal 15 amino acid residues from region v of p21 abolished its ability to bind factor h. at the same time, however, synthetic peptides corresponding to the c-termini of ospe, p21 and erpp did not inhibit factor h binding to ospe. thus, the c-terminal-binding region v appears to be necessary but not sufficient for factor h binding. when a more specific mutation strategy was employed, where single amino acid residues in peptides spanning over the factor h-binding regions were mutated to alanines, we observed that lysines in the factor h-binding regions of ospe were required for factor h binding. the combined data have revealed that key lysine residues in ospe-related lipoproteins and ionic interactions are crucial for factor h interactions. furthermore, binding of ospe to the c-termini of both mouse and human factor h suggests that borrelia spirochetes utilize analogous complement resistance mechanisms in both rodents and man. in borrelia garinii strains, which in in vitro analyses have been found to be sensitive to complement killing, differences in the ospe sequences as well as in the expression of factor h-binding proteins may account for their susceptibility to serum lysis. role of yada, ail and lipopolysaccharide in serum resistance of yersinia enterocolitica serotype o:3 mannan-binding lectin (mbl), l-ficolin and h-ficolin are pattern recognition molecules of the innate immune system. we investigated the ability of these molecules to bind to different serotypes and noncapsulated variants of streptococcus pneumonia and staphylococcus aureus. we found that mbl binds to noncapsulated s. aureus strain (wood) but not any of the examined s. pneumoniae serotypes. l-ficolin binds to some capsulated s. pneumoniae serotypes (11a, 11d and 11f) as well as some capsulated s. aureus serotypes (type-1, -8, -9, -11 and -12). h-ficolin does not bind to any of the examined s. pneumoniae and s. aureus serotypes included in this study but did bind to a strain of aerococcus viridans. when bound to bacteria, mbl and h-ficolin initiated activation of complement factor c4, whereas l-ficolin did not. during this study, quantitative assays for the three proteins were developed and the concentration in 97 plasma samples were determined and the median values were estimated at 0.8 mg of mbl/ml, 3.3 mg of l-ficolin/ml and 18.4 mg of h-ficolin/ ml, respectively. the absence of early complement components (c1, c4 and c2 but not c3) is a predisposing factor for systemic lupus erythematosus (sle). recently, we demonstrated that, in c4-deficient (c4 def.) mice, igm-containing immune complexes (igm-ic) are filtered by the splenic barrier of marginal zone macrophages (mzm), resulting in an increased immune response against antigens within these igm-ic, but this could not be observed in wildtype or c3 def. mice. we hypothesized that splenic cd11b þ mzm play an important role in the induction of autoimmunity, and we therefore analysed their cytokine profile after isolation with the help of magnetic antibody cell sorting. mrna was isolated, and real-time pcr was performed with specific primers for murine ifn-g (ifn-g), interleukin-12 (il-12) and ifn-a (ifn-a). we observe a moderate increase of il-12 and ifn-g mrna in cd11b þ cells of c4 def. mice compared to wildtype cells. surprisingly, the concentration of ifn-a mrna is six times higher in c4 def. mice. preliminary results suggest that mrna in cd11b þ cells of c3 def. mice is even lower than that in wt. six hours following i.v. application of 20 mg of a abstracts 625 .................................................................................................................................................................................................. murine monoclonal igm anti-dsdna antibody, production of il-12, ifn-g and ifn-a mrna is increased in cd11b þ cells of both c4 def. and wt mice. several references described increased levels of inf-a in patients with sle. dendritic cells are discussed as a major source of ifn-a. our observation that c4-deficient, sle-susceptible mice demonstrate an increased spontaneous ifn-a production by splenic cd11b þ marginal zone macrophages could be an early sign and a trigger for the development of sle. this is supported by the fact that the absence of c3 is not a predisposing factor for sle and our observation that c3 def. animals display low levels of ifn-a mrna. 200-400 million people worldwide and represents one of the leading causes for liver cirrhosis and hepatocellular carcinoma. control over the hbv infection is achieved mainly by vaccination with hepatitis b surface antigen (hbsag). hbsag contains n-linked glycosylation side and is recognized by both mbl-a and mbl-c in a cadependent manner. hbsag-mbl complexes activate complement and may thus affect humoural immunity. to investigate the role of mbl in humoural responses to hbsag, we immununized mice that lack both mbl-a and mbl-c proteins with soluble hbsag. it has been shown that deficiencies in other complement components like c1q, c4 and c3 result in decreased antibody responses. however, mbl double ko animals mounted dramatically increased humoural responses. after priming, mbl double kos mounted hbsag-specific igm responses, which were threefold higher than wt controls. after boosting the hbsag, total igg was 10-fold higher in mbl ko than in wt control animals. similar to the response to hbsag, other glycosylated soluble antigens (e.g. invertase) induced better humoural responses in mbl double ko animals, suggesting that mbl plays an important role in a negative feedback regulation of adaptive immunity. reconstitution experiments with rmbl partially rescued the ko phenotype. we propose that the clearance of glycoprotein antigens in mbl ko is handled differently from the wt, resulting in better stimulation of humoural responses. alternatively, glycoprotein-ag-mbl-rich complexes inhibit b-cell responsiveness via putative mbl receptors. the complement system is an important part of the innate immune system. the activation of complement proceeds through three different pathways that converge in the generation of c3-activating enzyme complexes. complement activation via the lectin pathway is initiated when recognition molecules, mannan-binding lectin (mbl) or ficolin, bind to carbohydrate structures characteristic for microbial surfaces. in the circulation, mbl and ficolins are found in association with three structurally related mblassociated serine proteases (masp)-1, -2 and -3 and a small, nonenzymatic component, map19. masp-2 has been shown to elicit complement activation through the sequential proteolytic cleavage of c4 and c2 upon binding of mbl/masp-2 complexes to microbial surfaces. we have recently uncovered a polymorphism in the masp-2/map19 gene in a patient shown to be deficient in the lectin pathway of complement activation. the polymorphism results in a single amino acid substitution in the n-terminal part of the masp-2 protein. recombinant wildtype masp-2 and masp-2 containing the amino acid substitution in question was produced, and the ability to activate complement was studied. the mutation had a profound impact on masp-2 function, resulting in the lack of complement activation through the lectin pathway. elisa-based experiments showed that the mutation leads to the impairment of complement activation through influencing the binding of masp-2 to mbl or ficolins. deficiencies in the lectin pathway of complement activation have so far been accounted for only by lack of functional mbl. the mutation described above is the first defect described affecting both activation through mbl and the ficolins. .................................................................................................................................................................................................. th1, th2 and treg cell balance. dcs are present in the gut mucosa and may thus be target for modulation by gut microbes, including ingested probiotics. here, we tested the hypothesis that species of lactic acid bacteria, important members of the gut flora, differentially activate dc. a large panel of human gut-derived lactobacillus and bifidobacterium spp. was screened for dc-polarizing capacity by exposing bone marrow-derived murine dc to lethally irradiated bacteria. cytokines in culture supernatants and dc-surface maturation markers were analysed. substantial differences were found among strains in the capacity to induce interleukin-12 (il-12) and tumour necrosis factor (tnf)-a, while the differences for il-10 and il-6 were less pronounced. bifidobacteria tended to be weak il-12 and tnf-a inducers, while both strong and weak il-12 inducers were found among the strains of lactobacillus. remarkably, strains weak in il-12 induction inhibited il-12 and tnf-a production induced by an otherwise strong cytokine-inducing strain of lactobacillus casei, while il-10 production remained unaltered. selected strains were tested for induction of dc maturation markers. those lactobacilli with greatest capacity to induce il-12 were most effective in upregulating surface mhc class ii and cd86. moreover, l. casei-induced upregulation of cd86 was reduced in the presence of a weak il-12inducing l. reuteri. in conclusion, human lactobacillus and bifidobacterium spp. polarize differentially dc maturation. thus, the potential exists for th1/th2/treg-driving capacities of the gut dc to be modulated according to composition of gut flora including ingested probiotics. the intestinal micro flora is indispensable in developing and maintaining homeostasis of the gut-associated immune system. evidence indicates that lactic acid bacteria (lab), e.g. lactobacilli and bifidobacteria, have beneficial effects on the host. established health effects include increased gut maturation, antagonisms towards pathogens and immune modulation. the objective of this study is to evaluate the immunomodulating properties of a range of lab of human origin. as dendritic cells (dcs) play a pivotal role in the balance between tolerance and immunity to commensal microorganisms, in vitro-generated immature dcs serve as a suitable model for studying the immunomodulating effects of lab. human immature dcs were generated in vitro from monocytes and exposed to lethally uv-irradiated lab. the effect of various species of lab on dcs in direct contact was evaluated. furthermore, the maturation pattern of dcs separated from the bacteria by an epithelial cell layer (caco-2 cells), which should mimic the intestinal environment, was studied. cytokine secretion (il-12, il-10 and tnf-a) and upregulation of maturation surface markers on dcs (cd83 and cd86) was measured. different lab induced diverse cytokine responses. some strains were strong il-12 and tnf-a inducers and others weak. all strains induced il-10. different lab also differentially modulated expression of cd83 and cd86 on dcs. although some variation in the response to lab of dcs generated from different blood donors was observed, general differences in the effect of the various lab was revealed. experiments with the dc caco-2 coculture system are ongoing. different species of lab differentially affect dc maturation; this suggets that the gut flora plays a pivotal role in polarization of the immune response. natural killer (nk) cells are cells of the nonspecific immune system lysing altered self-cells. a noncytolytic subset of nk cells may serve a regulatory role by secreting cytokines. bacteria translocating across the gastrointestinal mucosa are presumed to gain access to nk cells, as consumption of certain lactic acid bacteria has been shown to increase in vivo nk cytotoxicity. here, we investigated how human gut flora-derived lactobacilli affect nk cells in vitro, by measuring proliferation and ifn-g production of human nk cells upon bacterial stimulation. cd3 -cd56 þ nk cells were isolated from buffy coats by negative isolation using non-nk lineage-specific antibodies and magnetic beads. nk cells were incubated with 10mg/ml uv-inactivated bacteria or 10mg/ml phytohemagglutinin (pha) for 4 days. proliferation was assessed by incorporation of radioactive thymidine into nk-cell dna. the ifn-g concentration was measured by elisa. incubation of nk cells with a lactobacillus acidophilus strain increased the proliferation of the nk cells and induced ifn-g production, both to levels comparable to pha stimulation. the proliferative response was further enhanced with autologous monocytes present, probably because cytokines, secreted by monocytes having engulfed bacteria, stimulated the nk cells. in contrast, a lactobacillus paracasei strain caused the nk cells to proliferate only in the presence of monocytes. these results demonstrate that various strains of lactobacilli have the capacity to activate nk cells in vitro, in a monocyte-dependent or -independent way. hence, the encounter of nk cells with lactic acid bacteria will affect nk-cell activation. such activation of nk cells may potentially skew an on-going or subsequent immune response towards a th1 response. lactobacilli are nonpathogenic gram-positive inhabitants of the normal human intestine known for their healthpromoting effects. in our earlier work, it is shown that human monoclonal antibody isolated from sera of a patient with waldenstrom macroglobulinaemia possess innate antibody characteristics and binds to lactic acid bacteria. according to the immune network model, immunization with this bacteria could induce the perturbations in immune system that might result in production of anti-lactobacillus antibodies, human monoclonal antibody like (ab1) and anti-idiotypic antibody (ab2). in this study, balb/c mice were immunized with two doses of bacteria lactobacillus acidophilus in complete and incomplete freund's adjuvant and phosphate-buffered saline (pbs), respectively. seven days after the last immunization, sera from immunized mice were collected and the presence of lactobacillus-specific ab1 and ab2 were determined by elisas. in the sera of immunized mice, antibodies specific to bacteria lactobacillus acidophilus were shown. the concentration of lactobacillus-specific antibodies was higher in the sera of hyperimmunized mice (mice immunized with 1 mg of igm dj) than in sera of mice immunized with 100 times lower doses of immunogen (0.01 mg per doses). moreover, ab1 and ab2 antibodies were detected in the sera of lactobacillus-hyperimmunized mice. in this study, we have shown the idiotypic network interactions in mice immunized with bacteria lactobacillus acidophilus. the normal gastrointestinal flora is crucial for the maturation of the acquired immunity via effects on antigenpresenting cells (apcs). here, we have investigated how two types of apcs, monocytes and dendritic cells (dcs), react to different bacterial strains typical of the commensal intestinal flora. purified monocytes and monocyte-derived dcs were stimulated with uv-inactivated gram-positive (lactobacillus plantarum and bifidobacterium adolescentis) and gram-negative (escherichia coli and veillonella parvula) bacterial strains. monocytes produced higher levels of il-12p70 and tnf, as detected by elisa, in response to l. plantarum than to e. coli and v. parvula. in contrast, dcs secreted high amounts of il-12p70, tnf, il-6 and il-10 in response to e. coli and v. parvula but were practically unresponsive to l. plantarum and b. adolescentis. the lack of response to the gram-positive strains correlated with a lower surface expression of toll-like reseptor 2 (tlr2) on dcs compared to monocytes. the surface expression of tlr4 on dcs was undetectable when analysed by flow cytometry, but blocking this receptor decreased the tnf production in response to v. parvula, indicating that low tlr4 expression on dcs is sufficient to mount an inflammatory response to gram-negative bacteria. ifn-g increased the expression of tlr4 on dcs and also potentiated the cytokine response to gram-negative bacteria. our results indicate that, when monocytes differentiate into dcs, their ability to respond to different commensal bacteria dramatically changes, thereby becoming unresponsive to probiotic gram-positive bacteria. these results may have important implications for the capacity of different groups of commensal bacteria to regulate mucosal and systemic immunity. probiotic bacteria, e.g. lactobacillus spp., may improve diseases such as chronic inflammatory bowel disease. we examined cytokine production and phenotypic change after in vitro stimulation of t cells from healthy volunteers using different probiotic strains. methods: t cells were cultured from colonic biopsies from eight healthy volunteers (agnholt and kaltoft, exp clin immunogenet 2001; 18:213-25) , and dendritic cells were matured from their peripheral blood mononuclear cells. t-cell cultures were stimulated with autologous bacterial sonicate or strains of lactobacillus spp., with and without the addition of dendritic cells. cytokine levels (tnf-a, ifn-g, il-10 and gm-csf) and phenotype (cd3, cd4, cd25 and cd69) were measured on day 4. results: lactobacillus spp. induced higher productions of tnf-a and il-10 than did autologous bacteria. in presence of dendritic cells, the production of all cytokines increased. however, the increases of ifn-g and tnf-a were more pronounced in wells with autologous bacteria than in wells with lactobacillus spp. the addition of dendritic cells upregulated cd25 expression without simultaneous upregulation of cd69. the upregulation was pronounced after stimulation with lactobacillus rhamnosus gg compared with autologous bacteria and other lactobacilli. discussion: in presence of dendritic cells, autologous bacteria induced inflammatory cytokines, while probiotics mainly induced regulatory cytokines. lactobacillus rhamnosus gg induced a regulatory phenotype (cd25 þ ), in part mediated by dendritic cells. future studies will address whether this shift to a cd25 þ phenotype represents a differentiation into competent regulatory t cells. in a clinical context, such cells might be used for treatment of inflammatory diseases. protein microarrays will play a key role in the postgenomic era and offer a unique possibility to perform highthroughput global proteome analysis. a chip can be printed with thousands of protein probes (e.g. antibodies), the biological sample added (e.g. a proteome) and any binding detected. we aim to develop protein microarrays based on human recombinant scfv antibody fragments for global proteome analysis. the concept of comparing proteomic maps of healthy versus diseased samples will allow diseasespecific proteins to be detected. in fact, antibody microarrays will allow us to perform comparative proteome analysis on any sample format in a species-independent manner, as long as a proteome can be isolated. however, the complexity of proteomes, containing several thousands of different proteins, is a problem. here, we have designed antibody microarrays targeting the water-soluble fraction of a proteome. to this end, an anticytokine antibody array was developed and human dendritic cells (aeactivation) was used as model system. the results showed that our antibody microarrays could be used to examine the cytokine profile in complex samples. furthermore, we have taken the first steps towards comparing our results with those of other technologies on both the protein and gene level. due to the complexity of the model proteome, we also examined the possibility to prefractionate the proteome in a simple one-step procedure (based on size) prior to the labelling step. in more detail, the sample proteome was fractionated into two fractions using membrane devices with different molecular weight cut-offs. the results showed that the fractionation considerably enhanced the assay sensitivity allowing cytokines in the pg/ml range to be readily detectable. the immunomodulatory effect of heat shock protein 70: immunization with a dna construct based on the malarial antigen fused with a fragment of hsp 70 primes for a th-1 type of response finding an appropriate adjuvant for human vaccination is crucial. heat shock proteins (hsps) act as adjuvants when coadministered with peptide antigens or given as fusion proteins. however, there is a potential risk of autoimmunity when using the complete molecules, because hsps are evolutionary conserved. to overcome this, we first evaluated the adjuvant effect against two different antigens of a less-conserved fraction of plasmodium falciparum hsp70 (pf70c) and compared it to the whole hsp70 molecule from trypanosoma cruzi (tchsp70). we found that pf70c exhibited similar adjuvant properties as the whole molecule. we later evaluated the adjuvant potential of pf70c against the malarial antigen eb200 in a chimeric dna construct. no appreciable levels of eb200-specific abstracts 629 .................................................................................................................................................................................................. antibodies were detected in mice immunized only with the dna constructs. however, dna primed the immune system, because subsequent challenge with the corresponding recombinant fusion proteins elicited a strong th-1 antibody response. in contrast, no priming effect was observed for ex vivo ifn-g production but stimulation with the hsp-chimeric fusion protein induced a stronger secretion of ifn-g in vitro than other proteins used. these results indicate that the use of hsps is promising in the design of new vaccines. high-throughput proteomics on antibody-based microarrays: the importance of probe and surface design in analogy to dna microarrays, protein microarrays offer a new distinct possibility to perform sensitive highthroughput global proteome analysis. however, the development of the protein microarray technology will place high demands upon the design of both probes and solid supports. the analysis of thousands of heterogeneous proteins on a single microarray requires the use of uniform probes, such as antibodies, directly designed for protein microarray applications. we have recently generated a human recombinant single-chain fv antibody library, genetically constructed around one framework, the ncoder-library, containing 2 â 1010 clones. single framework antibody fragments (sinfabs) selected from this library were successfully applied as probes for microarrays providing sensitive detection in the 600 attomol (mass spectrometry) and the 300 zeptomole range (fluorescence). however, the choice of framework is critical. we have shown that the selected ncoder framework displayed excellent functional on-chip stability and arrayed dehydrated probes retained their activity for several months. furthermore, we have addressed the issues of biocompatibility of the solid support and immobilization strategies for our microarray setup. an in-house-designed substrate, macroporous silicon coated with nitrocellulose (map3-nc7), displayed properties equal to, or better than, those of five commercially available supports used as reference surfaces. we have also evaluated different coupling strategies, such as adsorption, covalent coupling, diffusion and affinity coupling. using a novel affinity tag, the double-(his)6-tag, we increased the binding efficiency of sinfab-molecules to ni2 þ -coated solid supports, thereby allowing nonpurified probes to be directly applied. the mannan-binding lectin (mbl) pathway is part of the innate immune system providing a first line of defence against infections. mbl and ficolins circulate in complexes with mbl-associated serine proteases (masp-1, -2 and -3). after recognition of a microorganism by mbl, activation of the complement system occurs. masp-1 and masp-3 share five domains (making up the so-called a-chain), whereas they have unique protease domains (b-chains). before the identification of masp-3, an assay for masp was presented, based on antibodies against the a-chain of masp-1. with the new knowledge of the three masps, and the sharing of domains by masp-1 and masp-3, assays specific for the protease domains have to be constructed, if one wishes to measure the proteins individually. we present an assay for quantifying total masp-3 in plasma and serum samples. the assay is a sandwich-type assay using as catching antibody a monoclonal antibody against the common a-chain of masp-1/3 and a developing secondary antibody against the c-terminal part of the protease domain of masp-3. we have used this assay for estimating the normal concentration of the protein as well as the concentration in patients and also for characterizing by gel permeation chromatography the masp-3 protein in serum. inducible costimulator ligand (icosl) is a costimulatory molecule related to b7.1 (cd80) and b7.2 (cd86). b cells, monocytes, dendritic cells and endothelial cells express icosl. inducible costimulator (icos) interacts with icosl, and this interaction leads to signals involved in isotype switching and the development of immunological memory. hitherto, no polymorphisms of this gene have been described. the aim of this study was to reveal variation of the icosl gene in normal individuals. all eight exons, except exon 1, were sequenced with flanking introns in 10 healthy blood donors. eight single nucleotide polymorphisms (snps) and two length polymorphisms were found. one of the snps was found in the coding regions of the gene. the base involved was located in exon 3 and caused a conservative amino acid change from valine (gtt) to isoleucine (att). three individuals were heterozygous g/a for the exon polymorphism, while the remaining seven individuals were homozygous for the wildtype g/g. exon 3 encodes the immunoglobulin variable (igv)-like domain of the molecule which is situated outside the cell. this means that the amino acid could be critical for the stability of the molecule or could constitute part of the binding site for icos. the results form the basis for further experiments to find possible associations of the alleles to diseases caused by immune dysregulation. especially, the exon 3 variant is interesting and could play a role for the development of immunological diseases. besides, it would be interesting to see whether both exon 3 alleles are expressed or only the wildtype allele is functional. interferon-a/b (ifn-a/b) is increasingly used as antiviral and immunomodulatory therapies. unfortunately, bioavailability varies with ifn species and mode of administration, and all ifn species are potentially immunogenic. assays for antiviral activity (ifn) and antiviral neutralization (antibodies, nab) have been used for some time to monitor patients on ifn biologicals. these assays require laborious titrations making them unsuitable for large-scale clinical use. myxovirus a (mxa) is a resistance gtpbinding protein that is specifically induced by treatment with type 1 ifns. for example, ifn-b-induced mxa in blood leucocytes has been used as a biomarker in ifn-btreated patients with multiple sclerosis. however, the degree of specificity of mxa in this regard is unclear, and measurements of mxa protein and/or mrna are not yet suitable for routine clinical use. in an attempt to find new and better reporter genes (and, hopefully, genes and gene products with proven specificity for ifn-a and -b), microarray screenings with u133a genechips (affymetrix) were carried using human blood leucocytes and the human lung carcinoma cell line a549. we studied the simultaneous expression of 22,000 transcripts before and after exposure to human recombinant ifn-a and ifn-b and other antiviral and immunomodulatory cytokines. the results will be presented at the conference. interferon-a/b (ifn-a/b) is increasingly used as antiviral and immunomodulatory therapies. unfortunately, bioavailability varies with ifn species and mode of administration, and all ifn species are potentially immunogenic. assays for antiviral activity (ifn) and antiviral neutralization (antibodies, nab) have been used for some time to monitor patients on ifn biologicals. these assays require laborious titrations making them unsuitable for large-scale clinical use. our laboratories have therefore modified the antiviral assays for ifn bioactivity and nab, so that they are suitable for large-scale screening in specialized laboratories. the read-out is survival of a subcloned a549 cell line in the presence of an otherwise lethal amount of virus. thus, survival increases in the presence of type 1 ifn and decreases in the presence of nab against the ifn added to the cells. mxa is induced by type 1 ifn and can be used for measuring the nab activity. in another assay, the mxa level in the a549 cell line is measured. in an attempt to find a new and better reporter gene for type 1 ifn than mxa and genes specific for either ifn-a or -b, a micro array screen was carried using the u133a chip from affymetrix. the expression of 22,000 genes can be studied simultaneous with this technology. the results will be presented at the conference. in our laboratory, we have developed a database system, which we believe is of immediate interest to the general scientific community. the database represents a computerbased replacement for the laboratory notebooks used in the majority of research laboratories worldwide. in addition, the database provides an effective tool for organizing and managing laboratory information at all levels, spanning from managing and revising standard operating procedures and producing documentation of research activities to keeping track of data and conclusions. using the commercially available database toolkit software filemaker pro, we have developed a relational database solution for management of laboratory information. the system consists of a hierarchy of five interrelated databases, each pertaining to a separate type of information, namely, overall project information, information relating to individual experiment setups, documentation of daily research activity, generated data and descriptions of standard operating procedures. like other databases, each individual database consists of a number of records, each comprised of a set of fields in which information is entered. in each record, a certain field is reserved to specify the relation of the record to a record in another database at a higher level. thus, the database is essentially five databases linked by a hierarchy of one-to-many relations, organizing information in a folder-like structure. importantly, the database system allows multiple users to access and edit records simultaneously, and the data entered in one database immediately becomes accessible through the other databases. the limitations of laboratory notebooks are apparent when looking for information, which is dispersed throughout one or more notebooks, or possibly on loose sheets of paper or printouts 'somewhere'. the often complicated process of gathering laboratory data or results when writing grant applications or research papers is made considerably easier with the database system. thus, the database solution presented should be broadly attractive to researchers, irrespective of their scientific discipline. an effective sars vaccine is likely to include components that can induce specific cytotoxic t-cell (ctl) responses. the specificities of such responses are governed by hlarestricted presentation of sars-derived peptide epitopes. exact knowledge of how the immune system handles protein antigens would allow for the identification of such linear sequences directly from genomic/proteomic sequence information. the latter was recently established when a causative coronavirus (sars cov) was isolated and full-length sequenced. here, we have combined advanced bioinformatics and high-throughput immunology to perform an hla supertype, genome-wide scan for sars-specific cytotoxic t cell epitopes. the scan includes all nine human hla supertypes in total covering >99% of all major human populations. for each hla supertype, we have selected the 15 top candidates for test in biochemical-binding assays. at this time (approximately 6 months after the genome was established), we have tested the majority of the hla supertypes and identified almost 100 potential vaccine candidates. these should be further validated in sars survivors and used for vaccine formulation. we suggest that immunobioinformatics may become a fast and valuable tool in rational vaccine design. rationale: major histocompatibility complex class i (mhc i) molecules monitor the protein content of the cell by binding small derived peptides and presenting them to cytotoxic cd8 þ t cells. the goal of the human mhc project is to predict the binding strength of any given peptide/mhc complex. this prediction allows the design of peptide-based vaccines. the prediction requires representative binding data from mhc alleles from all the nine hla supertypes. here, we describe the genetic construction, protein production and purification as well as the establishment-binding assays for two recombinant mhc supertype alleles, hla-b*1501 and hla-b*5801. methods: using the quikchange multisite directed mutagenesis kit (stratagene), codon-optimized genes encoding hla-b*1501 and hla-b*5801 are created. the two mhc i molecules are fermented and purified by ion exchange chromatography, hydrophobic interaction chromatography and size exclusion chromatography. the binding (kd) of natural t-cell epitopes, as well as predicted peptide ligands, is described by radioactive immunoassays (rias) and enzyme-linked immunosorbent assays (elisas). the mhc molecules are biotinylated during expression. results: the expression of mhc i resulted in multiple disulfide bond isomers, which are separated by hydrophobic interaction chromatography and used in subsequent binding studies resulting in the determination of kd for various peptide ligands ranging from strong binders we have previously demonstrated that bioinformatics tools such as artificial neural networks (anns) are capable of performing pathogen-, genome-and hlawide predictions of peptide-hla interactions. these tools may therefore enable a fast and rational approach to epitope identification and thereby assist in the development of vaccines and immunotherapy. a crucial step in the generation of such bioinformatics tools is the selection of data representing the event in question (in casu peptide-hla interaction). this is particularly important when it is difficult and expensive to obtain data. herein, we demonstrate the importance in selecting information-rich data and we develop a computational method, query-bycommittee, which can perform a global identification of such information-rich data in an unbiased and automated manner. furthermore, we demonstrate how this method can be applied to an efficient iterative development strategy for these bioinformatics tools. methods: a large panel of binding affinities of peptides binding to hla a*0204 was measured by a radioimmunoassay (ria). this data was used to develop multiple first generation anns, which formed a virtual committee. this committee was used to screen (or 'queried') for peptides, where the anns agreed ('low-qbc'), or disagreed ('high-qbc'), on their hla-binding potential. seventeen low-qbc peptides and 17 high-qbc peptides were synthesized and tested. the high-or low-qbc data were added to the original data, and new high-or low-qbc second generation anns were developed, respectively. this procedure was repeated 40 times. the high-qbc-enriched ann performed significantly better than the low-qbc-enriched ann in 37 of the 40 tests. conclusion: these results demonstrate that high-qbcenriched networks perform better than low-qbc-enriched networks in selecting informative data for developing peptide-mhc-binding predictors. this improvement in selecting data is not due to differences in network training performance but due to the difference in information content in the high-qbc experiment and in the low-qbc experiment. finally, it should be noted that this strategy could be used in many contexts where generation of data is difficult and costly. interleukin-18 (il-18), a pro-inflammatory cytokine that is produced by both lymphoid and nonlymphoid cells, has a critical role in modulation of innate and adaptive immunity. its primary function in stimulation of ifn-g production and stimulation of nk-cell-cytotoxic activities makes this cytokine a candidate for cancer immunotherapy. in oral cavity, this cytokine is produced by oral epithelia and carcinoma cells and is related to tumour regression in nude mice bearing salivary adenocarcinoma. however, direct effects of this cytokine on oral cancer cells have not been elucidated. in this project, we investigated il-18 effect on an oral carcinoma (kb) cell line. with rt-pcr technique, kb-cell line was found to express il-18 receptors (il-18ra and il-18rb), indicating that this oral carcinoma line is a target for il-18 study. we showed that recombinant human il-18 inhibited kb-cell proliferation by 17% at concentration of 100 ng/ml (p < 0.05), whereas ldh release by these cells in treatment group and control groups was comparable, indicating that il-18 suppression of cell proliferation was not mediated by the induction of cell death. to further address this hypothesis, we found that il-18 treatment did not induce apoptotic cell death, as studied by dna laddering and tunel assays. in addition, expression pattern of cell death-controlling genes (bcl-2 and bax) was not altered by this cytokine. findings in these studies indicated that suppression of kb-cell proliferation may be attributed to control of cell cycle, growth arrest or induction of cell differentiation. the data presented in this project could provide an insight of how cancer cell directly responds to il-18, as this cytokine is an important regulator of anticancer mechanisms. aloe emodin (ae) is a naturally occurring compound with wide spectrum of biological properties, including antimicrobial, vasorelaxant, immunosuppressive and anticancer actions. this anthraquinone induces apoptosis in several tumour cell lines with special affinity to tumours of neuroectodermal origin. high amounts of nitric oxide (no) released by activated macrophages induce tumour cell death. therefore, we explored the capacity of ae to modulate no-mediated antitumour response in vitro. interestingly, while ae markedly suppressed no release from macrophages alone, it significantly potentiated no production in cocultures of macrophages and c6 cells, after 48 h of cultivation. accordingly, the viability of c6 cells cocultivated with macrophages was reduced in the presence of ae. moreover, the observed ae-imposed potentiation of no production in macrophages was closely related to macrophage culture cell density. according to these data, we proposed that no modulator capacity of ae strongly depended on intercellular contact, indicating that macrophage antitumour response was not compromised but even potentiated by ae. immunotherapy represents an attractive fourth-modality therapeutic approach, especially in the light of the shortcomings of conventional surgery, radiation and chemotherapies in the management of metastatic cancer. to this end, a large number of peptide antigens derived from taa have been applied in immunotherapeutic trials for the treatment of various malignancies, e.g. cancers of the breast, prostate and kidney, in addition to haematological cancers. in some cases the response rates have been impressive and no adverse autoimmunity have been observed. a major strategic difficulty associated with these trials relates to the choice of best-suited peptide antigens. the vast majority of the antigens described thus far is not vital for survival and growth of the tumour cells, and immunoselection of antigen-loss variants may therefore prove to be an additional obstacle for the clinical applicability of most of the known peptide epitopes. in this respect, the development of acquired antigen loss during immunotherapy has been demonstrated in several cases. obviously, the development of loss-variant tumour cells implies that these cells acquire a pronounced growth advantage and are left unaffected by further treatment. ideally, target antigens should be derived from proteins required for survival and growth of tumour cells, as antigens with these characteristics would not be inflicted by the development of loss-variant tumour cells. in this respect, several inhibitors of apoptosis proteins (iaps) are universally expressed among tumours and play an important role in tumour cell escape from apoptosis. we have characterized spontaneous t-cell reactivity against iapderived peptides in cancer patients. from the iap survivin, we have characterized peptides restricted to the class i molecules hla-a1, a2, a3, a11, b7 and b35. furthermore, we have demonstrated that survivin-specific t cells infiltrate metastatic lesions and that isolated survivinspecific ctls are capable of killing hla-matched tumour cells. survivin-derived peptides are now in clinical trial, and continued work in our lab has demonstrated that other iaps are targets for spontaneous t-cell reactivity in cancer patients. we previously reported that in mice with large progressing t-cell lymphoma tumours, dysfunctions in the antitumour ctl activity occur, associated with an accumulation of splenic arginase-producing myeloid suppressor cells (mscs). in this study, we first demonstrate that both the presence and the activation state of these msc depends on tumour evolution. while in tumour regressors hardly any arginase-producing msc can be found, both the amount and the arginase activity of this population expands from early over late progressors. this gradual induction of mscs is paralleled by an increasing suppression of ctl activity and th1, but not th2, cytokine production. upon analysing the molecular repertoire of msc in vitro, we found, besides arginase1, a well-established marker for alternatively activated myeloid cells or m2, a strong upregulation of fizz1 and ym, two additional recently identified markers for m2. further evaluation of molecular markers by microarray analysis in msc yielded genes involved in wound healing (e.g. coagulation factor xiiia), anti-inflammation (e.g. selenoprotein p), immunomodulation (e.g. pd-l2) and fat and sugar metabolism (e.g. leptin receptor). of note, many of these genes are regulated by type 2 cytokines (il-4, il-13 and il-10) and are therefore rather m2 associated. overall, our data provide new markers for msc in cancer and further establish their m2 activation state. study. only sp-a showed a significant expression in normal mucosa which was downregulated in crc. as the absolute signal level was below the noise threshold, these results have to be interpreted with caution and require confirmation by direct measurenment of the proteins. our results suggest that there is no major role for the human collectins in colorectal cancer. tetramerization is visualized by sds-page. conclusion: an effective method for the production of highly pure mhc i molecules has been applied to hla-b*1501 and hla-b*5801, and ria and elisa binding assays for those alleles have been established background: proliferation, differentiation and apoptosis are essential processes in the normal functions of the mammary epithelium. the hypothesis examined in this study is that the transcription factor bcl-6 is critically important not only for regulating b-cell growth and development but also for mammary epithelial apoptosis. methodology: twenty breast cancer cases and 31 healthy controls were used to investigate whether bcl-6 protein in involved in breast cancer (grade iii). full length bcl-6 cdna was retrovirally transduced into eph-4 cell line. we then used flow cytometry of brdurd-stained cells to investigate the cell-cycle duration of the control and transduced cell lines. tunel was used as a marker of apoptosis to find out differences in the frequencies of apoptotic cells in the control and transduced cell lines. finally, immunohistochemistry staining was performed to detect bcl-6 in breast cancer (iii). results: restoration of bcl-6 into eph-4 cells not only inhibits apoptosis but also prolongs the cell cycle and results in increased cell size and protein content. the results also indicated that the cell-cycle time of bcl-6-transduced eph-4 cells is prolonged by about 3 h, presumably as a result of the action of bcl-6 at the bcl-6 at the g1/s transition. we found differences in the frequencies of viable and apoptotic cells in cultures of the parent eph-4 cells, control-transduced eph-4 cells and bcl-6-transduced eph-4 cells. consistently, we demonstrated that bcl-6 is expressed in 90% of high grade of breast carcinoma, which is considered as the most aggressive of tumours. conclusion: together, these results suggest that bcl-6 is likely to be involved in mammary gland development and carcinogenesis. inflammatory cytokines have a critical role in modulation of both innate and adaptive immunity in response to foreign antigen. they also play an important role in anticancer immunity. for example, they can promote cell-mediated immunity against cancer cells. with their immunostimulatory effects, these cytokines are being tested for cancer treatment in the form of dna vaccine or adjuvant or therapeutic cytokines. direct effect of these cytokines on cancer cell, however, is still unclear. in this project, we investigated whether il-1( and il-18 can modulate cancer cell proliferation. we employed a simple nonradioactive proliferation (mtt) assay and detection of lactate dehydrogenase (ldh) to test the effect of these recombinant human cytokines on various cancer cell lines, including breast cancer cell line (mcf-7), oral carcinoma cell line (kb), colon cancer cell line (caco-2) and choriocarcinoma cell line (jar). cytokines used in this study had both inhibitory and stimulatory effect on cell proliferation. findings in this project could provide an insight of cancer cell response to these cytokines and this could lead to a consideration on using cytokine as immunotherapy for cancer treatment.capacity of ae to modulate nitric oxide production depended on intercellular contact donor t cells are involved in the antitumour effects observed after bmt. thus, patients receiving t-celldepleted bmt have a higher risk of leukaemia relapse compared to patients receiving nonmanipulated bmt, and patients experiencing graft-versus-host disease (gvhd) have a lower risk of disease relapse than patients who do not experience gvhd. although the importance of donor t cells for the curative action of bmt has been established, the exact mechanisms and molecules involved in this graft-versus-tumour effect remain largely unknown. in a recently initiated project, we have conducted a longitudinal study of t-cell clonotypes in patients who received peripheral blood stem cell grafts after nonmyeloablative conditioning. peripheral blood samples were obtained sequentially after transplant, and the mononuclear cells (mncs) were isolated and cryopreserved. cd8 þ t cells were isolated from the mncs by use of immunomagnetic beads or facs and analysed for the presence of clonally expanded cells by t-cell receptor clonotype mapping based on rt-pcr and denaturing gradient gel electrophoresis (dgge). using this gel-based methodology, clonally expanded t cells were monitored after transplant and compared to the clinical data of the patients. the preliminary results demonstrates the presence of clonally expanded cd8 þ t cells at all time points analysed. furthermore, a number of clonotypes persisted for more than 6 months, and other clonotypes emerged during this period. the appearance of newly emerged clonotypes which coincided with clinical gvhd could indicate a role for these t cells in the pathogenesis of gvhd. background: deficiency of the mannan-binding lectin (mbl) pathway of innate immunity leads to increased susceptibility to infections. in patients with colorectal cancer, postoperative infection is associated with poor prognosis. the aim of the present study was to evaluate (1) the relation between the mbl pathway and postoperative infectious complications and survival of patients resected for colorectal cancer and (2) the role of mbl as acute phase reactant compared to crp. methods: preoperative mbl concentration, mbl/mblassociated serine protease (masp) activity and crp were determined in serum from 611 patients and 150 healthy controls. the patients were observed for 8 years. postoperative infections, recurrence and survival were recorded. results: the mbl pathway components were increased in the patients (p < 0.0001) compared to healthy controls. low mbl levels were predictive of pneumonia (p ¼ 0.01), and pneumonia (n ¼ 87) was associated with poor survival (p ¼ 0.003, hr ¼ 1.5, 95% ci 1.1-1.9). mbl and mbl/ masp activity could not predict postoperative overall infections. mbl showed no correlation (spearman's r ¼ 0.02, 95% ci à0.06-0.10) with crp. conclusions: low preoperative mbl levels are predictive of pneumonia, which is associated with poorer survival. mbl concentration and mbl/masp activity was not predictive of other postoperative infections or long-term prognosis. mbl apparently is not a surrogate measure of crp. department of surgery, university hospital of erlangen, erlangen, germany. e-mail: michael.siassi@rzmail.uni-erlangen.de introduction: the human collectins, mannan-binding lectin (mbl), surfactant protein-a (sp-a) and surfactantprotein-d (sp-d) play a central role in the innate immune system. immunological responses to malignant transformation of epithelial cells gained increasing interest recently. a former study could demonstrate binding of mbl to certain colorectal carcinoma (crc) cell lines in vitro. we therefore examined the expression of human collectins in normal colon mucosa and in colorectal carcinomas. materials and methods: colon samples from 20 crc patients and 10 normal mucosa samples were collected immediately after surgery. the tissue was microdissected and rna isolated (qiagen, rneasy-kit). gene expression profiles were analysed using gene-chips (affymetrix, hg-u133). we analysed the data for the expression of mbl, its associated serine proteases mannan-binding lectinassociated serine protease 1/2 (masp 1/2), sp-a and sp-d. the signal intensity of the genes of interest was compared using the mann-whitney u-test. results: the expression of human collectins in normal human colon mucosa was generally low. only the expression of sp-a and masp-2 reached the noise threshold of 250 signals. these genes were significantly downregulated in crc specimens. the expression of the other proteins showed no difference in normal mucosa and crc. conclusion: as demonstrated before, the expression of human collectins in normal colon was low in this being the first lymph node to receive drainage from the tumour area, the sentinel node offers a unique possibility to obtain tumour-reactive lymphocytes. we investigated antitumour immune responses in sentinel nodes from patients with bladder cancer, by assaying tumour-specific proliferation and tcr vb repertoires. during tumour surgery, sentinel lymph nodes were identified by peritumoural injection of blue dye. fresh specimens of tumour, sentinel and nonsentinel lymph nodes were obtained, and single-cell suspensions were prepared. cells were assayed for reactivity against autologous tumour extract in [ 3 h]-thymidine incorporation assays and characterized by flow cytometry. parallel analyses of the expression of vb gene families were performed with padlock probes, linear oligonucleotides which upon target recognition can be converted to circular molecules by a ligase. probes were reacted with cdna prepared from magnetically separated cd4 þ cells, and the tcr repertoire was determined by hybridizing the products to oligonucleotide microarrays. dose-dependent proliferation in response to tumour extract could be detected in sentinel lymph nodes. common clonal expansions were detected among tumourinfiltrating lymphocytes and in sentinel lymph nodes. nonsentinel lymph nodes displayed a divergent tcr vb repertoire. these results indicate an ongoing immune response against tumour antigens in sentinel nodes, draining urinary bladder cancer. identification of sentinel lymph nodes makes it possible to obtain tumour-reactive lymphocytes for use in adoptive immunotherapy. key: cord-023375-x4p187u7 authors: alitalo, a.; meri, t.; lankinen, h.; cheng, z.‐z.; jokiranta, s.; seppälä, i.; lahdenne, p.; brooks, c.; hefty, p. s.; akins, d. r.; meri, s. title: lysine‐dependent binding of ospe to the c‐terminus of factor h mediates complement resistance in borrelia burgdorferi date: 2008-06-28 journal: scand j immunol doi: 10.1111/j.0300-9475.2004.01423aj.x sha: doc_id: 23375 cord_uid: x4p187u7 serum resistance of borrelia burgdorferi strains belonging to the b. afzelii and b. burgdorferi sensu stricto genospecies is dependent on binding of complement inhibitor factor h. we recently reported that factor h binding by b. burgdorferi is due to inducible expression of several approximately 20 kda plasmid‐encoded, surface‐exposed lipoproteins related to ospe (e.g. erpa, erpp and p21). in addition, a second class of factor h‐binding proteins of approximately 27–35 kda has been described. the ospe‐related lipoproteins are dramatically induced by b. burgdorferi during transmission from its tick vector into the mammalian host. the induction of ospe‐related lipoproteins during mammalian infection may play a key a role in the borrelial evasion of the host's immune system. the goal of the present study was to define the factor h‐binding regions of ospe‐related proteins using mutagenesis, peptide mapping and surface plasmon resonance analysis (biacore). the combined studies revealed that the c‐terminal regions of both human and mouse factor h (scrs 18–20) specifically bind to ospe‐related lipoproteins. we also found fhr‐1, whose c‐terminal scrs 3–5 are homologous to scrs 18–20 of factor h, to bind to ospe. peptide mapping revealed five putative regions (designated i‐v) in ospe that could directly interact with factor h. deleting the c‐terminal 15 amino acid residues from region v of p21 abolished its ability to bind factor h. at the same time, however, synthetic peptides corresponding to the c‐termini of ospe, p21 and erpp did not inhibit factor h binding to ospe. thus, the c‐terminal‐binding region v appears to be necessary but not sufficient for factor h binding. when a more specific mutation strategy was employed, where single amino acid residues in peptides spanning over the factor h‐binding regions were mutated to alanines, we observed that lysines in the factor h‐binding regions of ospe were required for factor h binding. the combined data have revealed that key lysine residues in ospe‐related lipoproteins and ionic interactions are crucial for factor h interactions. furthermore, binding of ospe to the c‐termini of both mouse and human factor h suggests that borrelia spirochetes utilize analogous complement resistance mechanisms in both rodents and man. in borrelia garinii strains, which in in vitro analyses have been found to be sensitive to complement killing, differences in the ospe sequences as well as in the expression of factor h‐binding proteins may account for their susceptibility to serum lysis. the nuclear receptor heterodimers of liver x receptors (lxrs) are recently identified as key transcriptional regulators of genes involved in lipid homeostasis and inflammation. lxrs and their ligands are negative regulators of macrophage inflammatory gene expression. multiple sclerosis (ms), a demyelinating disease of the central nervous system of unknown cause, is characterized by recurrent inflammation involving macrophages and their inflammatory mediators. sweden belongs to the countries with a high ms incidence. in italy, incidence is lower, with an exception for sardinia where the incidence is even higher than that in sweden. subjects from sardinia are ethnically more homogeneous and differ from swedes, also regarding genetic background and environment. we studied lxrs and their related molecules of blood mononuclear cells (mncs) from female patients with untreated relapsing-remitting ms from sassari, sardinia and stockholm, sweden. sex-and age-matched healthy controls (hcs) were from both areas. mrna expression was evaluated by real-time pcr. lxr-a was lower (p < 0.05) in ms (mean ae sem: 3.1 ae 0.2; n ¼ 37) compared to hc (3.6 ae 0.1; n ¼ 37). lxr-a was lower in ms from stockholm (2.6 ae 0.2; n ¼ 22) compared to corresponding hc (3.4 ae 0.1; n ¼ 22; p < 0.01) and compared to ms (3.8 ae 0.2; n ¼ 15; p < 0.001) and hc (4 ae 0.2; n ¼ 15; p < 0.001) from sardinia. ms patients from stockholm, but not from sassari, also expressed lower (p < 0.05) lxr-b (à4.1 ae 0.4) compared to corresponding hc (à2.9 ae 0.3). ms from stockholm was associated with higher abca-1 (6.1 ae 0.4 versus 5.0 ae 0.3; p < 0.05) and higher estrogen receptor-b-cx (2.4 ae 0.4 versus 0.8 ae 0.4; p < 0.01) compared to corresponding hc. the hc from sassari had higher androgen receptor (2.9 ae 0.2) compared to ms from sassari (1.4 ae 0.3; p < 0.01), ms (1.3 ae 0.4; p < 0.01) and hc from stockholm (1.2 ae 0.3; p < 0.01). ms from sassari had lower cyclooxygenase-1 compared to corresponding hc (5.1 ae 0.4 versus 6.6 ae 0.3; p < 0.01) and lower prostaglandin-e (à0.03 ae 0.5) compared to the hc (1.4 ae 0.5; p < 0.05) and ms (2.7 ae 0.4; p < 0.05) and hc from stockholm (1.9 ae 0.4, p < 0.001). our findings identify lxrs and their related molecules as being involved in ms from stockholm but not from sassari, while sex hormone receptors seem to be involved in ms in sassari. multiple sclerosis: ifn-b induces cd123 + bdca2 -dendritic cells that produce il-6 and il-10 and have no enhanced type i interferon production y. m. huang, 1 s. adikari, 1 u. båve, 2 a. sanna 1,3 & g. alm 4 dc antigens (bdca) and investigate their ability to produce type i ifn in response to virus stimulation. we show that ifn-b induces development of cd123 þ dc from human blood monocytes, which coexpress bdca4 þ but are negative for bdca2 -, a specific marker for plasmacytoid dc. such ifn-b-modulated dc produce large amounts of il-6 and il-10, but no il-12p40 and have no enhanced ifn-b and ifn-b production. the findings indicate that ifn-bmodulated dc represent a myeloid dc subset with diminished cd11c, bdca-1 and cd1a expression, having potent th2-promoting function but lacking antiviral capacity. the association of psoriasis with throat infections by streptococcus pyogenes suggests a potential antigenic target for the t cells that are known to infiltrate dermis and epidermis of psoriatic skin. streptococcal m protein shares an extensive sequence homology with human epidermal keratins. keratins 16 (k16) and 17 (k17) are mostly absent from uninvolved skin but are upregulated in psoriatic lesions. there is increasing evidence that cd8 þ t cells play an important effector role in psoriasis and m proteinprimed t cells may recognize these shared epitopes in skin via molecular mimicry. to identify candidate epitopes, peptides with sequences from k17 were selected on the basis of predicted binding to hla-cw6 and sequence similarities with m6 protein. matched peptides from the sequence of m6 protein and a set of peptides with poor predicted binding were also selected. cw6 þ individuals with psoriasis and cw6 þ healthy controls, having a family history of psoriasis, were recruited. pbmcs were incubated with the peptide antigens. t-cell activation in the cd4 þ , cd8 þ and later the skin-homing cutaneous lymphocyteassociated antigen (cla)-expressing subset of cd8 þ t cells was evaluated by cd69 expression and intracellular ifn-g accumulation using flow cytometry. we demonstrate that cw6 þ psoriasis patients had significant cd8 þ t-cell ifn-g responses to peptides from k17 and m6 protein selected on the basis of sequence homology and predicted hla-cw*0602 binding. these responses were about 10 times more frequent in the skin-homing cutaneous lymphocyte-associated antigen-expressing (cla þ ) subset of cd8 þ t cells. cd4 þ t cells showed only borderline responses. cd8 þ t cells from cw6 þ nonpsoriatic individuals responded to some m6 peptides but very rarely to k17 peptides, and this also applied to the cla þ cd8 þ subset. these findings indicate that psoriatic individuals have cd8 þ t cells that recognize keratin self-antigens and that epitopes shared by streptococcal m protein and human keratin may be targets for the cd8 þ t cells that infiltrate psoriatic skin lesions. autoantibodies directed against citrulline-containing proteins have an impressive specificity of nearly 100% in ra patients and a suggestive involvement in the pathogenesis. the targeted epitopes are generated by a post-translational modification catalysed by the calcium-dependent enzyme peptidyl arginine deaminase that converts the positively charged arginine to polar but uncharged citrullin. the aim of this study was to analyse the presence of citrulline in the joints at different time points of collagen-induced arthritis in da rats by immunohistochemistry and to investigate how immunogenicity and arthritogenicity was affected by citrullination of rat serum albumin (rsa) and collagen type ii (cii). our results indicate that citrulline could be detected in joints of arthritic animals, first appearance at the onset of disease and increasing as disease progressed into a chronic state. unimmunized animals or time points before clinical signs of arthritis were negative. by morphology, we state that some infiltrating macrophages as well as the cartilage surface stain positive for citrulline, while the major source of citrullinated proteins appears to be fibrin depositions. a specific cit-rsa t-cell response was observed in animals challenged by citrullinated rsa, no response was recorded when rsa was used as a stimulus. the igg analysis reveals not only a response towards the modified protein but also cross-reactivity to native rsa. no t-cell or b-cell response was noted in animals injected with unmodified rsa. cit-cii induced a disease with higher incidence and earlier onset than did the native counterpart. we conclude that, in contrast to the human disease, citrulline does not seem to appear before clinical signs. as inflammation proceeds, citrulline is detected specifically in the joints. all other organs investigated were negative. we also conclude that citrullination of a protein can break tolerance and increase its arthritogenic properties. ectopic germinal centers (gcs) can be detected in the salivary glands of approximately 1/5 of patients with sjögren's syndrome (ss) and appear in both primary and secondary ss. previously, ectopic gc have been associated with increased local autoantibody production. the aim of this study was to determine whether gc in primary sjögren's syndrome (pss) defines a distinct seroimmunological phenotype. retrospectively, a material of 130 haematoxylin and eosin-stained paraffin-embedded tissue sections of minor salivary gland tissue from patients with pss was morphologically screened for the presence of ectopic gc. gc-like lesions were detected in 33/130 (25%) of the pss patients. seventy-two pss patients lacking these structures (gc-) were randomly selected for comparison. focus score was significantly increased in the gc þ patients compared to the gcpatients (p ¼ 0.035). in the gc þ group, 54.5% of the patients presented with anti-ro/ssa compared to 43.7% in the gcgroup. anti-la/ssb was detected in 31.3% of the gc þ patients compared to 25.7% of the gcpatients. sixty-one percentage of gc þ patients presented with increased levels of igg, a nonsignificant difference when compared to 39.4% in the gcpatients (p ¼ 0.089). levels of rf, ana, ena, igm and iga were similar in both patient groups, as were esr and crp. in conclusion, patients with ectopic gc have a higher focus score and more often present with autoantibodies and increased levels of igg compared to pss patients with regular focal infiltration (gc -). our findings may indicate a certain seroimmunological phenotype and warrant for further prospective studies. association between mannose-binding lectin and vascular complications in type 1 diabetes complement activation and inflammation have been suggested in the pathogenesis of diabetic vascular lesions. we investigated serum mannose-binding lectin (mbl) levels and polymorphisms in the mbl gene in type 1 diabetic (t1dm) patients with and without diabetic nephropathy and associated macrovascular complications. polymorphisms in the mbl gene and serum mbl levels were determined in 199 t1dm patients with overt nephropathy and 192 t1dm patients with persistent normoalbuminuria matched for age, sex and duration of diabetes as well as in 100 healthy control subjects. the frequencies of high and low expression mbl genotypes were similar in patients with t1dm and healthy controls. high mbl genotypes were significantly more frequent in diabetic patients with nephropathy than in the normoalbuminuric group, and the risk of having nephropathy, given a high mbl genotype, assessed by odds ratio was 1.52 (1.02-2.27), p ¼ 0.04. median serum mbl concentrations were significantly higher in patients with nephropathy than in patients with normoalbuminuria [2306 mg/l (iqr 753-4867 mg/l) versus 1491 mg/l (iqr 577-2944), p ¼ 0.0003], and even when comparing patients with identical genotypes, serum mbl levels were higher in the nephropathy group than in the normoalbuminuric group. patients with a history of cardiovascular disease had significantly elevated mbl levels independently of nephropathy status [3178 mg/l (iqr 636-5231 mg/l) versus 1741 mg/l (iqr 656-3149 mg/l), p ¼ 0.02]. the differences in mbl levels between patients with and without vascular complications were driven primarily by pronounced differences among carriers of high mbl genotypes (p < 0.0001). our findings suggest that mbl may be involved in the pathogenesis of microvascular and macrovascular complications in type 1 diabetes and that determination of mbl status might be used to identify patients at increased risk of developing these complications. neuroimmunology unit, center for molecular medicine, karolinska institutet, stockholm, sweden. e-mail: judit.wefer@cmm.ki.se dna vaccine coding for the encephalitogenic peptide mog 91-108 protects lew.1av1 from subsequent development of experimental autoimmune encephalomyelitis (eae). protection is associated with a type 1 immune response and is dependent on the presence of cpg dna motifs. the mechanisms underlying the observed reduction of eae development in protected rats have not been fully clarified. we investigated immunological characteristics of lymphocytes after dna vaccinaton and subsequent eae induction. we confirm that protection was not associated with suppression of t1 cells, as transcription of the novel molecule rat t-cell immunoglobulin-and mucindomain-containing molecule (tim-3), reported to be exclusively expressed on differentiated t1 cells, was not altered by dna vaccination. we did not note any clonal deletion upon tolerization, but detected an antigen-specific lymphocyte population upregulating ifng upon recall stimulation 3 weeks after protective dna vaccination. in protected rats, we observed (1) no alterations in antigenspecific th2 or th3 responses, (2) reduced mhc ii expression on splenocytes early after eae induction, (3) antigen-specific upregulation of ifnb upon recall stimulation and (4) reduced il-12rb2 on lymphocytes. we thus demonstrate an association of the protective effect of dna vaccination with expression of ifnb. we are currently investigating the cellular mechanisms behind this ifnb-mediated protection. multiple sclerosis (ms) is an autoimmune condition characterized by degeneration of nerve fibre myelin sheets. a candidate autoantigen, myelin basic protein (mbp), has especially attracted attention. the presence of anti-mbp antibodies is a predictor of definite ms, but their role in the pathogenesis remains obscure. t cells have long been known to play a pivotal role in the pathogenesis of ms. recently, an important role for b cells as autoantigen-presenting cells has been demonstrated in other autoimmune diseases, including rheumatoid arthritis and diabetes. the uptake of mbp by b cells and the presentation of mbp-derive peptides to t helper (th) cells by b cells may be promoted by the formation of complement (c) activating immune complexes (ics) between mbp and natural autoantibodies in healthy individuals and disease-associated anti-mbp antibodies in ms patients, respectively. we have investigated the formation of mbp-containing ic, the binding of mbp to b cells, the mbp-elicited induction of th-cell and b-cell proliferation and the cytokine production in peripheral blood mononuclear cells (pbmcs) from healthy donors grown in the presence of intact or c-inactivated serum from healthy donors or patients with ms. while mbp did not induce measurable proliferation of b cells nor cd4 þ t cells, we observed the production of tnf-a, ifn-g and il-10 by pbmc in response to incubation with mbp in the presence of sera from healthy controls as well as sera from ms patients. by contrast, no production of il-2, il-4 and il-5 was detected. we are currently investigating the capability of ms sera to promote the formation of mbp-containing ic and thereby enhance the cytokine responses, by virtue of elevated anti-mbp contents. the phagolysosomally localized acid sphingomyelinase (asmase) activated by proinflammatory cytokines such as tnf and ifn-g generates the signalling molecule ceramide which in turn results in the activation of proteases like cathepsin d. these characteristics of asmase suggest a possible role of this molecule in the phagocytotic uptake and phagosomal degradation processes of antigens or in antigen presentation. we show here that asmase -/mice fail to eliminate the noncytopathic lymphocytic choriomeningitis (lcm) virus as rapidly as littermate wildtype mice. investigation of the immune response revealed a reduced expansion of cd8 þ t cells. the secretion of ifn-g in response to contact with target cells as well as the cytolytic activity of virus-specific cd8 þ t cells was severely impaired. additionally, both phases of the lcm virus-specific dth response, mediated by cd8 þ and cd4 þ t cells consecutively, were diminished in asmase -/mice. however, the secondary memory response of virus-specific ctl was not altered, and the 614 abstracts ................................................................................................................................................................................................. virus was effectively controlled for at least 3 months by asmase -/mice. in conclusion, the results of this study suggest an involvement of the asmase in the activation, expansion or maturation of virus-specific cd8 þ t cells during the acute infection of mice with the lcm virus. novel markers for alternative activation of macrophages: macrophage galactose-type c-type lectins 1 and 2 in parallel with the th1/th2 dichotomy, macrophages are capable of developing into functionally and molecularly distinct subpopulations, due to differences in, for example cytokine environment and pathological conditions. while the best-studied, classically activated macrophage is induced by type i stimuli such as ifn-g, a type ii cytokine environment antagonizes the classical activation of macrophages and is capable of alternatively activating macrophages. however, molecular markers associated with these type ii cytokine-dependent, alternatively activated macrophages remain scarce. besides the earlier documented markers macrophage mannose receptor and arginase 1, we recently demonstrated that murine alternatively activated macrophages are characterized by increased expression of fizz1 and ym. we now report that expression of the two members of the mouse macrophage galactose-type c-type lectin gene family, termed mmgl1 and mmgl2, is induced in diverse populations of alternatively activated macrophages, including peritoneal macrophages elicited during infection with the protozoan trypanosoma brucei or the helminth taenia crassiceps, and alveolar macrophages elicited in a mouse model of allergic asthma. we also demonstrate that, in vitro, interleukin-4 and interleukin-13 upregulate mmgl1 and mmgl2 expression and that, in vivo, induction of mmgl1 and mmgl2 is dependent on interleukin-4 receptor signalling. moreover, we show that regulation of mgl expression is similar in human monocytes and monocyte-derived macrophages. hence, macrophage galactose-type c-type lectins represent novel markers for both murine and human alternatively activated macrophages; thus, paving the way for further characterization of the phenotype of macrophages occurring in th2 conditions. background: human parvovirus b19 (b19) is a ubiquitous pathogen, normally causing a mild self-limiting disease, but also capable of causing both significant pathology and long-term persistence. the small size and stability of the virus makes it suitable for mapping of the full breath and the kinetics of the cellular immune responses following acute viral infection. methods: five patients with acute primary b19 infection were included in the study and followed consecutively for up to 200 weeks. cellular immune responses were mapped by ifng enzyme-linked immunospot to overlapping peptides spanning the whole b19 genome. results: in all five acutely infected patients, we were able to monitor the kinetics of a strong specific cellular immune reaction. responses peaked at levels of 850-1850 sfc/ million pbmcs, roughly corresponding to 0.3-0.6% b19specific cd8 þ cells circulating in peripheral blood at 10-80 weeks post-infection. the responses in individual patients were directed to three or four different peptide pools, and the specificity was confined to the same cd8 epitopes present in the pools throughout the follow-up period. the majority of responses were directed to the virus nonstructural protein, only two patients showed any response to the capsid proteins, elicited by the same epitope in both cases. conclusion: the cellular immune responses to acute b19 infection are surprisingly narrow in distribution and remain at high levels for up to 80 weeks post-infection. the initial epitope specificity is maintained, and the majority of responses target the virus nonstructural protein, which is not included in vaccine preparations, evaluated against the infection. the relationship between malnutrition and malaria is controversial. on one hand, malaria may cause malnutrition, while on the other, malnutrition itself may modulate susceptibility to the disease. we investigated the association between plasmodium falciparum malaria and malnutrition in a cohort of children living on the coast of kenya. the study involved longitudinal follow-up for clinical malaria episodes and anthropometric measurements at four cross-sectional surveys. we used poisson regression analysis to investigate the association between malaria and nutritional status. compared to baseline (children with a waz or haz score of !à2), the crude incidence rate ratios (irrs) for malaria in children with low haz or waz scores (<à2) during the period prior to assessment were 1.17 (95% ci 0.91-1.50; 0 ¼ 0.21) and 0.94 (0.71-1.25; 0.67), respectively, suggesting no association between malaria and the subsequent development of pem. however, we found that age was acting as an effect modifier in the association between malaria and malnutrition. the irr for malaria in children 0-2 years old who were subsequently characterized as wasted was 1.65 (1.10-2.20; p ¼ 0.01), and a significant overall relationship between malaria and low-haz was found on regression analysis when adjusting for the interaction with age (irr 1.89; 1.01-3.53; p < 0.05). although children living on the coast of kenya continue to suffer clinical episodes of uncomplicated malaria throughout their first decade, the association between malaria and malnutrition appears to be limited to the first 2 years of life. a. astrinidou-vakaloudi, 1 s. xytsas, 1 i. diamanti, 1 h. ioannidis 2 & p. pangidis 2 1 microbiology department of general hospital of thessaloniki 'agios pavlos', thessaloniki, greece, and 2 nefrology, 2 nd ika hospital of thessaloniki, thessaloniki, greece. e-mail: stasa@hol.gr aim: renal dysfunction may influence the colonization of gastric mucosa by urea-splitting bacteria such as helicobacter pylori, by increasing urea concentrations in the gastric juice. our aim was to investigate the prevalence of h. pylori in patients with end-stage renal disease (esrd), receiving long-term haemodialysis treatment. methods: this study included 40 sera from patients with esrd (29 male and 11 female) undergoing periodic haemodialysis; mean time of treatment was 42.6 months. using elisa technique, we investigated the presence of igg and iga antibodies against h. pylori as well as igg caga (antibodies specific for caga(þ) strains of h. pylori). sera from 40 healthy blood donors were used as a control group. results: h. pylori igg antibodies were detected in 32 out of 40 (80%) patients in the dialysis group, while 31/40 (77.5%) tested positive for iga. igg caga antibodies were present in 13 out of 40 (32.5%). prevalence of h. pylori igg, iga and caga igg antibodies in the control group was 33, 7 and 15%, respectively. conclusions: although international data suggest that prevalence of h. pylori infection is the same in esrd patients as in healthy individuals, in our study that seems not to be the case. the higher blood and gastric juice urea levels may be a risk factor (among many others), but more studies are required in order to understand the relation of h. pylori infection in this group of patients. flanders interuniversity institute for biotechnology, department of molecular and cellular interactions, free university of brussels, brussels, and 2 pasteur institute of brussels, mycobacterial immunology, brussels, belgium. e-mail: tgartner@vub.ac.be immunity against tuberculosis (tb), caused by mycobacterium tuberculosis, depends largely on activation and maintenance of strong cell-mediated immune responses involving both cd4 þ and cd8 þ t cells and the ability to respond with th1-type cytokines, particularly ifn-g. recent studies suggested that bcg, the only licensed vaccine against m. tuberculosis, may fail to induce t-cell responses in the lung mucosa and may therefore not protect against pulmonary tb. a decrease in tb mortality may be achieved by enhancing immunity in the lung. the present study evaluated the induction of antigen-specific immunity in the lung by intranasal (i.n.) delivery of the lipoprotein i (opri) from pseudomonas aeruginosa. opri has shown to be a toll-like receptor 2/4 agonist that, when given subcutaneously, induces type-1 immune responses against heterologous antigens. here, a fusion of opri to ag85a of mtb (opri-ag85a) was used as a subunit vaccine in homologous prime-boost immunizations. in addition, opri-ag85a was combined with an ag85a-encoding dna vaccine (ag85a dna) or with bcg in heterologous prime-boost vaccinations. intranasal and parenteral delivery with opri-ag85a elicited comparable t-cell responses in the spleen; in addition, i.n. delivery elicited specific t-cell responses in the lung lymph nodes (llns). intramuscular delivery of ag85a dna induced significant systemic th1 immune responses. intranasal boosting with opri-ag85a enhanced this response and in addition induced an antigen-specific ifn-g response in lln. opri may therefore be an efficient adjuvant for mucosal boosting. we continue to evaluate the protection induced by opri-based prime-boost vaccinations against pulmonary tb. results on the immunogenicity and protection against intravenous mtb h37rv infection will be presented. toll-like receptors (tlrs) are pattern recognition receptors of the innate immune system, which recognize molecular structures on pathogens or cellular stress-associated molecules. tlr-ligand interactions trigger activation of inflammatory signal transduction and expression of genes involved in host defense. in this study, we have examined the requirement for different tlr adaptor molecules in virus-induced chemokine expression and are currently trying to identify the tlr involved. we have found that both a herpesvirus [herpes simplex virus (hsv)] and a paramyxovirus (sendai virus) require a functional genome to induce expression or proinflammatory chemokines in human and murine monocytic cell lines. for both viruses, this is independent of the tlr adaptor molecules trif and mal. however, overexpression of the vaccinia virus-encoded inhibitor of tlr-signalling a52r or dominant-negative myd88 totally inhibited hsv-induced rantes expression but only partially prevented sendai virus from inducing this chemokine. this suggests that hsv-induced rantes expression occurs via a tlr pathways, whereas sendai virus utilizes both tlr-dependent and -independent pathways to stimulate expression of rantes. we are currently trying to identify the tlrs involved. data from these studies will also be presented at the meeting. 2 0 -5 0 -oligoadenylate synthetases are interferon-induced, double-stranded rna-activated antiviral enzymes which are the only proteins known to catalyse 2 0 -specific nucleotidyl transfer. this first crystal structure of a 2 0 -5 0oligoadenylate synthetase reveals a structural conservation with the 3 0 -specific poly(a) polymerase that, coupled with structure-guided mutagenesis, supports a conserved catalytic mechanism for the 2 0 -and 3 0 -specific nucleotidyl transferases. comparison with structures of other superfamily members indicates that the donor substrates are bound by conserved active site features while the acceptor substrates are oriented by nonconserved regions. the 2 0 -5 0oligoadenylate synthetases are activated by viral doublestranded rna in infected cells and initiate a cellular response by synthesizing 2 0 -5 0 -oligoadenylates, that in turn activate rnase l. this crystal structure suggests that activation involves a domain-domain shift and identifies a putative dsrna activation site that is probed by mutagenesis. we demonstrated that this site is required both for the binding of dsrna and for the subsequent activation of oas. this rna-binding site is different from known rna-binding site; rather than forming a defined three-dimensional domain, it is located at the interface of the two major domains in oas. this novel architecture ensures that the dsrna helix can make simultaneously contact with both domains of oas and ensure the subsequent structural rearrangement leading to the activation of oas. our work provides structural insight into cellular recognition of double-stranded rna of viral origin and identifies a novel rna-binding motif. bacteria-specific iga antibodies are efficient opsonins for neutrophils and mononuclear phagocytes, provided that the phagocytes express the fca receptor (cd89). expression of cd89 can be stimulated by inflammatory cytokines, activated complement factors and certain microbial components. in one study, unstimulated phagocytes were able to ingest iga antibody-treated pneumococci, but only in the presence of complement, which was found to be activated by the iga antibodies along the alternative pathway. pneumococci produce iga1 protease that cleaves human iga1, but not iga2, molecules in the hinge region. this leaves iga1 as faba (monovalent) deprived of fca which contains the docking site for cd89. iga1 is the vastly predominant subclass of iga in the upper airways and circulation of humans. aims: to examine the effects of iga1 protease activity and complement on phagocytosis of iga antibody-coated pneumococci by an unstimulated human phagocytic cell line (hl60). materials and methods: iga1 and iga2 monoclonal antibodies to serotype 4 pneumococcal capsular polysaccharide (ps) were generated by heterohybridoma technique involving b cells from human vaccinees. isogenic serotype 4 pneumococci with and without iga1 protease activity, respectively, were obtained after inactivation of the iga gene of the tigr4 strain. opsonophagocytosis was quantitated using the assay described by romero-steiner et al. based on enumeration of surviving bacteria by culture. the integrity of iga molecules was examined by western blotting. results: both iga1 and iga2 antibody to type-4 polysaccharide-induced phagocytosis of iga1 protease-deficient type-4 pneumococci equally well in the absence as in the presence of complement. iga1 antibody to type-4 polysaccharide displayed a fourfold higher opsonophagocytosis titer against iga1 protease deficient compared to homologous wildtype target bacteria. a similar effect of iga1 protease activity of the target bacteria was not observed in a parallel experiment where iga2 antibody to type-4 polysaccharide served as opsonin. iga1 antibody extracted from iga1 protease-producing target bacteria was almost exclusively in the form of faba. conversely, iga1 from protease-deficient bacteria and iga2 from both types of bacteria were intact. conclusions: these results indicate that the iga1 protease activity of s. neumoniae may help the bacteria escape iga1 antibody-mediated opsonophagocytosis. besides, in these experiments, iga-mediated opsonophagocytosis was independent of complement. vitamins e and c have been found to increase the cellular and humeral immunity of pigs. vitamin e deficiency has also been found to predispose pigs to different diseases, e. coli infection is one among them. after weaning, the vitamin e status of pigs often decreases to a critical low level. in this experiment, we studied whether vitamin c supplementation would be a possible feeding strategy to optimize the immune status of weaners. the interaction between vitamin e and c is interesting due to the reported sparing action on vitamin e or synergism between these to vitamins. piglets were weaned at day 28 of age from sows fed increasing dietary vitamin e during lactation, and piglets were during the following 3 weeks fed either a control diet or this diet supplemented with 500 mg stay-c per kg. blood sampling was obtained weekly from day 28 and until day 49 of age. on the same days, one piglet per dietary treatment was killed and alveolar macrophages (am) were harvested. vitamin c supplementation increased the concentration of igm in serum of piglets throughout the weaning period. although the vitamin e concentration in am decreased with increasing age of the piglets, the concentration was numerically higher in piglets of sows fed the high dietary level of vitamin e. however, vitamin c supplementation tended to increase the total am concentration of vitamin e after weaning and increased the proportion of the biologically most active isomer of vitamin e [rrr-(a-tocopherol)] in the am. the eicosanoid synthesis by am was not influenced by the vitamin c supplementation, but the synthesis of leukotriene b4 was decreased 2 weeks after weaning compared to other days of am harvesting. in conclusion, dietary vitamin c supplementation improved the immune responses of piglets after weaning. a whole blood stimulation assay with escherichia coli (o111:b4) endotoxin was established to measure the capacity of dairy cows to produce the proinflammatory cytokine tumour necrosis factor-a (tnf-a) ex vivo. initially, a time-and dose-dependent study was carried out to find the optimal stimulation conditions for the tnf-a response. the tnf-a response peaked between 3 and 4 h at 38.5 c. a dose in the range of 5-10 g of e. coli lipopolysaccharide (lps)/ml whole blood was found to give the maximum tnf-a response. thirty-eight danish-holstein dairy cows were investigated for their tnf-a responsiveness ex vivo in the periparturient period. heparin-stabilized blood samples were collected seven times over a period of 4 months (weeks à3, à1, 2, 3, 5, 9 and 13 around calving) and stimulated with 5 g/ml of e. coli lps. indeed, fluctuations in the tnf-a responsiveness occurred over time. moreover, the mean tnf-a responsiveness of 38 cows was found to be significantly increased (p < 0.001) in the weeks close to calving. however, in the more stabile physiological periods, some cows had a consistently low tnf-a response, whereas others had high a tnf-a response. we are currently investigating whether high and low tnf-a responders to e. coli lps also exist in dairy cows in vivo. moreover, the importance of tnf-a responsiveness ex vivo to dairy cows' susceptibility and clinical response to experimental e. coli infections in the udder is being investigated. coelomic cytolytic factor (ccf) is a 42 kda invertebrate pattern recognition molecule isolated from the coelomic fluid of the earthworm eisenia foetida (oligochaeta, annelida). ccf displays a number of similarities with the mammalian cytokine tumour necrosis factor-a (tnfa) as a result of a shared n,n 0 -diacetylchitobiose lectin-like domain. however, these similarities are solely functional and are not based on any (dna or amino acid) sequence homology, thus suggesting a form of convergent evolution. in particular, the lectin-like domain of tnf-a has been shown to induce membrane depolarization in various mammalian cell types, through interactions with endogenous amiloride-sensitive ion channels. this nonreceptor-mediated activity of tnf-a has been reported to be involved in the resorption of oedema. likewise, the lectin-like domain of ccf also induces membrane depolarization in mammalian cells. here, we show that ccf appears to be able to induce oedema resorption in an alveolar epithelial cell line through its lectin-like domain. this lectin-like domain of ccf interacts (directly or indirectly) with endogenous sodium and/or chloride channels, and not potassium channels, on mammalian cells. additionally, we suggest that the jnk/sapk and erk1/2 pathways are involved in ccf-induced macrophage activation. these results further establish the functional analogy between an invertebrate pattern recognition molecule and a mammalian cytokine and, from a more applied point of view, suggest the possibility of utilizing ccf in the treatment of oedema. release of svegf and svegfr1 from white blood cells and platelets during surgery and stimulation with bacterial antigens introduction: the influence of surgery on release of soluble vascular endothelial growth factor (svegf) and the soluble vascular endothelial growth factor inhibitory receptor 1 (svegfr1) is unknown. we studied the effect of major and minor surgery on potential variations in svegf and svegfr1 concentrations in vivo and on bacterial antigen-induced release of svegf and svegfr1 from whole blood in vitro. methods: sixty-one patients with abdominal diseases undergoing five different surgical procedures were included. blood samples were drawn from anaesthetized patients before and after the operation. white blood cells and platelets were counted, and plasma svegf and svegfr1 was determined by an elisa method. whole blood from each blood sample was stimulated in vitro with bacteria-derived antigens (lps or protein-a) and svegf and svegfr1 levels were subsequently determined in the supernatants. stimulation with isotonic saline served as control assay. neither svegf or svegfr1 in plasma changed during surgery. in vitro stimulation of blood samples with bacteria-derived antigens resulted in a significant increase in svegf (p < 0.0001) and a less pronounced but still significant increase in svegfr1. release of svegf due to stimulation was significantly higher after the operation (nonsignificant), whereas svegfr1 release remained largely unchanged after surgery. correlation between bacterial antigen-induced release of svegf and neutrophile cell count was highly significant (p < 0.0001). there was no correlation between svegf and platelet cell count, and bacterial antigen-induced svegfr1 release did not correlate with counts of neutrophils and platelets. conclusions: plasma svegf and svegfr1 concentrations did not change during surgery. in vitro bacterial stimulation led to increased release of svegf and svegfr1, which was not significantly amplified during surgery and which may be related to number of circulating neutrophils. natural killer cell functions and subsets after in vitro stimulation with il-2 and il-12, with special emphasis on intracellular ifn-g and nk-cell cytotoxicity r. nyboe, 1,2 t. rix, 1,2 j. krog, 1,2 e. tønnesen 1 & m. hokland 2 1 department of anaesthesiology and intensive care, aarhus university hospital, and 2 institute of medical microbiology, and immunology, university of aarhus, aarhus, denmark. e-mail: rnsr@studmed.au.dk materials and methods: isolated cryopreserved human peripheral blood mononuclear cells (pbmcs) were stimulated with il-2 and il-12. this stimulation has previously been shown to activate nk cells. cell cytotoxicity was measured by flow cytometry after incubation with k562 cells. this method was compared to the current standard 51cr release assay. cells were treated with bfa to accumulate ifn-g, stained for surface markers, permeabilized and stained for intracellular ifn-g. flow cytometry was then performed to measure intracellular ifn-g production in pbmc, especially in nk cells. results: we have demonstrated that stimulation with il-2 and il-12 is effective in increasing the number of ifn-gpositive cells. there is a distinct difference between the cd3-cd56dim and the cd3-cd56bright subsets, with a much greater proportion of ifn-g-positive cells in the cd3-cd56bright subset. the effects of stimulation with il-2 and il-12 on cytotoxicity will be presented, as will the relation between ifn-g production and cytotoxicity. in addition, we will present results of these assays applied to porcine cells. discussion: in combination, these tests will address nk cell function by combining cytotoxicity with ifn-g production in nk cell subsets. the results will demonstrate whether this could serve as a useful tool in describing nkcell function, which could be of value in clinical and experimental settings. culture of regulatory t-cell lines from bronchial mucosa t lymphocytes play a major role in many immune responses. in the last decade, special focus has been on the function of th1 and th2 effector cells. now the importance of regulatory cd4 þ cd25 þ t cells in maintenance of the immunological homeostasis emerges. sarcoidosis is a multisystem granulomatous disorder often affecting the lungs. the typical sarcoid granulomas consists of epitheloid cells, macrophages and lymphocytes, mainly cd4 þ t cells of th1 phenotype. we have cultured t cells from bronchial biopsies of patients with sarcoidosis as well as from controls in high levels of interleukin 2 (il-2) and il-4 and demonstrate spontaneously arising cd4 þ cd25 þ populations and high concentrations of il-10 in these cultures. the main difference between cultures of sarcoid origin compared to controls is a very much higher concentration of the inflammatory cytokines il-6 and tnf-a in cultures of sarcoid origin. the effects of hyperbaric exposure on human peripheral blood mononuclear cells, with special emphasis on natural killer cell cytotoxicity and subsets materials and methods: as an experimental physiological stress model, we examined the effects of hyperbaric exposure on peripheral blood mononuclear cells (pbmcs) obtained from venous blood drawn from eight divers during a simulated heliox saturation dive. eight persons working in normobar atmosphere outside the pressurized chamber served as control donors. the spontaneous cytotoxicity of the pbmcs was estimated in a 4 h 51cr-release assay using k562 as nk-sensitive target cells. the pbmcs were characterized, using 4-colour flow cytometry, with special emphasis on the nk-cell subsets. the data were statistically analysed using a multivariate regression model (stata 8.2). p values <0.05 was considered statistically significant. results: the estimated cytotoxicity increased significantly in both the group of divers and control donors during the dive (pdivers < 0.01 and pcontrols < 0.01). although the cytotoxicity increased relatively more (p < 0.01) in the group of divers compared to the group of control donors between day 1 and 2. discussion: the increased cytotoxicity of pbmc estimated in the group of divers indicate that parts of the cellular immune system are affected during the extreme physiological conditions induced during the initial phase of the presented experimental hyperbaric setup. the increase in cytotoxicity observed in the group of control donors could hypothetically reflect the stress level in persons working outside the pressurized chamber during the dive. the interaction with adhesion molecules expressed by vascular endothelium is the first step in lymphocyte infiltration into tissues. at both cutaneous and mucosal sites interleukin-10 (il-10), il-12 and transforming growth factor (tgf)-b are important regulators of chronic inflammatory disease, where cutaneous lymphocyteassociated antigen (cla) and ae integrin (cd103) may be expressed. unlike cla, cd103 is not believed to play a role in tissue-specific homing but may help to retain t cells within epithelial layers. we have previously shown that il-12 alone can together with an unknown cofactor increase the expression of cla. stimulation with streptococcal pyrogenic exotoxin c (spec) increased the expression of cd103 by cd8 þ but not cd4 þ t cells. while il-12 increased superantigen-stimulated expression of cla, this cytokine strongly inhibited the cd103 expres-sion, and a combination of il-12 and tgf-b completely abrogated the induced cd103 expression. conversely, il-10 suppressed cla but increased cd103 expression. these findings indicate that, in addition to suppressing the development of th1-mediated inflammatory responses, il-10 may also inhibit the migration of cd8 þ t cells into the skin while il-12 promotes such migration. thus, the expression of cla and cd103 may be antagonistically regulated by il-10 and il-12, and the balance between these cytokines could influence the t-cell migration of inflammatory cells into epithelial tissues. during contact sensitivity reaction, immune cells proliferate. in order to study the histological picture of these proliferation phases, we used a mouse model of contact sensitivity in the oral mucosa and on skin. we also used bromodeoxyuridin (brdu, an analogue to thymidin) that is incorporated into the nucleus during cell replication. the hapten oxazolone (oxa) was used to sensitize and elicit the oral mucosa and/or the ear skin. mice were killed at various times after elicitation, and unsensitized animals were also exposed to the hapten as controls. brdu (25 mg/ kg animal) was injected i.p. 2 h before the kill. specimens from the oral mucosa, ear skin and submandibular and auricular lymph nodes were cut and fixed in 4% paraformaldehyde. they were then treated with acid and biotinylated anti-brdu antibody and developed using abc-kit and dab. the analyses were performed using a leica light microscope and the computer program analysis. in the oral mucosa, the frequency of proliferating cells were increasing during the observation period, 4-24 h after elicitation, regardless of site of sensitization. the proliferating cells were found mainly in the basal cell layer of the epithelium. similar patterns were found in ear skin. the regional lymph nodes demonstrated a few scattered proliferating cells 4 h after elicitation. after 24 h, these cells were found frequently in the whole lymph node. control animals exhibited considerable less proliferating cells at all times. we conclude that most proliferating cells were found 24 h after elicitation locally at the hapten-exposed sites (the oral mucosa or the ear skin) as well as in the regional lymph nodes. the endogenous nucleoside adenosine is released in excess during inflammation or other metabolic stress and is generally known to deliver tissue protective anti-inflammatory effects. adenosine acts via four adenosine receptors of which the a2a receptor is the predominant form in t cells. adenosine levels are elevated in asthmatic lung, and adenosine can directly induce mast cell degranulation and bronchoconstriction in these patients. instead, the role of anti-inflammatory mechanisms of adenosine on t cells in asthma is unclear. aim: to study the a2a receptor expression in peripheral blood cd4 þ t cells in asthmatic and healthy individuals using flow cytometric and quantitative real-time pcr methods. results: unstimulated cd4 þ cells of asthmatic patients expressed significantly lower levels (p < 0.001) of a2a receptor in protein level (mean percentage of cells positive ae sem: 76.8 ae 1.2, n ¼ 6) compared to healthy individuals (90.4% ae 1.9, n ¼ 4). double staining for cd69 expression showed that stimulation of cd4 þ cells decreased a2a expression in both groups but indicated that the detected lower levels of a2a in unstimulated cells of asthmatics was not due to preactivation in these patients. surprisingly, a2a mrna expression in unstimulated cd4 þ cells was significantly higher (p < 0.05) in asthmatics (n ¼ 28) compared to healthy controls (n ¼ 7). the expression did not correlate with serum total ige levels. conclusions: asthmatic individuals express less a2a adenosine receptor on their peripheral cd4 þ t cells. the higher mrna levels instead may point to a negative feedback regulation in the receptor expression. the role of possibly decreased adenosine-mediated anti-inflammatory effects in asthma pathogenesis require further studies on this t-cell mediated disease. the chronic inflammatory skin disease atopic eczema (ae) affects almost 15% of the population in many countries today. the pathogenesis of ae is not fully understood. a combination of genetic predisposition and environmental factors like microorganisms seems to contribute to the symptoms. the yeast malassezia sympodialis is part of our normal skin micro flora but can act as an allergen and elicit specific ige and t-cell reactivity in patients with ae. recently, we identified a novel major m. sympodialis allergen, designated mala s 11 (22.4 kda), with sequence similarity to the mitochondrial enzyme manganese superoxide dismutase (mnsod). interestingly, mala s 11 has a high degree of homology to human mnsod. the aim of this study was to examine the effects of recombinant mala s 11 on antigen-presenting dendritic cells. monocytederived dendritic cells (mddcs) from healthy blood donors were cultured with or without mala s 11 for different time periods. it was found that the maturation marker cd83 and the costimulatory molecules cd80 and cd86 were upregulated on the mddcs exposed to mala s 11 for 24 h, as demonstrated by flow cytometry. furthermore, coculture of mddcs with mala s 11 for 9 h induced an increased production of the inflammatory cytokines il-6 (200-fold), tnf-a (100-fold) and il-8 (sixfold), as detected by the cytometric bead array (cba) analysis. our results suggest that mala s 11 affects the immune response through dc maturation and production of inflammatory cytokines. the potential cross-reactivity with human mnsod needs to be explored and the exact role of mala s 11 in the pathogenesis of ae assessed in clinical studies involving skin prick and atopy patch tests. allergen-specific immunotherapy (sit) is commonly conducted with allergen extracts adsorbed to aluminium hydroxide (alum). drawbacks linked to the use of alum, such as the formation of granuloma at the site of injection, have led to suggestions of novel allergen carriers. an alternative carrier is 2 mm carbohydrate-based particles (cbps). in mouse, allergen-coupled cbps have been demonstrated to skew the allergen-specific immune response towards a th1-like activity (grönlund et al. immunology, 2002) . we here coupled the recombinant major cat allergen fel d 1 to cbps (cbp-fel d 1) by cyanogen-bromide activation, resulting in covalent binding. the effect of cbp-fel d 1 on monocyte-derived dendritic cells (mddcs) from healthy human blood donors was studied. we found that the majority of the cd1a þ mddcs were capable of taking up fitc-labelled cbp-fel d 1, as demonstrated by flow cytometry and confocal laser scanning microscopy. furthermore, incubation with cbp-fel d 1 resulted in an upregulation of the costimulatory molecule cd86 on the mddcs, which was not observed with fel d 1 or cbps alone. finally, cbp-fel d 1 induced a fivefold increase in the release of the pro-inflammatory cytokine tumour necrosis factor (tnf)-a and a fourfold increase in the release of the chemokine interleukin-8 from mddcs. taken together, the effects cbps possess make them interesting as novel allergen carriers for sit. the cysteine protease der p1 from dust mite of the genus dermatophagoides pteronyssinus is a major type i allergen. about 80% of house dust mite (hdm) allergic individuals are reactive to this protease in standard assays for detection of ige. a curative treatment for atopic allergy is immunotherapy (it) with hdm extracts which are complex mixtures occasionally resulting in anaphylactic reactions. novozymes focuses on developing a recombinant variant of der p1 which exhibit lowered risk of ige-mediated allergic reactions, while maintaining its ability to trigger proper th-cell responses. this may provide a safer alternative for specific it of hdm allergy. a secreted recombinant form of pro-der p 1 expressed by saccharamyces cerevisiae was obtained by fusion of the pro-enzyme to a fungal signal peptide. the n-glycosylation site of der p1 was mutated resulting in a deglycosylated pro-enzyme with a molecular mass of 35 kda. protein purification procedure was developed to obtain nearly pure der p1 protein followed by determination of concentration by active-site-titration with the cysteine protease inhibitor e64. the deglycosylated recombinant pro-der p 1 revealed immunologic similarity to the native der p 1 molecule when compared in basophile histamine release, ige-binding assays and t-cell proliferation assays. by in silico epitope mapping of a modelled 3-dimensional structure of der p1, five putative igg and ige epitopes were predicted. by protein engineering, the predicted epitopes were removed one by one in der p1 and screening for hypoallergenic variants was performed. combining inhaled long-acting b-2 agonist (laba) and inhaled corticosteroid (ics) seems to offer asthma control at a lower dose of ics than achieved by ics alone. fine mapping of t-cell surface markers by flow cytometry offers a detailed status of the individual's inflammatory response. the frequency of mt2 (cd4 þ cd45ra -cd62l þ cd11adim) and mt1 (cd4 þ cd45ra -cd62l -cd11abright) cells in peripheral blood, and their ratio, has been shown to differ predictably in atopics and patients with leprosy, where mt2 correlates with a th2 phenotype and mt1 with a th1 phenotype. stable asthmatics, requiring fluticasone propionate (fp) 750-1000 mg daily or equivalent, were randomized to receive, double-blinded, either seretide 1 [salmeterol and fluticasone propionate (sfc, n ¼ 16)] 50 mg/500 mg bd or fp 500 mg bd (n ¼ 17). if asthma was controlled based on lung function and symptoms at clinic visits every 6 weeks, ics dose was tapered until asthma exacerbated or 0 mg was reached. the frequency and ratio of mt2 and mt1 t cells of the patients was monitored at 6 week intervals. as treatment tapered, the frequency of mt2 cells decreased (p ¼ 0038 from first to final visit), whereas that of mt1 cells increased. the ratio of mt2/mt1 decreased (p ¼ 0049 from first to final visit). in patients receiving laba þ ics, the fall in mt2/mt1 ratio appeared to be more pronounced than in patients receiving ics alone. thus, the mt2 phenotype may be associated with stable asthma, whereas an imminent exacerbation may associate with an increase in the mt1 phenotype. laba may allow for a greater effect of fp on the mt ratio. activation of complement pathways, leading to production of c3a and c5a anaphylatoxins, has been postulated in the pathogenesis of asthma and allergic airway inflammation. the present study was undertaken to investigate the role of mannan-binding lectin (mbl), an initiator of the lectin pathway of complement, in asthma and allergic rhinitis. mbl levels and mbl-induced complement activity were determined in 19 patients of bronchial asthma with allergic rhinitis and 20 unrelated, age-matched controls of indian origin. mbl levels and activity were correlated with percent eosinophilia and percent predicted fev1 values of the patients. association of single nucleotide polymorphisms (snps) in exon 1 and intron 1 of the mbl with the disease, clinical markers, mbl levels and mbl-induced complement activity was analysed using standard statistical tools. significantly higher mbl levels and activity were observed in patients of bronchial asthma with allergic rhinitis as compared to the controls. we identified five snps, of which two, a816g in exon 1 and g1011a in intron 1 of the mbl, were novel. snp g1011a was significantly associated with the disease (p ¼ 0.0024, or ¼ 5.8696, 95% ci: 1.7316 < or < 19.8963). individuals with 'a' allele at position 1011 showed increased mbl levels, activity and disease severity. our results suggest that 'a' allele at position 1011 leading to high mbl levels and complement activity may be contributing to the severity of bronchial asthma and allergic airway inflammation. serum resistance of borrelia burgdorferi strains belonging to the b. afzelii and b. burgdorferi sensu stricto genospecies is dependent on binding of complement inhibitor factor h. we recently reported that factor h binding by b. burgdorferi is due to inducible expression of several approximately 20 kda plasmid-encoded, surface-exposed lipoproteins related to ospe (e.g. erpa, erpp and p21). in addition, a second class of factor h-binding proteins of approximately 27-35 kda has been described. the ospe-related lipoproteins are dramatically induced by b. burgdorferi during transmission from its tick vector into the mammalian host. the induction of ospe-related lipoproteins during mammalian infection may play a key a role in the borrelial evasion of the host's immune system. the goal of the present study was to define the factor h-binding regions of ospe-related proteins using mutagenesis, peptide mapping and surface plasmon resonance analysis (biacore). the combined studies revealed that the c-terminal regions of both human and mouse factor h (scrs 18-20) specifically bind to ospe-related lipoproteins. we also found fhr-1, whose c-terminal scrs 3-5 are homologous to scrs 18-20 of factor h, to bind to ospe. peptide mapping revealed five putative regions (designated i-v) in ospe that could directly interact with factor h. deleting the c-terminal 15 amino acid residues from region v of p21 abolished its ability to bind factor h. at the same time, however, synthetic peptides corresponding to the c-termini of ospe, p21 and erpp did not inhibit factor h binding to ospe. thus, the c-terminal-binding region v appears to be necessary but not sufficient for factor h binding. when a more specific mutation strategy was employed, where single amino acid residues in peptides spanning over the factor h-binding regions were mutated to alanines, we observed that lysines in the factor h-binding regions of ospe were required for factor h binding. the combined data have revealed that key lysine residues in ospe-related lipoproteins and ionic interactions are crucial for factor h interactions. furthermore, binding of ospe to the c-termini of both mouse and human factor h suggests that borrelia spirochetes utilize analogous complement resistance mechanisms in both rodents and man. in borrelia garinii strains, which in in vitro analyses have been found to be sensitive to complement killing, differences in the ospe sequences as well as in the expression of factor h-binding proteins may account for their susceptibility to serum lysis. role of yada, ail and lipopolysaccharide in serum resistance of yersinia enterocolitica serotype o:3 mannan-binding lectin (mbl), l-ficolin and h-ficolin are pattern recognition molecules of the innate immune system. we investigated the ability of these molecules to bind to different serotypes and noncapsulated variants of streptococcus pneumonia and staphylococcus aureus. we found that mbl binds to noncapsulated s. aureus strain (wood) but not any of the examined s. pneumoniae serotypes. l-ficolin binds to some capsulated s. pneumoniae serotypes (11a, 11d and 11f) as well as some capsulated s. aureus serotypes (type-1, -8, -9, -11 and -12). h-ficolin does not bind to any of the examined s. pneumoniae and s. aureus serotypes included in this study but did bind to a strain of aerococcus viridans. when bound to bacteria, mbl and h-ficolin initiated activation of complement factor c4, whereas l-ficolin did not. during this study, quantitative assays for the three proteins were developed and the concentration in 97 plasma samples were determined and the median values were estimated at 0.8 mg of mbl/ml, 3.3 mg of l-ficolin/ml and 18.4 mg of h-ficolin/ ml, respectively. the absence of early complement components (c1, c4 and c2 but not c3) is a predisposing factor for systemic lupus erythematosus (sle). recently, we demonstrated that, in c4-deficient (c4 def.) mice, igm-containing immune complexes (igm-ic) are filtered by the splenic barrier of marginal zone macrophages (mzm), resulting in an increased immune response against antigens within these igm-ic, but this could not be observed in wildtype or c3 def. mice. we hypothesized that splenic cd11b þ mzm play an important role in the induction of autoimmunity, and we therefore analysed their cytokine profile after isolation with the help of magnetic antibody cell sorting. mrna was isolated, and real-time pcr was performed with specific primers for murine ifn-g (ifn-g), interleukin-12 (il-12) and ifn-a (ifn-a). we observe a moderate increase of il-12 and ifn-g mrna in cd11b þ cells of c4 def. mice compared to wildtype cells. surprisingly, the concentration of ifn-a mrna is six times higher in c4 def. mice. preliminary results suggest that mrna in cd11b þ cells of c3 def. mice is even lower than that in wt. six hours following i.v. application of 20 mg of a abstracts 625 .................................................................................................................................................................................................. murine monoclonal igm anti-dsdna antibody, production of il-12, ifn-g and ifn-a mrna is increased in cd11b þ cells of both c4 def. and wt mice. several references described increased levels of inf-a in patients with sle. dendritic cells are discussed as a major source of ifn-a. our observation that c4-deficient, sle-susceptible mice demonstrate an increased spontaneous ifn-a production by splenic cd11b þ marginal zone macrophages could be an early sign and a trigger for the development of sle. this is supported by the fact that the absence of c3 is not a predisposing factor for sle and our observation that c3 def. animals display low levels of ifn-a mrna. 200-400 million people worldwide and represents one of the leading causes for liver cirrhosis and hepatocellular carcinoma. control over the hbv infection is achieved mainly by vaccination with hepatitis b surface antigen (hbsag). hbsag contains n-linked glycosylation side and is recognized by both mbl-a and mbl-c in a cadependent manner. hbsag-mbl complexes activate complement and may thus affect humoural immunity. to investigate the role of mbl in humoural responses to hbsag, we immununized mice that lack both mbl-a and mbl-c proteins with soluble hbsag. it has been shown that deficiencies in other complement components like c1q, c4 and c3 result in decreased antibody responses. however, mbl double ko animals mounted dramatically increased humoural responses. after priming, mbl double kos mounted hbsag-specific igm responses, which were threefold higher than wt controls. after boosting the hbsag, total igg was 10-fold higher in mbl ko than in wt control animals. similar to the response to hbsag, other glycosylated soluble antigens (e.g. invertase) induced better humoural responses in mbl double ko animals, suggesting that mbl plays an important role in a negative feedback regulation of adaptive immunity. reconstitution experiments with rmbl partially rescued the ko phenotype. we propose that the clearance of glycoprotein antigens in mbl ko is handled differently from the wt, resulting in better stimulation of humoural responses. alternatively, glycoprotein-ag-mbl-rich complexes inhibit b-cell responsiveness via putative mbl receptors. the complement system is an important part of the innate immune system. the activation of complement proceeds through three different pathways that converge in the generation of c3-activating enzyme complexes. complement activation via the lectin pathway is initiated when recognition molecules, mannan-binding lectin (mbl) or ficolin, bind to carbohydrate structures characteristic for microbial surfaces. in the circulation, mbl and ficolins are found in association with three structurally related mblassociated serine proteases (masp)-1, -2 and -3 and a small, nonenzymatic component, map19. masp-2 has been shown to elicit complement activation through the sequential proteolytic cleavage of c4 and c2 upon binding of mbl/masp-2 complexes to microbial surfaces. we have recently uncovered a polymorphism in the masp-2/map19 gene in a patient shown to be deficient in the lectin pathway of complement activation. the polymorphism results in a single amino acid substitution in the n-terminal part of the masp-2 protein. recombinant wildtype masp-2 and masp-2 containing the amino acid substitution in question was produced, and the ability to activate complement was studied. the mutation had a profound impact on masp-2 function, resulting in the lack of complement activation through the lectin pathway. elisa-based experiments showed that the mutation leads to the impairment of complement activation through influencing the binding of masp-2 to mbl or ficolins. deficiencies in the lectin pathway of complement activation have so far been accounted for only by lack of functional mbl. the mutation described above is the first defect described affecting both activation through mbl and the ficolins. .................................................................................................................................................................................................. th1, th2 and treg cell balance. dcs are present in the gut mucosa and may thus be target for modulation by gut microbes, including ingested probiotics. here, we tested the hypothesis that species of lactic acid bacteria, important members of the gut flora, differentially activate dc. a large panel of human gut-derived lactobacillus and bifidobacterium spp. was screened for dc-polarizing capacity by exposing bone marrow-derived murine dc to lethally irradiated bacteria. cytokines in culture supernatants and dc-surface maturation markers were analysed. substantial differences were found among strains in the capacity to induce interleukin-12 (il-12) and tumour necrosis factor (tnf)-a, while the differences for il-10 and il-6 were less pronounced. bifidobacteria tended to be weak il-12 and tnf-a inducers, while both strong and weak il-12 inducers were found among the strains of lactobacillus. remarkably, strains weak in il-12 induction inhibited il-12 and tnf-a production induced by an otherwise strong cytokine-inducing strain of lactobacillus casei, while il-10 production remained unaltered. selected strains were tested for induction of dc maturation markers. those lactobacilli with greatest capacity to induce il-12 were most effective in upregulating surface mhc class ii and cd86. moreover, l. casei-induced upregulation of cd86 was reduced in the presence of a weak il-12inducing l. reuteri. in conclusion, human lactobacillus and bifidobacterium spp. polarize differentially dc maturation. thus, the potential exists for th1/th2/treg-driving capacities of the gut dc to be modulated according to composition of gut flora including ingested probiotics. the intestinal micro flora is indispensable in developing and maintaining homeostasis of the gut-associated immune system. evidence indicates that lactic acid bacteria (lab), e.g. lactobacilli and bifidobacteria, have beneficial effects on the host. established health effects include increased gut maturation, antagonisms towards pathogens and immune modulation. the objective of this study is to evaluate the immunomodulating properties of a range of lab of human origin. as dendritic cells (dcs) play a pivotal role in the balance between tolerance and immunity to commensal microorganisms, in vitro-generated immature dcs serve as a suitable model for studying the immunomodulating effects of lab. human immature dcs were generated in vitro from monocytes and exposed to lethally uv-irradiated lab. the effect of various species of lab on dcs in direct contact was evaluated. furthermore, the maturation pattern of dcs separated from the bacteria by an epithelial cell layer (caco-2 cells), which should mimic the intestinal environment, was studied. cytokine secretion (il-12, il-10 and tnf-a) and upregulation of maturation surface markers on dcs (cd83 and cd86) was measured. different lab induced diverse cytokine responses. some strains were strong il-12 and tnf-a inducers and others weak. all strains induced il-10. different lab also differentially modulated expression of cd83 and cd86 on dcs. although some variation in the response to lab of dcs generated from different blood donors was observed, general differences in the effect of the various lab was revealed. experiments with the dc caco-2 coculture system are ongoing. different species of lab differentially affect dc maturation; this suggets that the gut flora plays a pivotal role in polarization of the immune response. natural killer (nk) cells are cells of the nonspecific immune system lysing altered self-cells. a noncytolytic subset of nk cells may serve a regulatory role by secreting cytokines. bacteria translocating across the gastrointestinal mucosa are presumed to gain access to nk cells, as consumption of certain lactic acid bacteria has been shown to increase in vivo nk cytotoxicity. here, we investigated how human gut flora-derived lactobacilli affect nk cells in vitro, by measuring proliferation and ifn-g production of human nk cells upon bacterial stimulation. cd3 -cd56 þ nk cells were isolated from buffy coats by negative isolation using non-nk lineage-specific antibodies and magnetic beads. nk cells were incubated with 10mg/ml uv-inactivated bacteria or 10mg/ml phytohemagglutinin (pha) for 4 days. proliferation was assessed by incorporation of radioactive thymidine into nk-cell dna. the ifn-g concentration was measured by elisa. incubation of nk cells with a lactobacillus acidophilus strain increased the proliferation of the nk cells and induced ifn-g production, both to levels comparable to pha stimulation. the proliferative response was further enhanced with autologous monocytes present, probably because cytokines, secreted by monocytes having engulfed bacteria, stimulated the nk cells. in contrast, a lactobacillus paracasei strain caused the nk cells to proliferate only in the presence of monocytes. these results demonstrate that various strains of lactobacilli have the capacity to activate nk cells in vitro, in a monocyte-dependent or -independent way. hence, the encounter of nk cells with lactic acid bacteria will affect nk-cell activation. such activation of nk cells may potentially skew an on-going or subsequent immune response towards a th1 response. lactobacilli are nonpathogenic gram-positive inhabitants of the normal human intestine known for their healthpromoting effects. in our earlier work, it is shown that human monoclonal antibody isolated from sera of a patient with waldenstrom macroglobulinaemia possess innate antibody characteristics and binds to lactic acid bacteria. according to the immune network model, immunization with this bacteria could induce the perturbations in immune system that might result in production of anti-lactobacillus antibodies, human monoclonal antibody like (ab1) and anti-idiotypic antibody (ab2). in this study, balb/c mice were immunized with two doses of bacteria lactobacillus acidophilus in complete and incomplete freund's adjuvant and phosphate-buffered saline (pbs), respectively. seven days after the last immunization, sera from immunized mice were collected and the presence of lactobacillus-specific ab1 and ab2 were determined by elisas. in the sera of immunized mice, antibodies specific to bacteria lactobacillus acidophilus were shown. the concentration of lactobacillus-specific antibodies was higher in the sera of hyperimmunized mice (mice immunized with 1 mg of igm dj) than in sera of mice immunized with 100 times lower doses of immunogen (0.01 mg per doses). moreover, ab1 and ab2 antibodies were detected in the sera of lactobacillus-hyperimmunized mice. in this study, we have shown the idiotypic network interactions in mice immunized with bacteria lactobacillus acidophilus. the normal gastrointestinal flora is crucial for the maturation of the acquired immunity via effects on antigenpresenting cells (apcs). here, we have investigated how two types of apcs, monocytes and dendritic cells (dcs), react to different bacterial strains typical of the commensal intestinal flora. purified monocytes and monocyte-derived dcs were stimulated with uv-inactivated gram-positive (lactobacillus plantarum and bifidobacterium adolescentis) and gram-negative (escherichia coli and veillonella parvula) bacterial strains. monocytes produced higher levels of il-12p70 and tnf, as detected by elisa, in response to l. plantarum than to e. coli and v. parvula. in contrast, dcs secreted high amounts of il-12p70, tnf, il-6 and il-10 in response to e. coli and v. parvula but were practically unresponsive to l. plantarum and b. adolescentis. the lack of response to the gram-positive strains correlated with a lower surface expression of toll-like reseptor 2 (tlr2) on dcs compared to monocytes. the surface expression of tlr4 on dcs was undetectable when analysed by flow cytometry, but blocking this receptor decreased the tnf production in response to v. parvula, indicating that low tlr4 expression on dcs is sufficient to mount an inflammatory response to gram-negative bacteria. ifn-g increased the expression of tlr4 on dcs and also potentiated the cytokine response to gram-negative bacteria. our results indicate that, when monocytes differentiate into dcs, their ability to respond to different commensal bacteria dramatically changes, thereby becoming unresponsive to probiotic gram-positive bacteria. these results may have important implications for the capacity of different groups of commensal bacteria to regulate mucosal and systemic immunity. probiotic bacteria, e.g. lactobacillus spp., may improve diseases such as chronic inflammatory bowel disease. we examined cytokine production and phenotypic change after in vitro stimulation of t cells from healthy volunteers using different probiotic strains. methods: t cells were cultured from colonic biopsies from eight healthy volunteers (agnholt and kaltoft, exp clin immunogenet 2001; 18:213-25) , and dendritic cells were matured from their peripheral blood mononuclear cells. t-cell cultures were stimulated with autologous bacterial sonicate or strains of lactobacillus spp., with and without the addition of dendritic cells. cytokine levels (tnf-a, ifn-g, il-10 and gm-csf) and phenotype (cd3, cd4, cd25 and cd69) were measured on day 4. results: lactobacillus spp. induced higher productions of tnf-a and il-10 than did autologous bacteria. in presence of dendritic cells, the production of all cytokines increased. however, the increases of ifn-g and tnf-a were more pronounced in wells with autologous bacteria than in wells with lactobacillus spp. the addition of dendritic cells upregulated cd25 expression without simultaneous upregulation of cd69. the upregulation was pronounced after stimulation with lactobacillus rhamnosus gg compared with autologous bacteria and other lactobacilli. discussion: in presence of dendritic cells, autologous bacteria induced inflammatory cytokines, while probiotics mainly induced regulatory cytokines. lactobacillus rhamnosus gg induced a regulatory phenotype (cd25 þ ), in part mediated by dendritic cells. future studies will address whether this shift to a cd25 þ phenotype represents a differentiation into competent regulatory t cells. in a clinical context, such cells might be used for treatment of inflammatory diseases. protein microarrays will play a key role in the postgenomic era and offer a unique possibility to perform highthroughput global proteome analysis. a chip can be printed with thousands of protein probes (e.g. antibodies), the biological sample added (e.g. a proteome) and any binding detected. we aim to develop protein microarrays based on human recombinant scfv antibody fragments for global proteome analysis. the concept of comparing proteomic maps of healthy versus diseased samples will allow diseasespecific proteins to be detected. in fact, antibody microarrays will allow us to perform comparative proteome analysis on any sample format in a species-independent manner, as long as a proteome can be isolated. however, the complexity of proteomes, containing several thousands of different proteins, is a problem. here, we have designed antibody microarrays targeting the water-soluble fraction of a proteome. to this end, an anticytokine antibody array was developed and human dendritic cells (aeactivation) was used as model system. the results showed that our antibody microarrays could be used to examine the cytokine profile in complex samples. furthermore, we have taken the first steps towards comparing our results with those of other technologies on both the protein and gene level. due to the complexity of the model proteome, we also examined the possibility to prefractionate the proteome in a simple one-step procedure (based on size) prior to the labelling step. in more detail, the sample proteome was fractionated into two fractions using membrane devices with different molecular weight cut-offs. the results showed that the fractionation considerably enhanced the assay sensitivity allowing cytokines in the pg/ml range to be readily detectable. the immunomodulatory effect of heat shock protein 70: immunization with a dna construct based on the malarial antigen fused with a fragment of hsp 70 primes for a th-1 type of response finding an appropriate adjuvant for human vaccination is crucial. heat shock proteins (hsps) act as adjuvants when coadministered with peptide antigens or given as fusion proteins. however, there is a potential risk of autoimmunity when using the complete molecules, because hsps are evolutionary conserved. to overcome this, we first evaluated the adjuvant effect against two different antigens of a less-conserved fraction of plasmodium falciparum hsp70 (pf70c) and compared it to the whole hsp70 molecule from trypanosoma cruzi (tchsp70). we found that pf70c exhibited similar adjuvant properties as the whole molecule. we later evaluated the adjuvant potential of pf70c against the malarial antigen eb200 in a chimeric dna construct. no appreciable levels of eb200-specific abstracts 629 .................................................................................................................................................................................................. antibodies were detected in mice immunized only with the dna constructs. however, dna primed the immune system, because subsequent challenge with the corresponding recombinant fusion proteins elicited a strong th-1 antibody response. in contrast, no priming effect was observed for ex vivo ifn-g production but stimulation with the hsp-chimeric fusion protein induced a stronger secretion of ifn-g in vitro than other proteins used. these results indicate that the use of hsps is promising in the design of new vaccines. high-throughput proteomics on antibody-based microarrays: the importance of probe and surface design in analogy to dna microarrays, protein microarrays offer a new distinct possibility to perform sensitive highthroughput global proteome analysis. however, the development of the protein microarray technology will place high demands upon the design of both probes and solid supports. the analysis of thousands of heterogeneous proteins on a single microarray requires the use of uniform probes, such as antibodies, directly designed for protein microarray applications. we have recently generated a human recombinant single-chain fv antibody library, genetically constructed around one framework, the ncoder-library, containing 2 â 1010 clones. single framework antibody fragments (sinfabs) selected from this library were successfully applied as probes for microarrays providing sensitive detection in the 600 attomol (mass spectrometry) and the 300 zeptomole range (fluorescence). however, the choice of framework is critical. we have shown that the selected ncoder framework displayed excellent functional on-chip stability and arrayed dehydrated probes retained their activity for several months. furthermore, we have addressed the issues of biocompatibility of the solid support and immobilization strategies for our microarray setup. an in-house-designed substrate, macroporous silicon coated with nitrocellulose (map3-nc7), displayed properties equal to, or better than, those of five commercially available supports used as reference surfaces. we have also evaluated different coupling strategies, such as adsorption, covalent coupling, diffusion and affinity coupling. using a novel affinity tag, the double-(his)6-tag, we increased the binding efficiency of sinfab-molecules to ni2 þ -coated solid supports, thereby allowing nonpurified probes to be directly applied. the mannan-binding lectin (mbl) pathway is part of the innate immune system providing a first line of defence against infections. mbl and ficolins circulate in complexes with mbl-associated serine proteases (masp-1, -2 and -3). after recognition of a microorganism by mbl, activation of the complement system occurs. masp-1 and masp-3 share five domains (making up the so-called a-chain), whereas they have unique protease domains (b-chains). before the identification of masp-3, an assay for masp was presented, based on antibodies against the a-chain of masp-1. with the new knowledge of the three masps, and the sharing of domains by masp-1 and masp-3, assays specific for the protease domains have to be constructed, if one wishes to measure the proteins individually. we present an assay for quantifying total masp-3 in plasma and serum samples. the assay is a sandwich-type assay using as catching antibody a monoclonal antibody against the common a-chain of masp-1/3 and a developing secondary antibody against the c-terminal part of the protease domain of masp-3. we have used this assay for estimating the normal concentration of the protein as well as the concentration in patients and also for characterizing by gel permeation chromatography the masp-3 protein in serum. inducible costimulator ligand (icosl) is a costimulatory molecule related to b7.1 (cd80) and b7.2 (cd86). b cells, monocytes, dendritic cells and endothelial cells express icosl. inducible costimulator (icos) interacts with icosl, and this interaction leads to signals involved in isotype switching and the development of immunological memory. hitherto, no polymorphisms of this gene have been described. the aim of this study was to reveal variation of the icosl gene in normal individuals. all eight exons, except exon 1, were sequenced with flanking introns in 10 healthy blood donors. eight single nucleotide polymorphisms (snps) and two length polymorphisms were found. one of the snps was found in the coding regions of the gene. the base involved was located in exon 3 and caused a conservative amino acid change from valine (gtt) to isoleucine (att). three individuals were heterozygous g/a for the exon polymorphism, while the remaining seven individuals were homozygous for the wildtype g/g. exon 3 encodes the immunoglobulin variable (igv)-like domain of the molecule which is situated outside the cell. this means that the amino acid could be critical for the stability of the molecule or could constitute part of the binding site for icos. the results form the basis for further experiments to find possible associations of the alleles to diseases caused by immune dysregulation. especially, the exon 3 variant is interesting and could play a role for the development of immunological diseases. besides, it would be interesting to see whether both exon 3 alleles are expressed or only the wildtype allele is functional. interferon-a/b (ifn-a/b) is increasingly used as antiviral and immunomodulatory therapies. unfortunately, bioavailability varies with ifn species and mode of administration, and all ifn species are potentially immunogenic. assays for antiviral activity (ifn) and antiviral neutralization (antibodies, nab) have been used for some time to monitor patients on ifn biologicals. these assays require laborious titrations making them unsuitable for large-scale clinical use. myxovirus a (mxa) is a resistance gtpbinding protein that is specifically induced by treatment with type 1 ifns. for example, ifn-b-induced mxa in blood leucocytes has been used as a biomarker in ifn-btreated patients with multiple sclerosis. however, the degree of specificity of mxa in this regard is unclear, and measurements of mxa protein and/or mrna are not yet suitable for routine clinical use. in an attempt to find new and better reporter genes (and, hopefully, genes and gene products with proven specificity for ifn-a and -b), microarray screenings with u133a genechips (affymetrix) were carried using human blood leucocytes and the human lung carcinoma cell line a549. we studied the simultaneous expression of 22,000 transcripts before and after exposure to human recombinant ifn-a and ifn-b and other antiviral and immunomodulatory cytokines. the results will be presented at the conference. interferon-a/b (ifn-a/b) is increasingly used as antiviral and immunomodulatory therapies. unfortunately, bioavailability varies with ifn species and mode of administration, and all ifn species are potentially immunogenic. assays for antiviral activity (ifn) and antiviral neutralization (antibodies, nab) have been used for some time to monitor patients on ifn biologicals. these assays require laborious titrations making them unsuitable for large-scale clinical use. our laboratories have therefore modified the antiviral assays for ifn bioactivity and nab, so that they are suitable for large-scale screening in specialized laboratories. the read-out is survival of a subcloned a549 cell line in the presence of an otherwise lethal amount of virus. thus, survival increases in the presence of type 1 ifn and decreases in the presence of nab against the ifn added to the cells. mxa is induced by type 1 ifn and can be used for measuring the nab activity. in another assay, the mxa level in the a549 cell line is measured. in an attempt to find a new and better reporter gene for type 1 ifn than mxa and genes specific for either ifn-a or -b, a micro array screen was carried using the u133a chip from affymetrix. the expression of 22,000 genes can be studied simultaneous with this technology. the results will be presented at the conference. in our laboratory, we have developed a database system, which we believe is of immediate interest to the general scientific community. the database represents a computerbased replacement for the laboratory notebooks used in the majority of research laboratories worldwide. in addition, the database provides an effective tool for organizing and managing laboratory information at all levels, spanning from managing and revising standard operating procedures and producing documentation of research activities to keeping track of data and conclusions. using the commercially available database toolkit software filemaker pro, we have developed a relational database solution for management of laboratory information. the system consists of a hierarchy of five interrelated databases, each pertaining to a separate type of information, namely, overall project information, information relating to individual experiment setups, documentation of daily research activity, generated data and descriptions of standard operating procedures. like other databases, each individual database consists of a number of records, each comprised of a set of fields in which information is entered. in each record, a certain field is reserved to specify the relation of the record to a record in another database at a higher level. thus, the database is essentially five databases linked by a hierarchy of one-to-many relations, organizing information in a folder-like structure. importantly, the database system allows multiple users to access and edit records simultaneously, and the data entered in one database immediately becomes accessible through the other databases. the limitations of laboratory notebooks are apparent when looking for information, which is dispersed throughout one or more notebooks, or possibly on loose sheets of paper or printouts 'somewhere'. the often complicated process of gathering laboratory data or results when writing grant applications or research papers is made considerably easier with the database system. thus, the database solution presented should be broadly attractive to researchers, irrespective of their scientific discipline. an effective sars vaccine is likely to include components that can induce specific cytotoxic t-cell (ctl) responses. the specificities of such responses are governed by hlarestricted presentation of sars-derived peptide epitopes. exact knowledge of how the immune system handles protein antigens would allow for the identification of such linear sequences directly from genomic/proteomic sequence information. the latter was recently established when a causative coronavirus (sars cov) was isolated and full-length sequenced. here, we have combined advanced bioinformatics and high-throughput immunology to perform an hla supertype, genome-wide scan for sars-specific cytotoxic t cell epitopes. the scan includes all nine human hla supertypes in total covering >99% of all major human populations. for each hla supertype, we have selected the 15 top candidates for test in biochemical-binding assays. at this time (approximately 6 months after the genome was established), we have tested the majority of the hla supertypes and identified almost 100 potential vaccine candidates. these should be further validated in sars survivors and used for vaccine formulation. we suggest that immunobioinformatics may become a fast and valuable tool in rational vaccine design. rationale: major histocompatibility complex class i (mhc i) molecules monitor the protein content of the cell by binding small derived peptides and presenting them to cytotoxic cd8 þ t cells. the goal of the human mhc project is to predict the binding strength of any given peptide/mhc complex. this prediction allows the design of peptide-based vaccines. the prediction requires representative binding data from mhc alleles from all the nine hla supertypes. here, we describe the genetic construction, protein production and purification as well as the establishment-binding assays for two recombinant mhc supertype alleles, hla-b*1501 and hla-b*5801. methods: using the quikchange multisite directed mutagenesis kit (stratagene), codon-optimized genes encoding hla-b*1501 and hla-b*5801 are created. the two mhc i molecules are fermented and purified by ion exchange chromatography, hydrophobic interaction chromatography and size exclusion chromatography. the binding (kd) of natural t-cell epitopes, as well as predicted peptide ligands, is described by radioactive immunoassays (rias) and enzyme-linked immunosorbent assays (elisas). the mhc molecules are biotinylated during expression. results: the expression of mhc i resulted in multiple disulfide bond isomers, which are separated by hydrophobic interaction chromatography and used in subsequent binding studies resulting in the determination of kd for various peptide ligands ranging from strong binders we have previously demonstrated that bioinformatics tools such as artificial neural networks (anns) are capable of performing pathogen-, genome-and hlawide predictions of peptide-hla interactions. these tools may therefore enable a fast and rational approach to epitope identification and thereby assist in the development of vaccines and immunotherapy. a crucial step in the generation of such bioinformatics tools is the selection of data representing the event in question (in casu peptide-hla interaction). this is particularly important when it is difficult and expensive to obtain data. herein, we demonstrate the importance in selecting information-rich data and we develop a computational method, query-bycommittee, which can perform a global identification of such information-rich data in an unbiased and automated manner. furthermore, we demonstrate how this method can be applied to an efficient iterative development strategy for these bioinformatics tools. methods: a large panel of binding affinities of peptides binding to hla a*0204 was measured by a radioimmunoassay (ria). this data was used to develop multiple first generation anns, which formed a virtual committee. this committee was used to screen (or 'queried') for peptides, where the anns agreed ('low-qbc'), or disagreed ('high-qbc'), on their hla-binding potential. seventeen low-qbc peptides and 17 high-qbc peptides were synthesized and tested. the high-or low-qbc data were added to the original data, and new high-or low-qbc second generation anns were developed, respectively. this procedure was repeated 40 times. the high-qbc-enriched ann performed significantly better than the low-qbc-enriched ann in 37 of the 40 tests. conclusion: these results demonstrate that high-qbcenriched networks perform better than low-qbc-enriched networks in selecting informative data for developing peptide-mhc-binding predictors. this improvement in selecting data is not due to differences in network training performance but due to the difference in information content in the high-qbc experiment and in the low-qbc experiment. finally, it should be noted that this strategy could be used in many contexts where generation of data is difficult and costly. interleukin-18 (il-18), a pro-inflammatory cytokine that is produced by both lymphoid and nonlymphoid cells, has a critical role in modulation of innate and adaptive immunity. its primary function in stimulation of ifn-g production and stimulation of nk-cell-cytotoxic activities makes this cytokine a candidate for cancer immunotherapy. in oral cavity, this cytokine is produced by oral epithelia and carcinoma cells and is related to tumour regression in nude mice bearing salivary adenocarcinoma. however, direct effects of this cytokine on oral cancer cells have not been elucidated. in this project, we investigated il-18 effect on an oral carcinoma (kb) cell line. with rt-pcr technique, kb-cell line was found to express il-18 receptors (il-18ra and il-18rb), indicating that this oral carcinoma line is a target for il-18 study. we showed that recombinant human il-18 inhibited kb-cell proliferation by 17% at concentration of 100 ng/ml (p < 0.05), whereas ldh release by these cells in treatment group and control groups was comparable, indicating that il-18 suppression of cell proliferation was not mediated by the induction of cell death. to further address this hypothesis, we found that il-18 treatment did not induce apoptotic cell death, as studied by dna laddering and tunel assays. in addition, expression pattern of cell death-controlling genes (bcl-2 and bax) was not altered by this cytokine. findings in these studies indicated that suppression of kb-cell proliferation may be attributed to control of cell cycle, growth arrest or induction of cell differentiation. the data presented in this project could provide an insight of how cancer cell directly responds to il-18, as this cytokine is an important regulator of anticancer mechanisms. aloe emodin (ae) is a naturally occurring compound with wide spectrum of biological properties, including antimicrobial, vasorelaxant, immunosuppressive and anticancer actions. this anthraquinone induces apoptosis in several tumour cell lines with special affinity to tumours of neuroectodermal origin. high amounts of nitric oxide (no) released by activated macrophages induce tumour cell death. therefore, we explored the capacity of ae to modulate no-mediated antitumour response in vitro. interestingly, while ae markedly suppressed no release from macrophages alone, it significantly potentiated no production in cocultures of macrophages and c6 cells, after 48 h of cultivation. accordingly, the viability of c6 cells cocultivated with macrophages was reduced in the presence of ae. moreover, the observed ae-imposed potentiation of no production in macrophages was closely related to macrophage culture cell density. according to these data, we proposed that no modulator capacity of ae strongly depended on intercellular contact, indicating that macrophage antitumour response was not compromised but even potentiated by ae. immunotherapy represents an attractive fourth-modality therapeutic approach, especially in the light of the shortcomings of conventional surgery, radiation and chemotherapies in the management of metastatic cancer. to this end, a large number of peptide antigens derived from taa have been applied in immunotherapeutic trials for the treatment of various malignancies, e.g. cancers of the breast, prostate and kidney, in addition to haematological cancers. in some cases the response rates have been impressive and no adverse autoimmunity have been observed. a major strategic difficulty associated with these trials relates to the choice of best-suited peptide antigens. the vast majority of the antigens described thus far is not vital for survival and growth of the tumour cells, and immunoselection of antigen-loss variants may therefore prove to be an additional obstacle for the clinical applicability of most of the known peptide epitopes. in this respect, the development of acquired antigen loss during immunotherapy has been demonstrated in several cases. obviously, the development of loss-variant tumour cells implies that these cells acquire a pronounced growth advantage and are left unaffected by further treatment. ideally, target antigens should be derived from proteins required for survival and growth of tumour cells, as antigens with these characteristics would not be inflicted by the development of loss-variant tumour cells. in this respect, several inhibitors of apoptosis proteins (iaps) are universally expressed among tumours and play an important role in tumour cell escape from apoptosis. we have characterized spontaneous t-cell reactivity against iapderived peptides in cancer patients. from the iap survivin, we have characterized peptides restricted to the class i molecules hla-a1, a2, a3, a11, b7 and b35. furthermore, we have demonstrated that survivin-specific t cells infiltrate metastatic lesions and that isolated survivinspecific ctls are capable of killing hla-matched tumour cells. survivin-derived peptides are now in clinical trial, and continued work in our lab has demonstrated that other iaps are targets for spontaneous t-cell reactivity in cancer patients. we previously reported that in mice with large progressing t-cell lymphoma tumours, dysfunctions in the antitumour ctl activity occur, associated with an accumulation of splenic arginase-producing myeloid suppressor cells (mscs). in this study, we first demonstrate that both the presence and the activation state of these msc depends on tumour evolution. while in tumour regressors hardly any arginase-producing msc can be found, both the amount and the arginase activity of this population expands from early over late progressors. this gradual induction of mscs is paralleled by an increasing suppression of ctl activity and th1, but not th2, cytokine production. upon analysing the molecular repertoire of msc in vitro, we found, besides arginase1, a well-established marker for alternatively activated myeloid cells or m2, a strong upregulation of fizz1 and ym, two additional recently identified markers for m2. further evaluation of molecular markers by microarray analysis in msc yielded genes involved in wound healing (e.g. coagulation factor xiiia), anti-inflammation (e.g. selenoprotein p), immunomodulation (e.g. pd-l2) and fat and sugar metabolism (e.g. leptin receptor). of note, many of these genes are regulated by type 2 cytokines (il-4, il-13 and il-10) and are therefore rather m2 associated. overall, our data provide new markers for msc in cancer and further establish their m2 activation state. study. only sp-a showed a significant expression in normal mucosa which was downregulated in crc. as the absolute signal level was below the noise threshold, these results have to be interpreted with caution and require confirmation by direct measurenment of the proteins. our results suggest that there is no major role for the human collectins in colorectal cancer. tetramerization is visualized by sds-page. conclusion: an effective method for the production of highly pure mhc i molecules has been applied to hla-b*1501 and hla-b*5801, and ria and elisa binding assays for those alleles have been established background: proliferation, differentiation and apoptosis are essential processes in the normal functions of the mammary epithelium. the hypothesis examined in this study is that the transcription factor bcl-6 is critically important not only for regulating b-cell growth and development but also for mammary epithelial apoptosis. methodology: twenty breast cancer cases and 31 healthy controls were used to investigate whether bcl-6 protein in involved in breast cancer (grade iii). full length bcl-6 cdna was retrovirally transduced into eph-4 cell line. we then used flow cytometry of brdurd-stained cells to investigate the cell-cycle duration of the control and transduced cell lines. tunel was used as a marker of apoptosis to find out differences in the frequencies of apoptotic cells in the control and transduced cell lines. finally, immunohistochemistry staining was performed to detect bcl-6 in breast cancer (iii). results: restoration of bcl-6 into eph-4 cells not only inhibits apoptosis but also prolongs the cell cycle and results in increased cell size and protein content. the results also indicated that the cell-cycle time of bcl-6-transduced eph-4 cells is prolonged by about 3 h, presumably as a result of the action of bcl-6 at the bcl-6 at the g1/s transition. we found differences in the frequencies of viable and apoptotic cells in cultures of the parent eph-4 cells, control-transduced eph-4 cells and bcl-6-transduced eph-4 cells. consistently, we demonstrated that bcl-6 is expressed in 90% of high grade of breast carcinoma, which is considered as the most aggressive of tumours. conclusion: together, these results suggest that bcl-6 is likely to be involved in mammary gland development and carcinogenesis. inflammatory cytokines have a critical role in modulation of both innate and adaptive immunity in response to foreign antigen. they also play an important role in anticancer immunity. for example, they can promote cell-mediated immunity against cancer cells. with their immunostimulatory effects, these cytokines are being tested for cancer treatment in the form of dna vaccine or adjuvant or therapeutic cytokines. direct effect of these cytokines on cancer cell, however, is still unclear. in this project, we investigated whether il-1( and il-18 can modulate cancer cell proliferation. we employed a simple nonradioactive proliferation (mtt) assay and detection of lactate dehydrogenase (ldh) to test the effect of these recombinant human cytokines on various cancer cell lines, including breast cancer cell line (mcf-7), oral carcinoma cell line (kb), colon cancer cell line (caco-2) and choriocarcinoma cell line (jar). cytokines used in this study had both inhibitory and stimulatory effect on cell proliferation. findings in this project could provide an insight of cancer cell response to these cytokines and this could lead to a consideration on using cytokine as immunotherapy for cancer treatment.capacity of ae to modulate nitric oxide production depended on intercellular contact donor t cells are involved in the antitumour effects observed after bmt. thus, patients receiving t-celldepleted bmt have a higher risk of leukaemia relapse compared to patients receiving nonmanipulated bmt, and patients experiencing graft-versus-host disease (gvhd) have a lower risk of disease relapse than patients who do not experience gvhd. although the importance of donor t cells for the curative action of bmt has been established, the exact mechanisms and molecules involved in this graft-versus-tumour effect remain largely unknown. in a recently initiated project, we have conducted a longitudinal study of t-cell clonotypes in patients who received peripheral blood stem cell grafts after nonmyeloablative conditioning. peripheral blood samples were obtained sequentially after transplant, and the mononuclear cells (mncs) were isolated and cryopreserved. cd8 þ t cells were isolated from the mncs by use of immunomagnetic beads or facs and analysed for the presence of clonally expanded cells by t-cell receptor clonotype mapping based on rt-pcr and denaturing gradient gel electrophoresis (dgge). using this gel-based methodology, clonally expanded t cells were monitored after transplant and compared to the clinical data of the patients. the preliminary results demonstrates the presence of clonally expanded cd8 þ t cells at all time points analysed. furthermore, a number of clonotypes persisted for more than 6 months, and other clonotypes emerged during this period. the appearance of newly emerged clonotypes which coincided with clinical gvhd could indicate a role for these t cells in the pathogenesis of gvhd. background: deficiency of the mannan-binding lectin (mbl) pathway of innate immunity leads to increased susceptibility to infections. in patients with colorectal cancer, postoperative infection is associated with poor prognosis. the aim of the present study was to evaluate (1) the relation between the mbl pathway and postoperative infectious complications and survival of patients resected for colorectal cancer and (2) the role of mbl as acute phase reactant compared to crp. methods: preoperative mbl concentration, mbl/mblassociated serine protease (masp) activity and crp were determined in serum from 611 patients and 150 healthy controls. the patients were observed for 8 years. postoperative infections, recurrence and survival were recorded. results: the mbl pathway components were increased in the patients (p < 0.0001) compared to healthy controls. low mbl levels were predictive of pneumonia (p ¼ 0.01), and pneumonia (n ¼ 87) was associated with poor survival (p ¼ 0.003, hr ¼ 1.5, 95% ci 1.1-1.9). mbl and mbl/ masp activity could not predict postoperative overall infections. mbl showed no correlation (spearman's r ¼ 0.02, 95% ci à0.06-0.10) with crp. conclusions: low preoperative mbl levels are predictive of pneumonia, which is associated with poorer survival. mbl concentration and mbl/masp activity was not predictive of other postoperative infections or long-term prognosis. mbl apparently is not a surrogate measure of crp. department of surgery, university hospital of erlangen, erlangen, germany. e-mail: michael.siassi@rzmail.uni-erlangen.de introduction: the human collectins, mannan-binding lectin (mbl), surfactant protein-a (sp-a) and surfactantprotein-d (sp-d) play a central role in the innate immune system. immunological responses to malignant transformation of epithelial cells gained increasing interest recently. a former study could demonstrate binding of mbl to certain colorectal carcinoma (crc) cell lines in vitro. we therefore examined the expression of human collectins in normal colon mucosa and in colorectal carcinomas. materials and methods: colon samples from 20 crc patients and 10 normal mucosa samples were collected immediately after surgery. the tissue was microdissected and rna isolated (qiagen, rneasy-kit). gene expression profiles were analysed using gene-chips (affymetrix, hg-u133). we analysed the data for the expression of mbl, its associated serine proteases mannan-binding lectinassociated serine protease 1/2 (masp 1/2), sp-a and sp-d. the signal intensity of the genes of interest was compared using the mann-whitney u-test. results: the expression of human collectins in normal human colon mucosa was generally low. only the expression of sp-a and masp-2 reached the noise threshold of 250 signals. these genes were significantly downregulated in crc specimens. the expression of the other proteins showed no difference in normal mucosa and crc. conclusion: as demonstrated before, the expression of human collectins in normal colon was low in this being the first lymph node to receive drainage from the tumour area, the sentinel node offers a unique possibility to obtain tumour-reactive lymphocytes. we investigated antitumour immune responses in sentinel nodes from patients with bladder cancer, by assaying tumour-specific proliferation and tcr vb repertoires. during tumour surgery, sentinel lymph nodes were identified by peritumoural injection of blue dye. fresh specimens of tumour, sentinel and nonsentinel lymph nodes were obtained, and single-cell suspensions were prepared. cells were assayed for reactivity against autologous tumour extract in [ 3 h]-thymidine incorporation assays and characterized by flow cytometry. parallel analyses of the expression of vb gene families were performed with padlock probes, linear oligonucleotides which upon target recognition can be converted to circular molecules by a ligase. probes were reacted with cdna prepared from magnetically separated cd4 þ cells, and the tcr repertoire was determined by hybridizing the products to oligonucleotide microarrays. dose-dependent proliferation in response to tumour extract could be detected in sentinel lymph nodes. common clonal expansions were detected among tumourinfiltrating lymphocytes and in sentinel lymph nodes. nonsentinel lymph nodes displayed a divergent tcr vb repertoire. these results indicate an ongoing immune response against tumour antigens in sentinel nodes, draining urinary bladder cancer. identification of sentinel lymph nodes makes it possible to obtain tumour-reactive lymphocytes for use in adoptive immunotherapy. key: cord-023430-5zuewjv2 authors: nilkaeo, a.; bhuvanath, s. title: interleukin‐18 inhibition of oral carcinoma cell proliferation date: 2008-06-28 journal: scand j immunol doi: 10.1111/j.0300-9475.2004.01423bg.x sha: doc_id: 23430 cord_uid: 5zuewjv2 interleukin‐18 (il‐18), a pro‐inflammatory cytokine that is produced by both lymphoid and nonlymphoid cells, has a critical role in modulation of innate and adaptive immunity. its primary function in stimulation of ifn‐γ production and stimulation of nk‐cell‐cytotoxic activities makes this cytokine a candidate for cancer immunotherapy. in oral cavity, this cytokine is produced by oral epithelia and carcinoma cells and is related to tumour regression in nude mice bearing salivary adenocarcinoma. however, direct effects of this cytokine on oral cancer cells have not been elucidated. in this project, we investigated il‐18 effect on an oral carcinoma (kb) cell line. with rt‐pcr technique, kb‐cell line was found to express il‐18 receptors (il‐18rα and il‐18rβ), indicating that this oral carcinoma line is a target for il‐18 study. we showed that recombinant human il‐18 inhibited kb‐cell proliferation by 17% at concentration of 100 ng/ml (p < 0.05), whereas ldh release by these cells in treatment group and control groups was comparable, indicating that il‐18 suppression of cell proliferation was not mediated by the induction of cell death. to further address this hypothesis, we found that il‐18 treatment did not induce apoptotic cell death, as studied by dna laddering and tunel assays. in addition, expression pattern of cell death‐controlling genes (bcl‐2 and bax) was not altered by this cytokine. findings in these studies indicated that suppression of kb‐cell proliferation may be attributed to control of cell cycle, growth arrest or induction of cell differentiation. the data presented in this project could provide an insight of how cancer cell directly responds to il‐18, as this cytokine is an important regulator of anticancer mechanisms. the nuclear receptor heterodimers of liver x receptors (lxrs) are recently identified as key transcriptional regulators of genes involved in lipid homeostasis and inflammation. lxrs and their ligands are negative regulators of macrophage inflammatory gene expression. multiple sclerosis (ms), a demyelinating disease of the central nervous system of unknown cause, is characterized by recurrent inflammation involving macrophages and their inflammatory mediators. sweden belongs to the countries with a high ms incidence. in italy, incidence is lower, with an exception for sardinia where the incidence is even higher than that in sweden. subjects from sardinia are ethnically more homogeneous and differ from swedes, also regarding genetic background and environment. we studied lxrs and their related molecules of blood mononuclear cells (mncs) from female patients with untreated relapsing-remitting ms from sassari, sardinia and stockholm, sweden. sex-and age-matched healthy controls (hcs) were from both areas. mrna expression was evaluated by real-time pcr. lxr-a was lower (p < 0.05) in ms (mean ae sem: 3.1 ae 0.2; n ¼ 37) compared to hc (3.6 ae 0.1; n ¼ 37). lxr-a was lower in ms from stockholm (2.6 ae 0.2; n ¼ 22) compared to corresponding hc (3.4 ae 0.1; n ¼ 22; p < 0.01) and compared to ms (3.8 ae 0.2; n ¼ 15; p < 0.001) and hc (4 ae 0.2; n ¼ 15; p < 0.001) from sardinia. ms patients from stockholm, but not from sassari, also expressed lower (p < 0.05) lxr-b (à4.1 ae 0.4) compared to corresponding hc (à2.9 ae 0.3). ms from stockholm was associated with higher abca-1 (6.1 ae 0.4 versus 5.0 ae 0.3; p < 0.05) and higher estrogen receptor-b-cx (2.4 ae 0.4 versus 0.8 ae 0.4; p < 0.01) compared to corresponding hc. the hc from sassari had higher androgen receptor (2.9 ae 0.2) compared to ms from sassari (1.4 ae 0.3; p < 0.01), ms (1.3 ae 0.4; p < 0.01) and hc from stockholm (1.2 ae 0.3; p < 0.01). ms from sassari had lower cyclooxygenase-1 compared to corresponding hc (5.1 ae 0.4 versus 6.6 ae 0.3; p < 0.01) and lower prostaglandin-e (à0.03 ae 0.5) compared to the hc (1.4 ae 0.5; p < 0.05) and ms (2.7 ae 0.4; p < 0.05) and hc from stockholm (1.9 ae 0.4, p < 0.001). our findings identify lxrs and their related molecules as being involved in ms from stockholm but not from sassari, while sex hormone receptors seem to be involved in ms in sassari. multiple sclerosis: ifn-b induces cd123 + bdca2 -dendritic cells that produce il-6 and il-10 and have no enhanced type i interferon production y. m. huang, 1 s. adikari, 1 u. båve, 2 a. sanna 1,3 & g. alm 4 dc antigens (bdca) and investigate their ability to produce type i ifn in response to virus stimulation. we show that ifn-b induces development of cd123 þ dc from human blood monocytes, which coexpress bdca4 þ but are negative for bdca2 -, a specific marker for plasmacytoid dc. such ifn-b-modulated dc produce large amounts of il-6 and il-10, but no il-12p40 and have no enhanced ifn-b and ifn-b production. the findings indicate that ifn-bmodulated dc represent a myeloid dc subset with diminished cd11c, bdca-1 and cd1a expression, having potent th2-promoting function but lacking antiviral capacity. the association of psoriasis with throat infections by streptococcus pyogenes suggests a potential antigenic target for the t cells that are known to infiltrate dermis and epidermis of psoriatic skin. streptococcal m protein shares an extensive sequence homology with human epidermal keratins. keratins 16 (k16) and 17 (k17) are mostly absent from uninvolved skin but are upregulated in psoriatic lesions. there is increasing evidence that cd8 þ t cells play an important effector role in psoriasis and m proteinprimed t cells may recognize these shared epitopes in skin via molecular mimicry. to identify candidate epitopes, peptides with sequences from k17 were selected on the basis of predicted binding to hla-cw6 and sequence similarities with m6 protein. matched peptides from the sequence of m6 protein and a set of peptides with poor predicted binding were also selected. cw6 þ individuals with psoriasis and cw6 þ healthy controls, having a family history of psoriasis, were recruited. pbmcs were incubated with the peptide antigens. t-cell activation in the cd4 þ , cd8 þ and later the skin-homing cutaneous lymphocyteassociated antigen (cla)-expressing subset of cd8 þ t cells was evaluated by cd69 expression and intracellular ifn-g accumulation using flow cytometry. we demonstrate that cw6 þ psoriasis patients had significant cd8 þ t-cell ifn-g responses to peptides from k17 and m6 protein selected on the basis of sequence homology and predicted hla-cw*0602 binding. these responses were about 10 times more frequent in the skin-homing cutaneous lymphocyte-associated antigen-expressing (cla þ ) subset of cd8 þ t cells. cd4 þ t cells showed only borderline responses. cd8 þ t cells from cw6 þ nonpsoriatic individuals responded to some m6 peptides but very rarely to k17 peptides, and this also applied to the cla þ cd8 þ subset. these findings indicate that psoriatic individuals have cd8 þ t cells that recognize keratin self-antigens and that epitopes shared by streptococcal m protein and human keratin may be targets for the cd8 þ t cells that infiltrate psoriatic skin lesions. autoantibodies directed against citrulline-containing proteins have an impressive specificity of nearly 100% in ra patients and a suggestive involvement in the pathogenesis. the targeted epitopes are generated by a post-translational modification catalysed by the calcium-dependent enzyme peptidyl arginine deaminase that converts the positively charged arginine to polar but uncharged citrullin. the aim of this study was to analyse the presence of citrulline in the joints at different time points of collagen-induced arthritis in da rats by immunohistochemistry and to investigate how immunogenicity and arthritogenicity was affected by citrullination of rat serum albumin (rsa) and collagen type ii (cii). our results indicate that citrulline could be detected in joints of arthritic animals, first appearance at the onset of disease and increasing as disease progressed into a chronic state. unimmunized animals or time points before clinical signs of arthritis were negative. by morphology, we state that some infiltrating macrophages as well as the cartilage surface stain positive for citrulline, while the major source of citrullinated proteins appears to be fibrin depositions. a specific cit-rsa t-cell response was observed in animals challenged by citrullinated rsa, no response was recorded when rsa was used as a stimulus. the igg analysis reveals not only a response towards the modified protein but also cross-reactivity to native rsa. no t-cell or b-cell response was noted in animals injected with unmodified rsa. cit-cii induced a disease with higher incidence and earlier onset than did the native counterpart. we conclude that, in contrast to the human disease, citrulline does not seem to appear before clinical signs. as inflammation proceeds, citrulline is detected specifically in the joints. all other organs investigated were negative. we also conclude that citrullination of a protein can break tolerance and increase its arthritogenic properties. ectopic germinal centers (gcs) can be detected in the salivary glands of approximately 1/5 of patients with sjögren's syndrome (ss) and appear in both primary and secondary ss. previously, ectopic gc have been associated with increased local autoantibody production. the aim of this study was to determine whether gc in primary sjögren's syndrome (pss) defines a distinct seroimmunological phenotype. retrospectively, a material of 130 haematoxylin and eosin-stained paraffin-embedded tissue sections of minor salivary gland tissue from patients with pss was morphologically screened for the presence of ectopic gc. gc-like lesions were detected in 33/130 (25%) of the pss patients. seventy-two pss patients lacking these structures (gc-) were randomly selected for comparison. focus score was significantly increased in the gc þ patients compared to the gcpatients (p ¼ 0.035). in the gc þ group, 54.5% of the patients presented with anti-ro/ssa compared to 43.7% in the gcgroup. anti-la/ssb was detected in 31.3% of the gc þ patients compared to 25.7% of the gcpatients. sixty-one percentage of gc þ patients presented with increased levels of igg, a nonsignificant difference when compared to 39.4% in the gcpatients (p ¼ 0.089). levels of rf, ana, ena, igm and iga were similar in both patient groups, as were esr and crp. in conclusion, patients with ectopic gc have a higher focus score and more often present with autoantibodies and increased levels of igg compared to pss patients with regular focal infiltration (gc -). our findings may indicate a certain seroimmunological phenotype and warrant for further prospective studies. association between mannose-binding lectin and vascular complications in type 1 diabetes complement activation and inflammation have been suggested in the pathogenesis of diabetic vascular lesions. we investigated serum mannose-binding lectin (mbl) levels and polymorphisms in the mbl gene in type 1 diabetic (t1dm) patients with and without diabetic nephropathy and associated macrovascular complications. polymorphisms in the mbl gene and serum mbl levels were determined in 199 t1dm patients with overt nephropathy and 192 t1dm patients with persistent normoalbuminuria matched for age, sex and duration of diabetes as well as in 100 healthy control subjects. the frequencies of high and low expression mbl genotypes were similar in patients with t1dm and healthy controls. high mbl genotypes were significantly more frequent in diabetic patients with nephropathy than in the normoalbuminuric group, and the risk of having nephropathy, given a high mbl genotype, assessed by odds ratio was 1.52 (1.02-2.27), p ¼ 0.04. median serum mbl concentrations were significantly higher in patients with nephropathy than in patients with normoalbuminuria [2306 mg/l (iqr 753-4867 mg/l) versus 1491 mg/l (iqr 577-2944), p ¼ 0.0003], and even when comparing patients with identical genotypes, serum mbl levels were higher in the nephropathy group than in the normoalbuminuric group. patients with a history of cardiovascular disease had significantly elevated mbl levels independently of nephropathy status [3178 mg/l (iqr 636-5231 mg/l) versus 1741 mg/l (iqr 656-3149 mg/l), p ¼ 0.02]. the differences in mbl levels between patients with and without vascular complications were driven primarily by pronounced differences among carriers of high mbl genotypes (p < 0.0001). our findings suggest that mbl may be involved in the pathogenesis of microvascular and macrovascular complications in type 1 diabetes and that determination of mbl status might be used to identify patients at increased risk of developing these complications. neuroimmunology unit, center for molecular medicine, karolinska institutet, stockholm, sweden. e-mail: judit.wefer@cmm.ki.se dna vaccine coding for the encephalitogenic peptide mog 91-108 protects lew.1av1 from subsequent development of experimental autoimmune encephalomyelitis (eae). protection is associated with a type 1 immune response and is dependent on the presence of cpg dna motifs. the mechanisms underlying the observed reduction of eae development in protected rats have not been fully clarified. we investigated immunological characteristics of lymphocytes after dna vaccinaton and subsequent eae induction. we confirm that protection was not associated with suppression of t1 cells, as transcription of the novel molecule rat t-cell immunoglobulin-and mucindomain-containing molecule (tim-3), reported to be exclusively expressed on differentiated t1 cells, was not altered by dna vaccination. we did not note any clonal deletion upon tolerization, but detected an antigen-specific lymphocyte population upregulating ifng upon recall stimulation 3 weeks after protective dna vaccination. in protected rats, we observed (1) no alterations in antigenspecific th2 or th3 responses, (2) reduced mhc ii expression on splenocytes early after eae induction, (3) antigen-specific upregulation of ifnb upon recall stimulation and (4) reduced il-12rb2 on lymphocytes. we thus demonstrate an association of the protective effect of dna vaccination with expression of ifnb. we are currently investigating the cellular mechanisms behind this ifnb-mediated protection. multiple sclerosis (ms) is an autoimmune condition characterized by degeneration of nerve fibre myelin sheets. a candidate autoantigen, myelin basic protein (mbp), has especially attracted attention. the presence of anti-mbp antibodies is a predictor of definite ms, but their role in the pathogenesis remains obscure. t cells have long been known to play a pivotal role in the pathogenesis of ms. recently, an important role for b cells as autoantigen-presenting cells has been demonstrated in other autoimmune diseases, including rheumatoid arthritis and diabetes. the uptake of mbp by b cells and the presentation of mbp-derive peptides to t helper (th) cells by b cells may be promoted by the formation of complement (c) activating immune complexes (ics) between mbp and natural autoantibodies in healthy individuals and disease-associated anti-mbp antibodies in ms patients, respectively. we have investigated the formation of mbp-containing ic, the binding of mbp to b cells, the mbp-elicited induction of th-cell and b-cell proliferation and the cytokine production in peripheral blood mononuclear cells (pbmcs) from healthy donors grown in the presence of intact or c-inactivated serum from healthy donors or patients with ms. while mbp did not induce measurable proliferation of b cells nor cd4 þ t cells, we observed the production of tnf-a, ifn-g and il-10 by pbmc in response to incubation with mbp in the presence of sera from healthy controls as well as sera from ms patients. by contrast, no production of il-2, il-4 and il-5 was detected. we are currently investigating the capability of ms sera to promote the formation of mbp-containing ic and thereby enhance the cytokine responses, by virtue of elevated anti-mbp contents. the phagolysosomally localized acid sphingomyelinase (asmase) activated by proinflammatory cytokines such as tnf and ifn-g generates the signalling molecule ceramide which in turn results in the activation of proteases like cathepsin d. these characteristics of asmase suggest a possible role of this molecule in the phagocytotic uptake and phagosomal degradation processes of antigens or in antigen presentation. we show here that asmase -/mice fail to eliminate the noncytopathic lymphocytic choriomeningitis (lcm) virus as rapidly as littermate wildtype mice. investigation of the immune response revealed a reduced expansion of cd8 þ t cells. the secretion of ifn-g in response to contact with target cells as well as the cytolytic activity of virus-specific cd8 þ t cells was severely impaired. additionally, both phases of the lcm virus-specific dth response, mediated by cd8 þ and cd4 þ t cells consecutively, were diminished in asmase -/mice. however, the secondary memory response of virus-specific ctl was not altered, and the 614 abstracts ................................................................................................................................................................................................. virus was effectively controlled for at least 3 months by asmase -/mice. in conclusion, the results of this study suggest an involvement of the asmase in the activation, expansion or maturation of virus-specific cd8 þ t cells during the acute infection of mice with the lcm virus. novel markers for alternative activation of macrophages: macrophage galactose-type c-type lectins 1 and 2 in parallel with the th1/th2 dichotomy, macrophages are capable of developing into functionally and molecularly distinct subpopulations, due to differences in, for example cytokine environment and pathological conditions. while the best-studied, classically activated macrophage is induced by type i stimuli such as ifn-g, a type ii cytokine environment antagonizes the classical activation of macrophages and is capable of alternatively activating macrophages. however, molecular markers associated with these type ii cytokine-dependent, alternatively activated macrophages remain scarce. besides the earlier documented markers macrophage mannose receptor and arginase 1, we recently demonstrated that murine alternatively activated macrophages are characterized by increased expression of fizz1 and ym. we now report that expression of the two members of the mouse macrophage galactose-type c-type lectin gene family, termed mmgl1 and mmgl2, is induced in diverse populations of alternatively activated macrophages, including peritoneal macrophages elicited during infection with the protozoan trypanosoma brucei or the helminth taenia crassiceps, and alveolar macrophages elicited in a mouse model of allergic asthma. we also demonstrate that, in vitro, interleukin-4 and interleukin-13 upregulate mmgl1 and mmgl2 expression and that, in vivo, induction of mmgl1 and mmgl2 is dependent on interleukin-4 receptor signalling. moreover, we show that regulation of mgl expression is similar in human monocytes and monocyte-derived macrophages. hence, macrophage galactose-type c-type lectins represent novel markers for both murine and human alternatively activated macrophages; thus, paving the way for further characterization of the phenotype of macrophages occurring in th2 conditions. background: human parvovirus b19 (b19) is a ubiquitous pathogen, normally causing a mild self-limiting disease, but also capable of causing both significant pathology and long-term persistence. the small size and stability of the virus makes it suitable for mapping of the full breath and the kinetics of the cellular immune responses following acute viral infection. methods: five patients with acute primary b19 infection were included in the study and followed consecutively for up to 200 weeks. cellular immune responses were mapped by ifng enzyme-linked immunospot to overlapping peptides spanning the whole b19 genome. results: in all five acutely infected patients, we were able to monitor the kinetics of a strong specific cellular immune reaction. responses peaked at levels of 850-1850 sfc/ million pbmcs, roughly corresponding to 0.3-0.6% b19specific cd8 þ cells circulating in peripheral blood at 10-80 weeks post-infection. the responses in individual patients were directed to three or four different peptide pools, and the specificity was confined to the same cd8 epitopes present in the pools throughout the follow-up period. the majority of responses were directed to the virus nonstructural protein, only two patients showed any response to the capsid proteins, elicited by the same epitope in both cases. conclusion: the cellular immune responses to acute b19 infection are surprisingly narrow in distribution and remain at high levels for up to 80 weeks post-infection. the initial epitope specificity is maintained, and the majority of responses target the virus nonstructural protein, which is not included in vaccine preparations, evaluated against the infection. the relationship between malnutrition and malaria is controversial. on one hand, malaria may cause malnutrition, while on the other, malnutrition itself may modulate susceptibility to the disease. we investigated the association between plasmodium falciparum malaria and malnutrition in a cohort of children living on the coast of kenya. the study involved longitudinal follow-up for clinical malaria episodes and anthropometric measurements at four cross-sectional surveys. we used poisson regression analysis to investigate the association between malaria and nutritional status. compared to baseline (children with a waz or haz score of !à2), the crude incidence rate ratios (irrs) for malaria in children with low haz or waz scores (<à2) during the period prior to assessment were 1.17 (95% ci 0.91-1.50; 0 ¼ 0.21) and 0.94 (0.71-1.25; 0.67), respectively, suggesting no association between malaria and the subsequent development of pem. however, we found that age was acting as an effect modifier in the association between malaria and malnutrition. the irr for malaria in children 0-2 years old who were subsequently characterized as wasted was 1.65 (1.10-2.20; p ¼ 0.01), and a significant overall relationship between malaria and low-haz was found on regression analysis when adjusting for the interaction with age (irr 1.89; 1.01-3.53; p < 0.05). although children living on the coast of kenya continue to suffer clinical episodes of uncomplicated malaria throughout their first decade, the association between malaria and malnutrition appears to be limited to the first 2 years of life. a. astrinidou-vakaloudi, 1 s. xytsas, 1 i. diamanti, 1 h. ioannidis 2 & p. pangidis 2 1 microbiology department of general hospital of thessaloniki 'agios pavlos', thessaloniki, greece, and 2 nefrology, 2 nd ika hospital of thessaloniki, thessaloniki, greece. e-mail: stasa@hol.gr aim: renal dysfunction may influence the colonization of gastric mucosa by urea-splitting bacteria such as helicobacter pylori, by increasing urea concentrations in the gastric juice. our aim was to investigate the prevalence of h. pylori in patients with end-stage renal disease (esrd), receiving long-term haemodialysis treatment. methods: this study included 40 sera from patients with esrd (29 male and 11 female) undergoing periodic haemodialysis; mean time of treatment was 42.6 months. using elisa technique, we investigated the presence of igg and iga antibodies against h. pylori as well as igg caga (antibodies specific for caga(þ) strains of h. pylori). sera from 40 healthy blood donors were used as a control group. results: h. pylori igg antibodies were detected in 32 out of 40 (80%) patients in the dialysis group, while 31/40 (77.5%) tested positive for iga. igg caga antibodies were present in 13 out of 40 (32.5%). prevalence of h. pylori igg, iga and caga igg antibodies in the control group was 33, 7 and 15%, respectively. conclusions: although international data suggest that prevalence of h. pylori infection is the same in esrd patients as in healthy individuals, in our study that seems not to be the case. the higher blood and gastric juice urea levels may be a risk factor (among many others), but more studies are required in order to understand the relation of h. pylori infection in this group of patients. flanders interuniversity institute for biotechnology, department of molecular and cellular interactions, free university of brussels, brussels, and 2 pasteur institute of brussels, mycobacterial immunology, brussels, belgium. e-mail: tgartner@vub.ac.be immunity against tuberculosis (tb), caused by mycobacterium tuberculosis, depends largely on activation and maintenance of strong cell-mediated immune responses involving both cd4 þ and cd8 þ t cells and the ability to respond with th1-type cytokines, particularly ifn-g. recent studies suggested that bcg, the only licensed vaccine against m. tuberculosis, may fail to induce t-cell responses in the lung mucosa and may therefore not protect against pulmonary tb. a decrease in tb mortality may be achieved by enhancing immunity in the lung. the present study evaluated the induction of antigen-specific immunity in the lung by intranasal (i.n.) delivery of the lipoprotein i (opri) from pseudomonas aeruginosa. opri has shown to be a toll-like receptor 2/4 agonist that, when given subcutaneously, induces type-1 immune responses against heterologous antigens. here, a fusion of opri to ag85a of mtb (opri-ag85a) was used as a subunit vaccine in homologous prime-boost immunizations. in addition, opri-ag85a was combined with an ag85a-encoding dna vaccine (ag85a dna) or with bcg in heterologous prime-boost vaccinations. intranasal and parenteral delivery with opri-ag85a elicited comparable t-cell responses in the spleen; in addition, i.n. delivery elicited specific t-cell responses in the lung lymph nodes (llns). intramuscular delivery of ag85a dna induced significant systemic th1 immune responses. intranasal boosting with opri-ag85a enhanced this response and in addition induced an antigen-specific ifn-g response in lln. opri may therefore be an efficient adjuvant for mucosal boosting. we continue to evaluate the protection induced by opri-based prime-boost vaccinations against pulmonary tb. results on the immunogenicity and protection against intravenous mtb h37rv infection will be presented. toll-like receptors (tlrs) are pattern recognition receptors of the innate immune system, which recognize molecular structures on pathogens or cellular stress-associated molecules. tlr-ligand interactions trigger activation of inflammatory signal transduction and expression of genes involved in host defense. in this study, we have examined the requirement for different tlr adaptor molecules in virus-induced chemokine expression and are currently trying to identify the tlr involved. we have found that both a herpesvirus [herpes simplex virus (hsv)] and a paramyxovirus (sendai virus) require a functional genome to induce expression or proinflammatory chemokines in human and murine monocytic cell lines. for both viruses, this is independent of the tlr adaptor molecules trif and mal. however, overexpression of the vaccinia virus-encoded inhibitor of tlr-signalling a52r or dominant-negative myd88 totally inhibited hsv-induced rantes expression but only partially prevented sendai virus from inducing this chemokine. this suggests that hsv-induced rantes expression occurs via a tlr pathways, whereas sendai virus utilizes both tlr-dependent and -independent pathways to stimulate expression of rantes. we are currently trying to identify the tlrs involved. data from these studies will also be presented at the meeting. 2 0 -5 0 -oligoadenylate synthetases are interferon-induced, double-stranded rna-activated antiviral enzymes which are the only proteins known to catalyse 2 0 -specific nucleotidyl transfer. this first crystal structure of a 2 0 -5 0oligoadenylate synthetase reveals a structural conservation with the 3 0 -specific poly(a) polymerase that, coupled with structure-guided mutagenesis, supports a conserved catalytic mechanism for the 2 0 -and 3 0 -specific nucleotidyl transferases. comparison with structures of other superfamily members indicates that the donor substrates are bound by conserved active site features while the acceptor substrates are oriented by nonconserved regions. the 2 0 -5 0oligoadenylate synthetases are activated by viral doublestranded rna in infected cells and initiate a cellular response by synthesizing 2 0 -5 0 -oligoadenylates, that in turn activate rnase l. this crystal structure suggests that activation involves a domain-domain shift and identifies a putative dsrna activation site that is probed by mutagenesis. we demonstrated that this site is required both for the binding of dsrna and for the subsequent activation of oas. this rna-binding site is different from known rna-binding site; rather than forming a defined three-dimensional domain, it is located at the interface of the two major domains in oas. this novel architecture ensures that the dsrna helix can make simultaneously contact with both domains of oas and ensure the subsequent structural rearrangement leading to the activation of oas. our work provides structural insight into cellular recognition of double-stranded rna of viral origin and identifies a novel rna-binding motif. bacteria-specific iga antibodies are efficient opsonins for neutrophils and mononuclear phagocytes, provided that the phagocytes express the fca receptor (cd89). expression of cd89 can be stimulated by inflammatory cytokines, activated complement factors and certain microbial components. in one study, unstimulated phagocytes were able to ingest iga antibody-treated pneumococci, but only in the presence of complement, which was found to be activated by the iga antibodies along the alternative pathway. pneumococci produce iga1 protease that cleaves human iga1, but not iga2, molecules in the hinge region. this leaves iga1 as faba (monovalent) deprived of fca which contains the docking site for cd89. iga1 is the vastly predominant subclass of iga in the upper airways and circulation of humans. aims: to examine the effects of iga1 protease activity and complement on phagocytosis of iga antibody-coated pneumococci by an unstimulated human phagocytic cell line (hl60). materials and methods: iga1 and iga2 monoclonal antibodies to serotype 4 pneumococcal capsular polysaccharide (ps) were generated by heterohybridoma technique involving b cells from human vaccinees. isogenic serotype 4 pneumococci with and without iga1 protease activity, respectively, were obtained after inactivation of the iga gene of the tigr4 strain. opsonophagocytosis was quantitated using the assay described by romero-steiner et al. based on enumeration of surviving bacteria by culture. the integrity of iga molecules was examined by western blotting. results: both iga1 and iga2 antibody to type-4 polysaccharide-induced phagocytosis of iga1 protease-deficient type-4 pneumococci equally well in the absence as in the presence of complement. iga1 antibody to type-4 polysaccharide displayed a fourfold higher opsonophagocytosis titer against iga1 protease deficient compared to homologous wildtype target bacteria. a similar effect of iga1 protease activity of the target bacteria was not observed in a parallel experiment where iga2 antibody to type-4 polysaccharide served as opsonin. iga1 antibody extracted from iga1 protease-producing target bacteria was almost exclusively in the form of faba. conversely, iga1 from protease-deficient bacteria and iga2 from both types of bacteria were intact. conclusions: these results indicate that the iga1 protease activity of s. neumoniae may help the bacteria escape iga1 antibody-mediated opsonophagocytosis. besides, in these experiments, iga-mediated opsonophagocytosis was independent of complement. vitamins e and c have been found to increase the cellular and humeral immunity of pigs. vitamin e deficiency has also been found to predispose pigs to different diseases, e. coli infection is one among them. after weaning, the vitamin e status of pigs often decreases to a critical low level. in this experiment, we studied whether vitamin c supplementation would be a possible feeding strategy to optimize the immune status of weaners. the interaction between vitamin e and c is interesting due to the reported sparing action on vitamin e or synergism between these to vitamins. piglets were weaned at day 28 of age from sows fed increasing dietary vitamin e during lactation, and piglets were during the following 3 weeks fed either a control diet or this diet supplemented with 500 mg stay-c per kg. blood sampling was obtained weekly from day 28 and until day 49 of age. on the same days, one piglet per dietary treatment was killed and alveolar macrophages (am) were harvested. vitamin c supplementation increased the concentration of igm in serum of piglets throughout the weaning period. although the vitamin e concentration in am decreased with increasing age of the piglets, the concentration was numerically higher in piglets of sows fed the high dietary level of vitamin e. however, vitamin c supplementation tended to increase the total am concentration of vitamin e after weaning and increased the proportion of the biologically most active isomer of vitamin e [rrr-(a-tocopherol)] in the am. the eicosanoid synthesis by am was not influenced by the vitamin c supplementation, but the synthesis of leukotriene b4 was decreased 2 weeks after weaning compared to other days of am harvesting. in conclusion, dietary vitamin c supplementation improved the immune responses of piglets after weaning. a whole blood stimulation assay with escherichia coli (o111:b4) endotoxin was established to measure the capacity of dairy cows to produce the proinflammatory cytokine tumour necrosis factor-a (tnf-a) ex vivo. initially, a time-and dose-dependent study was carried out to find the optimal stimulation conditions for the tnf-a response. the tnf-a response peaked between 3 and 4 h at 38.5 c. a dose in the range of 5-10 g of e. coli lipopolysaccharide (lps)/ml whole blood was found to give the maximum tnf-a response. thirty-eight danish-holstein dairy cows were investigated for their tnf-a responsiveness ex vivo in the periparturient period. heparin-stabilized blood samples were collected seven times over a period of 4 months (weeks à3, à1, 2, 3, 5, 9 and 13 around calving) and stimulated with 5 g/ml of e. coli lps. indeed, fluctuations in the tnf-a responsiveness occurred over time. moreover, the mean tnf-a responsiveness of 38 cows was found to be significantly increased (p < 0.001) in the weeks close to calving. however, in the more stabile physiological periods, some cows had a consistently low tnf-a response, whereas others had high a tnf-a response. we are currently investigating whether high and low tnf-a responders to e. coli lps also exist in dairy cows in vivo. moreover, the importance of tnf-a responsiveness ex vivo to dairy cows' susceptibility and clinical response to experimental e. coli infections in the udder is being investigated. coelomic cytolytic factor (ccf) is a 42 kda invertebrate pattern recognition molecule isolated from the coelomic fluid of the earthworm eisenia foetida (oligochaeta, annelida). ccf displays a number of similarities with the mammalian cytokine tumour necrosis factor-a (tnfa) as a result of a shared n,n 0 -diacetylchitobiose lectin-like domain. however, these similarities are solely functional and are not based on any (dna or amino acid) sequence homology, thus suggesting a form of convergent evolution. in particular, the lectin-like domain of tnf-a has been shown to induce membrane depolarization in various mammalian cell types, through interactions with endogenous amiloride-sensitive ion channels. this nonreceptor-mediated activity of tnf-a has been reported to be involved in the resorption of oedema. likewise, the lectin-like domain of ccf also induces membrane depolarization in mammalian cells. here, we show that ccf appears to be able to induce oedema resorption in an alveolar epithelial cell line through its lectin-like domain. this lectin-like domain of ccf interacts (directly or indirectly) with endogenous sodium and/or chloride channels, and not potassium channels, on mammalian cells. additionally, we suggest that the jnk/sapk and erk1/2 pathways are involved in ccf-induced macrophage activation. these results further establish the functional analogy between an invertebrate pattern recognition molecule and a mammalian cytokine and, from a more applied point of view, suggest the possibility of utilizing ccf in the treatment of oedema. release of svegf and svegfr1 from white blood cells and platelets during surgery and stimulation with bacterial antigens introduction: the influence of surgery on release of soluble vascular endothelial growth factor (svegf) and the soluble vascular endothelial growth factor inhibitory receptor 1 (svegfr1) is unknown. we studied the effect of major and minor surgery on potential variations in svegf and svegfr1 concentrations in vivo and on bacterial antigen-induced release of svegf and svegfr1 from whole blood in vitro. methods: sixty-one patients with abdominal diseases undergoing five different surgical procedures were included. blood samples were drawn from anaesthetized patients before and after the operation. white blood cells and platelets were counted, and plasma svegf and svegfr1 was determined by an elisa method. whole blood from each blood sample was stimulated in vitro with bacteria-derived antigens (lps or protein-a) and svegf and svegfr1 levels were subsequently determined in the supernatants. stimulation with isotonic saline served as control assay. neither svegf or svegfr1 in plasma changed during surgery. in vitro stimulation of blood samples with bacteria-derived antigens resulted in a significant increase in svegf (p < 0.0001) and a less pronounced but still significant increase in svegfr1. release of svegf due to stimulation was significantly higher after the operation (nonsignificant), whereas svegfr1 release remained largely unchanged after surgery. correlation between bacterial antigen-induced release of svegf and neutrophile cell count was highly significant (p < 0.0001). there was no correlation between svegf and platelet cell count, and bacterial antigen-induced svegfr1 release did not correlate with counts of neutrophils and platelets. conclusions: plasma svegf and svegfr1 concentrations did not change during surgery. in vitro bacterial stimulation led to increased release of svegf and svegfr1, which was not significantly amplified during surgery and which may be related to number of circulating neutrophils. natural killer cell functions and subsets after in vitro stimulation with il-2 and il-12, with special emphasis on intracellular ifn-g and nk-cell cytotoxicity r. nyboe, 1,2 t. rix, 1,2 j. krog, 1,2 e. tønnesen 1 & m. hokland 2 1 department of anaesthesiology and intensive care, aarhus university hospital, and 2 institute of medical microbiology, and immunology, university of aarhus, aarhus, denmark. e-mail: rnsr@studmed.au.dk materials and methods: isolated cryopreserved human peripheral blood mononuclear cells (pbmcs) were stimulated with il-2 and il-12. this stimulation has previously been shown to activate nk cells. cell cytotoxicity was measured by flow cytometry after incubation with k562 cells. this method was compared to the current standard 51cr release assay. cells were treated with bfa to accumulate ifn-g, stained for surface markers, permeabilized and stained for intracellular ifn-g. flow cytometry was then performed to measure intracellular ifn-g production in pbmc, especially in nk cells. results: we have demonstrated that stimulation with il-2 and il-12 is effective in increasing the number of ifn-gpositive cells. there is a distinct difference between the cd3-cd56dim and the cd3-cd56bright subsets, with a much greater proportion of ifn-g-positive cells in the cd3-cd56bright subset. the effects of stimulation with il-2 and il-12 on cytotoxicity will be presented, as will the relation between ifn-g production and cytotoxicity. in addition, we will present results of these assays applied to porcine cells. discussion: in combination, these tests will address nk cell function by combining cytotoxicity with ifn-g production in nk cell subsets. the results will demonstrate whether this could serve as a useful tool in describing nkcell function, which could be of value in clinical and experimental settings. culture of regulatory t-cell lines from bronchial mucosa t lymphocytes play a major role in many immune responses. in the last decade, special focus has been on the function of th1 and th2 effector cells. now the importance of regulatory cd4 þ cd25 þ t cells in maintenance of the immunological homeostasis emerges. sarcoidosis is a multisystem granulomatous disorder often affecting the lungs. the typical sarcoid granulomas consists of epitheloid cells, macrophages and lymphocytes, mainly cd4 þ t cells of th1 phenotype. we have cultured t cells from bronchial biopsies of patients with sarcoidosis as well as from controls in high levels of interleukin 2 (il-2) and il-4 and demonstrate spontaneously arising cd4 þ cd25 þ populations and high concentrations of il-10 in these cultures. the main difference between cultures of sarcoid origin compared to controls is a very much higher concentration of the inflammatory cytokines il-6 and tnf-a in cultures of sarcoid origin. the effects of hyperbaric exposure on human peripheral blood mononuclear cells, with special emphasis on natural killer cell cytotoxicity and subsets materials and methods: as an experimental physiological stress model, we examined the effects of hyperbaric exposure on peripheral blood mononuclear cells (pbmcs) obtained from venous blood drawn from eight divers during a simulated heliox saturation dive. eight persons working in normobar atmosphere outside the pressurized chamber served as control donors. the spontaneous cytotoxicity of the pbmcs was estimated in a 4 h 51cr-release assay using k562 as nk-sensitive target cells. the pbmcs were characterized, using 4-colour flow cytometry, with special emphasis on the nk-cell subsets. the data were statistically analysed using a multivariate regression model (stata 8.2). p values <0.05 was considered statistically significant. results: the estimated cytotoxicity increased significantly in both the group of divers and control donors during the dive (pdivers < 0.01 and pcontrols < 0.01). although the cytotoxicity increased relatively more (p < 0.01) in the group of divers compared to the group of control donors between day 1 and 2. discussion: the increased cytotoxicity of pbmc estimated in the group of divers indicate that parts of the cellular immune system are affected during the extreme physiological conditions induced during the initial phase of the presented experimental hyperbaric setup. the increase in cytotoxicity observed in the group of control donors could hypothetically reflect the stress level in persons working outside the pressurized chamber during the dive. the interaction with adhesion molecules expressed by vascular endothelium is the first step in lymphocyte infiltration into tissues. at both cutaneous and mucosal sites interleukin-10 (il-10), il-12 and transforming growth factor (tgf)-b are important regulators of chronic inflammatory disease, where cutaneous lymphocyteassociated antigen (cla) and ae integrin (cd103) may be expressed. unlike cla, cd103 is not believed to play a role in tissue-specific homing but may help to retain t cells within epithelial layers. we have previously shown that il-12 alone can together with an unknown cofactor increase the expression of cla. stimulation with streptococcal pyrogenic exotoxin c (spec) increased the expression of cd103 by cd8 þ but not cd4 þ t cells. while il-12 increased superantigen-stimulated expression of cla, this cytokine strongly inhibited the cd103 expres-sion, and a combination of il-12 and tgf-b completely abrogated the induced cd103 expression. conversely, il-10 suppressed cla but increased cd103 expression. these findings indicate that, in addition to suppressing the development of th1-mediated inflammatory responses, il-10 may also inhibit the migration of cd8 þ t cells into the skin while il-12 promotes such migration. thus, the expression of cla and cd103 may be antagonistically regulated by il-10 and il-12, and the balance between these cytokines could influence the t-cell migration of inflammatory cells into epithelial tissues. during contact sensitivity reaction, immune cells proliferate. in order to study the histological picture of these proliferation phases, we used a mouse model of contact sensitivity in the oral mucosa and on skin. we also used bromodeoxyuridin (brdu, an analogue to thymidin) that is incorporated into the nucleus during cell replication. the hapten oxazolone (oxa) was used to sensitize and elicit the oral mucosa and/or the ear skin. mice were killed at various times after elicitation, and unsensitized animals were also exposed to the hapten as controls. brdu (25 mg/ kg animal) was injected i.p. 2 h before the kill. specimens from the oral mucosa, ear skin and submandibular and auricular lymph nodes were cut and fixed in 4% paraformaldehyde. they were then treated with acid and biotinylated anti-brdu antibody and developed using abc-kit and dab. the analyses were performed using a leica light microscope and the computer program analysis. in the oral mucosa, the frequency of proliferating cells were increasing during the observation period, 4-24 h after elicitation, regardless of site of sensitization. the proliferating cells were found mainly in the basal cell layer of the epithelium. similar patterns were found in ear skin. the regional lymph nodes demonstrated a few scattered proliferating cells 4 h after elicitation. after 24 h, these cells were found frequently in the whole lymph node. control animals exhibited considerable less proliferating cells at all times. we conclude that most proliferating cells were found 24 h after elicitation locally at the hapten-exposed sites (the oral mucosa or the ear skin) as well as in the regional lymph nodes. the endogenous nucleoside adenosine is released in excess during inflammation or other metabolic stress and is generally known to deliver tissue protective anti-inflammatory effects. adenosine acts via four adenosine receptors of which the a2a receptor is the predominant form in t cells. adenosine levels are elevated in asthmatic lung, and adenosine can directly induce mast cell degranulation and bronchoconstriction in these patients. instead, the role of anti-inflammatory mechanisms of adenosine on t cells in asthma is unclear. aim: to study the a2a receptor expression in peripheral blood cd4 þ t cells in asthmatic and healthy individuals using flow cytometric and quantitative real-time pcr methods. results: unstimulated cd4 þ cells of asthmatic patients expressed significantly lower levels (p < 0.001) of a2a receptor in protein level (mean percentage of cells positive ae sem: 76.8 ae 1.2, n ¼ 6) compared to healthy individuals (90.4% ae 1.9, n ¼ 4). double staining for cd69 expression showed that stimulation of cd4 þ cells decreased a2a expression in both groups but indicated that the detected lower levels of a2a in unstimulated cells of asthmatics was not due to preactivation in these patients. surprisingly, a2a mrna expression in unstimulated cd4 þ cells was significantly higher (p < 0.05) in asthmatics (n ¼ 28) compared to healthy controls (n ¼ 7). the expression did not correlate with serum total ige levels. conclusions: asthmatic individuals express less a2a adenosine receptor on their peripheral cd4 þ t cells. the higher mrna levels instead may point to a negative feedback regulation in the receptor expression. the role of possibly decreased adenosine-mediated anti-inflammatory effects in asthma pathogenesis require further studies on this t-cell mediated disease. the chronic inflammatory skin disease atopic eczema (ae) affects almost 15% of the population in many countries today. the pathogenesis of ae is not fully understood. a combination of genetic predisposition and environmental factors like microorganisms seems to contribute to the symptoms. the yeast malassezia sympodialis is part of our normal skin micro flora but can act as an allergen and elicit specific ige and t-cell reactivity in patients with ae. recently, we identified a novel major m. sympodialis allergen, designated mala s 11 (22.4 kda), with sequence similarity to the mitochondrial enzyme manganese superoxide dismutase (mnsod). interestingly, mala s 11 has a high degree of homology to human mnsod. the aim of this study was to examine the effects of recombinant mala s 11 on antigen-presenting dendritic cells. monocytederived dendritic cells (mddcs) from healthy blood donors were cultured with or without mala s 11 for different time periods. it was found that the maturation marker cd83 and the costimulatory molecules cd80 and cd86 were upregulated on the mddcs exposed to mala s 11 for 24 h, as demonstrated by flow cytometry. furthermore, coculture of mddcs with mala s 11 for 9 h induced an increased production of the inflammatory cytokines il-6 (200-fold), tnf-a (100-fold) and il-8 (sixfold), as detected by the cytometric bead array (cba) analysis. our results suggest that mala s 11 affects the immune response through dc maturation and production of inflammatory cytokines. the potential cross-reactivity with human mnsod needs to be explored and the exact role of mala s 11 in the pathogenesis of ae assessed in clinical studies involving skin prick and atopy patch tests. allergen-specific immunotherapy (sit) is commonly conducted with allergen extracts adsorbed to aluminium hydroxide (alum). drawbacks linked to the use of alum, such as the formation of granuloma at the site of injection, have led to suggestions of novel allergen carriers. an alternative carrier is 2 mm carbohydrate-based particles (cbps). in mouse, allergen-coupled cbps have been demonstrated to skew the allergen-specific immune response towards a th1-like activity (grönlund et al. immunology, 2002) . we here coupled the recombinant major cat allergen fel d 1 to cbps (cbp-fel d 1) by cyanogen-bromide activation, resulting in covalent binding. the effect of cbp-fel d 1 on monocyte-derived dendritic cells (mddcs) from healthy human blood donors was studied. we found that the majority of the cd1a þ mddcs were capable of taking up fitc-labelled cbp-fel d 1, as demonstrated by flow cytometry and confocal laser scanning microscopy. furthermore, incubation with cbp-fel d 1 resulted in an upregulation of the costimulatory molecule cd86 on the mddcs, which was not observed with fel d 1 or cbps alone. finally, cbp-fel d 1 induced a fivefold increase in the release of the pro-inflammatory cytokine tumour necrosis factor (tnf)-a and a fourfold increase in the release of the chemokine interleukin-8 from mddcs. taken together, the effects cbps possess make them interesting as novel allergen carriers for sit. the cysteine protease der p1 from dust mite of the genus dermatophagoides pteronyssinus is a major type i allergen. about 80% of house dust mite (hdm) allergic individuals are reactive to this protease in standard assays for detection of ige. a curative treatment for atopic allergy is immunotherapy (it) with hdm extracts which are complex mixtures occasionally resulting in anaphylactic reactions. novozymes focuses on developing a recombinant variant of der p1 which exhibit lowered risk of ige-mediated allergic reactions, while maintaining its ability to trigger proper th-cell responses. this may provide a safer alternative for specific it of hdm allergy. a secreted recombinant form of pro-der p 1 expressed by saccharamyces cerevisiae was obtained by fusion of the pro-enzyme to a fungal signal peptide. the n-glycosylation site of der p1 was mutated resulting in a deglycosylated pro-enzyme with a molecular mass of 35 kda. protein purification procedure was developed to obtain nearly pure der p1 protein followed by determination of concentration by active-site-titration with the cysteine protease inhibitor e64. the deglycosylated recombinant pro-der p 1 revealed immunologic similarity to the native der p 1 molecule when compared in basophile histamine release, ige-binding assays and t-cell proliferation assays. by in silico epitope mapping of a modelled 3-dimensional structure of der p1, five putative igg and ige epitopes were predicted. by protein engineering, the predicted epitopes were removed one by one in der p1 and screening for hypoallergenic variants was performed. combining inhaled long-acting b-2 agonist (laba) and inhaled corticosteroid (ics) seems to offer asthma control at a lower dose of ics than achieved by ics alone. fine mapping of t-cell surface markers by flow cytometry offers a detailed status of the individual's inflammatory response. the frequency of mt2 (cd4 þ cd45ra -cd62l þ cd11adim) and mt1 (cd4 þ cd45ra -cd62l -cd11abright) cells in peripheral blood, and their ratio, has been shown to differ predictably in atopics and patients with leprosy, where mt2 correlates with a th2 phenotype and mt1 with a th1 phenotype. stable asthmatics, requiring fluticasone propionate (fp) 750-1000 mg daily or equivalent, were randomized to receive, double-blinded, either seretide 1 [salmeterol and fluticasone propionate (sfc, n ¼ 16)] 50 mg/500 mg bd or fp 500 mg bd (n ¼ 17). if asthma was controlled based on lung function and symptoms at clinic visits every 6 weeks, ics dose was tapered until asthma exacerbated or 0 mg was reached. the frequency and ratio of mt2 and mt1 t cells of the patients was monitored at 6 week intervals. as treatment tapered, the frequency of mt2 cells decreased (p ¼ 0038 from first to final visit), whereas that of mt1 cells increased. the ratio of mt2/mt1 decreased (p ¼ 0049 from first to final visit). in patients receiving laba þ ics, the fall in mt2/mt1 ratio appeared to be more pronounced than in patients receiving ics alone. thus, the mt2 phenotype may be associated with stable asthma, whereas an imminent exacerbation may associate with an increase in the mt1 phenotype. laba may allow for a greater effect of fp on the mt ratio. activation of complement pathways, leading to production of c3a and c5a anaphylatoxins, has been postulated in the pathogenesis of asthma and allergic airway inflammation. the present study was undertaken to investigate the role of mannan-binding lectin (mbl), an initiator of the lectin pathway of complement, in asthma and allergic rhinitis. mbl levels and mbl-induced complement activity were determined in 19 patients of bronchial asthma with allergic rhinitis and 20 unrelated, age-matched controls of indian origin. mbl levels and activity were correlated with percent eosinophilia and percent predicted fev1 values of the patients. association of single nucleotide polymorphisms (snps) in exon 1 and intron 1 of the mbl with the disease, clinical markers, mbl levels and mbl-induced complement activity was analysed using standard statistical tools. significantly higher mbl levels and activity were observed in patients of bronchial asthma with allergic rhinitis as compared to the controls. we identified five snps, of which two, a816g in exon 1 and g1011a in intron 1 of the mbl, were novel. snp g1011a was significantly associated with the disease (p ¼ 0.0024, or ¼ 5.8696, 95% ci: 1.7316 < or < 19.8963). individuals with 'a' allele at position 1011 showed increased mbl levels, activity and disease severity. our results suggest that 'a' allele at position 1011 leading to high mbl levels and complement activity may be contributing to the severity of bronchial asthma and allergic airway inflammation. serum resistance of borrelia burgdorferi strains belonging to the b. afzelii and b. burgdorferi sensu stricto genospecies is dependent on binding of complement inhibitor factor h. we recently reported that factor h binding by b. burgdorferi is due to inducible expression of several approximately 20 kda plasmid-encoded, surface-exposed lipoproteins related to ospe (e.g. erpa, erpp and p21). in addition, a second class of factor h-binding proteins of approximately 27-35 kda has been described. the ospe-related lipoproteins are dramatically induced by b. burgdorferi during transmission from its tick vector into the mammalian host. the induction of ospe-related lipoproteins during mammalian infection may play a key a role in the borrelial evasion of the host's immune system. the goal of the present study was to define the factor h-binding regions of ospe-related proteins using mutagenesis, peptide mapping and surface plasmon resonance analysis (biacore). the combined studies revealed that the c-terminal regions of both human and mouse factor h (scrs 18-20) specifically bind to ospe-related lipoproteins. we also found fhr-1, whose c-terminal scrs 3-5 are homologous to scrs 18-20 of factor h, to bind to ospe. peptide mapping revealed five putative regions (designated i-v) in ospe that could directly interact with factor h. deleting the c-terminal 15 amino acid residues from region v of p21 abolished its ability to bind factor h. at the same time, however, synthetic peptides corresponding to the c-termini of ospe, p21 and erpp did not inhibit factor h binding to ospe. thus, the c-terminal-binding region v appears to be necessary but not sufficient for factor h binding. when a more specific mutation strategy was employed, where single amino acid residues in peptides spanning over the factor h-binding regions were mutated to alanines, we observed that lysines in the factor h-binding regions of ospe were required for factor h binding. the combined data have revealed that key lysine residues in ospe-related lipoproteins and ionic interactions are crucial for factor h interactions. furthermore, binding of ospe to the c-termini of both mouse and human factor h suggests that borrelia spirochetes utilize analogous complement resistance mechanisms in both rodents and man. in borrelia garinii strains, which in in vitro analyses have been found to be sensitive to complement killing, differences in the ospe sequences as well as in the expression of factor h-binding proteins may account for their susceptibility to serum lysis. role of yada, ail and lipopolysaccharide in serum resistance of yersinia enterocolitica serotype o:3 mannan-binding lectin (mbl), l-ficolin and h-ficolin are pattern recognition molecules of the innate immune system. we investigated the ability of these molecules to bind to different serotypes and noncapsulated variants of streptococcus pneumonia and staphylococcus aureus. we found that mbl binds to noncapsulated s. aureus strain (wood) but not any of the examined s. pneumoniae serotypes. l-ficolin binds to some capsulated s. pneumoniae serotypes (11a, 11d and 11f) as well as some capsulated s. aureus serotypes (type-1, -8, -9, -11 and -12). h-ficolin does not bind to any of the examined s. pneumoniae and s. aureus serotypes included in this study but did bind to a strain of aerococcus viridans. when bound to bacteria, mbl and h-ficolin initiated activation of complement factor c4, whereas l-ficolin did not. during this study, quantitative assays for the three proteins were developed and the concentration in 97 plasma samples were determined and the median values were estimated at 0.8 mg of mbl/ml, 3.3 mg of l-ficolin/ml and 18.4 mg of h-ficolin/ ml, respectively. the absence of early complement components (c1, c4 and c2 but not c3) is a predisposing factor for systemic lupus erythematosus (sle). recently, we demonstrated that, in c4-deficient (c4 def.) mice, igm-containing immune complexes (igm-ic) are filtered by the splenic barrier of marginal zone macrophages (mzm), resulting in an increased immune response against antigens within these igm-ic, but this could not be observed in wildtype or c3 def. mice. we hypothesized that splenic cd11b þ mzm play an important role in the induction of autoimmunity, and we therefore analysed their cytokine profile after isolation with the help of magnetic antibody cell sorting. mrna was isolated, and real-time pcr was performed with specific primers for murine ifn-g (ifn-g), interleukin-12 (il-12) and ifn-a (ifn-a). we observe a moderate increase of il-12 and ifn-g mrna in cd11b þ cells of c4 def. mice compared to wildtype cells. surprisingly, the concentration of ifn-a mrna is six times higher in c4 def. mice. preliminary results suggest that mrna in cd11b þ cells of c3 def. mice is even lower than that in wt. six hours following i.v. application of 20 mg of a abstracts 625 .................................................................................................................................................................................................. murine monoclonal igm anti-dsdna antibody, production of il-12, ifn-g and ifn-a mrna is increased in cd11b þ cells of both c4 def. and wt mice. several references described increased levels of inf-a in patients with sle. dendritic cells are discussed as a major source of ifn-a. our observation that c4-deficient, sle-susceptible mice demonstrate an increased spontaneous ifn-a production by splenic cd11b þ marginal zone macrophages could be an early sign and a trigger for the development of sle. this is supported by the fact that the absence of c3 is not a predisposing factor for sle and our observation that c3 def. animals display low levels of ifn-a mrna. 200-400 million people worldwide and represents one of the leading causes for liver cirrhosis and hepatocellular carcinoma. control over the hbv infection is achieved mainly by vaccination with hepatitis b surface antigen (hbsag). hbsag contains n-linked glycosylation side and is recognized by both mbl-a and mbl-c in a cadependent manner. hbsag-mbl complexes activate complement and may thus affect humoural immunity. to investigate the role of mbl in humoural responses to hbsag, we immununized mice that lack both mbl-a and mbl-c proteins with soluble hbsag. it has been shown that deficiencies in other complement components like c1q, c4 and c3 result in decreased antibody responses. however, mbl double ko animals mounted dramatically increased humoural responses. after priming, mbl double kos mounted hbsag-specific igm responses, which were threefold higher than wt controls. after boosting the hbsag, total igg was 10-fold higher in mbl ko than in wt control animals. similar to the response to hbsag, other glycosylated soluble antigens (e.g. invertase) induced better humoural responses in mbl double ko animals, suggesting that mbl plays an important role in a negative feedback regulation of adaptive immunity. reconstitution experiments with rmbl partially rescued the ko phenotype. we propose that the clearance of glycoprotein antigens in mbl ko is handled differently from the wt, resulting in better stimulation of humoural responses. alternatively, glycoprotein-ag-mbl-rich complexes inhibit b-cell responsiveness via putative mbl receptors. the complement system is an important part of the innate immune system. the activation of complement proceeds through three different pathways that converge in the generation of c3-activating enzyme complexes. complement activation via the lectin pathway is initiated when recognition molecules, mannan-binding lectin (mbl) or ficolin, bind to carbohydrate structures characteristic for microbial surfaces. in the circulation, mbl and ficolins are found in association with three structurally related mblassociated serine proteases (masp)-1, -2 and -3 and a small, nonenzymatic component, map19. masp-2 has been shown to elicit complement activation through the sequential proteolytic cleavage of c4 and c2 upon binding of mbl/masp-2 complexes to microbial surfaces. we have recently uncovered a polymorphism in the masp-2/map19 gene in a patient shown to be deficient in the lectin pathway of complement activation. the polymorphism results in a single amino acid substitution in the n-terminal part of the masp-2 protein. recombinant wildtype masp-2 and masp-2 containing the amino acid substitution in question was produced, and the ability to activate complement was studied. the mutation had a profound impact on masp-2 function, resulting in the lack of complement activation through the lectin pathway. elisa-based experiments showed that the mutation leads to the impairment of complement activation through influencing the binding of masp-2 to mbl or ficolins. deficiencies in the lectin pathway of complement activation have so far been accounted for only by lack of functional mbl. the mutation described above is the first defect described affecting both activation through mbl and the ficolins. .................................................................................................................................................................................................. th1, th2 and treg cell balance. dcs are present in the gut mucosa and may thus be target for modulation by gut microbes, including ingested probiotics. here, we tested the hypothesis that species of lactic acid bacteria, important members of the gut flora, differentially activate dc. a large panel of human gut-derived lactobacillus and bifidobacterium spp. was screened for dc-polarizing capacity by exposing bone marrow-derived murine dc to lethally irradiated bacteria. cytokines in culture supernatants and dc-surface maturation markers were analysed. substantial differences were found among strains in the capacity to induce interleukin-12 (il-12) and tumour necrosis factor (tnf)-a, while the differences for il-10 and il-6 were less pronounced. bifidobacteria tended to be weak il-12 and tnf-a inducers, while both strong and weak il-12 inducers were found among the strains of lactobacillus. remarkably, strains weak in il-12 induction inhibited il-12 and tnf-a production induced by an otherwise strong cytokine-inducing strain of lactobacillus casei, while il-10 production remained unaltered. selected strains were tested for induction of dc maturation markers. those lactobacilli with greatest capacity to induce il-12 were most effective in upregulating surface mhc class ii and cd86. moreover, l. casei-induced upregulation of cd86 was reduced in the presence of a weak il-12inducing l. reuteri. in conclusion, human lactobacillus and bifidobacterium spp. polarize differentially dc maturation. thus, the potential exists for th1/th2/treg-driving capacities of the gut dc to be modulated according to composition of gut flora including ingested probiotics. the intestinal micro flora is indispensable in developing and maintaining homeostasis of the gut-associated immune system. evidence indicates that lactic acid bacteria (lab), e.g. lactobacilli and bifidobacteria, have beneficial effects on the host. established health effects include increased gut maturation, antagonisms towards pathogens and immune modulation. the objective of this study is to evaluate the immunomodulating properties of a range of lab of human origin. as dendritic cells (dcs) play a pivotal role in the balance between tolerance and immunity to commensal microorganisms, in vitro-generated immature dcs serve as a suitable model for studying the immunomodulating effects of lab. human immature dcs were generated in vitro from monocytes and exposed to lethally uv-irradiated lab. the effect of various species of lab on dcs in direct contact was evaluated. furthermore, the maturation pattern of dcs separated from the bacteria by an epithelial cell layer (caco-2 cells), which should mimic the intestinal environment, was studied. cytokine secretion (il-12, il-10 and tnf-a) and upregulation of maturation surface markers on dcs (cd83 and cd86) was measured. different lab induced diverse cytokine responses. some strains were strong il-12 and tnf-a inducers and others weak. all strains induced il-10. different lab also differentially modulated expression of cd83 and cd86 on dcs. although some variation in the response to lab of dcs generated from different blood donors was observed, general differences in the effect of the various lab was revealed. experiments with the dc caco-2 coculture system are ongoing. different species of lab differentially affect dc maturation; this suggets that the gut flora plays a pivotal role in polarization of the immune response. natural killer (nk) cells are cells of the nonspecific immune system lysing altered self-cells. a noncytolytic subset of nk cells may serve a regulatory role by secreting cytokines. bacteria translocating across the gastrointestinal mucosa are presumed to gain access to nk cells, as consumption of certain lactic acid bacteria has been shown to increase in vivo nk cytotoxicity. here, we investigated how human gut flora-derived lactobacilli affect nk cells in vitro, by measuring proliferation and ifn-g production of human nk cells upon bacterial stimulation. cd3 -cd56 þ nk cells were isolated from buffy coats by negative isolation using non-nk lineage-specific antibodies and magnetic beads. nk cells were incubated with 10mg/ml uv-inactivated bacteria or 10mg/ml phytohemagglutinin (pha) for 4 days. proliferation was assessed by incorporation of radioactive thymidine into nk-cell dna. the ifn-g concentration was measured by elisa. incubation of nk cells with a lactobacillus acidophilus strain increased the proliferation of the nk cells and induced ifn-g production, both to levels comparable to pha stimulation. the proliferative response was further enhanced with autologous monocytes present, probably because cytokines, secreted by monocytes having engulfed bacteria, stimulated the nk cells. in contrast, a lactobacillus paracasei strain caused the nk cells to proliferate only in the presence of monocytes. these results demonstrate that various strains of lactobacilli have the capacity to activate nk cells in vitro, in a monocyte-dependent or -independent way. hence, the encounter of nk cells with lactic acid bacteria will affect nk-cell activation. such activation of nk cells may potentially skew an on-going or subsequent immune response towards a th1 response. lactobacilli are nonpathogenic gram-positive inhabitants of the normal human intestine known for their healthpromoting effects. in our earlier work, it is shown that human monoclonal antibody isolated from sera of a patient with waldenstrom macroglobulinaemia possess innate antibody characteristics and binds to lactic acid bacteria. according to the immune network model, immunization with this bacteria could induce the perturbations in immune system that might result in production of anti-lactobacillus antibodies, human monoclonal antibody like (ab1) and anti-idiotypic antibody (ab2). in this study, balb/c mice were immunized with two doses of bacteria lactobacillus acidophilus in complete and incomplete freund's adjuvant and phosphate-buffered saline (pbs), respectively. seven days after the last immunization, sera from immunized mice were collected and the presence of lactobacillus-specific ab1 and ab2 were determined by elisas. in the sera of immunized mice, antibodies specific to bacteria lactobacillus acidophilus were shown. the concentration of lactobacillus-specific antibodies was higher in the sera of hyperimmunized mice (mice immunized with 1 mg of igm dj) than in sera of mice immunized with 100 times lower doses of immunogen (0.01 mg per doses). moreover, ab1 and ab2 antibodies were detected in the sera of lactobacillus-hyperimmunized mice. in this study, we have shown the idiotypic network interactions in mice immunized with bacteria lactobacillus acidophilus. the normal gastrointestinal flora is crucial for the maturation of the acquired immunity via effects on antigenpresenting cells (apcs). here, we have investigated how two types of apcs, monocytes and dendritic cells (dcs), react to different bacterial strains typical of the commensal intestinal flora. purified monocytes and monocyte-derived dcs were stimulated with uv-inactivated gram-positive (lactobacillus plantarum and bifidobacterium adolescentis) and gram-negative (escherichia coli and veillonella parvula) bacterial strains. monocytes produced higher levels of il-12p70 and tnf, as detected by elisa, in response to l. plantarum than to e. coli and v. parvula. in contrast, dcs secreted high amounts of il-12p70, tnf, il-6 and il-10 in response to e. coli and v. parvula but were practically unresponsive to l. plantarum and b. adolescentis. the lack of response to the gram-positive strains correlated with a lower surface expression of toll-like reseptor 2 (tlr2) on dcs compared to monocytes. the surface expression of tlr4 on dcs was undetectable when analysed by flow cytometry, but blocking this receptor decreased the tnf production in response to v. parvula, indicating that low tlr4 expression on dcs is sufficient to mount an inflammatory response to gram-negative bacteria. ifn-g increased the expression of tlr4 on dcs and also potentiated the cytokine response to gram-negative bacteria. our results indicate that, when monocytes differentiate into dcs, their ability to respond to different commensal bacteria dramatically changes, thereby becoming unresponsive to probiotic gram-positive bacteria. these results may have important implications for the capacity of different groups of commensal bacteria to regulate mucosal and systemic immunity. probiotic bacteria, e.g. lactobacillus spp., may improve diseases such as chronic inflammatory bowel disease. we examined cytokine production and phenotypic change after in vitro stimulation of t cells from healthy volunteers using different probiotic strains. methods: t cells were cultured from colonic biopsies from eight healthy volunteers (agnholt and kaltoft, exp clin immunogenet 2001; 18:213-25) , and dendritic cells were matured from their peripheral blood mononuclear cells. t-cell cultures were stimulated with autologous bacterial sonicate or strains of lactobacillus spp., with and without the addition of dendritic cells. cytokine levels (tnf-a, ifn-g, il-10 and gm-csf) and phenotype (cd3, cd4, cd25 and cd69) were measured on day 4. results: lactobacillus spp. induced higher productions of tnf-a and il-10 than did autologous bacteria. in presence of dendritic cells, the production of all cytokines increased. however, the increases of ifn-g and tnf-a were more pronounced in wells with autologous bacteria than in wells with lactobacillus spp. the addition of dendritic cells upregulated cd25 expression without simultaneous upregulation of cd69. the upregulation was pronounced after stimulation with lactobacillus rhamnosus gg compared with autologous bacteria and other lactobacilli. discussion: in presence of dendritic cells, autologous bacteria induced inflammatory cytokines, while probiotics mainly induced regulatory cytokines. lactobacillus rhamnosus gg induced a regulatory phenotype (cd25 þ ), in part mediated by dendritic cells. future studies will address whether this shift to a cd25 þ phenotype represents a differentiation into competent regulatory t cells. in a clinical context, such cells might be used for treatment of inflammatory diseases. protein microarrays will play a key role in the postgenomic era and offer a unique possibility to perform highthroughput global proteome analysis. a chip can be printed with thousands of protein probes (e.g. antibodies), the biological sample added (e.g. a proteome) and any binding detected. we aim to develop protein microarrays based on human recombinant scfv antibody fragments for global proteome analysis. the concept of comparing proteomic maps of healthy versus diseased samples will allow diseasespecific proteins to be detected. in fact, antibody microarrays will allow us to perform comparative proteome analysis on any sample format in a species-independent manner, as long as a proteome can be isolated. however, the complexity of proteomes, containing several thousands of different proteins, is a problem. here, we have designed antibody microarrays targeting the water-soluble fraction of a proteome. to this end, an anticytokine antibody array was developed and human dendritic cells (aeactivation) was used as model system. the results showed that our antibody microarrays could be used to examine the cytokine profile in complex samples. furthermore, we have taken the first steps towards comparing our results with those of other technologies on both the protein and gene level. due to the complexity of the model proteome, we also examined the possibility to prefractionate the proteome in a simple one-step procedure (based on size) prior to the labelling step. in more detail, the sample proteome was fractionated into two fractions using membrane devices with different molecular weight cut-offs. the results showed that the fractionation considerably enhanced the assay sensitivity allowing cytokines in the pg/ml range to be readily detectable. the immunomodulatory effect of heat shock protein 70: immunization with a dna construct based on the malarial antigen fused with a fragment of hsp 70 primes for a th-1 type of response finding an appropriate adjuvant for human vaccination is crucial. heat shock proteins (hsps) act as adjuvants when coadministered with peptide antigens or given as fusion proteins. however, there is a potential risk of autoimmunity when using the complete molecules, because hsps are evolutionary conserved. to overcome this, we first evaluated the adjuvant effect against two different antigens of a less-conserved fraction of plasmodium falciparum hsp70 (pf70c) and compared it to the whole hsp70 molecule from trypanosoma cruzi (tchsp70). we found that pf70c exhibited similar adjuvant properties as the whole molecule. we later evaluated the adjuvant potential of pf70c against the malarial antigen eb200 in a chimeric dna construct. no appreciable levels of eb200-specific abstracts 629 .................................................................................................................................................................................................. antibodies were detected in mice immunized only with the dna constructs. however, dna primed the immune system, because subsequent challenge with the corresponding recombinant fusion proteins elicited a strong th-1 antibody response. in contrast, no priming effect was observed for ex vivo ifn-g production but stimulation with the hsp-chimeric fusion protein induced a stronger secretion of ifn-g in vitro than other proteins used. these results indicate that the use of hsps is promising in the design of new vaccines. high-throughput proteomics on antibody-based microarrays: the importance of probe and surface design in analogy to dna microarrays, protein microarrays offer a new distinct possibility to perform sensitive highthroughput global proteome analysis. however, the development of the protein microarray technology will place high demands upon the design of both probes and solid supports. the analysis of thousands of heterogeneous proteins on a single microarray requires the use of uniform probes, such as antibodies, directly designed for protein microarray applications. we have recently generated a human recombinant single-chain fv antibody library, genetically constructed around one framework, the ncoder-library, containing 2 â 1010 clones. single framework antibody fragments (sinfabs) selected from this library were successfully applied as probes for microarrays providing sensitive detection in the 600 attomol (mass spectrometry) and the 300 zeptomole range (fluorescence). however, the choice of framework is critical. we have shown that the selected ncoder framework displayed excellent functional on-chip stability and arrayed dehydrated probes retained their activity for several months. furthermore, we have addressed the issues of biocompatibility of the solid support and immobilization strategies for our microarray setup. an in-house-designed substrate, macroporous silicon coated with nitrocellulose (map3-nc7), displayed properties equal to, or better than, those of five commercially available supports used as reference surfaces. we have also evaluated different coupling strategies, such as adsorption, covalent coupling, diffusion and affinity coupling. using a novel affinity tag, the double-(his)6-tag, we increased the binding efficiency of sinfab-molecules to ni2 þ -coated solid supports, thereby allowing nonpurified probes to be directly applied. the mannan-binding lectin (mbl) pathway is part of the innate immune system providing a first line of defence against infections. mbl and ficolins circulate in complexes with mbl-associated serine proteases (masp-1, -2 and -3). after recognition of a microorganism by mbl, activation of the complement system occurs. masp-1 and masp-3 share five domains (making up the so-called a-chain), whereas they have unique protease domains (b-chains). before the identification of masp-3, an assay for masp was presented, based on antibodies against the a-chain of masp-1. with the new knowledge of the three masps, and the sharing of domains by masp-1 and masp-3, assays specific for the protease domains have to be constructed, if one wishes to measure the proteins individually. we present an assay for quantifying total masp-3 in plasma and serum samples. the assay is a sandwich-type assay using as catching antibody a monoclonal antibody against the common a-chain of masp-1/3 and a developing secondary antibody against the c-terminal part of the protease domain of masp-3. we have used this assay for estimating the normal concentration of the protein as well as the concentration in patients and also for characterizing by gel permeation chromatography the masp-3 protein in serum. inducible costimulator ligand (icosl) is a costimulatory molecule related to b7.1 (cd80) and b7.2 (cd86). b cells, monocytes, dendritic cells and endothelial cells express icosl. inducible costimulator (icos) interacts with icosl, and this interaction leads to signals involved in isotype switching and the development of immunological memory. hitherto, no polymorphisms of this gene have been described. the aim of this study was to reveal variation of the icosl gene in normal individuals. all eight exons, except exon 1, were sequenced with flanking introns in 10 healthy blood donors. eight single nucleotide polymorphisms (snps) and two length polymorphisms were found. one of the snps was found in the coding regions of the gene. the base involved was located in exon 3 and caused a conservative amino acid change from valine (gtt) to isoleucine (att). three individuals were heterozygous g/a for the exon polymorphism, while the remaining seven individuals were homozygous for the wildtype g/g. exon 3 encodes the immunoglobulin variable (igv)-like domain of the molecule which is situated outside the cell. this means that the amino acid could be critical for the stability of the molecule or could constitute part of the binding site for icos. the results form the basis for further experiments to find possible associations of the alleles to diseases caused by immune dysregulation. especially, the exon 3 variant is interesting and could play a role for the development of immunological diseases. besides, it would be interesting to see whether both exon 3 alleles are expressed or only the wildtype allele is functional. interferon-a/b (ifn-a/b) is increasingly used as antiviral and immunomodulatory therapies. unfortunately, bioavailability varies with ifn species and mode of administration, and all ifn species are potentially immunogenic. assays for antiviral activity (ifn) and antiviral neutralization (antibodies, nab) have been used for some time to monitor patients on ifn biologicals. these assays require laborious titrations making them unsuitable for large-scale clinical use. myxovirus a (mxa) is a resistance gtpbinding protein that is specifically induced by treatment with type 1 ifns. for example, ifn-b-induced mxa in blood leucocytes has been used as a biomarker in ifn-btreated patients with multiple sclerosis. however, the degree of specificity of mxa in this regard is unclear, and measurements of mxa protein and/or mrna are not yet suitable for routine clinical use. in an attempt to find new and better reporter genes (and, hopefully, genes and gene products with proven specificity for ifn-a and -b), microarray screenings with u133a genechips (affymetrix) were carried using human blood leucocytes and the human lung carcinoma cell line a549. we studied the simultaneous expression of 22,000 transcripts before and after exposure to human recombinant ifn-a and ifn-b and other antiviral and immunomodulatory cytokines. the results will be presented at the conference. interferon-a/b (ifn-a/b) is increasingly used as antiviral and immunomodulatory therapies. unfortunately, bioavailability varies with ifn species and mode of administration, and all ifn species are potentially immunogenic. assays for antiviral activity (ifn) and antiviral neutralization (antibodies, nab) have been used for some time to monitor patients on ifn biologicals. these assays require laborious titrations making them unsuitable for large-scale clinical use. our laboratories have therefore modified the antiviral assays for ifn bioactivity and nab, so that they are suitable for large-scale screening in specialized laboratories. the read-out is survival of a subcloned a549 cell line in the presence of an otherwise lethal amount of virus. thus, survival increases in the presence of type 1 ifn and decreases in the presence of nab against the ifn added to the cells. mxa is induced by type 1 ifn and can be used for measuring the nab activity. in another assay, the mxa level in the a549 cell line is measured. in an attempt to find a new and better reporter gene for type 1 ifn than mxa and genes specific for either ifn-a or -b, a micro array screen was carried using the u133a chip from affymetrix. the expression of 22,000 genes can be studied simultaneous with this technology. the results will be presented at the conference. in our laboratory, we have developed a database system, which we believe is of immediate interest to the general scientific community. the database represents a computerbased replacement for the laboratory notebooks used in the majority of research laboratories worldwide. in addition, the database provides an effective tool for organizing and managing laboratory information at all levels, spanning from managing and revising standard operating procedures and producing documentation of research activities to keeping track of data and conclusions. using the commercially available database toolkit software filemaker pro, we have developed a relational database solution for management of laboratory information. the system consists of a hierarchy of five interrelated databases, each pertaining to a separate type of information, namely, overall project information, information relating to individual experiment setups, documentation of daily research activity, generated data and descriptions of standard operating procedures. like other databases, each individual database consists of a number of records, each comprised of a set of fields in which information is entered. in each record, a certain field is reserved to specify the relation of the record to a record in another database at a higher level. thus, the database is essentially five databases linked by a hierarchy of one-to-many relations, organizing information in a folder-like structure. importantly, the database system allows multiple users to access and edit records simultaneously, and the data entered in one database immediately becomes accessible through the other databases. the limitations of laboratory notebooks are apparent when looking for information, which is dispersed throughout one or more notebooks, or possibly on loose sheets of paper or printouts 'somewhere'. the often complicated process of gathering laboratory data or results when writing grant applications or research papers is made considerably easier with the database system. thus, the database solution presented should be broadly attractive to researchers, irrespective of their scientific discipline. an effective sars vaccine is likely to include components that can induce specific cytotoxic t-cell (ctl) responses. the specificities of such responses are governed by hlarestricted presentation of sars-derived peptide epitopes. exact knowledge of how the immune system handles protein antigens would allow for the identification of such linear sequences directly from genomic/proteomic sequence information. the latter was recently established when a causative coronavirus (sars cov) was isolated and full-length sequenced. here, we have combined advanced bioinformatics and high-throughput immunology to perform an hla supertype, genome-wide scan for sars-specific cytotoxic t cell epitopes. the scan includes all nine human hla supertypes in total covering >99% of all major human populations. for each hla supertype, we have selected the 15 top candidates for test in biochemical-binding assays. at this time (approximately 6 months after the genome was established), we have tested the majority of the hla supertypes and identified almost 100 potential vaccine candidates. these should be further validated in sars survivors and used for vaccine formulation. we suggest that immunobioinformatics may become a fast and valuable tool in rational vaccine design. rationale: major histocompatibility complex class i (mhc i) molecules monitor the protein content of the cell by binding small derived peptides and presenting them to cytotoxic cd8 þ t cells. the goal of the human mhc project is to predict the binding strength of any given peptide/mhc complex. this prediction allows the design of peptide-based vaccines. the prediction requires representative binding data from mhc alleles from all the nine hla supertypes. here, we describe the genetic construction, protein production and purification as well as the establishment-binding assays for two recombinant mhc supertype alleles, hla-b*1501 and hla-b*5801. methods: using the quikchange multisite directed mutagenesis kit (stratagene), codon-optimized genes encoding hla-b*1501 and hla-b*5801 are created. the two mhc i molecules are fermented and purified by ion exchange chromatography, hydrophobic interaction chromatography and size exclusion chromatography. the binding (kd) of natural t-cell epitopes, as well as predicted peptide ligands, is described by radioactive immunoassays (rias) and enzyme-linked immunosorbent assays (elisas). the mhc molecules are biotinylated during expression. results: the expression of mhc i resulted in multiple disulfide bond isomers, which are separated by hydrophobic interaction chromatography and used in subsequent binding studies resulting in the determination of kd for various peptide ligands ranging from strong binders we have previously demonstrated that bioinformatics tools such as artificial neural networks (anns) are capable of performing pathogen-, genome-and hlawide predictions of peptide-hla interactions. these tools may therefore enable a fast and rational approach to epitope identification and thereby assist in the development of vaccines and immunotherapy. a crucial step in the generation of such bioinformatics tools is the selection of data representing the event in question (in casu peptide-hla interaction). this is particularly important when it is difficult and expensive to obtain data. herein, we demonstrate the importance in selecting information-rich data and we develop a computational method, query-bycommittee, which can perform a global identification of such information-rich data in an unbiased and automated manner. furthermore, we demonstrate how this method can be applied to an efficient iterative development strategy for these bioinformatics tools. methods: a large panel of binding affinities of peptides binding to hla a*0204 was measured by a radioimmunoassay (ria). this data was used to develop multiple first generation anns, which formed a virtual committee. this committee was used to screen (or 'queried') for peptides, where the anns agreed ('low-qbc'), or disagreed ('high-qbc'), on their hla-binding potential. seventeen low-qbc peptides and 17 high-qbc peptides were synthesized and tested. the high-or low-qbc data were added to the original data, and new high-or low-qbc second generation anns were developed, respectively. this procedure was repeated 40 times. the high-qbc-enriched ann performed significantly better than the low-qbc-enriched ann in 37 of the 40 tests. conclusion: these results demonstrate that high-qbcenriched networks perform better than low-qbc-enriched networks in selecting informative data for developing peptide-mhc-binding predictors. this improvement in selecting data is not due to differences in network training performance but due to the difference in information content in the high-qbc experiment and in the low-qbc experiment. finally, it should be noted that this strategy could be used in many contexts where generation of data is difficult and costly. interleukin-18 (il-18), a pro-inflammatory cytokine that is produced by both lymphoid and nonlymphoid cells, has a critical role in modulation of innate and adaptive immunity. its primary function in stimulation of ifn-g production and stimulation of nk-cell-cytotoxic activities makes this cytokine a candidate for cancer immunotherapy. in oral cavity, this cytokine is produced by oral epithelia and carcinoma cells and is related to tumour regression in nude mice bearing salivary adenocarcinoma. however, direct effects of this cytokine on oral cancer cells have not been elucidated. in this project, we investigated il-18 effect on an oral carcinoma (kb) cell line. with rt-pcr technique, kb-cell line was found to express il-18 receptors (il-18ra and il-18rb), indicating that this oral carcinoma line is a target for il-18 study. we showed that recombinant human il-18 inhibited kb-cell proliferation by 17% at concentration of 100 ng/ml (p < 0.05), whereas ldh release by these cells in treatment group and control groups was comparable, indicating that il-18 suppression of cell proliferation was not mediated by the induction of cell death. to further address this hypothesis, we found that il-18 treatment did not induce apoptotic cell death, as studied by dna laddering and tunel assays. in addition, expression pattern of cell death-controlling genes (bcl-2 and bax) was not altered by this cytokine. findings in these studies indicated that suppression of kb-cell proliferation may be attributed to control of cell cycle, growth arrest or induction of cell differentiation. the data presented in this project could provide an insight of how cancer cell directly responds to il-18, as this cytokine is an important regulator of anticancer mechanisms. aloe emodin (ae) is a naturally occurring compound with wide spectrum of biological properties, including antimicrobial, vasorelaxant, immunosuppressive and anticancer actions. this anthraquinone induces apoptosis in several tumour cell lines with special affinity to tumours of neuroectodermal origin. high amounts of nitric oxide (no) released by activated macrophages induce tumour cell death. therefore, we explored the capacity of ae to modulate no-mediated antitumour response in vitro. interestingly, while ae markedly suppressed no release from macrophages alone, it significantly potentiated no production in cocultures of macrophages and c6 cells, after 48 h of cultivation. accordingly, the viability of c6 cells cocultivated with macrophages was reduced in the presence of ae. moreover, the observed ae-imposed potentiation of no production in macrophages was closely related to macrophage culture cell density. according to these data, we proposed that no modulator capacity of ae strongly depended on intercellular contact, indicating that macrophage antitumour response was not compromised but even potentiated by ae. immunotherapy represents an attractive fourth-modality therapeutic approach, especially in the light of the shortcomings of conventional surgery, radiation and chemotherapies in the management of metastatic cancer. to this end, a large number of peptide antigens derived from taa have been applied in immunotherapeutic trials for the treatment of various malignancies, e.g. cancers of the breast, prostate and kidney, in addition to haematological cancers. in some cases the response rates have been impressive and no adverse autoimmunity have been observed. a major strategic difficulty associated with these trials relates to the choice of best-suited peptide antigens. the vast majority of the antigens described thus far is not vital for survival and growth of the tumour cells, and immunoselection of antigen-loss variants may therefore prove to be an additional obstacle for the clinical applicability of most of the known peptide epitopes. in this respect, the development of acquired antigen loss during immunotherapy has been demonstrated in several cases. obviously, the development of loss-variant tumour cells implies that these cells acquire a pronounced growth advantage and are left unaffected by further treatment. ideally, target antigens should be derived from proteins required for survival and growth of tumour cells, as antigens with these characteristics would not be inflicted by the development of loss-variant tumour cells. in this respect, several inhibitors of apoptosis proteins (iaps) are universally expressed among tumours and play an important role in tumour cell escape from apoptosis. we have characterized spontaneous t-cell reactivity against iapderived peptides in cancer patients. from the iap survivin, we have characterized peptides restricted to the class i molecules hla-a1, a2, a3, a11, b7 and b35. furthermore, we have demonstrated that survivin-specific t cells infiltrate metastatic lesions and that isolated survivinspecific ctls are capable of killing hla-matched tumour cells. survivin-derived peptides are now in clinical trial, and continued work in our lab has demonstrated that other iaps are targets for spontaneous t-cell reactivity in cancer patients. we previously reported that in mice with large progressing t-cell lymphoma tumours, dysfunctions in the antitumour ctl activity occur, associated with an accumulation of splenic arginase-producing myeloid suppressor cells (mscs). in this study, we first demonstrate that both the presence and the activation state of these msc depends on tumour evolution. while in tumour regressors hardly any arginase-producing msc can be found, both the amount and the arginase activity of this population expands from early over late progressors. this gradual induction of mscs is paralleled by an increasing suppression of ctl activity and th1, but not th2, cytokine production. upon analysing the molecular repertoire of msc in vitro, we found, besides arginase1, a well-established marker for alternatively activated myeloid cells or m2, a strong upregulation of fizz1 and ym, two additional recently identified markers for m2. further evaluation of molecular markers by microarray analysis in msc yielded genes involved in wound healing (e.g. coagulation factor xiiia), anti-inflammation (e.g. selenoprotein p), immunomodulation (e.g. pd-l2) and fat and sugar metabolism (e.g. leptin receptor). of note, many of these genes are regulated by type 2 cytokines (il-4, il-13 and il-10) and are therefore rather m2 associated. overall, our data provide new markers for msc in cancer and further establish their m2 activation state. study. only sp-a showed a significant expression in normal mucosa which was downregulated in crc. as the absolute signal level was below the noise threshold, these results have to be interpreted with caution and require confirmation by direct measurenment of the proteins. our results suggest that there is no major role for the human collectins in colorectal cancer. tetramerization is visualized by sds-page. conclusion: an effective method for the production of highly pure mhc i molecules has been applied to hla-b*1501 and hla-b*5801, and ria and elisa binding assays for those alleles have been established background: proliferation, differentiation and apoptosis are essential processes in the normal functions of the mammary epithelium. the hypothesis examined in this study is that the transcription factor bcl-6 is critically important not only for regulating b-cell growth and development but also for mammary epithelial apoptosis. methodology: twenty breast cancer cases and 31 healthy controls were used to investigate whether bcl-6 protein in involved in breast cancer (grade iii). full length bcl-6 cdna was retrovirally transduced into eph-4 cell line. we then used flow cytometry of brdurd-stained cells to investigate the cell-cycle duration of the control and transduced cell lines. tunel was used as a marker of apoptosis to find out differences in the frequencies of apoptotic cells in the control and transduced cell lines. finally, immunohistochemistry staining was performed to detect bcl-6 in breast cancer (iii). results: restoration of bcl-6 into eph-4 cells not only inhibits apoptosis but also prolongs the cell cycle and results in increased cell size and protein content. the results also indicated that the cell-cycle time of bcl-6-transduced eph-4 cells is prolonged by about 3 h, presumably as a result of the action of bcl-6 at the bcl-6 at the g1/s transition. we found differences in the frequencies of viable and apoptotic cells in cultures of the parent eph-4 cells, control-transduced eph-4 cells and bcl-6-transduced eph-4 cells. consistently, we demonstrated that bcl-6 is expressed in 90% of high grade of breast carcinoma, which is considered as the most aggressive of tumours. conclusion: together, these results suggest that bcl-6 is likely to be involved in mammary gland development and carcinogenesis. inflammatory cytokines have a critical role in modulation of both innate and adaptive immunity in response to foreign antigen. they also play an important role in anticancer immunity. for example, they can promote cell-mediated immunity against cancer cells. with their immunostimulatory effects, these cytokines are being tested for cancer treatment in the form of dna vaccine or adjuvant or therapeutic cytokines. direct effect of these cytokines on cancer cell, however, is still unclear. in this project, we investigated whether il-1( and il-18 can modulate cancer cell proliferation. we employed a simple nonradioactive proliferation (mtt) assay and detection of lactate dehydrogenase (ldh) to test the effect of these recombinant human cytokines on various cancer cell lines, including breast cancer cell line (mcf-7), oral carcinoma cell line (kb), colon cancer cell line (caco-2) and choriocarcinoma cell line (jar). cytokines used in this study had both inhibitory and stimulatory effect on cell proliferation. findings in this project could provide an insight of cancer cell response to these cytokines and this could lead to a consideration on using cytokine as immunotherapy for cancer treatment.capacity of ae to modulate nitric oxide production depended on intercellular contact donor t cells are involved in the antitumour effects observed after bmt. thus, patients receiving t-celldepleted bmt have a higher risk of leukaemia relapse compared to patients receiving nonmanipulated bmt, and patients experiencing graft-versus-host disease (gvhd) have a lower risk of disease relapse than patients who do not experience gvhd. although the importance of donor t cells for the curative action of bmt has been established, the exact mechanisms and molecules involved in this graft-versus-tumour effect remain largely unknown. in a recently initiated project, we have conducted a longitudinal study of t-cell clonotypes in patients who received peripheral blood stem cell grafts after nonmyeloablative conditioning. peripheral blood samples were obtained sequentially after transplant, and the mononuclear cells (mncs) were isolated and cryopreserved. cd8 þ t cells were isolated from the mncs by use of immunomagnetic beads or facs and analysed for the presence of clonally expanded cells by t-cell receptor clonotype mapping based on rt-pcr and denaturing gradient gel electrophoresis (dgge). using this gel-based methodology, clonally expanded t cells were monitored after transplant and compared to the clinical data of the patients. the preliminary results demonstrates the presence of clonally expanded cd8 þ t cells at all time points analysed. furthermore, a number of clonotypes persisted for more than 6 months, and other clonotypes emerged during this period. the appearance of newly emerged clonotypes which coincided with clinical gvhd could indicate a role for these t cells in the pathogenesis of gvhd. background: deficiency of the mannan-binding lectin (mbl) pathway of innate immunity leads to increased susceptibility to infections. in patients with colorectal cancer, postoperative infection is associated with poor prognosis. the aim of the present study was to evaluate (1) the relation between the mbl pathway and postoperative infectious complications and survival of patients resected for colorectal cancer and (2) the role of mbl as acute phase reactant compared to crp. methods: preoperative mbl concentration, mbl/mblassociated serine protease (masp) activity and crp were determined in serum from 611 patients and 150 healthy controls. the patients were observed for 8 years. postoperative infections, recurrence and survival were recorded. results: the mbl pathway components were increased in the patients (p < 0.0001) compared to healthy controls. low mbl levels were predictive of pneumonia (p ¼ 0.01), and pneumonia (n ¼ 87) was associated with poor survival (p ¼ 0.003, hr ¼ 1.5, 95% ci 1.1-1.9). mbl and mbl/ masp activity could not predict postoperative overall infections. mbl showed no correlation (spearman's r ¼ 0.02, 95% ci à0.06-0.10) with crp. conclusions: low preoperative mbl levels are predictive of pneumonia, which is associated with poorer survival. mbl concentration and mbl/masp activity was not predictive of other postoperative infections or long-term prognosis. mbl apparently is not a surrogate measure of crp. department of surgery, university hospital of erlangen, erlangen, germany. e-mail: michael.siassi@rzmail.uni-erlangen.de introduction: the human collectins, mannan-binding lectin (mbl), surfactant protein-a (sp-a) and surfactantprotein-d (sp-d) play a central role in the innate immune system. immunological responses to malignant transformation of epithelial cells gained increasing interest recently. a former study could demonstrate binding of mbl to certain colorectal carcinoma (crc) cell lines in vitro. we therefore examined the expression of human collectins in normal colon mucosa and in colorectal carcinomas. materials and methods: colon samples from 20 crc patients and 10 normal mucosa samples were collected immediately after surgery. the tissue was microdissected and rna isolated (qiagen, rneasy-kit). gene expression profiles were analysed using gene-chips (affymetrix, hg-u133). we analysed the data for the expression of mbl, its associated serine proteases mannan-binding lectinassociated serine protease 1/2 (masp 1/2), sp-a and sp-d. the signal intensity of the genes of interest was compared using the mann-whitney u-test. results: the expression of human collectins in normal human colon mucosa was generally low. only the expression of sp-a and masp-2 reached the noise threshold of 250 signals. these genes were significantly downregulated in crc specimens. the expression of the other proteins showed no difference in normal mucosa and crc. conclusion: as demonstrated before, the expression of human collectins in normal colon was low in this being the first lymph node to receive drainage from the tumour area, the sentinel node offers a unique possibility to obtain tumour-reactive lymphocytes. we investigated antitumour immune responses in sentinel nodes from patients with bladder cancer, by assaying tumour-specific proliferation and tcr vb repertoires. during tumour surgery, sentinel lymph nodes were identified by peritumoural injection of blue dye. fresh specimens of tumour, sentinel and nonsentinel lymph nodes were obtained, and single-cell suspensions were prepared. cells were assayed for reactivity against autologous tumour extract in [ 3 h]-thymidine incorporation assays and characterized by flow cytometry. parallel analyses of the expression of vb gene families were performed with padlock probes, linear oligonucleotides which upon target recognition can be converted to circular molecules by a ligase. probes were reacted with cdna prepared from magnetically separated cd4 þ cells, and the tcr repertoire was determined by hybridizing the products to oligonucleotide microarrays. dose-dependent proliferation in response to tumour extract could be detected in sentinel lymph nodes. common clonal expansions were detected among tumourinfiltrating lymphocytes and in sentinel lymph nodes. nonsentinel lymph nodes displayed a divergent tcr vb repertoire. these results indicate an ongoing immune response against tumour antigens in sentinel nodes, draining urinary bladder cancer. identification of sentinel lymph nodes makes it possible to obtain tumour-reactive lymphocytes for use in adoptive immunotherapy. key: cord-023394-ptfjxpo6 authors: isa, a.; norbeck, o.; pöhlmann, c.; tolfvenstam, t. title: mapping of the ex vivo cellular immune response against the complete human parvovirus b19 genome during acute infection date: 2008-06-28 journal: scand j immunol doi: 10.1111/j.0300-9475.2004.01423n.x sha: doc_id: 23394 cord_uid: ptfjxpo6 background: human parvovirus b19 (b19) is a ubiquitous pathogen, normally causing a mild self‐limiting disease, but also capable of causing both significant pathology and long‐term persistence. the small size and stability of the virus makes it suitable for mapping of the full breath and the kinetics of the cellular immune responses following acute viral infection. methods: five patients with acute primary b19 infection were included in the study and followed consecutively for up to 200 weeks. cellular immune responses were mapped by ifnγ enzyme‐linked immunospot to overlapping peptides spanning the whole b19 genome. results: in all five acutely infected patients, we were able to monitor the kinetics of a strong specific cellular immune reaction. responses peaked at levels of 850–1850 sfc/million pbmcs, roughly corresponding to 0.3–0.6% b19‐specific cd8(+) cells circulating in peripheral blood at 10–80 weeks post‐infection. the responses in individual patients were directed to three or four different peptide pools, and the specificity was confined to the same cd8 epitopes present in the pools throughout the follow‐up period. the majority of responses were directed to the virus nonstructural protein, only two patients showed any response to the capsid proteins, elicited by the same epitope in both cases. conclusion: the cellular immune responses to acute b19 infection are surprisingly narrow in distribution and remain at high levels for up to 80 weeks post‐infection. the initial epitope specificity is maintained, and the majority of responses target the virus nonstructural protein, which is not included in vaccine preparations, evaluated against the infection. the nuclear receptor heterodimers of liver x receptors (lxrs) are recently identified as key transcriptional regulators of genes involved in lipid homeostasis and inflammation. lxrs and their ligands are negative regulators of macrophage inflammatory gene expression. multiple sclerosis (ms), a demyelinating disease of the central nervous system of unknown cause, is characterized by recurrent inflammation involving macrophages and their inflammatory mediators. sweden belongs to the countries with a high ms incidence. in italy, incidence is lower, with an exception for sardinia where the incidence is even higher than that in sweden. subjects from sardinia are ethnically more homogeneous and differ from swedes, also regarding genetic background and environment. we studied lxrs and their related molecules of blood mononuclear cells (mncs) from female patients with untreated relapsing-remitting ms from sassari, sardinia and stockholm, sweden. sex-and age-matched healthy controls (hcs) were from both areas. mrna expression was evaluated by real-time pcr. lxr-a was lower (p < 0.05) in ms (mean ae sem: 3.1 ae 0.2; n ¼ 37) compared to hc (3.6 ae 0.1; n ¼ 37). lxr-a was lower in ms from stockholm (2.6 ae 0.2; n ¼ 22) compared to corresponding hc (3.4 ae 0.1; n ¼ 22; p < 0.01) and compared to ms (3.8 ae 0.2; n ¼ 15; p < 0.001) and hc (4 ae 0.2; n ¼ 15; p < 0.001) from sardinia. ms patients from stockholm, but not from sassari, also expressed lower (p < 0.05) lxr-b (à4.1 ae 0.4) compared to corresponding hc (à2.9 ae 0.3). ms from stockholm was associated with higher abca-1 (6.1 ae 0.4 versus 5.0 ae 0.3; p < 0.05) and higher estrogen receptor-b-cx (2.4 ae 0.4 versus 0.8 ae 0.4; p < 0.01) compared to corresponding hc. the hc from sassari had higher androgen receptor (2.9 ae 0.2) compared to ms from sassari (1.4 ae 0.3; p < 0.01), ms (1.3 ae 0.4; p < 0.01) and hc from stockholm (1.2 ae 0.3; p < 0.01). ms from sassari had lower cyclooxygenase-1 compared to corresponding hc (5.1 ae 0.4 versus 6.6 ae 0.3; p < 0.01) and lower prostaglandin-e (à0.03 ae 0.5) compared to the hc (1.4 ae 0.5; p < 0.05) and ms (2.7 ae 0.4; p < 0.05) and hc from stockholm (1.9 ae 0.4, p < 0.001). our findings identify lxrs and their related molecules as being involved in ms from stockholm but not from sassari, while sex hormone receptors seem to be involved in ms in sassari. multiple sclerosis: ifn-b induces cd123 + bdca2 -dendritic cells that produce il-6 and il-10 and have no enhanced type i interferon production y. m. huang, 1 s. adikari, 1 u. båve, 2 a. sanna 1,3 & g. alm 4 dc antigens (bdca) and investigate their ability to produce type i ifn in response to virus stimulation. we show that ifn-b induces development of cd123 þ dc from human blood monocytes, which coexpress bdca4 þ but are negative for bdca2 -, a specific marker for plasmacytoid dc. such ifn-b-modulated dc produce large amounts of il-6 and il-10, but no il-12p40 and have no enhanced ifn-b and ifn-b production. the findings indicate that ifn-bmodulated dc represent a myeloid dc subset with diminished cd11c, bdca-1 and cd1a expression, having potent th2-promoting function but lacking antiviral capacity. the association of psoriasis with throat infections by streptococcus pyogenes suggests a potential antigenic target for the t cells that are known to infiltrate dermis and epidermis of psoriatic skin. streptococcal m protein shares an extensive sequence homology with human epidermal keratins. keratins 16 (k16) and 17 (k17) are mostly absent from uninvolved skin but are upregulated in psoriatic lesions. there is increasing evidence that cd8 þ t cells play an important effector role in psoriasis and m proteinprimed t cells may recognize these shared epitopes in skin via molecular mimicry. to identify candidate epitopes, peptides with sequences from k17 were selected on the basis of predicted binding to hla-cw6 and sequence similarities with m6 protein. matched peptides from the sequence of m6 protein and a set of peptides with poor predicted binding were also selected. cw6 þ individuals with psoriasis and cw6 þ healthy controls, having a family history of psoriasis, were recruited. pbmcs were incubated with the peptide antigens. t-cell activation in the cd4 þ , cd8 þ and later the skin-homing cutaneous lymphocyteassociated antigen (cla)-expressing subset of cd8 þ t cells was evaluated by cd69 expression and intracellular ifn-g accumulation using flow cytometry. we demonstrate that cw6 þ psoriasis patients had significant cd8 þ t-cell ifn-g responses to peptides from k17 and m6 protein selected on the basis of sequence homology and predicted hla-cw*0602 binding. these responses were about 10 times more frequent in the skin-homing cutaneous lymphocyte-associated antigen-expressing (cla þ ) subset of cd8 þ t cells. cd4 þ t cells showed only borderline responses. cd8 þ t cells from cw6 þ nonpsoriatic individuals responded to some m6 peptides but very rarely to k17 peptides, and this also applied to the cla þ cd8 þ subset. these findings indicate that psoriatic individuals have cd8 þ t cells that recognize keratin self-antigens and that epitopes shared by streptococcal m protein and human keratin may be targets for the cd8 þ t cells that infiltrate psoriatic skin lesions. autoantibodies directed against citrulline-containing proteins have an impressive specificity of nearly 100% in ra patients and a suggestive involvement in the pathogenesis. the targeted epitopes are generated by a post-translational modification catalysed by the calcium-dependent enzyme peptidyl arginine deaminase that converts the positively charged arginine to polar but uncharged citrullin. the aim of this study was to analyse the presence of citrulline in the joints at different time points of collagen-induced arthritis in da rats by immunohistochemistry and to investigate how immunogenicity and arthritogenicity was affected by citrullination of rat serum albumin (rsa) and collagen type ii (cii). our results indicate that citrulline could be detected in joints of arthritic animals, first appearance at the onset of disease and increasing as disease progressed into a chronic state. unimmunized animals or time points before clinical signs of arthritis were negative. by morphology, we state that some infiltrating macrophages as well as the cartilage surface stain positive for citrulline, while the major source of citrullinated proteins appears to be fibrin depositions. a specific cit-rsa t-cell response was observed in animals challenged by citrullinated rsa, no response was recorded when rsa was used as a stimulus. the igg analysis reveals not only a response towards the modified protein but also cross-reactivity to native rsa. no t-cell or b-cell response was noted in animals injected with unmodified rsa. cit-cii induced a disease with higher incidence and earlier onset than did the native counterpart. we conclude that, in contrast to the human disease, citrulline does not seem to appear before clinical signs. as inflammation proceeds, citrulline is detected specifically in the joints. all other organs investigated were negative. we also conclude that citrullination of a protein can break tolerance and increase its arthritogenic properties. ectopic germinal centers (gcs) can be detected in the salivary glands of approximately 1/5 of patients with sjögren's syndrome (ss) and appear in both primary and secondary ss. previously, ectopic gc have been associated with increased local autoantibody production. the aim of this study was to determine whether gc in primary sjögren's syndrome (pss) defines a distinct seroimmunological phenotype. retrospectively, a material of 130 haematoxylin and eosin-stained paraffin-embedded tissue sections of minor salivary gland tissue from patients with pss was morphologically screened for the presence of ectopic gc. gc-like lesions were detected in 33/130 (25%) of the pss patients. seventy-two pss patients lacking these structures (gc-) were randomly selected for comparison. focus score was significantly increased in the gc þ patients compared to the gcpatients (p ¼ 0.035). in the gc þ group, 54.5% of the patients presented with anti-ro/ssa compared to 43.7% in the gcgroup. anti-la/ssb was detected in 31.3% of the gc þ patients compared to 25.7% of the gcpatients. sixty-one percentage of gc þ patients presented with increased levels of igg, a nonsignificant difference when compared to 39.4% in the gcpatients (p ¼ 0.089). levels of rf, ana, ena, igm and iga were similar in both patient groups, as were esr and crp. in conclusion, patients with ectopic gc have a higher focus score and more often present with autoantibodies and increased levels of igg compared to pss patients with regular focal infiltration (gc -). our findings may indicate a certain seroimmunological phenotype and warrant for further prospective studies. association between mannose-binding lectin and vascular complications in type 1 diabetes complement activation and inflammation have been suggested in the pathogenesis of diabetic vascular lesions. we investigated serum mannose-binding lectin (mbl) levels and polymorphisms in the mbl gene in type 1 diabetic (t1dm) patients with and without diabetic nephropathy and associated macrovascular complications. polymorphisms in the mbl gene and serum mbl levels were determined in 199 t1dm patients with overt nephropathy and 192 t1dm patients with persistent normoalbuminuria matched for age, sex and duration of diabetes as well as in 100 healthy control subjects. the frequencies of high and low expression mbl genotypes were similar in patients with t1dm and healthy controls. high mbl genotypes were significantly more frequent in diabetic patients with nephropathy than in the normoalbuminuric group, and the risk of having nephropathy, given a high mbl genotype, assessed by odds ratio was 1.52 (1.02-2.27), p ¼ 0.04. median serum mbl concentrations were significantly higher in patients with nephropathy than in patients with normoalbuminuria [2306 mg/l (iqr 753-4867 mg/l) versus 1491 mg/l (iqr 577-2944), p ¼ 0.0003], and even when comparing patients with identical genotypes, serum mbl levels were higher in the nephropathy group than in the normoalbuminuric group. patients with a history of cardiovascular disease had significantly elevated mbl levels independently of nephropathy status [3178 mg/l (iqr 636-5231 mg/l) versus 1741 mg/l (iqr 656-3149 mg/l), p ¼ 0.02]. the differences in mbl levels between patients with and without vascular complications were driven primarily by pronounced differences among carriers of high mbl genotypes (p < 0.0001). our findings suggest that mbl may be involved in the pathogenesis of microvascular and macrovascular complications in type 1 diabetes and that determination of mbl status might be used to identify patients at increased risk of developing these complications. neuroimmunology unit, center for molecular medicine, karolinska institutet, stockholm, sweden. e-mail: judit.wefer@cmm.ki.se dna vaccine coding for the encephalitogenic peptide mog 91-108 protects lew.1av1 from subsequent development of experimental autoimmune encephalomyelitis (eae). protection is associated with a type 1 immune response and is dependent on the presence of cpg dna motifs. the mechanisms underlying the observed reduction of eae development in protected rats have not been fully clarified. we investigated immunological characteristics of lymphocytes after dna vaccinaton and subsequent eae induction. we confirm that protection was not associated with suppression of t1 cells, as transcription of the novel molecule rat t-cell immunoglobulin-and mucindomain-containing molecule (tim-3), reported to be exclusively expressed on differentiated t1 cells, was not altered by dna vaccination. we did not note any clonal deletion upon tolerization, but detected an antigen-specific lymphocyte population upregulating ifng upon recall stimulation 3 weeks after protective dna vaccination. in protected rats, we observed (1) no alterations in antigenspecific th2 or th3 responses, (2) reduced mhc ii expression on splenocytes early after eae induction, (3) antigen-specific upregulation of ifnb upon recall stimulation and (4) reduced il-12rb2 on lymphocytes. we thus demonstrate an association of the protective effect of dna vaccination with expression of ifnb. we are currently investigating the cellular mechanisms behind this ifnb-mediated protection. multiple sclerosis (ms) is an autoimmune condition characterized by degeneration of nerve fibre myelin sheets. a candidate autoantigen, myelin basic protein (mbp), has especially attracted attention. the presence of anti-mbp antibodies is a predictor of definite ms, but their role in the pathogenesis remains obscure. t cells have long been known to play a pivotal role in the pathogenesis of ms. recently, an important role for b cells as autoantigen-presenting cells has been demonstrated in other autoimmune diseases, including rheumatoid arthritis and diabetes. the uptake of mbp by b cells and the presentation of mbp-derive peptides to t helper (th) cells by b cells may be promoted by the formation of complement (c) activating immune complexes (ics) between mbp and natural autoantibodies in healthy individuals and disease-associated anti-mbp antibodies in ms patients, respectively. we have investigated the formation of mbp-containing ic, the binding of mbp to b cells, the mbp-elicited induction of th-cell and b-cell proliferation and the cytokine production in peripheral blood mononuclear cells (pbmcs) from healthy donors grown in the presence of intact or c-inactivated serum from healthy donors or patients with ms. while mbp did not induce measurable proliferation of b cells nor cd4 þ t cells, we observed the production of tnf-a, ifn-g and il-10 by pbmc in response to incubation with mbp in the presence of sera from healthy controls as well as sera from ms patients. by contrast, no production of il-2, il-4 and il-5 was detected. we are currently investigating the capability of ms sera to promote the formation of mbp-containing ic and thereby enhance the cytokine responses, by virtue of elevated anti-mbp contents. the phagolysosomally localized acid sphingomyelinase (asmase) activated by proinflammatory cytokines such as tnf and ifn-g generates the signalling molecule ceramide which in turn results in the activation of proteases like cathepsin d. these characteristics of asmase suggest a possible role of this molecule in the phagocytotic uptake and phagosomal degradation processes of antigens or in antigen presentation. we show here that asmase -/mice fail to eliminate the noncytopathic lymphocytic choriomeningitis (lcm) virus as rapidly as littermate wildtype mice. investigation of the immune response revealed a reduced expansion of cd8 þ t cells. the secretion of ifn-g in response to contact with target cells as well as the cytolytic activity of virus-specific cd8 þ t cells was severely impaired. additionally, both phases of the lcm virus-specific dth response, mediated by cd8 þ and cd4 þ t cells consecutively, were diminished in asmase -/mice. however, the secondary memory response of virus-specific ctl was not altered, and the 614 abstracts ................................................................................................................................................................................................. virus was effectively controlled for at least 3 months by asmase -/mice. in conclusion, the results of this study suggest an involvement of the asmase in the activation, expansion or maturation of virus-specific cd8 þ t cells during the acute infection of mice with the lcm virus. novel markers for alternative activation of macrophages: macrophage galactose-type c-type lectins 1 and 2 in parallel with the th1/th2 dichotomy, macrophages are capable of developing into functionally and molecularly distinct subpopulations, due to differences in, for example cytokine environment and pathological conditions. while the best-studied, classically activated macrophage is induced by type i stimuli such as ifn-g, a type ii cytokine environment antagonizes the classical activation of macrophages and is capable of alternatively activating macrophages. however, molecular markers associated with these type ii cytokine-dependent, alternatively activated macrophages remain scarce. besides the earlier documented markers macrophage mannose receptor and arginase 1, we recently demonstrated that murine alternatively activated macrophages are characterized by increased expression of fizz1 and ym. we now report that expression of the two members of the mouse macrophage galactose-type c-type lectin gene family, termed mmgl1 and mmgl2, is induced in diverse populations of alternatively activated macrophages, including peritoneal macrophages elicited during infection with the protozoan trypanosoma brucei or the helminth taenia crassiceps, and alveolar macrophages elicited in a mouse model of allergic asthma. we also demonstrate that, in vitro, interleukin-4 and interleukin-13 upregulate mmgl1 and mmgl2 expression and that, in vivo, induction of mmgl1 and mmgl2 is dependent on interleukin-4 receptor signalling. moreover, we show that regulation of mgl expression is similar in human monocytes and monocyte-derived macrophages. hence, macrophage galactose-type c-type lectins represent novel markers for both murine and human alternatively activated macrophages; thus, paving the way for further characterization of the phenotype of macrophages occurring in th2 conditions. background: human parvovirus b19 (b19) is a ubiquitous pathogen, normally causing a mild self-limiting disease, but also capable of causing both significant pathology and long-term persistence. the small size and stability of the virus makes it suitable for mapping of the full breath and the kinetics of the cellular immune responses following acute viral infection. methods: five patients with acute primary b19 infection were included in the study and followed consecutively for up to 200 weeks. cellular immune responses were mapped by ifng enzyme-linked immunospot to overlapping peptides spanning the whole b19 genome. results: in all five acutely infected patients, we were able to monitor the kinetics of a strong specific cellular immune reaction. responses peaked at levels of 850-1850 sfc/ million pbmcs, roughly corresponding to 0.3-0.6% b19specific cd8 þ cells circulating in peripheral blood at 10-80 weeks post-infection. the responses in individual patients were directed to three or four different peptide pools, and the specificity was confined to the same cd8 epitopes present in the pools throughout the follow-up period. the majority of responses were directed to the virus nonstructural protein, only two patients showed any response to the capsid proteins, elicited by the same epitope in both cases. conclusion: the cellular immune responses to acute b19 infection are surprisingly narrow in distribution and remain at high levels for up to 80 weeks post-infection. the initial epitope specificity is maintained, and the majority of responses target the virus nonstructural protein, which is not included in vaccine preparations, evaluated against the infection. the relationship between malnutrition and malaria is controversial. on one hand, malaria may cause malnutrition, while on the other, malnutrition itself may modulate susceptibility to the disease. we investigated the association between plasmodium falciparum malaria and malnutrition in a cohort of children living on the coast of kenya. the study involved longitudinal follow-up for clinical malaria episodes and anthropometric measurements at four cross-sectional surveys. we used poisson regression analysis to investigate the association between malaria and nutritional status. compared to baseline (children with a waz or haz score of !à2), the crude incidence rate ratios (irrs) for malaria in children with low haz or waz scores (<à2) during the period prior to assessment were 1.17 (95% ci 0.91-1.50; 0 ¼ 0.21) and 0.94 (0.71-1.25; 0.67), respectively, suggesting no association between malaria and the subsequent development of pem. however, we found that age was acting as an effect modifier in the association between malaria and malnutrition. the irr for malaria in children 0-2 years old who were subsequently characterized as wasted was 1.65 (1.10-2.20; p ¼ 0.01), and a significant overall relationship between malaria and low-haz was found on regression analysis when adjusting for the interaction with age (irr 1.89; 1.01-3.53; p < 0.05). although children living on the coast of kenya continue to suffer clinical episodes of uncomplicated malaria throughout their first decade, the association between malaria and malnutrition appears to be limited to the first 2 years of life. a. astrinidou-vakaloudi, 1 s. xytsas, 1 i. diamanti, 1 h. ioannidis 2 & p. pangidis 2 1 microbiology department of general hospital of thessaloniki 'agios pavlos', thessaloniki, greece, and 2 nefrology, 2 nd ika hospital of thessaloniki, thessaloniki, greece. e-mail: stasa@hol.gr aim: renal dysfunction may influence the colonization of gastric mucosa by urea-splitting bacteria such as helicobacter pylori, by increasing urea concentrations in the gastric juice. our aim was to investigate the prevalence of h. pylori in patients with end-stage renal disease (esrd), receiving long-term haemodialysis treatment. methods: this study included 40 sera from patients with esrd (29 male and 11 female) undergoing periodic haemodialysis; mean time of treatment was 42.6 months. using elisa technique, we investigated the presence of igg and iga antibodies against h. pylori as well as igg caga (antibodies specific for caga(þ) strains of h. pylori). sera from 40 healthy blood donors were used as a control group. results: h. pylori igg antibodies were detected in 32 out of 40 (80%) patients in the dialysis group, while 31/40 (77.5%) tested positive for iga. igg caga antibodies were present in 13 out of 40 (32.5%). prevalence of h. pylori igg, iga and caga igg antibodies in the control group was 33, 7 and 15%, respectively. conclusions: although international data suggest that prevalence of h. pylori infection is the same in esrd patients as in healthy individuals, in our study that seems not to be the case. the higher blood and gastric juice urea levels may be a risk factor (among many others), but more studies are required in order to understand the relation of h. pylori infection in this group of patients. flanders interuniversity institute for biotechnology, department of molecular and cellular interactions, free university of brussels, brussels, and 2 pasteur institute of brussels, mycobacterial immunology, brussels, belgium. e-mail: tgartner@vub.ac.be immunity against tuberculosis (tb), caused by mycobacterium tuberculosis, depends largely on activation and maintenance of strong cell-mediated immune responses involving both cd4 þ and cd8 þ t cells and the ability to respond with th1-type cytokines, particularly ifn-g. recent studies suggested that bcg, the only licensed vaccine against m. tuberculosis, may fail to induce t-cell responses in the lung mucosa and may therefore not protect against pulmonary tb. a decrease in tb mortality may be achieved by enhancing immunity in the lung. the present study evaluated the induction of antigen-specific immunity in the lung by intranasal (i.n.) delivery of the lipoprotein i (opri) from pseudomonas aeruginosa. opri has shown to be a toll-like receptor 2/4 agonist that, when given subcutaneously, induces type-1 immune responses against heterologous antigens. here, a fusion of opri to ag85a of mtb (opri-ag85a) was used as a subunit vaccine in homologous prime-boost immunizations. in addition, opri-ag85a was combined with an ag85a-encoding dna vaccine (ag85a dna) or with bcg in heterologous prime-boost vaccinations. intranasal and parenteral delivery with opri-ag85a elicited comparable t-cell responses in the spleen; in addition, i.n. delivery elicited specific t-cell responses in the lung lymph nodes (llns). intramuscular delivery of ag85a dna induced significant systemic th1 immune responses. intranasal boosting with opri-ag85a enhanced this response and in addition induced an antigen-specific ifn-g response in lln. opri may therefore be an efficient adjuvant for mucosal boosting. we continue to evaluate the protection induced by opri-based prime-boost vaccinations against pulmonary tb. results on the immunogenicity and protection against intravenous mtb h37rv infection will be presented. toll-like receptors (tlrs) are pattern recognition receptors of the innate immune system, which recognize molecular structures on pathogens or cellular stress-associated molecules. tlr-ligand interactions trigger activation of inflammatory signal transduction and expression of genes involved in host defense. in this study, we have examined the requirement for different tlr adaptor molecules in virus-induced chemokine expression and are currently trying to identify the tlr involved. we have found that both a herpesvirus [herpes simplex virus (hsv)] and a paramyxovirus (sendai virus) require a functional genome to induce expression or proinflammatory chemokines in human and murine monocytic cell lines. for both viruses, this is independent of the tlr adaptor molecules trif and mal. however, overexpression of the vaccinia virus-encoded inhibitor of tlr-signalling a52r or dominant-negative myd88 totally inhibited hsv-induced rantes expression but only partially prevented sendai virus from inducing this chemokine. this suggests that hsv-induced rantes expression occurs via a tlr pathways, whereas sendai virus utilizes both tlr-dependent and -independent pathways to stimulate expression of rantes. we are currently trying to identify the tlrs involved. data from these studies will also be presented at the meeting. 2 0 -5 0 -oligoadenylate synthetases are interferon-induced, double-stranded rna-activated antiviral enzymes which are the only proteins known to catalyse 2 0 -specific nucleotidyl transfer. this first crystal structure of a 2 0 -5 0oligoadenylate synthetase reveals a structural conservation with the 3 0 -specific poly(a) polymerase that, coupled with structure-guided mutagenesis, supports a conserved catalytic mechanism for the 2 0 -and 3 0 -specific nucleotidyl transferases. comparison with structures of other superfamily members indicates that the donor substrates are bound by conserved active site features while the acceptor substrates are oriented by nonconserved regions. the 2 0 -5 0oligoadenylate synthetases are activated by viral doublestranded rna in infected cells and initiate a cellular response by synthesizing 2 0 -5 0 -oligoadenylates, that in turn activate rnase l. this crystal structure suggests that activation involves a domain-domain shift and identifies a putative dsrna activation site that is probed by mutagenesis. we demonstrated that this site is required both for the binding of dsrna and for the subsequent activation of oas. this rna-binding site is different from known rna-binding site; rather than forming a defined three-dimensional domain, it is located at the interface of the two major domains in oas. this novel architecture ensures that the dsrna helix can make simultaneously contact with both domains of oas and ensure the subsequent structural rearrangement leading to the activation of oas. our work provides structural insight into cellular recognition of double-stranded rna of viral origin and identifies a novel rna-binding motif. bacteria-specific iga antibodies are efficient opsonins for neutrophils and mononuclear phagocytes, provided that the phagocytes express the fca receptor (cd89). expression of cd89 can be stimulated by inflammatory cytokines, activated complement factors and certain microbial components. in one study, unstimulated phagocytes were able to ingest iga antibody-treated pneumococci, but only in the presence of complement, which was found to be activated by the iga antibodies along the alternative pathway. pneumococci produce iga1 protease that cleaves human iga1, but not iga2, molecules in the hinge region. this leaves iga1 as faba (monovalent) deprived of fca which contains the docking site for cd89. iga1 is the vastly predominant subclass of iga in the upper airways and circulation of humans. aims: to examine the effects of iga1 protease activity and complement on phagocytosis of iga antibody-coated pneumococci by an unstimulated human phagocytic cell line (hl60). materials and methods: iga1 and iga2 monoclonal antibodies to serotype 4 pneumococcal capsular polysaccharide (ps) were generated by heterohybridoma technique involving b cells from human vaccinees. isogenic serotype 4 pneumococci with and without iga1 protease activity, respectively, were obtained after inactivation of the iga gene of the tigr4 strain. opsonophagocytosis was quantitated using the assay described by romero-steiner et al. based on enumeration of surviving bacteria by culture. the integrity of iga molecules was examined by western blotting. results: both iga1 and iga2 antibody to type-4 polysaccharide-induced phagocytosis of iga1 protease-deficient type-4 pneumococci equally well in the absence as in the presence of complement. iga1 antibody to type-4 polysaccharide displayed a fourfold higher opsonophagocytosis titer against iga1 protease deficient compared to homologous wildtype target bacteria. a similar effect of iga1 protease activity of the target bacteria was not observed in a parallel experiment where iga2 antibody to type-4 polysaccharide served as opsonin. iga1 antibody extracted from iga1 protease-producing target bacteria was almost exclusively in the form of faba. conversely, iga1 from protease-deficient bacteria and iga2 from both types of bacteria were intact. conclusions: these results indicate that the iga1 protease activity of s. neumoniae may help the bacteria escape iga1 antibody-mediated opsonophagocytosis. besides, in these experiments, iga-mediated opsonophagocytosis was independent of complement. vitamins e and c have been found to increase the cellular and humeral immunity of pigs. vitamin e deficiency has also been found to predispose pigs to different diseases, e. coli infection is one among them. after weaning, the vitamin e status of pigs often decreases to a critical low level. in this experiment, we studied whether vitamin c supplementation would be a possible feeding strategy to optimize the immune status of weaners. the interaction between vitamin e and c is interesting due to the reported sparing action on vitamin e or synergism between these to vitamins. piglets were weaned at day 28 of age from sows fed increasing dietary vitamin e during lactation, and piglets were during the following 3 weeks fed either a control diet or this diet supplemented with 500 mg stay-c per kg. blood sampling was obtained weekly from day 28 and until day 49 of age. on the same days, one piglet per dietary treatment was killed and alveolar macrophages (am) were harvested. vitamin c supplementation increased the concentration of igm in serum of piglets throughout the weaning period. although the vitamin e concentration in am decreased with increasing age of the piglets, the concentration was numerically higher in piglets of sows fed the high dietary level of vitamin e. however, vitamin c supplementation tended to increase the total am concentration of vitamin e after weaning and increased the proportion of the biologically most active isomer of vitamin e [rrr-(a-tocopherol)] in the am. the eicosanoid synthesis by am was not influenced by the vitamin c supplementation, but the synthesis of leukotriene b4 was decreased 2 weeks after weaning compared to other days of am harvesting. in conclusion, dietary vitamin c supplementation improved the immune responses of piglets after weaning. a whole blood stimulation assay with escherichia coli (o111:b4) endotoxin was established to measure the capacity of dairy cows to produce the proinflammatory cytokine tumour necrosis factor-a (tnf-a) ex vivo. initially, a time-and dose-dependent study was carried out to find the optimal stimulation conditions for the tnf-a response. the tnf-a response peaked between 3 and 4 h at 38.5 c. a dose in the range of 5-10 g of e. coli lipopolysaccharide (lps)/ml whole blood was found to give the maximum tnf-a response. thirty-eight danish-holstein dairy cows were investigated for their tnf-a responsiveness ex vivo in the periparturient period. heparin-stabilized blood samples were collected seven times over a period of 4 months (weeks à3, à1, 2, 3, 5, 9 and 13 around calving) and stimulated with 5 g/ml of e. coli lps. indeed, fluctuations in the tnf-a responsiveness occurred over time. moreover, the mean tnf-a responsiveness of 38 cows was found to be significantly increased (p < 0.001) in the weeks close to calving. however, in the more stabile physiological periods, some cows had a consistently low tnf-a response, whereas others had high a tnf-a response. we are currently investigating whether high and low tnf-a responders to e. coli lps also exist in dairy cows in vivo. moreover, the importance of tnf-a responsiveness ex vivo to dairy cows' susceptibility and clinical response to experimental e. coli infections in the udder is being investigated. coelomic cytolytic factor (ccf) is a 42 kda invertebrate pattern recognition molecule isolated from the coelomic fluid of the earthworm eisenia foetida (oligochaeta, annelida). ccf displays a number of similarities with the mammalian cytokine tumour necrosis factor-a (tnfa) as a result of a shared n,n 0 -diacetylchitobiose lectin-like domain. however, these similarities are solely functional and are not based on any (dna or amino acid) sequence homology, thus suggesting a form of convergent evolution. in particular, the lectin-like domain of tnf-a has been shown to induce membrane depolarization in various mammalian cell types, through interactions with endogenous amiloride-sensitive ion channels. this nonreceptor-mediated activity of tnf-a has been reported to be involved in the resorption of oedema. likewise, the lectin-like domain of ccf also induces membrane depolarization in mammalian cells. here, we show that ccf appears to be able to induce oedema resorption in an alveolar epithelial cell line through its lectin-like domain. this lectin-like domain of ccf interacts (directly or indirectly) with endogenous sodium and/or chloride channels, and not potassium channels, on mammalian cells. additionally, we suggest that the jnk/sapk and erk1/2 pathways are involved in ccf-induced macrophage activation. these results further establish the functional analogy between an invertebrate pattern recognition molecule and a mammalian cytokine and, from a more applied point of view, suggest the possibility of utilizing ccf in the treatment of oedema. release of svegf and svegfr1 from white blood cells and platelets during surgery and stimulation with bacterial antigens introduction: the influence of surgery on release of soluble vascular endothelial growth factor (svegf) and the soluble vascular endothelial growth factor inhibitory receptor 1 (svegfr1) is unknown. we studied the effect of major and minor surgery on potential variations in svegf and svegfr1 concentrations in vivo and on bacterial antigen-induced release of svegf and svegfr1 from whole blood in vitro. methods: sixty-one patients with abdominal diseases undergoing five different surgical procedures were included. blood samples were drawn from anaesthetized patients before and after the operation. white blood cells and platelets were counted, and plasma svegf and svegfr1 was determined by an elisa method. whole blood from each blood sample was stimulated in vitro with bacteria-derived antigens (lps or protein-a) and svegf and svegfr1 levels were subsequently determined in the supernatants. stimulation with isotonic saline served as control assay. neither svegf or svegfr1 in plasma changed during surgery. in vitro stimulation of blood samples with bacteria-derived antigens resulted in a significant increase in svegf (p < 0.0001) and a less pronounced but still significant increase in svegfr1. release of svegf due to stimulation was significantly higher after the operation (nonsignificant), whereas svegfr1 release remained largely unchanged after surgery. correlation between bacterial antigen-induced release of svegf and neutrophile cell count was highly significant (p < 0.0001). there was no correlation between svegf and platelet cell count, and bacterial antigen-induced svegfr1 release did not correlate with counts of neutrophils and platelets. conclusions: plasma svegf and svegfr1 concentrations did not change during surgery. in vitro bacterial stimulation led to increased release of svegf and svegfr1, which was not significantly amplified during surgery and which may be related to number of circulating neutrophils. natural killer cell functions and subsets after in vitro stimulation with il-2 and il-12, with special emphasis on intracellular ifn-g and nk-cell cytotoxicity r. nyboe, 1,2 t. rix, 1,2 j. krog, 1,2 e. tønnesen 1 & m. hokland 2 1 department of anaesthesiology and intensive care, aarhus university hospital, and 2 institute of medical microbiology, and immunology, university of aarhus, aarhus, denmark. e-mail: rnsr@studmed.au.dk materials and methods: isolated cryopreserved human peripheral blood mononuclear cells (pbmcs) were stimulated with il-2 and il-12. this stimulation has previously been shown to activate nk cells. cell cytotoxicity was measured by flow cytometry after incubation with k562 cells. this method was compared to the current standard 51cr release assay. cells were treated with bfa to accumulate ifn-g, stained for surface markers, permeabilized and stained for intracellular ifn-g. flow cytometry was then performed to measure intracellular ifn-g production in pbmc, especially in nk cells. results: we have demonstrated that stimulation with il-2 and il-12 is effective in increasing the number of ifn-gpositive cells. there is a distinct difference between the cd3-cd56dim and the cd3-cd56bright subsets, with a much greater proportion of ifn-g-positive cells in the cd3-cd56bright subset. the effects of stimulation with il-2 and il-12 on cytotoxicity will be presented, as will the relation between ifn-g production and cytotoxicity. in addition, we will present results of these assays applied to porcine cells. discussion: in combination, these tests will address nk cell function by combining cytotoxicity with ifn-g production in nk cell subsets. the results will demonstrate whether this could serve as a useful tool in describing nkcell function, which could be of value in clinical and experimental settings. culture of regulatory t-cell lines from bronchial mucosa t lymphocytes play a major role in many immune responses. in the last decade, special focus has been on the function of th1 and th2 effector cells. now the importance of regulatory cd4 þ cd25 þ t cells in maintenance of the immunological homeostasis emerges. sarcoidosis is a multisystem granulomatous disorder often affecting the lungs. the typical sarcoid granulomas consists of epitheloid cells, macrophages and lymphocytes, mainly cd4 þ t cells of th1 phenotype. we have cultured t cells from bronchial biopsies of patients with sarcoidosis as well as from controls in high levels of interleukin 2 (il-2) and il-4 and demonstrate spontaneously arising cd4 þ cd25 þ populations and high concentrations of il-10 in these cultures. the main difference between cultures of sarcoid origin compared to controls is a very much higher concentration of the inflammatory cytokines il-6 and tnf-a in cultures of sarcoid origin. the effects of hyperbaric exposure on human peripheral blood mononuclear cells, with special emphasis on natural killer cell cytotoxicity and subsets materials and methods: as an experimental physiological stress model, we examined the effects of hyperbaric exposure on peripheral blood mononuclear cells (pbmcs) obtained from venous blood drawn from eight divers during a simulated heliox saturation dive. eight persons working in normobar atmosphere outside the pressurized chamber served as control donors. the spontaneous cytotoxicity of the pbmcs was estimated in a 4 h 51cr-release assay using k562 as nk-sensitive target cells. the pbmcs were characterized, using 4-colour flow cytometry, with special emphasis on the nk-cell subsets. the data were statistically analysed using a multivariate regression model (stata 8.2). p values <0.05 was considered statistically significant. results: the estimated cytotoxicity increased significantly in both the group of divers and control donors during the dive (pdivers < 0.01 and pcontrols < 0.01). although the cytotoxicity increased relatively more (p < 0.01) in the group of divers compared to the group of control donors between day 1 and 2. discussion: the increased cytotoxicity of pbmc estimated in the group of divers indicate that parts of the cellular immune system are affected during the extreme physiological conditions induced during the initial phase of the presented experimental hyperbaric setup. the increase in cytotoxicity observed in the group of control donors could hypothetically reflect the stress level in persons working outside the pressurized chamber during the dive. the interaction with adhesion molecules expressed by vascular endothelium is the first step in lymphocyte infiltration into tissues. at both cutaneous and mucosal sites interleukin-10 (il-10), il-12 and transforming growth factor (tgf)-b are important regulators of chronic inflammatory disease, where cutaneous lymphocyteassociated antigen (cla) and ae integrin (cd103) may be expressed. unlike cla, cd103 is not believed to play a role in tissue-specific homing but may help to retain t cells within epithelial layers. we have previously shown that il-12 alone can together with an unknown cofactor increase the expression of cla. stimulation with streptococcal pyrogenic exotoxin c (spec) increased the expression of cd103 by cd8 þ but not cd4 þ t cells. while il-12 increased superantigen-stimulated expression of cla, this cytokine strongly inhibited the cd103 expres-sion, and a combination of il-12 and tgf-b completely abrogated the induced cd103 expression. conversely, il-10 suppressed cla but increased cd103 expression. these findings indicate that, in addition to suppressing the development of th1-mediated inflammatory responses, il-10 may also inhibit the migration of cd8 þ t cells into the skin while il-12 promotes such migration. thus, the expression of cla and cd103 may be antagonistically regulated by il-10 and il-12, and the balance between these cytokines could influence the t-cell migration of inflammatory cells into epithelial tissues. during contact sensitivity reaction, immune cells proliferate. in order to study the histological picture of these proliferation phases, we used a mouse model of contact sensitivity in the oral mucosa and on skin. we also used bromodeoxyuridin (brdu, an analogue to thymidin) that is incorporated into the nucleus during cell replication. the hapten oxazolone (oxa) was used to sensitize and elicit the oral mucosa and/or the ear skin. mice were killed at various times after elicitation, and unsensitized animals were also exposed to the hapten as controls. brdu (25 mg/ kg animal) was injected i.p. 2 h before the kill. specimens from the oral mucosa, ear skin and submandibular and auricular lymph nodes were cut and fixed in 4% paraformaldehyde. they were then treated with acid and biotinylated anti-brdu antibody and developed using abc-kit and dab. the analyses were performed using a leica light microscope and the computer program analysis. in the oral mucosa, the frequency of proliferating cells were increasing during the observation period, 4-24 h after elicitation, regardless of site of sensitization. the proliferating cells were found mainly in the basal cell layer of the epithelium. similar patterns were found in ear skin. the regional lymph nodes demonstrated a few scattered proliferating cells 4 h after elicitation. after 24 h, these cells were found frequently in the whole lymph node. control animals exhibited considerable less proliferating cells at all times. we conclude that most proliferating cells were found 24 h after elicitation locally at the hapten-exposed sites (the oral mucosa or the ear skin) as well as in the regional lymph nodes. the endogenous nucleoside adenosine is released in excess during inflammation or other metabolic stress and is generally known to deliver tissue protective anti-inflammatory effects. adenosine acts via four adenosine receptors of which the a2a receptor is the predominant form in t cells. adenosine levels are elevated in asthmatic lung, and adenosine can directly induce mast cell degranulation and bronchoconstriction in these patients. instead, the role of anti-inflammatory mechanisms of adenosine on t cells in asthma is unclear. aim: to study the a2a receptor expression in peripheral blood cd4 þ t cells in asthmatic and healthy individuals using flow cytometric and quantitative real-time pcr methods. results: unstimulated cd4 þ cells of asthmatic patients expressed significantly lower levels (p < 0.001) of a2a receptor in protein level (mean percentage of cells positive ae sem: 76.8 ae 1.2, n ¼ 6) compared to healthy individuals (90.4% ae 1.9, n ¼ 4). double staining for cd69 expression showed that stimulation of cd4 þ cells decreased a2a expression in both groups but indicated that the detected lower levels of a2a in unstimulated cells of asthmatics was not due to preactivation in these patients. surprisingly, a2a mrna expression in unstimulated cd4 þ cells was significantly higher (p < 0.05) in asthmatics (n ¼ 28) compared to healthy controls (n ¼ 7). the expression did not correlate with serum total ige levels. conclusions: asthmatic individuals express less a2a adenosine receptor on their peripheral cd4 þ t cells. the higher mrna levels instead may point to a negative feedback regulation in the receptor expression. the role of possibly decreased adenosine-mediated anti-inflammatory effects in asthma pathogenesis require further studies on this t-cell mediated disease. the chronic inflammatory skin disease atopic eczema (ae) affects almost 15% of the population in many countries today. the pathogenesis of ae is not fully understood. a combination of genetic predisposition and environmental factors like microorganisms seems to contribute to the symptoms. the yeast malassezia sympodialis is part of our normal skin micro flora but can act as an allergen and elicit specific ige and t-cell reactivity in patients with ae. recently, we identified a novel major m. sympodialis allergen, designated mala s 11 (22.4 kda), with sequence similarity to the mitochondrial enzyme manganese superoxide dismutase (mnsod). interestingly, mala s 11 has a high degree of homology to human mnsod. the aim of this study was to examine the effects of recombinant mala s 11 on antigen-presenting dendritic cells. monocytederived dendritic cells (mddcs) from healthy blood donors were cultured with or without mala s 11 for different time periods. it was found that the maturation marker cd83 and the costimulatory molecules cd80 and cd86 were upregulated on the mddcs exposed to mala s 11 for 24 h, as demonstrated by flow cytometry. furthermore, coculture of mddcs with mala s 11 for 9 h induced an increased production of the inflammatory cytokines il-6 (200-fold), tnf-a (100-fold) and il-8 (sixfold), as detected by the cytometric bead array (cba) analysis. our results suggest that mala s 11 affects the immune response through dc maturation and production of inflammatory cytokines. the potential cross-reactivity with human mnsod needs to be explored and the exact role of mala s 11 in the pathogenesis of ae assessed in clinical studies involving skin prick and atopy patch tests. allergen-specific immunotherapy (sit) is commonly conducted with allergen extracts adsorbed to aluminium hydroxide (alum). drawbacks linked to the use of alum, such as the formation of granuloma at the site of injection, have led to suggestions of novel allergen carriers. an alternative carrier is 2 mm carbohydrate-based particles (cbps). in mouse, allergen-coupled cbps have been demonstrated to skew the allergen-specific immune response towards a th1-like activity (grönlund et al. immunology, 2002) . we here coupled the recombinant major cat allergen fel d 1 to cbps (cbp-fel d 1) by cyanogen-bromide activation, resulting in covalent binding. the effect of cbp-fel d 1 on monocyte-derived dendritic cells (mddcs) from healthy human blood donors was studied. we found that the majority of the cd1a þ mddcs were capable of taking up fitc-labelled cbp-fel d 1, as demonstrated by flow cytometry and confocal laser scanning microscopy. furthermore, incubation with cbp-fel d 1 resulted in an upregulation of the costimulatory molecule cd86 on the mddcs, which was not observed with fel d 1 or cbps alone. finally, cbp-fel d 1 induced a fivefold increase in the release of the pro-inflammatory cytokine tumour necrosis factor (tnf)-a and a fourfold increase in the release of the chemokine interleukin-8 from mddcs. taken together, the effects cbps possess make them interesting as novel allergen carriers for sit. the cysteine protease der p1 from dust mite of the genus dermatophagoides pteronyssinus is a major type i allergen. about 80% of house dust mite (hdm) allergic individuals are reactive to this protease in standard assays for detection of ige. a curative treatment for atopic allergy is immunotherapy (it) with hdm extracts which are complex mixtures occasionally resulting in anaphylactic reactions. novozymes focuses on developing a recombinant variant of der p1 which exhibit lowered risk of ige-mediated allergic reactions, while maintaining its ability to trigger proper th-cell responses. this may provide a safer alternative for specific it of hdm allergy. a secreted recombinant form of pro-der p 1 expressed by saccharamyces cerevisiae was obtained by fusion of the pro-enzyme to a fungal signal peptide. the n-glycosylation site of der p1 was mutated resulting in a deglycosylated pro-enzyme with a molecular mass of 35 kda. protein purification procedure was developed to obtain nearly pure der p1 protein followed by determination of concentration by active-site-titration with the cysteine protease inhibitor e64. the deglycosylated recombinant pro-der p 1 revealed immunologic similarity to the native der p 1 molecule when compared in basophile histamine release, ige-binding assays and t-cell proliferation assays. by in silico epitope mapping of a modelled 3-dimensional structure of der p1, five putative igg and ige epitopes were predicted. by protein engineering, the predicted epitopes were removed one by one in der p1 and screening for hypoallergenic variants was performed. combining inhaled long-acting b-2 agonist (laba) and inhaled corticosteroid (ics) seems to offer asthma control at a lower dose of ics than achieved by ics alone. fine mapping of t-cell surface markers by flow cytometry offers a detailed status of the individual's inflammatory response. the frequency of mt2 (cd4 þ cd45ra -cd62l þ cd11adim) and mt1 (cd4 þ cd45ra -cd62l -cd11abright) cells in peripheral blood, and their ratio, has been shown to differ predictably in atopics and patients with leprosy, where mt2 correlates with a th2 phenotype and mt1 with a th1 phenotype. stable asthmatics, requiring fluticasone propionate (fp) 750-1000 mg daily or equivalent, were randomized to receive, double-blinded, either seretide 1 [salmeterol and fluticasone propionate (sfc, n ¼ 16)] 50 mg/500 mg bd or fp 500 mg bd (n ¼ 17). if asthma was controlled based on lung function and symptoms at clinic visits every 6 weeks, ics dose was tapered until asthma exacerbated or 0 mg was reached. the frequency and ratio of mt2 and mt1 t cells of the patients was monitored at 6 week intervals. as treatment tapered, the frequency of mt2 cells decreased (p ¼ 0038 from first to final visit), whereas that of mt1 cells increased. the ratio of mt2/mt1 decreased (p ¼ 0049 from first to final visit). in patients receiving laba þ ics, the fall in mt2/mt1 ratio appeared to be more pronounced than in patients receiving ics alone. thus, the mt2 phenotype may be associated with stable asthma, whereas an imminent exacerbation may associate with an increase in the mt1 phenotype. laba may allow for a greater effect of fp on the mt ratio. activation of complement pathways, leading to production of c3a and c5a anaphylatoxins, has been postulated in the pathogenesis of asthma and allergic airway inflammation. the present study was undertaken to investigate the role of mannan-binding lectin (mbl), an initiator of the lectin pathway of complement, in asthma and allergic rhinitis. mbl levels and mbl-induced complement activity were determined in 19 patients of bronchial asthma with allergic rhinitis and 20 unrelated, age-matched controls of indian origin. mbl levels and activity were correlated with percent eosinophilia and percent predicted fev1 values of the patients. association of single nucleotide polymorphisms (snps) in exon 1 and intron 1 of the mbl with the disease, clinical markers, mbl levels and mbl-induced complement activity was analysed using standard statistical tools. significantly higher mbl levels and activity were observed in patients of bronchial asthma with allergic rhinitis as compared to the controls. we identified five snps, of which two, a816g in exon 1 and g1011a in intron 1 of the mbl, were novel. snp g1011a was significantly associated with the disease (p ¼ 0.0024, or ¼ 5.8696, 95% ci: 1.7316 < or < 19.8963). individuals with 'a' allele at position 1011 showed increased mbl levels, activity and disease severity. our results suggest that 'a' allele at position 1011 leading to high mbl levels and complement activity may be contributing to the severity of bronchial asthma and allergic airway inflammation. serum resistance of borrelia burgdorferi strains belonging to the b. afzelii and b. burgdorferi sensu stricto genospecies is dependent on binding of complement inhibitor factor h. we recently reported that factor h binding by b. burgdorferi is due to inducible expression of several approximately 20 kda plasmid-encoded, surface-exposed lipoproteins related to ospe (e.g. erpa, erpp and p21). in addition, a second class of factor h-binding proteins of approximately 27-35 kda has been described. the ospe-related lipoproteins are dramatically induced by b. burgdorferi during transmission from its tick vector into the mammalian host. the induction of ospe-related lipoproteins during mammalian infection may play a key a role in the borrelial evasion of the host's immune system. the goal of the present study was to define the factor h-binding regions of ospe-related proteins using mutagenesis, peptide mapping and surface plasmon resonance analysis (biacore). the combined studies revealed that the c-terminal regions of both human and mouse factor h (scrs 18-20) specifically bind to ospe-related lipoproteins. we also found fhr-1, whose c-terminal scrs 3-5 are homologous to scrs 18-20 of factor h, to bind to ospe. peptide mapping revealed five putative regions (designated i-v) in ospe that could directly interact with factor h. deleting the c-terminal 15 amino acid residues from region v of p21 abolished its ability to bind factor h. at the same time, however, synthetic peptides corresponding to the c-termini of ospe, p21 and erpp did not inhibit factor h binding to ospe. thus, the c-terminal-binding region v appears to be necessary but not sufficient for factor h binding. when a more specific mutation strategy was employed, where single amino acid residues in peptides spanning over the factor h-binding regions were mutated to alanines, we observed that lysines in the factor h-binding regions of ospe were required for factor h binding. the combined data have revealed that key lysine residues in ospe-related lipoproteins and ionic interactions are crucial for factor h interactions. furthermore, binding of ospe to the c-termini of both mouse and human factor h suggests that borrelia spirochetes utilize analogous complement resistance mechanisms in both rodents and man. in borrelia garinii strains, which in in vitro analyses have been found to be sensitive to complement killing, differences in the ospe sequences as well as in the expression of factor h-binding proteins may account for their susceptibility to serum lysis. role of yada, ail and lipopolysaccharide in serum resistance of yersinia enterocolitica serotype o:3 mannan-binding lectin (mbl), l-ficolin and h-ficolin are pattern recognition molecules of the innate immune system. we investigated the ability of these molecules to bind to different serotypes and noncapsulated variants of streptococcus pneumonia and staphylococcus aureus. we found that mbl binds to noncapsulated s. aureus strain (wood) but not any of the examined s. pneumoniae serotypes. l-ficolin binds to some capsulated s. pneumoniae serotypes (11a, 11d and 11f) as well as some capsulated s. aureus serotypes (type-1, -8, -9, -11 and -12). h-ficolin does not bind to any of the examined s. pneumoniae and s. aureus serotypes included in this study but did bind to a strain of aerococcus viridans. when bound to bacteria, mbl and h-ficolin initiated activation of complement factor c4, whereas l-ficolin did not. during this study, quantitative assays for the three proteins were developed and the concentration in 97 plasma samples were determined and the median values were estimated at 0.8 mg of mbl/ml, 3.3 mg of l-ficolin/ml and 18.4 mg of h-ficolin/ ml, respectively. the absence of early complement components (c1, c4 and c2 but not c3) is a predisposing factor for systemic lupus erythematosus (sle). recently, we demonstrated that, in c4-deficient (c4 def.) mice, igm-containing immune complexes (igm-ic) are filtered by the splenic barrier of marginal zone macrophages (mzm), resulting in an increased immune response against antigens within these igm-ic, but this could not be observed in wildtype or c3 def. mice. we hypothesized that splenic cd11b þ mzm play an important role in the induction of autoimmunity, and we therefore analysed their cytokine profile after isolation with the help of magnetic antibody cell sorting. mrna was isolated, and real-time pcr was performed with specific primers for murine ifn-g (ifn-g), interleukin-12 (il-12) and ifn-a (ifn-a). we observe a moderate increase of il-12 and ifn-g mrna in cd11b þ cells of c4 def. mice compared to wildtype cells. surprisingly, the concentration of ifn-a mrna is six times higher in c4 def. mice. preliminary results suggest that mrna in cd11b þ cells of c3 def. mice is even lower than that in wt. six hours following i.v. application of 20 mg of a abstracts 625 .................................................................................................................................................................................................. murine monoclonal igm anti-dsdna antibody, production of il-12, ifn-g and ifn-a mrna is increased in cd11b þ cells of both c4 def. and wt mice. several references described increased levels of inf-a in patients with sle. dendritic cells are discussed as a major source of ifn-a. our observation that c4-deficient, sle-susceptible mice demonstrate an increased spontaneous ifn-a production by splenic cd11b þ marginal zone macrophages could be an early sign and a trigger for the development of sle. this is supported by the fact that the absence of c3 is not a predisposing factor for sle and our observation that c3 def. animals display low levels of ifn-a mrna. 200-400 million people worldwide and represents one of the leading causes for liver cirrhosis and hepatocellular carcinoma. control over the hbv infection is achieved mainly by vaccination with hepatitis b surface antigen (hbsag). hbsag contains n-linked glycosylation side and is recognized by both mbl-a and mbl-c in a cadependent manner. hbsag-mbl complexes activate complement and may thus affect humoural immunity. to investigate the role of mbl in humoural responses to hbsag, we immununized mice that lack both mbl-a and mbl-c proteins with soluble hbsag. it has been shown that deficiencies in other complement components like c1q, c4 and c3 result in decreased antibody responses. however, mbl double ko animals mounted dramatically increased humoural responses. after priming, mbl double kos mounted hbsag-specific igm responses, which were threefold higher than wt controls. after boosting the hbsag, total igg was 10-fold higher in mbl ko than in wt control animals. similar to the response to hbsag, other glycosylated soluble antigens (e.g. invertase) induced better humoural responses in mbl double ko animals, suggesting that mbl plays an important role in a negative feedback regulation of adaptive immunity. reconstitution experiments with rmbl partially rescued the ko phenotype. we propose that the clearance of glycoprotein antigens in mbl ko is handled differently from the wt, resulting in better stimulation of humoural responses. alternatively, glycoprotein-ag-mbl-rich complexes inhibit b-cell responsiveness via putative mbl receptors. the complement system is an important part of the innate immune system. the activation of complement proceeds through three different pathways that converge in the generation of c3-activating enzyme complexes. complement activation via the lectin pathway is initiated when recognition molecules, mannan-binding lectin (mbl) or ficolin, bind to carbohydrate structures characteristic for microbial surfaces. in the circulation, mbl and ficolins are found in association with three structurally related mblassociated serine proteases (masp)-1, -2 and -3 and a small, nonenzymatic component, map19. masp-2 has been shown to elicit complement activation through the sequential proteolytic cleavage of c4 and c2 upon binding of mbl/masp-2 complexes to microbial surfaces. we have recently uncovered a polymorphism in the masp-2/map19 gene in a patient shown to be deficient in the lectin pathway of complement activation. the polymorphism results in a single amino acid substitution in the n-terminal part of the masp-2 protein. recombinant wildtype masp-2 and masp-2 containing the amino acid substitution in question was produced, and the ability to activate complement was studied. the mutation had a profound impact on masp-2 function, resulting in the lack of complement activation through the lectin pathway. elisa-based experiments showed that the mutation leads to the impairment of complement activation through influencing the binding of masp-2 to mbl or ficolins. deficiencies in the lectin pathway of complement activation have so far been accounted for only by lack of functional mbl. the mutation described above is the first defect described affecting both activation through mbl and the ficolins. .................................................................................................................................................................................................. th1, th2 and treg cell balance. dcs are present in the gut mucosa and may thus be target for modulation by gut microbes, including ingested probiotics. here, we tested the hypothesis that species of lactic acid bacteria, important members of the gut flora, differentially activate dc. a large panel of human gut-derived lactobacillus and bifidobacterium spp. was screened for dc-polarizing capacity by exposing bone marrow-derived murine dc to lethally irradiated bacteria. cytokines in culture supernatants and dc-surface maturation markers were analysed. substantial differences were found among strains in the capacity to induce interleukin-12 (il-12) and tumour necrosis factor (tnf)-a, while the differences for il-10 and il-6 were less pronounced. bifidobacteria tended to be weak il-12 and tnf-a inducers, while both strong and weak il-12 inducers were found among the strains of lactobacillus. remarkably, strains weak in il-12 induction inhibited il-12 and tnf-a production induced by an otherwise strong cytokine-inducing strain of lactobacillus casei, while il-10 production remained unaltered. selected strains were tested for induction of dc maturation markers. those lactobacilli with greatest capacity to induce il-12 were most effective in upregulating surface mhc class ii and cd86. moreover, l. casei-induced upregulation of cd86 was reduced in the presence of a weak il-12inducing l. reuteri. in conclusion, human lactobacillus and bifidobacterium spp. polarize differentially dc maturation. thus, the potential exists for th1/th2/treg-driving capacities of the gut dc to be modulated according to composition of gut flora including ingested probiotics. the intestinal micro flora is indispensable in developing and maintaining homeostasis of the gut-associated immune system. evidence indicates that lactic acid bacteria (lab), e.g. lactobacilli and bifidobacteria, have beneficial effects on the host. established health effects include increased gut maturation, antagonisms towards pathogens and immune modulation. the objective of this study is to evaluate the immunomodulating properties of a range of lab of human origin. as dendritic cells (dcs) play a pivotal role in the balance between tolerance and immunity to commensal microorganisms, in vitro-generated immature dcs serve as a suitable model for studying the immunomodulating effects of lab. human immature dcs were generated in vitro from monocytes and exposed to lethally uv-irradiated lab. the effect of various species of lab on dcs in direct contact was evaluated. furthermore, the maturation pattern of dcs separated from the bacteria by an epithelial cell layer (caco-2 cells), which should mimic the intestinal environment, was studied. cytokine secretion (il-12, il-10 and tnf-a) and upregulation of maturation surface markers on dcs (cd83 and cd86) was measured. different lab induced diverse cytokine responses. some strains were strong il-12 and tnf-a inducers and others weak. all strains induced il-10. different lab also differentially modulated expression of cd83 and cd86 on dcs. although some variation in the response to lab of dcs generated from different blood donors was observed, general differences in the effect of the various lab was revealed. experiments with the dc caco-2 coculture system are ongoing. different species of lab differentially affect dc maturation; this suggets that the gut flora plays a pivotal role in polarization of the immune response. natural killer (nk) cells are cells of the nonspecific immune system lysing altered self-cells. a noncytolytic subset of nk cells may serve a regulatory role by secreting cytokines. bacteria translocating across the gastrointestinal mucosa are presumed to gain access to nk cells, as consumption of certain lactic acid bacteria has been shown to increase in vivo nk cytotoxicity. here, we investigated how human gut flora-derived lactobacilli affect nk cells in vitro, by measuring proliferation and ifn-g production of human nk cells upon bacterial stimulation. cd3 -cd56 þ nk cells were isolated from buffy coats by negative isolation using non-nk lineage-specific antibodies and magnetic beads. nk cells were incubated with 10mg/ml uv-inactivated bacteria or 10mg/ml phytohemagglutinin (pha) for 4 days. proliferation was assessed by incorporation of radioactive thymidine into nk-cell dna. the ifn-g concentration was measured by elisa. incubation of nk cells with a lactobacillus acidophilus strain increased the proliferation of the nk cells and induced ifn-g production, both to levels comparable to pha stimulation. the proliferative response was further enhanced with autologous monocytes present, probably because cytokines, secreted by monocytes having engulfed bacteria, stimulated the nk cells. in contrast, a lactobacillus paracasei strain caused the nk cells to proliferate only in the presence of monocytes. these results demonstrate that various strains of lactobacilli have the capacity to activate nk cells in vitro, in a monocyte-dependent or -independent way. hence, the encounter of nk cells with lactic acid bacteria will affect nk-cell activation. such activation of nk cells may potentially skew an on-going or subsequent immune response towards a th1 response. lactobacilli are nonpathogenic gram-positive inhabitants of the normal human intestine known for their healthpromoting effects. in our earlier work, it is shown that human monoclonal antibody isolated from sera of a patient with waldenstrom macroglobulinaemia possess innate antibody characteristics and binds to lactic acid bacteria. according to the immune network model, immunization with this bacteria could induce the perturbations in immune system that might result in production of anti-lactobacillus antibodies, human monoclonal antibody like (ab1) and anti-idiotypic antibody (ab2). in this study, balb/c mice were immunized with two doses of bacteria lactobacillus acidophilus in complete and incomplete freund's adjuvant and phosphate-buffered saline (pbs), respectively. seven days after the last immunization, sera from immunized mice were collected and the presence of lactobacillus-specific ab1 and ab2 were determined by elisas. in the sera of immunized mice, antibodies specific to bacteria lactobacillus acidophilus were shown. the concentration of lactobacillus-specific antibodies was higher in the sera of hyperimmunized mice (mice immunized with 1 mg of igm dj) than in sera of mice immunized with 100 times lower doses of immunogen (0.01 mg per doses). moreover, ab1 and ab2 antibodies were detected in the sera of lactobacillus-hyperimmunized mice. in this study, we have shown the idiotypic network interactions in mice immunized with bacteria lactobacillus acidophilus. the normal gastrointestinal flora is crucial for the maturation of the acquired immunity via effects on antigenpresenting cells (apcs). here, we have investigated how two types of apcs, monocytes and dendritic cells (dcs), react to different bacterial strains typical of the commensal intestinal flora. purified monocytes and monocyte-derived dcs were stimulated with uv-inactivated gram-positive (lactobacillus plantarum and bifidobacterium adolescentis) and gram-negative (escherichia coli and veillonella parvula) bacterial strains. monocytes produced higher levels of il-12p70 and tnf, as detected by elisa, in response to l. plantarum than to e. coli and v. parvula. in contrast, dcs secreted high amounts of il-12p70, tnf, il-6 and il-10 in response to e. coli and v. parvula but were practically unresponsive to l. plantarum and b. adolescentis. the lack of response to the gram-positive strains correlated with a lower surface expression of toll-like reseptor 2 (tlr2) on dcs compared to monocytes. the surface expression of tlr4 on dcs was undetectable when analysed by flow cytometry, but blocking this receptor decreased the tnf production in response to v. parvula, indicating that low tlr4 expression on dcs is sufficient to mount an inflammatory response to gram-negative bacteria. ifn-g increased the expression of tlr4 on dcs and also potentiated the cytokine response to gram-negative bacteria. our results indicate that, when monocytes differentiate into dcs, their ability to respond to different commensal bacteria dramatically changes, thereby becoming unresponsive to probiotic gram-positive bacteria. these results may have important implications for the capacity of different groups of commensal bacteria to regulate mucosal and systemic immunity. probiotic bacteria, e.g. lactobacillus spp., may improve diseases such as chronic inflammatory bowel disease. we examined cytokine production and phenotypic change after in vitro stimulation of t cells from healthy volunteers using different probiotic strains. methods: t cells were cultured from colonic biopsies from eight healthy volunteers (agnholt and kaltoft, exp clin immunogenet 2001; 18:213-25) , and dendritic cells were matured from their peripheral blood mononuclear cells. t-cell cultures were stimulated with autologous bacterial sonicate or strains of lactobacillus spp., with and without the addition of dendritic cells. cytokine levels (tnf-a, ifn-g, il-10 and gm-csf) and phenotype (cd3, cd4, cd25 and cd69) were measured on day 4. results: lactobacillus spp. induced higher productions of tnf-a and il-10 than did autologous bacteria. in presence of dendritic cells, the production of all cytokines increased. however, the increases of ifn-g and tnf-a were more pronounced in wells with autologous bacteria than in wells with lactobacillus spp. the addition of dendritic cells upregulated cd25 expression without simultaneous upregulation of cd69. the upregulation was pronounced after stimulation with lactobacillus rhamnosus gg compared with autologous bacteria and other lactobacilli. discussion: in presence of dendritic cells, autologous bacteria induced inflammatory cytokines, while probiotics mainly induced regulatory cytokines. lactobacillus rhamnosus gg induced a regulatory phenotype (cd25 þ ), in part mediated by dendritic cells. future studies will address whether this shift to a cd25 þ phenotype represents a differentiation into competent regulatory t cells. in a clinical context, such cells might be used for treatment of inflammatory diseases. protein microarrays will play a key role in the postgenomic era and offer a unique possibility to perform highthroughput global proteome analysis. a chip can be printed with thousands of protein probes (e.g. antibodies), the biological sample added (e.g. a proteome) and any binding detected. we aim to develop protein microarrays based on human recombinant scfv antibody fragments for global proteome analysis. the concept of comparing proteomic maps of healthy versus diseased samples will allow diseasespecific proteins to be detected. in fact, antibody microarrays will allow us to perform comparative proteome analysis on any sample format in a species-independent manner, as long as a proteome can be isolated. however, the complexity of proteomes, containing several thousands of different proteins, is a problem. here, we have designed antibody microarrays targeting the water-soluble fraction of a proteome. to this end, an anticytokine antibody array was developed and human dendritic cells (aeactivation) was used as model system. the results showed that our antibody microarrays could be used to examine the cytokine profile in complex samples. furthermore, we have taken the first steps towards comparing our results with those of other technologies on both the protein and gene level. due to the complexity of the model proteome, we also examined the possibility to prefractionate the proteome in a simple one-step procedure (based on size) prior to the labelling step. in more detail, the sample proteome was fractionated into two fractions using membrane devices with different molecular weight cut-offs. the results showed that the fractionation considerably enhanced the assay sensitivity allowing cytokines in the pg/ml range to be readily detectable. the immunomodulatory effect of heat shock protein 70: immunization with a dna construct based on the malarial antigen fused with a fragment of hsp 70 primes for a th-1 type of response finding an appropriate adjuvant for human vaccination is crucial. heat shock proteins (hsps) act as adjuvants when coadministered with peptide antigens or given as fusion proteins. however, there is a potential risk of autoimmunity when using the complete molecules, because hsps are evolutionary conserved. to overcome this, we first evaluated the adjuvant effect against two different antigens of a less-conserved fraction of plasmodium falciparum hsp70 (pf70c) and compared it to the whole hsp70 molecule from trypanosoma cruzi (tchsp70). we found that pf70c exhibited similar adjuvant properties as the whole molecule. we later evaluated the adjuvant potential of pf70c against the malarial antigen eb200 in a chimeric dna construct. no appreciable levels of eb200-specific abstracts 629 .................................................................................................................................................................................................. antibodies were detected in mice immunized only with the dna constructs. however, dna primed the immune system, because subsequent challenge with the corresponding recombinant fusion proteins elicited a strong th-1 antibody response. in contrast, no priming effect was observed for ex vivo ifn-g production but stimulation with the hsp-chimeric fusion protein induced a stronger secretion of ifn-g in vitro than other proteins used. these results indicate that the use of hsps is promising in the design of new vaccines. high-throughput proteomics on antibody-based microarrays: the importance of probe and surface design in analogy to dna microarrays, protein microarrays offer a new distinct possibility to perform sensitive highthroughput global proteome analysis. however, the development of the protein microarray technology will place high demands upon the design of both probes and solid supports. the analysis of thousands of heterogeneous proteins on a single microarray requires the use of uniform probes, such as antibodies, directly designed for protein microarray applications. we have recently generated a human recombinant single-chain fv antibody library, genetically constructed around one framework, the ncoder-library, containing 2 â 1010 clones. single framework antibody fragments (sinfabs) selected from this library were successfully applied as probes for microarrays providing sensitive detection in the 600 attomol (mass spectrometry) and the 300 zeptomole range (fluorescence). however, the choice of framework is critical. we have shown that the selected ncoder framework displayed excellent functional on-chip stability and arrayed dehydrated probes retained their activity for several months. furthermore, we have addressed the issues of biocompatibility of the solid support and immobilization strategies for our microarray setup. an in-house-designed substrate, macroporous silicon coated with nitrocellulose (map3-nc7), displayed properties equal to, or better than, those of five commercially available supports used as reference surfaces. we have also evaluated different coupling strategies, such as adsorption, covalent coupling, diffusion and affinity coupling. using a novel affinity tag, the double-(his)6-tag, we increased the binding efficiency of sinfab-molecules to ni2 þ -coated solid supports, thereby allowing nonpurified probes to be directly applied. the mannan-binding lectin (mbl) pathway is part of the innate immune system providing a first line of defence against infections. mbl and ficolins circulate in complexes with mbl-associated serine proteases (masp-1, -2 and -3). after recognition of a microorganism by mbl, activation of the complement system occurs. masp-1 and masp-3 share five domains (making up the so-called a-chain), whereas they have unique protease domains (b-chains). before the identification of masp-3, an assay for masp was presented, based on antibodies against the a-chain of masp-1. with the new knowledge of the three masps, and the sharing of domains by masp-1 and masp-3, assays specific for the protease domains have to be constructed, if one wishes to measure the proteins individually. we present an assay for quantifying total masp-3 in plasma and serum samples. the assay is a sandwich-type assay using as catching antibody a monoclonal antibody against the common a-chain of masp-1/3 and a developing secondary antibody against the c-terminal part of the protease domain of masp-3. we have used this assay for estimating the normal concentration of the protein as well as the concentration in patients and also for characterizing by gel permeation chromatography the masp-3 protein in serum. inducible costimulator ligand (icosl) is a costimulatory molecule related to b7.1 (cd80) and b7.2 (cd86). b cells, monocytes, dendritic cells and endothelial cells express icosl. inducible costimulator (icos) interacts with icosl, and this interaction leads to signals involved in isotype switching and the development of immunological memory. hitherto, no polymorphisms of this gene have been described. the aim of this study was to reveal variation of the icosl gene in normal individuals. all eight exons, except exon 1, were sequenced with flanking introns in 10 healthy blood donors. eight single nucleotide polymorphisms (snps) and two length polymorphisms were found. one of the snps was found in the coding regions of the gene. the base involved was located in exon 3 and caused a conservative amino acid change from valine (gtt) to isoleucine (att). three individuals were heterozygous g/a for the exon polymorphism, while the remaining seven individuals were homozygous for the wildtype g/g. exon 3 encodes the immunoglobulin variable (igv)-like domain of the molecule which is situated outside the cell. this means that the amino acid could be critical for the stability of the molecule or could constitute part of the binding site for icos. the results form the basis for further experiments to find possible associations of the alleles to diseases caused by immune dysregulation. especially, the exon 3 variant is interesting and could play a role for the development of immunological diseases. besides, it would be interesting to see whether both exon 3 alleles are expressed or only the wildtype allele is functional. interferon-a/b (ifn-a/b) is increasingly used as antiviral and immunomodulatory therapies. unfortunately, bioavailability varies with ifn species and mode of administration, and all ifn species are potentially immunogenic. assays for antiviral activity (ifn) and antiviral neutralization (antibodies, nab) have been used for some time to monitor patients on ifn biologicals. these assays require laborious titrations making them unsuitable for large-scale clinical use. myxovirus a (mxa) is a resistance gtpbinding protein that is specifically induced by treatment with type 1 ifns. for example, ifn-b-induced mxa in blood leucocytes has been used as a biomarker in ifn-btreated patients with multiple sclerosis. however, the degree of specificity of mxa in this regard is unclear, and measurements of mxa protein and/or mrna are not yet suitable for routine clinical use. in an attempt to find new and better reporter genes (and, hopefully, genes and gene products with proven specificity for ifn-a and -b), microarray screenings with u133a genechips (affymetrix) were carried using human blood leucocytes and the human lung carcinoma cell line a549. we studied the simultaneous expression of 22,000 transcripts before and after exposure to human recombinant ifn-a and ifn-b and other antiviral and immunomodulatory cytokines. the results will be presented at the conference. interferon-a/b (ifn-a/b) is increasingly used as antiviral and immunomodulatory therapies. unfortunately, bioavailability varies with ifn species and mode of administration, and all ifn species are potentially immunogenic. assays for antiviral activity (ifn) and antiviral neutralization (antibodies, nab) have been used for some time to monitor patients on ifn biologicals. these assays require laborious titrations making them unsuitable for large-scale clinical use. our laboratories have therefore modified the antiviral assays for ifn bioactivity and nab, so that they are suitable for large-scale screening in specialized laboratories. the read-out is survival of a subcloned a549 cell line in the presence of an otherwise lethal amount of virus. thus, survival increases in the presence of type 1 ifn and decreases in the presence of nab against the ifn added to the cells. mxa is induced by type 1 ifn and can be used for measuring the nab activity. in another assay, the mxa level in the a549 cell line is measured. in an attempt to find a new and better reporter gene for type 1 ifn than mxa and genes specific for either ifn-a or -b, a micro array screen was carried using the u133a chip from affymetrix. the expression of 22,000 genes can be studied simultaneous with this technology. the results will be presented at the conference. in our laboratory, we have developed a database system, which we believe is of immediate interest to the general scientific community. the database represents a computerbased replacement for the laboratory notebooks used in the majority of research laboratories worldwide. in addition, the database provides an effective tool for organizing and managing laboratory information at all levels, spanning from managing and revising standard operating procedures and producing documentation of research activities to keeping track of data and conclusions. using the commercially available database toolkit software filemaker pro, we have developed a relational database solution for management of laboratory information. the system consists of a hierarchy of five interrelated databases, each pertaining to a separate type of information, namely, overall project information, information relating to individual experiment setups, documentation of daily research activity, generated data and descriptions of standard operating procedures. like other databases, each individual database consists of a number of records, each comprised of a set of fields in which information is entered. in each record, a certain field is reserved to specify the relation of the record to a record in another database at a higher level. thus, the database is essentially five databases linked by a hierarchy of one-to-many relations, organizing information in a folder-like structure. importantly, the database system allows multiple users to access and edit records simultaneously, and the data entered in one database immediately becomes accessible through the other databases. the limitations of laboratory notebooks are apparent when looking for information, which is dispersed throughout one or more notebooks, or possibly on loose sheets of paper or printouts 'somewhere'. the often complicated process of gathering laboratory data or results when writing grant applications or research papers is made considerably easier with the database system. thus, the database solution presented should be broadly attractive to researchers, irrespective of their scientific discipline. an effective sars vaccine is likely to include components that can induce specific cytotoxic t-cell (ctl) responses. the specificities of such responses are governed by hlarestricted presentation of sars-derived peptide epitopes. exact knowledge of how the immune system handles protein antigens would allow for the identification of such linear sequences directly from genomic/proteomic sequence information. the latter was recently established when a causative coronavirus (sars cov) was isolated and full-length sequenced. here, we have combined advanced bioinformatics and high-throughput immunology to perform an hla supertype, genome-wide scan for sars-specific cytotoxic t cell epitopes. the scan includes all nine human hla supertypes in total covering >99% of all major human populations. for each hla supertype, we have selected the 15 top candidates for test in biochemical-binding assays. at this time (approximately 6 months after the genome was established), we have tested the majority of the hla supertypes and identified almost 100 potential vaccine candidates. these should be further validated in sars survivors and used for vaccine formulation. we suggest that immunobioinformatics may become a fast and valuable tool in rational vaccine design. rationale: major histocompatibility complex class i (mhc i) molecules monitor the protein content of the cell by binding small derived peptides and presenting them to cytotoxic cd8 þ t cells. the goal of the human mhc project is to predict the binding strength of any given peptide/mhc complex. this prediction allows the design of peptide-based vaccines. the prediction requires representative binding data from mhc alleles from all the nine hla supertypes. here, we describe the genetic construction, protein production and purification as well as the establishment-binding assays for two recombinant mhc supertype alleles, hla-b*1501 and hla-b*5801. methods: using the quikchange multisite directed mutagenesis kit (stratagene), codon-optimized genes encoding hla-b*1501 and hla-b*5801 are created. the two mhc i molecules are fermented and purified by ion exchange chromatography, hydrophobic interaction chromatography and size exclusion chromatography. the binding (kd) of natural t-cell epitopes, as well as predicted peptide ligands, is described by radioactive immunoassays (rias) and enzyme-linked immunosorbent assays (elisas). the mhc molecules are biotinylated during expression. results: the expression of mhc i resulted in multiple disulfide bond isomers, which are separated by hydrophobic interaction chromatography and used in subsequent binding studies resulting in the determination of kd for various peptide ligands ranging from strong binders we have previously demonstrated that bioinformatics tools such as artificial neural networks (anns) are capable of performing pathogen-, genome-and hlawide predictions of peptide-hla interactions. these tools may therefore enable a fast and rational approach to epitope identification and thereby assist in the development of vaccines and immunotherapy. a crucial step in the generation of such bioinformatics tools is the selection of data representing the event in question (in casu peptide-hla interaction). this is particularly important when it is difficult and expensive to obtain data. herein, we demonstrate the importance in selecting information-rich data and we develop a computational method, query-bycommittee, which can perform a global identification of such information-rich data in an unbiased and automated manner. furthermore, we demonstrate how this method can be applied to an efficient iterative development strategy for these bioinformatics tools. methods: a large panel of binding affinities of peptides binding to hla a*0204 was measured by a radioimmunoassay (ria). this data was used to develop multiple first generation anns, which formed a virtual committee. this committee was used to screen (or 'queried') for peptides, where the anns agreed ('low-qbc'), or disagreed ('high-qbc'), on their hla-binding potential. seventeen low-qbc peptides and 17 high-qbc peptides were synthesized and tested. the high-or low-qbc data were added to the original data, and new high-or low-qbc second generation anns were developed, respectively. this procedure was repeated 40 times. the high-qbc-enriched ann performed significantly better than the low-qbc-enriched ann in 37 of the 40 tests. conclusion: these results demonstrate that high-qbcenriched networks perform better than low-qbc-enriched networks in selecting informative data for developing peptide-mhc-binding predictors. this improvement in selecting data is not due to differences in network training performance but due to the difference in information content in the high-qbc experiment and in the low-qbc experiment. finally, it should be noted that this strategy could be used in many contexts where generation of data is difficult and costly. interleukin-18 (il-18), a pro-inflammatory cytokine that is produced by both lymphoid and nonlymphoid cells, has a critical role in modulation of innate and adaptive immunity. its primary function in stimulation of ifn-g production and stimulation of nk-cell-cytotoxic activities makes this cytokine a candidate for cancer immunotherapy. in oral cavity, this cytokine is produced by oral epithelia and carcinoma cells and is related to tumour regression in nude mice bearing salivary adenocarcinoma. however, direct effects of this cytokine on oral cancer cells have not been elucidated. in this project, we investigated il-18 effect on an oral carcinoma (kb) cell line. with rt-pcr technique, kb-cell line was found to express il-18 receptors (il-18ra and il-18rb), indicating that this oral carcinoma line is a target for il-18 study. we showed that recombinant human il-18 inhibited kb-cell proliferation by 17% at concentration of 100 ng/ml (p < 0.05), whereas ldh release by these cells in treatment group and control groups was comparable, indicating that il-18 suppression of cell proliferation was not mediated by the induction of cell death. to further address this hypothesis, we found that il-18 treatment did not induce apoptotic cell death, as studied by dna laddering and tunel assays. in addition, expression pattern of cell death-controlling genes (bcl-2 and bax) was not altered by this cytokine. findings in these studies indicated that suppression of kb-cell proliferation may be attributed to control of cell cycle, growth arrest or induction of cell differentiation. the data presented in this project could provide an insight of how cancer cell directly responds to il-18, as this cytokine is an important regulator of anticancer mechanisms. aloe emodin (ae) is a naturally occurring compound with wide spectrum of biological properties, including antimicrobial, vasorelaxant, immunosuppressive and anticancer actions. this anthraquinone induces apoptosis in several tumour cell lines with special affinity to tumours of neuroectodermal origin. high amounts of nitric oxide (no) released by activated macrophages induce tumour cell death. therefore, we explored the capacity of ae to modulate no-mediated antitumour response in vitro. interestingly, while ae markedly suppressed no release from macrophages alone, it significantly potentiated no production in cocultures of macrophages and c6 cells, after 48 h of cultivation. accordingly, the viability of c6 cells cocultivated with macrophages was reduced in the presence of ae. moreover, the observed ae-imposed potentiation of no production in macrophages was closely related to macrophage culture cell density. according to these data, we proposed that no modulator capacity of ae strongly depended on intercellular contact, indicating that macrophage antitumour response was not compromised but even potentiated by ae. immunotherapy represents an attractive fourth-modality therapeutic approach, especially in the light of the shortcomings of conventional surgery, radiation and chemotherapies in the management of metastatic cancer. to this end, a large number of peptide antigens derived from taa have been applied in immunotherapeutic trials for the treatment of various malignancies, e.g. cancers of the breast, prostate and kidney, in addition to haematological cancers. in some cases the response rates have been impressive and no adverse autoimmunity have been observed. a major strategic difficulty associated with these trials relates to the choice of best-suited peptide antigens. the vast majority of the antigens described thus far is not vital for survival and growth of the tumour cells, and immunoselection of antigen-loss variants may therefore prove to be an additional obstacle for the clinical applicability of most of the known peptide epitopes. in this respect, the development of acquired antigen loss during immunotherapy has been demonstrated in several cases. obviously, the development of loss-variant tumour cells implies that these cells acquire a pronounced growth advantage and are left unaffected by further treatment. ideally, target antigens should be derived from proteins required for survival and growth of tumour cells, as antigens with these characteristics would not be inflicted by the development of loss-variant tumour cells. in this respect, several inhibitors of apoptosis proteins (iaps) are universally expressed among tumours and play an important role in tumour cell escape from apoptosis. we have characterized spontaneous t-cell reactivity against iapderived peptides in cancer patients. from the iap survivin, we have characterized peptides restricted to the class i molecules hla-a1, a2, a3, a11, b7 and b35. furthermore, we have demonstrated that survivin-specific t cells infiltrate metastatic lesions and that isolated survivinspecific ctls are capable of killing hla-matched tumour cells. survivin-derived peptides are now in clinical trial, and continued work in our lab has demonstrated that other iaps are targets for spontaneous t-cell reactivity in cancer patients. we previously reported that in mice with large progressing t-cell lymphoma tumours, dysfunctions in the antitumour ctl activity occur, associated with an accumulation of splenic arginase-producing myeloid suppressor cells (mscs). in this study, we first demonstrate that both the presence and the activation state of these msc depends on tumour evolution. while in tumour regressors hardly any arginase-producing msc can be found, both the amount and the arginase activity of this population expands from early over late progressors. this gradual induction of mscs is paralleled by an increasing suppression of ctl activity and th1, but not th2, cytokine production. upon analysing the molecular repertoire of msc in vitro, we found, besides arginase1, a well-established marker for alternatively activated myeloid cells or m2, a strong upregulation of fizz1 and ym, two additional recently identified markers for m2. further evaluation of molecular markers by microarray analysis in msc yielded genes involved in wound healing (e.g. coagulation factor xiiia), anti-inflammation (e.g. selenoprotein p), immunomodulation (e.g. pd-l2) and fat and sugar metabolism (e.g. leptin receptor). of note, many of these genes are regulated by type 2 cytokines (il-4, il-13 and il-10) and are therefore rather m2 associated. overall, our data provide new markers for msc in cancer and further establish their m2 activation state. study. only sp-a showed a significant expression in normal mucosa which was downregulated in crc. as the absolute signal level was below the noise threshold, these results have to be interpreted with caution and require confirmation by direct measurenment of the proteins. our results suggest that there is no major role for the human collectins in colorectal cancer. tetramerization is visualized by sds-page. conclusion: an effective method for the production of highly pure mhc i molecules has been applied to hla-b*1501 and hla-b*5801, and ria and elisa binding assays for those alleles have been established background: proliferation, differentiation and apoptosis are essential processes in the normal functions of the mammary epithelium. the hypothesis examined in this study is that the transcription factor bcl-6 is critically important not only for regulating b-cell growth and development but also for mammary epithelial apoptosis. methodology: twenty breast cancer cases and 31 healthy controls were used to investigate whether bcl-6 protein in involved in breast cancer (grade iii). full length bcl-6 cdna was retrovirally transduced into eph-4 cell line. we then used flow cytometry of brdurd-stained cells to investigate the cell-cycle duration of the control and transduced cell lines. tunel was used as a marker of apoptosis to find out differences in the frequencies of apoptotic cells in the control and transduced cell lines. finally, immunohistochemistry staining was performed to detect bcl-6 in breast cancer (iii). results: restoration of bcl-6 into eph-4 cells not only inhibits apoptosis but also prolongs the cell cycle and results in increased cell size and protein content. the results also indicated that the cell-cycle time of bcl-6-transduced eph-4 cells is prolonged by about 3 h, presumably as a result of the action of bcl-6 at the bcl-6 at the g1/s transition. we found differences in the frequencies of viable and apoptotic cells in cultures of the parent eph-4 cells, control-transduced eph-4 cells and bcl-6-transduced eph-4 cells. consistently, we demonstrated that bcl-6 is expressed in 90% of high grade of breast carcinoma, which is considered as the most aggressive of tumours. conclusion: together, these results suggest that bcl-6 is likely to be involved in mammary gland development and carcinogenesis. inflammatory cytokines have a critical role in modulation of both innate and adaptive immunity in response to foreign antigen. they also play an important role in anticancer immunity. for example, they can promote cell-mediated immunity against cancer cells. with their immunostimulatory effects, these cytokines are being tested for cancer treatment in the form of dna vaccine or adjuvant or therapeutic cytokines. direct effect of these cytokines on cancer cell, however, is still unclear. in this project, we investigated whether il-1( and il-18 can modulate cancer cell proliferation. we employed a simple nonradioactive proliferation (mtt) assay and detection of lactate dehydrogenase (ldh) to test the effect of these recombinant human cytokines on various cancer cell lines, including breast cancer cell line (mcf-7), oral carcinoma cell line (kb), colon cancer cell line (caco-2) and choriocarcinoma cell line (jar). cytokines used in this study had both inhibitory and stimulatory effect on cell proliferation. findings in this project could provide an insight of cancer cell response to these cytokines and this could lead to a consideration on using cytokine as immunotherapy for cancer treatment.capacity of ae to modulate nitric oxide production depended on intercellular contact donor t cells are involved in the antitumour effects observed after bmt. thus, patients receiving t-celldepleted bmt have a higher risk of leukaemia relapse compared to patients receiving nonmanipulated bmt, and patients experiencing graft-versus-host disease (gvhd) have a lower risk of disease relapse than patients who do not experience gvhd. although the importance of donor t cells for the curative action of bmt has been established, the exact mechanisms and molecules involved in this graft-versus-tumour effect remain largely unknown. in a recently initiated project, we have conducted a longitudinal study of t-cell clonotypes in patients who received peripheral blood stem cell grafts after nonmyeloablative conditioning. peripheral blood samples were obtained sequentially after transplant, and the mononuclear cells (mncs) were isolated and cryopreserved. cd8 þ t cells were isolated from the mncs by use of immunomagnetic beads or facs and analysed for the presence of clonally expanded cells by t-cell receptor clonotype mapping based on rt-pcr and denaturing gradient gel electrophoresis (dgge). using this gel-based methodology, clonally expanded t cells were monitored after transplant and compared to the clinical data of the patients. the preliminary results demonstrates the presence of clonally expanded cd8 þ t cells at all time points analysed. furthermore, a number of clonotypes persisted for more than 6 months, and other clonotypes emerged during this period. the appearance of newly emerged clonotypes which coincided with clinical gvhd could indicate a role for these t cells in the pathogenesis of gvhd. background: deficiency of the mannan-binding lectin (mbl) pathway of innate immunity leads to increased susceptibility to infections. in patients with colorectal cancer, postoperative infection is associated with poor prognosis. the aim of the present study was to evaluate (1) the relation between the mbl pathway and postoperative infectious complications and survival of patients resected for colorectal cancer and (2) the role of mbl as acute phase reactant compared to crp. methods: preoperative mbl concentration, mbl/mblassociated serine protease (masp) activity and crp were determined in serum from 611 patients and 150 healthy controls. the patients were observed for 8 years. postoperative infections, recurrence and survival were recorded. results: the mbl pathway components were increased in the patients (p < 0.0001) compared to healthy controls. low mbl levels were predictive of pneumonia (p ¼ 0.01), and pneumonia (n ¼ 87) was associated with poor survival (p ¼ 0.003, hr ¼ 1.5, 95% ci 1.1-1.9). mbl and mbl/ masp activity could not predict postoperative overall infections. mbl showed no correlation (spearman's r ¼ 0.02, 95% ci à0.06-0.10) with crp. conclusions: low preoperative mbl levels are predictive of pneumonia, which is associated with poorer survival. mbl concentration and mbl/masp activity was not predictive of other postoperative infections or long-term prognosis. mbl apparently is not a surrogate measure of crp. department of surgery, university hospital of erlangen, erlangen, germany. e-mail: michael.siassi@rzmail.uni-erlangen.de introduction: the human collectins, mannan-binding lectin (mbl), surfactant protein-a (sp-a) and surfactantprotein-d (sp-d) play a central role in the innate immune system. immunological responses to malignant transformation of epithelial cells gained increasing interest recently. a former study could demonstrate binding of mbl to certain colorectal carcinoma (crc) cell lines in vitro. we therefore examined the expression of human collectins in normal colon mucosa and in colorectal carcinomas. materials and methods: colon samples from 20 crc patients and 10 normal mucosa samples were collected immediately after surgery. the tissue was microdissected and rna isolated (qiagen, rneasy-kit). gene expression profiles were analysed using gene-chips (affymetrix, hg-u133). we analysed the data for the expression of mbl, its associated serine proteases mannan-binding lectinassociated serine protease 1/2 (masp 1/2), sp-a and sp-d. the signal intensity of the genes of interest was compared using the mann-whitney u-test. results: the expression of human collectins in normal human colon mucosa was generally low. only the expression of sp-a and masp-2 reached the noise threshold of 250 signals. these genes were significantly downregulated in crc specimens. the expression of the other proteins showed no difference in normal mucosa and crc. conclusion: as demonstrated before, the expression of human collectins in normal colon was low in this being the first lymph node to receive drainage from the tumour area, the sentinel node offers a unique possibility to obtain tumour-reactive lymphocytes. we investigated antitumour immune responses in sentinel nodes from patients with bladder cancer, by assaying tumour-specific proliferation and tcr vb repertoires. during tumour surgery, sentinel lymph nodes were identified by peritumoural injection of blue dye. fresh specimens of tumour, sentinel and nonsentinel lymph nodes were obtained, and single-cell suspensions were prepared. cells were assayed for reactivity against autologous tumour extract in [ 3 h]-thymidine incorporation assays and characterized by flow cytometry. parallel analyses of the expression of vb gene families were performed with padlock probes, linear oligonucleotides which upon target recognition can be converted to circular molecules by a ligase. probes were reacted with cdna prepared from magnetically separated cd4 þ cells, and the tcr repertoire was determined by hybridizing the products to oligonucleotide microarrays. dose-dependent proliferation in response to tumour extract could be detected in sentinel lymph nodes. common clonal expansions were detected among tumourinfiltrating lymphocytes and in sentinel lymph nodes. nonsentinel lymph nodes displayed a divergent tcr vb repertoire. these results indicate an ongoing immune response against tumour antigens in sentinel nodes, draining urinary bladder cancer. identification of sentinel lymph nodes makes it possible to obtain tumour-reactive lymphocytes for use in adoptive immunotherapy. key: cord-023431-zjyrhlxn authors: sigmundsdóttir, h.; johnston, a.; gudjónsson, j. e.; valdimarsson, h. title: differential effects of interleukin‐12 and interleukin‐10 on superantigen‐induced expression of cutaneous lymphocyte‐associated antigen and αeβ7 integrin (cd103) by cd8(+) t cells date: 2008-06-28 journal: scand j immunol doi: 10.1111/j.0300-9475.2004.01423ab.x sha: doc_id: 23431 cord_uid: zjyrhlxn the interaction with adhesion molecules expressed by vascular endothelium is the first step in lymphocyte infiltration into tissues. at both cutaneous and mucosal sites interleukin‐10 (il‐10), il‐12 and transforming growth factor (tgf)‐β are important regulators of chronic inflammatory disease, where cutaneous lymphocyte‐associated antigen (cla) and αe integrin (cd103) may be expressed. unlike cla, cd103 is not believed to play a role in tissue‐specific homing but may help to retain t cells within epithelial layers. we have previously shown that il‐12 alone can together with an unknown cofactor increase the expression of cla. stimulation with streptococcal pyrogenic exotoxin c (spec) increased the expression of cd103 by cd8(+) but not cd4(+) t cells. while il‐12 increased superantigen‐stimulated expression of cla, this cytokine strongly inhibited the cd103 expression, and a combination of il‐12 and tgf‐β completely abrogated the induced cd103 expression. conversely, il‐10 suppressed cla but increased cd103 expression. these findings indicate that, in addition to suppressing the development of th1‐mediated inflammatory responses, il‐10 may also inhibit the migration of cd8(+) t cells into the skin while il‐12 promotes such migration. thus, the expression of cla and cd103 may be antagonistically regulated by il‐10 and il‐12, and the balance between these cytokines could influence the t‐cell migration of inflammatory cells into epithelial tissues. the nuclear receptor heterodimers of liver x receptors (lxrs) are recently identified as key transcriptional regulators of genes involved in lipid homeostasis and inflammation. lxrs and their ligands are negative regulators of macrophage inflammatory gene expression. multiple sclerosis (ms), a demyelinating disease of the central nervous system of unknown cause, is characterized by recurrent inflammation involving macrophages and their inflammatory mediators. sweden belongs to the countries with a high ms incidence. in italy, incidence is lower, with an exception for sardinia where the incidence is even higher than that in sweden. subjects from sardinia are ethnically more homogeneous and differ from swedes, also regarding genetic background and environment. we studied lxrs and their related molecules of blood mononuclear cells (mncs) from female patients with untreated relapsing-remitting ms from sassari, sardinia and stockholm, sweden. sex-and age-matched healthy controls (hcs) were from both areas. mrna expression was evaluated by real-time pcr. lxr-a was lower (p < 0.05) in ms (mean ae sem: 3.1 ae 0.2; n ¼ 37) compared to hc (3.6 ae 0.1; n ¼ 37). lxr-a was lower in ms from stockholm (2.6 ae 0.2; n ¼ 22) compared to corresponding hc (3.4 ae 0.1; n ¼ 22; p < 0.01) and compared to ms (3.8 ae 0.2; n ¼ 15; p < 0.001) and hc (4 ae 0.2; n ¼ 15; p < 0.001) from sardinia. ms patients from stockholm, but not from sassari, also expressed lower (p < 0.05) lxr-b (à4.1 ae 0.4) compared to corresponding hc (à2.9 ae 0.3). ms from stockholm was associated with higher abca-1 (6.1 ae 0.4 versus 5.0 ae 0.3; p < 0.05) and higher estrogen receptor-b-cx (2.4 ae 0.4 versus 0.8 ae 0.4; p < 0.01) compared to corresponding hc. the hc from sassari had higher androgen receptor (2.9 ae 0.2) compared to ms from sassari (1.4 ae 0.3; p < 0.01), ms (1.3 ae 0.4; p < 0.01) and hc from stockholm (1.2 ae 0.3; p < 0.01). ms from sassari had lower cyclooxygenase-1 compared to corresponding hc (5.1 ae 0.4 versus 6.6 ae 0.3; p < 0.01) and lower prostaglandin-e (à0.03 ae 0.5) compared to the hc (1.4 ae 0.5; p < 0.05) and ms (2.7 ae 0.4; p < 0.05) and hc from stockholm (1.9 ae 0.4, p < 0.001). our findings identify lxrs and their related molecules as being involved in ms from stockholm but not from sassari, while sex hormone receptors seem to be involved in ms in sassari. multiple sclerosis: ifn-b induces cd123 + bdca2 -dendritic cells that produce il-6 and il-10 and have no enhanced type i interferon production y. m. huang, 1 s. adikari, 1 u. båve, 2 a. sanna 1,3 & g. alm 4 dc antigens (bdca) and investigate their ability to produce type i ifn in response to virus stimulation. we show that ifn-b induces development of cd123 þ dc from human blood monocytes, which coexpress bdca4 þ but are negative for bdca2 -, a specific marker for plasmacytoid dc. such ifn-b-modulated dc produce large amounts of il-6 and il-10, but no il-12p40 and have no enhanced ifn-b and ifn-b production. the findings indicate that ifn-bmodulated dc represent a myeloid dc subset with diminished cd11c, bdca-1 and cd1a expression, having potent th2-promoting function but lacking antiviral capacity. the association of psoriasis with throat infections by streptococcus pyogenes suggests a potential antigenic target for the t cells that are known to infiltrate dermis and epidermis of psoriatic skin. streptococcal m protein shares an extensive sequence homology with human epidermal keratins. keratins 16 (k16) and 17 (k17) are mostly absent from uninvolved skin but are upregulated in psoriatic lesions. there is increasing evidence that cd8 þ t cells play an important effector role in psoriasis and m proteinprimed t cells may recognize these shared epitopes in skin via molecular mimicry. to identify candidate epitopes, peptides with sequences from k17 were selected on the basis of predicted binding to hla-cw6 and sequence similarities with m6 protein. matched peptides from the sequence of m6 protein and a set of peptides with poor predicted binding were also selected. cw6 þ individuals with psoriasis and cw6 þ healthy controls, having a family history of psoriasis, were recruited. pbmcs were incubated with the peptide antigens. t-cell activation in the cd4 þ , cd8 þ and later the skin-homing cutaneous lymphocyteassociated antigen (cla)-expressing subset of cd8 þ t cells was evaluated by cd69 expression and intracellular ifn-g accumulation using flow cytometry. we demonstrate that cw6 þ psoriasis patients had significant cd8 þ t-cell ifn-g responses to peptides from k17 and m6 protein selected on the basis of sequence homology and predicted hla-cw*0602 binding. these responses were about 10 times more frequent in the skin-homing cutaneous lymphocyte-associated antigen-expressing (cla þ ) subset of cd8 þ t cells. cd4 þ t cells showed only borderline responses. cd8 þ t cells from cw6 þ nonpsoriatic individuals responded to some m6 peptides but very rarely to k17 peptides, and this also applied to the cla þ cd8 þ subset. these findings indicate that psoriatic individuals have cd8 þ t cells that recognize keratin self-antigens and that epitopes shared by streptococcal m protein and human keratin may be targets for the cd8 þ t cells that infiltrate psoriatic skin lesions. autoantibodies directed against citrulline-containing proteins have an impressive specificity of nearly 100% in ra patients and a suggestive involvement in the pathogenesis. the targeted epitopes are generated by a post-translational modification catalysed by the calcium-dependent enzyme peptidyl arginine deaminase that converts the positively charged arginine to polar but uncharged citrullin. the aim of this study was to analyse the presence of citrulline in the joints at different time points of collagen-induced arthritis in da rats by immunohistochemistry and to investigate how immunogenicity and arthritogenicity was affected by citrullination of rat serum albumin (rsa) and collagen type ii (cii). our results indicate that citrulline could be detected in joints of arthritic animals, first appearance at the onset of disease and increasing as disease progressed into a chronic state. unimmunized animals or time points before clinical signs of arthritis were negative. by morphology, we state that some infiltrating macrophages as well as the cartilage surface stain positive for citrulline, while the major source of citrullinated proteins appears to be fibrin depositions. a specific cit-rsa t-cell response was observed in animals challenged by citrullinated rsa, no response was recorded when rsa was used as a stimulus. the igg analysis reveals not only a response towards the modified protein but also cross-reactivity to native rsa. no t-cell or b-cell response was noted in animals injected with unmodified rsa. cit-cii induced a disease with higher incidence and earlier onset than did the native counterpart. we conclude that, in contrast to the human disease, citrulline does not seem to appear before clinical signs. as inflammation proceeds, citrulline is detected specifically in the joints. all other organs investigated were negative. we also conclude that citrullination of a protein can break tolerance and increase its arthritogenic properties. ectopic germinal centers (gcs) can be detected in the salivary glands of approximately 1/5 of patients with sjögren's syndrome (ss) and appear in both primary and secondary ss. previously, ectopic gc have been associated with increased local autoantibody production. the aim of this study was to determine whether gc in primary sjögren's syndrome (pss) defines a distinct seroimmunological phenotype. retrospectively, a material of 130 haematoxylin and eosin-stained paraffin-embedded tissue sections of minor salivary gland tissue from patients with pss was morphologically screened for the presence of ectopic gc. gc-like lesions were detected in 33/130 (25%) of the pss patients. seventy-two pss patients lacking these structures (gc-) were randomly selected for comparison. focus score was significantly increased in the gc þ patients compared to the gcpatients (p ¼ 0.035). in the gc þ group, 54.5% of the patients presented with anti-ro/ssa compared to 43.7% in the gcgroup. anti-la/ssb was detected in 31.3% of the gc þ patients compared to 25.7% of the gcpatients. sixty-one percentage of gc þ patients presented with increased levels of igg, a nonsignificant difference when compared to 39.4% in the gcpatients (p ¼ 0.089). levels of rf, ana, ena, igm and iga were similar in both patient groups, as were esr and crp. in conclusion, patients with ectopic gc have a higher focus score and more often present with autoantibodies and increased levels of igg compared to pss patients with regular focal infiltration (gc -). our findings may indicate a certain seroimmunological phenotype and warrant for further prospective studies. association between mannose-binding lectin and vascular complications in type 1 diabetes complement activation and inflammation have been suggested in the pathogenesis of diabetic vascular lesions. we investigated serum mannose-binding lectin (mbl) levels and polymorphisms in the mbl gene in type 1 diabetic (t1dm) patients with and without diabetic nephropathy and associated macrovascular complications. polymorphisms in the mbl gene and serum mbl levels were determined in 199 t1dm patients with overt nephropathy and 192 t1dm patients with persistent normoalbuminuria matched for age, sex and duration of diabetes as well as in 100 healthy control subjects. the frequencies of high and low expression mbl genotypes were similar in patients with t1dm and healthy controls. high mbl genotypes were significantly more frequent in diabetic patients with nephropathy than in the normoalbuminuric group, and the risk of having nephropathy, given a high mbl genotype, assessed by odds ratio was 1.52 (1.02-2.27), p ¼ 0.04. median serum mbl concentrations were significantly higher in patients with nephropathy than in patients with normoalbuminuria [2306 mg/l (iqr 753-4867 mg/l) versus 1491 mg/l (iqr 577-2944), p ¼ 0.0003], and even when comparing patients with identical genotypes, serum mbl levels were higher in the nephropathy group than in the normoalbuminuric group. patients with a history of cardiovascular disease had significantly elevated mbl levels independently of nephropathy status [3178 mg/l (iqr 636-5231 mg/l) versus 1741 mg/l (iqr 656-3149 mg/l), p ¼ 0.02]. the differences in mbl levels between patients with and without vascular complications were driven primarily by pronounced differences among carriers of high mbl genotypes (p < 0.0001). our findings suggest that mbl may be involved in the pathogenesis of microvascular and macrovascular complications in type 1 diabetes and that determination of mbl status might be used to identify patients at increased risk of developing these complications. neuroimmunology unit, center for molecular medicine, karolinska institutet, stockholm, sweden. e-mail: judit.wefer@cmm.ki.se dna vaccine coding for the encephalitogenic peptide mog 91-108 protects lew.1av1 from subsequent development of experimental autoimmune encephalomyelitis (eae). protection is associated with a type 1 immune response and is dependent on the presence of cpg dna motifs. the mechanisms underlying the observed reduction of eae development in protected rats have not been fully clarified. we investigated immunological characteristics of lymphocytes after dna vaccinaton and subsequent eae induction. we confirm that protection was not associated with suppression of t1 cells, as transcription of the novel molecule rat t-cell immunoglobulin-and mucindomain-containing molecule (tim-3), reported to be exclusively expressed on differentiated t1 cells, was not altered by dna vaccination. we did not note any clonal deletion upon tolerization, but detected an antigen-specific lymphocyte population upregulating ifng upon recall stimulation 3 weeks after protective dna vaccination. in protected rats, we observed (1) no alterations in antigenspecific th2 or th3 responses, (2) reduced mhc ii expression on splenocytes early after eae induction, (3) antigen-specific upregulation of ifnb upon recall stimulation and (4) reduced il-12rb2 on lymphocytes. we thus demonstrate an association of the protective effect of dna vaccination with expression of ifnb. we are currently investigating the cellular mechanisms behind this ifnb-mediated protection. multiple sclerosis (ms) is an autoimmune condition characterized by degeneration of nerve fibre myelin sheets. a candidate autoantigen, myelin basic protein (mbp), has especially attracted attention. the presence of anti-mbp antibodies is a predictor of definite ms, but their role in the pathogenesis remains obscure. t cells have long been known to play a pivotal role in the pathogenesis of ms. recently, an important role for b cells as autoantigen-presenting cells has been demonstrated in other autoimmune diseases, including rheumatoid arthritis and diabetes. the uptake of mbp by b cells and the presentation of mbp-derive peptides to t helper (th) cells by b cells may be promoted by the formation of complement (c) activating immune complexes (ics) between mbp and natural autoantibodies in healthy individuals and disease-associated anti-mbp antibodies in ms patients, respectively. we have investigated the formation of mbp-containing ic, the binding of mbp to b cells, the mbp-elicited induction of th-cell and b-cell proliferation and the cytokine production in peripheral blood mononuclear cells (pbmcs) from healthy donors grown in the presence of intact or c-inactivated serum from healthy donors or patients with ms. while mbp did not induce measurable proliferation of b cells nor cd4 þ t cells, we observed the production of tnf-a, ifn-g and il-10 by pbmc in response to incubation with mbp in the presence of sera from healthy controls as well as sera from ms patients. by contrast, no production of il-2, il-4 and il-5 was detected. we are currently investigating the capability of ms sera to promote the formation of mbp-containing ic and thereby enhance the cytokine responses, by virtue of elevated anti-mbp contents. the phagolysosomally localized acid sphingomyelinase (asmase) activated by proinflammatory cytokines such as tnf and ifn-g generates the signalling molecule ceramide which in turn results in the activation of proteases like cathepsin d. these characteristics of asmase suggest a possible role of this molecule in the phagocytotic uptake and phagosomal degradation processes of antigens or in antigen presentation. we show here that asmase -/mice fail to eliminate the noncytopathic lymphocytic choriomeningitis (lcm) virus as rapidly as littermate wildtype mice. investigation of the immune response revealed a reduced expansion of cd8 þ t cells. the secretion of ifn-g in response to contact with target cells as well as the cytolytic activity of virus-specific cd8 þ t cells was severely impaired. additionally, both phases of the lcm virus-specific dth response, mediated by cd8 þ and cd4 þ t cells consecutively, were diminished in asmase -/mice. however, the secondary memory response of virus-specific ctl was not altered, and the 614 abstracts ................................................................................................................................................................................................. virus was effectively controlled for at least 3 months by asmase -/mice. in conclusion, the results of this study suggest an involvement of the asmase in the activation, expansion or maturation of virus-specific cd8 þ t cells during the acute infection of mice with the lcm virus. novel markers for alternative activation of macrophages: macrophage galactose-type c-type lectins 1 and 2 in parallel with the th1/th2 dichotomy, macrophages are capable of developing into functionally and molecularly distinct subpopulations, due to differences in, for example cytokine environment and pathological conditions. while the best-studied, classically activated macrophage is induced by type i stimuli such as ifn-g, a type ii cytokine environment antagonizes the classical activation of macrophages and is capable of alternatively activating macrophages. however, molecular markers associated with these type ii cytokine-dependent, alternatively activated macrophages remain scarce. besides the earlier documented markers macrophage mannose receptor and arginase 1, we recently demonstrated that murine alternatively activated macrophages are characterized by increased expression of fizz1 and ym. we now report that expression of the two members of the mouse macrophage galactose-type c-type lectin gene family, termed mmgl1 and mmgl2, is induced in diverse populations of alternatively activated macrophages, including peritoneal macrophages elicited during infection with the protozoan trypanosoma brucei or the helminth taenia crassiceps, and alveolar macrophages elicited in a mouse model of allergic asthma. we also demonstrate that, in vitro, interleukin-4 and interleukin-13 upregulate mmgl1 and mmgl2 expression and that, in vivo, induction of mmgl1 and mmgl2 is dependent on interleukin-4 receptor signalling. moreover, we show that regulation of mgl expression is similar in human monocytes and monocyte-derived macrophages. hence, macrophage galactose-type c-type lectins represent novel markers for both murine and human alternatively activated macrophages; thus, paving the way for further characterization of the phenotype of macrophages occurring in th2 conditions. background: human parvovirus b19 (b19) is a ubiquitous pathogen, normally causing a mild self-limiting disease, but also capable of causing both significant pathology and long-term persistence. the small size and stability of the virus makes it suitable for mapping of the full breath and the kinetics of the cellular immune responses following acute viral infection. methods: five patients with acute primary b19 infection were included in the study and followed consecutively for up to 200 weeks. cellular immune responses were mapped by ifng enzyme-linked immunospot to overlapping peptides spanning the whole b19 genome. results: in all five acutely infected patients, we were able to monitor the kinetics of a strong specific cellular immune reaction. responses peaked at levels of 850-1850 sfc/ million pbmcs, roughly corresponding to 0.3-0.6% b19specific cd8 þ cells circulating in peripheral blood at 10-80 weeks post-infection. the responses in individual patients were directed to three or four different peptide pools, and the specificity was confined to the same cd8 epitopes present in the pools throughout the follow-up period. the majority of responses were directed to the virus nonstructural protein, only two patients showed any response to the capsid proteins, elicited by the same epitope in both cases. conclusion: the cellular immune responses to acute b19 infection are surprisingly narrow in distribution and remain at high levels for up to 80 weeks post-infection. the initial epitope specificity is maintained, and the majority of responses target the virus nonstructural protein, which is not included in vaccine preparations, evaluated against the infection. the relationship between malnutrition and malaria is controversial. on one hand, malaria may cause malnutrition, while on the other, malnutrition itself may modulate susceptibility to the disease. we investigated the association between plasmodium falciparum malaria and malnutrition in a cohort of children living on the coast of kenya. the study involved longitudinal follow-up for clinical malaria episodes and anthropometric measurements at four cross-sectional surveys. we used poisson regression analysis to investigate the association between malaria and nutritional status. compared to baseline (children with a waz or haz score of !à2), the crude incidence rate ratios (irrs) for malaria in children with low haz or waz scores (<à2) during the period prior to assessment were 1.17 (95% ci 0.91-1.50; 0 ¼ 0.21) and 0.94 (0.71-1.25; 0.67), respectively, suggesting no association between malaria and the subsequent development of pem. however, we found that age was acting as an effect modifier in the association between malaria and malnutrition. the irr for malaria in children 0-2 years old who were subsequently characterized as wasted was 1.65 (1.10-2.20; p ¼ 0.01), and a significant overall relationship between malaria and low-haz was found on regression analysis when adjusting for the interaction with age (irr 1.89; 1.01-3.53; p < 0.05). although children living on the coast of kenya continue to suffer clinical episodes of uncomplicated malaria throughout their first decade, the association between malaria and malnutrition appears to be limited to the first 2 years of life. a. astrinidou-vakaloudi, 1 s. xytsas, 1 i. diamanti, 1 h. ioannidis 2 & p. pangidis 2 1 microbiology department of general hospital of thessaloniki 'agios pavlos', thessaloniki, greece, and 2 nefrology, 2 nd ika hospital of thessaloniki, thessaloniki, greece. e-mail: stasa@hol.gr aim: renal dysfunction may influence the colonization of gastric mucosa by urea-splitting bacteria such as helicobacter pylori, by increasing urea concentrations in the gastric juice. our aim was to investigate the prevalence of h. pylori in patients with end-stage renal disease (esrd), receiving long-term haemodialysis treatment. methods: this study included 40 sera from patients with esrd (29 male and 11 female) undergoing periodic haemodialysis; mean time of treatment was 42.6 months. using elisa technique, we investigated the presence of igg and iga antibodies against h. pylori as well as igg caga (antibodies specific for caga(þ) strains of h. pylori). sera from 40 healthy blood donors were used as a control group. results: h. pylori igg antibodies were detected in 32 out of 40 (80%) patients in the dialysis group, while 31/40 (77.5%) tested positive for iga. igg caga antibodies were present in 13 out of 40 (32.5%). prevalence of h. pylori igg, iga and caga igg antibodies in the control group was 33, 7 and 15%, respectively. conclusions: although international data suggest that prevalence of h. pylori infection is the same in esrd patients as in healthy individuals, in our study that seems not to be the case. the higher blood and gastric juice urea levels may be a risk factor (among many others), but more studies are required in order to understand the relation of h. pylori infection in this group of patients. flanders interuniversity institute for biotechnology, department of molecular and cellular interactions, free university of brussels, brussels, and 2 pasteur institute of brussels, mycobacterial immunology, brussels, belgium. e-mail: tgartner@vub.ac.be immunity against tuberculosis (tb), caused by mycobacterium tuberculosis, depends largely on activation and maintenance of strong cell-mediated immune responses involving both cd4 þ and cd8 þ t cells and the ability to respond with th1-type cytokines, particularly ifn-g. recent studies suggested that bcg, the only licensed vaccine against m. tuberculosis, may fail to induce t-cell responses in the lung mucosa and may therefore not protect against pulmonary tb. a decrease in tb mortality may be achieved by enhancing immunity in the lung. the present study evaluated the induction of antigen-specific immunity in the lung by intranasal (i.n.) delivery of the lipoprotein i (opri) from pseudomonas aeruginosa. opri has shown to be a toll-like receptor 2/4 agonist that, when given subcutaneously, induces type-1 immune responses against heterologous antigens. here, a fusion of opri to ag85a of mtb (opri-ag85a) was used as a subunit vaccine in homologous prime-boost immunizations. in addition, opri-ag85a was combined with an ag85a-encoding dna vaccine (ag85a dna) or with bcg in heterologous prime-boost vaccinations. intranasal and parenteral delivery with opri-ag85a elicited comparable t-cell responses in the spleen; in addition, i.n. delivery elicited specific t-cell responses in the lung lymph nodes (llns). intramuscular delivery of ag85a dna induced significant systemic th1 immune responses. intranasal boosting with opri-ag85a enhanced this response and in addition induced an antigen-specific ifn-g response in lln. opri may therefore be an efficient adjuvant for mucosal boosting. we continue to evaluate the protection induced by opri-based prime-boost vaccinations against pulmonary tb. results on the immunogenicity and protection against intravenous mtb h37rv infection will be presented. toll-like receptors (tlrs) are pattern recognition receptors of the innate immune system, which recognize molecular structures on pathogens or cellular stress-associated molecules. tlr-ligand interactions trigger activation of inflammatory signal transduction and expression of genes involved in host defense. in this study, we have examined the requirement for different tlr adaptor molecules in virus-induced chemokine expression and are currently trying to identify the tlr involved. we have found that both a herpesvirus [herpes simplex virus (hsv)] and a paramyxovirus (sendai virus) require a functional genome to induce expression or proinflammatory chemokines in human and murine monocytic cell lines. for both viruses, this is independent of the tlr adaptor molecules trif and mal. however, overexpression of the vaccinia virus-encoded inhibitor of tlr-signalling a52r or dominant-negative myd88 totally inhibited hsv-induced rantes expression but only partially prevented sendai virus from inducing this chemokine. this suggests that hsv-induced rantes expression occurs via a tlr pathways, whereas sendai virus utilizes both tlr-dependent and -independent pathways to stimulate expression of rantes. we are currently trying to identify the tlrs involved. data from these studies will also be presented at the meeting. 2 0 -5 0 -oligoadenylate synthetases are interferon-induced, double-stranded rna-activated antiviral enzymes which are the only proteins known to catalyse 2 0 -specific nucleotidyl transfer. this first crystal structure of a 2 0 -5 0oligoadenylate synthetase reveals a structural conservation with the 3 0 -specific poly(a) polymerase that, coupled with structure-guided mutagenesis, supports a conserved catalytic mechanism for the 2 0 -and 3 0 -specific nucleotidyl transferases. comparison with structures of other superfamily members indicates that the donor substrates are bound by conserved active site features while the acceptor substrates are oriented by nonconserved regions. the 2 0 -5 0oligoadenylate synthetases are activated by viral doublestranded rna in infected cells and initiate a cellular response by synthesizing 2 0 -5 0 -oligoadenylates, that in turn activate rnase l. this crystal structure suggests that activation involves a domain-domain shift and identifies a putative dsrna activation site that is probed by mutagenesis. we demonstrated that this site is required both for the binding of dsrna and for the subsequent activation of oas. this rna-binding site is different from known rna-binding site; rather than forming a defined three-dimensional domain, it is located at the interface of the two major domains in oas. this novel architecture ensures that the dsrna helix can make simultaneously contact with both domains of oas and ensure the subsequent structural rearrangement leading to the activation of oas. our work provides structural insight into cellular recognition of double-stranded rna of viral origin and identifies a novel rna-binding motif. bacteria-specific iga antibodies are efficient opsonins for neutrophils and mononuclear phagocytes, provided that the phagocytes express the fca receptor (cd89). expression of cd89 can be stimulated by inflammatory cytokines, activated complement factors and certain microbial components. in one study, unstimulated phagocytes were able to ingest iga antibody-treated pneumococci, but only in the presence of complement, which was found to be activated by the iga antibodies along the alternative pathway. pneumococci produce iga1 protease that cleaves human iga1, but not iga2, molecules in the hinge region. this leaves iga1 as faba (monovalent) deprived of fca which contains the docking site for cd89. iga1 is the vastly predominant subclass of iga in the upper airways and circulation of humans. aims: to examine the effects of iga1 protease activity and complement on phagocytosis of iga antibody-coated pneumococci by an unstimulated human phagocytic cell line (hl60). materials and methods: iga1 and iga2 monoclonal antibodies to serotype 4 pneumococcal capsular polysaccharide (ps) were generated by heterohybridoma technique involving b cells from human vaccinees. isogenic serotype 4 pneumococci with and without iga1 protease activity, respectively, were obtained after inactivation of the iga gene of the tigr4 strain. opsonophagocytosis was quantitated using the assay described by romero-steiner et al. based on enumeration of surviving bacteria by culture. the integrity of iga molecules was examined by western blotting. results: both iga1 and iga2 antibody to type-4 polysaccharide-induced phagocytosis of iga1 protease-deficient type-4 pneumococci equally well in the absence as in the presence of complement. iga1 antibody to type-4 polysaccharide displayed a fourfold higher opsonophagocytosis titer against iga1 protease deficient compared to homologous wildtype target bacteria. a similar effect of iga1 protease activity of the target bacteria was not observed in a parallel experiment where iga2 antibody to type-4 polysaccharide served as opsonin. iga1 antibody extracted from iga1 protease-producing target bacteria was almost exclusively in the form of faba. conversely, iga1 from protease-deficient bacteria and iga2 from both types of bacteria were intact. conclusions: these results indicate that the iga1 protease activity of s. neumoniae may help the bacteria escape iga1 antibody-mediated opsonophagocytosis. besides, in these experiments, iga-mediated opsonophagocytosis was independent of complement. vitamins e and c have been found to increase the cellular and humeral immunity of pigs. vitamin e deficiency has also been found to predispose pigs to different diseases, e. coli infection is one among them. after weaning, the vitamin e status of pigs often decreases to a critical low level. in this experiment, we studied whether vitamin c supplementation would be a possible feeding strategy to optimize the immune status of weaners. the interaction between vitamin e and c is interesting due to the reported sparing action on vitamin e or synergism between these to vitamins. piglets were weaned at day 28 of age from sows fed increasing dietary vitamin e during lactation, and piglets were during the following 3 weeks fed either a control diet or this diet supplemented with 500 mg stay-c per kg. blood sampling was obtained weekly from day 28 and until day 49 of age. on the same days, one piglet per dietary treatment was killed and alveolar macrophages (am) were harvested. vitamin c supplementation increased the concentration of igm in serum of piglets throughout the weaning period. although the vitamin e concentration in am decreased with increasing age of the piglets, the concentration was numerically higher in piglets of sows fed the high dietary level of vitamin e. however, vitamin c supplementation tended to increase the total am concentration of vitamin e after weaning and increased the proportion of the biologically most active isomer of vitamin e [rrr-(a-tocopherol)] in the am. the eicosanoid synthesis by am was not influenced by the vitamin c supplementation, but the synthesis of leukotriene b4 was decreased 2 weeks after weaning compared to other days of am harvesting. in conclusion, dietary vitamin c supplementation improved the immune responses of piglets after weaning. a whole blood stimulation assay with escherichia coli (o111:b4) endotoxin was established to measure the capacity of dairy cows to produce the proinflammatory cytokine tumour necrosis factor-a (tnf-a) ex vivo. initially, a time-and dose-dependent study was carried out to find the optimal stimulation conditions for the tnf-a response. the tnf-a response peaked between 3 and 4 h at 38.5 c. a dose in the range of 5-10 g of e. coli lipopolysaccharide (lps)/ml whole blood was found to give the maximum tnf-a response. thirty-eight danish-holstein dairy cows were investigated for their tnf-a responsiveness ex vivo in the periparturient period. heparin-stabilized blood samples were collected seven times over a period of 4 months (weeks à3, à1, 2, 3, 5, 9 and 13 around calving) and stimulated with 5 g/ml of e. coli lps. indeed, fluctuations in the tnf-a responsiveness occurred over time. moreover, the mean tnf-a responsiveness of 38 cows was found to be significantly increased (p < 0.001) in the weeks close to calving. however, in the more stabile physiological periods, some cows had a consistently low tnf-a response, whereas others had high a tnf-a response. we are currently investigating whether high and low tnf-a responders to e. coli lps also exist in dairy cows in vivo. moreover, the importance of tnf-a responsiveness ex vivo to dairy cows' susceptibility and clinical response to experimental e. coli infections in the udder is being investigated. coelomic cytolytic factor (ccf) is a 42 kda invertebrate pattern recognition molecule isolated from the coelomic fluid of the earthworm eisenia foetida (oligochaeta, annelida). ccf displays a number of similarities with the mammalian cytokine tumour necrosis factor-a (tnfa) as a result of a shared n,n 0 -diacetylchitobiose lectin-like domain. however, these similarities are solely functional and are not based on any (dna or amino acid) sequence homology, thus suggesting a form of convergent evolution. in particular, the lectin-like domain of tnf-a has been shown to induce membrane depolarization in various mammalian cell types, through interactions with endogenous amiloride-sensitive ion channels. this nonreceptor-mediated activity of tnf-a has been reported to be involved in the resorption of oedema. likewise, the lectin-like domain of ccf also induces membrane depolarization in mammalian cells. here, we show that ccf appears to be able to induce oedema resorption in an alveolar epithelial cell line through its lectin-like domain. this lectin-like domain of ccf interacts (directly or indirectly) with endogenous sodium and/or chloride channels, and not potassium channels, on mammalian cells. additionally, we suggest that the jnk/sapk and erk1/2 pathways are involved in ccf-induced macrophage activation. these results further establish the functional analogy between an invertebrate pattern recognition molecule and a mammalian cytokine and, from a more applied point of view, suggest the possibility of utilizing ccf in the treatment of oedema. release of svegf and svegfr1 from white blood cells and platelets during surgery and stimulation with bacterial antigens introduction: the influence of surgery on release of soluble vascular endothelial growth factor (svegf) and the soluble vascular endothelial growth factor inhibitory receptor 1 (svegfr1) is unknown. we studied the effect of major and minor surgery on potential variations in svegf and svegfr1 concentrations in vivo and on bacterial antigen-induced release of svegf and svegfr1 from whole blood in vitro. methods: sixty-one patients with abdominal diseases undergoing five different surgical procedures were included. blood samples were drawn from anaesthetized patients before and after the operation. white blood cells and platelets were counted, and plasma svegf and svegfr1 was determined by an elisa method. whole blood from each blood sample was stimulated in vitro with bacteria-derived antigens (lps or protein-a) and svegf and svegfr1 levels were subsequently determined in the supernatants. stimulation with isotonic saline served as control assay. neither svegf or svegfr1 in plasma changed during surgery. in vitro stimulation of blood samples with bacteria-derived antigens resulted in a significant increase in svegf (p < 0.0001) and a less pronounced but still significant increase in svegfr1. release of svegf due to stimulation was significantly higher after the operation (nonsignificant), whereas svegfr1 release remained largely unchanged after surgery. correlation between bacterial antigen-induced release of svegf and neutrophile cell count was highly significant (p < 0.0001). there was no correlation between svegf and platelet cell count, and bacterial antigen-induced svegfr1 release did not correlate with counts of neutrophils and platelets. conclusions: plasma svegf and svegfr1 concentrations did not change during surgery. in vitro bacterial stimulation led to increased release of svegf and svegfr1, which was not significantly amplified during surgery and which may be related to number of circulating neutrophils. natural killer cell functions and subsets after in vitro stimulation with il-2 and il-12, with special emphasis on intracellular ifn-g and nk-cell cytotoxicity r. nyboe, 1,2 t. rix, 1,2 j. krog, 1,2 e. tønnesen 1 & m. hokland 2 1 department of anaesthesiology and intensive care, aarhus university hospital, and 2 institute of medical microbiology, and immunology, university of aarhus, aarhus, denmark. e-mail: rnsr@studmed.au.dk materials and methods: isolated cryopreserved human peripheral blood mononuclear cells (pbmcs) were stimulated with il-2 and il-12. this stimulation has previously been shown to activate nk cells. cell cytotoxicity was measured by flow cytometry after incubation with k562 cells. this method was compared to the current standard 51cr release assay. cells were treated with bfa to accumulate ifn-g, stained for surface markers, permeabilized and stained for intracellular ifn-g. flow cytometry was then performed to measure intracellular ifn-g production in pbmc, especially in nk cells. results: we have demonstrated that stimulation with il-2 and il-12 is effective in increasing the number of ifn-gpositive cells. there is a distinct difference between the cd3-cd56dim and the cd3-cd56bright subsets, with a much greater proportion of ifn-g-positive cells in the cd3-cd56bright subset. the effects of stimulation with il-2 and il-12 on cytotoxicity will be presented, as will the relation between ifn-g production and cytotoxicity. in addition, we will present results of these assays applied to porcine cells. discussion: in combination, these tests will address nk cell function by combining cytotoxicity with ifn-g production in nk cell subsets. the results will demonstrate whether this could serve as a useful tool in describing nkcell function, which could be of value in clinical and experimental settings. culture of regulatory t-cell lines from bronchial mucosa t lymphocytes play a major role in many immune responses. in the last decade, special focus has been on the function of th1 and th2 effector cells. now the importance of regulatory cd4 þ cd25 þ t cells in maintenance of the immunological homeostasis emerges. sarcoidosis is a multisystem granulomatous disorder often affecting the lungs. the typical sarcoid granulomas consists of epitheloid cells, macrophages and lymphocytes, mainly cd4 þ t cells of th1 phenotype. we have cultured t cells from bronchial biopsies of patients with sarcoidosis as well as from controls in high levels of interleukin 2 (il-2) and il-4 and demonstrate spontaneously arising cd4 þ cd25 þ populations and high concentrations of il-10 in these cultures. the main difference between cultures of sarcoid origin compared to controls is a very much higher concentration of the inflammatory cytokines il-6 and tnf-a in cultures of sarcoid origin. the effects of hyperbaric exposure on human peripheral blood mononuclear cells, with special emphasis on natural killer cell cytotoxicity and subsets materials and methods: as an experimental physiological stress model, we examined the effects of hyperbaric exposure on peripheral blood mononuclear cells (pbmcs) obtained from venous blood drawn from eight divers during a simulated heliox saturation dive. eight persons working in normobar atmosphere outside the pressurized chamber served as control donors. the spontaneous cytotoxicity of the pbmcs was estimated in a 4 h 51cr-release assay using k562 as nk-sensitive target cells. the pbmcs were characterized, using 4-colour flow cytometry, with special emphasis on the nk-cell subsets. the data were statistically analysed using a multivariate regression model (stata 8.2). p values <0.05 was considered statistically significant. results: the estimated cytotoxicity increased significantly in both the group of divers and control donors during the dive (pdivers < 0.01 and pcontrols < 0.01). although the cytotoxicity increased relatively more (p < 0.01) in the group of divers compared to the group of control donors between day 1 and 2. discussion: the increased cytotoxicity of pbmc estimated in the group of divers indicate that parts of the cellular immune system are affected during the extreme physiological conditions induced during the initial phase of the presented experimental hyperbaric setup. the increase in cytotoxicity observed in the group of control donors could hypothetically reflect the stress level in persons working outside the pressurized chamber during the dive. the interaction with adhesion molecules expressed by vascular endothelium is the first step in lymphocyte infiltration into tissues. at both cutaneous and mucosal sites interleukin-10 (il-10), il-12 and transforming growth factor (tgf)-b are important regulators of chronic inflammatory disease, where cutaneous lymphocyteassociated antigen (cla) and ae integrin (cd103) may be expressed. unlike cla, cd103 is not believed to play a role in tissue-specific homing but may help to retain t cells within epithelial layers. we have previously shown that il-12 alone can together with an unknown cofactor increase the expression of cla. stimulation with streptococcal pyrogenic exotoxin c (spec) increased the expression of cd103 by cd8 þ but not cd4 þ t cells. while il-12 increased superantigen-stimulated expression of cla, this cytokine strongly inhibited the cd103 expres-sion, and a combination of il-12 and tgf-b completely abrogated the induced cd103 expression. conversely, il-10 suppressed cla but increased cd103 expression. these findings indicate that, in addition to suppressing the development of th1-mediated inflammatory responses, il-10 may also inhibit the migration of cd8 þ t cells into the skin while il-12 promotes such migration. thus, the expression of cla and cd103 may be antagonistically regulated by il-10 and il-12, and the balance between these cytokines could influence the t-cell migration of inflammatory cells into epithelial tissues. during contact sensitivity reaction, immune cells proliferate. in order to study the histological picture of these proliferation phases, we used a mouse model of contact sensitivity in the oral mucosa and on skin. we also used bromodeoxyuridin (brdu, an analogue to thymidin) that is incorporated into the nucleus during cell replication. the hapten oxazolone (oxa) was used to sensitize and elicit the oral mucosa and/or the ear skin. mice were killed at various times after elicitation, and unsensitized animals were also exposed to the hapten as controls. brdu (25 mg/ kg animal) was injected i.p. 2 h before the kill. specimens from the oral mucosa, ear skin and submandibular and auricular lymph nodes were cut and fixed in 4% paraformaldehyde. they were then treated with acid and biotinylated anti-brdu antibody and developed using abc-kit and dab. the analyses were performed using a leica light microscope and the computer program analysis. in the oral mucosa, the frequency of proliferating cells were increasing during the observation period, 4-24 h after elicitation, regardless of site of sensitization. the proliferating cells were found mainly in the basal cell layer of the epithelium. similar patterns were found in ear skin. the regional lymph nodes demonstrated a few scattered proliferating cells 4 h after elicitation. after 24 h, these cells were found frequently in the whole lymph node. control animals exhibited considerable less proliferating cells at all times. we conclude that most proliferating cells were found 24 h after elicitation locally at the hapten-exposed sites (the oral mucosa or the ear skin) as well as in the regional lymph nodes. the endogenous nucleoside adenosine is released in excess during inflammation or other metabolic stress and is generally known to deliver tissue protective anti-inflammatory effects. adenosine acts via four adenosine receptors of which the a2a receptor is the predominant form in t cells. adenosine levels are elevated in asthmatic lung, and adenosine can directly induce mast cell degranulation and bronchoconstriction in these patients. instead, the role of anti-inflammatory mechanisms of adenosine on t cells in asthma is unclear. aim: to study the a2a receptor expression in peripheral blood cd4 þ t cells in asthmatic and healthy individuals using flow cytometric and quantitative real-time pcr methods. results: unstimulated cd4 þ cells of asthmatic patients expressed significantly lower levels (p < 0.001) of a2a receptor in protein level (mean percentage of cells positive ae sem: 76.8 ae 1.2, n ¼ 6) compared to healthy individuals (90.4% ae 1.9, n ¼ 4). double staining for cd69 expression showed that stimulation of cd4 þ cells decreased a2a expression in both groups but indicated that the detected lower levels of a2a in unstimulated cells of asthmatics was not due to preactivation in these patients. surprisingly, a2a mrna expression in unstimulated cd4 þ cells was significantly higher (p < 0.05) in asthmatics (n ¼ 28) compared to healthy controls (n ¼ 7). the expression did not correlate with serum total ige levels. conclusions: asthmatic individuals express less a2a adenosine receptor on their peripheral cd4 þ t cells. the higher mrna levels instead may point to a negative feedback regulation in the receptor expression. the role of possibly decreased adenosine-mediated anti-inflammatory effects in asthma pathogenesis require further studies on this t-cell mediated disease. the chronic inflammatory skin disease atopic eczema (ae) affects almost 15% of the population in many countries today. the pathogenesis of ae is not fully understood. a combination of genetic predisposition and environmental factors like microorganisms seems to contribute to the symptoms. the yeast malassezia sympodialis is part of our normal skin micro flora but can act as an allergen and elicit specific ige and t-cell reactivity in patients with ae. recently, we identified a novel major m. sympodialis allergen, designated mala s 11 (22.4 kda), with sequence similarity to the mitochondrial enzyme manganese superoxide dismutase (mnsod). interestingly, mala s 11 has a high degree of homology to human mnsod. the aim of this study was to examine the effects of recombinant mala s 11 on antigen-presenting dendritic cells. monocytederived dendritic cells (mddcs) from healthy blood donors were cultured with or without mala s 11 for different time periods. it was found that the maturation marker cd83 and the costimulatory molecules cd80 and cd86 were upregulated on the mddcs exposed to mala s 11 for 24 h, as demonstrated by flow cytometry. furthermore, coculture of mddcs with mala s 11 for 9 h induced an increased production of the inflammatory cytokines il-6 (200-fold), tnf-a (100-fold) and il-8 (sixfold), as detected by the cytometric bead array (cba) analysis. our results suggest that mala s 11 affects the immune response through dc maturation and production of inflammatory cytokines. the potential cross-reactivity with human mnsod needs to be explored and the exact role of mala s 11 in the pathogenesis of ae assessed in clinical studies involving skin prick and atopy patch tests. allergen-specific immunotherapy (sit) is commonly conducted with allergen extracts adsorbed to aluminium hydroxide (alum). drawbacks linked to the use of alum, such as the formation of granuloma at the site of injection, have led to suggestions of novel allergen carriers. an alternative carrier is 2 mm carbohydrate-based particles (cbps). in mouse, allergen-coupled cbps have been demonstrated to skew the allergen-specific immune response towards a th1-like activity (grönlund et al. immunology, 2002) . we here coupled the recombinant major cat allergen fel d 1 to cbps (cbp-fel d 1) by cyanogen-bromide activation, resulting in covalent binding. the effect of cbp-fel d 1 on monocyte-derived dendritic cells (mddcs) from healthy human blood donors was studied. we found that the majority of the cd1a þ mddcs were capable of taking up fitc-labelled cbp-fel d 1, as demonstrated by flow cytometry and confocal laser scanning microscopy. furthermore, incubation with cbp-fel d 1 resulted in an upregulation of the costimulatory molecule cd86 on the mddcs, which was not observed with fel d 1 or cbps alone. finally, cbp-fel d 1 induced a fivefold increase in the release of the pro-inflammatory cytokine tumour necrosis factor (tnf)-a and a fourfold increase in the release of the chemokine interleukin-8 from mddcs. taken together, the effects cbps possess make them interesting as novel allergen carriers for sit. the cysteine protease der p1 from dust mite of the genus dermatophagoides pteronyssinus is a major type i allergen. about 80% of house dust mite (hdm) allergic individuals are reactive to this protease in standard assays for detection of ige. a curative treatment for atopic allergy is immunotherapy (it) with hdm extracts which are complex mixtures occasionally resulting in anaphylactic reactions. novozymes focuses on developing a recombinant variant of der p1 which exhibit lowered risk of ige-mediated allergic reactions, while maintaining its ability to trigger proper th-cell responses. this may provide a safer alternative for specific it of hdm allergy. a secreted recombinant form of pro-der p 1 expressed by saccharamyces cerevisiae was obtained by fusion of the pro-enzyme to a fungal signal peptide. the n-glycosylation site of der p1 was mutated resulting in a deglycosylated pro-enzyme with a molecular mass of 35 kda. protein purification procedure was developed to obtain nearly pure der p1 protein followed by determination of concentration by active-site-titration with the cysteine protease inhibitor e64. the deglycosylated recombinant pro-der p 1 revealed immunologic similarity to the native der p 1 molecule when compared in basophile histamine release, ige-binding assays and t-cell proliferation assays. by in silico epitope mapping of a modelled 3-dimensional structure of der p1, five putative igg and ige epitopes were predicted. by protein engineering, the predicted epitopes were removed one by one in der p1 and screening for hypoallergenic variants was performed. combining inhaled long-acting b-2 agonist (laba) and inhaled corticosteroid (ics) seems to offer asthma control at a lower dose of ics than achieved by ics alone. fine mapping of t-cell surface markers by flow cytometry offers a detailed status of the individual's inflammatory response. the frequency of mt2 (cd4 þ cd45ra -cd62l þ cd11adim) and mt1 (cd4 þ cd45ra -cd62l -cd11abright) cells in peripheral blood, and their ratio, has been shown to differ predictably in atopics and patients with leprosy, where mt2 correlates with a th2 phenotype and mt1 with a th1 phenotype. stable asthmatics, requiring fluticasone propionate (fp) 750-1000 mg daily or equivalent, were randomized to receive, double-blinded, either seretide 1 [salmeterol and fluticasone propionate (sfc, n ¼ 16)] 50 mg/500 mg bd or fp 500 mg bd (n ¼ 17). if asthma was controlled based on lung function and symptoms at clinic visits every 6 weeks, ics dose was tapered until asthma exacerbated or 0 mg was reached. the frequency and ratio of mt2 and mt1 t cells of the patients was monitored at 6 week intervals. as treatment tapered, the frequency of mt2 cells decreased (p ¼ 0038 from first to final visit), whereas that of mt1 cells increased. the ratio of mt2/mt1 decreased (p ¼ 0049 from first to final visit). in patients receiving laba þ ics, the fall in mt2/mt1 ratio appeared to be more pronounced than in patients receiving ics alone. thus, the mt2 phenotype may be associated with stable asthma, whereas an imminent exacerbation may associate with an increase in the mt1 phenotype. laba may allow for a greater effect of fp on the mt ratio. activation of complement pathways, leading to production of c3a and c5a anaphylatoxins, has been postulated in the pathogenesis of asthma and allergic airway inflammation. the present study was undertaken to investigate the role of mannan-binding lectin (mbl), an initiator of the lectin pathway of complement, in asthma and allergic rhinitis. mbl levels and mbl-induced complement activity were determined in 19 patients of bronchial asthma with allergic rhinitis and 20 unrelated, age-matched controls of indian origin. mbl levels and activity were correlated with percent eosinophilia and percent predicted fev1 values of the patients. association of single nucleotide polymorphisms (snps) in exon 1 and intron 1 of the mbl with the disease, clinical markers, mbl levels and mbl-induced complement activity was analysed using standard statistical tools. significantly higher mbl levels and activity were observed in patients of bronchial asthma with allergic rhinitis as compared to the controls. we identified five snps, of which two, a816g in exon 1 and g1011a in intron 1 of the mbl, were novel. snp g1011a was significantly associated with the disease (p ¼ 0.0024, or ¼ 5.8696, 95% ci: 1.7316 < or < 19.8963). individuals with 'a' allele at position 1011 showed increased mbl levels, activity and disease severity. our results suggest that 'a' allele at position 1011 leading to high mbl levels and complement activity may be contributing to the severity of bronchial asthma and allergic airway inflammation. serum resistance of borrelia burgdorferi strains belonging to the b. afzelii and b. burgdorferi sensu stricto genospecies is dependent on binding of complement inhibitor factor h. we recently reported that factor h binding by b. burgdorferi is due to inducible expression of several approximately 20 kda plasmid-encoded, surface-exposed lipoproteins related to ospe (e.g. erpa, erpp and p21). in addition, a second class of factor h-binding proteins of approximately 27-35 kda has been described. the ospe-related lipoproteins are dramatically induced by b. burgdorferi during transmission from its tick vector into the mammalian host. the induction of ospe-related lipoproteins during mammalian infection may play a key a role in the borrelial evasion of the host's immune system. the goal of the present study was to define the factor h-binding regions of ospe-related proteins using mutagenesis, peptide mapping and surface plasmon resonance analysis (biacore). the combined studies revealed that the c-terminal regions of both human and mouse factor h (scrs 18-20) specifically bind to ospe-related lipoproteins. we also found fhr-1, whose c-terminal scrs 3-5 are homologous to scrs 18-20 of factor h, to bind to ospe. peptide mapping revealed five putative regions (designated i-v) in ospe that could directly interact with factor h. deleting the c-terminal 15 amino acid residues from region v of p21 abolished its ability to bind factor h. at the same time, however, synthetic peptides corresponding to the c-termini of ospe, p21 and erpp did not inhibit factor h binding to ospe. thus, the c-terminal-binding region v appears to be necessary but not sufficient for factor h binding. when a more specific mutation strategy was employed, where single amino acid residues in peptides spanning over the factor h-binding regions were mutated to alanines, we observed that lysines in the factor h-binding regions of ospe were required for factor h binding. the combined data have revealed that key lysine residues in ospe-related lipoproteins and ionic interactions are crucial for factor h interactions. furthermore, binding of ospe to the c-termini of both mouse and human factor h suggests that borrelia spirochetes utilize analogous complement resistance mechanisms in both rodents and man. in borrelia garinii strains, which in in vitro analyses have been found to be sensitive to complement killing, differences in the ospe sequences as well as in the expression of factor h-binding proteins may account for their susceptibility to serum lysis. role of yada, ail and lipopolysaccharide in serum resistance of yersinia enterocolitica serotype o:3 mannan-binding lectin (mbl), l-ficolin and h-ficolin are pattern recognition molecules of the innate immune system. we investigated the ability of these molecules to bind to different serotypes and noncapsulated variants of streptococcus pneumonia and staphylococcus aureus. we found that mbl binds to noncapsulated s. aureus strain (wood) but not any of the examined s. pneumoniae serotypes. l-ficolin binds to some capsulated s. pneumoniae serotypes (11a, 11d and 11f) as well as some capsulated s. aureus serotypes (type-1, -8, -9, -11 and -12). h-ficolin does not bind to any of the examined s. pneumoniae and s. aureus serotypes included in this study but did bind to a strain of aerococcus viridans. when bound to bacteria, mbl and h-ficolin initiated activation of complement factor c4, whereas l-ficolin did not. during this study, quantitative assays for the three proteins were developed and the concentration in 97 plasma samples were determined and the median values were estimated at 0.8 mg of mbl/ml, 3.3 mg of l-ficolin/ml and 18.4 mg of h-ficolin/ ml, respectively. the absence of early complement components (c1, c4 and c2 but not c3) is a predisposing factor for systemic lupus erythematosus (sle). recently, we demonstrated that, in c4-deficient (c4 def.) mice, igm-containing immune complexes (igm-ic) are filtered by the splenic barrier of marginal zone macrophages (mzm), resulting in an increased immune response against antigens within these igm-ic, but this could not be observed in wildtype or c3 def. mice. we hypothesized that splenic cd11b þ mzm play an important role in the induction of autoimmunity, and we therefore analysed their cytokine profile after isolation with the help of magnetic antibody cell sorting. mrna was isolated, and real-time pcr was performed with specific primers for murine ifn-g (ifn-g), interleukin-12 (il-12) and ifn-a (ifn-a). we observe a moderate increase of il-12 and ifn-g mrna in cd11b þ cells of c4 def. mice compared to wildtype cells. surprisingly, the concentration of ifn-a mrna is six times higher in c4 def. mice. preliminary results suggest that mrna in cd11b þ cells of c3 def. mice is even lower than that in wt. six hours following i.v. application of 20 mg of a abstracts 625 .................................................................................................................................................................................................. murine monoclonal igm anti-dsdna antibody, production of il-12, ifn-g and ifn-a mrna is increased in cd11b þ cells of both c4 def. and wt mice. several references described increased levels of inf-a in patients with sle. dendritic cells are discussed as a major source of ifn-a. our observation that c4-deficient, sle-susceptible mice demonstrate an increased spontaneous ifn-a production by splenic cd11b þ marginal zone macrophages could be an early sign and a trigger for the development of sle. this is supported by the fact that the absence of c3 is not a predisposing factor for sle and our observation that c3 def. animals display low levels of ifn-a mrna. 200-400 million people worldwide and represents one of the leading causes for liver cirrhosis and hepatocellular carcinoma. control over the hbv infection is achieved mainly by vaccination with hepatitis b surface antigen (hbsag). hbsag contains n-linked glycosylation side and is recognized by both mbl-a and mbl-c in a cadependent manner. hbsag-mbl complexes activate complement and may thus affect humoural immunity. to investigate the role of mbl in humoural responses to hbsag, we immununized mice that lack both mbl-a and mbl-c proteins with soluble hbsag. it has been shown that deficiencies in other complement components like c1q, c4 and c3 result in decreased antibody responses. however, mbl double ko animals mounted dramatically increased humoural responses. after priming, mbl double kos mounted hbsag-specific igm responses, which were threefold higher than wt controls. after boosting the hbsag, total igg was 10-fold higher in mbl ko than in wt control animals. similar to the response to hbsag, other glycosylated soluble antigens (e.g. invertase) induced better humoural responses in mbl double ko animals, suggesting that mbl plays an important role in a negative feedback regulation of adaptive immunity. reconstitution experiments with rmbl partially rescued the ko phenotype. we propose that the clearance of glycoprotein antigens in mbl ko is handled differently from the wt, resulting in better stimulation of humoural responses. alternatively, glycoprotein-ag-mbl-rich complexes inhibit b-cell responsiveness via putative mbl receptors. the complement system is an important part of the innate immune system. the activation of complement proceeds through three different pathways that converge in the generation of c3-activating enzyme complexes. complement activation via the lectin pathway is initiated when recognition molecules, mannan-binding lectin (mbl) or ficolin, bind to carbohydrate structures characteristic for microbial surfaces. in the circulation, mbl and ficolins are found in association with three structurally related mblassociated serine proteases (masp)-1, -2 and -3 and a small, nonenzymatic component, map19. masp-2 has been shown to elicit complement activation through the sequential proteolytic cleavage of c4 and c2 upon binding of mbl/masp-2 complexes to microbial surfaces. we have recently uncovered a polymorphism in the masp-2/map19 gene in a patient shown to be deficient in the lectin pathway of complement activation. the polymorphism results in a single amino acid substitution in the n-terminal part of the masp-2 protein. recombinant wildtype masp-2 and masp-2 containing the amino acid substitution in question was produced, and the ability to activate complement was studied. the mutation had a profound impact on masp-2 function, resulting in the lack of complement activation through the lectin pathway. elisa-based experiments showed that the mutation leads to the impairment of complement activation through influencing the binding of masp-2 to mbl or ficolins. deficiencies in the lectin pathway of complement activation have so far been accounted for only by lack of functional mbl. the mutation described above is the first defect described affecting both activation through mbl and the ficolins. .................................................................................................................................................................................................. th1, th2 and treg cell balance. dcs are present in the gut mucosa and may thus be target for modulation by gut microbes, including ingested probiotics. here, we tested the hypothesis that species of lactic acid bacteria, important members of the gut flora, differentially activate dc. a large panel of human gut-derived lactobacillus and bifidobacterium spp. was screened for dc-polarizing capacity by exposing bone marrow-derived murine dc to lethally irradiated bacteria. cytokines in culture supernatants and dc-surface maturation markers were analysed. substantial differences were found among strains in the capacity to induce interleukin-12 (il-12) and tumour necrosis factor (tnf)-a, while the differences for il-10 and il-6 were less pronounced. bifidobacteria tended to be weak il-12 and tnf-a inducers, while both strong and weak il-12 inducers were found among the strains of lactobacillus. remarkably, strains weak in il-12 induction inhibited il-12 and tnf-a production induced by an otherwise strong cytokine-inducing strain of lactobacillus casei, while il-10 production remained unaltered. selected strains were tested for induction of dc maturation markers. those lactobacilli with greatest capacity to induce il-12 were most effective in upregulating surface mhc class ii and cd86. moreover, l. casei-induced upregulation of cd86 was reduced in the presence of a weak il-12inducing l. reuteri. in conclusion, human lactobacillus and bifidobacterium spp. polarize differentially dc maturation. thus, the potential exists for th1/th2/treg-driving capacities of the gut dc to be modulated according to composition of gut flora including ingested probiotics. the intestinal micro flora is indispensable in developing and maintaining homeostasis of the gut-associated immune system. evidence indicates that lactic acid bacteria (lab), e.g. lactobacilli and bifidobacteria, have beneficial effects on the host. established health effects include increased gut maturation, antagonisms towards pathogens and immune modulation. the objective of this study is to evaluate the immunomodulating properties of a range of lab of human origin. as dendritic cells (dcs) play a pivotal role in the balance between tolerance and immunity to commensal microorganisms, in vitro-generated immature dcs serve as a suitable model for studying the immunomodulating effects of lab. human immature dcs were generated in vitro from monocytes and exposed to lethally uv-irradiated lab. the effect of various species of lab on dcs in direct contact was evaluated. furthermore, the maturation pattern of dcs separated from the bacteria by an epithelial cell layer (caco-2 cells), which should mimic the intestinal environment, was studied. cytokine secretion (il-12, il-10 and tnf-a) and upregulation of maturation surface markers on dcs (cd83 and cd86) was measured. different lab induced diverse cytokine responses. some strains were strong il-12 and tnf-a inducers and others weak. all strains induced il-10. different lab also differentially modulated expression of cd83 and cd86 on dcs. although some variation in the response to lab of dcs generated from different blood donors was observed, general differences in the effect of the various lab was revealed. experiments with the dc caco-2 coculture system are ongoing. different species of lab differentially affect dc maturation; this suggets that the gut flora plays a pivotal role in polarization of the immune response. natural killer (nk) cells are cells of the nonspecific immune system lysing altered self-cells. a noncytolytic subset of nk cells may serve a regulatory role by secreting cytokines. bacteria translocating across the gastrointestinal mucosa are presumed to gain access to nk cells, as consumption of certain lactic acid bacteria has been shown to increase in vivo nk cytotoxicity. here, we investigated how human gut flora-derived lactobacilli affect nk cells in vitro, by measuring proliferation and ifn-g production of human nk cells upon bacterial stimulation. cd3 -cd56 þ nk cells were isolated from buffy coats by negative isolation using non-nk lineage-specific antibodies and magnetic beads. nk cells were incubated with 10mg/ml uv-inactivated bacteria or 10mg/ml phytohemagglutinin (pha) for 4 days. proliferation was assessed by incorporation of radioactive thymidine into nk-cell dna. the ifn-g concentration was measured by elisa. incubation of nk cells with a lactobacillus acidophilus strain increased the proliferation of the nk cells and induced ifn-g production, both to levels comparable to pha stimulation. the proliferative response was further enhanced with autologous monocytes present, probably because cytokines, secreted by monocytes having engulfed bacteria, stimulated the nk cells. in contrast, a lactobacillus paracasei strain caused the nk cells to proliferate only in the presence of monocytes. these results demonstrate that various strains of lactobacilli have the capacity to activate nk cells in vitro, in a monocyte-dependent or -independent way. hence, the encounter of nk cells with lactic acid bacteria will affect nk-cell activation. such activation of nk cells may potentially skew an on-going or subsequent immune response towards a th1 response. lactobacilli are nonpathogenic gram-positive inhabitants of the normal human intestine known for their healthpromoting effects. in our earlier work, it is shown that human monoclonal antibody isolated from sera of a patient with waldenstrom macroglobulinaemia possess innate antibody characteristics and binds to lactic acid bacteria. according to the immune network model, immunization with this bacteria could induce the perturbations in immune system that might result in production of anti-lactobacillus antibodies, human monoclonal antibody like (ab1) and anti-idiotypic antibody (ab2). in this study, balb/c mice were immunized with two doses of bacteria lactobacillus acidophilus in complete and incomplete freund's adjuvant and phosphate-buffered saline (pbs), respectively. seven days after the last immunization, sera from immunized mice were collected and the presence of lactobacillus-specific ab1 and ab2 were determined by elisas. in the sera of immunized mice, antibodies specific to bacteria lactobacillus acidophilus were shown. the concentration of lactobacillus-specific antibodies was higher in the sera of hyperimmunized mice (mice immunized with 1 mg of igm dj) than in sera of mice immunized with 100 times lower doses of immunogen (0.01 mg per doses). moreover, ab1 and ab2 antibodies were detected in the sera of lactobacillus-hyperimmunized mice. in this study, we have shown the idiotypic network interactions in mice immunized with bacteria lactobacillus acidophilus. the normal gastrointestinal flora is crucial for the maturation of the acquired immunity via effects on antigenpresenting cells (apcs). here, we have investigated how two types of apcs, monocytes and dendritic cells (dcs), react to different bacterial strains typical of the commensal intestinal flora. purified monocytes and monocyte-derived dcs were stimulated with uv-inactivated gram-positive (lactobacillus plantarum and bifidobacterium adolescentis) and gram-negative (escherichia coli and veillonella parvula) bacterial strains. monocytes produced higher levels of il-12p70 and tnf, as detected by elisa, in response to l. plantarum than to e. coli and v. parvula. in contrast, dcs secreted high amounts of il-12p70, tnf, il-6 and il-10 in response to e. coli and v. parvula but were practically unresponsive to l. plantarum and b. adolescentis. the lack of response to the gram-positive strains correlated with a lower surface expression of toll-like reseptor 2 (tlr2) on dcs compared to monocytes. the surface expression of tlr4 on dcs was undetectable when analysed by flow cytometry, but blocking this receptor decreased the tnf production in response to v. parvula, indicating that low tlr4 expression on dcs is sufficient to mount an inflammatory response to gram-negative bacteria. ifn-g increased the expression of tlr4 on dcs and also potentiated the cytokine response to gram-negative bacteria. our results indicate that, when monocytes differentiate into dcs, their ability to respond to different commensal bacteria dramatically changes, thereby becoming unresponsive to probiotic gram-positive bacteria. these results may have important implications for the capacity of different groups of commensal bacteria to regulate mucosal and systemic immunity. probiotic bacteria, e.g. lactobacillus spp., may improve diseases such as chronic inflammatory bowel disease. we examined cytokine production and phenotypic change after in vitro stimulation of t cells from healthy volunteers using different probiotic strains. methods: t cells were cultured from colonic biopsies from eight healthy volunteers (agnholt and kaltoft, exp clin immunogenet 2001; 18:213-25) , and dendritic cells were matured from their peripheral blood mononuclear cells. t-cell cultures were stimulated with autologous bacterial sonicate or strains of lactobacillus spp., with and without the addition of dendritic cells. cytokine levels (tnf-a, ifn-g, il-10 and gm-csf) and phenotype (cd3, cd4, cd25 and cd69) were measured on day 4. results: lactobacillus spp. induced higher productions of tnf-a and il-10 than did autologous bacteria. in presence of dendritic cells, the production of all cytokines increased. however, the increases of ifn-g and tnf-a were more pronounced in wells with autologous bacteria than in wells with lactobacillus spp. the addition of dendritic cells upregulated cd25 expression without simultaneous upregulation of cd69. the upregulation was pronounced after stimulation with lactobacillus rhamnosus gg compared with autologous bacteria and other lactobacilli. discussion: in presence of dendritic cells, autologous bacteria induced inflammatory cytokines, while probiotics mainly induced regulatory cytokines. lactobacillus rhamnosus gg induced a regulatory phenotype (cd25 þ ), in part mediated by dendritic cells. future studies will address whether this shift to a cd25 þ phenotype represents a differentiation into competent regulatory t cells. in a clinical context, such cells might be used for treatment of inflammatory diseases. protein microarrays will play a key role in the postgenomic era and offer a unique possibility to perform highthroughput global proteome analysis. a chip can be printed with thousands of protein probes (e.g. antibodies), the biological sample added (e.g. a proteome) and any binding detected. we aim to develop protein microarrays based on human recombinant scfv antibody fragments for global proteome analysis. the concept of comparing proteomic maps of healthy versus diseased samples will allow diseasespecific proteins to be detected. in fact, antibody microarrays will allow us to perform comparative proteome analysis on any sample format in a species-independent manner, as long as a proteome can be isolated. however, the complexity of proteomes, containing several thousands of different proteins, is a problem. here, we have designed antibody microarrays targeting the water-soluble fraction of a proteome. to this end, an anticytokine antibody array was developed and human dendritic cells (aeactivation) was used as model system. the results showed that our antibody microarrays could be used to examine the cytokine profile in complex samples. furthermore, we have taken the first steps towards comparing our results with those of other technologies on both the protein and gene level. due to the complexity of the model proteome, we also examined the possibility to prefractionate the proteome in a simple one-step procedure (based on size) prior to the labelling step. in more detail, the sample proteome was fractionated into two fractions using membrane devices with different molecular weight cut-offs. the results showed that the fractionation considerably enhanced the assay sensitivity allowing cytokines in the pg/ml range to be readily detectable. the immunomodulatory effect of heat shock protein 70: immunization with a dna construct based on the malarial antigen fused with a fragment of hsp 70 primes for a th-1 type of response finding an appropriate adjuvant for human vaccination is crucial. heat shock proteins (hsps) act as adjuvants when coadministered with peptide antigens or given as fusion proteins. however, there is a potential risk of autoimmunity when using the complete molecules, because hsps are evolutionary conserved. to overcome this, we first evaluated the adjuvant effect against two different antigens of a less-conserved fraction of plasmodium falciparum hsp70 (pf70c) and compared it to the whole hsp70 molecule from trypanosoma cruzi (tchsp70). we found that pf70c exhibited similar adjuvant properties as the whole molecule. we later evaluated the adjuvant potential of pf70c against the malarial antigen eb200 in a chimeric dna construct. no appreciable levels of eb200-specific abstracts 629 .................................................................................................................................................................................................. antibodies were detected in mice immunized only with the dna constructs. however, dna primed the immune system, because subsequent challenge with the corresponding recombinant fusion proteins elicited a strong th-1 antibody response. in contrast, no priming effect was observed for ex vivo ifn-g production but stimulation with the hsp-chimeric fusion protein induced a stronger secretion of ifn-g in vitro than other proteins used. these results indicate that the use of hsps is promising in the design of new vaccines. high-throughput proteomics on antibody-based microarrays: the importance of probe and surface design in analogy to dna microarrays, protein microarrays offer a new distinct possibility to perform sensitive highthroughput global proteome analysis. however, the development of the protein microarray technology will place high demands upon the design of both probes and solid supports. the analysis of thousands of heterogeneous proteins on a single microarray requires the use of uniform probes, such as antibodies, directly designed for protein microarray applications. we have recently generated a human recombinant single-chain fv antibody library, genetically constructed around one framework, the ncoder-library, containing 2 â 1010 clones. single framework antibody fragments (sinfabs) selected from this library were successfully applied as probes for microarrays providing sensitive detection in the 600 attomol (mass spectrometry) and the 300 zeptomole range (fluorescence). however, the choice of framework is critical. we have shown that the selected ncoder framework displayed excellent functional on-chip stability and arrayed dehydrated probes retained their activity for several months. furthermore, we have addressed the issues of biocompatibility of the solid support and immobilization strategies for our microarray setup. an in-house-designed substrate, macroporous silicon coated with nitrocellulose (map3-nc7), displayed properties equal to, or better than, those of five commercially available supports used as reference surfaces. we have also evaluated different coupling strategies, such as adsorption, covalent coupling, diffusion and affinity coupling. using a novel affinity tag, the double-(his)6-tag, we increased the binding efficiency of sinfab-molecules to ni2 þ -coated solid supports, thereby allowing nonpurified probes to be directly applied. the mannan-binding lectin (mbl) pathway is part of the innate immune system providing a first line of defence against infections. mbl and ficolins circulate in complexes with mbl-associated serine proteases (masp-1, -2 and -3). after recognition of a microorganism by mbl, activation of the complement system occurs. masp-1 and masp-3 share five domains (making up the so-called a-chain), whereas they have unique protease domains (b-chains). before the identification of masp-3, an assay for masp was presented, based on antibodies against the a-chain of masp-1. with the new knowledge of the three masps, and the sharing of domains by masp-1 and masp-3, assays specific for the protease domains have to be constructed, if one wishes to measure the proteins individually. we present an assay for quantifying total masp-3 in plasma and serum samples. the assay is a sandwich-type assay using as catching antibody a monoclonal antibody against the common a-chain of masp-1/3 and a developing secondary antibody against the c-terminal part of the protease domain of masp-3. we have used this assay for estimating the normal concentration of the protein as well as the concentration in patients and also for characterizing by gel permeation chromatography the masp-3 protein in serum. inducible costimulator ligand (icosl) is a costimulatory molecule related to b7.1 (cd80) and b7.2 (cd86). b cells, monocytes, dendritic cells and endothelial cells express icosl. inducible costimulator (icos) interacts with icosl, and this interaction leads to signals involved in isotype switching and the development of immunological memory. hitherto, no polymorphisms of this gene have been described. the aim of this study was to reveal variation of the icosl gene in normal individuals. all eight exons, except exon 1, were sequenced with flanking introns in 10 healthy blood donors. eight single nucleotide polymorphisms (snps) and two length polymorphisms were found. one of the snps was found in the coding regions of the gene. the base involved was located in exon 3 and caused a conservative amino acid change from valine (gtt) to isoleucine (att). three individuals were heterozygous g/a for the exon polymorphism, while the remaining seven individuals were homozygous for the wildtype g/g. exon 3 encodes the immunoglobulin variable (igv)-like domain of the molecule which is situated outside the cell. this means that the amino acid could be critical for the stability of the molecule or could constitute part of the binding site for icos. the results form the basis for further experiments to find possible associations of the alleles to diseases caused by immune dysregulation. especially, the exon 3 variant is interesting and could play a role for the development of immunological diseases. besides, it would be interesting to see whether both exon 3 alleles are expressed or only the wildtype allele is functional. interferon-a/b (ifn-a/b) is increasingly used as antiviral and immunomodulatory therapies. unfortunately, bioavailability varies with ifn species and mode of administration, and all ifn species are potentially immunogenic. assays for antiviral activity (ifn) and antiviral neutralization (antibodies, nab) have been used for some time to monitor patients on ifn biologicals. these assays require laborious titrations making them unsuitable for large-scale clinical use. myxovirus a (mxa) is a resistance gtpbinding protein that is specifically induced by treatment with type 1 ifns. for example, ifn-b-induced mxa in blood leucocytes has been used as a biomarker in ifn-btreated patients with multiple sclerosis. however, the degree of specificity of mxa in this regard is unclear, and measurements of mxa protein and/or mrna are not yet suitable for routine clinical use. in an attempt to find new and better reporter genes (and, hopefully, genes and gene products with proven specificity for ifn-a and -b), microarray screenings with u133a genechips (affymetrix) were carried using human blood leucocytes and the human lung carcinoma cell line a549. we studied the simultaneous expression of 22,000 transcripts before and after exposure to human recombinant ifn-a and ifn-b and other antiviral and immunomodulatory cytokines. the results will be presented at the conference. interferon-a/b (ifn-a/b) is increasingly used as antiviral and immunomodulatory therapies. unfortunately, bioavailability varies with ifn species and mode of administration, and all ifn species are potentially immunogenic. assays for antiviral activity (ifn) and antiviral neutralization (antibodies, nab) have been used for some time to monitor patients on ifn biologicals. these assays require laborious titrations making them unsuitable for large-scale clinical use. our laboratories have therefore modified the antiviral assays for ifn bioactivity and nab, so that they are suitable for large-scale screening in specialized laboratories. the read-out is survival of a subcloned a549 cell line in the presence of an otherwise lethal amount of virus. thus, survival increases in the presence of type 1 ifn and decreases in the presence of nab against the ifn added to the cells. mxa is induced by type 1 ifn and can be used for measuring the nab activity. in another assay, the mxa level in the a549 cell line is measured. in an attempt to find a new and better reporter gene for type 1 ifn than mxa and genes specific for either ifn-a or -b, a micro array screen was carried using the u133a chip from affymetrix. the expression of 22,000 genes can be studied simultaneous with this technology. the results will be presented at the conference. in our laboratory, we have developed a database system, which we believe is of immediate interest to the general scientific community. the database represents a computerbased replacement for the laboratory notebooks used in the majority of research laboratories worldwide. in addition, the database provides an effective tool for organizing and managing laboratory information at all levels, spanning from managing and revising standard operating procedures and producing documentation of research activities to keeping track of data and conclusions. using the commercially available database toolkit software filemaker pro, we have developed a relational database solution for management of laboratory information. the system consists of a hierarchy of five interrelated databases, each pertaining to a separate type of information, namely, overall project information, information relating to individual experiment setups, documentation of daily research activity, generated data and descriptions of standard operating procedures. like other databases, each individual database consists of a number of records, each comprised of a set of fields in which information is entered. in each record, a certain field is reserved to specify the relation of the record to a record in another database at a higher level. thus, the database is essentially five databases linked by a hierarchy of one-to-many relations, organizing information in a folder-like structure. importantly, the database system allows multiple users to access and edit records simultaneously, and the data entered in one database immediately becomes accessible through the other databases. the limitations of laboratory notebooks are apparent when looking for information, which is dispersed throughout one or more notebooks, or possibly on loose sheets of paper or printouts 'somewhere'. the often complicated process of gathering laboratory data or results when writing grant applications or research papers is made considerably easier with the database system. thus, the database solution presented should be broadly attractive to researchers, irrespective of their scientific discipline. an effective sars vaccine is likely to include components that can induce specific cytotoxic t-cell (ctl) responses. the specificities of such responses are governed by hlarestricted presentation of sars-derived peptide epitopes. exact knowledge of how the immune system handles protein antigens would allow for the identification of such linear sequences directly from genomic/proteomic sequence information. the latter was recently established when a causative coronavirus (sars cov) was isolated and full-length sequenced. here, we have combined advanced bioinformatics and high-throughput immunology to perform an hla supertype, genome-wide scan for sars-specific cytotoxic t cell epitopes. the scan includes all nine human hla supertypes in total covering >99% of all major human populations. for each hla supertype, we have selected the 15 top candidates for test in biochemical-binding assays. at this time (approximately 6 months after the genome was established), we have tested the majority of the hla supertypes and identified almost 100 potential vaccine candidates. these should be further validated in sars survivors and used for vaccine formulation. we suggest that immunobioinformatics may become a fast and valuable tool in rational vaccine design. rationale: major histocompatibility complex class i (mhc i) molecules monitor the protein content of the cell by binding small derived peptides and presenting them to cytotoxic cd8 þ t cells. the goal of the human mhc project is to predict the binding strength of any given peptide/mhc complex. this prediction allows the design of peptide-based vaccines. the prediction requires representative binding data from mhc alleles from all the nine hla supertypes. here, we describe the genetic construction, protein production and purification as well as the establishment-binding assays for two recombinant mhc supertype alleles, hla-b*1501 and hla-b*5801. methods: using the quikchange multisite directed mutagenesis kit (stratagene), codon-optimized genes encoding hla-b*1501 and hla-b*5801 are created. the two mhc i molecules are fermented and purified by ion exchange chromatography, hydrophobic interaction chromatography and size exclusion chromatography. the binding (kd) of natural t-cell epitopes, as well as predicted peptide ligands, is described by radioactive immunoassays (rias) and enzyme-linked immunosorbent assays (elisas). the mhc molecules are biotinylated during expression. results: the expression of mhc i resulted in multiple disulfide bond isomers, which are separated by hydrophobic interaction chromatography and used in subsequent binding studies resulting in the determination of kd for various peptide ligands ranging from strong binders we have previously demonstrated that bioinformatics tools such as artificial neural networks (anns) are capable of performing pathogen-, genome-and hlawide predictions of peptide-hla interactions. these tools may therefore enable a fast and rational approach to epitope identification and thereby assist in the development of vaccines and immunotherapy. a crucial step in the generation of such bioinformatics tools is the selection of data representing the event in question (in casu peptide-hla interaction). this is particularly important when it is difficult and expensive to obtain data. herein, we demonstrate the importance in selecting information-rich data and we develop a computational method, query-bycommittee, which can perform a global identification of such information-rich data in an unbiased and automated manner. furthermore, we demonstrate how this method can be applied to an efficient iterative development strategy for these bioinformatics tools. methods: a large panel of binding affinities of peptides binding to hla a*0204 was measured by a radioimmunoassay (ria). this data was used to develop multiple first generation anns, which formed a virtual committee. this committee was used to screen (or 'queried') for peptides, where the anns agreed ('low-qbc'), or disagreed ('high-qbc'), on their hla-binding potential. seventeen low-qbc peptides and 17 high-qbc peptides were synthesized and tested. the high-or low-qbc data were added to the original data, and new high-or low-qbc second generation anns were developed, respectively. this procedure was repeated 40 times. the high-qbc-enriched ann performed significantly better than the low-qbc-enriched ann in 37 of the 40 tests. conclusion: these results demonstrate that high-qbcenriched networks perform better than low-qbc-enriched networks in selecting informative data for developing peptide-mhc-binding predictors. this improvement in selecting data is not due to differences in network training performance but due to the difference in information content in the high-qbc experiment and in the low-qbc experiment. finally, it should be noted that this strategy could be used in many contexts where generation of data is difficult and costly. interleukin-18 (il-18), a pro-inflammatory cytokine that is produced by both lymphoid and nonlymphoid cells, has a critical role in modulation of innate and adaptive immunity. its primary function in stimulation of ifn-g production and stimulation of nk-cell-cytotoxic activities makes this cytokine a candidate for cancer immunotherapy. in oral cavity, this cytokine is produced by oral epithelia and carcinoma cells and is related to tumour regression in nude mice bearing salivary adenocarcinoma. however, direct effects of this cytokine on oral cancer cells have not been elucidated. in this project, we investigated il-18 effect on an oral carcinoma (kb) cell line. with rt-pcr technique, kb-cell line was found to express il-18 receptors (il-18ra and il-18rb), indicating that this oral carcinoma line is a target for il-18 study. we showed that recombinant human il-18 inhibited kb-cell proliferation by 17% at concentration of 100 ng/ml (p < 0.05), whereas ldh release by these cells in treatment group and control groups was comparable, indicating that il-18 suppression of cell proliferation was not mediated by the induction of cell death. to further address this hypothesis, we found that il-18 treatment did not induce apoptotic cell death, as studied by dna laddering and tunel assays. in addition, expression pattern of cell death-controlling genes (bcl-2 and bax) was not altered by this cytokine. findings in these studies indicated that suppression of kb-cell proliferation may be attributed to control of cell cycle, growth arrest or induction of cell differentiation. the data presented in this project could provide an insight of how cancer cell directly responds to il-18, as this cytokine is an important regulator of anticancer mechanisms. aloe emodin (ae) is a naturally occurring compound with wide spectrum of biological properties, including antimicrobial, vasorelaxant, immunosuppressive and anticancer actions. this anthraquinone induces apoptosis in several tumour cell lines with special affinity to tumours of neuroectodermal origin. high amounts of nitric oxide (no) released by activated macrophages induce tumour cell death. therefore, we explored the capacity of ae to modulate no-mediated antitumour response in vitro. interestingly, while ae markedly suppressed no release from macrophages alone, it significantly potentiated no production in cocultures of macrophages and c6 cells, after 48 h of cultivation. accordingly, the viability of c6 cells cocultivated with macrophages was reduced in the presence of ae. moreover, the observed ae-imposed potentiation of no production in macrophages was closely related to macrophage culture cell density. according to these data, we proposed that no modulator capacity of ae strongly depended on intercellular contact, indicating that macrophage antitumour response was not compromised but even potentiated by ae. immunotherapy represents an attractive fourth-modality therapeutic approach, especially in the light of the shortcomings of conventional surgery, radiation and chemotherapies in the management of metastatic cancer. to this end, a large number of peptide antigens derived from taa have been applied in immunotherapeutic trials for the treatment of various malignancies, e.g. cancers of the breast, prostate and kidney, in addition to haematological cancers. in some cases the response rates have been impressive and no adverse autoimmunity have been observed. a major strategic difficulty associated with these trials relates to the choice of best-suited peptide antigens. the vast majority of the antigens described thus far is not vital for survival and growth of the tumour cells, and immunoselection of antigen-loss variants may therefore prove to be an additional obstacle for the clinical applicability of most of the known peptide epitopes. in this respect, the development of acquired antigen loss during immunotherapy has been demonstrated in several cases. obviously, the development of loss-variant tumour cells implies that these cells acquire a pronounced growth advantage and are left unaffected by further treatment. ideally, target antigens should be derived from proteins required for survival and growth of tumour cells, as antigens with these characteristics would not be inflicted by the development of loss-variant tumour cells. in this respect, several inhibitors of apoptosis proteins (iaps) are universally expressed among tumours and play an important role in tumour cell escape from apoptosis. we have characterized spontaneous t-cell reactivity against iapderived peptides in cancer patients. from the iap survivin, we have characterized peptides restricted to the class i molecules hla-a1, a2, a3, a11, b7 and b35. furthermore, we have demonstrated that survivin-specific t cells infiltrate metastatic lesions and that isolated survivinspecific ctls are capable of killing hla-matched tumour cells. survivin-derived peptides are now in clinical trial, and continued work in our lab has demonstrated that other iaps are targets for spontaneous t-cell reactivity in cancer patients. we previously reported that in mice with large progressing t-cell lymphoma tumours, dysfunctions in the antitumour ctl activity occur, associated with an accumulation of splenic arginase-producing myeloid suppressor cells (mscs). in this study, we first demonstrate that both the presence and the activation state of these msc depends on tumour evolution. while in tumour regressors hardly any arginase-producing msc can be found, both the amount and the arginase activity of this population expands from early over late progressors. this gradual induction of mscs is paralleled by an increasing suppression of ctl activity and th1, but not th2, cytokine production. upon analysing the molecular repertoire of msc in vitro, we found, besides arginase1, a well-established marker for alternatively activated myeloid cells or m2, a strong upregulation of fizz1 and ym, two additional recently identified markers for m2. further evaluation of molecular markers by microarray analysis in msc yielded genes involved in wound healing (e.g. coagulation factor xiiia), anti-inflammation (e.g. selenoprotein p), immunomodulation (e.g. pd-l2) and fat and sugar metabolism (e.g. leptin receptor). of note, many of these genes are regulated by type 2 cytokines (il-4, il-13 and il-10) and are therefore rather m2 associated. overall, our data provide new markers for msc in cancer and further establish their m2 activation state. study. only sp-a showed a significant expression in normal mucosa which was downregulated in crc. as the absolute signal level was below the noise threshold, these results have to be interpreted with caution and require confirmation by direct measurenment of the proteins. our results suggest that there is no major role for the human collectins in colorectal cancer. tetramerization is visualized by sds-page. conclusion: an effective method for the production of highly pure mhc i molecules has been applied to hla-b*1501 and hla-b*5801, and ria and elisa binding assays for those alleles have been established background: proliferation, differentiation and apoptosis are essential processes in the normal functions of the mammary epithelium. the hypothesis examined in this study is that the transcription factor bcl-6 is critically important not only for regulating b-cell growth and development but also for mammary epithelial apoptosis. methodology: twenty breast cancer cases and 31 healthy controls were used to investigate whether bcl-6 protein in involved in breast cancer (grade iii). full length bcl-6 cdna was retrovirally transduced into eph-4 cell line. we then used flow cytometry of brdurd-stained cells to investigate the cell-cycle duration of the control and transduced cell lines. tunel was used as a marker of apoptosis to find out differences in the frequencies of apoptotic cells in the control and transduced cell lines. finally, immunohistochemistry staining was performed to detect bcl-6 in breast cancer (iii). results: restoration of bcl-6 into eph-4 cells not only inhibits apoptosis but also prolongs the cell cycle and results in increased cell size and protein content. the results also indicated that the cell-cycle time of bcl-6-transduced eph-4 cells is prolonged by about 3 h, presumably as a result of the action of bcl-6 at the bcl-6 at the g1/s transition. we found differences in the frequencies of viable and apoptotic cells in cultures of the parent eph-4 cells, control-transduced eph-4 cells and bcl-6-transduced eph-4 cells. consistently, we demonstrated that bcl-6 is expressed in 90% of high grade of breast carcinoma, which is considered as the most aggressive of tumours. conclusion: together, these results suggest that bcl-6 is likely to be involved in mammary gland development and carcinogenesis. inflammatory cytokines have a critical role in modulation of both innate and adaptive immunity in response to foreign antigen. they also play an important role in anticancer immunity. for example, they can promote cell-mediated immunity against cancer cells. with their immunostimulatory effects, these cytokines are being tested for cancer treatment in the form of dna vaccine or adjuvant or therapeutic cytokines. direct effect of these cytokines on cancer cell, however, is still unclear. in this project, we investigated whether il-1( and il-18 can modulate cancer cell proliferation. we employed a simple nonradioactive proliferation (mtt) assay and detection of lactate dehydrogenase (ldh) to test the effect of these recombinant human cytokines on various cancer cell lines, including breast cancer cell line (mcf-7), oral carcinoma cell line (kb), colon cancer cell line (caco-2) and choriocarcinoma cell line (jar). cytokines used in this study had both inhibitory and stimulatory effect on cell proliferation. findings in this project could provide an insight of cancer cell response to these cytokines and this could lead to a consideration on using cytokine as immunotherapy for cancer treatment.capacity of ae to modulate nitric oxide production depended on intercellular contact donor t cells are involved in the antitumour effects observed after bmt. thus, patients receiving t-celldepleted bmt have a higher risk of leukaemia relapse compared to patients receiving nonmanipulated bmt, and patients experiencing graft-versus-host disease (gvhd) have a lower risk of disease relapse than patients who do not experience gvhd. although the importance of donor t cells for the curative action of bmt has been established, the exact mechanisms and molecules involved in this graft-versus-tumour effect remain largely unknown. in a recently initiated project, we have conducted a longitudinal study of t-cell clonotypes in patients who received peripheral blood stem cell grafts after nonmyeloablative conditioning. peripheral blood samples were obtained sequentially after transplant, and the mononuclear cells (mncs) were isolated and cryopreserved. cd8 þ t cells were isolated from the mncs by use of immunomagnetic beads or facs and analysed for the presence of clonally expanded cells by t-cell receptor clonotype mapping based on rt-pcr and denaturing gradient gel electrophoresis (dgge). using this gel-based methodology, clonally expanded t cells were monitored after transplant and compared to the clinical data of the patients. the preliminary results demonstrates the presence of clonally expanded cd8 þ t cells at all time points analysed. furthermore, a number of clonotypes persisted for more than 6 months, and other clonotypes emerged during this period. the appearance of newly emerged clonotypes which coincided with clinical gvhd could indicate a role for these t cells in the pathogenesis of gvhd. background: deficiency of the mannan-binding lectin (mbl) pathway of innate immunity leads to increased susceptibility to infections. in patients with colorectal cancer, postoperative infection is associated with poor prognosis. the aim of the present study was to evaluate (1) the relation between the mbl pathway and postoperative infectious complications and survival of patients resected for colorectal cancer and (2) the role of mbl as acute phase reactant compared to crp. methods: preoperative mbl concentration, mbl/mblassociated serine protease (masp) activity and crp were determined in serum from 611 patients and 150 healthy controls. the patients were observed for 8 years. postoperative infections, recurrence and survival were recorded. results: the mbl pathway components were increased in the patients (p < 0.0001) compared to healthy controls. low mbl levels were predictive of pneumonia (p ¼ 0.01), and pneumonia (n ¼ 87) was associated with poor survival (p ¼ 0.003, hr ¼ 1.5, 95% ci 1.1-1.9). mbl and mbl/ masp activity could not predict postoperative overall infections. mbl showed no correlation (spearman's r ¼ 0.02, 95% ci à0.06-0.10) with crp. conclusions: low preoperative mbl levels are predictive of pneumonia, which is associated with poorer survival. mbl concentration and mbl/masp activity was not predictive of other postoperative infections or long-term prognosis. mbl apparently is not a surrogate measure of crp. department of surgery, university hospital of erlangen, erlangen, germany. e-mail: michael.siassi@rzmail.uni-erlangen.de introduction: the human collectins, mannan-binding lectin (mbl), surfactant protein-a (sp-a) and surfactantprotein-d (sp-d) play a central role in the innate immune system. immunological responses to malignant transformation of epithelial cells gained increasing interest recently. a former study could demonstrate binding of mbl to certain colorectal carcinoma (crc) cell lines in vitro. we therefore examined the expression of human collectins in normal colon mucosa and in colorectal carcinomas. materials and methods: colon samples from 20 crc patients and 10 normal mucosa samples were collected immediately after surgery. the tissue was microdissected and rna isolated (qiagen, rneasy-kit). gene expression profiles were analysed using gene-chips (affymetrix, hg-u133). we analysed the data for the expression of mbl, its associated serine proteases mannan-binding lectinassociated serine protease 1/2 (masp 1/2), sp-a and sp-d. the signal intensity of the genes of interest was compared using the mann-whitney u-test. results: the expression of human collectins in normal human colon mucosa was generally low. only the expression of sp-a and masp-2 reached the noise threshold of 250 signals. these genes were significantly downregulated in crc specimens. the expression of the other proteins showed no difference in normal mucosa and crc. conclusion: as demonstrated before, the expression of human collectins in normal colon was low in this being the first lymph node to receive drainage from the tumour area, the sentinel node offers a unique possibility to obtain tumour-reactive lymphocytes. we investigated antitumour immune responses in sentinel nodes from patients with bladder cancer, by assaying tumour-specific proliferation and tcr vb repertoires. during tumour surgery, sentinel lymph nodes were identified by peritumoural injection of blue dye. fresh specimens of tumour, sentinel and nonsentinel lymph nodes were obtained, and single-cell suspensions were prepared. cells were assayed for reactivity against autologous tumour extract in [ 3 h]-thymidine incorporation assays and characterized by flow cytometry. parallel analyses of the expression of vb gene families were performed with padlock probes, linear oligonucleotides which upon target recognition can be converted to circular molecules by a ligase. probes were reacted with cdna prepared from magnetically separated cd4 þ cells, and the tcr repertoire was determined by hybridizing the products to oligonucleotide microarrays. dose-dependent proliferation in response to tumour extract could be detected in sentinel lymph nodes. common clonal expansions were detected among tumourinfiltrating lymphocytes and in sentinel lymph nodes. nonsentinel lymph nodes displayed a divergent tcr vb repertoire. these results indicate an ongoing immune response against tumour antigens in sentinel nodes, draining urinary bladder cancer. identification of sentinel lymph nodes makes it possible to obtain tumour-reactive lymphocytes for use in adoptive immunotherapy. key: cord-001808-47496o0e authors: bayat, ahmad; burbelo, peter d.; browne, sarah k.; quinlivan, mark; martinez, bianca; holland, steven m.; buvanendran, asokumar; kroin, jeffrey s.; mannes, andrew j.; breuer, judith; cohen, jeffrey i.; iadarola, michael j. title: anti-cytokine autoantibodies in postherpetic neuralgia date: 2015-10-20 journal: j transl med doi: 10.1186/s12967-015-0695-6 sha: doc_id: 1808 cord_uid: 47496o0e background: the mechanisms by which varicella zoster virus (vzv) reactivation causes postherpetic neuralgia (phn), a debilitating chronic pain condition, have not been fully elucidated. based on previous studies identifying a causative role for anti-cytokine autoantibodies in patients with opportunistic infections, we explored this possibility in phn. methods: sera from herpes zoster (hz) patients without and with phn (n = 115 and 83, respectively) were examined for the presence of autoantibodies against multiple cytokines, and other known autoantigens. in addition, a cohort of patients with complex regional pain syndrome or neuropathic pain was tested for autoantibodies against selected cytokines. antibody levels against vzv, epstein barr virus, and herpes simplex virus-2 were also measured in the hz and phn patients. patient sera with high levels of anti-cytokine autoantibodies were functionally tested for in vitro neutralizing activity. results: six phn subjects demonstrated markedly elevated levels of single, autoantibodies against interferon-α, interferon-γ, gm-csf, or interleukin-6. in contrast, the hz and the pain control group showed low or no autoantibodies, respectively, against these four cytokines. further analysis revealed that one phn patient with high levels of anti-interleukin-6 autoantibodies had a markedly depressed antibody level to vzv, potentially reflecting poor t cell immunity against vzv. in vitro functional testing revealed that three of the five anti-cytokine autoantibody positive phn subjects had neutralizing autoantibodies against interferon-α, gm-csf or interleukin-6. in contrast, none of the hz patients without phn had neutralizing autoantibodies. conclusions: these results suggest the possibility that sporadic anti-cytokine autoantibodies in some subjects may cause an autoimmune immunodeficiency syndrome leading to uncontrolled vzv reactivation, nerve damage and subsequent phn. varicella-zoster virus (vzv) is a member of the alphaherpes virus subfamily that causes chickenpox early in life and then remains latent in neurons of sensory ganglia for the lifetime of the individual [1] . in persons with immune deficiencies and in older individuals with a waning immune system, vzv can reactivate causing herpes zoster (hz), also known as shingles [2] . hz patients have vesicular, unilateral, painful, dermatomally demarcated skin eruptions that typically resolve in less than 1 month. hz is quite frequent, and approximately one third of the population may experience zoster once in their lifetime [3] . adults over the age of 50 are particularly susceptible to hz [1] , implicating a stronger role of acquired factors compared to genetic influences [4] . one potential consequence of hz is the development of postherpetic neuralgia (phn), defined as pain that lasts for greater than 3 months and which originates in the same area as the hz rash. the frequency of developing phn has been reported to be approximately 10 % after zoster [5, 6] . immunocompromised individuals are at higher risk of hz [7, 8] , suggesting an important role of immune surveillance in controlling vzv infection. the shingles prevention study showed that vaccination of elderly persons with a live attenuated varicella-zoster virus vaccine reduced the rates of both zoster and phn [9] . in a followup study, weinberg et al. showed higher cell-mediated immunity at hz onset, which correlated with reduced hz severity and less phn; in contrast humoral immunity, as reflected by antibodies to vzv at onset of hz did not correlate with severity of disease [10] . these results indicate a critical function of t-cell-mediated immunity for protection against hz and phn. recently, we explored the role of anti-cytokine autoantibodies in several diseases by screening patient sera with a panel of recombinant renilla luciferase cytokine fusion proteins as antigenic probes using the luciferase immunoprecipitation systems (lips) technology [11] [12] [13] . using this approach, thymoma patients with opportunistic infections, including some with disseminated vzv infection, demonstrated autoantibodies against interferon-α (ifn-α), interleukin 12p35 (il-12p35), and several other cytokines [12] . high levels of neutralizing anti-ifn-γ autoantibodies were also detected in patients with disseminated nontuberculous mycobacteria and other opportunistic infections, including both with localized and disseminated vzv reactivation [14, 15] . based on the late age of onset of phn, we explored in this study whether anti-cytokine and other autoantibodies, might be associated with phn. from screening a cohort of hz patients with and without phn, high levels of autoantibodies against several different cytokines were detected in six phn patients. further analysis revealed that three of the phn patients had neutralizing anti-cytokine autoantibodies. in one phn patient with high level anti-il-6 autoantibodies, antibody responses against vzv were completely absent. the finding that several patients each harbored single, neutralizing autoantibodies against interferon-α, gm-csf or il-6 suggests that anti-cytokine immunodeficiency may contribute to development of phn. informed written consent was obtained from all subjects with vzv reactivation in accordance with the human experimentation guidelines of university college london, (east london and the city research ethics committee lrec r&wf2002/38). a total of 198 hz subjects were studied: 115 without phn (hereafter referred to as hz) and 83 with phn (hereafter referred to as phn). there were 28 subjects with hz who had a dysesthesia, but no phn; using our measured endpoints there were no differences between patients with hz and those with hz and dysesthesia and these two groups were analyzed together. two additional subject groups were studied as controls. first, a small group of healthy blood donor controls (n = 8) were used to standardize the assay. second, a phn agematched disease control group (n = 50), from patients having either complex regional pain syndrome (crps) or neuropathic pain (np) were tested for selected anticytokine autoantibodies. the crps/np subjects were collected at rush university under an irb approved protocol and with patient written consent. the clinical characteristics of these four different groups of subjects including the age, gender, diagnosis, and the presence of other associated immunodeficiencies, are described in table 1 . the antigen targets used for lips testing have been previously described [12] [13] [14] . an initial autoantibody screen of twenty-four potential autoantigens was performed with a pilot set of 33 phn patients and 8 healthy controls. these included 13 cytokines (gm-csf, il-6, ifn-α, il-12-p35, ifn-γ, ifn-ω, ifn-λ, il-12-p40, il-10, tnf-α, tnf-β, il-17 and il-1α), six known autoantigens (ro52, ro60, la, rnp-a, sm-d3 and rnp-70), four neuro-glial proteins, glutamic acid decarboxylase (gad-65), tyrosine hydroxylase, s100-β, and aquaporin-4, and trim-15. based on the results of the pilot study, 168 additional hz patients with and without phn were tested against the most informative targets (ro52, ro60, ifn-α, ifn-γ, il-6, and gm-csf). all the subjects in the cohort also were evaluated for antibodies against blrf2 of ebv (p23 capsid), the ge glycoprotein of vzv [16] and the gg-2 glycoprotein of hsv-2 [17] . for lips autoantibody testing, serum samples were diluted 1:10 in assay buffer a (20 mm tris, ph 7.5, 150 mm nacl, 5 mm mgcl 2 , 1 % triton x-100), arrayed in 96 deep well microtiter plates, and tested as described [17] . buffer blanks were used to monitor the performance and background binding activity of the assays. light units (lu) were measured using a berthold luminometer and all lu data were obtained from the average of at least two separate experiments. to evaluate the neutralizing capacity of anti-cytokine autoantibodies, control pbmcs from individual, healthy blood donors, that were distinct from the 8 controls used for antibody analysis, were incubated in the presence of 10 % healthy control or patient sera and left unstimulated or stimulated with the cytokine recognized by the autoantibodies in that particular patient sample. the pbmc were fixed, permeabilized and stained for an increase in phosphorylation of the specific downstream signal transducer and activator of transcription (pstat) molecules by flow cytometry as described previously [14] . thus, for patients with anti-ifn-α autoantibodies, cells were stimulated with ifn-α (1000 u/ml) and assessed for ifn-α-induced pstat-1 in cd14 + monocytes; those with anti-ifnγautoantibodies were stimulated with ifn-γ (1000 u/ ml) induced pstat-1 in cd14 + monocytes; those with anti-gm-csf autoantibodies for gm-csf (10 ng/ ml)-induced pstat-5 in cd14+ monocytes; those with anti-il-6 autoantibodies for il-6 (10 ng/ml) induced pstat-3 in cd3+ lymphocytes. antibodies for flow cytometry were purchased from bd biosciences. data were collected using facscanto (bd biosciences) and analyzed using flowjo version 9.1 (treestar). using this approach as described [12, 14, 18, 19] , the magnitude of pstat production due to cytokine stimulation is extremely reproducible and sensitive to incremental changes in the amount of cytokine added and sera was categorized as not neutralizing, partially neutralizing and neutralizing. graphpad prism software (san diego, ca, usa) was used for analyzing the antibody levels in this study. geometric mean antibody levels, expressed as mean log (10) lu and 95 % confidence intervals (ci), were calculated and presented as antilog values. the nonparametric mann-whitney u statistical test was used for comparison of antibody levels in the control and patient groups. the fischer exact test was used to evaluate the statistical significance of the prevalence of anti-cytokine autoantibodies in the hz and phn subjects. an initial autoantibody screen of twenty-four potential autoantigens was performed with a pilot set of 33 phn patients and 8 healthy controls. seropositivity was observed as follows: 15 % (5/33) for ro52 and la and 12 % (4/33) for anti ro60 autoantibodies. antibody levels for these three autoantigens were often 1000-fold higher than the buffer blanks or other seronegative samples. we also observed that four phn patients showed unusually high levels of autoantibodies against single cytokines including ifn-γ, gm-csf and il-6. additional weak positive autoantibodies were detected against ifn-ω and aqp-4 in several healthy controls and phn samples. autoantibodies against rnp-70, sm-d3, ifn-λ, tnf-α, tnf-β, il-17, il-10, il-12-p35, il-12-p40, s100β and gad-65 were not found in the pilot cohort. based on these findings, an additional 115 subjects with only hz and 50 phn subjects were evaluated with the most informative autoantigens (ifn-α, ifn-γ, gmcsf, il-6, ro52 and ro60) identified in the pilot cohort. in this larger group of samples, ten more of the phn patients were seropositive for anti-cytokine autoantibodies. for analysis and presentation, both the pilot and second set of samples were merged. as shown in fig. 1a , only three hz patients demonstrated single, seropositive anti-cytokine autoantibodies that were directed against ifn-α, ifn-γ, and gm-csf. in total, the phn subjects contained thirteen seropositive anti-cytokine autoantibodies, which as a group was statistically different than the hz (fischer's exact test p = 0.001). moreover, an age-matched disease control cohort of crps/np patients (n = 50) failed to harbor any statistically significant autoantibody responses against ifn-α, ifn-γ, gm-csf or il-6 ( fig. 1) . particularly relevant was the finding of six phn patients (one against anti-ifn-α, three against ifn-γ, one against gm-csf, and one against il-6) with high levels of autoantibodies greater than 100,000 lu that might have potential neutralizing activity against the target (fig. 1) . besides cytokine autoantibodies, five phn patients had robust autoantibodies against ro52 and four phn patients with ro60 autoantibodies, in which three of the phn patients were co-positive for both ro52 and ro60 (data not shown). inspection of the individual patients revealed that only one of the low il-6 autoantibody seropositive phn subjects was copositive for ro52 or ro60, suggesting that general polyreactivity did not underlie the immunoreactivity seen in anti-cytokine autoantibody patients. lastly, review of the patients' charts in subjects with anticytokine autoantibodies did not document the presence of thymoma, staphylococcus infection, or any other unusual condition to explain these autoantibodies. none of the patients had other known autoimmune diseases associated with anti-cytokine autoantibodies including autoimmune polyendocrinopathy candidiasis ectodermal dystrophy (apeced) or pulmonary alveolar proteinosis (pap). in addition to studying autoantibodies, we evaluated the possibility that the levels of vzv antibodies might be altered between hz and phn. for serologic testing, we measured antibodies against ge, a known viral glycoprotein target of natural and vaccine-mediated immunity [16] . as shown in fig. 2a , there were no statistical differences in the ge antibody levels in subjects with hz compared to phn. several hz and phn subjects had very low or no anti-vzv antibodies (fig. 2a) . however, further inspection of the individual patients with anti-cytokine autoantibodies revealed that one phn patient, #226, with high levels of anti-il-6 autoantibodies was seronegative, having the lowest level of antibodies against vzv in the phn patients (fig. 2a) . the two other subjects with neutralizing autoantibodies against ifn-α and gm-csf had vzv antibodies in the normal range (fig. 2a) . to explore whether these blunted antibody responses were unique only to vzv, antibodies against another herpes virus, ebv, using the p23 capsid protein were measured. the average antibody level to the p23 capsid of ebv was significantly higher (p = 0.01) in the phn group compared to the hz group (fig. 2b ). antibodies to a third herpes virus, hsv-2, were too sporadic to be informative (only 13 % of the entire cohort (26/198 patients) exhibited hsv-2 seropositivity (data not shown). taken together, viral antibody profiling suggests that, as a group, these patients are not globally immunocompromised yet at a more fine-grained level, patient #226, who showed severely blunted vzv antibodies, had high levels of anti-ebv antibodies. based on our previous studies [12, 14] , the eight patients in fig. 1a with high levels of anti-cytokine autoantibodies (>100,000 lu) were selected to test for their in vitro inhibitory capacities to block the activity of the cognate cytokine ( table 2) . serum from hz patient #6 partially inhibited ifnα-induced pstat-1, and serum from phn patient #56 completely prevented ifn-α-induced pstat-1 ( table 2) . for the three patients with high levels of ifn-γ autoantibodies, phn patient #123 and #322 did not block ifnγ-induced pstat-1 whereas serum from the third phn patient, #207, was partially blocking ( table 2) . phn patient #205 with anti-gm-csf autoantibodies prevented gm-csf-induced pstat-5 production, while serum from hz patient #215 with lower levels of anti-gm-csf autoantibodies did not. testing the phn patient #226 with the sole anti-il-6 autoantibodies revealed that serum from this individual prevented il-6-induced pstat-3. in total, only three phn patients had serum autoantibodies that had complete neutralizing activity against the cognate cytokine. chronic pain conditions are frequently life-long problems that are difficult to treat, in part, because the underlying factors that contribute to human pain disorders are unclear. for phn, the mechanisms that contribute to the transition from zoster to phn remain nebulous. while animal models of phn have been explored they have many limitations in reproducing the multiple factors encountered in the human disease [20] . thus, clinical research efforts based directly in human subjects are needed to obtain mechanistic insights into phn. based on our earlier work [12, 14, 18, 19] , the potential contributions of humoral immune processes to hz and phn were investigated with a [16] . the red square corresponds to patient #226 (see table 2 ) who is the il-6 seropositive phn patient who was seronegative for vzv antibodies focus on autoantibodies against cytokines and several known neural and non-neural autoantigens. we further hypothesized that damage to the central or peripheral nervous systems may expose autoantigenic epitopes thereby provoking an autoimmune response [21] . several reports have appeared examining the presence of autoantigens to peripheral nerve in complex regional pain syndrome [22, 23] and to central nervous system proteins in disorders such multiple sclerosis, although results for some antigens are controversial [24] . in phn, significant shrinkage in the dorsal horn of the spinal cord has been observed that might be attributable to injury and potential autoimmune attack [25] . using lips, no autoantibodies were detected against two neural antigens, gad-65 and tyrosine hydroxylase, or two astrocyte antigens s100-β and aquaporin-4 in any of the groups examined. while autoantibodies to four of the six sjögren's/systemic lupus erythematosus antigens tested (rnp-a, rnp-70, sm-d3, and la), were also seronegative, a few patients exhibited elevated autoantibodies against ro52 and ro60. these data suggest that phn, while a painful, stressful sequelae to an infectious disease process, does not induce an autoimmune state to the proteins tested. previously, the induction of autoantibodies in acute respiratory distress syndrome (ards) and septic shock, conditions characterized by severe inflammation and cytokine storm were observed [26] . we hypothesized that the autoantibody induction was due to the ongoing systemic inflammation and associated tissue destruction that may mediate a break in tolerance against these self-proteins. in the ards study the levels of autoantibodies were substantially lower than those seen in the anti-cytokine positive patients identified here. this quantitative difference implies that in the phn patients these anti-cytokine autoantibodies may be pathogenic rather than biomarkers for inflammation. moreover, the antiviral humoral response against vzv and other hsvs was similar between the hz and phn patients. with the lips fluidphase immunoassay used, our data indicate that the humoral response to vzv is intact in most subjects with hz and phn. the novel finding of our study was the presence of sporadic anti-cytokine autoantibodies that were associated with phn but not with hz per se. we found six patients with phn who had high levels (>100,000 lu) of anti-cytokine autoantibodies, but only two patients with hz and none with crps/np. in neutralization assays, only three subjects had serum antibodies that blocked cytokine signaling, all of whom had phn. two additional subjects, one with hz and one with phn, had partially neutralizing activities. the anti-ifn-α and anti-gm-csf autoantibodies observed in two phn patients may be associated with inflammation, as previously observed in ards and septic shock [26] . if the neutralizing levels of autoantibodies against these cytokines were present before or at the time of initial reactivation, then one might expect that they predispose to more severe vzv infection. consistent with this hypothesis, patients with thymoma have both neutralizing anti-cytokine autoantibodies against ifn-α and a predisposition to both localized and disseminated varicella reactivation, raising the possibility that anti-cytokine autoantibodies might contribute to vzv reactivation in some cases [12] . the potential role of anti-gmcsf autoantibodies in generation of phn is intriguing. up until recently, anti-gmcsf autoantibodies were only thought to cause pulmonary alveolar proteinosis [27] . however, in the last several years, anti-gmcsf autoantibodies have been found in patients with cryptococcal meningitis [18, 28] and cns infections with nocardia bacteria [19] . in light of the finding of two subjects with vzv reactivation having elevated gmcsf autoantibodies, further investigations of the relationships between gmcsf and vzv infection are warranted. the one phn patient with very high levels of anti-il-6 autoantibodies (i.e., 2,300,000 light units) that were neutralizing is potentially instructive. the presence of il-6 autoantibodies in the general population, based on sampling of over 300 samples [12, 14] is quite rare. consistent with a potential role for anti-il-6 autoantibodies in promoting vzv reactivation, this phn patient lacked antibodies against vzv, but showed normal humoral responses to ebv. it is possible that impaired il-6 signaling blunts the inflammatory t-cell reaction and impairs adequate signaling to the relevant plasma cells for anti-vzv antibody production. high levels of anti-il-6 autoantibodies were also noted in a child who developed an acute vzv infection complicated by multiple cellulitis lesions and subcutaneous staphylococcus aureus abscesses [29] . together these two patients implicate il-6 autoantibodies in the modulation of primary and reactivation vzv infection. il-6 is secreted by t cells and macrophages to stimulate the immune response and is known to be increased in the serum of hz [30] . mechanistically, vzv signals through toll-like receptor-2-dependent activation of nf-kappa b to induce an efficient inflammatory response [31] . low levels of il-6 may lead to impaired anti-vzv immune responses, causing ineffective containment of vzv, more tissue damage and a stronger "peripheral generator" that then drives stronger central sensitization, all of which can contribute to phn pain. additional studies in more subjects with phn are warranted to examine the possibility that il-6 and other cytokine autoantibodies contribute to phn. there are several clinical implications of our findings. first, in sporadic cases, anti-cytokine autoantibodies may potentially contribute to phn, highlighting the complex interactions between humoral immunity, t-cell immunity and cytokines. based on the time line for the development of phn from initial vzv reactivation, screening for anti-cytokine autoantibodies is theoretically possible, yet it is not clear how this might impact the course of treatment. the finding that the il-6 cytokine might be involved in controlling vzv reactivation and thereby might be useful for early stage treatment of immunocompromised individuals warrants further investigation. it is likely that the development of phn is a highly complex, multifactorial process driven by many subtle genetic, environmental and acquired factors. based on the present observations, one such acquired factor is the presence of anti-cytokine autoantibodies, which could act as potential drivers of phn by creating deficits in immune signaling. while the frequency of phn patients harboring anticytokine autoantibodies found in our study was small, it is important to point out that phn is quite common in adults over 60 and these findings would translate into explaining potentially many cases per year. since autoantibodies and decline in immune system function are known to occur more commonly in older subjects [32] [33] [34] , the present observations suggest the likelihood that there may be additional, unrecognized pathogenic autoantibodies against other immune components in older subjects that may increase susceptibility to phn. varicella-zoster the impact of varicella zoster virus: chronic pain herpes zoster disentangling inborn and acquired immunity in human twins diagnosing and managing postherpetic neuralgia management of herpes zoster (shingles) and postherpetic neuralgia a population-based study of the incidence and complication rates of herpes zoster before zoster vaccine introduction predictors of postherpetic neuralgia among patients with herpes zoster: a prospective study a vaccine to prevent herpes zoster and postherpetic neuralgia in older adults varicella-zoster virus-specific immune responses to herpes zoster in elderly participants in a trial of a clinically effective zoster vaccine anticytokine autoantibody-associated immunodeficiency anti-cytokine autoantibodies are associated with opportunistic infection in patients with thymic neoplasia two major autoantibody clusters in systemic lupus erythematosus adult-onset immunodeficiency in thailand and taiwan anti-ifn-gamma autoantibodies in adults with disseminated nontuberculous mycobacterial infections are associated with hla-drb1*16:02 and hla-dqb1*05:02 and the reactivation of latent varicella-zoster virus infection detection of antibodies to varicella-zoster virus in recipients of the varicella vaccine by using a luciferase immunoprecipitation system assay serological diagnosis of human herpes simplex virus type 1 and 2 infections by luciferase immunoprecipitation system assay anti-gm-csf autoantibodies in patients with cryptococcal meningitis nocardia-induced granulocyte macrophage colony-stimulating factor is neutralized by autoantibodies in disseminated/extrapulmonary nocardiosis varicella zoster virus-induced pain and postherpetic neuralgia in the human host and in rodent animal models peripheral nerve proteins as potential autoantigens in acute and chronic inflammatory demyelinating polyneuropathies longstanding complex regional pain syndrome is associated with activating autoantibodies against alpha-1a adrenoceptors autoimmunity against the beta2 adrenergic receptor and muscarinic-2 receptor in complex regional pain syndrome targets of the humoral autoimmune response in multiple sclerosis post-herpetic neuralgia: further post-mortem studies of cases with and without pain rapid induction of autoantibodies during ards and septic shock the molecular basis of pulmonary alveolar proteinosis anti-granulocytemacrophage colony-stimulating factor autoantibodies are a risk factor for central nervous system infection by cryptococcus gattii in otherwise immunocompetent patients recurrent staphylococcal cellulitis and subcutaneous abscesses in a child with autoantibodies against il-6 local immune responses and systemic cytokine responses in zoster: relationship to the development of postherpetic neuralgia varicella-zoster virus activates inflammatory cytokines in human monocytes and macrophages via toll-like receptor 2 increased production of autoantibodies and specific antibodies in response to influenza virus vaccination in physically active older individuals age-related increase in autoantibodies submit your next manuscript to biomed central and take full advantage of: • convenient online submission • thorough peer review • no space constraints or color figure charges • immediate publication on acceptance • inclusion in pubmed, cas, scopus and google scholar • research which is freely available for redistribution this work was supported by the intramural research programs of the national institutes of health clinical center, national institute of dental and craniofacial research, the national institute of allergy and infectious diseases, and the vzv research foundation. jb receives funding from the uk national institutes for health research ucl/uclh biomedical research centre. the authors declare no financial interests that may represent a competing interest. the authors declare that they have no competing interests.received: 11 august 2015 accepted: 10 october 2015 authors' contributions pdb, ajm, jic and mji were involved in study design. ab and pdb were involved in lips testing, data analysis, and manuscript drafting and editing. ab and jk collected and provided the crps/np cohort. skb, bm and smh were involved in the neutralization experiments. mq and jb provided hz and phn patient samples and clinical data. all authors read and approved final manuscript. key: cord-007646-yh8zi1ee authors: weiss, r.c.; oostrom-ram, t. title: effect of recombinant human interferon-alpha in vitro and in vivo on mitogen-induced lymphocyte blastogenesis in cats() date: 2002-11-12 journal: vet immunol immunopathol doi: 10.1016/0165-2427(90)90017-m sha: doc_id: 7646 cord_uid: yh8zi1ee the effect of recombinant human interferon-alpha (rhuifn-α) in vitro and in vivo on mitogen-induced lymphocyte blastogenesis was evaluated in specific-pathogen-free cats. pre-incubation of isolated feline peripheral blood lymphocytes (pbl) in vitro with either 10(4) or 10(3) international units (u) of rhuifn-α for 24 h significantly suppressed (p<0.001 and 0.01, respectively) blastogenic responses to the phytomitogens concanavalin a (con a) and pokeweed mitogen (pwm). lower doses of ifn (range, 10–10(−3) u/ml) neither suppressed nor enhanced mitogenesis. in the absence of phytomitogens, incubation of pbl with 10(4)–10(2) u (p<0.001) or 10 u (p<0.05) of rhuifn-α/ml resulted in a significant decrease in incorporation of [methyl-(3)h] thymidine into newly synthesized cellular dna. cultures of pbl exposed continuously for 4 days to rhuifn-α doses of 10(4) u/ml or less did not demonstrate specific reductions in cell viability, indicating that the observed antiproliferative actions of ifn apparently were independent of any direct cytotoxic effects. to investigate the dose-response effects of rhuifn-α in vivo on lymphocyte blastogenesis, individual groups of cats were evaluated on 3 consecutive days before and then 24 h after each cat was inoculated intramuscularly with either a high dose (10(6) u/kg), moderate dose (10(4) u/kg), or a relatively low dose (10(2) u/kg) of rhuifn-α. cats inoculated with 10(6) u ofrhuifn-α/kg had significantly reduced (p=0.037) blastogenic responses to con a at 24 h postinoculation compared to preinoculation values; mean pwm responses were also decreased, but this effect was not statistically significant. in contrast, inoculation of cats with either 10(4) or 10(2) u of rhuifn-α/kg significantly enhanced (p=0.05 or 0.008, respectively) con a-induced blastogenesis and had no discernible effect on pwm responses. these findings suggest that very high doses of rhuifn-α given parenterally may be associated with suppression of certain t-cell responses in cats; conversely, much lower doses may be immunoenhancing. gen-induced lymphocyte blastogenesis was evaluated in specific-pathogen-free cats. pre-incubation of isolated feline peripheral blood lymphocytes (pbl) in vitro with either 104 or 103 international units (u) of rhuifn-o~ for 24 h significantly suppressed (p < 0.001 and 0.01, respectively) blastogenic responses to the phytomitogens concanavalin a (con a) and pokeweed mitogen (pwm). lower doses of ifn (range, 10-10 -3 u/ml) neither suppressed nor enhanced mitogenesis. in the absence of phytomitogens, incubation of pbl with 104-102 u (p < 0.001 ) or 10 u (p< 0.05) of rhuifn-o~/ml resulted in a significant decrease in incorporation of [methyl-3h] thymidine into newly synthesized cellular dna. cultures of pbl exposed continuously for 4 days to rhuifn-o~ doses of 104 u/ml or less did not demonstrate specific reductions in cell viability, indicating that the observed antiproliferative actions of ifn apparently were independent of any direct cytotoxic effects. to investigate the dose-response effects of rhuifn-~ in vivo on lymphocyte blastogenesis, individual groups of cats were evaluated on 3 consecutive days before and then 24 h after each cat was inoculated intramuscularly with either a high dose ( 106 u/kg), moderate dose ( 104 u/kg), or a relatively low dose ( l0 s u/kg) of rhuifn-ol. cats inoculated with 108 u of rhuifn-~fkg had significantly reduced (p = 0.037 ) blastogenic responses to con a at 24 h postinoculation compared to preinoculation values; mean pwm responses were also decreased, but this effect was not statistically significant. in contrast, inoculation of cats with either 104 or 102 u of rhuifn-o~/kg significantly enhanced (p--0.05 or 0.008, respectively) con a-induced blastogenesis and had no discernible effect on pwm responses. these findings suggest that very high doses of rhuifn-o! given parenterally may be associated with suppression of certain t-cell responses in cats; conversely, much lower doses may be immunoenhancing. introduction interferons (ifn) are a group of cellular proteins that inhibit viral replication, modulate immune responses, inhibit normal cellular division and have antitumor activity (borden and ball, 1981 ) . there are several classes of ifn ( a, fl and y) which differ on the basis of their antigenic, biological and physic ochemical properties (mannering and deloria, 1986) . because ifn (particularly human ifn-c~ ) has antiviral and immunomodulatory activities that cross species lines (pallikoff et al., 1962; desmyter et al., 1968; gresser et al., 1974; krakowka et al., 1988) and because recombinant dna-produced human ifn is now available in relatively large quantity, the use of human ifn in veterinary medicine has increased. human ifn-c~ has been used clinically in cattle afflicted with respiratory diseases (roney et al., 1985; cummins and hutcheson, 1986) , in cats infected with either feline leukemia virus (felv) or feline infectious peritonitis virus (fipv) , and in dogs infected with canine parvovirus (dr. j. cummins, unpublished data, 1986) . dosages of ifn used clinically in domestic animals have been empirical and extrapolated largely from studies in persons or mice using interferon preparations that can differ in purity and potency. unfortunately, studies of the immunological effects of homologous or heterologous ifn in domestic animals have been lacking; the effects of human ifn-c~ in vitro on canine immune responses, however, have recently been reported (krakowka et al., 1988) . our interest in studying ifn is related to its application clinically as an antiviral and immunomodulating drug in cats with viral diseases. the effects of heterologous ifns on feline immune responses in general are not known. resistance to diseases such as fip is associated with effective cell-mediated immunity (cmi) (pedersen and floyd, 1985; ; cats infected with other viruses like felv may have severely impaired t-cell responses (rojko and olsen, 1984 ) . obviously, an understanding of the relationship between ifn dosage and modulation of cmi is necessary prior to recommending ifn as treatment for viral diseases where stimulation of cmi is required. mitogen-induced lymphocyte blastogenesis, which is a widely used in vitro assay for evaluation of cmi (oppenheim and schechter, 1976) , has been used previously in cats to assess alterations in cmi induced by immunomodulating agents such as cyclosporin (gregory et al., 1987 ) . in the studies reported here, the effects of doses of human ifn-c~ in vitro and in vivo on lymphocyte blastogenesis in normal cats was investigated. the cats used in these studies were healthy, 10-12-month-old specific-path-ogen-free (spf) males and females, weighing approximately 3.5 kg. the cats were purchased from a commercial breeder (liberty laboratories, liberty corners, nj ) and were felv test-negative (by elisa) and feline coronavirus antibody-negative prior to the studies. the cats were housed separately in cages located in the scott-ritchey animal isolation facility and were tested and cared for according to humane standards as set forth in the "guide for the care and use of laboratory animals" (publication no. 85-23, national institutes of health, bethesda, md). all experimental protocols were approved by an independent animal welfare committee prior to the studies. the recombinant dna-derived (bgl-ii restriction endonuclease-specified) human leukocyte (alpha) hybrid {subtypes a/d) ifn (rhuifn-a), lot no. ro-23-1740, was kindly supplied by dr. richard cordts, hoffman laroche, nutley, nj. the ifn was stored lyophilized (at a concentration of 50x 106 international units (u)/vial) at -80°c prior to use. the ifn was diluted before use in hank's balanced salt solution (hbss; ph 7.2) and was sterilefiltered through a 0.2 ]~m cellulose acetate membrane (nalge co, rochester, ny ). the biological activity of the rhuifn-c~ (expressed in u/ml of antiviral activity) was determined in the manufacturer's laboratory, using an international reference standard for human leukocyte ifn. cats were lightly anesthetized with an intramuscular injection of ketamine hydrochloride (vetalar; parke, davis & co, detroit, mi) and blood collected by jugular venipuncture into glass syringes containing heparin (10 units/ml of blood). peripheral blood lymphocytes (pbl) were isolated and prepared as previously described (cockerell et al., 1975; tham et al., 1982) . briefly, pbl were separated by ficoll-diatrizoate (histopaque-1077; sigma chemical co, st louis, mo) gradient separation and the interface mononuclear cells collected and washed in hbss. the washed cells were resuspended to 2 x 106 viable cells/ml in tissue culture growth medium (gm) consisting of rpmi-1640 (gibco, grand island, ny) supplemented with 10% heat-inactivated fetal bovine serum, 100 u of penicillin/ml and 100/lg of streptomycin/ml along with 1.0 mm l-glutamine and 25 mm hepes. for the in vitro ifn studies, pbl (2.0 x 105 cells, 0.1 ml) were pipetted into wells of sterile 96-well flat-bottom microtitration plates (corning glass works, corning, ny) and then incubated with gm containing varying amounts of rhuifn-a or gm only (0.1 ml/well) for 24 h at 37°c in humidified air containing 5% co2. for the in vivo studies, preincubation of pbl with ifn was omitted. the cell suspensions were then cultured 72 h with either concanavalin a (con a) (0.6 ttg/well) or pokeweed mitogen (pwm) (0.5 ttg/well), or they were not treated with mitogens (6 replicates/treatment). previous titrations in feline pbl indicated that the concentrations of mitogen used were optimal for our assay. to each well, 0.5/~ci (0.025 ml ) of [methyl-3h ]thymidine (specific activity 6.7 ci/mmol) (dupont, nen research products, boston, ma) was added for the last 20 h of incubation. the cultures were then stored at -80 ° c until harvested. cells were harvested using a semi-automatic multiple cell microharvester (bellco glass, vineland, nj) so that the cellular proteins were collected onto glass fiber filter paper strips (bellco). the paper strips were dried at 60 °c for 30 min and the filter discs transferred to scintillation vials into which 5 ml of toluene base cocktail (scinti verse ii; fischer scientific, fairlawn, nj) was added. the incorporation of [methyl-3h]thymidine into newly synthesized cellular dna was quantitated in a liquid scintillation spectrometer (lkb-wallac oy, turku, finland) using channels ratio method of quench correction. the net counts per minute (c.p.m.) of a total of 6 wells for each variable were averaged to obtain the mean c.p.m. the stimulation index (s.i.) was determined by dividing the mean c.p.m, of the mitogen-stimulated (or rhuifn-~-stimulated only) cells by the mean c.p.m, of unstimulated (media control) cells. stock solutions of ifn, thymidine and fetal calf serum were prepared from the same lots, respectively, prior to the studies and only fresh reagents were used. to determine the potential cytotoxic effect of rhuifn-c~ on feline pbl, lymphocytes were isolated from eight normal cats as described before and were exposed to varying amounts of rhuifn-c~ continuously for several days. briefly, freshly isolated pbl (2.0 × 105 cells, 0.1 ml) were cultured with gm (0.1 ml/ well) containing rhuifn-c~ (ranges, 104-1.0 u/ml) or gm only for 96 h at 37°c in 96-well microtitration plates (6 replicates/dose). the nonadherent cells were aspirated from a total of 6 wells for each ifn dose; the number of viable cells, determined by trypan blue dye exclusion, were counted in duplicate using a neubauer hemocytometer. results were expressed as mean viable cell density (number of viable cells/ml) in rhuifn-c~-treated or medium control cultures. a study was designed to evaluate the effects of giving varying amounts of rhuifn-c~ parenterally on lymphocyte blastogenesis in cats. cats were randomly assigned to one of three experimental groups and were treated as follows: a high-dose ifn group (n= 10 cats) received a single injection i.m. of rhuifn-~ at a dosage of 106 u/kg; a moderate-dose ifn group (n=4 cats) likewise received 104 u of rhuifn-c~/kg; and a low-dose ifn group (n=4 cats) similarly received 102 u of rhuifn-c~/kg. three additional cats were inoculated similarly with hbss (diluent controls). cats were evaluated by lymphocyte blastogenesis in response to phytomitogens at 24 h postinoculation. in order to minimize normal diurnal variation in test results, all cats were evaluated by lymphocyte blastogenesis at 24-h intervals on 3 successive days preceding inoculation and then 24 h postinoculation. for each group of cats, a single preinoculation mean con a or pwm response (3-day average) was determined and then compared statistically against the corresponding postinoculation group mean. each group of cats was evaluated separately on alternate weeks. the one-tailed student's t test was used to determine statistical significance between groups. all data analysis was performed using a computerized statistical analysis program (abstat; anderson-bell, canon city, co). a conservative number of degrees of freedom was used (i.e., one for each animal rather than one for each well). p values of ~< 0.05 were considered significant. incubation of normal feline pbl with doses of rhuifn-a in vitro suppressed lymphocyte proliferative responses to con a and pwm (table 1) . suppression of blastogenesis was dose-dependent; significant inhibition occurred at relatively high in vitro doses of rhuifn-c~ (104 or 103 u/ml). doses of 102 u of rhuifn-c~/ml also decreased mean con a or pwm responses, but this effect was not statistically significant. lower doses of rhuifn-a (10.0-10-3 u/ml) neither suppressed nor enhanced mitogenesis. incorporation of [methyl-3h ] thymidine into newly synthesized cellular dna in the absence of phytomitogens was significantly inhibited in feline pbl cultures exposed to rhuifn-~ doses of 104-10.0 u/ml compared to untreated pbl (table 2) . mean thymidine incorporation (c.p.m.) in cultures treated with 104-102 or with 10.0 u/ml was approximately 14% or 50%, respectively, of the thymidine incorporation measured in untreated cells. varying amounts of rhuifn-c~ (104-1.0 u/ml) were added to pbl cultures to determine whether direct cytotoxic effects from the ifn itself may have contributed to the dose-related suppression of blastogenesis observed in vitro. differences in mean viable cell density between untreated pbl cultures and those treated with ifn, however, were not observed. (fig. 1) . to determine the effects of varying amounts of rhuifn-a in vivo on mitogen-induced blastogenesis, cats in each of three groups were given a single injection of either a high (106 u/kg), moderate (104 u/kg), or relatively low dose (102 u/kg) of rhuifn-~ and then evaluated 24 h later. to minimize normal diurnal variation in responses, a 3-day average of daily mean responses was determined preinoculation for each group. day-to-day variation in blastogenic responses of individual cats evaluated on 3 consecutive days of the same week was not significant (p > 0.05, n---18; data not shown). cats inoculated with a single high dose of rhuifn-~ had a significant suppression (p= 0.037) in con a-induced lymphocyte blastogenesis 24 h postinoculation compared to preinoculation values ( fig. 2a) . mean pwm responses were also decreased, but this effect was not statistically significant. in contrast to the cats inoculated with a high dose of rhuifn-~, cats given 104 or 102 u of rhuifn-c~/kg had significantly enhanced (p=0.008 or 0.05, respectively) blastogenic responses to con a; pwm responses, however, were unaffected ( fig. 2b and c ) . inoculation of cats with hbss alone had no significant effect on mitogen-induced blastogenesis 24 h postinoculation (data not shown). the effects of ifn in vitro or in vivo on the immune system are manyfold and sometimes apparently contradictory. overall, it appears that ifn can either stimulate or suppress various arms of the immune response, depending on the timing of administration and dosage (epstein, 1977) . ifn in general seems to inhibit immunologic responses when given prior to an immunogen but enhances responses if given some days later (white and fenner, 1986) . moreover, high doses of ifn can produce opposite effects or annul the responses obtained with much lower doses. for example, very low doses of ifn-c~ in vitro or in vivo may stimulate development of antibody-forming spleen cells in mice, whereas high doses are immunosuppressive (braun and levy, 1972; epstein, 1977) . the addition of low doses of ifn (10 or 100 units/ml) in the primary mixed lymphocyte reaction increases the cytotoxic response in mice severalfold, but larger doses (104 units/ml ) depress the response (fradelizi and gresser, 1982 ) . similarly, low doses of ifn can enhance lymphocyte blastogenesis in mice or persons, whereas large doses are suppressive (miorner et al., 1978; taylor-papadimitriou, 1980) . depending on the timing of administration, extremely low doses of ifn-~, fl (2 × 10-lo u) in mice significantly increase the number of antibody-secreting cells in spleen and strongly stimulate cytotoxic activities of allospecific t-cells (daurat et al., 1988) . the results-of this study showed that very high doses of rhuifn-~ either in vitro or in vivo suppressed mitogen-induced lymphocyte proliferative responses in cats. the rhuifn-~ also directly inhibited the in vitro incorporation of [methyl-3h ]thymidine into newly synthesized cellular dna of unstimulated pbl and at much lower ifn concentrations than those required to suppress mitogen-induced blastogenesis. the inhibitory effects of various types of ifn on dna synthesis and lymphocyte blastogenesis after mitogenic stimulation have been described previously in different species, including mice, cattle, and persons (lindahl-magnusson et al., 1972; blomgren et al., 1974; bielefeldt-ohman and babiuk, 1986; kim et al., 1988; roth and frank, 1989 ). ifn has also been shown to directly inhibit thymidine incorporation into the dna of normal cells, including both lymphoid and epithelial cells, in the absence of mitogenic stimulation (brouty-boye and tovey, 1978; stadler et al., 1986; roth and frank, 1989) . although the suppressive mechanism (s) associated with rhuifn-c~ on dna synthesis and lymphocyte blastogenesis were not investigated in our study, it is possible that high doses of ifn in vitro enhanced lectin-binding on lymphocytes and stimulated suppressor cell activities. ifn can markedly enhance the binding of lectins to lymphocyte membranes (miorner et al., 1978) , and suppressor cells are activated by high doses of mitogen, particularly con a (piguet et al., 1976) . the ifn-mediated suppression of lectin-induced blastogenesis observed when feline pbl were preincubated 24 h with relatively high doses of rhuifna was also seen 24 h after cats were inoculated i.m. with very high doses (106 u/kg) of rhuifn-a. significant decreases in blastogenic responses to t-cell mitogens (con a) in particular were observed after parenteral administration of high-dose rhuifn-a. although responses to pwm (which acts both as a b-and t-cell mitogen) (heegaard and muller, 1988) were diminished, this effect was not statistically significant, suggesting that t-cell responses perhaps were somewhat more sensitive to the suppressive actions of high doses of ifn in vivo. curiously, lower parenteral doses of rhuifn-a (104-102 u/kg) significantly enhanced the blastogenic responses of pbl after stimulation with t-cell mitogens (this phenomenon, however, was not observed when low doses of ifn were incubated in vitro with pbl). an inverse relationship between ifn dose and lymphocyte proliferative responses similar to that which we observed in cats has been documented previously in vitro and in vivo in mice and persons (miorner et al., 1978; taylor-papadimitriou, 1980; kim, 1988) . seemingly, a mechanism associated with this phenomenon may have been ifninduced activation of suppressor cells. bielefeldt-ohmann and babiuk (1986) reported that in vitro treatment of bovine pbl with recombinant bovine ifngamma (rboifn-7) induced suppressor cells which may have competed with interleukin (il)-2; moreover, the suppression in lymphocyte blastogenesis after in vivo administration of high doses of rboifn-7 was found to be reversible by addition of human il-2 to the lymphocyte cultures. lower amounts of ifn, however, may actually enhance the cellular immune response by selectively blocking suppressor pathways (knop et al., 1984) . in support of this theory, daurat et al. (1988) demonstrated enhanced cytotoxic activities of allospecific t-cells and enhanced cytotoxic responses of nk cells in mice inoculated several times with ifn-a, fl at dosages as low as 2.0 or 2 × 10-lo u. the inverse dose-response effects of ifn, particularly its biologic activity at very low pharmacologic doses, has suggested a predominantly hormone-like action of ifn as a homeostatic regulator of immune functions (daurat et al., 1988) . undoubtedly, an understanding of the dose-response effects of ifn on normal feline immune responses is imperative when considering ifn therapy in cats with disease. additional in vitro and in vivo studies on the effects of ifn on t-and also b-cell responses in cats will be required so that specific recommendations concerning the use of ifn in feline viral or other diseases rationally can be established. alteration of some leukocyte functions following in vivo and in vitro exposure to recombinant bovine alpha-and gamma-interferon effect of human leukocyte interferon on the response of lymphocytes to mitogenic stimuli in vitro interferon: biochemical, cell growth inhibitory, and immunologic effects interferon preparations as modifiers of the immune response inhibition by interferon of thymidine uptake in chemostat cultures of l1210 cells phytomitogen-and antigeninduced blast transformation of feline lymphocytes low dosage of interferon to enhance vaccine efficiency in feedlot calves oral use of human alpha interferon in cats immunomodulatory activity of low doses of interferon o~, fl in mice human interferon that crosses species lines the effects of interferons on the immune response in vitro and in vivo interferon inhibits the generation of allospecific suppressor tlymphocytes response to isoantigens and mitogens in the cat: effects of cyclosporin a pronounced antiviral activity of human interferon on bovine and porcine cells lectins and the immune system interferon inhibition of il-2-mediated lymphocyte proliferation inhibition of the t-suppressor circuit of delayed-type hypersensitivity by interferon the effects of human interferon-alpha upon in vitro canine immune responses interferon inhibits dna synthesis induced in mouse lymphocyte suspensions by phytohaemagglutinin or by allogeneic cells the pharmacology and toxicology of the interferons: an overview regulation of mitogen-induced lymphocyte dna synthesis by human interferon of different origins manual of clinical immunology tissue specificity of interferons prepared in various tissue cultures experimental studies with three new strains of feline infectious virus: fipv-ucd2, fipv-ucd3, and fipv-ucd4 induction or suppression of b cell proliferation and differentiation by phytohemagglutinin or concanavalin a in mouse spleen cell cultures the immunobiology of the feline leukemia virus effect of human leukocyte interferon on prevention of infectious bovine rhinotracheitis virus infection recombinant bovine interferon-y as an immunomodulator in dexamethasone-treated and non-treated cattle effect of recombinant alpha a-interferon on dna synthesis and differentiation of human keratinocytes in vitro effects of interferons on cell growth and function optimal conditions for in vitro blastogenesis of feline peripheral blood lymphocytes evaluation of immunity to feline infectious peritonitis in cats with cutaneous viral-induced delayed hypersensitivity effect of interferon or propionibacterium acnes on the course of experimentally induced feline infectious peritonitis medical virology (3rd edn research program. the authors thank ms. lewanne french and dr. mitzi martinez for their assistance. key: cord-000103-3zh8jmc2 authors: caignard, grégory; komarova, anastassia v.; bouraï, mehdi; mourez, thomas; jacob, yves; jones, louis m.; rozenberg, flore; vabret, astrid; freymuth, françois; tangy, frédéric; vidalain, pierre-olivier title: differential regulation of type i interferon and epidermal growth factor pathways by a human respirovirus virulence factor date: 2009-09-18 journal: plos pathog doi: 10.1371/journal.ppat.1000587 sha: doc_id: 103 cord_uid: 3zh8jmc2 a number of paramyxoviruses are responsible for acute respiratory infections in children, elderly and immuno-compromised individuals, resulting in airway inflammation and exacerbation of chronic diseases like asthma. to understand the molecular pathogenesis of these infections, we searched for cellular targets of the virulence protein c of human parainfluenza virus type 3 (hpiv3-c). we found that hpiv3-c interacts directly through its c-terminal domain with stat1 and grb2, whereas c proteins from measles or nipah viruses failed to do so. binding to stat1 explains the previously reported capacity of hpiv3-c to block type i interferon signaling, but the interaction with grb2 was unexpected. this adaptor protein bridges epidermal growth factor (egf) receptor to mapk/erk pathway, a signaling cascade recently found to be involved in airway inflammatory response. we report that either hpiv3 infection or transient expression of hpiv3-c both increase cellular response to egf, as assessed by elk1 transactivation and phosphorylation levels of erk1/2, 40s ribosomal subunit protein s6 and translation initiation factor 4e (eif4e). furthermore, inhibition of mapk/erk pathway with u0126 prevented viral protein expression in infected cells. altogether, our data provide molecular basis to explain the role of hpiv3-c as a virulence factor and determinant of pathogenesis and demonstrate that paramyxoviridae have evolved a single virulence factor to block type i interferon signaling and to boost simultaneous cellular response to growth factors. viruses need to interact with host macromolecules to hijack the cellular machinery and replicate. these interactions are essential for viruses to target endocytic pathways and penetrate host cells, to recruit cellular transcription and/or translation machinery, and to achieve intracellular migration and viral particles assembly. but viruses also encode virulence factors that induce a substantial alteration of host cell functions and genetic programs to increase virus replication and spreading. for example, specific viral factors stimulate survival pathways to prevent apoptosis of infected cells or inhibit cell signaling involved in immune response. among these pathways, ifn-a/b signaling represents a prime target for viruses because of its critical role in the induction of both innate and adaptive antiviral immune responses [1] . ifn-a/b transduce signals through direct binding to a cell surface receptor composed of two transmembrane subunits, ifnar1 and ifnar2c [2] . this interaction activates ifnar1/ifnar2c associated kinases tyk2 and jak1 that subsequently phosphorylate stat2 and stat1 transcription factors. activated stat1 and stat2, altogether with irf9, form the interferon-stimulated gene factor 3 that binds ifn-stimulated response element (isre) promoter sequences to induce a large antiviral gene cluster. as a consequence, most viruses that are pathogenic in vertebrates have evolved virulence factors both to block ifn-a/b expression and signal transduction downstream of ifn-a/b receptor. human parainfluenza virus type 1 (hpiv1) and human parainfluenza virus type 3 (hpiv3) are important human pathogens that belong to respirovirus genus (paramyxoviridae family; [3] ). these viruses are responsible for upper respiratory tract infections and colds, but often spread to the lower respiratory tract causing bronchitis, bronchiolitis and pneumonia in young children and immuno-compromised patients. hpiv3 infection is also suspected to exacerbate chronic airway inflammatory diseases like asthma [4] . sendai virus and bovine parainfluenza virus type 3 (bpiv3) are animal counterparts of hpiv1 and hpiv3 that infect mouse and cattle, respectively. respirovirus genome is a single-strand, negativesense rna molecule that encodes six structural proteins (mononegavirales order). while hemagglutinin-neuraminidase (hn) and fusion (f) are membrane glycoproteins associated with the envelop of hpiv3 particles, the nucleoprotein (n), the phosphoprotein (p) and the viral polymerase (l) form the ribonucleocapsid complex. the matrix protein (m) is at the interface between glycoprotein tails and ribonucleocapsids. the p gene of respirovirus encodes for p but also for a panel of accessory proteins by site-specific editing of p mrna and usage of overlapping open reading frames (orfs). in all respirovirus except hpiv1, the co-transcriptional insertion of one g residue at an editing motif midway of p mrna leads to the expression of a chimeric protein called v. the v proteins of bpiv3 and sendai virus bind mda5 and suppress double-stranded rna-stimulated ifn-b production, thereby contributing to the virus evasion of host immune response [5] . surprisingly in hpiv3, multiple stop codons localized downstream of the editing site prevent the normal expression of a full-length v protein. as a result, p mrna molecules edited by the addition of one g residue encode for the 242 amino acid (aa)-long n-terminal residues of p followed by only six additional aa (see materials and methods and [6] ). but p mrna molecules edited by the addition of five g residues encode for d, a protein exhibiting a large and specific c-terminal domain of unknown function ( figure 1a ). besides co-transcriptional edition, an overlapping orf embedded in the first half of the p mrna allows the expression of a single c protein (hpiv3 and bpiv3) or a nested set of four proteins called c9, c, y1 and y2 (sendai virus and hpiv1). the c proteins of sendai virus and hpiv1 have a high degree of sequence homology and have been studied in details. they are involved in the regulation of viral rna synthesis [7, 8] , the inhibition of innate immune response [9] and potentially contribute to the budding of viral particles [10] [11] [12] . in particular, the c protein of sendai virus both inhibits ifn-b production [13, 14] and blocks interferon signaling downstream of ifn-a/b and ifn-c receptors [15] [16] [17] . the c proteins of hpiv3 and bpiv3 only share ,35% of sequence homology with the c proteins of sendai virus and hpiv1, but they have also been shown to target interferon expression and signaling [5, 18] . although expression of the c protein of hpiv3 (hpiv3-c) is essential to virulence in vitro and in vivo [19] and explains hpiv3 ability to block ifn-a/b signaling [20] , host proteins that bind hpiv3-c remain unknown. (a) organization of the gene p of hpiv3 that encodes for three proteins: p, d and c. whereas conventional transcription and translation lead to the expression of the phosphoprotein p, co-transcriptional insertion of five g residues at the editing site by the virus rna polymerase leads to the expression of a chimeric protein called d. insertion of one g residue can also occur during transcription but two stop codons immediately downstream of the editing site prevent the expression of the protein v that is specific of paramyxoviridae. the protein c is encoded by an overlapping opened reading frame (orf) embedded in p mrna. (b and c) hek-293t cells were transfected with expression vectors encoding gst alone or fused to the c proteins of measles virus (mv-c), hpiv3 (hpiv3-c), or nipah virus (nipah-c), and tested for the interaction with endogenous stat1 (b) or grb2 (c). total cell lysates were prepared 48 h post transfection (cell lysate; middle and lower panels), and copurifications of endogenous cellular proteins were assayed by pulldown using glutathione-sepharose beads (gst pull-down; upper panel). gst-tagged c proteins were detected by immunoblotting using anti-gst antibody, while endogenous stat1 and grb2 were detected with specific monoclonal antibodies. doi:10.1371/journal.ppat.1000587.g001 respiroviruses are important pathogens responsible for acute respiratory tract infections associated with severe airway inflammation in children, elderly and immunocompromised individuals. their rna genome encodes for structural proteins that compose viral particles, but also for virulence factors that alter cell biology to enhance virus replication and spreading. among them, the c protein plays a critical role by blocking cellular response to type i interferons, the main antiviral cytokines secreted during virus infections. to provide molecular basis to this activity, we found that the c protein of human parainfluenza virus type 3 (hpiv3-c), the most frequent human respirovirus, interacts with stat1, a key component of type i interferon receptor complex. but hpiv3-c was also found to interact with grb2, an adaptor molecule involved in cellular response to epidermal growth factor (egf), and to enhance cell response to this cytokine. this pathway increases protein translation, promotes cell survival and contributes to airway inflammation and mucus secretion. thus, our findings show that hpiv3-c not only inhibits the antiviral response but also stimulates cellular response to egf, which benefits virus replication and induces an excessive inflammation of airways during infection. in an attempt to answer this question, we performed a yeast two-hybrid screen and we report here the identification of stat1 and grb2 as direct interactors of hpiv3-c. although binding to stat1 accounts for hpiv3-c ability to block ifn-a/b signaling, the interaction with grb2 was unexpected. this adaptor protein bridges growth factor receptor tyrosine kinases (rtks), like epidermal growth factor (egf) receptor, to the mitogenactivated protein kinase/extracellular signal-regulated kinase (mapk/erk) pathway. upon engagement by their ligands, rtks autophosphorylate on tyrosine residues to recruit adaptor proteins containing phosphotyrosine binding (ptb) or src homology 2 (sh2) domains like grb2 [21] . once associated to rtks by its sh2 domain, grb2 recruits the guanine nucleotidereleasing factor son-of-sevenless (sos) to activate ras. downstream events include mapk/erk kinase (mek1/2) activation, which in turn phosphorylates erk1/2. finally, phosphorylated erk1/2 directly or indirectly activates numerous cellular targets including transcription factors (e.g. elk1, sap1, sap2, c-fos, creb, srf) but also cellular factors that control mrna translation like eukaryotic initiation factor 4e (eif4e) or small ribosomal subunit s6 protein [22, 23] . growth factor binding to rtks regulates a multiplicity of cellular processes including proliferation, differentiation and survival. in the respiratory tract, this signaling cascade has been shown to trigger inflammation and mucus secretion by epithelial cells [24] [25] [26] [27] , suggesting a critical role in innate immunity [28] . however, excessive activation of this pathway could benefit to virus replication by inhibiting ifn-a/b signaling [29] and promoting infected cell survival [25] . altogether, these data provided a rational to investigate the functional impact of hpiv3-c expression on ifn-a/b vs egf receptor and mapk/erk signaling pathways. the c protein of hpiv3 interacts directly with stat1 and grb2 to identify cellular targets of hpiv3-c, this viral protein was used as bait in the yeast two-hybrid system to screen a human cdna library. the screen was performed at saturation with a 10-fold coverage of the library (50.10 +6 diploids), and positive yeast colonies growing on selective medium were analyzed by pcr and sequencing to identify binding partners of hpiv3-c. stat1 and grb2 were the main interactors of hpiv3-c identified in the screen with 5 and 150 yeast colonies corresponding to these cellular proteins, respectively. in both cases, cdna clones retrieved from the screen corresponded to full-length stat1 and grb2 in frame with the gal4-ad transactivation domain. to validate these interactions in human cells, gst-tagged hpiv3-c was expressed in hek-293t cells and purified with glutathion-sepharose beads. as shown in figure 1b and 1c, endogenous stat1 and grb2 co-purified with hpiv3-c. highly divergent c proteins from measles virus (mv-c) and nipah virus (nipah-c) failed to do so, thereby demonstrating the specificity of identified interactions. binding to stat1 provides molecular basis to the inhibition of ifn-a/b signaling by hpiv3-c [18] , and parallels the interaction previously identified between sendai virus c protein and mouse stat1 [15] . altogether, this suggests that stat1 is a specific cellular interactor of respirovirus c proteins. in contrast, binding to grb2 is unexpected and suggests a new function for hpiv3-c that we decided to investigate. hpiv3-c has opposite effects on ifn-a/b and egf signaling pathways the adaptor protein grb2 plays a critical role in coupling signal from growth factor receptors to mapk/erk signaling pathway. to address the question of hpiv3-c interference with this pathway, we used a trans-reporter gene assay that measures elk1 activation by erk1/2. in this system, elk1 transcription factor is fused to the dna binding domain of gal4 (gal4-db) and binds the promoter sequence of a luciferase reporter gene. upon stimulation with a growth factor like egf, elk1 is activated as assessed by a significant increase in luciferase expression. surprisingly, we observed a 6-fold enhancement in this cellular response to egf when 36flag-tagged hpiv3-c was expressed in hek-293t cells (figure 2a ). same results were obtained when using hpiv3-c without a tag (14-fold enhancement) or tagged with the red fluorescent protein cherry (7-fold enhancement). in contrast to hpiv3-c, neither mv-c nor nipah-c enhanced elk1 activity upon egf stimulation (figure 2a ) whereas expression levels of hpiv3-c, mv-c and nipah-c were similar in this system ( figure 2f , left panel). elk1 activity was also enhanced by hpiv3-c expression in vero and hela cells as well as beas-2b and a549, two epithelial cell lines that originate from the respiratory tract, which is the tissue targeted by hpiv3 in vivo ( table 1 ). the effect of hpiv3-c in these different cell lines was highly significant (see p-values in table 1 ) although relatively modest when compared to hek-293t cells. this is probably because our reporter system requires the co-transfection of four plasmids and vero, hela, beas-2b and a549 cells are more difficult to transfect than hek-293t. in parallel experiments, cellular response to ifn-a/b was monitored using a cis-reporter gene, of which expression is controlled by five isres. as previously reported [18] , we found that hpiv3-c efficiently blocked ifn-a/b signaling ( figure 2b ) as opposed to what we observed for the egf pathway. again, mv-c or nipah-c was unable to do so. altogether, these results show that hpiv3-c enhances the cellular response to egf in addition to its ability to block ifn-a/b signaling. we also determined if similar effects on the egf pathway were observed in infected cells. hek-293t cells were infected with hpiv3 (moi = 3) and then transfected with elk1 activity reporter plasmids. infection of hek-293t cells was confirmed by anti-hpiv3 hemagglutinin-neuraminidase (hpiv3-hn) immunostaining and flow cytometry analysis ( figure 2e ). like hpiv3-c alone, hpiv3 infection enhanced elk1 activity upon egf stimulation ( figure 2c ). interestingly, hpiv3 infection induced a significant level of elk1 activity in the absence of egf stimulation. this suggests that in addition to hpiv3-c interaction with grb2, other mechanisms modulate mapk/erk pathway during hpiv3 infection. finally, to demonstrate that enhancement of elk1 activation by hpiv3-c is completely dependent on erk1/2 activation, hek-293t cells were pre-treated with mek1/2 inhibitor u0126 before stimulation with egf. this molecule targets mek1/2 and totally abrogates downstream phosphorylation and activation of erk1/2 [30] . as shown in figure 2d , elk1 activation was blocked by u0126, whereas hpiv3-c expression was maintained ( figure 2f , right panel). this demonstrates that hpiv3-c is acting through erk1/2 stimulation. altogether, these results support a model where hpiv3-c interaction with grb2 enhances cellular response to growth factors as assessed by an increased activation of mapk/ erk pathway. phosphorylation of erk1/2, eif4e and small ribosomal subunit s6 protein are stimulated by hpiv3-c expression or hpiv3 infection to further document hpiv3-c impact on mapk/erk signaling pathway, we compared the kinetic of erk1/2 phosphorylation in hek-293t cells expressing hpiv3-c or not. cells were transfected hpiv3-c impact on ifn-a/b and egf pathways in addition to these three plasmids, cells were co-transfected with an expression vector encoding 36flag-tagged mv-c, hpiv3-c or nipah-c or the corresponding empty vector pci-neo-36flag. 12 h after transfection, cells were starved and 6 h later egf was added at a final concentration of 100 ng/ml. after 24 h, relative luciferase activity was determined. (b) hek-293t cells were transfected with pisre-luc, a plasmid containing a luciferase gene of which expression is controlled by five isres, and prl-cmv. in addition to these two plasmids, cells were co-transfected with an expression plasmid encoding 36flag-tagged mv-c, hpiv3-c or nipah-c or the corresponding empty vector pci-neo-36flag. 24 h after transfection, 1000 iu/ml of recombinant ifn-b were added. after 24 h, relative luciferase activity was determined. (c) hek-293t cells were infected with hpiv3 (moi = 3) and then transfected with pfa2-elk1, pgal4-uas-luc, prl-cmv vectors. 12 h later, cells were starved during 6 h and stimulated with egf at a final concentration of 100 ng/ml. after 24 h, relative luciferase activity was determined. (d) same experiment as (a) but 20 mm of mek1/2 specific inhibitor u0126 was added as indicated. (a-d) all experiments were achieved in triplicate, and data represent means6sd. (e) hek-293t cells were infected as in (c), and hpiv3-hn expression determined by immunostaining and flow cytometry analysis. (f) hek-293t cells were transfected to express 36flag-tagged mv-c, hpiv3-c or nipah-c as described in (a) and (b), and relative expression levels were determined 36 h later by western blot analysis (left panel). in a parallel experiment, hek-293t cells were transfected to express hpiv3-c and were cultured with or without egf in the presence or absence of u0126 as described in (d). hpiv3-c expression level was determined by western blot analysis (right panel). doi:10.1371/journal.ppat.1000587.g002 with 36flag-tagged hpiv3-c or a control plasmid and 24 h post transfection, they were starved before stimulation with egf. erk1/ 2 phosphorylation was determined at 10, 30 and 120 min after stimulation. as illustrated by one representative experiment in figure 3a , egf stimulation induced erk1/2 phosphorylation in control cells but signal was markedly and reproducibly increased by hpiv3-c expression at maximum phosphorylation time point (1.4 to 2.8 fold increase; p = 0.005; n = 4). we then determined the phosphorylation level of two downstream targets of this pathway that are involved in the control of mrna translation, the translation initiation factor eif4e and the ribosomal protein s6 ( figure 3b ). before egf stimulation, low levels of phosphorylated eif4e and s6 were detectable in mock-treated cells ( figure 3b and 3d). hpiv3-c expression had virtually no effects on this background. thus, eif4e and s6 phosphorylation levels were determined at different time-points after egf stimulation. because erk1/2 activation precedes eif4e and s6 phosphorylation, maximal phosphorylation occurs at later time points and was determined at 30 min, 2 h, 6 h and 24 h after stimulation. as observed for erk1/2, phosphorylation levels of eif4e and s6 were enhanced by hpiv3-c expression when stimulating the cells with egf. to validate these observations in infected cells, hek-293t cells were infected with hpiv3 (moi = 3) and 24 h later, cells were starved for 12 h before stimulation with egf. like hpiv3-c expression alone, hpiv3 infection enhanced erk1/2 phosphorylation at the peak of induction, i.e. 10 min after adding egf to the cells ( figure 3c ). interestingly, hpiv3 infection of a549 cells also enhanced erk1/2 phosphorylation but the induction profile was different. indeed, erk1/2 phosphorylation was not significantly increased at the peak of induction, but the signal was boosted by hpiv3 infection at late time points ( figure s1 ). the same profile was observed when eif4e and s6 phosphorylation levels were analyzed in infected hek-293t cells. hpiv3 infection sustained the phosphorylation of these two translation factors at late time points, but showed no increase at the peak of stimulation, i.e. 30 min after adding egf to the cells ( figure 3d ). this could relate to the fact that hpiv3 infection also induces low levels of eif4e and s6 phosphorylation in the absence of egf stimulation ( figure 3d ). this is reminiscent to what was observed for elk1 ( figure 2c ), and suggests that hpiv3 infection induces a basal activation of mapk/erk pathway leading to the constitutive phosphorylation of downstream targets. altogether, these data demonstrate that hpiv3 infection or hpiv3-c expression alone both enhance mapk/erk pathway activation in egf-stimulated cells. several rna viruses require an activated mapk/erk pathway to produce viral components and replicate properly (for review see [31] ). to test if the same was true for hpiv3, cells were treated for 2 h with mapk/erk pathway inhibitor u0126 and infected with hpiv3 (moi = 1). two days after infection, cell surface expression of hpiv3-hn was detected by immunostaining and flow cytometry. u0126 completely blocked the expression of hpiv3-hn in hpiv3-infected cells (figure 4 ), whereas the same inhibitor had no effect when cells were infected with mv ( figure s2 ). altogether, this demonstrates that mapk/erk signaling is essential for the expression of hpiv3 proteins and suggests that hpiv3 manipulates this pathway to increase replication efficiency. the c-terminal region of hpiv3-c binds stat1 and grb2 to better understand how hpiv3-c targets both the ifn-a/b and egf signaling pathways, we characterized the functional domains of hpiv3-c that bind stat1 and grb2. to do so, we generated by pcr a full matrix of hpiv3-c overlapping fragments and tested their ability to interact with either stat1 or grb2 in the yeast two-hybrid system ( figure 5 and 6 ). both forward and reverse primers were designed every 75 nucleotides along hpiv3-c sequence and fused to appropriate tails to allow gap-repair recombination with linearized gal4-db yeast two-hybrid vector ( figure 5a ). all possible combinations of forward and reverse primers were used to amplify hpiv3-c fragments. finally, corresponding pcr products were transformed in a yeast strain expressing gal4-ad fused to either stat1 or grb2, and growth on selective medium was used to detect potential interactions. a 124 (aa)-long peptide encompassing position 76 to 199 located in the c-terminal half of hpiv3-c was sufficient to bind stat1 ( figure 5b ) or grb2 ( figure 6a ). in an iterative process, we then generated a second, a third and a fourth set of hpiv3-c fragments corresponding to one-by-one aa deletions ( figure 5c -e and figure 6b -d), allowing to further reduce the stat1 and grb2 binding motifs to minimal peptides. a 106 aa peptide encompassing residues 90 to 195 of hpiv3-c was sufficient to observe the interaction with stat1 ( figure 5e ). the binding region to grb2 was virtually the same, encompassing aa 97 to 195 ( figure 6d ). the c-terminal region of hpiv3-c required to bind stat1 and grb2 in the yeast two-hybrid system is highly conserved among respiroviruses ( figure 7a ) and suspected to fold into a structured coiled-coil domain [18] . furthermore, virtually the same c-terminal region of sendai virus c protein (aa 85-204) was previously reported to mediate the interaction with mouse stat1 [32] . to further validate our observations performed in the yeast two-hybrid system, we retested by co-affinity purification the ability of hpiv3-c fragment encompassing aa 90-195 (hpiv3-c 90-195 ) to interact with stat1 and grb2 in hek-293t cells. gst-tagged hpiv3-c 90-195 was expressed together with 36-flag-tagged stat1 or grb2, and purified with glutathionsepharose beads. full-length hpiv3-c and the n-terminal region encompassing aa 1-89 (hpiv3-c 1-89 ) were used as positive and negative controls, respectively. as shown in figure 7b and 7c, hpiv3-c 90-195 interacted with stat1 and grb2, whereas hpiv3-c 1-89 did not. although hpiv3-c 90-195 interacted with grb2 as efficiently as full-length hpiv3-c ( figure 7c ), interaction with stat1 was weaker suggesting that more residues contribute to the stabilization of this interaction ( figure 7b ). altogether, these results confirm that aa 90-195 of hpiv-3 include both stat1 and grb2 binding sites. as described in figure 2 , hek-293t, hela, vero, a549 or beas-2b cells were transfected with pfa2-elk1, pgal4-uas-luc, prl-cmv to measure the activation level of mapk/erk signaling pathway. cells were co-transfected with plasmids encoding 36flag-tagged mv-c, hpiv3-c or nipah-c or the corresponding empty vector pci-neo-36flag. 12 h after transfection, cells were starved and stimulated 6 h later with 100 ng/ml of egf. after 24 h, expression of luciferase was quantified. results were normalized so that reporter activity in cells transfected with a control vector equals 1. experiments were performed in triplicates and data represent means6sd. doi:10.1371/journal.ppat.1000587.t001 hpiv3-c impact on ifn-a/b and egf pathways although stat1 and grb2 essentially bind to the same region of hpiv3-c as demonstrated above, it remained unclear whether these interactions are mutually exclusive. to answer this question, a competition experiment was designed where gst-tagged hpiv3-c was co-expressed with stat1 in the presence or absence of grb2 ( figure 7d ). in this setting, grb2 expression prevents stat1 copurification together with gst-tagged hpiv3-c. this validates our finding that stat1 and grb2 interact with the same region of hpiv3-c, and demonstrates that stat1 and grb2 compete for hpiv3-c binding. interestingly, grb2 interaction with hpiv3-c was not affected by stat1 expression (figure 7d and data not shown), suggesting that grb2 has a higher affinity for hpiv3-c than stat1. both the n-and c-terminal domains of hpiv3-c are required for its activity we finally tested if hpiv3-c 90-195 was able, like full-length hpiv3-c, to block ifn-a/b signaling and enhance cellular response to egf stimulation. first, cells were transfected with 36flag-tagged hpiv3-c, hpiv3-c 90-195 or hpiv3-c 1-89 together with the ifn-a/b reporter plasmid, and stimulated 24 h later with recombinant ifn-b. reporter gene expression was determined 24 h post transfection and found to be inhibited exclusively by full-length hpiv3-c ( figure 8a ). although this may reflect the weakness of hpiv3-c interaction with stat1 ( figure 7b ), this also indicates that both the n-terminal and c-terminal regions of hpiv3-c are required to block ifn-a/b signaling, even if only the c-terminal region is required for the binding to stat1. the same constructs were tested using the elk1 activity reporter plasmids ( figure 8b ). again, only full-length hpiv3-c was able to enhance elk1 activation upon egf stimulation whereas full-length hpiv3-c and hpiv3-c 90-195 were expressed at similar levels in transfected cells ( figure 8b , upper right panel). because grb2 binding to hpiv3-c and hpiv3-c 90-195 were essentially equivalent in co-affinity purification experiments, we hypothesized that the n-terminal region of hpiv3-c was required for its proper subcellular localization. thus, hpiv3-c, hpiv3-c 90-195 and hpiv3-c 1-89 were expressed in fusion downstream of the red fluorescent protein cherry. as shown in figure 8c , cherry alone or fused to hpiv3-c 90-195 localized both in the nucleus and the cytoplasm of transfected cells. in contrast, full-length hpiv3-c essentially accumulated at the cellular membrane whereas hpiv3-c 1-89 was in the nucleus. although we have no explanation for this unexpected localization of hpiv3-c 1-89 , these observations show that only full-length hpiv3-c is able to target the cellular membrane where both ifn-a/b and egf signaling are triggered. paramyxoviridae have evolved various mechanisms to block ifna/b response, in particular signaling downstream ifnar1/ ifnar2c receptor [20, 33] . although members of pneumovirinae subfamily have specific genes to encode inhibitors of ifn-a/b signaling pathway, those expressed by other paramyxoviridae (i.e. paramyxovirinae subfamily) are encoded by overlapping reading frames embedded within the gene p. rubulaviruses express v proteins that target stat1 and/or stat2 for ubiquitination and degradation, while morbilliviruses and henipaviruses v proteins essentially impair stat1/2 phosphorylation, activation and nuclear translocation. in addition, morbilliviruses and henipaviruses also encode for c proteins of which role in the inhibition of ifna/b response has been a matter of debates [34] [35] [36] [37] . recent reports showed that morbillivirus c proteins only have a minor role in the inhibition of ifn-a/b signaling [37] , but are essential to block ifn-a/b induction [38] . whether henipavirus c proteins can directly block ifn-a/b or promote viral replication through alternative mechanisms is unclear [35] . in contrast, it has been clearly established that respirovirus c proteins are potent inhibitors of ifn-a/b signaling [5, 18, [39] [40] [41] . in this report, we show that hpiv3-c, but not mv-c or nipah-c, directly interacts with stat1 and efficiently inhibits ifn-a/b signaling. in addition, we identified a minimal stat1 binding domain that encompasses aa 90-195 of hpiv3-c, a region suspected to fold into a coiled coil. interestingly, this conserved domain is localized within the stat1 binding region shared by all four isoforms of sendai virus c protein [32] . together, these results confirm the capacity of hpiv3-c to block ifn-a/b signaling pathway [18] , provide molecular basis to this inhibition and clarify the fact that respirovirus c proteins are functionally distinct from morbillivirus and henipavirus c proteins. in addition to stat1, we show that hpiv3-c interacts directly with grb2 and enhances mapk/erk signaling downstream of egf receptor (egfr). our data give the first example of a paramyxoviridae protein that contributes to the stimulation of egfr and mapk/erk pathway and provides molecular basis to this activity. this pathway has been known for decades as a prime target of dna tumor viruses and oncogenic retroviruses, and its activation represents an essential step toward carcinogenesis [3, 42] . but recent data demonstrate that non-oncogenic rna viruses also activate this signaling cascade to support viral replication and spreading [31] . whether it is activated upon egfr engagement or other means, mapk/erk pathway regulates a multiplicity of cellular processes including proliferation, differentiation, development, cell survival and inflammation. as a consequence, how the activation of mapk/erk pathway promotes viral replication is a complex question. interestingly, two non-oncogenic rna viruses associated with acute respiratory tract infections have been recently reported to modulate the egfr pathway. both human respiratory syncytial virus (hrsv), a member of paramyxoviridae like hpiv3, and a rhinovirus that belongs to picornaviridae family activate egfr and mapk/erk pathway [25, 27] . infection of epithelial cells by these viruses stimulates the processing and activation of egfr ligands by membrane matrix metalloproteinase and subsequent engagement of egfr through autocrine/paracrine mechanisms. experiments performed on rhinovirus show that viral replication and tlr3 engagement by viral rna are both required to activate the egfr and mapk/erk pathway [27] . in this report, we show that hpiv3 infection also activates mapk/erk pathway in the absence of external stimuli, a phenomenon that possibly relies on the engagement of pathogen recognition receptors. although hpiv3-c alone is unable to activate this pathway, our data suggest that expression of this virulence factor enhances mapk/erk activation above normal level in infected cells, thereby contributing to viral replication and pathogenesis. induction of mapk/erk pathway by rna viruses has numerous consequences on cell biology. first, it results in increased expression of inflammatory factors, in particular cytokines and chemokines that recruit cellular effectors of immunity [27, [43] [44] [45] [46] [47] . mapk/erk pathway was also reported to block the antiviral response induced by ifn-a/b, making its activation beneficial to virus replication [29] . another consequence of mapk/erk pathway activation is the induction of mucin production by infected epithelial cells [24, 27] . although mucin expression is a critical innate defense system, excessive production of mucus results in the obstruction of airways and delays the elimination of pathogens. finally, it has been demonstrated in vitro that upon hrsv infection, activation of egfr and mapk/erk pathway sustains viral replication by retarding the death of infected cells [25] . altogether, these data suggest that although a moderate activation of mapk/erk pathway contributes to the innate response against viruses, an excessive activation leads to deleterious inflammation, inhibition of ifn-a/b response, airway obstruction and infected cell survival figure 6 . identification of a minimal hpiv3-c region interacting with grb2. fragments of hpiv3-c were generated and tested for their ability to interact with grb2 following the procedure described in figure 5a . (a) the first iteration identified a grb2 binding region of 124 aa. after two (b), three (c) and four additional rounds (d), this domain was finally reduced to a minimal grb2 binding motif of 99 aa. this binding domain, encompassing position 97 to 195, is contained in the stat1 binding region of hpiv3-c previously identified in figure 5 . doi:10.1371/journal.ppat.1000587.g006 hpiv3-c impact on ifn-a/b and egf pathways [28] . therefore, it is tempting to speculate that hpiv3-c interaction with grb2 and egfr pathway participates in such deregulation of airway epithelium homeostasis to promote hpiv3 replication and spreading. a consequence of such perturbations could be an aggravation of chronic inflammatory airway diseases like asthma or chronic obstructive pulmonary disease as already suggested by epidemiological links with paramyxoviridae infections and in vivo models [48, 49] . besides its effects on immune response, activation of mapk/ erk pathway has direct consequences on viral replication as assessed by in vitro experiments. it is now well documented that mapk/erk pathway inhibition with u0126 or pd098059 deeply impairs the replication of numerous rna viruses including hrsv ( [50] ; and for review see [31] ). similarly, we show in this report that mapk/erk pathway inhibition prevents hpiv3 protein expression in infected cells as assessed by hpiv3-hn detection. in influenza virus infected cells, membrane accumulation of influenza virus hemagglutinin (ha) induces lipid-rafts clustering that leads to mapk/erk pathway activation and nuclear export of viral ribonucleoprotein complexes to achieve a and b) hpiv3-c 90-195 and hpiv3-c 1-89 were tested for their ability to modulate ifn-a/b or egf signaling. (a) as described in figure 2 , hek-293t cells were transfected with pisre-luc and prl-cmv to determine the activation level of ifn-a/b signaling pathway. cells were co-transfected with expression plasmids encoding 36flag-tagged full-length hpiv3-c (c fl ) or fragments. 24 h after transfection, 1000 iu/ml of recombinant ifn-b were added. after 24 h, relative luciferase activity was determined. experiments were performed in triplicates, and data represent means6sd. (b) as described in figure 2 , cells were transfected with pfa2-elk1, pgal4-uas-luc and prl-cmv to determine the activation level of mapk/erk signaling pathway. cells were co-transfected with expression plasmids encoding full-length hpiv3-c (c fl ) or fragments. 12 h later, cells were starved during 6 h and stimulated with egf at a final concentration of 100 ng/ml. after 24 h, relative luciferase activity was determined. experiments were performed in triplicate, and data represent means6sd. as a control, relative expression levels of c fl , c 90-195 and c 1-89 were determined by western blot analysis (upper right panel). (c) full-length hpiv3-c, hpiv3-c 90-195 or hpiv3-c 1-89 was expressed in fusion downstream of the red fluorescent protein cherry to determine subcellular localization in hek-293t cells. 24 h after transfection, cells were fixed with pfa, permeabilized and labeled with dapi to stain nuclei. single confocal sections show cherrytagged protein fluorescence in red and dapi staining in blue. doi:10.1371/journal.ppat.1000587.g008 viral particles assembly [51] [52] [53] . because hpiv3 replication cycle is only cytoplasmic, mechanisms involved are necessarily distinct. a possible link between mapk/erk pathway and hpiv3 protein expression lies in the fact that among downstream targets of this pathway are essential factors of cellular translational machinery. we show that hpiv3-c expression enhances the phosphorylation of s6 and eif4e. the small ribosomal subunit protein s6 is the major phosphoprotein of eukaryotic ribosomes with five phosphorylation sites (ser235, ser236, ser240, ser244, and ser247). two families of serine/threonine kinases phosphorylate s6 in vitro: s6k1/2 and p90 ribosomal s6 kinase (rsk). recently it has been shown that mapk/erk signaling pathway activates rsk family members that contribute to s6 phosphorylation on ser235/236 thereby stimulating cap-dependent translation [23] . in addition, eif4e that interacts with the cap structure and brings translation initiation factors together with the small ribosomal subunit via the scaffold protein eif4g, undergoes regulated phosphorylation on ser209 upon mapk/erk pathway activation. this phosphorylation event is dependent on eif4gassociated mapk signal-integrating kinases, mnk1 and mnk2 [22] . eif4e is believed to be the least abundant of all initiation factors and therefore considered as a perfect target to regulate protein synthesis. even though there is no direct link between eif4e phosphorylation and the enhanced translation observed, the fraction of phosphorylated eif4e dramatically increases following treatment of the cells with growth factors, hormones and mitogens. therefore, eif4e phosphorylation has been associated with increased translation rates. hpiv3 mrnas are capped and polyadenylated like their host counterparts. thus, s6 and eif4e phosphorylation together with a high level of viral gene transcription may contribute to a rapid switch toward viral protein synthesis within infected cells. specific biochemical investigations are still required to decipher how hpiv3-c can both inhibit ifn-a/b signaling and enhance egfr and mapk/erk pathway. when searching the literature for viral proteins that target grb2, we found specific reports on ns5a from hepatitis c virus and orf3 from hepatitis e virus [54, 55] . although ns5a inhibits mapk/erk activation induced by exogenous egf, orf3 was described as an activator of mapk/erk pathway like hpiv3-c. both ns5a and orf3 exhibit a proline-rich motif (pxxp) to bind the src homology 3 (sh3) domains of grb2, but there is no such motif in hpiv3-c suggesting that other mechanisms mediate stat1 and grb2 binding. interestingly, these two cellular proteins exhibit sh2 domains. such domains typically bind a phosphorylated tyrosine residue in the context of a longer peptide motif within a target protein. although there is no evidence that hpiv3-c becomes phosphorylated, we have tested hpiv3-c interaction with mutant stat1 and grb2 exhibiting sh2 domains disabled for the interaction with phosphotyrosine residues. these mutants were not affected for the interaction with hpiv3-c (data not shown). this suggests that hpiv3-c either binds distinct regions of stat1 and grb2, or interacts with a region of the sh2 domain that does not involve the phosphotyrosine binding site. finally, our results also show that full-length hpiv3-c is required to modulate ifna/b and mapk/erk pathways since aa 90-195 that bind stat1 and grb2 are unable to do so when expressed alone. full-length hpiv3-c was also required to observe a localization at the cell membrane, suggesting a link with its activity. interestingly, the n-terminal 23 residues of sendai virus c protein act as a membrane targeting signal [56] . but the n-terminal residues of hpiv3-c (aa 1-89) were unable to do so, and sequence analysis did not show any conservation with the c protein of sendai virus. thus, hpiv3-c tertiary structure is apparently required to target this protein at the cell membrane. this specific localization could both sequester stat1 to prevent the stimulation of ifn-target genes and contribute to the aggregation of grb2-sos complexes to enhance mapk/erk signaling [57] . altogether, this suggests that hpiv3-c interaction with stat1 and grb2 represents a potential target for the development of antiviral molecules against hpiv3 and possibly other members of respirovirus genus. plasmid dna constructs p-encoding sequence from hpiv3 wild-type strain (df042505) was amplified by rt-pcr (titan one tube; roche applied science) from total rna purified from infected cells (rneasy kit; qiagen). amplification was performed using the following hpiv3-p specific primers flanked with gateway cloning sites: 59-gggga-caactttgtacaaaaaagttggcatggaaagcgatgctaaaaactatc-aaa and 59-ggggacaactttgtacaagaaagttggttattggcaattatt-gacatcttcattgaac. pcr products were cloned using topo ta cloning kit (invitrogen) into topo vector. a total of 21 clones were analyzed to establish the sequence of hpiv3-p (genbank id: eu719627). interestingly, 8 clones were not edited, 11 clones were edited by the addition of one g residue, and 2 clones were edited by the addition of 5 g residues. one of the plasmids containing the unedited sequence of hpiv3-p was selected and subsequently used as a template to clone hpiv3-c. dna sequences encoding full-length hpiv3-c or fragments corresponding to aa 1-89 or 90-195 were amplified by pcr from p(hpiv3-p)-topo and cloned by in vitro recombination into pdonr207 (gateway system; invitrogen) as previously described [58] . similarly, mv-c was amplified from p(+)mv323 that contains the full genome of measles virus wild-type strain ichinose (kindly provided by dr. k. takeuchi, [59] ). nipah-c was amplified from niv-p plasmid (kindly provided by dr. tf. wild; [60] ). grb2 coding sequence was amplified from the human spleen cdna library used to perform the yeast two-hybrid screen (invitrogen). the pdonr207 plasmid containing stat1 was previously described [58] . viral or cellular coding sequences were subsequently transferred by in vitro recombination from pdonr207 into different gateway-compatible destination vectors (see below) following manufacturer's recommendation (lr cloning reaction, invitrogen). to perform yeast two-hybrid experiments, coding sequences were recombined into ppc86 (invitrogen) to be expressed in fusion downstream of the activation domain of gal4 (gal4-ad) or into pdest32 to be expressed in fusion downstream of the dna binding domain of gal4 (gal4-db). in mammalian cells, gst-tag and 36flag-tag fusions were achieved using pdest27 (invitrogen) or pci-neo-36flag vector, respectively [61] . we also used pci-neo (promega) and pmcherry-c1 (clontech) to express proteins without a tag or in fusion downstream of cherry, respectively. these two plasmids were made gateway-compatible using the gateway vector conversion system (invitrogen). hek-293t, hela and vero cells were maintained in dulbecco's modified eagle's medium (dmem; gibco-invitrogen) containing 10% fetal bovine serum, penicillin, and streptomycin at 37uc and 5% co 2 . a549 and beas-2b cells were maintained in f-12k medium (gibco-invitrogen) containing 10% fetal bovine serum, penicillin, and streptomycin at 37uc and 5% co 2 . hpiv3 (strain c243) was amplified and titrated on vero cells following recommendations of atcc (american type culture collection). recombinant mv-egfp virus used in figure s2 has been hpiv3-c impact on ifn-a/b and egf pathways previously described [62] . infections were performed for 2 h at 37uc in optimem (gibco-invitrogen). later on, cells were washed and incubated in fresh culture medium for 24 or 48 h. to detect viral replication, cells were recovered and incubated in pbsparaformaldehyde 3.2% for 20 min. after extensive washing with pbs, cells were permeabilized with pbs-triton 0.05% for 15 min, and then incubated with a monoclonal antibody specific to hpiv3-hn (m02122321, abcam). cells were washed and incubated in the presence of an anti-mouse cy3-conjugated antibody (jackson immunoresearch). after extensive washing, cellular immunostaining was analyzed using a facscalibur flow cytometer (bd). when specified, cells were pre-treated with mek1/2 specific inhibitor u0126 (20 mm final; promega) for 2 h before, during and after infection to study the impact on hpiv3 infection. to perform co-affinity purification experiments, cloned orfs were transferred from pdonr207 to pdest27 expression vector (invitrogen) to achieve gst fusion, and to pci-neo-36flag vector [61] for 36flag-fusion. cell transfections were performed using lipofectamine 2000 (invitrogen). unless specified otherwise, 5610 5 hek-293t cells were dispensed in each well of a 6-well plate, and transfected 24 h later with 600 ng of each plasmid dna per well. two days post transfection, hek-293t cells were washed in pbs, then resuspended in lysis buffer (0.5% nonidet p-40, 20 mm tris-hcl at ph 8, 120 mm nacl and 1 mm edta) supplemented with complete protease inhibitor cocktail (roche). cell lysates were incubated on ice for 20 min, and then clarified by centrifugation at 14,0006g for 10 min. for pull-down analysis, 400 mg of protein extracts were incubated for 1 h at 4uc with 25 ml of glutathione-sepharose beads (amersham biosciences) to purify gst-tagged proteins. beads were then washed 3 times in ice-cold lysis buffer and proteins were recovered by boiling in denaturing loading buffer (invitrogen). purified complexes and protein extracts were resolved by sdspolyacrylamide gel electrophoresis (sds-page) on 4-12% nupage bis-tris gels with mops running buffer (invitrogen), and transferred to a nitrocellulose membrane. proteins were detected using standard immunoblotting techniques. 36flagand gst-tagged proteins were detected with a mouse monoclonal hrp-conjugated anti-36flag antibody (m2; sigma-aldrich) and a rabbit polyclonal anti-gst antibody (sigma-aldrich), respectively. specific antibodies were used to detect endogenous stat1 (clone-1; bd biosciences), grb2 (clone-81; bd biosciences), phospho-erk1/2 (clone-12d4; upstate), erk1/2 (ct; upstate), phospho-eif4e (ser209; cell signaling), eif4e (cell signaling), phospho-s6 235-236 (ser235/236; cell signaling), s6 (clone-54d2; cell signaling) and hpiv3-hn (m02122321; abcam). secondary anti-mouse and anti-rabbit hrp-conjugated antibodies were from ge-healthcare. densitometric analysis of the gels was performed using a specific module of photoshop cs3 extended (adobe systems inc.). yeast two-hybrid screening and gap-repair procedure our yeast two-hybrid protocols have been described in details elsewhere [58] . briefly, pdest32 plasmid encoding gal4-db fused to hpiv3-c was transformed in ah109 yeast strain (clontech), and used to screen by mating a human spleen cdna library cloned in the gal4-ad ppc86 vector (invitrogen) and previously established in y187 yeast strain (clontech). yeast cells were plated on a selective medium lacking histidine and supplemented with 10 mm 3-amino-triazole (3-at; sigma-aldrich) to select for interaction-dependent transactivation of his3 reporter gene. ad-cdnas from [his+] colonies were amplified by pcr and sequenced to identify the host proteins interacting with hpiv3-c. the gap-repair procedure was used to map the minimal portion of hpiv3-c interacting with stat1 and grb2. as previously described [63] , both forward and reverse pcr primers were designed along the sequence of hpiv3-c and fused to specific tails allowing yeast-based recombination in gal4-db two-hybrid vector. matrix combinations of forward and reverse primers were used to amplify fragments of hpiv3-c by pcr. ah109 yeast cells expressing ad-fused stat1 or grb2 were co-transformed with 5 ml of each pcr product in the presence 50 ng of linearized pdest32 vector to achieve recombinatorial cloning by gaprepair. fragments of hpiv3-c fused to gal4-db were then tested for interaction with ad-stat1 or ad-grb2 by plating yeast cells on selective medium lacking histidine and supplemented with 10 mm of 3-at. luciferase reporter gene assay hek-293t, hela or vero cells were plated in 24-well plates (2610 5 cells per well). one day later, cells were transfected with either pisre-luc (0.3 mg/well; stratagene) or pfa2-elk1 (0.3 mg/well; stratagene) and pgal4-uas-luc plasmids (0.3 mg/ well; provided by dr. y. jacob) together with prl-cmv reference plasmid (0.03 mg/well; promega). cells were simultaneously cotransfected with 0.3 mg/well of pci-neo-36flag, pci-neo or pmcherryc1 expression vectors encoding viral proteins as specified. 24 h after transfection, cells were stimulated with ifnb (biosource) at 1000 iu/ml or starved for 6 h then stimulated with egf (upstate) at 100 ng/ml. 24 h post transfection, cells were lysed, and both firefly and renilla luciferase activities in the lysate were determined using the dual-luciferase reporter assay system (promega). reporter activity was calculated as the ratio of firefly luciferase activity to reference renilla luciferase activity, and normalized so that positive control activity equals 100. when indicated, cells were treated with u0126 (promega) at 20 mm final concentration upon egf stimulation. subcellular localization of cherry-tagged hpiv3-c fl , c 1-89 and c 24-well plates containing coverslips were seeded with hek-293t cells (2610 5 cells per well). one day later, cells were transfected with pmcherryc1 expression vector alone or encoding hpiv3-c fl , hpiv3-c 1-89 or hpiv3-c 90-195 . 36 h after transfection, cells were incubated with pbs-pfa 4% for 20 min at rt, then treated with pbs-triton 0.05% for 15 min at rt to permeabilize the cells. finally, cells were incubated for 10 min at rt in a pbs-pfa 4% solution containing dapi (49-6-diamidino-2-phenylindole) at 10 mg/ml. preparations were mounted using fluoromount-g (southernbiotech), and imaging performed using a sp5 confocal miscroscope (leica). figure s1 erk1/2 phosphorylation is enhanced by hpiv3 infection in a549 cells. a549 cells were infected with hpiv3 (moi = 3) and after 24 h, cells were starved for 12 h before stimulation with 100 ng/ml of egf. phosphorylation of erk1/2 was determined by western blot analysis at 10 min, 30 min and 2 h post stimulation. hpiv3 infection was confirmed by anti-hpiv3 hemagglutinin-neuraminidase (hpiv3-hn) immunoblotting. found at: doi:10.1371/journal.ppat.1000587.s001 (7.34 mb tif) hpiv3-c impact on ifn-a/b and egf pathways figure s2 mek1/2 inhibitor u0126 has no effect on mv protein synthesis. hek-293t cells were left untreated (a) or treated with 20 mm of u0126 for 2 h (b). then, cells were mocktreated or infected with a recombinant mv strain expressing egfp (moi = 1) and cultured with or without u0126 (a and b, respectively). 48 h after infection, egfp expression was quantified by flow cytometry analysis. found at: doi:10.1371/journal.ppat.1000587.s002 (7.34 mb tif) pathogenic viruses: smart manipulators of the interferon system how cells respond to interferons ) fields' virology naturally occurring parainfluenza virus 3 infection in adults induces mild exacerbation of asthma associated with increased sputum concentrations of cysteinyl leukotrienes bovine parainfluenza virus type 3 accessory proteins that suppress beta interferon production rna editing in the phosphoprotein gene of the human parainfluenza virus type 3 sendai virus wild-type and mutant c proteins show a direct correlation between l polymerase binding and inhibition of viral rna synthesis paramyxovirus sendai virus c proteins are essential for maintenance of negative-sense rna genome in virus particles sendai virus c protein plays a role in restricting pkr activation by limiting the generation of intracellular double-stranded rna aip1/alix is a binding partner of sendai virus c protein and facilitates virus budding sendai virus budding in the course of an infection does not require alix and vps4a host factors recruitment of alix/aip1 to the plasma membrane by sendai virus c protein facilitates budding of virus-like particles c and v proteins of sendai virus target signaling pathways leading to irf-3 activation for the negative regulation of interferon-beta production activation of the beta interferon promoter by unnatural sendai virus infection requires rig-i and is inhibited by viral c proteins sendai virus c protein physically associates with stat1 all four sendai virus c proteins bind stat1, but only the larger forms also induce its mono-ubiquitination and degradation importance of the anti-interferon capacity of sendai virus c protein for pathogenicity in mice inhibition of stat 1 phosphorylation by human parainfluenza virus type 3 c protein mutations in the c, d, and v open reading frames of human parainfluenza virus type 3 attenuate replication in rodents and primates paramyxoviridae use distinct virus-specific mechanisms to circumvent the interferon response specificity in signal transduction: from phosphotyrosine-sh2 domain interactions to complex cellular systems phosphorylation of the cap-binding protein eif4e by the mapk-activated protein kinase mnk1 ras/erk signaling promotes site-specific ribosomal protein s6 phosphorylation via rsk and stimulates cap-dependent translation epidermal growth factor system regulates mucin production in airways activation of the epidermal growth factor receptor by respiratory syncytial virus results in increased inflammation and delayed apoptosis multiple tlrs activate egfr via a signaling cascade to produce innate immune responses in airway epithelium rhinovirus-induced major airway mucin production involves a novel tlr3-egfr-dependent pathway epidermal growth factor receptor-mediated innate immune responses and their roles in airway diseases negative regulation of the alpha interferon-induced antiviral response by the ras/raf/mek pathway identification of a novel inhibitor of mitogen-activated protein kinase kinase rna viruses and the mitogenic raf/mek/erk signal transduction cascade the stat2 activation process is a crucial target of sendai virus c protein for the blockade of alpha interferon signaling inhibition of interferon induction and signaling by paramyxoviruses the c protein of measles virus inhibits the type i interferon response newcastle disease virus (ndv)-based assay demonstrates interferon-antagonist activity for the ndv v protein and the nipah virus v, w, and c proteins stringent requirement for the c protein of wild-type measles virus for growth both in vitro and in macaques regulation of interferon signaling by the c and v proteins from attenuated and wild-type strains of measles virus the rinderpest virus non-structural c protein blocks the induction of type 1 interferon sendai virus c proteins counteract the interferon-mediated induction of an antiviral state knockout of the sendai virus c gene eliminates the viral ability to prevent the interferonalpha/beta-mediated responses human parainfluenza virus type 1 but not sendai virus replicates in human respiratory cells despite ifn treatment the egfr as a target for viral oncoproteins mapk activation is involved in posttranscriptional regulation of rsv-induced rantes gene expression role of mitogen-activated protein kinases in influenza virus induction of prostaglandin e2 from arachidonic acid in bronchial epithelial cells ebola virus-like particle-induced activation of nf-kappab and erk signaling in human dendritic cells requires the glycoprotein mucin domain japanese encephalitis virus infection stimulates src tyrosine kinase in neuron/glia spike protein of sars-cov stimulates cyclooxygenase-2 expression via both calcium-dependent and calcium-independent protein kinase c pathways the causal direction in the association between respiratory syncytial virus hospitalization and asthma persistent activation of an innate immune response translates respiratory viral infection into chronic lung disease erk-1/2 activity is required for efficient rsv infection influenza virus propagation is impaired by inhibition of the raf/mek/erk signalling cascade membrane accumulation of influenza a virus hemagglutinin triggers nuclear export of the viral genome via protein kinase calpha-mediated activation of erk signaling clustering of raftassociated proteins in the external membrane leaflet modulates internal leaflet h-ras diffusion and signaling the orf3 protein of hepatitis e virus binds to src homology 3 domains and activates mapk ns5a, a nonstructural protein of hepatitis c virus, binds growth factor receptor-bound protein 2 adaptor protein in a src homology 3 domain/ligand-dependent manner and perturbs mitogenic signaling targeting of the sendai virus c protein to the plasma membrane via a peptide-only membrane anchor aggregation of membrane proteins by cytosolic cross-linkers: theory and simulation of the lat-grb2-sos1 system measles virus v protein blocks jak1-mediated phosphorylation of stat1 to escape ifn-alpha/beta signaling recovery of pathogenic measles virus from cloned cdna establishment of a nipah virus rescue system human papillomavirus type 5 e6 oncoprotein represses the transforming growth factor beta signaling pathway by binding to smad3 a molecularly cloned schwarz strain of measles virus vaccine induces strong immune responses in macaques and transgenic mice inhibition of ifn-alpha/beta signaling by two discrete peptides within measles virus v protein that specifically bind stat1 and stat2 we would like to thank dr. k. takeuchi and dr. tf. wild for kindly providing p(+)mv323 and niv-p plasmids, respectively. we thank all members of pf1-pasteur genopole sequencing core facility. we thank members of the infection-mapping project i-map for fruitful discussions. we thank dr. m. mesel-lemoine and dr. j. pothlichet for their technical support. we thank miss r. parker for proofreading the manuscript. key: cord-001236-cgiok0ce authors: binjawadagi, basavaraj; dwivedi, varun; manickam, cordelia; ouyang, kang; torrelles, jordi b; renukaradhya, gourapura j title: an innovative approach to induce cross-protective immunity against porcine reproductive and respiratory syndrome virus in the lungs of pigs through adjuvanted nanotechnology-based vaccination date: 2014-03-24 journal: int j nanomedicine doi: 10.2147/ijn.s59924 sha: doc_id: 1236 cord_uid: cgiok0ce porcine reproductive and respiratory syndrome (prrs) is an economically devastating respiratory disease of pigs. the disease is caused by the prrs virus (prrsv), an arterivirus which is a highly mutating rna virus. widely used modified live prrsv vaccines have failed to prevent prrs outbreaks and reinfections; moreover, safety of the live virus vaccines is questionable. though poorly immunogenic, inactivated prrsv vaccine is safe. the prrsv infects primarily the lung macrophages. therefore, we attempted to strengthen the immunogenicity of inactivated/killed prrsv vaccine antigens (kag), especially in the pig respiratory system, through: 1) entrapping the kag in biodegradable poly(lactic-co-glycolic acid) nanoparticles (np-kag); 2) coupling the np-kag with a potent mucosal adjuvant, whole cell lysate of mycobacterium tuberculosis (m. tb wcl); and 3) delivering the vaccine formulation twice intranasally to growing pigs. we have previously shown that a single dose of np-kag partially cleared the challenged heterologous prrsv. recently, we reported that np-kag coupled with unentrapped m. tb wcl significantly cleared the viremia of challenged heterologous prrsv. since prrsv is primarily a lung disease, our goal in this study was to investigate lung viral load and various immune correlates of protection at the lung mucosal surfaces and its parenchyma in vaccinated heterologous prrsv-challenged pigs. our results indicated that out of five different vaccine-adjuvant formulations, the combination of np-kag and unentrapped m. tb wcl significantly cleared detectable replicating infective prrsv with a tenfold reduction in viral rna load in the lungs, associated with substantially reduced gross and microscopic lung pathology. immunologically, strong humoral (enhanced virus neutralization titers by high avidity antibodies) and cell-mediated immune responses (augmented population of interferon-γ secreting cd4(+) and cd8(+) lymphocytes and reduced secretion of immunosuppressive cytokines) in the lungs were observed. in conclusion, combination of np-kag and soluble m. tb wcl elicits broadly cross-protective anti-prrsv immunity in the pig respiratory system. porcine reproductive and respiratory syndrome (prrs) is an economically devastating disease in pigs causing an estimated direct loss of greater than $664 million annually to the us pork industry. 1 prrs is caused by prrs virus (prrsv), an enveloped positive-sense rna virus belongs to the family arteriviridae. there are broadly two distinct prrsv genotypes, the european (type i) and the north american (type ii), dovepress dovepress 1520 binjawadagi et al which possess a wide range of intra-and intergenotypic, genetic, and antigenic diversity. 2 therefore, developing preventive measures to control prrs outbreaks has been a challenge to the global swine industry. though both modified live virus (mlv) and inactivated prrsv vaccines have been in use since 1994, control of disease outbreaks has remained unsuccessful. live virus vaccines are successful in reducing the clinical disease, but are invariably implicated in spreading the mutated viruses to susceptible pigs. 3 in contrast, available inactivated prrsv vaccines are safe, but they have failed to elicit protective immunity even against homologous infections. 4 in addition, killed vaccine antigens do not undergo intracellular antigen presentation pathways to induce a strong cytotoxic t-cell (ctl) response, which is necessary for clearance of intracellular pathogens like viruses. [5] [6] [7] thus, research aimed at developing better cross-protective inactivated prrsv vaccines is warranted. therefore, several innovative strategies should be adopted to strengthen potency and efficacy of inactivated/killed prrsv vaccine antigens (kag), with respect to suitable methods of viral inactivation and purification, use of potent adjuvants, route, and efficient delivery of vaccine to protect ags from rapid enzymatic degradation in the body. since prrsv infects primarily the pig respiratory tract and the target cells are lung alveolar and interstitial macrophages, 8 induction of strong local mucosal immunity in the respiratory tract is important. the intranasal route of delivery of vaccines to control primary respiratory infections has shown great promise in induction of protective mucosal (ie, local) as well as systemic immunity. 5, 9, 10 poly(lactide-co-glycolide) (plga) is a synthetic biodegradable polymer used successfully in particulate delivery of inactivated vaccines. [11] [12] [13] the adjuvant mycobacterium tuberculosis whole cell lysate (m. tb wcl) was shown to augment immunogenicity of both live prrsv vaccine and plga-nanoparticles entrapped with killed prrsv antigens (np-kag) without causing any side effects in pigs [14] [15] [16] [17] and with other vaccines in rodents, guinea pigs and rabbits. 18, 19 unlike complete freund's adjuvant (cfa), m. tb wcl is free from water-insoluble toxic cell wall components of the bacterium, 20, 21 and it is endotoxin free and contains only water-soluble components. 19 therefore, unlike cfa, m. tb wcl does not cause any toxicity or granulomatous lesions at the site of inoculation. 19 previously, we have shown that a single dose of prrsv kag-entrapped in plga (50:50) nanoparticle (np-kag) elicits both mucosal and systemic immune responses. 22, 23 recently, np-kag coadministered intranasally twice with m. tb wcl induced cross-protective anti-prrsv immune response in blood to a challenged heterologous prrsv, associated with a significant reduction in viremia. 14 in this report, we made use of various types of the lung samples of that recent study 14 to evaluate viral load and local mucosal immunity both at airway surfaces and in the lung parenchyma, and also microscopic lung histopathology in vaccinated, heterologous prrsv-challenged pigs. killed prrsv vaccine antigens (kag) were prepared as described earlier. 22 briefly, north american prototype prrsv strain vr2332 24 was grown in marc 145 cells, freeze-thawed three times, and the harvested cell culture supernatant was subjected for clarification followed by ultracentrifugation to pellet the virus. resuspended pellet in sterile phosphate-buffered saline (pbs) was subjected to ultraviolet inactivation (254 nm for 1 hour) to prepare kag for use in vaccine preparation. for restimulation experiments, similarly prepared kag of the challenge virus, prrsv strain mn184, 24 was used. preparation of whole cell lysate of m. tb m. tb was grown in agar medium and wcl was prepared as described previously. 25 briefly, h37rv strain of m. tb was grown in oleic acid-albumine-dextrose-catalase enriched 7h11 (difco) agar plates (becton, dickinson and company, franklin lakes, nj, usa). the bacterial cells were harvested by centrifugation of colony scrapings at 3000 ×g and washed with pbs (ph 7.4). live bacterial cells were suspended [2 g (wet weight)/ml] in pbs containing 8 mm ethylenediaminetetraacetic acid (edta) (becton, dickinson and company), proteinase inhibitors (emd millipore, billerica, ma, usa), dnase and rnase (sigma-aldrich, st louis, mo, usa), and the bacterial cell wall was disrupted by bead beater until .90% cell breakage was obtained (confirmed by acid fast staining). the cell lysate was centrifuged at 3000 ×g to pellet unbroken cells and insoluble broken cell wall components. the clear supernatant-containing water soluble fraction of the bacterium was harvested and sterilized through 0.2 µm low protein binding membrane filter. further, endotoxin levels in every batch of m. tb wcl was confirmed to be less than the acceptable levels (,0.002 µg/mg conventional large white-duroc crossbred 3-4 weeks old weaned pigs were procured from a swine herd seronegative for prrsv, porcine respiratory coro navirus, transmissible gastroenteritis virus, and porcine circo virus 2-specific antibodies. a total of 30 pigs were randomly divided into one of the ten groups (n=3 pigs/group) and vaccinated with the indicated vaccine formulation, intranasally (2 ml/pig), twice at 2-week intervals ( table 1 ). all the vaccinated pigs were intranasally challenged on postvaccination day 28 with a virulent heterologous north american prrsv (type ii) strain mn184 (5 × 10 5 median tissue culture infective dose [tcid 50 ]/pig). 24 adjuvant and vaccine were entrapped separately and combined before administering to pigs, and the dose of adjuvant (1 mg/dose/pig) and kag were either 100 (low dose) or 500 (high dose) µg/dose/pig of semipurified viral protein containing ∼ 0.5 × 10 6 or 2.5 × 10 6 tcid 50 of killed prrsv, respectively, either entrapped in nps or unentrapped. the vaccine and adjuvant doses were tested to be efficacious in pigs earlier. 16, 22 pigs were monitored daily for the respiratory symptoms, and rectal temperatures and body weights were recorded every third day postchallenge (pc); animals were euthanized on day pc15 as per the approved protocol of the institutional animal care and use committee, the ohio state university, and indicated samples were collected during the necropsy. the lungs were examined for gross lesions in all the lobes. the lung samples collected from the right cranial lobe were fixed in 10% neutral buffered formalin and sections (5 µm) were made and stained with hematoxylin and eosin as described previously. 26 the slides were examined by an unbiased certified veterinary pathologist to score prrsvinduced inflammation. collection of bronchoalveolar lavage fluid, preparation of lung homogenate, and isolation of lung mononuclear cells during necropsy, the lungs were harvested and the bronchoalveolar lavage (bal) fluid was collected by washing the airways using 25-30 ml of cold pbs containing antibiotics and edta; the harvested fluid was centrifuged at 2,851 ×g for 15 minutes at 4°c and the clarified bal fluid was aliquoted and stored at -70°c. the lung homogenate was prepared as described previously. 27 briefly, 1 gram of lung tissue from the right cranial lobe of every pig was collected in 5 ml ice-cold dulbecco's modified eagle's medium and minced and homogenized using a stomacher 400 laboratory blender (seward limited, worthing, west sussex, uk) for 10 minutes, and the clarified supernatant (lung homogenate/lung lysate) was aliquoted and stored at -70°c. the lung mononuclear cells (lmncs) from the individual pig lungs were isolated by treating the perfused and minced lung tissue using collagenase and dnase as described previously. 22 estimation of total immunoglobulin (ig) total isotype specific pig ig levels were estimated by elisa as described previously, 28 with a few modifications. briefly, 96-well enzyme-linked immunosorbent assay (elisa) plates were coated with pretitrated dilution of (1:1,000) of goat the assay was performed as previously described. 29 avidity of the prrsv-specific antibodies in the lungs prrsv-specific antibody avidity was determined as described previously, 31,32 with a few modifications. briefly, bal fluid (1:1) and lung homogenate (1:10) samples were added to prrsv-ag-coated and -blocked plates. after 2 hours incubation of test samples at room temperature, washed plates (4×) were treated with serially twofold diluted ammonium thiocyanate (nh 4 cn) (100 µl/well) solution at 5 to 0.313 mol concentration and incubated at room temperature for 10 minutes. the plates were washed (4×) and the remaining steps of elisa carried out as described above. for calculation purpose, od value of the test sample from nh 4 cn-untreated (0 m) well was considered as 100% absorbance (contributed by prrsvspecific antigen-antibody interaction), and the od value of a test sample at different molar concentration of nh 4 cn was used to calculate the percent remained antigen-antibody interaction compared to its 100% absorbance value. estimation of prrsv-specific igg1 and igg2 antibody subtypes total prrsv-specific igg1 and igg2 subtypes in the lung homogenate samples were analyzed as described previously, 33 with a few modifications. briefly, kag-coated plates were blocked and a serial tenfold diluted lung homogenate was added; mouse antipig igg1 or igg2 (abd serotec, raleigh, nc, usa) (1:250 dilution) secondary antibody was added to detect virus-specific igg1or igg2 subtype, respectively. further, the washed plate was treated with goat antimouse igg-hrp (sigma-aldrich) (1:10,000 dilution). 34 the reaction was developed using tmb (3,3',5,5'-tetramethylbenzidine) substrate (kpl, gaithersburg, md, usa), stopped using 1 m phosphoric acid, and the plates were read at od 450nm . for calculation of the prrsv-specific igg1 and igg2 antibody levels of test samples, the od values obtained at 1:500 dilution was considered. determination of prrsv-specific inter feron gamma (ifn-γ ) secreting cells by enzyme-linked immunospot (elispot) assay the elispot assay was performed as described previously. 35 adjuvanted plga nanoparticle prrsv vaccine immunity in the pig lungs lated were included as positive and negative controls, respectively. lung homogenates were analyzed for pig cytokines, th1 (ifn-γ and interleukin [il-12]), proinflammatory (il-6), and immunosuppressive (il-10 and transforming growth factor beta [tgf-β]) by elisa as described previously. 15 flow-cytometric analyses the phenotypes and frequencies of lymphoid and myeloid cells populations from 50,000 events of immunostained lmncs were determined by flow cytometry as described previously. 23 for intracellular ifn-γ staining, monensin (golgiplug, bd biosciences) was added during the last 6 hours of the 48-hour incubation of lmncs that were unstimulated or stimulated with prrsv mn184 kag as described above. lmncs were first immunostained using pig lymphocyte-specific monoclonal antibodies (cd3ε, cd4α, cd8α, cd56, and tcr1n4) conjugated with different fluorochromes. cells were fixed with 1% paraformaldehyde and permeabilized with a cell-permeabilization buffer (85.9% deionized water, 11% pbs with no ca or mg, 3% formaldehyde solution, and 0.1% saponin) overnight at 4°c. cells were washed and stained with a fluorochrome-conjugated antipig ifn-γ or its isotype control monoclonal antibodies (bd biosciences) in 0.1% saponin containing fluorescence activated cell sorting (facs) buffer. immunostained lmncs were acquired using a facs aria ii (bd biosciences) flow cytometer and analyzed using the flowjo software (treestar inc., ashland, or, usa). all the specific immune cell frequencies were presented as the percent of total lymphocytes or myeloid cells. determination of prrsv load, virus-neutralizing antibody titer, and rna copies prrsv titer and virus-neutralizing (vn) titer in bal fluid and lung homogenate samples were analyzed by indirect immunofluorescence assay as previously described. 15 extracted rna was reverse transcribed into complimentary deoxyribonucleic acid (cdna) using quantitect reverse transcription kit (qiagen, venlo, the netherlands). the cdna was subjected to quantitative real-time polymerase chain reaction (qpcr) using primers against prrsv orf6 in perfecta sybr green fast mix (quanta biosciences, gaithersburg, md, usa) with forward primer (gataac-cacgcatttgtcgtc) and reverse primer (tgccgtt-gttatttggcata). standard curve was generated using serial tenfold dilution of prrsv vr2332 stock virus starting at 10 7 tcid 50 per µl for viral rna quantification. 39 the data were expressed as the mean ± standard error of mean of three pigs. statistical analyses were performed by one-way analysis of variance followed by tukey's multiple comparison test (or unpaired t-test for figure binjawadagi et al cated that sham or entrapped np particles were spherical with a smooth surface. dynamic light scattering of nps indicated the mean diameter ± standard deviation was 480±53 nm for sham, 520±41 nm for np-kag, and 650±98 nm for np-m. tb wcl, respectively. however, all nps possessed a uniform surface electrostatic potential of -26 mv. 14 other in vitro studies revealed that the entrapped antigen was pulse-released over a period of several weeks in normal physiological conditions, and was efficiently uptaken by porcine alveolar macrophages. a detailed physical and biological characterization of np-kag vaccine used in this study was published recently. 14 in the serum of all the virus-challenged pigs (groups 2 to 6), irrespective of vaccination history at 2 weeks postchallenge, a twofold to threefold increase in total igg amount was observed (data not shown). the total igm and iga amounts in the lung homogenate were comparable in most of the prrsvchallenged pig groups ( figure 1a and c). however, the levels of igg (both in lung homogenate and bal fluid) ( figure 1b and e), and igm and iga (in bal fluid) ( figure 1d and f) in pig groups 2 to 5 were four-to fivefold higher compared to mock enhanced prrsv-specific antibody response in adjuvanted np-kag-vaccinated pigs prrsv-specific iga response was significantly higher both in the bal fluid and lung homogenate of both low (100 µg/pig) and high (500 µg/pig) doses of adjuvanted np-kag-vaccinated pigs, compared to most of the vaccine trial groups ( figure 1g and h). in contrast, levels of virusspecific igg were comparable in the bal fluid of all the virus-challenged pig groups ( figure 1i) . surprisingly, in the lung homogenate, significantly higher levels of virusspecific igg was observed in group 4 and 6 pigs vaccinated with either dose ( figure 1j ). this suggests that in adjuvanted np-kag-vaccinated pigs (group 6), virus-specific iga is the major antibody isotype in the airway surfaces (bal fluid), while both iga and igg isotypes appear to play important roles in the lung parenchyma. the binding strength of heterogeneous antibodies to their cognate ag is defined as avidity. 32 in adjuvanted np-kagvaccinated pigs, increased avidity of virus-specific iga was detected in both bal fluid (both low and high doses) and lung homogenate (only low dose) samples compared to other tested groups (figure 2a-c) . interestingly, the virus-specific iga avidity in lung homogenate of pigs receiving the highdose vaccine was comparable among all the tested pig groups ( figure 2d ). comparable levels of avidity of prrsv-specific igg antibody response was observed in lung homogenate of all the tested groups (data not shown). these data suggested that enhanced avidity of prrsv-specific iga antibody persisted for a relatively longer period at the airway surfaces of adjuvanted np-kag-vaccinated pigs. balanced th1 and th2 antibody responses in adjuvanted np-kag-vaccinated pigs t helper type (th)1-or th2-biased immune response is measured by quantifying antigen-specific igg antibody subtypes. in case of pigs, higher levels of igg2 and igg1 indicate th1and th2-biased responses, respectively. 33 our results indicated that in group 6 pigs, comparable levels of both igg2 and igg1 subtypes were secreted in lung homogenate ( figure 3a and c). the ratio of igg1:igg2, if greater or less than one, indicates th2-or th1-biased response, respectively. in the lungs of adjuvanted np-kag-vaccinated pigs at day pc15 a balanced th1 and th2 response (ratio close to one) was detected ( figure 3b and d). in pig groups 3 and 5, although the ratio was close to one, the detected amount of virus-specific igg1 and igg2 subtypes were low ( figure 3a-d) . enhanced prrsv-neutralizing antibody response in adjuvanted np-kag-vaccinated pigs except group 1 (mock), all the other pigs were challenged using a heterologous prrsv (strain mn184), which is genetically highly divergent (∼15%) compared to the vaccine strain vr2332. 24 prrsv-specific vn titers were analyzed both in the bal fluid and lung homogenate samples against mn184 strain, and against another variant type 2 strain, prrsv 1-4-4 (accession #10-16734), is genetically distinct from both mn184 and vr2332 strains. 37 in addition, vn titers were also analyzed against an antigenically highly divergent (∼40%) heterogenotypic (type 1) prrsv strain, sd03-15 ( figure 3e -j). 38 in group 6 pigs, the lung vn titers against mn184 were significantly higher with mean titers of 16 and 27 with low and high vaccine doses, respectively, compared to other tested groups ( figure 3e and f). the vn titers against both prrsv 1-4-4 and sd03-15 strains were significantly greater in group 6 pigs receiving the high-dose vaccine compared to group 5 and group 3, respectively ( figure 3h and j). the kag and soluble wcl-vaccinated pigs (group 4) also had significantly higher vn titer against only prrsv 1-4-4 strain compared to group 5 animals ( figure 3h ). these results indicated that soluble m. tb wcl-adjuvanted np-kag vaccine elicits broadly cross-reactive vn titers. the vn titers observed in bal fluid were low (data not shown). a standard reference for measuring cmi response against prrsv is by determining the frequency of virus-specific iscs by elispot assay. 40 in low-dose vaccinated group 6 pigs, significantly increased iscs in lmncs was detected compared to three other tested groups ( figure 4a ) and compared to all the tested groups in the high-dose category ( figure 4g ). the quantity of ifn-γ detected in the lung homogenate was significantly higher in group 6 pigs compared to group 5 (low dose) and groups 4 and 5 (high dose) categories ( figure 4b and h) . another important th1 cytokine, il-12, was not significantly modulated among the tested pig groups in the high vaccine dose category ( figure 4j ), but in low-dose groups 4 and 6, significantly higher levels of il-12 were detected compared to groups 2, 3, and 5 ( figure 4d ). one of the important proinflammatory cytokines, il-6, was significantly reduced in the lungs of all the vaccine trial groups compared to mock-challenged animals ( figure 4c and i). cytokines il-10 and tgf-β are immunosuppressive in nature, and they play a vital role in prrsv pathogenesis. 15, 40 the quantity of tgf-β was significantly reduced in group 6 pigs compared to group 2 ( figure 4e and k) , and the quantity of il-10 among all the tested vaccine trial pig groups was comparable ( figure 4f and l). figure 5a and b) . the frequencies of intracellular ifn-γ + cells in lmncs either unstimulated or stimulated with mn184 ags identified the virus-specific memory lymphocyte response. significantly increased prrsvspecific recall ifn-γ response was detected both in cd4 + and cd8 + lymphocyte subsets of group 6 pigs vaccinated with a high dose of vaccine ( figure 5c and d) . these data suggest that both t-helper and ctls were potentially primed in the lungs of only group 6 pigs. when prrsv-specific ifn-γ secreting lymphocyte subsets in only restimulated cells were compared among different vaccine trial groups, only in group 6 animals (both vaccines doses) significantly increased cd4 + cd8 -ifn-γ + cells compared to group 2 animals ( figure 5f and n) , while cd4 -cd8 + ifn-γ + cell frequency was significantly enhanced in group 6 pigs were present compared to groups 2, 3, and 5 with low vaccine dose and groups 2 to 5 with high vaccine dose ( figure 5g and o). similarly, cd4 + cd8 + ifn-γ + and ifn-γ + γδ t cells were significantly higher in group 6 pigs compared to pigs in groups 2, 3, and 5 ( figure 5h -i, p-q). an increased frequency of activated γδ t cells was detected in group 6 pigs compared to other groups (table 2a and b) . although there was no significant difference in total natural killer (nk) (cd56 + ) cell frequency ( figure 5j and r) , an increase in ifn-γ + nk cell frequency was significant in group 6 pigs compared to other tested groups ( figure 5k and s) . in addition, macrophage (cd172 + cd163 + slaii + ) and dendritic cells (cd172 + cd11c + slaii + ) rich apc populations were significantly higher in group 2 and 6 pigs in the high-dose vaccine category compared to group 5 animals ( figure 5t and u); in the low-dose category, though a similar trend was detected, the data was not statistically significant ( figure 5l and m). in the bal fluid of pig groups 4 and 6 (low vaccine dose), detectable replicating prrsv was relatively reduced (but not significant) compared to other vaccine groups ( figure 6a ); in the same pig groups with the high vaccine dose, detectable replicating virus was absent, and the data was statistically significant compared to pig group 2 ( figure 6b ). in the lung homogenate of group 6 pigs receiving the low-dose vaccine, detectable replicating virus load was significantly reduced compared to mock-challenged pigs ( figure 6e) ; detectable virus was absent in the high-dose group 6 pigs, and the data was statistically significant compared to pig groups 2 and 3 ( figure 6f ). further, prrsv rna copy numbers in bal fluid and lung homogenate were quantified by qpcr. in both the lung sample adjuvanted plga nanoparticle prrsv vaccine immunity in the pig lungs types of group 4 and 6 pigs in the low-dose vaccine category, reduction (but not significant) in viral rna copy numbers was observed compared to other vaccine trial groups ( figure 6c and g); and in the same pig groups vaccinated with a high dose, a significant reduction (5-10 fold) in rna load was detected compared to group 2 animals ( figure 6d and h). gross lung lesions revealed marked consolidation in pig groups 2, 3, and 5, while the group 6 pig lungs were comparable to mock animals and the group 4 pig lungs were in between mock and the group 6 animals. microscopic lmncs isolated on the day of necropsy were unstimulated or stimulated with killed prrsv mn184 ags and immunostained using a combination of indicated pig lymphocytespecific cell surface markers followed by intracellular ifn-γ and analyzed by flow cytometry. representative histograms showing stimulated total lymphocytes in lmncs with intracellular ifn-γ + (a and b) . the dotted line: isotype control and solid line: ifn-γ + -specific staining. unstimulated (clear bars) or stimulated (black bars) lmncs with killed prrsv mn184 ags were analyzed for total ifn-γ + cd4-, cd8-, and cd56-expressing lymphocytes (c-e). only stimulated lmncs were compared for indicated ifn-γ + lymphocyte subsets: cd4 + cd8 -ifn-γ + (f and n) ; cd4 -cd8 + ifn-γ + (g and o); cd4 + cd8 + ifn-γ + (h and p); γδ + ifn-γ + (i and q); and total nk (cd56 + ) (j and r); cd56 + ifn-γ + cells (k and s). also immunostained for potential apcs; macrophage-rich (cd172 + cd163 + sla-ii + ) (l and t) and dendritic cell-rich (cd172 + cd11c + sla-ii + ) (m and u) populations. each bar indicates the average frequency of indicated cells from three pigs ± standard error of mean. asterisk indicates statistically significant (p,0.05) difference between the two indicated pig groups. the unpaired t-test was applied to compare the data of only (c-e); for the other data, one-way anova followed by tukey's t-test was used. figure 6i ii, iii, and v). these data were consistent with the gross lung lesions, especially in pig groups 2 and 3; also, these pig groups had irregular fever with reduced feed intake during first week postchallenge. in contrast, the absence of any clinical prrs disease and gross lung lesions in group 6 pigs was associated with the absence of detectable microscopic lung pathology, and the lung architecture was comparable to mock pigs ( figure 6i vi) . the other pig group which had moderately reduced lung lesions was group 4 (kag + m. tb wcl) ( figure 6i iv), suggesting the adjuvant mediated protective immune response in pigs. similar but severe lung pathology was observed in pigs receiving the low vaccine dose in groups 2, 3, and 5 (data not shown). potent mucosal vaccines against pathogens which predominantly cause disease at mucosal sites have been proven efficacious, with vaccines against influenza, parainfluenza, adjuvanted plga nanoparticle prrsv vaccine immunity in the pig lungs respiratory syncytial virus, rotavirus, and hiv/siv. [41] [42] [43] [44] [45] since prrsv primarily infects the lungs of pigs, its effective control appears to be possible by induction of strong respiratory mucosal immunity. thus, development of a novel prrsv mucosal vaccine is warranted. further, among the mucosal routes, intranasal delivery of np-based vaccine elicits a higher and longer duration of igg and iga antibody responses compared to rectal, oral, or intramuscular routes. 46 in the lungs, prrsv infects both alveolar and interstitial macrophages, 47 and greater than 90% of bal cells are macrophages. 48 we investigated anti-prrsv response both in bal cells and lmncs. cd11c + apcs are richly present in the lungs in both bal cells and lmncs, but they differ in their antigen presentation potential. apcs present in the lung mucosal surfaces (represented by bal cells) activate only antigen-primed t-cells, while apcs in the lung parenchyma (represented by lmncs) activate both naïve and antigen-primed t-cells. 49 therefore, our study comprehensively investigated overall immune responses and viral load in the lungs of pigs vaccinated and challenged through the intranasal route. a couple of earlier studies showed hypergammaglobulinemia in the serum of prrsv-infected pigs, indicated by a four-to fivefold increase in total igg levels at 2-3 weeks postinfection. 50, 51 consistent with that observation, in our study, an approximately fourfold increase in total igg amounts was observed both in the serum and lung samples (bal fluid and lung homogenate); however, in the lung homogenate of adjuvanted np-kag-vaccinated pigs, the total igg levels were only twofold more compared to mock animals. these data suggest that adjuvanted np-kag has a strong positive influence on humoral response, further indicated by a significant increase in production of anti-prrsv antibodies and virusspecific high avidity vn antibody titers. inactivated vaccines are safe, but suitable adjuvant and delivery system are critical to boost their efficacy. several unsuccessful attempts were made to develop protective killed prrsv vaccines. 29, 52 a recent study using uv-or bei-inactivated prrsv (1 × 10 8 tcid 50 per dose) coadministered with either freund's incomplete or suvaxyn oil/water adjuvant elicited the vn titer of greater than 16 associated with partial clearance of homologous viral challenge. 53 in that study, cmi response was not investigated. plga is the food and drug administration (fda)-approved agent, and plga-based intranasal delivery of prrsv vaccine was found safe in pigs. 14, 22, 23 one of the strategies to augment uptake of particulate antigens by apcs is by increasing its surface hydrophilic nature of the particles by coating with a nonionic surfactant, polaxamer 188. 54, 55 efficient th1-immunity-inducing adjuvants are critical to promote a strong cmi response to subunit and inactivated virus vaccines. 56 the water-soluble components of m. tb wcl, such as heat shock protein-70 57 and pro-glu/ppe, 58 are potent adjuvants. in addition, four other water-soluble components in m. tb wcl, such as short-and long-chain poly-peptidoglycans, acetylated peptidoglycans, and tetrasaccharide-heptapeptide, have potent adjuvant effects comparable to cfa in inducing production of antibodies in rabbits coadministered with an inactivated influenza virus vaccine. 19 plga-np vaccine coadministered with a potent adjuvant elicits a protective immune response. 59 we demonstrated potent adjuvant effects of m. tb wcl to prrs-mlv, [15] [16] [17] 60, 62 and also to np-kag, with no side effects. 14 our initial studies using a single dose of np-kag elicited partial cross-protective immune response in pigs. 22, 23 to potentiate the efficacy of np-kag vaccine, in a recent study np-kag was evaluated by coadministering intranasally twice with either entrapped or unentrapped m. tb wcl. our results suggested that combination of np-kag with unentrapped m. tb wcl significantly cleared the challenged heterologous virus from the circulation, supported with strong humoral and cmi responses in the blood. the enhanced t-and b-cell responses in adjuvanted np-kag-vaccinated pigs were attributed to concerted effects of both plga and m. tb wcl. 14 induction of strong local mucosal immunity and clearance of heterologous prrsv from vaccinated pig lungs is important for effective control of prrs. therefore, in this study we investigated the immune responses exclusively both at mucosal surfaces and parenchyma of pig lungs. our results indicated that adjuvanted np-kag (group 6 pigs) did potentiate anti-prrsv immune response in the lungs, as indicated by the following parameters: 1) increased prrsv-specific igg and iga response with enhanced antibody avidity and vn titers, and balanced th1 and th2 immune responses; 2) upregulated secretion of th1 (il-12 and ifn-γ) and downregulated immunosuppressive (tgf-β and il-10) cytokines; 3) enhanced frequency of iscs and ifn-γ producing cd4 + , cd8 + , cd4 + cd8 + t cells, γδ + t cells, and nk cells, and expanded frequency of apcs; and most importantly, 4) complete clearance of detectable replicating challenged heterologous prrsv and tenfold reduction in viral rna load in the lungs. further, the microscopic lung lesions ( figure 6i ) strongly supported the observed virus clearance and immunological responses. in our previous study, in pigs immunized with the adjuvanted np-kag, increased frequency of only cd4 -cd8 + ifn-γ + cells in restimulated pbmcs was detected, 14 but in stimulated lmncs, increased populations of both cd4 + cd8 -ifn-γ + and cd4 -cd8 + ifn-γ + cells was observed, perhaps indicating the induction of both t-helper and ctl memory responses in the lungs. like in pbmcs, increased but comparable frequency of other ifn-γ + lymphocyte subsets in both restimulated and unstimulated cells was observed in lmncs of group 6 pigs, suggesting that other t-effector and nk cells were actively secreting ifn-γ in the lungs to prrsv-challenge infection. due to lack of similar data on ifn-γ + lymphocytes in vaccinated pigs isolated prior to challenge, it is difficult to demarcate the vaccine-alone induced response. np-based delivery of vaccine facilitates affinity maturation and activation of b cells, leading to high avidity antibody production and also cmi response. 63, 64 avidity of prrsvspecific iga antibody isotype was significantly higher in the bal fluid of adjuvanted np-kag-vaccinated pigs. in the lung homogenates of low-dose (but not high-dose) adjuvanted np-kag-vaccinated pigs, high avidity iga response was observed; the reason for this discrepancy could be the time of lung sample collection. overall, our results suggested that virus-specific functional iga response at mucosal tissues (lungs) was enhanced in adjuvanted np-kag-vaccinated pigs. further, our results indicated that in the lung mucosal surfaces, iga isotype appears to play an important role, while in the lung parenchyma both iga and igg isotypes contribute to protective immune response. vn antibodies against putative neutralizing epitopes on prrsv gp5 and m glycoproteins play an important role in prrsv clearance. 65 in group 6 pigs, high levels of cross-reactive vn titers were detected against a challenged prrsv mn184 strain, another heterologous type ii viral strain, and most importantly-even against a highly variant heterogenotypic strain (prrsv sd03-15), confirming the broadly cross-protective nature of adjuvanted np-kag vaccine formulation. further, prrsv antibody avidity results were positively correlated with vn titers, in agreement with a previous report. 31 typically, killed vaccines elicit a predominantly th2 response, but np-based vaccines drive either a balanced or th1-biased response, 46 required for efficient clearance of virus. 66 in group 6 pigs, enhanced and balanced th1-th2 humoral and cmi responses were detected. a robust cmi response is essential for complete protection against prrsv. 17 a crucial th1 cytokine, ifn-γ, is produced by nk cells, γδ t cells, cd4 + and cd8 + t cells, and cd4 + cd8 + t cells. enhanced secretion of ifn-γ and the presence of increased frequencies of ifn-γ + memory cd4 + and cd8 + cells in the lungs of adjuvanted np-kag-vaccinated pigs have confirmed the additive effect of plga-mediated delivery and adjuvanticity of m. tb wcl. a possible mechanism of improved cmi response in group 6 pigs was mediated by cross-presentation of plga-entrapped ags to cd8 + t cells through mhc class i molecules, of dendritic cells and macrophages. 67 thus, our results were consistent with the previous reports on plga np-based vaccines, which elicited strong effector and memory cmi responses. 68, 69 in addition, increased levels of another important th1 cytokine, il-12, 70 was detected in the lungs of adjuvanted np-kag-vaccinated pigs, associated with reduced production of immunosuppressive cytokines, il-10 and tgf-β, which play a vital role in prrsv pathogenesis. 17 the γδ t cells are present in high frequency in pigs and are involved in both innate and adaptive immunity at mucosal tissues. 71 enhanced frequency of activated γδ t cells in the lungs of group 6 pigs was observed. significantly reduced production of il-6 in group 6 pigs indicated the absence of inflammatory response in the lungs of group 6 pigs. importance of immediate availability of unentrapped potent adjuvant to intranasally delivered np-kag vaccine was critical in our study, because inadequate immune response and incomplete viral clearance was observed in other vaccine formulations pigs received. plga-entrapped hepatitis b subunit vaccine coadministered with an encapsulated adjuvant failed to induce strong antibody response, 72 consistent with our results observed in group 5 pigs (np-kag + np-wcl). further, pigs vaccinated with both adjuvant and kag unentrapped formulation (group 4) exhibited th2-biased and weak ifn-γ response, associated with partial clearance of replicating prrsv. the presence of 5-10 fold fewer prrsv rna copies in the lungs of adjuvanted np-kag-vaccinated pigs and the absence of detectable replicating challenged virus is consistent with a previous report, wherein qpcr failed to differentiate infectious and noninfectious virus, and inactivated prrsv is relatively stable in the environment. 73 further, in samples with low levels of prrsv rna copies, cell culture method has failed to detect the replicating virus. 74 plga-based vaccine delivery is shown to dramatically reduce the required vaccine dose by up to 63 times. 69 plga-np-based vaccine delivery is getting global recognition for effective delivery of subunit/inactivated mucosal vaccines, because their size, contents, and cell targeting properties can be engineered. 59 our study has demonstrated that plga-based adjuvanted np-kag vaccine induced strong anti-prrsv immunity both systemically 14 and locally at the lungs. in conclusion, intranasal coadministration of plga-np-entrapped inactivated prrsv vaccine with a potent adjuvant has the potential to induce superior cross-protective international journal of nanomedicine 2014:9 submit your manuscript | www.dovepress.com adjuvanted plga nanoparticle prrsv vaccine immunity in the pig lungs immunity in pigs. future studies should aim to fractionate m. tb wcl to identify adjuvant components to np-kag and to identify alternate adjuvants to reduce the cost of vaccine formulation, and perform field trials to validate efficacy of this innovative vaccine delivery approach. our study not only establishes the utility of nanotechnology-based vaccines in large animals, it also envisages its potential application against important human respiratory pathogens. prrs costs industry $664 million annually porcine reproductive and respiratory syndrome virus productively infects monocyte-derived dendritic cells and compromises their antigen-presenting ability reversion of a live porcine reproductive and respiratory syndrome virus vaccine investigated by parallel mutations challenges for porcine reproductive and respiratory syndrome virus (prrsv) vaccinology nanotechnology solutions for mucosal immunization role of nanotechnology in pharmaceutical product development vaccine manufacturing: challenges and solutions immunological responses of swine to porcine reproductive and respiratory syndrome virus infection polymeric nano-and microparticle technologies for oral gene delivery is intranasal vaccination a feasible solution for tuberculosis? nanotechnology for the biologist biodegradable nanoparticles for drug and gene delivery to cells and tissue mucosal delivery of vaccines: role of mucoadhesive/biodegradable polymers adjuvanted poly(lactic-co-glycolic) acid nanoparticle-entrapped inactivated porcine reproductive and respiratory syndrome virus vaccine elicits cross-protective immune response in pigs cross-protective immunity to porcine reproductive and respiratory syndrome virus by intranasal delivery of a live virus vaccine with a potent adjuvant intranasal delivery of whole cell lysate of mycobacterium tuberculosis induces protective immune responses to a modified live porcine reproductive and respiratory syndrome virus vaccine in pigs mucosal vaccines to prevent porcine reproductive and respiratory syndrome: a new perspective interaction of heat shock proteins with peptides and antigen presenting cells: chaperoning of the innate and adaptive immune responses adjuvant and immunostimulating activities of water-soluble substances extracted from mycobacterium tuberculosis (var. hominis) symposium on relationship of structure of microorganisms to their immunological properties. ii. host-reactive properties of cell walls and protoplasm from mycobacteria the influence of components of m. tuberculosis and other mycobacteria upon antibody production to ovalbumin biodegradable nanoparticle-entrapped vaccine induces cross-protective immune response against a virulent heterologous respiratory viral infection in pigs plga nanoparticle entrapped killed porcine reproductive and respiratory syndrome virus vaccine helps in viral clearance in pigs effect of genotypic and biotypic differences among prrs viruses on the serologic assessment of pigs for virus infection site of inhibitory action of isoniazid in the synthesis of mycolic acids in mycobacterium tuberculosis altered pathogenesis of porcine respiratory coronavirus in pigs due to immunosuppressive effects of dexamethasone: implications for corticosteroid use in treatment of severe acute respiratory syndrome coronavirus porcine reproductive and respiratory syndrome virus-induced immunosuppression exacerbates the inflammatory response to porcine respiratory coronavirus in pigs measurement of immunoglobulin g, a and m concentrations in boar seminal plasma intranasal administration of cpg oligonucleotides induces mucosal and systemic type 1 immune responses and adjuvant activity to porcine reproductive and respiratory syndrome killed virus vaccine in piglets in vivo antigen-specific b-cell responses to porcine reproductive and respiratory syndrome virus infection maximal adjuvant activity of nasally delivered il-1α requires adjuvant-responsive cd11c(+) cells and does not correlate with adjuvant-induced in vivo cytokine production the assessment of antibody affinity distribution by thiocyanate elution: a simple dose-response approach protection of pigs against taenia solium cysticercosis using recombinant antigen or in combination with dna vaccine isolation and properties of a macromolecular, water-soluble, immuno-adjuvant fraction from the cell wall of mycobacterium smegmatis cytokine responses in gnotobiotic pigs after infection with virulent or attenuated human rotavirus detection and duration of porcine reproductive and respiratory syndrome virus in semen, serum, peripheral blood mononuclear cells, and tissues from yorkshire, hampshire, and landrace boars restriction fragment length polymorphism of porcine reproductive and respiratory syndrome viruses recovered from ontario farms heterogeneity in nsp2 of european-like porcine reproductive and respiratory syndrome viruses isolated in the united states porcine reproductive and respiratory syndrome virus induces pronounced immune modulatory responses at mucosal tissues in the parental vaccine strain vr2332 infected pigs certainties, doubts and hypotheses in porcine reproductive and respiratory syndrome virus immunobiology mechanism of nkt cell activation by intranasal coadministration of alpha-galactosylceramide, which can induce cross-protection against influenza viruses combined nkt cell activation and influenza virus vaccination boosts memory ctl generation and protective immunity mucosal immune response to poliovirus vaccines in childhood mucosal immunization of rhesus monkeys against respiratory syncytial virus subgroups a and b and human parainfluenza virus type 3 by using a live cdna-derived vaccine based on a host range-attenuated bovine parainfluenza virus type 3 vector backbone nasal immunization of nonhuman primates with simian immunodeficiency virus p55gag and cholera toxin adjuvant induces th1/th2 help for virus-specific immune responses in reproductive tissues enhanced mucosal and systemic immune response with intranasal immunization of mice with hiv peptides entrapped in plg microparticles in combination with ulex europaeus-i lectin as m cell target chronological immunohistochemical detection and localization of porcine reproductive and respiratory syndrome virus in gnotobiotic pigs cellular variables in bronchoalveolar lavage fluids (balf) in selected healthy pigs cd11c+ antigen presenting cells from the alveolar space, lung parenchyma and spleen differ in their phenotype and capabilities to activate naïve and antigen-primed t cells polyclonal hypergammaglobulinemia and formation of hydrophobic immune complexes in porcine reproductive and respiratory syndrome virus-infected and uninfected pigs porcine reproductive and respiratory syndrome virus subverts repertoire development by proliferation of germline-encoded b cells of all isotypes bearing hydrophobic heavy chain cdr3 the assessment of efficacy of porcine reproductive respiratory syndrome virus inactivated vaccine based on the viral quantity and inactivation methods development of an experimental inactivated prrsv vaccine that induces virus-neutralizing antibodies intranasal m cell uptake of nanoparticles is independently influenced by targeting ligands and buffer ionic strength influence of the surfactant concentration on the body distribution of nanoparticles adjuvants and delivery systems in veterinary vaccinology: current state and future developments the adjuvant effects of mycobacterium tuberculosis heat shock protein 70 result from the rapid and prolonged activation of antigen-specific cd8+ t cells in vivo pe_pgrs antigens of mycobacterium tuberculosis induce maturation and activation of human dendritic cells recent progress in mucosal vaccine development: potential and limitations intranasal delivery of an adjuvanted modified live porcine reproductive and respiratory syndrome virus vaccine reduces ros production evaluation of immune responses to porcine reproductive and respiratory syndrome virus in pigs during early stage of infection under farm conditions mycobacterium tuberculosis whole cell lysate enhances proliferation of cd8 positive lymphocytes and nitric oxide secretion in the lungs of live porcine respiratory and reproductive syndrome virus vaccinated pigs towards tailored vaccine delivery: needs, challenges and perspectives vaccine delivery: a matter of size, geometry, kinetics and molecular patterns role of neutralizing antibodies in prrsv protective immunity type 1/type 2 immunity in infectious diseases international-journal-of-nanomedicine-journal the international journal of nanomedicine is an international, peerreviewed journal focusing on the application of nanotechnology in diagnostics, therapeutics, and drug delivery systems throughout the biomedical field role of sustained antigen release from nanoparticle vaccines in shaping the t cell memory phenotype plga nanoparticle-mediated delivery of tumor antigenic peptides elicits effective immune responses debugging how bacteria manipulate the immune response gammadelta lymphocyte response to porcine reproductive and respiratory syndrome virus enhancement of t helper type 1 immune responses against hepatitis b virus core antigen by plga nanoparticle vaccine delivery porcine reproductive and respiratory syndrome virus (porcine arterivirus) porcine reproductive and respiratory syndrome virus: a persistent infection the authors report no conflicts of interest in this work. international journal of nanomedicine 2014:9 submit your manuscript | www.dovepress.com key: cord-013280-kczj24se authors: yang, bo; zhang, xiaohui; zhang, dajun; hou, jing; xu, guowei; sheng, chaochao; choudhury, sk mohiuddin; zhu, zixiang; li, dan; zhang, keshan; zheng, haixue; liu, xiangtao title: molecular mechanisms of immune escape for foot-and-mouth disease virus date: 2020-09-04 journal: pathogens doi: 10.3390/pathogens9090729 sha: doc_id: 13280 cord_uid: kczj24se foot-and-mouth disease virus (fmdv) causes a highly contagious vesicular disease in cloven-hoofed livestock that results in severe consequences for international trade, posing a great economic threat to agriculture. the fmdv infection antagonizes the host immune responses via different signaling pathways to achieve immune escape. strategies to escape the cell immune system are key to effective infection and pathogenesis. this review is focused on summarizing the recent advances to understand how the proteins encoded by fmdv antagonize the host innate and adaptive immune responses. foot-and-mouth disease (fmd) is an acute and highly contagious disease affecting the cloven-hoofed animals, such as pigs and cattle. the pathogen that causes fmd is known as fmd virus (fmdv), a single-stranded positive-sense rna virus that is classified into the genus aphthovirus in the family picornaviridae [1] [2] [3] . the pathogen causes vesicular disease of mouth and feet in susceptible animals [4] . the high mutation rate of the genome of fmdv and the rapid proliferation has led to the rapid evolution of the virus and the formation of seven main serotypes [5] [6] [7] . the antigenic diversity among the serotypes poses challenges to the research of efficient and cross-protective vaccines [8] . the genome of fmdv contains an open reading frame (orf) that encodes a polyprotein precursor, and it is cleaved into four structural proteins and 10 non-structural proteins by viral autoproteases and host protease [2, 9] (figure 1 ). upon infection of the host, a virus will face the attack from the host's immune response. in the long-term battle with the host immune response, the virus has evolved and developed a series of immune escape mechanisms to overcome the killing and inhibition from the host immune system. the mechanism of virus immune escape can be divided into three categories: (1) enable the virus to avoid the recognition of humoral immune response; (2) interfere with the function of cellular immune response; (3) interfere with the host's immune response to the virus [10] . all these strategies would be exploited by the virus for replication and spreading to other hosts. as a highly contagious and fast-spreading virus, fmdv has multiple ways to evade the killing by the immune system [11] , which makes it difficult for controlling the virus. viral capsid protein vp1 and leading protein l pro can inhibit the production of interferon (ifn) and innate immune response by interacting with soluble resistance-related calcium-binding protein (sorcin) or host transcription factor adnp [12, 13] . recently, new mechanisms and functions of fmdv proteins inhibiting innate immunity have been discovered. ddx56 (a kind of rna helicase), participate in rna metabolism and ribosome synthesis is reported to involve in this new mechanism. the interaction between fmdv 3a and ddx56 suppresses the host innate immunity by reducing the phosphorylation of irf3 [14] . in addition, nucleotide-binding oligomerization domain 2 (nod2), a member of the nucleotide-binding oligomerization domain-like receptor (nlr) family [15] , activates the nf-κb and ifn-β signaling pathways during fmdv infection and inhibits the replication of fmdv in infected cells [16] . fmdv 2b, 2c, and 3c pro inhibit the expression of nod2 protein, which antagonizes the antiviral response [16] . reportedly, multiple structural and non-structural proteins of fmdv escape the killing of the host immune system. this review summrized the molecular mechanisms of immune evasion caused by fmdv proteins. the present study aimed to fill the gaps of knowledge on fmdv immune evasion mechanism, providing the basis for the prevention and control strategies for fmdv. pathogens 2020, 9, x for peer review 2 of 25 figure 1 . schematic of the genome and polypeptide processing of fmdv [3, 9] . the fmdv genome contains an orf of about 7 kbp, indicated by the shaded rectangle. each region within the orf rectangle represents a single protein. the flank of orf is a long 5' untranslated region (5'-utr) and a short 3'-utr. 3b covalently binds to the 5'-end. upon infection of the host, a virus will face the attack from the host's immune response. in the long-term battle with the host immune response, the virus has evolved and developed a series of immune escape mechanisms to overcome the killing and inhibition from the host immune system. the mechanism of virus immune escape can be divided into three categories: (1) enable the virus to avoid the recognition of humoral immune response; (2) interfere with the function of cellular immune response; (3) interfere with the host's immune response to the virus [10] . all these strategies would be exploited by the virus for replication and spreading to other hosts. as a highly contagious and fast-spreading virus, fmdv has multiple ways to evade the killing by the immune system [11] , which makes it difficult for controlling the virus. viral capsid protein vp1 and leading protein l pro can inhibit the production of interferon (ifn) and innate immune response by interacting with soluble resistance-related calcium-binding protein (sorcin) or host transcription factor adnp [12, 13] . recently, new mechanisms and functions of fmdv proteins inhibiting innate immunity have been discovered. ddx56 (a kind of rna helicase), participate in rna metabolism and ribosome synthesis is reported to involve in this new mechanism. the interaction between fmdv 3a and ddx56 suppresses the host innate immunity by reducing the phosphorylation of irf3 [14] . in addition, nucleotide-binding oligomerization domain 2 (nod2), a figure 1 . schematic of the genome and polypeptide processing of fmdv [3, 9] . the fmdv genome contains an orf of about 7 kbp, indicated by the shaded rectangle. each region within the orf rectangle represents a single protein. the flank of orf is a long 5 untranslated region (5 -utr) and a short 3 -utr. 3b covalently binds to the 5 -end. the p1 structural protein of fmdv was cleaved into three main viral structural proteins vp0, vp1 and vp3 by 3c pro protease during the later translation and modification. vp0 protein was further cleaved into vp4 and vp2 proteins by protease 3c. interestingly, the vp0 protein is a cleavage precursor of vp2 and vp4 [2] . the last step in the production of mature virions is the cleavage of 1ab (vp0), which converts 85 residues of the n-terminal into vp4 and the remaining into vp2, although some copies of vp0 may be retained in the intact virions [17] . previous studies reported that vp0 protein of fmdv inhibits the activation of type i ifn signaling pathway by interacting with irf3 [18] (figure 2 , table 1 ). however, further studies are essential to assess the combination of vp0 to irf3 to restrain the production of ifns. since then, it is reported that vp0 proteins of fmdv interact with poly (rc) binding protein 2 (pcbp2) to promote the replication of fmdv [19] (figure 2 , table 1 ). the pcbp2 can recruit e3 ligase aip4 that contains the hect domain into the polyubiquitin and degrades mav [20] . the vp0 protein of fmdv suppresses the host's innate immunity by cooperating with pcbp2 to suppress the activation of ifn-β promoter. vp0 protein promotes the formation of pcbp2-virus-induced signaling adapter (visa) complex, enhances the degradation of visa mediated by pcbp2, and promotes the replication of fmdv [19] . in addition, the vp0 structural protein of fmdv is necessary for the correct assembly of the virus [21] . pathogens 2020, 9, the fmdv capsid protein, vp0, was further cleaved into vp4 and vp2 proteins by protease 3c. according to the structure of the virus, the vp4 protein of fmdv is localized on the inner surface of the capsid [22] . although vp4 does not stimulate the production of neutralizing antibodies independently, it contains t and b cell epitopes, which could be recognized by a variety of haplotype mhc molecules and exhibit high immunogenicity [23] [24] [25] . therefore, the combination of fmdv vp4 and vp1 could be used as a backup antigen for the development of a universal vaccine [26, 27] . in addition, the vp4 protein plays a crucial role in immunosuppression. the recombinant fmdv vp1-vp4 protein has been reported to have an inhibitory effect on the innate immune function of mouse peritoneal mast cells, putatively mediated by mannose receptor [28] . furthermore, nucleoside diphosphate kinase 1 (nme1) regulates the function of p53 to prevent tumor metastasis and progression and inhibit the metastasis of several malignant tumors [29] [30] [31] . the role of nme1 in viral infection is not yet clarified. recent studies have demonstrated that nme1 has antiviral activity and enhances p53-mediated transcription, while p53 regulates the expression of many antiviral genes to perform antiviral functions. however, fmdv vp4 does not directly interact but degrades nme1 through macroautophagy [32] [33] [34] (figure 2 , table 1 ). this phenomenon promotes the interaction between p53 and mdm2 (mdm2 is a negative regulatory factor of p53), while on the other hand, it enhances the mif-mediated inhibition of p53 activity, thereby impeding the antiviral response [34] . autophagy is an ancient and conservative biological process, which exists in almost all eukaryotes. through continuous research, it has been found that autophagy can selectively degrade intracellular redundant or harmful substances [35] [36] [37] , thus affecting the pathogenesis of some diseases. autophagy can also degrade invading microorganisms (such as bacteria, virus, and parasites) [38] , and is one of the immune mechanisms against pathogenic infection. it has been proved that a variety of viruses can activate autophagy and be swallowed and degraded [39] . not only that, after virus infection, host cells resist virus infection by releasing inflammatory factors and activating innate and adaptive immune responses, and autophagy also plays an important role in these defense responses [40] . in the long process of coexistence of virus and host, autophagy pathway has become one of the targets of virus-versus-host immunity. the inhibitory effect of virus on autophagy is also in many ways. for example, some studies have shown that after prrsv infection, the type i microtubule-associated protein light chain 3 (lc3-i) is transformed into lc3-ii, which activates the autophagy mechanism and leads to the accumulation of autophagosomes by preventing the fusion of autophagosomes and lysosomes. autophagosomes can act as replication sites to enhance prrsv replication [41, 42] . vp2 is one of the structural proteins of fmdv, localized on the surface of the virus. types o, a, and c fmdv vp2 contain several antigenic sites that present immunological significance [24, [43] [44] [45] . in addition, the amino acid substitution on the vp2 b-c loop of fmdv type asia1 not only mediates the significant antigenic diversity but also alters the replication ability and pathogenicity of the virus. for example, the single asp-to-asn substitution at vp2 72 position will reduce the virus replication ability and virulence [46] . however, the exact reasons for this result need to be further studied. a recent study demonstrated that the interaction between fmdv vp2 and hspb1 activates the eif2s1-atf4 pathway, which in turn, inhibits the akt-mtor pathway, leads to autophagy, and promotes virus replication [47] (figure 2 , table 1 ). autophagy has been proposed to provide a membrane platform for virus replication complexes or mediate the virus assembly and release [48] . thus, autophagy plays a crucial role in the replication of fmdv, and the expression of related vp2 mutants decreases the level of autophagy. therefore, vp2-induced autophagy may be one of the mechanisms of fmdv infection. autophagy regulates type i ifn signaling machinery and plays a vital role in antiviral innate immunity [49, 50] , and atg12-atg5 conjugate inhibits the production of type i ifn during vsv infection [51] . thus, it can be speculated that fmdv vp2 induces autophagy to increase the replication of fmdv, which might be achieved by blocking type i ifn signal. also, the correlation between vp2-induced autophagy and host antiviral immunity of fmdv needs to be investigated further. the changes in some sites on the surface of the vp2 protein of fmdv affects the cellular tropism and adaptability of the virus; for instance, the replacement of (glu82 to gly) alters the binding characteristics of the virus to cells [52] . the positively charged lysine residue at the vp2131 site of fmdv a can increase the adaptability of bhk-21 cells [53] . furthermore, the change in some sites of vp2 would also affect the stability and immunogenicity of fmdv. for example, vp2 h145y replacement reduces the acid sensitivity of the capsid of fmdv type asia 1, makes the h145y mutant of fmdv resistant to acid, and enhances the immunogenicity of virions [54] . the tyrosine at position 98 of vp2 mutated to phenylalanine (y98f) enhanced the thermal stability of the virus. this mutant presented optimal immunogenicity, and neutralizing antibodies could be induced by immunizing guinea pigs [55] . thus, identifying these specific sites of vp2 protein in fmdv provides an idea for the preparation of a heat-resistant and immunogenetically superior fmd antigen. the vp1 protein is the major surface protein on the fmdv and the primary antigen that elicits the neutralizing antibody response. the fmdv vp1 stimulates the host to produce cd8+ t cell responses with cross-protection against multiple serotypes of fmdv. therefore, vp1 protein or its antigenic determinants have become a research hotspot in the development of novel vaccines [2, 56, 57] . fmdv protein aqueous soluble recombinant dna-derived vp1 (rvp1) binds to integrin induces apoptosis, and fmdv rvp1 may selectively act as an effective human tumor apoptosis factor by regulating akt signal pathway [58] . the vp1 that interacts with host proteins can either enhance or inhibit the production of ifn in cells. first, it was found that vp1 interacts with soluble resistance-related calcium-binding protein (sorcin), a negative regulator in the innate immune signaling pathway, through yeast two-hybrid and immunoprecipitation experiments. also, vp1 binds to sorcin and activates the transcription factor stat3. stat3 inhibits the activation of ikk and nf-κb pathway, thus inhibiting the expression of type i ifn and cytokines [12] (figure 2 , table 1 ). second, the host protein kinases are essential regulators of virus interaction and play a crucial role in virus replication. tpl2 (tumor progression locus 2), a serine/threonine-protein kinase, promotes the activation of ifn-β signaling pathway by increasing the phosphorylation of irf3. tpl2 phosphorylation site thr290 is vital for promoting irf3-induced ifn-β signal activation. vp1 inhibits the protein expression of tpl2 phosphorylated at thr290, thereby inhibiting the irf3-activated ifn-β signaling, while vp1 reduces the mrna levels of tpl2-mediated ifn-β and some isgs ( figure 2 , table 1 ). third, another study proved that vp1 suppresses the ifn-β signaling pathway at the irf3 level by inhibiting the irf3 phosphorylation, dimerization, and nuclear translocation ( figure 2 , table 1 ). another study suggested that the activation of the mitogen-activated protein kinase (mapk) pathway is essential for fmdv replication. fmdv vp1 interacts with host ribosomal protein sa (rpsa) to continually activate the mapk signal pathway and promote virus replication by inhibiting the rpsa-mediated function [59] (figure 2 , table 1 ). as a structural protein of fmdv, vp3 plays a crucial role in virus assembly [9] . it blocks the ifn signal transduction, promotes the replication of fmdv, and inhibits the host immune response. in addition, vp3 inhibits the protein and mrna expression of innate immune junction molecule, visa. it interacts with the visa protein to inhibit the formation of visa-regulated complex, thereby inhibiting the dimerization and phosphorylation of irf3, inhibiting the expression of antiviral genes induced by ifn-β, and promoting fmdv replication [60] (figure 2 , table 1 ). previous studies have focussed on the effect of fmdv after treatment of type i ifn. also, it was demonstrated that fmdv vp3 inhibits the type ii ifn signaling pathway. furthermore, vp3 interacts with the host protein kinase jak1 protein and degrades the jak1 protein through lysosome pathway, inhibits the activation of the jak-stat pathway, and reduces the ifn-induced antiviral gene expression [61] (figure 2 , table 1 ). during evolution, some host substances can act on viral proteins, inhibit viral replication, and resist infection. reportedly, the fmdv infection stimulates the expression of mir-1307, which indirectly induces the degradation of fmdv vp3 protein through the proteasome pathway and strengthens the host immune response to inhibit the replication of fmdv [62] . moreover, the induction of mir-1307 is earlier than the full activation of nf-κb and irf3/7 [62] . nonetheless, fmdv infection-stimulated expression of mir-1307 would be a research hotspot in the future. however, the direct goal of mir-1307 has not yet been determined. recently, it has been reported that tank-binding kinase1 (tbk1) degrades the vp3 protein of several types of small ribonucleic acid virus, including fmdv, through its kinase and e3 ubiquitin ligase activity, while p-tbk1 is highly enriched in the mir-1307 overexpression cells [63] . thus, mir-1307 may target negative regulatory factors of tbk1. it has been reported that single or co-transfection of micrornamir-203a-3p and mir-203a-5p in porcine cell lines followed by infection of fmdv resulted in a decrease in viral protein synthesis and virus production. so, mir-203a-3p and mir-203a-5p are potential natural biotherapies against foot-and-mouth disease virus [64] . interaction between vp2 and hspb1 activates the eif2s1-atf4 pathway, which leads to autophagy and promotes virus replication [47] . sorcin soluble resistance-related calcium binding protein vp1 can bind to sorcin to inhibit the activation of ikk and nf-κ b pathway [12] . tumor progression locus 2; a serine/threonine protein kinase vp1 inhibits the protein expression of tpl2 phosphorylation site thr290, thereby inhibiting the promotion of irf3-activated ifn-β signal by tpl2. interferon regulatory factor 3 vp1 suppresses ifn-β signaling pathway at irf3 level by inhibiting irf3 phosphorylation, dimerization, and nuclear translocation. ribosomal protein sa vp1 interacts with rpsa to maintain the activation of mapk signal pathway and promote virus replication [59] . visa innate immune junction molecule vp3 inhibits the expression of visa protein mrna, and interacts with visa protein to inhibit the formation of visa-regulated complex, thereby inhibiting the dimerization and phosphorylation of irf3 [60] . janus kinase 1 vp3 can interact with jak1 protein and degrade jak1 protein to inhibit the activation of jak-stat pathway [61] . fmdv l pro , a papain-like protease, is the first translated protein from the fmdv genome and coexists in two forms in vivo and in vitro, lab pro and lb pro [65] [66] [67] . l pro , a key virulence factor of fmdv, suppresses the host immune response and achieves immune escape. l pro can cleave host translation-related proteins or interact with host transcription factors to inhibit the synthesis of antiviral factors. l pro can target cleavage/degradation pattern recognition receptors, the proteins of the interferon pathway, nf-κb pathway, and stress-related pathway. in addition, l pro acts as a deubiquitinase (dub) and deisgylase. first, l pro specifically cleaves the eukaryotic initiation factors (eifs), 4gi and 4gii. the loss of integrity of eif4gi and eif4gii hinders the formation of eukaryotic cellular translation initiation factor 4f(eif4f) complex, while eif4f complex significantly affects the cell cap-dependent translation [68] , thereby preventing the recruitment of capped mrna in host cells and inhibiting the synthesis of antiviral molecules of innate immunity [69] . fmdv rna starts translation in a cap-independent manner through the internal ribosome entry site (ires) elements [70] [71] [72] . therefore, fmdv can make use of host protein synthesis mechanism to quickly synthesize virus protein and complete virus reproduction. the interaction between l pro and activity-dependent neuroprotective protein (adnp) is crucial in the process of infection and promotes fmdv replication by inhibiting the expression of ifn and ifn stimulated gene (isg) [13] . however, whether the processing of adnp by l pro is performed directly by l pro or by related enzymes induced or activated by l pro is yet to be deduced. the present study further elucidates the mechanism though which fmdv evades the immune response by interaction with transcription factors. second, fmdv l pro downregulates the expression of nf-κb and irf3/7, which in turn, interferes with the transcription of ifn-α1/β mrna. fmdv l pro induces the degradation of p65/rela, the core component of nf-κb, which destroys the integrity of nf-κb and downregulates the transcription of ifn-β in host cells, thus inhibiting the host immune response [73] . however, the degradation mechanism of p65/rela by l pro is still unclear, and the products of p65/rela digested by l pro have not been identified. in addition to destroying the integrity of nf-κb, l pro also decreased the expression of irf3/7, the key factors of virus-triggered ifn-α/β secretion that inhibit the production of dsrna-induced type i ifn [74] . third, porcine ifn-λ1, a type iii ifn, inhibits the replication of fmdv. however, after screening, the lead protease l pro exerts a robust inhibitory effect on the activity of ifn-λ1 promoter induced by poly(i:c) by inhibiting the rig-i/mda5 pathway and interfering with the activation of interferon regulatory factors (irfs) and nf-κb. fmdv l pro alone can inhibit the expression of dsrna-induced ifn-λ1, suggesting a new mechanism of fmdv antagonizing ifn-λ1-mediated innate immune response [75] . fourth, in addition to its conventional papain-like protease activity, l pro acts as a deubiquitinase (dub) and deisgylase. lb pro has deubiquitination activity, and the deubiquitination functional sites are highly conserved among the seven serotypes of fmdv. these motifs could significantly inhibit the ubiquitination of key molecules of innate immune signaling pathway such as retinoic acid-inducible gene i (rig-i), tank-binding kinase 1 (tbk1), tnf receptor-associated factor 6 (traf6), and traf3, thereby inhibiting the innate immune response and achieving immune escape [76] . lb pro selectively cleaves the c-terminal peptide bond of isg15 and exposes an easily detected glygly epitope on the substrate of the modifier, which provides a new method for monitoring fmdv [77] . a new study shows that abolishing/reducing the deisgylase/dub activity of l pro causes viral attenuation independently of its ability to block the expression of ifn and isg mrna [78] . the latest research shows that l pro 's ability to cleave rlr signaling proteins but not its deubiquitination/deisgylation activity correlates with the reduced ifn-β gene transcription [79] . fifth, laboratory of genetics and physiology 2 (lgp2), an innate immune sensor promotes the interaction between viral rna and mda5, thus producing antiviral signals [80] . recently, it has been reported that lgp2 is the biphasic main activator of many innate immune genes that induce the production of ifn by a cascade effect [81] . however, fmdv l pro cleaves lgp2 and blocks the effect of lgp2-mediated ifn-β induction [82] . these features represent a new approach of immune escape and provide a basis for in-depth research on the role of lgp2 in anti-fmdv response and the interaction between mda5 and lgp2-l pro . sixth, recent studies have demonstrated that the stress response was inhibited during fmdv infection. fmdv l pro targets the sg (stress granule) scaffold proteins g3bp1 and g3bp2 to antagonize the formation of sg, a potentially significant antiviral signaling platform [83] [84] [85] [86] that regulates the integrated stress response [87] . however, the l pro -mediated sg inhibition mechanism of fmdv might not be the only one in the cells infected with fmdv. although several studies have suggested the antiviral effect of sgs, their exact role as an antiviral signal platform is not yet clarified. therefore, these studies demonstrated that fmdv l pro inhibits the host immune response and promotes the fmdv replication through various mechanisms ( figure 3 , table 2 ). fmdv 2b protein is a hydrophobic transmembrane viroporin with oligomeric transmembrane pores that can destabilize the integrity of the host cell membrane, disrupt host cell ca 2+ balance, induce host cell autophagy, and promote virion release [88, 89] . fmdv 2b may play an active role in virus immune escape because of its viroporin characteristics. previous studies have shown three pattern recognition receptors, retinoic acid-inducible gene i (rig-i), melanoma differentiation-associated factor 5 (mda5), and lgp2 that bind to viral rna. of these, rig-i and mda5 recognize different structures of rna to activate the antiviral signal transduction, and lgp2 regulates this process [80, 90, 91] . targeted studies have found that rig-i and lgp2 inhibit the fmdv replication, and lgp2 significantly inhibits the inflammatory response of fmdv-infected cells. fmdv 2b protein suppresses the expression of pattern recognition receptors rig-i, mda5, and lgp2, inhibiting the host antiviral response and promoting fmdv replication. 2b protein directly interacts, reduces the protein levels, and inhibits the antiviral effect mediated by rig-i, mda5, and lgp2 ( figure 3 , table 2 ). this reduction does not depend on proteasome, lysosome, or autophagy pathway, and the specific molecular mechanism is yet unclear. in addition, the study on whether 2b reduces the level of other junction molecules in rig-i like receptor (rlr) signaling pathway showed that although fmdv 2b does not mediate the decline in tbk1 and irf3 in the rlr signaling pathway, it inhibits the phosphorylation of tbk1 and irf3, followed by suppression of the expression of type i ifn [92] [93] [94] (figure 3 , table 2 ). the results of yeast two-hybrid screening and immunoprecipitation showed that one of the two host proteins that could interact with fmdv 2b protein is the eukaryotic translation elongation factor1 γ (eef1g) [95] . a previous study confirmed that the decrease in eef1g affects the synthesis of some membrane proteins necessary for vesicle formation, and its mislocation reduces the synthesis of membrane proteins [96] . the 2b protein of fmdv is associated with increased cell membrane integrity and membrane permeability [88] . therefore, it can be speculated that eef1g assists 2b in producing virus-induced vesicles and inducing cell lysis. however, further studies are required to substantiate these findings. the other host protein is cyclophilin a, which degrades fmdv l pro and 3a protein that suppresses the innate immune. strikingly, the interaction between 2b protein and cyclophilin a directly inhibits the degradation of l pro and 3a protein by cyclophilin a, thereby inhibiting the host immune response [97] (figure 3 , table 2 ). recent studies have shown that cyclophilin a also promotes the ubiquitination of rig-i, and thus enhances the innate immune response [98] . however, whether the interaction between 2b protein and cyclophilin a affects the ubiquitination of rig-i is yet to be elucidated. another study showed that fmdv 2b protein interacts with nod2 to reduce the expression of nod2 protein, for which the 2b carboxyl-terminal 105-114 region was essential, thus inhibiting the activation of nf-κb and ifn-β signaling pathways ( figure 3 , table 2 ). the decrease in nod2 is not related to the cleavage of eif4g, induction of apoptosis or proteasome, nor lysosome or caspase pathways [16] . fmdv protein 2c is a highly conserved polypeptide of 318 amino acids (aa) [99, 100] . subsequent studies proved that the 2c protein plays a critical role in virus replication. guanidine hydrochloride, a molecular antagonist of protein 2c, suppresses the synthesis of viral genetic material in small ribonucleic acid virus-infected cells [101, 102] . three host proteins beclin1, vimentin, and nod2 interacting with fmdv 2c, were screened by yeast two-hybrid assay. beclin1 is related to the formation of autophagosomes and the fusion of autophagosomes to lysosomes [103, 104] . protein 2c interacts and inactivates beclin1, which in turn, inhibits the fusion of autophagosomes containing fmdv and lysosomes, thereby preventing the degradation of the virus [105] (figure 3 , table 2 ). vimentin plays a role in the lysosomal degradation of proteins and has been shown to be related to autophagosomes [106, 107] . in the early stage of fmdv infection, vimentin forms a cage-like structure around 2c in order to facilitate virus replication. additionally, the expression of the dominant-negative (dn) form of vimentin significantly reduces the replication ability of fmdv. the replication of fmdv requires a complete vimentin network. however, the exact mechanism of governing vimentin cage formation and dissolution remains to be elucidated [108] . the interaction between fmdv protein 2c and nod2 reduces the level of nod2 protein to help the virus evade the immune response, and the carboxyl-terminal 116-209 and 210-260 regions of 2c were essential for the interaction [16] (figure 3 , table 2 ). strikingly, the interaction between 2c and beclin1 or nod2 evades immune response through different pathways, which contributes to the replication of fmdv. the interaction between 2c and cellular vimentin is crucial for the replication of fmdv, albeit the specific pathway is not yet clarified. the 3a protein is a conserved 153-aa polypeptide of fmdv, larger than other picornaviruses [9] . fmdv 3a protein is related to host tropism and virulence, and the deletion of 3a alters the virus tropism and virulence. reportedly, a single amino acid change in 3a endowed fmdv with a new adaptive phenotype, and fmdv 3a protein is associated with membrane correlation and regulation of host protein secretion [9, 109] . plaque assay and virus titeration showed that the stable expression of 3a or 3ab protein enhances the replication of fmdv. however, the infection level of fmdv decreased after the transient expression of 3ab protein. these findings suggested that 3a and 3ab play a crucial role in the replication of fmdv [110] . fmdv 3a has been proved to interacts with cellular protein dctn3 by the two-hybrid method. dctn3 is a subunit of the dynactin complex, a cofactor for dynein. dynactin-dynein complexes are related to the transport of intracellular organelles. the overexpression of dctn3 and the disruption of dynactin-dynein complex significantly reduces the production of fmdv in infected cells. fmdv replication seems to require a complete dynamic protein cell pathway [111] . in the previous study, fmdv 3a protein mutantageness study based on reverse genetic technique revealed the effect of amino acid 89 mutation in 3a protein on the interaction between 3a protein and dctn3 was detected by yeast two-hybrid technique. the data show that 3a could be bound to dctn3 when amino acid 89 was alanine or leucine, but when amino acid 89 was mutated to proline, which destroy the binding between 3a and dctn3 [111] . interestingly, both the fmdv o/taw/97 strain with pldg peptide from 89-92 aa on 3a and the recombinant fmdv o1c3a virus with deletion of residues 87-106 on 3a lacked the binding site of the host protein dctn3. also, the replication rate in primary bovine cells was slower than that of parental virus strains. however, no significant change was detected in the replication rate of either of the two viruses and their parent virus strains in porcine cells [112, 113] . thus, it could be speculated that the binding of fmdv 3a and host dctn3 might be related to the host tropism of the virus, but the slight difference in dctn3 among species could be attributed to the range of hosts of fmdv. the recombinant fmdv with pldg residues 89-92 on 3a produced a delayed and mild disease in cattle, suggesting that 3a-dctn3 interaction might play a role in the virulence of the bovine virus. in addition, the virus recovered rapidly during infection and regained dctn3 binding, suggesting that the interaction between fmdv 3a and dctn3 is vital for virus replication in cattle [111] (figure 3 , table 2 ). however, the 3a-dctn3 combination needs further exploration. the type i ifn reporting system was utilized to confirm that the 3a protein inhibits the activation of the ifn-β signaling pathway. further studies demonstrated that 3a protein interacted with innate immune molecules rig-i, mda5, and visa, inhibited the expression of innate immune junction molecules, such as rig-i, mda5, and visa, and inhibited the formation of signal transduction complex, thereby escaping the host innate immune system [114] (figure 3 , table 2 ). the overexpression of the fmdv 3a inhibited the sendai virus-triggered activation of irf3 [114] . a recent study found that the interaction between dead-box helicase 56 (ddx56) and fmdv protein 3a increases the interaction between ddx56 and irf3 and enhances the ability of fmdv 3a to inhibit irf3 phosphorylation ( figure 3 , table 2 ). also, fmdv 3a inhibits the activation of the ifn-β promoter and isre by reducing the phosphorylation of irf3 and increasing the replication of fmdv. however, the overexpression of ddx56 cannot significantly reduce the phosphorylation of irf3. thus, we speculated that ddx56 also inhibits the production of ifn through another different pathway, thereby promoting virus replication [14] . unlike other picornaviruses that encode a single 3b copy, fmdv encodes three similar but different 3b proteins (3b1, 3b2, and 3b3), which are ubiquitous in all fmdv isolates [115] . the effective replication of fmdv in bovine cells requires a full-length 3a and three vpg (3b). the deletion of the 3b3 coding sequence adversely affects the rna replication of fmdv, and the viral activity requires highly conserved 3b3 protein [109, 116] . although fmdv lacking 3b1 and 3b2 can also reduce the viral rna synthesis, the growth of the virus on pig-derived cells is only slightly reduced, and the disease of pigs has also been slightly alleviated [109, 117] . these studies showed that 3b3 is more critical than 3b1 and 3b2 in maintaining virus rna replication. the efficiency of rna replication is maximal when three 3b copies coexist, and the absence of 3b1 and 3b2 might affect the virulence and host range of fmdv. however, the integration of the three proteins in replication needs to be studied further. fmdv 3b significantly reduces the levels of ifn-β, isg15, and il-6 levels and the activation of ifn-β, nf-κb, and isre promoters induced by poly(i:c), indicating that fmdv 3b is also a viral escape protein, which inhibits the response of type i ifn in cells. subsequently, 3b blocked the interaction between rig-i and trim25, thus inhibiting the ubiquitination of rig-1 and the formation of the rig-i-visa complex, which inhibits the ifn signaling pathway [118] (figure 3 , table 2 ). further studies have shown that fmdv 3b reduces the expression of type i ifn by inhibiting the visa signaling pathway via interaction with the visa protein ( figure 3 , table 2 ). fmdv 3c protein has been identified as a protease. it cleaves not only most of the virus precursor proteins [9] but also the host proteins to block/inhibit the cellular defense mechanism and promote virus replication. fmdv protein 3c pro cleaves the related host proteins, inhibits the transcription and translation of host cells, or promotes the translation of virus rna, thus promoting virus replication. for example, fmdv protein 3c pro is related to the cleavage of histone h3, as it removes 20 n-terminal amino acid residues from histone h3. the amino terminal of h3 is related to the regulation of chromosomal transcriptional activity in eukaryotic cells. the cleavage of 3cpro to h3 inhibits the transcription of host cells and ultimately hinders the translation of host cells [119] , which is beneficial for the virus to escape antiviral immune response ( figure 3 , table 2 ). in addition, fmdv protein 3c pro also cleaves the host translation initiation factors, eif4g and eif4a, which are components of the cap-binding complex eif4f, which inhibits the synthesis of host-related antiviral proteins [120] ( figure 3 , table 2 ). however, it can only partially cleave eif4g and eif4a but not completely block the translation of host cells [121, 122] . unlike the cleavage of eif4g by l pro in the early stage of infection, the cleavage of eif4g and eif4a by fmdv protein 3cpro requires the accumulation of 3c protein. hence, it occurs in the later stage of infection cycle, and the cutting site of eif4g by 3c pro is different from that mediated by l pro [120] . in addition, the cleaving of eif4a may be disadvantageous to the virus, and the translation of viral rna requires eif4a [123] . the fmdv protein 3c pro can also cleave the 68-kda src-associated substrate during mitosis (sam68), a unique rna-binding protein (figure 3 , table 2 ). truncated sam68 spreads to the cytoplasm, combines with fmdv rna and attaches to ires to enhance the translation of the virus rna. however, the titer of fmdv was reduced 1000-fold after transfection of sam68-targeted sirna molecules, indicating sam68 might not limit to enhancing virus translation, and sam68 may play a variety of roles in fmdv infection [124] . fmdv protein 3c pro directly or indirectly degrades vital immune molecules, thus inhibiting/blocking the expression of ifn and antiviral genes. fmdv protein 3c pro can degrade pattern recognition receptors, rig-i and lgp2, thus inhibiting the production of antiviral factors and promoting fmdv replication [92, 93] ( figure 3 , table 2 ). it also degraded the modulator necessary for innate immune key molecule nemo, the vital regulator of nf-κb, a junction protein necessary for activating nf-κb, and the ifn regulatory factor signaling pathway. this cleavage impaired the activation of irfs and nf-κb and inhibited the expression of downstream antiviral genes ( figure 3 , table 2 ). the cysteine protease activity of 3c pro is necessary for 3c pro to cleave nemo [125] . reportedly, fmdv 3c suppresses the jak-stat signaling pathway, thus inhibiting the antiviral response induced by ifn. another study found that 3c protease activity promoted the degradation of kpna1, the nuclear localization signal receptor for tyrosine-phosphorylated stat1, to block the nuclear translocation of stat1/stat2 to inhibit the jak-stat signaling pathway [126] (figure 3 , table 2 ). autophagy-associated protein atg5-atg12 is shown to be associated with the replication of fmdv. in the process of fmdv infection, atg5-atg12 upregulates the anti-virus nf-κb and irf3 signaling pathways, thereby inhibiting the proliferation of fmdv. fmdv protein 3c pro antagonizes the host antiviral immunity and suppresses autophagy by degrading atg5-atg12 [127] (figure 3 , table 2 ). furthermore, atg5-atg12 enhances the expression of host protein kinase pkr, a serine-threonine kinase, which can be induced by ifn and activated by double-stranded rna (dsrna). consequently, it blocks the synthesis of cell and virus protein, inhibits the replication of fmdv, and exerts a significant antiviral effect [128] . moreover, fmdv 3c pro protein induces pkr degradation through the lysosome pathway and inhibits the pkr-mediated antiviral effect by downregulating the pkr protein. no interaction occurred between fmdv 3c pro and pkr [129] (figure 3 , table 2 ). recent studies have shown that 3c pro decreases the level of nod2, thus inhibiting the antiviral effect induced by nod2, and the reduction of nod2 induced by 3c pro depends on its protease activity ( figure 3 , table 2 ). no interaction occurred between fmdv 3c pro and nod2 [16] . taken together, fmdv 3c pro exerts its immunosuppressive function and suppresses the innate immunity of the host via a myriad of cascades. can directly or indirectly act on retinoic acid-induced gene i-like receptor (rlr) to inhibit innate immunity. fmdv 3a and 3b reduce the expression of junction protein visa at the transcriptional or protein level. l pro , 2b and 3a can directly or indirectly target irf3 to inhibit interferon production. 3c proteins inhibit jak-stat signaling pathway, thus inhibiting isgs production. l pro and 3c inhibit the synthesis of antiviral molecules by cutting related factors of host transcription and translation. l pro protein can not only induce apoptosis, but also inhibit host cell apoptosis and promote virus replication, which is achieved by blocking the translation of α-ifn and inhibiting pkr synthesis. 2c can promote virus replication by regulating autophagy. eif4g: eukaryotic initiation factor 4g; nf-κb: nuclear factor kappa b; irf3: interferon regulatory factor 3; irf7: interferon regulatory factor 7; rig-i: retinoic acid inducible gene i; tbk1: tank binding kinase i; traf6: tnf receptor-associated factor 6; traf3: tnf receptor-associated factor 3; adnp: activity-dependent neuroprotective protein; lgp2: laboratory of genetics and physiology 2; mda5: melanoma differentiation associated factor 5; cypa: cyclophilin a; nod2: nucleotide-binding oligomerization domain 2; dctn3: dynactin 3; ddx56: dead-box helicase 56; sam68: 68 kda src-associated substrate during mitosis; visa: virus-induced signaling adapter. can directly or indirectly act on retinoic acid-induced gene i-like receptor (rlr) to inhibit innate immunity. fmdv 3a and 3b reduce the expression of junction protein visa at the transcriptional or protein level. l pro , 2b and 3a can directly or indirectly target irf3 to inhibit interferon production. 3c proteins inhibit jak-stat signaling pathway, thus inhibiting isgs production. l pro and 3c inhibit the synthesis of antiviral molecules by cutting related factors of host transcription and translation. l pro protein can not only induce apoptosis, but also inhibit host cell apoptosis and promote virus replication, which is achieved by blocking the translation of α-ifn and inhibiting pkr synthesis. 2c can promote virus replication by regulating autophagy. eif4g: eukaryotic initiation factor 4g; nf-κb: nuclear factor kappa b; irf3: interferon regulatory factor 3; irf7: interferon regulatory factor 7; rig-i: retinoic acid inducible gene i; tbk1: tank binding kinase i; traf6: tnf receptor-associated factor 6; traf3: tnf receptor-associated factor 3; adnp: activity-dependent neuroprotective protein; lgp2: laboratory of genetics and physiology 2; mda5: melanoma differentiation associated factor 5; cypa: cyclophilin a; nod2: nucleotide-binding oligomerization domain 2; dctn3: dynactin 3; ddx56: dead-box helicase 56; sam68: 68 kda src-associated substrate during mitosis; visa: virus-induced signaling adapter. l pro cut eif4gi and eif4gii, thus preventing the recruitment of capped mrna and inhibiting the synthesis of antiviral molecules [68, 69] . nf-κb nuclear factor kappa b l pro induce the degradation of p65/rela, which is the core component of nf-κb [73] . interferon regulatory factor 3/7 l pro decreased the expression of irf3/7 to inhibit the production of type i ifn induced by dsrna [74] . rig-i tbk1 traf6 traf3 retinoic acid inducible gene i; tank binding kinase i; tnf receptor associated factor 6 tnf receptor associated factor 3 l pro can significantly inhibit the ubiquitination of key molecules of innate immune signaling pathway such as rig-i, tbk1, traf6, and traf3 [76] . adnp activity dependent neuroprotective protein l pro and adnp interact to promote the replication of fmdv by inhibiting the expression of ifn and isg [13] . lgp2 laboratory of genetics and physiology2 l pro can cleave lgp2 and block the effect of lgp2-mediated the production of ifn-β [82] . g3bp1 and g3bp2 stress granule scaffold proteins l pro targets to cleave the sg scaffold proteins g3bp1 and g3bp2 to antagonize the formation of sg [87] . retinoic acid inducible gene i; melanoma differentiation associated factor 5; laboratory of genetics and physiology 2; 2b protein suppress the expression of rig-i, mda5 and lgp2, inhibiting host antiviral response [93, 94] . tbk1; irf3 tank binding kinase i; interferon regulatory factor 3 2b suppress the phosphorylation of tbk1 and irf3, and then inhibit the expression of type i interferon [93] . cypa cyclophilin a the interaction between 2b protein and cyclophilin a directly inhibits the degradation of l pro and 3a protein by cyclophilin a [97] . nod2 nucleotide-binding oligomerization domain 2 2b protein can interact with nod2 to reduce the protein level of nod2, which inhibit the activation of nf-κb and ifn-β signal pathways [16] . beclin1 involve in the fusion of autophagosomes to lysosomes 2c interacts with beclin1 to induce beclin1 inactivation, which inhibits the fusion of autophagosomes of containing fmdv and lysosomes [105] . nod2 nucleotide-binding oligomerization domain 2 the interaction between fmdv protein 2c and nod2 reduces nod2 at the protein level to help the virus evade immune response [16] . 3a protein increases the interaction between ddx56 to inhibits the activation of ifn-β promoter and isre by reducing the phosphorylation of irf3 [14] . retinoic acid inducible gene i; 3b blocked the interaction between rig-i and trim25, thus inhibiting the interferon signal pathway [118] . visa virus-induced signaling adapter 3b reduces the expression of type i interferon by inhibiting visa signal pathway by interacting with visa protein. histone h3 related to the transcription of host cells the cleavage of 3c pro to h3 inhibits the transcription of host cells and ultimately hinders the translation of host cells [119] . eif4g and eif4a host translation initiation factors 3c pro is involved in the cleavage of eif4g and eif4a, thus inhibiting the synthesis of host-related antiviral proteins [120] . 68 kda src-associated substrate during mitosis 3c pro can also cleave sam68. truncated sam68 spreads to the cytoplasm and meets the fmdv rna and attaches to the ires to enhance the translation of the virus rna [124] . retinoic acid inducible gene i; laboratory of genetics and physiology 2; protein 3c pro can degrade rig-i and lgp2 [92, 93] . nemo nf-κb necessary regulator 3c can also degrade nemo to impaired activation of irfs and nf-κb [125] . kpna1 the nuclear localization signal receptor for tyrosine-phosphorylated stat1 3c promoted the degradation of kpna1 to block the nuclear translocation of stat1/stat2 to inhibit jak-stat signal pathway [126] . autophagy associated protein fmdv protein 3c pro antagonizes host antiviral immunity and suppresses autophagy by degrading atg5-atg12 [127] . pkr a serine-threonine kinase 3c pro induces pkr degradation and inhibits pkr-mediated antiviral effect by down-regulating pkr protein [129] . nod2 nucleotide-binding oligomerization domain 2 3c pro induces the reduction of nod2, thus inhibiting the antiviral effect induced by nod2 [16] . the 5 untranslated region (5 utr) of fmdv is about 1300 bp, which is larger than that of several small rna virus of the family picornaviridae. it contains s-fragment, poly (c), pseudoknots (pks), the cis-acting replication element (cre) and internal ribosome entry site (ires). studies have shown that the special structure of the 5 untranslated region is essential for the translation and replication of the virus genome [130] . the first element of the 5 end is the s-fragment with approximately 350 nt long, the sequence can fold and form the stem ring. it is speculated that this structure can block the function of host exonuclease, thereby maintaining the stability and replication of the virus genome [9] . s fragment is related to the interaction between virus and host protein. some studies have confirmed the direct correlation between the degree of s fragment deletion mutation and the attenuated phenotype. the fmdv mutant with 164bp deletion in the upper part of the s fragment loop was highly attenuated in vivo [131] . fmdv with deletion of s fragment induces higher expression of ifn-β and isg mrna, so it is concluded that s fragment of fmdv is necessary for host cell replication and regulation of innate immune response [131] . downstream of s-fragment is the variable length poly (c) region. studies on the viral genome have shown that a certain threshold length of poly (c) is correlated to the viability of the virus, but there is no evidence that the length of the poly (c) chain is directly related to virulence [132] . the 3 end of ploy (c) is pseudoknots. some studies have shown that the deletion of 86-nt in pks reduces the pathogenicity of o/cha/7/2011 strain of fmdv in bovine cells and bovines, and the artificial deletion of 43 bases will reduce the pathogenicity of o/me-sa/panasia strain fmdv in bovine. this deletion occurs naturally in the region of the porcinophilic cathay topotype fmdv genome. it is suggested that the natural absence of pks area may be the reason for the transformation from bovines to pigs as a vector for the transmission of fmdv. it is concluded that the pseudoknots region of fmdv 5 untranslated region is the decisive factor of virus tendency and virulence [133] . the cis-acting replication element (cre) is a stem-loop structure containing conserved aaaca motifs [134, 135] . it has been confirmed that fmdv cre is necessary for genome replication, and cre has been found to be adjacent to ires, which indicates that cre may play a role in coordinating translation and replication, but this still needs further confirmation [136] . fmdv 3 -utr consists of a 90nt structural sequence folded into two independent stem loops and a poly (a) tail of variable length [137, 138] . related studies have shown that the 3 -utr directly binds to the s-fragment and ires elements of the 5 -utr at different sites, and fmdv 3 -utr affects virus replication and virulence by enhancing the activity of ires elements [139] . moreover, genetic studies have shown that the recombinant fmdv with missing structural sequence of 3 -utr cannot be recovered [140] , and it is concluded that the structural region of 3 -utr is essential for the infectivity and replication of fmdv. in addition to the direct rna-rna interaction, 5 -3 -end bridging, which is also related to protein-protein and protein-rna interactions, it has been found that cellular proteins pcbps and p47 can directly bind to s-fragment and 3-utr [138] . it is inferred that 5 -3 -end bridging may play an important role in the replication of fmdv. fmdv is a highly contagious virus that infects almost all cloven-hoofed animals, showing vesicles on the foot and mouth, skin erosion on the mucous membranes, fever, weight loss, pacing, and salivation, severely threatening the development of animal husbandry. however, in addition to causing acute infections and diseases, fmdv can be asymptomatic carriers in some cases, which might lead to another outbreak of fmd, making prevention and control challenging and costly. high infectivity, wide geographical distribution, wide host range, short-term immunity without serotype cross-protection, multiple modes of transmission, and persistent infection render the control and eradication of this disease rather difficult. therefore, study the molecular mechanism underlying fmdv evading immunity is imperative for the control of an epidemic situation. the immune system includes innate immunity and acquired immunity, which is a major protective system against the invasion of pathogenic microorganisms, surveillance, and removal of foreign bodies. fmdv suppresses the function of the immune system at the initial stage of infection, such that the virus can proliferate rapidly in the respiratory system and spread to its natural infection site [141] . in terms of evading the humoral immune system, each serotype of fmdv is prone to antigenic variation, which makes the virus escape from the neutralizing antibodies [142] . in the aspect of inhibiting cellular immune response, fmdv infection can cause the decrease of host lymphocytes and is accompanied by severe viremia, which will eventually lead to the destruction of t cells and fmdv infection inhibits the function of dendritic cells and weakens the ability of dendritic cells to process them into antigens [143, 144] . previous studies have shown that, mhc class i molecule expression on the surface of cells was suppressed at 30 min after fmdv infection, indicating that the cells infected with fmdv will immediately lose the ability to present mhc-i-related viral peptides to t lymphocytes. this mechanism would facilitate the virus escape from the host's cytotoxic immune response. limiting the killing effect mediated by nk cells is also an important mechanism for fmdv to evade the cellular immune response. some studies have shown that the responsiveness of porcine nk cells decreases significantly 2-3 days after fmdv infection, and then returns to normal [145] . strikingly, nk cells isolated from infected pigs could not secrete ifn-γ [146] . the research on fmdv interference with immune effect and suppression of innate immunity has been widely studied. some proteins of fmdv (l pro ,2b, 3a, 3b, 3c) can directly or indirectly act on retinoic acid-induced gene i-like receptor (rlr) to inhibit innate immunity [82, [92] [93] [94] 114] . fmdv vp0, vp3, 3a, and 3b reduce the expression of junction protein visa at the transcriptional or protein level [19, 60, 114] . fmdv l pro , vp0, vp1, 2b, and 3a can directly or indirectly target irf3 to inhibit interferon production [18, 74, 93, 114] . vp3 and 3c proteins inhibit jak-stat signaling pathway, thus inhibiting isgs production [61, 126] . fmdv proteins l pro and 3c inhibit the synthesis of antiviral molecules by cutting related factors of host transcription and translation [13, 68, 69, 120, 124] . in addition, it is interesting that l pro protein can not only induce apoptosis, but also inhibit host cell apoptosis and promote virus replication, which is achieved by blocking the translation of α-ifn and inhibiting pkr synthesis [147] . fmdv protein vp2 and 2c can promote virus replication by regulating autophagy [47, 105] . these mechanisms provide opportunities for rapid transcription and translation of fmdv. in the previous studies on fmdv, hek293 cells have been widely used in in vitro experiments because of its highly transfected efficiency. however, hek293 cells are not fmdv susceptible cells and there are species differences between hek293 cells and fmdv susceptible cells. therefore, the use of hek293 cells for fmdv-related research has some limitations. in summary, fmdv has evolved a variety of ways to evade the immune response in the long-term combat with the host immune system. although there are many breakthroughs in the research on the immune escape of fmdv, many mechanisms underlying the fmdv-affected host immunity have not yet been elucidated, and the interaction between fmdv protein and host protein need to be explored further. in addition to the interaction between the virus and host protein, exploring the mechanism of synergistic inhibition of immune response by multiple viral proteins is of great significance for the development of specific drugs and new vaccines. previous studies mainly focused on the effect of fmdv with respect to innate immunity. however, there are a few studies on acquired immunity, and these need to be supplemented further. also, persistent infection of fmdv needs to be investigated intensively in the future. it was the goal of the literature study to summarize the current knowledge and to point out future research directions and define the scientific questions that remain to be elucidated to gain better knowledge of immune responses against fmdv and its immune escape mechanisms. foot-and-mouth disease: host range and pathogenesis foot-and-mouth disease foot-and-mouth disease: past, present and future molecular epidemiology of foot-and-mouth disease virus the generation and persistence of genetic variation in foot-and-mouth disease virus foot-and-mouth disease virus evolution: exploring pathways towards virus extinction emergence and selection of rna virus variants: memory and extinction understanding the molecular epidemiology of foot-and-mouth-disease virus molecular basis of pathogenesis of fmdv viral immune evasion: a masterpiece of evolution immune evasion during foot-and-mouth disease virus infection of swine engagement of soluble resistance-related calcium binding protein (sorcin) with foot-and-mouth disease virus (fmdv) vp1 inhibits type i interferon response in cells interaction between fmdv lpro and transcription factor adnp is required for optimal viral replication ddx56 cooperates with fmdv 3a to enhance fmdv replication by inhibiting the phosphorylation of irf3 activation of innate immune antiviral responses by nod2 foot-and-mouth disease virus antagonizes nod2-mediated antiviral effects by inhibiting nod2 protein expression the structure of foot-and-mouth disease virus structural protein vp0 of foot-and-mouth disease virus inhibits type i interferon signaling pathway poly (rc) binding protein 2 interacts with vp0 and increases the replication of the foot-and-mouth disease virus pcbp2 mediates degradation of the adaptor mavs via the hect ubiquitin ligase aip4 dissecting the roles of vp0 cleavage and rna packaging in picornavirus capsid stabilization: the structure of empty capsids of foot-and-mouth disease virus the structure and function of a foot-and-mouth disease virus-oligosaccharide receptor complex construction and immunogenicity of dna vaccine plasmid expressing vp1 and vp4 genes of foot-and-mouth disease virus. chin detection and characterization of functional t-cell epitopes on the structural proteins vp2, vp3, and vp4 of foot and mouth disease virus o1 campos molecular design and immunogenicity of a multiple-epitope fmdv antigen and dna vaccination the influence of mhc polymorphism on the selection of t-cell determinants of fmdv in cattle induction of protective immunity by dna vaccination with toxoplasma gondii hsp70, hsp30 and sag1 genes protein expression profile of mast cells in response to recombinant vp1-vp4 of foot-and-mouth disease virus nm23-h1 suppresses metastasis by inhibiting expression of the lysophosphatidic acid receptor edg2 cell-permeable nm23 blocks the maintenance and progression of established pulmonary metastasis implication of metastasis suppressor nm23-h1 in maintaining adherens junctions and limiting the invasive potential of human cancer cells emerging roles of p53 and other tumour-suppressor genes in immune regulation transcriptional role of p53 in interferon-mediated antiviral immunity foot-and-mouth disease virus induces lysosomal degradation of nme1 to impair p53-regulated interferon-inducible antiviral genes expression autophagy: more than a nonselective pathway selective autophagy: ubiquitin-mediated recognition and beyond autophagy, immunity, and microbial adaptations autophagy, antiviral immunity, and viral countermeasures autophagy and viruses: adversaries or allies? coronavirus nsp6 restricts autophagosome expansion porcine reproductive and respiratory syndrome virus induces autophagy to promote virus replication identification of neutralizing antigenic sites on vp1 and vp2 of type a5 foot-and-mouth disease virus, defined by neutralization-resistant variants antigenic heterogeneity of a foot-and-mouth disease virus serotype in the field is mediated by very limited sequence variation at several antigenic sites neutralizing epitopes of type o foot-and-mouth disease virus. i. identification and characterization of three functionally independent, conformational sites effects of amino acid substitutions in the vp2 b-c loop on antigenicity and pathogenicity of serotype asia1 foot-and-mouth disease virus foot-and-mouth disease virus capsid protein vp2 activates the cellular eif2s1-atf4 pathway and induces autophagy via hspb1 divergent roles of autophagy in virus infection autophagy in antiviral innate immunity autophagy and innate immunity: triggering, targeting and tuning the atg5-atg12 conjugate associates with innate antiviral immune responses perturbations in the surface structure of a22 iraq foot-and-mouth disease virus accompanying coupled changes in host cell specificity and antigenicity a positively charged lysine residue at vp2 131 position allows for the enhanced adaptability of foot-and-mouth disease virus serotype a in bhk-21 cells single amino acid substitution of vp1 n17d or vp2 h145y confers acid-resistant phenotype of type asia1 foot-and-mouth disease virus mutation in the vp2 gene of p1-2a capsid protein increases the thermostability of virus-like particles of foot-and-mouth disease virus serotype o enhanced immune response of dna vaccine (vp1-pcdna) adsorbed on cationic plg for foot and mouth disease in guinea pigs induction of a cross-reactive cd8+ t cell response following foot-and-mouth disease virus vaccination vp1 of foot-and-mouth disease virus induces apoptosis via the akt signaling pathway foot-and-mouth disease virus capsid protein vp1 interacts with host ribosomal protein sa to maintain activation of the mapk signal pathway and promote virus replication the vp3 structural protein of foot-and-mouth disease virus inhibits the ifn-β signaling pathway foot-and-mouth disease virus structural protein vp3 degrades janus kinase 1 to inhibit ifn-gamma signal transduction pathways host microrna mir-1307 suppresses foot-and-mouth disease virus replication by promoting vp3 degradation and enhancing innate immune response the e3 ubiquitin ligase tbk1 mediates the degradation of multiple picornavirus vp3 proteins by phosphorylation and ubiquitination host microrna-203a is antagonistic to the progression of foot-and-mouth disease virus infection two initiation sites for foot-and-mouth disease virus polyprotein in vivo the two species of the foot-and-mouth disease virus leader protein, expressed individually, exhibit the same activities identification of critical amino acids within the foot-and-mouth disease virus leader protein, a cysteine protease sonenberg, n. eif4 initiation factors: effectors of mrna recruitment to ribosomes and regulators of translation foot-and-mouth disease virus leader proteinase involvement of c-terminal residues in self-processing and cleavage of eif4gi divergent picornavirus ires elements extremely efficient cleavage of eif4g by picornaviral proteinases l and 2a in vitro l protease from foot and mouth disease virus confers eif2-independent translation for mrnas bearing picornavirus ires degradation of nuclear factor kappa b during foot-and-mouth disease virus infection foot-and-mouth disease virus leader proteinase inhibits dsrna-induced type i interferon transcription by decreasing interferon regulatory factor 3/7 in protein levels foot-and-mouth disease virus (fmdv) leader proteinase negatively regulates the porcine interferon-λ1 pathway the leader proteinase of foot-and-mouth disease virus negatively regulates the type i interferon pathway by acting as a viral deubiquitinase irreversible inactivation of isg15 by a viral leader protease enables alternative infection detection strategies impairment of the deisgylation activity of foot-and-mouth disease virus lpro causes attenuation in vitro and in vivo dissecting distinct proteolytic activities of fmdv lpro implicates cleavage and degradation of rlr signaling proteins, not its deisgylase/dub activity, in type i interferon suppression lgp2 synergy with mda5 in rlr-mediated rna recognition and antiviral signaling pum1 is a biphasic negative regulator of innate immunity genes by suppressing lgp2 innate immune sensor lgp2 is cleaved by the leader protease of foot-and-mouth disease virus stress granules and cell signaling: more than just a passing phase? translation inhibition and stress granules in the antiviral immune response stress granules regulate double-stranded rna-dependent protein kinase activation through a complex containing g3bp1 and caprin1 the stress granule protein g3bp1 recruits protein kinase r to promote multiple innate immune antiviral responses foot-and-mouth disease virus leader protease cleaves g3bp1 and g3bp2 and inhibits stress granule formation viroporin activity of the foot-and-mouth disease virus non-structural 2b protein functional analysis of picornavirus 2b proteins: effects on calcium homeostasis and intracellular protein trafficking differential roles of mda5 and rig-i helicases in the recognition of rna viruses viral rna detection by rig-i-like receptors foot-and-mouth disease virus viroporin 2b antagonizes rig-i-mediated antiviral effects by inhibition of its protein expression foot-and-mouth disease virus infection inhibits lgp2 protein expression to exaggerate inflammatory response and promote viral replication foot-and-mouth disease virus non-structural protein 2b negatively regulates the rlr-mediated ifn-β induction eef1g interaction with foot-and-mouth disease virus nonstructural protein 2b: identification by yeast two-hybrid system kinectin-dependent assembly of translation elongation factor-1 complex on endoplasmic reticulum regulates protein synthesis foot-and-mouth disease virus nonstructural protein 2b interacts with cyclophilin a, modulating virus replication cyclophilin a-regulated ubiquitination is critical for rig-i-mediated antiviral immune responses the picornavirus replication inhibitors hbb and guanidine in the echovirus-9 system: the significance of viral protein 2c testing the modularity of the n-terminal amphipathic helix conserved in picornavirus 2c proteins and hepatitis c ns5a protein guanidine-resistant mutants of aphthovirus induce the synthesis of an altered nonstructural polypeptide, p34 recombination and oligonucleotide analysis of guanidine-resistant foot-and-mouth disease virus mutants targeting beclin 1 for viral subversion of macroautophagy the beclin 1 network regulates autophagy and apoptosis foot-and-mouth disease virus nonstructural protein 2c interacts with beclin1, modulating virus replication a putative protein-sequestration site involving intermediate filaments for protein degradation by autophagy. studies with transplanted sendai-viral envelope proteins in htc cells intracellular protein catabolism: evidence for sequestration of proteins into an intermediate-filament fraction before lysosomal degradation foot-and-mouth disease virus modulates cellular vimentin for virus survival role of nonstructural proteins 3a and 3b in host rangeand pathogenicity of foot-and-mouth diseasevirus susceptibility to viral infection is enhanced by stable expression of 3a or 3ab proteins from foot-and-mouth disease virus interaction of foot-and-mouth disease virus nonstructural protein 3a with host protein dctn3 is important for viral virulence in cattle a partial deletion in non-structural protein 3a can attenuate foot-and-mouth disease virus in cattle evaluation of infectivity and transmission of different asian foot-and-mouth disease viruses in swine foot-and-mouth disease virus non-structural protein 3a inhibits the interferon-β signaling pathway comparative genomics of foot-and-mouth disease virus vpg gene amplification correlates with infective particle formation in foot-and-mouth disease virus domain disruptions of individual 3b proteins of foot-and-mouth disease virus do not alter growth in cell culture or virulence in cattle foot-and-mouth disease virus 3b protein inhibits type i interferon pathway signaling by blocking the interaction of rig-i with trim25 foot-and-mouth disease virus protease 3c induces specific proteolytic cleavage of host cell histone h3 foot-and-mouth disease virus 3c protease induces cleavage of translation initiation factors eif4a and eif4g within infected cells cleavage of translation initiation factor 4ai (eif4ai) but not eif4aii by foot-and-mouth disease virus 3c protease: identification of the eif4ai cleavage site sequential modification of translation initiation factor eif4gi by two different foot-and-mouth disease virus proteases within infected baby hamster kidney cells: identification of the 3cpro cleavage site dominant negative mutants of mammalian translation initiation factor eif-4a define a critical role for eif-4f in cap-dependent and cap-independent initiation of translation the nuclear protein sam68 is cleaved by the fmdv 3c protease redistributing sam68 to the cytoplasm during fmdv infection of host cells foot-and-mouth disease virus 3c protease cleaves nemo to impair innate immune signaling 3cpro of foot-and-mouth disease virus antagonizes the interferon signaling pathway by blocking stat1/stat2 nuclear translocation foot-and-mouth disease virus infection suppresses autophagy and nf-kb antiviral responses via degradation of atg5-atg12 by 3cpro regulation of innate immunity through rna structure and the protein kinase pkr foot-and-mouth disease virus induces lysosomal degradation of host protein kinase pkr by 3c proteinase to facilitate virus replication biological function of foot-and-mouth disease virus non-structural proteins and non-coding elements foot-and-mouth disease virus 5 -terminal s fragment is required for replication and modulation of the innate immune response in host cells infectious foot-and-mouth disease virus derived from a cloned full-length cdna the pseudoknot region of the 5 untranslated region is a determinant of viral tropism and virulence of foot-and-mouth disease virus the rhinovirus type 14 genome contains an internally located rna structure that is required for viral replication identification of an rna hairpin in poliovirus rna that serves as the primary template in the in vitro uridylylation of vpg identification and characterization of a cis-acting replication element (cre) adjacent to the internal ribosome entry site of foot-and-mouth disease virus translation and replication of fmdv rna the 3 end of the foot-and-mouth disease virus genome establishes two distinct long-range rna-rna interactions with the 5 end region enhanced ires activity by the 3 utr element determines the virulence of fmdv isolates deletion or substitution of the aphthovirus 3 ncr abrogates infectivity and virus replication the pathogenesis of foot-and-mouth disease in pigs morphologic and phenotypic characteristics of myocarditis in two pigs infected by foot-and mouth disease virus strains of serotypes o or a. acta veter effect of foot-and-mouth disease virus infection on the frequency, phenotype and function of circulating dendritic cells in cattle loss of plasmacytoid dendritic cell function coincides with lymphopenia and viremia during foot-and-mouth disease virus infection natural killer cell dysfunction during acute infection with foot-and-mouth disease virus cell mediated innate responses of cattle and swine are diverse during foot-and-mouth disease virus (fmdv) infection: a unique landscape of innate immunity discriminating self and non-self by rna: roles for rna structure, misfolding, and modification in regulating the innate immune sensor pkr the authors would like to thank the anonymous editors and reviewers for their valuable comments and suggestions that helped improve the quality of this manuscript. the authors declare no conflict of interest. key: cord-002238-fyztb8d9 authors: young, d. f.; andrejeva, j.; li, x.; inesta-vaquera, f.; dong, c.; cowling, v. h.; goodbourn, s.; randall, r. e. title: human ifit1 inhibits mrna translation of rubulaviruses but not other members of the paramyxoviridae family date: 2016-09-29 journal: j virol doi: 10.1128/jvi.01056-16 sha: doc_id: 2238 cord_uid: fyztb8d9 we have previously shown that ifit1 is primarily responsible for the antiviral action of interferon (ifn) alpha/beta against parainfluenza virus type 5 (piv5), selectively inhibiting the translation of piv5 mrnas. here we report that while piv2, piv5, and mumps virus (muv) are sensitive to ifit1, nonrubulavirus members of the paramyxoviridae such as piv3, sendai virus (sev), and canine distemper virus (cdv) are resistant. the ifit1 sensitivity of piv5 was not rescued by coinfection with an ifit1-resistant virus (piv3), demonstrating that piv3 does not specifically inhibit the antiviral activity of ifit1 and that the inhibition of piv5 mrnas is regulated by cis-acting elements. we developed an in vitro translation system using purified human ifit1 to further investigate the mechanism of action of ifit1. while the translations of piv2, piv5, and muv mrnas were directly inhibited by ifit1, the translations of piv3, sev, and cdv mrnas were not. using purified human mrna-capping enzymes, we show biochemically that efficient inhibition by ifit1 is dependent upon a 5′ guanosine nucleoside cap (which need not be n7 methylated) and that this sensitivity is partly abrogated by 2′o methylation of the cap 1 ribose. intriguingly, piv5 m mrna, in contrast to np mrna, remained sensitive to inhibition by ifit1 following in vitro 2′o methylation, suggesting that other structural features of mrnas may influence their sensitivity to ifit1. thus, surprisingly, the viral polymerases (which have 2′-o-methyltransferase activity) of rubulaviruses do not protect these viruses from inhibition by ifit1. possible biological consequences of this are discussed. importance paramyxoviruses cause a wide variety of diseases, and yet most of their genes encode structural proteins and proteins involved in their replication cycle. thus, the amount of genetic information that determines the type of disease that paramyxoviruses cause is relatively small. one factor that will influence disease outcomes is how they interact with innate host cell defenses, including the interferon (ifn) system. here we show that different paramyxoviruses interact in distinct ways with cells in a preexisting ifn-induced antiviral state. strikingly, all the rubulaviruses tested were sensitive to the antiviral action of isg56/ifit1, while all the other paramyxoviruses tested were resistant. we developed novel in vitro biochemical assays to investigate the mechanism of action of ifit1, demonstrating that the mrnas of rubulaviruses can be directly inhibited by ifit1 and that this is at least partially because their mrnas are not correctly methylated. paramyxoviruses cause a wide variety of diseases, and yet most of their genes encode structural proteins and proteins involved in their replication cycle. thus, the amount of genetic information that determines the type of disease that paramyxoviruses cause is relatively small. one factor that will influence disease outcomes is how they interact with innate host cell defenses, including the interferon (ifn) system. here we show that different paramyxoviruses interact in distinct ways with cells in a preexisting ifn-induced antiviral state. strikingly, all the rubulaviruses tested were sensitive to the antiviral action of isg56/ifit1, while all the other paramyxoviruses tested were resistant. we developed novel in vitro biochemical assays to investigate the mechanism of action of ifit1, demonstrating that the mrnas of rubulaviruses can be directly inhibited by ifit1 and that this is at least partially because their mrnas are not correctly methylated. p aramyxoviruses are a large group of negative-sense singlestranded rna viruses that cause a wide variety of animal and human diseases. the paramyxoviridae family is divided into two subfamilies, the paramyxovirinae and the pneumovirinae subfamilies. the paramyxovirinae are further subdivided into a number of genera, including morbillivirus (e.g., measles virus [mev] and canine distemper virus [cdv] ), respirovirus (e.g., sendai virus [sev] and parainfluenza virus type 3 [piv3]), and rubulavirus (e.g., mumps virus [muv] , piv2, and piv5). paramyxoviruses are enveloped viruses; the viral glycoproteins protrude from the outer surface of the envelope and function to attach the viruses to their target cells. on the inner surface of the envelope is the matrix (m) protein, which is required for the structural integrity of the virion. the envelope surrounds a helical nucleocapsid, in which the nucleocapsid protein (np) encapsidates genomic or antigenomic rna. associated with the nucleocapsid is the virally encoded polymerase complex. the viral polymerase both transcribes and replicates the viral genome. viral mrnas are capped and polyadenylated by the viral polymerase (for reviews of the molecular biology of paramyxoviruses, see references 1 and 2). despite their limited genetic information, the majority of paramyxoviruses encode small multifunctional accessory proteins that function to aid virus multiplication and block cellular antiviral defense mechanisms; typically, these proteins can block both the production of, and the signaling response to, interferons (ifns) (for reviews, see references 3, 4, 5, 6, and 7). significantly, the mechanisms of action of these multifunctional ifn antagonists differ from one virus to another. undoubtedly, these properties and in general the manner in which paramyxoviruses interact with the ifn system and other innate defense mechanisms are likely to be major factors in determining the type of disease that each virus causes (8) . the ifn response is an extremely powerful antiviral defense system that, unless counteracted by viruses, will limit their replication to such a degree that they will not cause disease or be efficiently transmitted between susceptible hosts (8, 9) . infected cells detect the presence of viruses due to the production by viruses of molecules with molecular signatures (pathogen-associated molecular patterns [pamps]) such as double-stranded rna (dsrna), which activate the ifn induction cascade and result in the secretion of ifn-␣/␤ from infected cells (9, 10) . the release of ifn induces an antiviral state in neighboring uninfected cells by upregulating the expression of hundreds of interferon-stimulated genes (isgs), many of which have direct or indirect antiviral activity (11) . most paramyxoviruses counteract the ifn responses by producing proteins that block ifn induction and/or ifn signaling by a variety of mechanisms (3) (4) (5) (6) (7) . furthermore, they tightly control viral transcription and replication, thereby limiting the production of pamps that may activate the ifn response (12, 13) . indeed, it is probably mistakes that viruses make during transcription and replication, such as the production of copy-back-defective interfering particles, that activate the ifn response (14-16; reviewed in reference 17). nevertheless, the ability of paramyxoviruses to block the ifn response both in tissue culture cells and in vivo is not absolute, and some ifn-␣/␤ will be produced (18, 19) . furthermore, ifn-␥, which can also induce an antiviral state in cells, will also be produced by activated subsets of lymphocytes (20) . therefore, it is inevitable that viruses will infect cells in a preexisting ifn-induced antiviral state, potentially limiting the speed of virus replication and spread. although ifns induce hundreds of isgs, several isgs with direct antiviral activity have been shown to be specific for families or groups of related viruses (11, 21, 22) . with regard to the paramyxoviridae family, we have previously shown that isg56/ifit1 (here referred to as ifit1), which selectively inhibits translation, is the primary effector of the ifninduced antiviral state that limits the replication of the rubulavirus piv5 (23) . pretreatment of cells with ifn-␣/␤ inhibits piv5 protein synthesis but not cellular protein synthesis. this is because ifit1 selectively inhibits the translation of piv5 mrnas but does not affect cellular mrnas (23) . mammalian mrnas have an n-7 methyl guanosine (m 7 gpppn), termed cap 0, at their 5= end that recruits factors involved in rna processing and translation initiation. the first and second nucleosides of mammalian mrnas are also methylated on the 2= hydroxyl group of the ribose ring, generating cap 1 and cap 2, respectively. while cap 1 and cap 2 are not required for efficient mrna translation, ifit1 can inhibit the translation of mrnas that lack cap 1 (24) (25) (26) (27) . ifit1 also binds uncapped, 5=triphosphorylated rna, characteristic of the 5= ends of the genomic and antigenomic rnas of some rna viruses, as well as those of some viral transcripts (28) ; for reviews on the mechanism of action of ifit1 and the ifit family of proteins, see references 21, 26, 27, and 29 . however, recent evidence suggests that there are differences in the mechanisms of action of the murine and human paralog ifit1 proteins. while murine ifit1 (ifit1b) inhibits the translation of mrnas that lack cap 1, it has been proposed that human ifit1 recognizes some other, as-yet-undefined structure near the cap or possibly that 5= mrna sequences may help define the specificity of inhibition by human ifit1 (30) . the rna-capping activity of viral rna polymerases often include 2=-o-methyltransferases (2=-o-mtases), which modify cap 1 and thus can avoid inhibition by ifit1(b), as evidenced by the sensitivity of virus mutants that lack 2=-o-mtase activity (for reviews, see references 21 and 26) . capping and methylation of viral rnas are also important, as such modifications can prevent the activation of rig-i, thereby reducing the amount of ifn produced by virally infected cells (for a review, see reference 31). here we have examined the ability of ifit1 to inhibit the translation of a variety of paramyxovirus mrnas and thus the replication of those viruses. we show that while all rubulaviruses tested were sensitive to ifit1, all nonrubulavirus members of the paramyxoviridae tested were insensitive. lack of 2= o-methylation of rubulavirus mrnas was at least partially responsible for their inhibition by ifit1. the possible biological consequences of differences in sensitivity of paramyxoviruses to ifit1 are discussed. cells, viruses, antibodies, and interferon. a549 cells and derivatives were grown as monolayers in 25-cm 2 , 75-cm 2 , or 300-cm 2 tissue culture flasks in dulbecco's modified eagle's medium supplemented with 10% fetal bovine serum at 37°c. when appropriate, cells were treated with human recombinant interferon (intron a; merck, sharpe and dohme) at 1,000 units/ml. viruses used in these studies were piv2 (strain colindale), piv3 (strains washington and js and recombinant ⌬c and ⌬d js viruses [32] ), piv5 (formerly known as sv5; strains w3 [33] and cpi ϩ and cpi ϫ [34] ), muv (enders [35] ), respiratory syncytial virus (rsv) (36) , sendai virus (strain cantell, free of defective interfering particles), and canine distemper virus (strain mill hill). plaque assays were performed by standard methods in six-well dishes that included 0.1% avicel (fmc biopolymer) in the overlay medium. plaques were visualized by immunostaining by using a pool of monoclonal antibodies or polyclonal antisera specific for the different viruses as described previously (37) , together with alkaline phosphatase-conjugated secondary antibody by using sigmafast bcip/ nbt as the substrate. preparation of l-[ 35 s]methionine-labeled total-cell extracts and sds-page. infected or uninfected cells that had or had not been pretreated with ifn for 12 h prior to infection were metabolically labeled for 1 h with l-[ 35 s]methionine (500 ci/mmol; mp biomedical, usa) at 18 h postinfection (p.i.). after labeling, cells were lysed in disruption buffer, sonicated, and heated for 5 min at 100°c and then analyzed by gel electrophoresis (sds-page). the gels were fixed, stained, and dried, and resolved bands were visualized by phosphorimager analysis. when appropriate, the same amounts of cell equivalents were run on page. furthermore, the amount of protein in each sample was monitored by staining the polyacrylamide gels (pags) with coomassie brilliant blue. immunofluorescence. cells to be stained for immunofluorescence were grown on 10-mm-diameter coverslips (mic3270; scientific laboratory supplies, united kingdom). cells were stained with specific monoclonal antibodies (mabs), as described in detail elsewhere (38) . briefly, monolayers were fixed with 5% formaldehyde-2% sucrose in phosphatebuffered saline (pbs) for 10 min at 20°c, permeabilized with 0.5% nonidet p-40 -10% sucrose in pbs for 5 min at 20°c, and washed three times in pbs containing 1% calf serum. piv5-and piv3-infected cells were detected by indirect immunofluorescence using a secondary goat antimouse ig texas red-conjugated antibody (catalog number ab6787; abcam). the primary antibodies were piv5-np-a and piv5-pe for piv5 (39) and 4721, 2281, and 4812 for piv3 (40) . after staining for immunofluorescence, the monolayers of cells were examined with a nikon microphot-fxa immunofluorescence microscope. rna selection and in vitro translation. rna for in vitro translations was isolated by sedimentation through cscl gradients by a modified method described by leppert et al. (41) . confluent monolayers of infected cells, grown in 300-cm 2 flasks, were resuspended in ice-cold lysis buffer (150 mm nacl, 50 mm tris-hcl [ph 7.5], 0.6% np-40, protease inhib-itor cocktail [complete mini edta-free, 1 tablet per 7 ml of buffer; roche]) at 1 ϫ 10 8 to 2 ϫ 10 8 cells per ml and left on ice for 5 min prior to vortexing for 2 min. nuclei were removed by centrifugation twice at 4,200 ϫ g for 5 min at 4°c. the supernatant (cytoplasmic extract) was collected, made up to 6 mm edta, and layered onto 35% (wt/wt) cscl in 25 mm tris-hcl (ph 7.5)-2 mm edta followed by centrifugation at 175,000 ϫ g at 12°c for 16 to 18 h. naked rna (including mrna) forms a pellet at the bottom of the gradient, while viral genomic and antigenomic rnas remain complexed with nucleoprotein and do not enter the 35% cscl cushion. the supernatant was discarded, and the pellet was resuspended in rnase-free water and adjusted to 1 g/l. selected rna was translated in vitro with a rabbit reticulocyte lysate kit (l4960; promega) in the presence of [ 35 s]methionine-cysteine (neg772, easytag express protein labeling mix; perkinelmer) using a modification to the manufacturer's instructions: methionine-cysteine-free medium (d0422; sigma) was used to provide other amino acids (1 l per 50 l reaction mixture). capping and methylation of mrna. human rna guanylyltransferase and 5=-phosphatase (rngtt), rna guanine-7 methyltransferase (rnmt), and cap methyltransferase 1 (cmtr1) were synthesized and purified according to the method of gonatopoulos-pournatzis et al. (42) . as described in that study, the enzymes were all verified as being active by in vitro reactions followed by thin-layer chromatography. capping and methylation reactions were carried out in 50 mm tris-hcl (ph 8.0), 6 mm kcl, 1.25 mm mgcl 2 , 1 mm dithiothreitol (dtt) buffer as follows: 1 l 10ϫ buffer, 1 l rngtt (2.5 mg/ml), 1 l rnmt (0.5 mg/ml), cmtr1 (0.28 mg/ml), 1 l sam (2 mm), 1 l gtp (1 mm), 0.5 l rnasin, 2 l rna (1 g/l). the reaction mixture was made up to 10 l with h 2 o, including in experiments in which rngtt, rnmt, or cmtr1 was omitted, and incubated at 37°c for 1 h. cloning and purification of ifit1. ifit1 was amplified with primer ifit1f/ifit1xho from the plasmid pgac-ha-ifit1, restricted with ncoi and xhoi, and ligated with a modified plou3, in which maltose binding protein (mpb) was replaced with sumo, while sali in the mutliple cloning site (mcs) was replaced with xhoi. the primers were as follows: ifit1f, ccgccatggctacaaatggtgatgatcatcagg; ifit1xho, gcgcctcgagctaaggaccttgtctcacagagtt. the fusion protein his-sumo-(tev)-ifit1 was expressed in escherichia coli strain rosetta in 6 liters lb-ampicillin-chloramphenicol (lb/ amp/cm). isopropyl-␤-d-thiogalactopyranoside (iptg; 0.2 mm) was added when an optical density (od) of 0.8 was reached. the expression was carried out at 18°c overnight. purification was carried out with a routine protocol for his-tagged protein. the binding buffer contained 20 mm tris-hcl (ph 8.0), 0.3 m nacl, and 10 mm imidazole; the washing buffer contained 30 mm imidazole; and protein was eluted with 300 mm imidazole. to remove nonspecifically bound rna, the columns were washed with 9 volumes of 0.2 m na 2 hpo 4 -4 m nacl (ph 7.5). after desalting into gf buffer (20 mm tris-hcl [ph 8], 150 mm nacl, 5% glycerol), the fusion was cleaved with tobacco etch virus (tev) protease (1:100) at room temperature overnight. gel filtration was carried out after passing through ni beads again and addition of 3 mm dtt. the ifit1 peak was collected and concentrated and had an a 260 /a 280 of 0.7 to 0.8. despite the fact that paramyxoviruses encode ifn antagonists that inhibit ifn production and signaling, their ability to block the ifn response is not absolute. thus, they form larger plaques on ifn-incompetent cells than ifn-competent cells ( fig. 1 ) (19) , showing that during virus replication and spread some ifn is produced and slows the spread of the viruses (see fig. 3 ). in the experiments shown in fig. 1 and below, we used naive a549, a549/npro, and a549/shifit1 cells; naive a549 cells can produce and respond to ifn in response to virus infection, and a549/npro cells respond to exogenous ifn but cannot produce ifn as they constitutively express npro from bovine viral diarrhea virus (bvdv), which targets irf-3 for degradation (43) . furthermore, because irf-3 is degraded in a549/npro cells, they cannot upregulate expression of ifit1 in an irf-3-dependent, ifn-independent manner in direct response to virus infection (29) . a549/shifit1 cells produce and respond to ifn, but expression of endogenous ifit1 in response to ifn or viral infection is inhibited due to constitutive expression of small hairpin rna (shrna) to ifit1 (23) . we previously showed that ifit1 is the major cellular protein responsible for the ifn sensitivity of the rubulavirus piv5 (23) . to further investigate the role of ifit1 and the ability of ifn to induce an antiviral state against other paramyxoviruses, we initially tested the ability of piv2, piv3, and piv5 to form plaques in a549, a549/npro, and a549/shifit1 cells. all three viruses induced ifn in a549 cells as the plaques developed, as observed by the induction of mxa in the uninfected cells surrounding the plaque (fig. 1a) . as previously observed (23), piv5 formed bigger plaques on a549/shifit1 cells than on a549 cells, but the plaques were not as large as those on a549/npro cells (fig. 1b) . while piv2 also produced slightly larger plaques on a549/shifit1 than on a549 cells, the plaques on a549/npro cells were obviously bigger (note that the center of mid-to large-sized piv2 plaques has fallen out of monolayers). piv3 produced similarly sized plaques on a549 and a549/shifit1 cells and slightly larger plaques on a549/npro cells. these results also support our previous conclusion that in a549 (and hep2) cells ifit1 is the primary isg effector to piv5 (23) and that the rubulavirus piv2 is also sensitive to ifit1. however, knocking down ifit1 did not have such a marked effect on piv2 plaque size as it did for piv5. this indicates that there are likely to be additional isgs that play an important role in ifn-mediated inhibition of piv2. in contrast, piv3 (washington strain) produced similarly sized plaques on a549 and a549/shifit1 cells and only slightly larger plaques on a549/npro cells; this suggests that the ifn response is capable of slowing the spread of piv3 to some degree (but not through the activity of ifit1), but not as dramatically as it does for piv2 or piv5. however, experiments on the js strain of piv3 showed it to be more sensitive to the antiviral effects of ifn, but this was not because js is sensitive to ifit1 (data not shown). we next compared the synthesis of viral proteins in cells infected with piv2, piv3, and piv5 that had, or had not, been pretreated with ifn prior to infection with piv2, piv3, and piv5. cells were infected at a high multiplicity of infection (moi; 10 to 20 pfu/cell), and the relative levels of np synthesis were visualized by radioactively labeling the cells for 1 h with [ 35 s]methionine at 18 h p.i. (fig. 2) . pretreatment of a549 and a549/npro cells with ifn in this assay reduced the expression of the np of piv2 and piv5 to barely detectable levels. however, ifn pretreatment had no discernible effect on the expression of the np protein of piv3 or on the expression of host cell proteins. strikingly, expression of np of piv2 and piv5 was largely rescued in ifn-pretreated a549/shifit1 cells, demonstrating that ifit1 plays a major role in the inhibition of piv2 and piv5 protein synthesis observed in a549 and a549/npro cells pretreated with ifn. figure 2 is an exemplar of many similar experiments that we have performed under different conditions (time course, moi, etc.) and that show the same results, namely, that piv2 and piv5 are inhibited by ifit1 while piv3 is not. having demonstrated that piv2 and piv5 are sensitive to ifit1 while piv3 is resistant, we tested the sensitivity of other members of the paramyxoviridae family, namely, mumps virus (muv strain enders), sendai virus (sev), and canine distemper virus (cdv). in a set of experiments similar to those illustrated in fig. 2 , a549/npro and a549/shifit1 cells were or were not pretreated with ifn prior to infection with high moi with these viruses. the relative levels of np synthesis were visualized by radioactively labeling the cells for 1 h with [ 35 s]methionine at 18 h p.i. (fig. 3a) . these experiments clearly demonstrated that, as was observed for piv2 and piv5, pretreating a549 cells with ifn inhibited muv strain enders protein synthesis but knocking down ifit1 expression could largely restore muv protein synthesis. in contrast, as was observed for piv3, although pretreatment of a549 cells with ifn slightly reduced the expression of sev and cdv protein synthesis, no increase in sev and cdv protein synthesis was observed in a549/shifit1 compared to a549 cells pretreated with ifn. these results therefore show that muv enders is sensitive to ifit1 but sev and cdv are not, the weak inhibition of sev and cdv protein synthesis observed in a549 and a549/shifit1 cells pretreated with ifn presumably being due to the action of other isgs induced by ifn. while muv is sensitive to ifit1, it forms pinpoint plaques on a549/npro cells only at 5 days p.i. (data not shown), strongly suggesting that there are host cell restrictions other than innate intracellular defense mechanisms on muv replication in a549 cells (44) . since in these experiments we used the attenuated enders strains of muv to test whether attenuation may be linked to sensitivity to ifit1, we tested a wild-type (wt) isolate of muv-london-1 (lo-1) for its sensitivity. at the same time, we also tested the sensitivity of another strain of piv5, termed cpi ϩ (fig. 3b) . muv-lo was as sensitive as muv enders, demonstrating that attenuation was not linked to differences in their relative sensitivity to ifit1. similarly, piv5 cpi ϩ was also sensitive to inhibition by ifit1. the ifit1 sensitivity of piv5 is not rescued by coinfection with an ifit1-resistant virus. from these results, it was clear that replication of the nonrubulaviruses piv3, sev, and cdv is not inhibited by ifit1. to investigate whether piv5 replication could be rescued by coinfections with an ifit1-resistant virus, mixed infections between piv3 and piv5 were undertaken. to avoid any possible synergistic effects between piv3 and piv5 in dismantling an ifn-induced antiviral state, the cpi ϫ strain of piv5 was used in these experiments because, due to mutations in its v protein, it does not block ifn signaling (45) . a549 or a549/shifit1 cells were or were not pretreated with ifn for 8 h prior to high-moi (10 to 20 pfu/cell) infection with piv5, piv3, or a mixture of the two viruses (fig. 4a) . the expression of the np protein of piv3 was resistant to ifn in both a549 and a549/shifit1 cells when they were infected with piv3 alone and when coinfected with piv5. in contrast, while the expression of piv5 np was resistant to ifn in a549/shifit1 cells, its expression was inhibited in a549 cells, even when the cells were coinfected with piv3. immunofluorescence was undertaken to ensure that in these experiments there was no exclusion of one virus by the other (fig. 4b and c) . these results confirmed that coinfection of piv3 with piv5 does not rescue the sensitivity of piv5 to ifit1 and strongly suggest that piv3 does not specifically inhibit the antiviral activity of ifit1 and that the inhibition of piv5 np expression is regulated by cis-acting elements. differential inhibition of translation of mrnas of different paramyxoviruses by purified ifit1. the data above show that the ifn sensitivity of rubulaviruses is at least in part due to the actions of ifit1. since this cellular protein has been shown to inhibit translation in a template-specific manner, we developed an in vitro translation system to study the ability of human ifit1 to selectively inhibit the translation of rubulavirus mrnas. the gene encoding human ifit1 was cloned as a sumo fusion protein expressed in escherichia coli, and the recombinant protein was purified (fig. 5a) . to determine whether the recombinant ifit1 was able to selectively inhibit piv5 mrnas, in vitro translation of mrna isolated from mock-and piv5-infected cells was carried out in the presence and absence of different concentrations of ifit1 (fig. 5b, c, and d) . in the absence of ifit1, expression of the np protein (and to a lesser extent the m protein) of piv5 was clearly visualized in the background of in vitro-translated cellular proteins ( fig. 5c and d) . increasing concentrations of ifit1 had no obvious effect on the efficiency of translation of host cell proteins, but in striking contrast, purified ifit1 selectively inhibited the translation of the np and m proteins of piv5 in a concentration-dependent manner. having established that the sensitivity of in vitro translation of piv5 mrna to inhibition by purified ifit1 correlated with the biological sensitivity of piv5 to ifit1, we next tested the ability of ifit1 to inhibit the translation of mrna isolated from cells in-fected with other paramyxoviruses (fig. 6) . these results clearly demonstrated that translation of (np) mrnas from piv2-and from muv-infected cells was inhibited by ifit1. in contrast, there was no obvious reduction in the amount of piv3 np synthesized when increasing amounts of ifit1 was added to the in vitro translation reaction mixtures. although there was a slight apparent reduction in the amount of sev and cdv np synthesis in the samples in which ifit1 was added, there was no increase in the inhibition observed by increasing the amount of ifit1 added to the in vitro translation reaction mixtures, strongly suggesting that the translations of sev and cdv mrnas are also resistant to inhibition by ifit1. lack of 2=-o methylation of the cap structure of muv and piv5 mrnas is partially responsible for their sensitivity to inhibition by ifit1. previous studies have shown that the absence of cap 1 on mrnas renders them sensitive to inhibition to ifit1. to investigate whether this was the case for rubulavirus mrnas, we developed an in vitro assay in which purified human mrnamodifying enzymes were used to progressively cap and add different methyl groups to the 5= ends of mrnas. purified human rna guanylyltransferase and 5=-phosphatase (rngtt), rna guanine-7 methyltransferase (rnmt), and cap methyltransferase 1 (cmtr1) were used in these assays. rngtt adds a 5= guanosine to rnas with 5=-ppp, while rnmt adds a methyl group to the 7 g of the guanine ring, generating (m 7 g) cap 0. cmtr1 adds a methyl group to the 2= oh position of the adjacent ribose, generating cap 1. to demonstrate the functionality of this system, we first tested the in vitro translation of luciferase mrna with a 5=triphosphate group. this rna was efficiently translated in a capindependent manner and was only weakly inhibited by ifit1 (fig. 7a, compare lanes 1 and 2) . when the luciferase mrna was capped with the addition of 5=-guanosine by rngtt (generating gppp-mrna), there was a slight decrease in the amount of luciferase made (fig. 7a, compare lanes 1 and 3) . this may have been due to rngtt destabilizing or blocking the translation of gppp-mrnas in the absence of 7 n methylation. however, strikingly, translation of this mrna was completely inhibited by ifit1 (fig. 7a , lane 4) despite this cap structure lacking n-7 methylation. as expected, the addition of a methyl group to the n-7 position of the guanine ring, generating m 7 gpppm 2 n, by rnmt increased the efficiency of translation, but m 7 gppp-luciferase remained completely sensitive to inhibition by ifit1 (fig. 7a, lanes 5 and 6) . addition of a methyl group to the 2= oh group of the adjacent ribose, generating cap 1, by cmtr1 did not affect the efficiency by which the mrna was translated, but it did clearly reduce the sensitivity of the mrna to inhibition by ifit1 (fig. 7a, compare lanes 7 and 8) . however, it should be noted that in these experiments, for reasons that are unclear, we were unable to completely restore full translation of the luciferase mrna in the presence of ifit1 by increasing the amount of cmtr1 or the length of incubation of the mrna with the enzyme (data not shown). to investigate how similar modifications to the cap of rubulavirus mrnas influenced their inhibition by ifit1, we initially used muv mrna in a parallel set of experiments. these results showed that treatment of the muv mrna with rngtt and rnmt did not increase the efficiency of in vitro translation of muv np mrna or its sensitivity to inhibition by ifit1 (fig. 7b, lanes 1 to 6) , consistent with the viral polymerase adding m 7 gppp-cap at (cap 0) to the 5= end of viral mrnas. however, surprisingly, since rubulavirus polymerases have conserved 2=-o mtase domains, addition of a methyl group to the 2=oh group of the adjacent ribose (cap 1) by cmtr1 clearly reduced the sensitivity of the np mrna to inhibition by ifit1 (fig. 7b, lanes 7 and 8) . as expected, the ifit1 sensitivity was dependent on the addition of s-adenosyl methionine (sam) to the reaction mixture (fig. 7c) . similarly, following 2=o methylation of piv5 mrna, in vitro translation of piv5 np became completely resistant to inhibition by ifit1 (fig. 7d) . strikingly, in contrast to np, the translation of piv5 m mrna remained completely sensitive to inhibition by ifit1 even after 2=o methylation of piv5 mrna by cmtr1 (fig. 7d and e) ; the basis for this is currently unknown, but we are investigating it further. over the past decade or so, it has become clear that the ways in which paramyxoviruses circumvent innate immune responses, including the ifn response, and differences in the multifunctional nature of their ifn antagonists are likely to influence the types of disease they cause. for example, the viral ifn antagonists within the rubulavirus genus, namely, the v proteins, as well as interacting with common targets such as mda 5 and lgp2 also have unique properties. the v protein of piv5 targets stat1 for degradation, piv2 targets stat2, and muv targets both stat1 and stat3. within the respirovirus and morbillivirus genera, it is a combination of the v and c proteins that counteract innate responses by different molecular mechanisms, and strikingly, although piv3 encodes a c protein, it does not encode a functional v protein. despite encoding of these powerful ifn antagonists, ifn is produced during virus spread both in tissue culture cells and in vivo, and thus undoubtedly paramyxoviruses will, during the course of an infection, infect cells in a preexisting ifn-induced antiviral state. here we show that different paramyxoviruses interact in distinct ways with cells in a preexisting ifn-induced antiviral state, and we suggest that this may influence the types of diseases caused. strikingly, in contrast to the sensitivity of rubulaviruses to ifit1, the other paramyxoviruses that we tested were resistant, strongly suggesting that this might be a distinguishing feature of rubulaviruses, although before this can be firmly concluded the sensitivity of more species of paramyxoviruses to ifit1 needs to be tested. even within the rubulavirus genus, it appears that there may be differences in how members interact with cells in an ifn-induced antiviral state. in a549 cells, ifit1 primarily is responsible for the ifn-induced antiviral state induced to counter piv5. however, although piv2 is sensitive to ifit1, there appear to be other isgs that have strong anti-piv2 activity. this conclusion comes from the observation that while there is a slight increase in the size of piv2 plaques on a549/shifit1 cells compared to a549 cells, it is not as obvious as that observed for piv5. furthermore, while plaques for piv5 were smaller on a549/shifit1 cells than on a549/npro cells, this difference was not as marked as that observed for piv2. muv strain enders is also sensitive to ifit1, but there are clearly other major constraints on the growth of muv enders in human cells, as the virus grows extremely poorly in ifn-incompetent human cells but replicates to high titers in vero cells (44) . it is striking that only rubulaviruses are sensitive to the antiviral activity of human ifit1. our data indicate that the inhibition of rubulavirus mrnas was produced by ifit1 in a cis-linked manner, implying that the restriction is associated with some feature of uncapped 5=-ppp mrna encoding luciferase synthesized by t7 polymerase (provided as a control in the promega in vitro translation kit) was translated in vitro in a rabbit reticulocyte lysate in the absence or presence of purified ifit1 (lanes 1 and 2). rngtt was used to add a 5= guanine cap (lanes 3 and 4); then, rnmt was used to methylate the cap at the n7 position (lanes 5 and 6), generating cap 0, and cmtr1 was used to methylate the adjacent ribose on the 2= oh position, generating cap 1. the modified mrnas were then in vitro translated in the absence (lanes 3, 5, and 7) or presence (lanes 4, 6, and 8) of ifit1. (b) mrna isolated from muv-infected cells was treated in parallel under the same conditions as those described for panel a. (c and d) mrna isolated from either muv (enders)or piv5 (w3)-infected cells was in vitro translated prior to (lanes 1 and 2) or following (lanes 3 and 4) modification by cmtr1 in the presence or absence (lanes 5 and 6) of sam. the mrna was also translated in the absence (lanes 1, 3, and 5) or presence (lanes 2, 4 and 6) of purified ifit1. (e) densitometry traces of lanes 1, 2, 3, and 4 of panel d. the numbers at the bottom of the gel indicate the fraction of either the host cell proteins or np proteins made in the in vitro translation mixes in the presence of purified ifit1 compared to those made in the absence of ifit1. the mrna sequence or structure. since ifit1 can selectively inhibit the translation of mrnas that are incorrectly capped or not methylated at the 2= oh group of the first ribose, i.e., cap 1 (24, 46, 47) , it was likely that rubulaviruses have a structural motif in their cap, not present or hidden in the mrna of other paramyxoviruses, that is recognized by ifit1. to investigate this further, we used purified human enzymes to modify the cap of mrnas. as a control for the activity of the enzymes, we used an uncapped 5=ppp mrna that encodes luciferase. the 5=-ppp luciferase mrna translated in a cap-independent manner in vitro using rabbit reticulocyte lysate, and this translation was only weakly inhibited by purified ifit1. while addition of a 5= guanosine nucleoside cap slightly decreased the amount of luciferase synthesized, probably because the enzyme rngtt destabilizes the mrna, addition of the (unmethylated) guanosine nucleoside to the 5= end of the mrna significantly increased the sensitivity of the mrna to inhibition by ifit1. furthermore, although methylation of the guanine ring at position n7 (m7gpppnp-rna) by rnmt increased the efficiency of translation of luciferase mrna, it did not appear to affect the sensitivity of inhibition by ifit1. these results are therefore consistent with the observation that human ifit1 binds with low affinity to 5=-ppp rna but more avidly to cap 0 rna lacking 2= o methylation. methylation at position n7 of the guanine ring has also been reported to increase the affinity of binding of ifit1 (24); however, the observation here that gppp-luciferase is inhibited as efficiently as m7gppp-luciferase suggests that the methyl group does not play a central role in the inhibition of mrnas by ifit1. in contrast, 2= o-methylation of the first ribose by cmtr1 to generate cap 1 partially prevented ifit1 from inhibiting the translation of the cap 0-modified mrna. however, even by increasing the amount of cmtr1 and the incubation time, we were unable to completely restore full translational activity of the luciferase mrna. the reasons for this are unclear, but it suggests that other structural features, for example, methylation of the penultimate ribose to generate cap 2 or sequences at the 5= end of mrnas, may also influence inhibition by ifit1, as has been suggested by daugherty et al. (30) . mrnas isolated from piv3-, sev-, and cdv-infected cells were not inhibited by ifit1, and neither was the replication of these viruses ( fig. 2 and 3 ). in contrast, 2= o-methylation of the terminal ribose by cmtr1 of muv mrnas partially alleviated inhibition of the np mrna by ifit1. with regards to piv5, our previous studies suggested that piv5 mrnas were 2= o-methylated (23) . furthermore, we never observed complete ifit1 inhibition of piv5 np synthesis in vitro, suggesting that at least a proportion of the piv5 np mrna was correctly capped. however, the fact that treating piv5 mrnas with cmtr1 rescued np synthesis in the presence of ifit1 suggests that a significant proportion of piv5 mrnas was also not fully methylated. it is also of potential significance that the m mrna of piv5 appears to be more sensitive than np mrna to inhibition by ifit1, and furthermore, translation inhibition of piv5 m mrna was not rescued by treatment with cmtr1. the differences in the relative sensitivity of the np and m mrnas clearly warrant further investigation but may be due to the fact that the viral methyltransferase differentially methylates the viral mrnas (as has been shown for vesicular stomatitis virus [vsv] [48] ), that cmtr1 does not recognize the untranslated region (utr) of the piv5 m mrna, or that inhibition by ifit1 is influenced by additional structural features present on piv5 m mrna but not np mrna. regarding the latter point, it is of note that the first three nucleotides of the utrs of np and m differ. furthermore, the 4 or 5 nucleotides downstream of cap 0 are thought to be bound by ifit1 and may thus modulate ifit1-rna interactions (49) , and some secondary rna structures, e.g., those found at the 5= end of some alphaviruses, can prevent ifit1 binding to rna independent of the cap methylation status (50) . most viruses successfully avoid inhibition by ifit1 by encoding their own 2=-o mtase, by cap snatching appropriately capped and 2=-o methylated structures from cellular mrnas, or by having cap-independent translation with the covalently linked viral protein vpg or a 5= rna secondary structure that blocks the activity of ifit1 (reviewed in reference 26). indeed, work on virus restriction by ifit1 has involved primarily the investigation of viruses in which the 2=-o-mtases have been mutated such that their mrnas do not have a cap 1 structure (25, (51) (52) (53) (54) . nevertheless, our results show that the viral polymerase of rubulaviruses, unlike other paramyxoviruses, does not fully protect the viral mrnas from inhibition by human ifit1. in this regard, it is of interest that although rubulaviruses have the conserved methyltransferase domain in their polymerase, they all have an alanine instead of the first glycine in a gxgxg motif present in the methyltransferase domain of other paramyxoviruses and mononegavirales, which has been shown to affect the efficiency of cap methylation (55) . most viruses, including other mononegavirales (56), appear to be naturally resistant to inhibition by ifit1. it is therefore intriguing that rubulaviruses have not evolved mechanisms to ensure that their mrnas are correctly capped and methylated or have the appropriate utrs to be resistant to ifit1. it is tempting to speculate that there is some unknown biological advantage to being sensitive to ifit1. for example, it may help some rubulaviruses (and perhaps hepatitis c virus [57] , which is also sensitive to ifit1) to establish prolonged or persistent infections. thus, following infection of cells in an ifn-induced antiviral state, ifit1 restricts piv5 replication. under such conditions, virus genomes are located in cytoplasmic foci, where, as we have previously suggested, they may remain hidden from intracellular and adaptive immune responses. furthermore, if viral mrna is produced in cells in an ifn-induced antiviral state, then viral protein synthesis will largely be inhibited by ifit1, thus reducing the amount of protein that may be processed and presented to cytotoxic t lymphocytes (ctls). eventually, in such cells, however, enough of the virus ifn antagonist, the v protein, will be produced or brought in by infecting virus particles to target stat1 for proteasomemediated degradation, and the cells will no longer be able to maintain their antiviral state, thus facilitating virus replication (58) . whether such a scenario occurs in vivo, these and other considerations emphasize that to fully understand the molecular pathogenesis of viruses, it will be necessary to understand the subtleties of how viruses interact with the ifn system and other host cell defense mechanisms. the biology of paramyxoviruses fields virology paramyxovirus activation and inhibition of innate immune responses the regulation of type i interferon production by paramyxoviruses paramyxovirus disruption of interferon signal transduction: status report inhibition of interferon induction and signaling by paramyxoviruses paramyxovirus evasion of innate immunity: diverse strategies for common targets interferons and viruses: an interplay between induction, signalling, antiviral responses and virus countermeasures newly described pattern recognition receptors team up against intracellular pathogens pattern recognition of viral nucleic acids by rig-i-like helicases interferon-stimulated genes and their antiviral effector functions plk1 down-regulates parainfluenza virus 5 gene expression role for the phosphoprotein p subunit of the paramyxovirus polymerase in limiting induction of host cell antiviral responses sendai virus defectiveinterfering genomes and the activation of interferon-beta deep sequencing analysis of defective genomes of parainfluenza virus 5 and their role in interferon induction defective viral genomes arising in vivo provide critical danger signals for the triggering of lung antiviral immunity defective viral genomes: critical danger signals of viral infections parainfluenza virus 5 genomes are located in viral cytoplasmic bodies whilst the virus dismantles the interferon-induced antiviral state of cells virus replication in engineered human cells that do not respond to interferons regulation of interferon-gamma during innate and adaptive immune responses interferon-induced ifit proteins: their role in viral pathogenesis a diverse range of gene products are effectors of the type i interferon antiviral response isg56/ifit1 is primarily responsible for interferon-induced changes to patterns of parainfluenza virus type 5 transcription and protein synthesis inhibition of translation by ifit family members is determined by their ability to interact selectively with the 5=-terminal regions of cap0-, cap1-and 5=ppp-mrnas 2=-o methylation of the viral mrna cap evades host restriction by ifit family members innate immune restriction and antagonism of viral rna lacking 2-o methylation ifits: emerging roles as key anti-viral proteins ifit1 is an antiviral protein that recognizes 5=-triphosphate rna the isg56/ifit1 gene family evolutionguided functional analyses reveal diverse antiviral specificities encoded by ifit1 genes in mammals when your cap matters: structural insights into self vs non-self recognition of 5= rna by immunomodulatory host proteins mutations in the c, d, and v open reading frames of human parainfluenza virus type 3 attenuate replication in rodents and primates multiplication of a myxovirus (sv5) with minimal cytopathic effects and without interference parainfluenza virus surface projections: glycoproteins with haemagglutinin and neuraminidase activities attenuation of virulence with retention of antigenicity of mumps virus after passage in the embryonated egg respiratory syncytial virus: reverse genetics and vaccine strategies heterocellular induction of interferon by negativesense rna viruses intranuclear localization of herpes simplex virus immediate-early and delayed-early proteins: evidence that icp 4 is associated with progeny virus dna isolation and characterization of monoclonal antibodies to simian virus 5 and their use in revealing antigenic differences between human, canine and simian isolates antigenic analysis of human and bovine parainfluenza virus type 3 strains with monoclonal antibodies plus and minus strand leader rnas in negative strand virus-infected cells ram/fam103a1 is required for mrna cap methylation the npro product of bovine viral diarrhea virus inhibits dna binding by interferon regulatory factor 3 and targets it for proteasomal degradation mumps virus enders strain is sensitive to interferon (ifn) despite encoding a functional ifn antagonist differences in interferon sensitivity and biological properties of two related isolates of simian virus 5: a model for virus persistence sequestration by ifit1 impairs translation of 2=o-unmethylated capped rna structural basis for viral 5=-ppp-rna recognition by human ifit proteins ribose 2=-o methylation of the vesicular stomatitis virus mrna cap precedes and facilitates subsequent guanine-n-7 methylation by the large polymerase protein characterization of transgenic mice with targeted disruption of the catalytic domain of the doublestranded rna-dependent protein kinase, pkr a viral rna structural element alters host recognition of nonself rna ifit1 inhibits japanese encephalitis virus replication through binding to 5= capped 2=-o unmethylated rna attenuation and restoration of severe acute respiratory syndrome coronavirus mutant lacking 2=-omethyltransferase activity 2=-o methylation of the viral mrna cap by west nile virus evades ifit1-dependent and -independent mechanisms of host restriction in vivo rational design of human metapneumovirus live attenuated vaccine candidates by inhibiting viral mrna cap methyltransferase sequence-function analysis of the sendai virus l protein domain vi human and murine ifit1 proteins do not restrict infection of negative-sense rna viruses of the orthomyxoviridae, bunyaviridae, and filoviridae families isg56 and ifitm1 proteins inhibit hepatitis c virus replication catalytic turnover of stat1 allows piv5 to dismantle the interferon-induced antiviral state of cells key: cord-007664-c5snhymz authors: mauerhoff, thekla; pujol-borrell, ricardo; mirakian, rita; bottazzo, gian franco title: differential expression and regulation of major histocompatibility complex (mhc) products in neural and glial cells of the human fetal brain date: 2002-11-13 journal: j neuroimmunol doi: 10.1016/0165-5728(88)90049-5 sha: doc_id: 7664 cord_uid: c5snhymz the cells of the central nervous system (cns) have the peculiarity of physiologically expressing very low levels of hla molecules. in multiple sclerosis (ms), however, as in endocrine autoimmune diseases, there is a marked increase of hla expression in the tissue (i.e. the plaques) and this is attributable not only to infiltrating cells but also to the astrocytes. to gain an insight into the regulation of hla in the different cell types in the cns and to compare it to that observed in the endocrine organs, we have studied the effect of the lympho/monokines interferon (ifn)-α and -γ, tumour necrosis factor (tnf)-α, and interleukin (il)-2 and other agents on this aspect of the biology of human fetal brain cells in culture. a two-colour immunofluorescence technique which combines antibodies to diverse cns cell markers and monoclonal antibodies (moabs) to the non-polymorphic region of hla molecules was used throughout this study. in control cultures, only astrocytes expressed mhc class i, but after incubation with either ifn-γ or tnf-α oligodendrocytes acquired class i expression. surprisingly, astrocytes became spontaneously class ii positive in culture and this was greatly enhanced by ifn-γ. other agents such as il-2, epidermal growth factor, phorbolmyristate acetate and lectins had no effect. the expression of hla molecules in the cells of the cns both in basal conditions and in response to lymphokines is therefore selective and highly heterogenous, thus reflecting their intrinsic biological diversity. these findings may help to explain the features of the immunopathology of ms and also of latent viral infections of neural cells. the level of cell surface expression of class i and class ii products of the major histocompatibility complex (mhc) (hla in humans) varies widely among different cell types and it seems to be determined not only by their ontogenic lineage but to be also influenced by the direct action of several inducers and modulators. of the latter, the best known are lymphokines and in particular those belonging to the interferon (ifn) family (review in pujol-borrell and . recently, tumour necrosis factor (tnf-a) and lymphotoxin (lt or tnf-fl) , produced by macrophages and t lymphocytes respectively, have also been found to induce and/or enhance hla molecule expression in certain systems (collins et al., 1986; pfizenmaier et al., 1987; . our previous work has clearly indicated that human endocrine cells have different sensitivities to the in vitro effect of lympho/monokines with regard to the induction of hla class ii expression. thus viable thyrocytes are easily inducible following incubation with mitogens (pujol-borrell et al., 1983) or ifn-3, , but pancreatic islet cells are much more resistant to the elicitation of this phenomenon when exposed to the same or other putative modulators . in fact, using islet cell cultures, it was only the combination of tnf or lt with ifn-'t which ultimately produced the first positive results . these latter studies were prompted by the interest generated by the hypothesis that endocrine cells inappropriately expressing hla class ii could present their own surface autoantigens to helper t cells and in this way generate an autoirnmune response . this postulation was based on the observation that thyroid cells have the capacity for de novo expression of class ii molecules (pujol-borrell et al., 1983) , and the finding that in thyroid glands affected by autoimmune disorders, the follicular cells strongly expressed class ii ). since the above hypothesis was proposed, the occurrence of inappropriate class ii expression has been documented in the target organs of many organ-specific autoimmune diseases (review in bottazzo et al., 1986) and the ability of class ii-positive thyroid cells to present antigen to t cells has been experimentally demonstrated (londei et al., 1984 (londei et al., , 1985 . the identification of class ii-positive astrocytes in the active 'plaques' of patients with multiple sclerosis (ms) has attracted a great deal of interest and has led to the suggestion that astrocytes inappropriately expressing class ii molecules in vivo could play a role relevant to the pathogenesis of ms (traugott et al., 1985) , a hypothesis which is somewhat parallel to that originally proposed by us . as an extension of our interests, we report here the results obtained in studies on the inducibility of hla molecules on human fetal brain cells in culture and their differential sensitivity to the action of a variety of stimuli known to be potent modulators of hla expression in other cell types. tissue was obtained from 26 fetal brain specimens (provided by the medical research council tissue bank, royal marsden hospital, london, u.k.), after legal terminations carried out by aspiration between 11 and 19 weeks of gestation. the experimental protocol described here was approved by the university college/middlesex hospital ethical committee. the meninges and visible vessels were carefully removed from the brain, tissue was washed with hanks' balanced salt solution containing 1% bovine serum albumin (bss-bsa), minced with scissors and forceps and digested for 15 min at 37°c in a solution which contained 0.25% trypsin (sigma, london, u.k.), and 0.04% dnase (dnase 25, sigma, london, u.k.) in dulbecco's modified eagle's medium (dmem, gibco, paisley, scotland, u.k.). digestion was stopped by the addition of 10% fetal calf serum (fcs, gibco). the undigested tissue was further disrupted by repeated pipetting. the digest was passed through a 300 #m nylon mesh to remove large fragments, pelleted by centrifugation at 800 rpm for 10 min and resuspended in 'complete medium'. this consisted of 45% dmem, 45% f12 (flow, irvine, scotland, u.k.), 10% fcs, glucose 500 mg/100 ml, glutamine 2 mm, gentamicin 40 gg/ml, and penicillin 50 gg/ml. cells were counted and viability, as assessed by differential staining with acridine orange/ethidium bromide under a uv microscope, was always close to 100%. aliquots of 105 cells were plated on glass coverslips (13 mm diameter), placed in 24-multiwell plates (nunc, kamstrud, denmark) and 'complete medium' added to a final volume of 0.5 ml/well. cultures were kept at 37°c in a 5% co 2 humidified incubator and left undisturbed for 4 days. in some instances small fragments of brain tissue were directly snap frozen and stored at -70°c until used for immunofluorescence studies on cryostat sections. given the complexity of the architecture of the brain tissue and the heterogeneity of cell types present in primary cultures prepared from it, a two-colour ifl technique was employed throughout the study combining monoclonal antibodies (moabs) to hla products with monoclonal or polyclonal antibodies to different cell markers (review in . this ensured the precise identification of positive and negative cells under observation. cryostat sections and monolayer cultures were stained similarly following the same protocols. for the identification of hla class i and ii molecules the following staining protocols were applied (for the specificity and source of the antibody used, see table 1 ). hla class l 1st layer: moab w6/32; 2nd layer: tritc-conjugated goat anti-mouse igg; 3rd layer and 4th layer: either rabbit anti-glial fibrillary acidic protein (astrocytes) or rabbit anti-factor viii (endothelial cells) followed by fitclabelled goat anti-rabbit ig or moab 04 (oligodendrocytes) followed by fitc goat anti-mouse igm. hla class 11. (using igg moabs to class ii) 1st layer: moab mid3 (class ii), tu22 (dq specific), b7/21 (dp specific) or vic-y1 (class ii invariant chain specific); 2nd layer: tritc-labelled goat anti-mouse igg; 3rd layer and 4th layer: either rabbit anti-glial fibrillary acidic protein (gfap) followed by fitc-labelled goat anti-rabbit ig or moab 04 followed by fitc-labelled goat anti-mouse igm. hla class ii. (using rfdr1 igm moab to class ii) 1st layer: moab rfdr1 (class ii); 2nd layer: fitc goat anti-mouse igm; 3rd layer: moabs gc (oligodendrocytes), 308 (astrocytes), or rt.97 (neurons); 4th layer: tritc-labelled goat anti-mouse igg. moabs were used at a final concentration of 10 /~g/ml. unstimulated control cultures were always stained in parallel with treated cultures. in addition, nonspecific binding was ruled out by staining parallel stimulated cultures with an unrelated moab of the same ig class. undesirable cross-reactivities among reagents derived from different species were assessed by omitting one of the layers in turn. to determine hla membrane expression, viable cultures were incubated with the corresponding moabs, stained by the appropriate conjugate and then, prior to counterstaining with antibodies to cell-specific cytoplasmic antigens, cultures were fixed with methane/acetone (50:50, v/v) for 10 min. when the second antibody was recolmizing membrane antigens distinct from hla molecules, the fixation step was done at the end of the entire procedure. for the detection of cytoplasmic class i, class ii and class ii invariant chain the cultures were fixed prior to the staining. all preparations were examined with a x 63 oil immersion objective using a zeiss iii uv photomicroscope equipped with epi-illumination and phase contrast. fluorescence intensity in both membrane and cytoplasm was evaluated in 200-500 cells per culture except in the case of oligodendrocytes where, given their small number, only fewer cells (around 50) could be evaluated. ifl intensity was arbitrarily scored from negative to 3 +. table 2 lists the various biological and chemical compounds used to induce hla product expression on human fetal brain cells in culture. • this compound was tested only for its effect on astrocyte mhc expression. in sections from three different human fetuses stained for neurofilaments with the moab rt.97, the soma of neurons was clearly visible among the complex network of fibres, while the antiserum to glial fibrillary acidic protein (anti-gfap) produced a very intricate reticular pattern which was denser around the vessels. in successive sections stained for class i, the only cells clearly positive were the endothelial cells lining the capillaries, these identified by antibodies to factor viii. in spite of careful and intensive search, no cells positive for class ii products were detected in the parenchyma or in the vessels of the brain when sections were stained with the appropriate moabs. cell attachment occurred as early as 24 h by the time most preparations were processed, between days 6 and 12, cultures showed a complex organization similar to that described by dickson et at. (1982) . cells with long processes identified as neurons (rt.97 +) and oligodendrocytes (04 +) (see fig. la-d) laid on a carpet of astrocytes (gfap +) (fig. 2b ) (raft et at., 1979) . the long processes of the neurons were arranged in dearly distinct bundles forming bridges among clusters of cells which seemed to serve as orga~i7jng centres. immature ofigodendrocytes were present in smaller numbers compared to neurons and were often located in the periphery of these clusters, their intricate branching processes completing the complex three-dimensional network in which all the cells were included. the number of neurons decreased during the period of culture but even after 30 days these cells were quite numerous in the monolayers. fibronectinpositive cells (fibroblasts) presumably derived from contaminating meningeal cells, were very scarce in the initial days and, although their number increased with time, they never overgrew the other cell types even after 4 weeks in culture. attempts were not made to identify microglial cells because we lacked specific reagents to recognize them. endothelial cells were not present in our monolayers, probably due to their inability to grow on glass surfaces (zetter, 1981) . (b) basal expression of hla molecules. class i molecules were consistently detected throughout the culture period on astrocytes but not on oligodendrocytes or neurons ( fig. 2a and b) . class ii molecules were seen in less than 1% of the cells in the culture up to day 3. however, astrocytes (identified by gfap-positive staining) became gradually class ii positive, and by day 20, around 50% of them showed a clear and bright staining ( fig. 2c and d, time-course in fig. 3 ). this 'spontaneous' class ii expression was observed in both the protoplasmic and the fibrous type of astrocytes (identified by their morphology under phase contrast and confirmed by gfap + staining) and also in the astrocyte subtype recently defined by the 308 moab (dickson et at., 1983) . astrocyte class ii expression included also hla-dp and dq subregion products and did not depend on the gestational age of the fetal brain. passive absorption of class ii molecules to the membrane of the astrocytes could be excluded by the concomitant detection of the non-secretory class ii invariant chain in the cytoplasm of these cells when they were stained with moab vic-y1. the other cell types (neurons and oligodendrocytes) remained negative throughout the culture period (up to 30 days). for the reasons mentioned above, microghal or endothelial cells could not be detected. table 2 were tested during the initial 8 days of culture when the percentage of spontaneously positive astrocytes was less than 15%, as to minimize the interference of the spontaneous expression of class ii in astrocytes in the reading of the preparations (see above). among the biological and chemical compounds used (table 2) , only ifn-a, ifn-7 and tnf-a had a clear effect on hla expression in the different cell types and the results are summarized in table 3 . neurons and oligodendrocytes both = astrocytes did not express class ii in the early stages of culture, but an increasing percentage were found spontaneously positive after 6 days in culture (see text and fig. 4) . lacked class i expression in basal conditions but, while the former remained always negative, oligodendrocytes acquired a clear membrane staining for class i after culture with either ifn-t or tnf-a (fig. 4) . ifn-a and ifn-t both produced an increase in class i expression in astrocytes and fibroblasts. none of the mediators or chemicals tested were able to induce class ii expression in neurons or oligodendrocytes, and this also applies to the combination of ifn-t and tnf-a. ifn-~, was able to produce a dramatic increase in the number of astrocytes positive for membrane and cytoplasmic class ii and 4 days after the addition of 1000 u/ml to the cultures, all gfap-positive cells were stained for class ii. this effect was dose related (see fig. 5 and legend) and was also detectable with the moab vic-y1 which reacts with the cytoplasmic invariant chain of class ii products. no apparent synergism was observed between ifn-~ and tnf-a in the induction of class ii in astrocytes (fig. 6 ). it has previously been reported that primary cultures of human fetal brain tissue can be a useful substrate for studying the expression of cell type-specific antigens, e.g. gangliosides, tetanus toxin receptors (dickson et al., 1982 (dickson et al., , 1985 , and the present data confirm that they can also be successfully employed to investigate the expression and modulation of hla molecules. our findings that neurons do not express hla class i or class ii products in basal conditions are in agreement with previous reports using a similar culture system of murine origin (wong et al., 1984; dubois et al., 1985; suzumura et al., 1985; tedeschi et al., 1986) . neither ifn-7 nor tnf-a separately or in combination were able to induce class i or class ii expression in human fetal neurons. with regard to class i expression our results differ from those reported by wong et al. (1985) who showed that ifn-y can induce class i in up to 40% of mouse neurons, and from a similar report by lampson et al. (1984 lampson et al. ( , 1986 ) on a human neuroblastoma cell line. stages of development, species differences and neoplastic transformation could account for these apparent discrepancies. referring to class ii molecules, our results agree with the generally accepted concept that cells of the neuronal lineage are refractory to the effect of available modulators, alone or in combinations, on the levels of hla class ii expression regardless of the species employed (wong et ai., 1985) . the inability of ifn-t to induce class i or class ii in neurons contrasts with the strong positive modulation it produces on most cell types including terminally differentiated human cells such as thyrocytes and melanocytes (houghton et al., 1984) . the biological meaning and molecular basis for this differential regulation are at present unknown. the potential of oligodendrocytes to express hla products has been extensively studied, especially as these cells seem to be the target of the postulated autoimmune response in ms and experimental allergic encephalomyelitis (eae). our finding that human oligodendrocytes are constitutively negative for both class i and class ii molecules but express class i after incubation with ifn-t is in agreement with numerous previous studies on cultures of murine brain cells and suggests the presence of receptors for ifn-t on the surface of these cells (wong et al., 1984; suzumura et al., 1985; tedeschi et al., 1986) . more interesting is the observation that tnf-a can also induce class i expression in oligodendrocytes from human fetal brain. reports of a similar action of tnf-a on other cell types (collins et al., 1986) have suggested that this may be one of the mechanisms through which this mediator exerts its anti-tumour action in vivo (pfizenmaier et ai., 1987) . neither ifn-t or tnf-a alone or the combination of the two were able, however, to induce class ii expression in oligodendrocytes in our system. this is in contrast with our recent findings which showed that human pancreatic islet cells, another cell type unresponsive to ifn-t alone, could be induced to express class ii products by the synergistic action of ifn-t and tnf-a or -fl . there is no consensus in the literature on whether adult human oligodendrocytes can express class ii molecules following incubation with ifn-t. lysak et al. (1983) , like us, did not find class ii expression in stimulated primary cultures while kim (1985) and kim et al. (1985) reported 4-16% of class ii-positive oligodendrocytes (although only in half of the culture preparations); the reason for these differences remains at present unclear. regulation of hla expression in astrocytes has also been extensively studied both in rodent (hirsch et al., 1983; wong et al., 1984; dubois et al., 1985; massa et al., 1986) and in human systems (shen et al., 1985; takiguchi et al., 1985) , again due to its potential importance in the pathogenesis of ms. astrocytes expressing class ii are present in the 'active' plaques in the brain of ms patients (traugott et al., 1985) and, most importantly, it has been shown that they can present antigens to t cell clones in vitro (fierz et al., 1985) . the postulate has accordingly been made that by presenting myelin basic protein (mbp) or related antigens to autoreactive t lymphocytes, class ii-positive astrocytes may initiate and possibly perpetuate an autoimmune recognition to mbp which subsequently leads to the demyelination process in active ms (fontana, 1984; sun and wekerle, 1986; takiguchi et al., 1986) . in short-term cultures we found that astrocytes express class i but not class ii molecules and this is in accordance with previous work (fierz et al., 1985) . however, we were surprised by the arousal of a population of astrocytes positive for class ii after 5-6 days while these cells were growing in culture. these results may substantiate the report by kim (1985) which showed that in cultures of human adult brain 9-27% of the astrocytes were class ii positive at day 10. similar findings were described on a human fetal astrocyte cell line, but the spontaneous class ii expression disappeared after subsequent passages (pulver et al., 1987) . this phenomenon seems to be a unique characteristic of the astrocytes since fibroblasts and other cell types present in our cultures remained negative, thus arguing against the possibility that it is mediated by ifns or other non-specific soluble factors generated during the culture period or by the medium employed, as previously suggested (pulver et al., 1987) . this spontaneous induction of class ii expression indicates that in vivo the expression of class ii by astrocytes should be constantly suppressed by still unidentified humoral or locally produced mediators. alternatively, it is also possible that the abundant debris which our cultures initially contained could have stimulated phagocytosis by the astrocytes and this then triggered the expression of class ii, as part of the general activation process undergoing in these cells. in this context, it is of interest to recall that it has recently been shown that the phagocytosis of coronavirus particles induced class ii expression in rat astrocytes (massa et al., 1986) . as expected, ifn-a and ifn-3, both produced an increase in class i expression in astrocytes. the studies of class ii modulation on astrocytes were carried out at the beginning of the culture period while the level of spontaneous expression was still low and in this way we were able to reproduce previous work which has shown that ifn-7 is indeed able to induce a rapid increase in the percentage of class ii-positive human fetal astrocytes (pulver et al., 1987) as well as enhance the intensity of the staining (fig. 5) . microglial cells were not easy to identify in our monolayers and this was most probably due to the early gestational age of the fetal tissue. however, even if these cells were present in small amounts, our lack of any specific marker made their identification, solely on morphological ground, very difficult. in summary, we have presented for the first time data on a large number of human fetal brains both in sections and in primary cultures indicating that the regulation of the expression of hla products in human fetal brain cells varies widely among the different cell types. there is a gradation in both constitutive expression and inducibility which ranges from neurons, which lack hla products completely and are refractory to all inducers and modulators tested so far, to astrocytes which in culture become spontaneously positive for class ii and are highly responsive to ifn-y. this heterogeneity of hla regulation among the brain cells is reminiscent of that observed for class ii in the human pancreas where ductal/exocrine cells are easily induced to express class ii while islet endocrine cells require a two-med~tor signal . considering the analogies between the different cell populations which form these two complex organs it seems that the cells with a high or intermediate turnover (ductal cells, astrocytes) are more easily induced to express hla molecules than those with a low turnover (e.g. islet cells) or no turnover (e.g. neurons). one may speculate on the biological significance of this differential regulation. one possibility is that the lack of inducibility of class ii expression may constitute one of the mechanisms which maintain tolerance to autoantigens present in highly specialized cells cowing, 1985) . on the other hand, absence of class i and inability to express it, may represent a more extreme way of excluding irreplaceable cells from the interaction with the immune system and in particular from the attack by cytotoxic t lymphocytes. this may help to explain the inability of the cellular immune mechanisms to eradicate certain neuronal viral infections, such as herpes simplex. it may well be that it is more advantageous for the organism to have some neurons latently infected than not to have them at all, with the consequence of an irreversible disability. if these postulates are correct, it follows that a better knowledge of the differential regulation of hla protein expression in the central nervous system may contribute to our understanding of the autoimmune processes and latent viral infections occurring in this organ. role of aberrant hla-dr expression and antigen presentation in the induction of endocrine autoimmunity organ-specific autoimmunity: a 1986 overview recombinant human tumour necrosis factor increases mrna levels and surface expression of hla, a, b, c antigens in vascular endothelial cells and dermal fibroblasts in vitro does t cell restriction to ia limit the need for self-tolerance? cell surface antigens of human foetal brain and dorsal root ganglion cells in tissue culture identification of cell surface antigens present exclusively on a subpopulation of astrocytes in human foetal brain cultures cellular distribution of 04 antigen and galactocerebroside in primary cultures of human foetal spinal cord expression of major histocompatibility complex antigens in neonate rat primary mixed glial cultures monoclonal antibody to a plasma membrane antigen of neurons astrocytes as antigen presenting cells. i. induction of la antigen expression on astrocytes by t cells via immune interferon and its effects on antigen presentation astrocytes present myelin basic protein to encephalitogenic t cell lines aberrant expression of hla-dr antigen on thyrocyte in graves' disease: relevance for autoimmunity expression of la antigens by cultured astrocytes with gamma interferon surface antigens of melanoma and melanocytes. specificity of induction of ia antigens by human gamma-interferon antigen expression by glial cells grown in culture expression of ia antigens on the surface of human oligodendrocytes and astrocytes in culture weak hla and beta2-microglobulin expression of neuronal cell lines can be modulated by interferon monoclonal antibody analysis of mhc expression in human brain biopsies: tissue ranging from 'histologically normal' to that showing different levels of glial tumour involvement epithelial ceils expressing aberrant mhc class li determinants can express antigen to cloned human t lymphocytes human t cell clones from autoimmune thyroid glands: specific recognition of autologous thyroid cells cultured human oligodendrocytes and rat schwann cells do not have immune response gene associated antigen (la) on their surface viral particles induce la antigen expression on astrocytes tumour necrosis factor enhanced hla-a, b, c and hla-dr gene expression in human tumour cell inappropriate class ii expression in endocrine cells. is it a primary event lectin induced expression of dr-antigens on human cultured follicular thyroid ceils differential expression and regulation of mhc products in the endocrine and exocrine cells of the human pancreas hla class li induction in human islet cells by ifn-gamma plus tnf or lt cultured human fetal astrocytes can be induced by interferon-y to express hla-dr cell-type specific markers for distinguishing and studying neurons and the major class of ghal cells in culture cellular and antigenic properties of cultured normal and fetal brain and ghoma cells monoclonal antibodies (o1 to 04) to oligodendrocyte cell surface. an immunocytochemical study in the central nervous system la restricted encephalitogenic t lymphocytes mediating eae lyse autoantigen-presenting astrocytes expression of h2 antigen on oligodendrocytes is induced by soluble factors from concanavalin a activated t cells induction of antigen presentation ability in purified cultures of astroglia by interferon-gamma response of glioma cells to gamma-interferon: increase in class ii mrna, protein and mlr stimulating ability astrocytes produce interferon that enhances the expression of h-2 antigens on a sub-population of brain cells laboratory investigation of autoimmune endocrine diseases interferon-gamma induces hla-dr expression by thyroid epithelium on the presence of ia positive endothelial cells and astrocytes in multiple sclerosis lesions and its relevance to antigen presentation inducible expression of the h2 and ia antigens on brain cells interferon-gamma induces the expression of h-2 and ia antigens on brain cells the endothelial cells of large and small blood vessels we are greatly indebted to all the scientists and commercial firms cited in the text and tables for their generous donations of monoclonal antibodies and other reagents and to the medical research council tissue bank, royal marsden hospital, london for the supply of fetal brain tissue. we thank our colleagues dr. a. belfiore and dr. i. todd for advice and help during the development of this work, professors deborah doniach and ivan roitt for their constant support, and dr. m.l. cuzner and dr. j.g. dickson for very useful discussion. we would also like to thank marian pine for her help in the preparation of the manuscript. key: cord-023433-d1b7qvhs authors: siassi, m.; hohenberger, w.; croner, r. title: expression of human collectins in colorectal carcinoma date: 2008-06-28 journal: scand j immunol doi: 10.1111/j.0300-9475.2004.01423bo.x sha: doc_id: 23433 cord_uid: d1b7qvhs introduction: the human collectins, mannan‐binding lectin (mbl), surfactant protein‐a (sp‐a) and surfactant‐protein‐d (sp‐d) play a central role in the innate immune system. immunological responses to malignant transformation of epithelial cells gained increasing interest recently. a former study could demonstrate binding of mbl to certain colorectal carcinoma (crc) cell lines in vitro. we therefore examined the expression of human collectins in normal colon mucosa and in colorectal carcinomas. materials and methods: colon samples from 20 crc patients and 10 normal mucosa samples were collected immediately after surgery. the tissue was microdissected and rna isolated (qiagen, rneasy‐kit). gene expression profiles were analysed using gene‐chips (affymetrix, hg‐u133). we analysed the data for the expression of mbl, its associated serine proteases mannan‐binding lectin‐associated serine protease 1/2 (masp 1/2), sp‐a and sp‐d. the signal intensity of the genes of interest was compared using the mann–whitney u‐test. results: the expression of human collectins in normal human colon mucosa was generally low. only the expression of sp‐a and masp‐2 reached the noise threshold of 250 signals. these genes were significantly downregulated in crc specimens. the expression of the other proteins showed no difference in normal mucosa and crc. conclusion: as demonstrated before, the expression of human collectins in normal colon was low in this study. only sp‐a showed a significant expression in normal mucosa which was downregulated in crc. as the absolute signal level was below the noise threshold, these results have to be interpreted with caution and require confirmation by direct measurenment of the proteins. our results suggest that there is no major role for the human collectins in colorectal cancer. the nuclear receptor heterodimers of liver x receptors (lxrs) are recently identified as key transcriptional regulators of genes involved in lipid homeostasis and inflammation. lxrs and their ligands are negative regulators of macrophage inflammatory gene expression. multiple sclerosis (ms), a demyelinating disease of the central nervous system of unknown cause, is characterized by recurrent inflammation involving macrophages and their inflammatory mediators. sweden belongs to the countries with a high ms incidence. in italy, incidence is lower, with an exception for sardinia where the incidence is even higher than that in sweden. subjects from sardinia are ethnically more homogeneous and differ from swedes, also regarding genetic background and environment. we studied lxrs and their related molecules of blood mononuclear cells (mncs) from female patients with untreated relapsing-remitting ms from sassari, sardinia and stockholm, sweden. sex-and age-matched healthy controls (hcs) were from both areas. mrna expression was evaluated by real-time pcr. lxr-a was lower (p < 0.05) in ms (mean ae sem: 3.1 ae 0.2; n ¼ 37) compared to hc (3.6 ae 0.1; n ¼ 37). lxr-a was lower in ms from stockholm (2.6 ae 0.2; n ¼ 22) compared to corresponding hc (3.4 ae 0.1; n ¼ 22; p < 0.01) and compared to ms (3.8 ae 0.2; n ¼ 15; p < 0.001) and hc (4 ae 0.2; n ¼ 15; p < 0.001) from sardinia. ms patients from stockholm, but not from sassari, also expressed lower (p < 0.05) lxr-b (à4.1 ae 0.4) compared to corresponding hc (à2.9 ae 0.3). ms from stockholm was associated with higher abca-1 (6.1 ae 0.4 versus 5.0 ae 0.3; p < 0.05) and higher estrogen receptor-b-cx (2.4 ae 0.4 versus 0.8 ae 0.4; p < 0.01) compared to corresponding hc. the hc from sassari had higher androgen receptor (2.9 ae 0.2) compared to ms from sassari (1.4 ae 0.3; p < 0.01), ms (1.3 ae 0.4; p < 0.01) and hc from stockholm (1.2 ae 0.3; p < 0.01). ms from sassari had lower cyclooxygenase-1 compared to corresponding hc (5.1 ae 0.4 versus 6.6 ae 0.3; p < 0.01) and lower prostaglandin-e (à0.03 ae 0.5) compared to the hc (1.4 ae 0.5; p < 0.05) and ms (2.7 ae 0.4; p < 0.05) and hc from stockholm (1.9 ae 0.4, p < 0.001). our findings identify lxrs and their related molecules as being involved in ms from stockholm but not from sassari, while sex hormone receptors seem to be involved in ms in sassari. multiple sclerosis: ifn-b induces cd123 + bdca2 -dendritic cells that produce il-6 and il-10 and have no enhanced type i interferon production y. m. huang, 1 s. adikari, 1 u. båve, 2 a. sanna 1,3 & g. alm 4 dc antigens (bdca) and investigate their ability to produce type i ifn in response to virus stimulation. we show that ifn-b induces development of cd123 þ dc from human blood monocytes, which coexpress bdca4 þ but are negative for bdca2 -, a specific marker for plasmacytoid dc. such ifn-b-modulated dc produce large amounts of il-6 and il-10, but no il-12p40 and have no enhanced ifn-b and ifn-b production. the findings indicate that ifn-bmodulated dc represent a myeloid dc subset with diminished cd11c, bdca-1 and cd1a expression, having potent th2-promoting function but lacking antiviral capacity. the association of psoriasis with throat infections by streptococcus pyogenes suggests a potential antigenic target for the t cells that are known to infiltrate dermis and epidermis of psoriatic skin. streptococcal m protein shares an extensive sequence homology with human epidermal keratins. keratins 16 (k16) and 17 (k17) are mostly absent from uninvolved skin but are upregulated in psoriatic lesions. there is increasing evidence that cd8 þ t cells play an important effector role in psoriasis and m proteinprimed t cells may recognize these shared epitopes in skin via molecular mimicry. to identify candidate epitopes, peptides with sequences from k17 were selected on the basis of predicted binding to hla-cw6 and sequence similarities with m6 protein. matched peptides from the sequence of m6 protein and a set of peptides with poor predicted binding were also selected. cw6 þ individuals with psoriasis and cw6 þ healthy controls, having a family history of psoriasis, were recruited. pbmcs were incubated with the peptide antigens. t-cell activation in the cd4 þ , cd8 þ and later the skin-homing cutaneous lymphocyteassociated antigen (cla)-expressing subset of cd8 þ t cells was evaluated by cd69 expression and intracellular ifn-g accumulation using flow cytometry. we demonstrate that cw6 þ psoriasis patients had significant cd8 þ t-cell ifn-g responses to peptides from k17 and m6 protein selected on the basis of sequence homology and predicted hla-cw*0602 binding. these responses were about 10 times more frequent in the skin-homing cutaneous lymphocyte-associated antigen-expressing (cla þ ) subset of cd8 þ t cells. cd4 þ t cells showed only borderline responses. cd8 þ t cells from cw6 þ nonpsoriatic individuals responded to some m6 peptides but very rarely to k17 peptides, and this also applied to the cla þ cd8 þ subset. these findings indicate that psoriatic individuals have cd8 þ t cells that recognize keratin self-antigens and that epitopes shared by streptococcal m protein and human keratin may be targets for the cd8 þ t cells that infiltrate psoriatic skin lesions. autoantibodies directed against citrulline-containing proteins have an impressive specificity of nearly 100% in ra patients and a suggestive involvement in the pathogenesis. the targeted epitopes are generated by a post-translational modification catalysed by the calcium-dependent enzyme peptidyl arginine deaminase that converts the positively charged arginine to polar but uncharged citrullin. the aim of this study was to analyse the presence of citrulline in the joints at different time points of collagen-induced arthritis in da rats by immunohistochemistry and to investigate how immunogenicity and arthritogenicity was affected by citrullination of rat serum albumin (rsa) and collagen type ii (cii). our results indicate that citrulline could be detected in joints of arthritic animals, first appearance at the onset of disease and increasing as disease progressed into a chronic state. unimmunized animals or time points before clinical signs of arthritis were negative. by morphology, we state that some infiltrating macrophages as well as the cartilage surface stain positive for citrulline, while the major source of citrullinated proteins appears to be fibrin depositions. a specific cit-rsa t-cell response was observed in animals challenged by citrullinated rsa, no response was recorded when rsa was used as a stimulus. the igg analysis reveals not only a response towards the modified protein but also cross-reactivity to native rsa. no t-cell or b-cell response was noted in animals injected with unmodified rsa. cit-cii induced a disease with higher incidence and earlier onset than did the native counterpart. we conclude that, in contrast to the human disease, citrulline does not seem to appear before clinical signs. as inflammation proceeds, citrulline is detected specifically in the joints. all other organs investigated were negative. we also conclude that citrullination of a protein can break tolerance and increase its arthritogenic properties. ectopic germinal centers (gcs) can be detected in the salivary glands of approximately 1/5 of patients with sjögren's syndrome (ss) and appear in both primary and secondary ss. previously, ectopic gc have been associated with increased local autoantibody production. the aim of this study was to determine whether gc in primary sjögren's syndrome (pss) defines a distinct seroimmunological phenotype. retrospectively, a material of 130 haematoxylin and eosin-stained paraffin-embedded tissue sections of minor salivary gland tissue from patients with pss was morphologically screened for the presence of ectopic gc. gc-like lesions were detected in 33/130 (25%) of the pss patients. seventy-two pss patients lacking these structures (gc-) were randomly selected for comparison. focus score was significantly increased in the gc þ patients compared to the gcpatients (p ¼ 0.035). in the gc þ group, 54.5% of the patients presented with anti-ro/ssa compared to 43.7% in the gcgroup. anti-la/ssb was detected in 31.3% of the gc þ patients compared to 25.7% of the gcpatients. sixty-one percentage of gc þ patients presented with increased levels of igg, a nonsignificant difference when compared to 39.4% in the gcpatients (p ¼ 0.089). levels of rf, ana, ena, igm and iga were similar in both patient groups, as were esr and crp. in conclusion, patients with ectopic gc have a higher focus score and more often present with autoantibodies and increased levels of igg compared to pss patients with regular focal infiltration (gc -). our findings may indicate a certain seroimmunological phenotype and warrant for further prospective studies. association between mannose-binding lectin and vascular complications in type 1 diabetes complement activation and inflammation have been suggested in the pathogenesis of diabetic vascular lesions. we investigated serum mannose-binding lectin (mbl) levels and polymorphisms in the mbl gene in type 1 diabetic (t1dm) patients with and without diabetic nephropathy and associated macrovascular complications. polymorphisms in the mbl gene and serum mbl levels were determined in 199 t1dm patients with overt nephropathy and 192 t1dm patients with persistent normoalbuminuria matched for age, sex and duration of diabetes as well as in 100 healthy control subjects. the frequencies of high and low expression mbl genotypes were similar in patients with t1dm and healthy controls. high mbl genotypes were significantly more frequent in diabetic patients with nephropathy than in the normoalbuminuric group, and the risk of having nephropathy, given a high mbl genotype, assessed by odds ratio was 1.52 (1.02-2.27), p ¼ 0.04. median serum mbl concentrations were significantly higher in patients with nephropathy than in patients with normoalbuminuria [2306 mg/l (iqr 753-4867 mg/l) versus 1491 mg/l (iqr 577-2944), p ¼ 0.0003], and even when comparing patients with identical genotypes, serum mbl levels were higher in the nephropathy group than in the normoalbuminuric group. patients with a history of cardiovascular disease had significantly elevated mbl levels independently of nephropathy status [3178 mg/l (iqr 636-5231 mg/l) versus 1741 mg/l (iqr 656-3149 mg/l), p ¼ 0.02]. the differences in mbl levels between patients with and without vascular complications were driven primarily by pronounced differences among carriers of high mbl genotypes (p < 0.0001). our findings suggest that mbl may be involved in the pathogenesis of microvascular and macrovascular complications in type 1 diabetes and that determination of mbl status might be used to identify patients at increased risk of developing these complications. neuroimmunology unit, center for molecular medicine, karolinska institutet, stockholm, sweden. e-mail: judit.wefer@cmm.ki.se dna vaccine coding for the encephalitogenic peptide mog 91-108 protects lew.1av1 from subsequent development of experimental autoimmune encephalomyelitis (eae). protection is associated with a type 1 immune response and is dependent on the presence of cpg dna motifs. the mechanisms underlying the observed reduction of eae development in protected rats have not been fully clarified. we investigated immunological characteristics of lymphocytes after dna vaccinaton and subsequent eae induction. we confirm that protection was not associated with suppression of t1 cells, as transcription of the novel molecule rat t-cell immunoglobulin-and mucindomain-containing molecule (tim-3), reported to be exclusively expressed on differentiated t1 cells, was not altered by dna vaccination. we did not note any clonal deletion upon tolerization, but detected an antigen-specific lymphocyte population upregulating ifng upon recall stimulation 3 weeks after protective dna vaccination. in protected rats, we observed (1) no alterations in antigenspecific th2 or th3 responses, (2) reduced mhc ii expression on splenocytes early after eae induction, (3) antigen-specific upregulation of ifnb upon recall stimulation and (4) reduced il-12rb2 on lymphocytes. we thus demonstrate an association of the protective effect of dna vaccination with expression of ifnb. we are currently investigating the cellular mechanisms behind this ifnb-mediated protection. multiple sclerosis (ms) is an autoimmune condition characterized by degeneration of nerve fibre myelin sheets. a candidate autoantigen, myelin basic protein (mbp), has especially attracted attention. the presence of anti-mbp antibodies is a predictor of definite ms, but their role in the pathogenesis remains obscure. t cells have long been known to play a pivotal role in the pathogenesis of ms. recently, an important role for b cells as autoantigen-presenting cells has been demonstrated in other autoimmune diseases, including rheumatoid arthritis and diabetes. the uptake of mbp by b cells and the presentation of mbp-derive peptides to t helper (th) cells by b cells may be promoted by the formation of complement (c) activating immune complexes (ics) between mbp and natural autoantibodies in healthy individuals and disease-associated anti-mbp antibodies in ms patients, respectively. we have investigated the formation of mbp-containing ic, the binding of mbp to b cells, the mbp-elicited induction of th-cell and b-cell proliferation and the cytokine production in peripheral blood mononuclear cells (pbmcs) from healthy donors grown in the presence of intact or c-inactivated serum from healthy donors or patients with ms. while mbp did not induce measurable proliferation of b cells nor cd4 þ t cells, we observed the production of tnf-a, ifn-g and il-10 by pbmc in response to incubation with mbp in the presence of sera from healthy controls as well as sera from ms patients. by contrast, no production of il-2, il-4 and il-5 was detected. we are currently investigating the capability of ms sera to promote the formation of mbp-containing ic and thereby enhance the cytokine responses, by virtue of elevated anti-mbp contents. the phagolysosomally localized acid sphingomyelinase (asmase) activated by proinflammatory cytokines such as tnf and ifn-g generates the signalling molecule ceramide which in turn results in the activation of proteases like cathepsin d. these characteristics of asmase suggest a possible role of this molecule in the phagocytotic uptake and phagosomal degradation processes of antigens or in antigen presentation. we show here that asmase -/mice fail to eliminate the noncytopathic lymphocytic choriomeningitis (lcm) virus as rapidly as littermate wildtype mice. investigation of the immune response revealed a reduced expansion of cd8 þ t cells. the secretion of ifn-g in response to contact with target cells as well as the cytolytic activity of virus-specific cd8 þ t cells was severely impaired. additionally, both phases of the lcm virus-specific dth response, mediated by cd8 þ and cd4 þ t cells consecutively, were diminished in asmase -/mice. however, the secondary memory response of virus-specific ctl was not altered, and the 614 abstracts ................................................................................................................................................................................................. virus was effectively controlled for at least 3 months by asmase -/mice. in conclusion, the results of this study suggest an involvement of the asmase in the activation, expansion or maturation of virus-specific cd8 þ t cells during the acute infection of mice with the lcm virus. novel markers for alternative activation of macrophages: macrophage galactose-type c-type lectins 1 and 2 in parallel with the th1/th2 dichotomy, macrophages are capable of developing into functionally and molecularly distinct subpopulations, due to differences in, for example cytokine environment and pathological conditions. while the best-studied, classically activated macrophage is induced by type i stimuli such as ifn-g, a type ii cytokine environment antagonizes the classical activation of macrophages and is capable of alternatively activating macrophages. however, molecular markers associated with these type ii cytokine-dependent, alternatively activated macrophages remain scarce. besides the earlier documented markers macrophage mannose receptor and arginase 1, we recently demonstrated that murine alternatively activated macrophages are characterized by increased expression of fizz1 and ym. we now report that expression of the two members of the mouse macrophage galactose-type c-type lectin gene family, termed mmgl1 and mmgl2, is induced in diverse populations of alternatively activated macrophages, including peritoneal macrophages elicited during infection with the protozoan trypanosoma brucei or the helminth taenia crassiceps, and alveolar macrophages elicited in a mouse model of allergic asthma. we also demonstrate that, in vitro, interleukin-4 and interleukin-13 upregulate mmgl1 and mmgl2 expression and that, in vivo, induction of mmgl1 and mmgl2 is dependent on interleukin-4 receptor signalling. moreover, we show that regulation of mgl expression is similar in human monocytes and monocyte-derived macrophages. hence, macrophage galactose-type c-type lectins represent novel markers for both murine and human alternatively activated macrophages; thus, paving the way for further characterization of the phenotype of macrophages occurring in th2 conditions. background: human parvovirus b19 (b19) is a ubiquitous pathogen, normally causing a mild self-limiting disease, but also capable of causing both significant pathology and long-term persistence. the small size and stability of the virus makes it suitable for mapping of the full breath and the kinetics of the cellular immune responses following acute viral infection. methods: five patients with acute primary b19 infection were included in the study and followed consecutively for up to 200 weeks. cellular immune responses were mapped by ifng enzyme-linked immunospot to overlapping peptides spanning the whole b19 genome. results: in all five acutely infected patients, we were able to monitor the kinetics of a strong specific cellular immune reaction. responses peaked at levels of 850-1850 sfc/ million pbmcs, roughly corresponding to 0.3-0.6% b19specific cd8 þ cells circulating in peripheral blood at 10-80 weeks post-infection. the responses in individual patients were directed to three or four different peptide pools, and the specificity was confined to the same cd8 epitopes present in the pools throughout the follow-up period. the majority of responses were directed to the virus nonstructural protein, only two patients showed any response to the capsid proteins, elicited by the same epitope in both cases. conclusion: the cellular immune responses to acute b19 infection are surprisingly narrow in distribution and remain at high levels for up to 80 weeks post-infection. the initial epitope specificity is maintained, and the majority of responses target the virus nonstructural protein, which is not included in vaccine preparations, evaluated against the infection. the relationship between malnutrition and malaria is controversial. on one hand, malaria may cause malnutrition, while on the other, malnutrition itself may modulate susceptibility to the disease. we investigated the association between plasmodium falciparum malaria and malnutrition in a cohort of children living on the coast of kenya. the study involved longitudinal follow-up for clinical malaria episodes and anthropometric measurements at four cross-sectional surveys. we used poisson regression analysis to investigate the association between malaria and nutritional status. compared to baseline (children with a waz or haz score of !à2), the crude incidence rate ratios (irrs) for malaria in children with low haz or waz scores (<à2) during the period prior to assessment were 1.17 (95% ci 0.91-1.50; 0 ¼ 0.21) and 0.94 (0.71-1.25; 0.67), respectively, suggesting no association between malaria and the subsequent development of pem. however, we found that age was acting as an effect modifier in the association between malaria and malnutrition. the irr for malaria in children 0-2 years old who were subsequently characterized as wasted was 1.65 (1.10-2.20; p ¼ 0.01), and a significant overall relationship between malaria and low-haz was found on regression analysis when adjusting for the interaction with age (irr 1.89; 1.01-3.53; p < 0.05). although children living on the coast of kenya continue to suffer clinical episodes of uncomplicated malaria throughout their first decade, the association between malaria and malnutrition appears to be limited to the first 2 years of life. a. astrinidou-vakaloudi, 1 s. xytsas, 1 i. diamanti, 1 h. ioannidis 2 & p. pangidis 2 1 microbiology department of general hospital of thessaloniki 'agios pavlos', thessaloniki, greece, and 2 nefrology, 2 nd ika hospital of thessaloniki, thessaloniki, greece. e-mail: stasa@hol.gr aim: renal dysfunction may influence the colonization of gastric mucosa by urea-splitting bacteria such as helicobacter pylori, by increasing urea concentrations in the gastric juice. our aim was to investigate the prevalence of h. pylori in patients with end-stage renal disease (esrd), receiving long-term haemodialysis treatment. methods: this study included 40 sera from patients with esrd (29 male and 11 female) undergoing periodic haemodialysis; mean time of treatment was 42.6 months. using elisa technique, we investigated the presence of igg and iga antibodies against h. pylori as well as igg caga (antibodies specific for caga(þ) strains of h. pylori). sera from 40 healthy blood donors were used as a control group. results: h. pylori igg antibodies were detected in 32 out of 40 (80%) patients in the dialysis group, while 31/40 (77.5%) tested positive for iga. igg caga antibodies were present in 13 out of 40 (32.5%). prevalence of h. pylori igg, iga and caga igg antibodies in the control group was 33, 7 and 15%, respectively. conclusions: although international data suggest that prevalence of h. pylori infection is the same in esrd patients as in healthy individuals, in our study that seems not to be the case. the higher blood and gastric juice urea levels may be a risk factor (among many others), but more studies are required in order to understand the relation of h. pylori infection in this group of patients. flanders interuniversity institute for biotechnology, department of molecular and cellular interactions, free university of brussels, brussels, and 2 pasteur institute of brussels, mycobacterial immunology, brussels, belgium. e-mail: tgartner@vub.ac.be immunity against tuberculosis (tb), caused by mycobacterium tuberculosis, depends largely on activation and maintenance of strong cell-mediated immune responses involving both cd4 þ and cd8 þ t cells and the ability to respond with th1-type cytokines, particularly ifn-g. recent studies suggested that bcg, the only licensed vaccine against m. tuberculosis, may fail to induce t-cell responses in the lung mucosa and may therefore not protect against pulmonary tb. a decrease in tb mortality may be achieved by enhancing immunity in the lung. the present study evaluated the induction of antigen-specific immunity in the lung by intranasal (i.n.) delivery of the lipoprotein i (opri) from pseudomonas aeruginosa. opri has shown to be a toll-like receptor 2/4 agonist that, when given subcutaneously, induces type-1 immune responses against heterologous antigens. here, a fusion of opri to ag85a of mtb (opri-ag85a) was used as a subunit vaccine in homologous prime-boost immunizations. in addition, opri-ag85a was combined with an ag85a-encoding dna vaccine (ag85a dna) or with bcg in heterologous prime-boost vaccinations. intranasal and parenteral delivery with opri-ag85a elicited comparable t-cell responses in the spleen; in addition, i.n. delivery elicited specific t-cell responses in the lung lymph nodes (llns). intramuscular delivery of ag85a dna induced significant systemic th1 immune responses. intranasal boosting with opri-ag85a enhanced this response and in addition induced an antigen-specific ifn-g response in lln. opri may therefore be an efficient adjuvant for mucosal boosting. we continue to evaluate the protection induced by opri-based prime-boost vaccinations against pulmonary tb. results on the immunogenicity and protection against intravenous mtb h37rv infection will be presented. toll-like receptors (tlrs) are pattern recognition receptors of the innate immune system, which recognize molecular structures on pathogens or cellular stress-associated molecules. tlr-ligand interactions trigger activation of inflammatory signal transduction and expression of genes involved in host defense. in this study, we have examined the requirement for different tlr adaptor molecules in virus-induced chemokine expression and are currently trying to identify the tlr involved. we have found that both a herpesvirus [herpes simplex virus (hsv)] and a paramyxovirus (sendai virus) require a functional genome to induce expression or proinflammatory chemokines in human and murine monocytic cell lines. for both viruses, this is independent of the tlr adaptor molecules trif and mal. however, overexpression of the vaccinia virus-encoded inhibitor of tlr-signalling a52r or dominant-negative myd88 totally inhibited hsv-induced rantes expression but only partially prevented sendai virus from inducing this chemokine. this suggests that hsv-induced rantes expression occurs via a tlr pathways, whereas sendai virus utilizes both tlr-dependent and -independent pathways to stimulate expression of rantes. we are currently trying to identify the tlrs involved. data from these studies will also be presented at the meeting. 2 0 -5 0 -oligoadenylate synthetases are interferon-induced, double-stranded rna-activated antiviral enzymes which are the only proteins known to catalyse 2 0 -specific nucleotidyl transfer. this first crystal structure of a 2 0 -5 0oligoadenylate synthetase reveals a structural conservation with the 3 0 -specific poly(a) polymerase that, coupled with structure-guided mutagenesis, supports a conserved catalytic mechanism for the 2 0 -and 3 0 -specific nucleotidyl transferases. comparison with structures of other superfamily members indicates that the donor substrates are bound by conserved active site features while the acceptor substrates are oriented by nonconserved regions. the 2 0 -5 0oligoadenylate synthetases are activated by viral doublestranded rna in infected cells and initiate a cellular response by synthesizing 2 0 -5 0 -oligoadenylates, that in turn activate rnase l. this crystal structure suggests that activation involves a domain-domain shift and identifies a putative dsrna activation site that is probed by mutagenesis. we demonstrated that this site is required both for the binding of dsrna and for the subsequent activation of oas. this rna-binding site is different from known rna-binding site; rather than forming a defined three-dimensional domain, it is located at the interface of the two major domains in oas. this novel architecture ensures that the dsrna helix can make simultaneously contact with both domains of oas and ensure the subsequent structural rearrangement leading to the activation of oas. our work provides structural insight into cellular recognition of double-stranded rna of viral origin and identifies a novel rna-binding motif. bacteria-specific iga antibodies are efficient opsonins for neutrophils and mononuclear phagocytes, provided that the phagocytes express the fca receptor (cd89). expression of cd89 can be stimulated by inflammatory cytokines, activated complement factors and certain microbial components. in one study, unstimulated phagocytes were able to ingest iga antibody-treated pneumococci, but only in the presence of complement, which was found to be activated by the iga antibodies along the alternative pathway. pneumococci produce iga1 protease that cleaves human iga1, but not iga2, molecules in the hinge region. this leaves iga1 as faba (monovalent) deprived of fca which contains the docking site for cd89. iga1 is the vastly predominant subclass of iga in the upper airways and circulation of humans. aims: to examine the effects of iga1 protease activity and complement on phagocytosis of iga antibody-coated pneumococci by an unstimulated human phagocytic cell line (hl60). materials and methods: iga1 and iga2 monoclonal antibodies to serotype 4 pneumococcal capsular polysaccharide (ps) were generated by heterohybridoma technique involving b cells from human vaccinees. isogenic serotype 4 pneumococci with and without iga1 protease activity, respectively, were obtained after inactivation of the iga gene of the tigr4 strain. opsonophagocytosis was quantitated using the assay described by romero-steiner et al. based on enumeration of surviving bacteria by culture. the integrity of iga molecules was examined by western blotting. results: both iga1 and iga2 antibody to type-4 polysaccharide-induced phagocytosis of iga1 protease-deficient type-4 pneumococci equally well in the absence as in the presence of complement. iga1 antibody to type-4 polysaccharide displayed a fourfold higher opsonophagocytosis titer against iga1 protease deficient compared to homologous wildtype target bacteria. a similar effect of iga1 protease activity of the target bacteria was not observed in a parallel experiment where iga2 antibody to type-4 polysaccharide served as opsonin. iga1 antibody extracted from iga1 protease-producing target bacteria was almost exclusively in the form of faba. conversely, iga1 from protease-deficient bacteria and iga2 from both types of bacteria were intact. conclusions: these results indicate that the iga1 protease activity of s. neumoniae may help the bacteria escape iga1 antibody-mediated opsonophagocytosis. besides, in these experiments, iga-mediated opsonophagocytosis was independent of complement. vitamins e and c have been found to increase the cellular and humeral immunity of pigs. vitamin e deficiency has also been found to predispose pigs to different diseases, e. coli infection is one among them. after weaning, the vitamin e status of pigs often decreases to a critical low level. in this experiment, we studied whether vitamin c supplementation would be a possible feeding strategy to optimize the immune status of weaners. the interaction between vitamin e and c is interesting due to the reported sparing action on vitamin e or synergism between these to vitamins. piglets were weaned at day 28 of age from sows fed increasing dietary vitamin e during lactation, and piglets were during the following 3 weeks fed either a control diet or this diet supplemented with 500 mg stay-c per kg. blood sampling was obtained weekly from day 28 and until day 49 of age. on the same days, one piglet per dietary treatment was killed and alveolar macrophages (am) were harvested. vitamin c supplementation increased the concentration of igm in serum of piglets throughout the weaning period. although the vitamin e concentration in am decreased with increasing age of the piglets, the concentration was numerically higher in piglets of sows fed the high dietary level of vitamin e. however, vitamin c supplementation tended to increase the total am concentration of vitamin e after weaning and increased the proportion of the biologically most active isomer of vitamin e [rrr-(a-tocopherol)] in the am. the eicosanoid synthesis by am was not influenced by the vitamin c supplementation, but the synthesis of leukotriene b4 was decreased 2 weeks after weaning compared to other days of am harvesting. in conclusion, dietary vitamin c supplementation improved the immune responses of piglets after weaning. a whole blood stimulation assay with escherichia coli (o111:b4) endotoxin was established to measure the capacity of dairy cows to produce the proinflammatory cytokine tumour necrosis factor-a (tnf-a) ex vivo. initially, a time-and dose-dependent study was carried out to find the optimal stimulation conditions for the tnf-a response. the tnf-a response peaked between 3 and 4 h at 38.5 c. a dose in the range of 5-10 g of e. coli lipopolysaccharide (lps)/ml whole blood was found to give the maximum tnf-a response. thirty-eight danish-holstein dairy cows were investigated for their tnf-a responsiveness ex vivo in the periparturient period. heparin-stabilized blood samples were collected seven times over a period of 4 months (weeks à3, à1, 2, 3, 5, 9 and 13 around calving) and stimulated with 5 g/ml of e. coli lps. indeed, fluctuations in the tnf-a responsiveness occurred over time. moreover, the mean tnf-a responsiveness of 38 cows was found to be significantly increased (p < 0.001) in the weeks close to calving. however, in the more stabile physiological periods, some cows had a consistently low tnf-a response, whereas others had high a tnf-a response. we are currently investigating whether high and low tnf-a responders to e. coli lps also exist in dairy cows in vivo. moreover, the importance of tnf-a responsiveness ex vivo to dairy cows' susceptibility and clinical response to experimental e. coli infections in the udder is being investigated. coelomic cytolytic factor (ccf) is a 42 kda invertebrate pattern recognition molecule isolated from the coelomic fluid of the earthworm eisenia foetida (oligochaeta, annelida). ccf displays a number of similarities with the mammalian cytokine tumour necrosis factor-a (tnfa) as a result of a shared n,n 0 -diacetylchitobiose lectin-like domain. however, these similarities are solely functional and are not based on any (dna or amino acid) sequence homology, thus suggesting a form of convergent evolution. in particular, the lectin-like domain of tnf-a has been shown to induce membrane depolarization in various mammalian cell types, through interactions with endogenous amiloride-sensitive ion channels. this nonreceptor-mediated activity of tnf-a has been reported to be involved in the resorption of oedema. likewise, the lectin-like domain of ccf also induces membrane depolarization in mammalian cells. here, we show that ccf appears to be able to induce oedema resorption in an alveolar epithelial cell line through its lectin-like domain. this lectin-like domain of ccf interacts (directly or indirectly) with endogenous sodium and/or chloride channels, and not potassium channels, on mammalian cells. additionally, we suggest that the jnk/sapk and erk1/2 pathways are involved in ccf-induced macrophage activation. these results further establish the functional analogy between an invertebrate pattern recognition molecule and a mammalian cytokine and, from a more applied point of view, suggest the possibility of utilizing ccf in the treatment of oedema. release of svegf and svegfr1 from white blood cells and platelets during surgery and stimulation with bacterial antigens introduction: the influence of surgery on release of soluble vascular endothelial growth factor (svegf) and the soluble vascular endothelial growth factor inhibitory receptor 1 (svegfr1) is unknown. we studied the effect of major and minor surgery on potential variations in svegf and svegfr1 concentrations in vivo and on bacterial antigen-induced release of svegf and svegfr1 from whole blood in vitro. methods: sixty-one patients with abdominal diseases undergoing five different surgical procedures were included. blood samples were drawn from anaesthetized patients before and after the operation. white blood cells and platelets were counted, and plasma svegf and svegfr1 was determined by an elisa method. whole blood from each blood sample was stimulated in vitro with bacteria-derived antigens (lps or protein-a) and svegf and svegfr1 levels were subsequently determined in the supernatants. stimulation with isotonic saline served as control assay. neither svegf or svegfr1 in plasma changed during surgery. in vitro stimulation of blood samples with bacteria-derived antigens resulted in a significant increase in svegf (p < 0.0001) and a less pronounced but still significant increase in svegfr1. release of svegf due to stimulation was significantly higher after the operation (nonsignificant), whereas svegfr1 release remained largely unchanged after surgery. correlation between bacterial antigen-induced release of svegf and neutrophile cell count was highly significant (p < 0.0001). there was no correlation between svegf and platelet cell count, and bacterial antigen-induced svegfr1 release did not correlate with counts of neutrophils and platelets. conclusions: plasma svegf and svegfr1 concentrations did not change during surgery. in vitro bacterial stimulation led to increased release of svegf and svegfr1, which was not significantly amplified during surgery and which may be related to number of circulating neutrophils. natural killer cell functions and subsets after in vitro stimulation with il-2 and il-12, with special emphasis on intracellular ifn-g and nk-cell cytotoxicity r. nyboe, 1,2 t. rix, 1,2 j. krog, 1,2 e. tønnesen 1 & m. hokland 2 1 department of anaesthesiology and intensive care, aarhus university hospital, and 2 institute of medical microbiology, and immunology, university of aarhus, aarhus, denmark. e-mail: rnsr@studmed.au.dk materials and methods: isolated cryopreserved human peripheral blood mononuclear cells (pbmcs) were stimulated with il-2 and il-12. this stimulation has previously been shown to activate nk cells. cell cytotoxicity was measured by flow cytometry after incubation with k562 cells. this method was compared to the current standard 51cr release assay. cells were treated with bfa to accumulate ifn-g, stained for surface markers, permeabilized and stained for intracellular ifn-g. flow cytometry was then performed to measure intracellular ifn-g production in pbmc, especially in nk cells. results: we have demonstrated that stimulation with il-2 and il-12 is effective in increasing the number of ifn-gpositive cells. there is a distinct difference between the cd3-cd56dim and the cd3-cd56bright subsets, with a much greater proportion of ifn-g-positive cells in the cd3-cd56bright subset. the effects of stimulation with il-2 and il-12 on cytotoxicity will be presented, as will the relation between ifn-g production and cytotoxicity. in addition, we will present results of these assays applied to porcine cells. discussion: in combination, these tests will address nk cell function by combining cytotoxicity with ifn-g production in nk cell subsets. the results will demonstrate whether this could serve as a useful tool in describing nkcell function, which could be of value in clinical and experimental settings. culture of regulatory t-cell lines from bronchial mucosa t lymphocytes play a major role in many immune responses. in the last decade, special focus has been on the function of th1 and th2 effector cells. now the importance of regulatory cd4 þ cd25 þ t cells in maintenance of the immunological homeostasis emerges. sarcoidosis is a multisystem granulomatous disorder often affecting the lungs. the typical sarcoid granulomas consists of epitheloid cells, macrophages and lymphocytes, mainly cd4 þ t cells of th1 phenotype. we have cultured t cells from bronchial biopsies of patients with sarcoidosis as well as from controls in high levels of interleukin 2 (il-2) and il-4 and demonstrate spontaneously arising cd4 þ cd25 þ populations and high concentrations of il-10 in these cultures. the main difference between cultures of sarcoid origin compared to controls is a very much higher concentration of the inflammatory cytokines il-6 and tnf-a in cultures of sarcoid origin. the effects of hyperbaric exposure on human peripheral blood mononuclear cells, with special emphasis on natural killer cell cytotoxicity and subsets materials and methods: as an experimental physiological stress model, we examined the effects of hyperbaric exposure on peripheral blood mononuclear cells (pbmcs) obtained from venous blood drawn from eight divers during a simulated heliox saturation dive. eight persons working in normobar atmosphere outside the pressurized chamber served as control donors. the spontaneous cytotoxicity of the pbmcs was estimated in a 4 h 51cr-release assay using k562 as nk-sensitive target cells. the pbmcs were characterized, using 4-colour flow cytometry, with special emphasis on the nk-cell subsets. the data were statistically analysed using a multivariate regression model (stata 8.2). p values <0.05 was considered statistically significant. results: the estimated cytotoxicity increased significantly in both the group of divers and control donors during the dive (pdivers < 0.01 and pcontrols < 0.01). although the cytotoxicity increased relatively more (p < 0.01) in the group of divers compared to the group of control donors between day 1 and 2. discussion: the increased cytotoxicity of pbmc estimated in the group of divers indicate that parts of the cellular immune system are affected during the extreme physiological conditions induced during the initial phase of the presented experimental hyperbaric setup. the increase in cytotoxicity observed in the group of control donors could hypothetically reflect the stress level in persons working outside the pressurized chamber during the dive. the interaction with adhesion molecules expressed by vascular endothelium is the first step in lymphocyte infiltration into tissues. at both cutaneous and mucosal sites interleukin-10 (il-10), il-12 and transforming growth factor (tgf)-b are important regulators of chronic inflammatory disease, where cutaneous lymphocyteassociated antigen (cla) and ae integrin (cd103) may be expressed. unlike cla, cd103 is not believed to play a role in tissue-specific homing but may help to retain t cells within epithelial layers. we have previously shown that il-12 alone can together with an unknown cofactor increase the expression of cla. stimulation with streptococcal pyrogenic exotoxin c (spec) increased the expression of cd103 by cd8 þ but not cd4 þ t cells. while il-12 increased superantigen-stimulated expression of cla, this cytokine strongly inhibited the cd103 expres-sion, and a combination of il-12 and tgf-b completely abrogated the induced cd103 expression. conversely, il-10 suppressed cla but increased cd103 expression. these findings indicate that, in addition to suppressing the development of th1-mediated inflammatory responses, il-10 may also inhibit the migration of cd8 þ t cells into the skin while il-12 promotes such migration. thus, the expression of cla and cd103 may be antagonistically regulated by il-10 and il-12, and the balance between these cytokines could influence the t-cell migration of inflammatory cells into epithelial tissues. during contact sensitivity reaction, immune cells proliferate. in order to study the histological picture of these proliferation phases, we used a mouse model of contact sensitivity in the oral mucosa and on skin. we also used bromodeoxyuridin (brdu, an analogue to thymidin) that is incorporated into the nucleus during cell replication. the hapten oxazolone (oxa) was used to sensitize and elicit the oral mucosa and/or the ear skin. mice were killed at various times after elicitation, and unsensitized animals were also exposed to the hapten as controls. brdu (25 mg/ kg animal) was injected i.p. 2 h before the kill. specimens from the oral mucosa, ear skin and submandibular and auricular lymph nodes were cut and fixed in 4% paraformaldehyde. they were then treated with acid and biotinylated anti-brdu antibody and developed using abc-kit and dab. the analyses were performed using a leica light microscope and the computer program analysis. in the oral mucosa, the frequency of proliferating cells were increasing during the observation period, 4-24 h after elicitation, regardless of site of sensitization. the proliferating cells were found mainly in the basal cell layer of the epithelium. similar patterns were found in ear skin. the regional lymph nodes demonstrated a few scattered proliferating cells 4 h after elicitation. after 24 h, these cells were found frequently in the whole lymph node. control animals exhibited considerable less proliferating cells at all times. we conclude that most proliferating cells were found 24 h after elicitation locally at the hapten-exposed sites (the oral mucosa or the ear skin) as well as in the regional lymph nodes. the endogenous nucleoside adenosine is released in excess during inflammation or other metabolic stress and is generally known to deliver tissue protective anti-inflammatory effects. adenosine acts via four adenosine receptors of which the a2a receptor is the predominant form in t cells. adenosine levels are elevated in asthmatic lung, and adenosine can directly induce mast cell degranulation and bronchoconstriction in these patients. instead, the role of anti-inflammatory mechanisms of adenosine on t cells in asthma is unclear. aim: to study the a2a receptor expression in peripheral blood cd4 þ t cells in asthmatic and healthy individuals using flow cytometric and quantitative real-time pcr methods. results: unstimulated cd4 þ cells of asthmatic patients expressed significantly lower levels (p < 0.001) of a2a receptor in protein level (mean percentage of cells positive ae sem: 76.8 ae 1.2, n ¼ 6) compared to healthy individuals (90.4% ae 1.9, n ¼ 4). double staining for cd69 expression showed that stimulation of cd4 þ cells decreased a2a expression in both groups but indicated that the detected lower levels of a2a in unstimulated cells of asthmatics was not due to preactivation in these patients. surprisingly, a2a mrna expression in unstimulated cd4 þ cells was significantly higher (p < 0.05) in asthmatics (n ¼ 28) compared to healthy controls (n ¼ 7). the expression did not correlate with serum total ige levels. conclusions: asthmatic individuals express less a2a adenosine receptor on their peripheral cd4 þ t cells. the higher mrna levels instead may point to a negative feedback regulation in the receptor expression. the role of possibly decreased adenosine-mediated anti-inflammatory effects in asthma pathogenesis require further studies on this t-cell mediated disease. the chronic inflammatory skin disease atopic eczema (ae) affects almost 15% of the population in many countries today. the pathogenesis of ae is not fully understood. a combination of genetic predisposition and environmental factors like microorganisms seems to contribute to the symptoms. the yeast malassezia sympodialis is part of our normal skin micro flora but can act as an allergen and elicit specific ige and t-cell reactivity in patients with ae. recently, we identified a novel major m. sympodialis allergen, designated mala s 11 (22.4 kda), with sequence similarity to the mitochondrial enzyme manganese superoxide dismutase (mnsod). interestingly, mala s 11 has a high degree of homology to human mnsod. the aim of this study was to examine the effects of recombinant mala s 11 on antigen-presenting dendritic cells. monocytederived dendritic cells (mddcs) from healthy blood donors were cultured with or without mala s 11 for different time periods. it was found that the maturation marker cd83 and the costimulatory molecules cd80 and cd86 were upregulated on the mddcs exposed to mala s 11 for 24 h, as demonstrated by flow cytometry. furthermore, coculture of mddcs with mala s 11 for 9 h induced an increased production of the inflammatory cytokines il-6 (200-fold), tnf-a (100-fold) and il-8 (sixfold), as detected by the cytometric bead array (cba) analysis. our results suggest that mala s 11 affects the immune response through dc maturation and production of inflammatory cytokines. the potential cross-reactivity with human mnsod needs to be explored and the exact role of mala s 11 in the pathogenesis of ae assessed in clinical studies involving skin prick and atopy patch tests. allergen-specific immunotherapy (sit) is commonly conducted with allergen extracts adsorbed to aluminium hydroxide (alum). drawbacks linked to the use of alum, such as the formation of granuloma at the site of injection, have led to suggestions of novel allergen carriers. an alternative carrier is 2 mm carbohydrate-based particles (cbps). in mouse, allergen-coupled cbps have been demonstrated to skew the allergen-specific immune response towards a th1-like activity (grönlund et al. immunology, 2002) . we here coupled the recombinant major cat allergen fel d 1 to cbps (cbp-fel d 1) by cyanogen-bromide activation, resulting in covalent binding. the effect of cbp-fel d 1 on monocyte-derived dendritic cells (mddcs) from healthy human blood donors was studied. we found that the majority of the cd1a þ mddcs were capable of taking up fitc-labelled cbp-fel d 1, as demonstrated by flow cytometry and confocal laser scanning microscopy. furthermore, incubation with cbp-fel d 1 resulted in an upregulation of the costimulatory molecule cd86 on the mddcs, which was not observed with fel d 1 or cbps alone. finally, cbp-fel d 1 induced a fivefold increase in the release of the pro-inflammatory cytokine tumour necrosis factor (tnf)-a and a fourfold increase in the release of the chemokine interleukin-8 from mddcs. taken together, the effects cbps possess make them interesting as novel allergen carriers for sit. the cysteine protease der p1 from dust mite of the genus dermatophagoides pteronyssinus is a major type i allergen. about 80% of house dust mite (hdm) allergic individuals are reactive to this protease in standard assays for detection of ige. a curative treatment for atopic allergy is immunotherapy (it) with hdm extracts which are complex mixtures occasionally resulting in anaphylactic reactions. novozymes focuses on developing a recombinant variant of der p1 which exhibit lowered risk of ige-mediated allergic reactions, while maintaining its ability to trigger proper th-cell responses. this may provide a safer alternative for specific it of hdm allergy. a secreted recombinant form of pro-der p 1 expressed by saccharamyces cerevisiae was obtained by fusion of the pro-enzyme to a fungal signal peptide. the n-glycosylation site of der p1 was mutated resulting in a deglycosylated pro-enzyme with a molecular mass of 35 kda. protein purification procedure was developed to obtain nearly pure der p1 protein followed by determination of concentration by active-site-titration with the cysteine protease inhibitor e64. the deglycosylated recombinant pro-der p 1 revealed immunologic similarity to the native der p 1 molecule when compared in basophile histamine release, ige-binding assays and t-cell proliferation assays. by in silico epitope mapping of a modelled 3-dimensional structure of der p1, five putative igg and ige epitopes were predicted. by protein engineering, the predicted epitopes were removed one by one in der p1 and screening for hypoallergenic variants was performed. combining inhaled long-acting b-2 agonist (laba) and inhaled corticosteroid (ics) seems to offer asthma control at a lower dose of ics than achieved by ics alone. fine mapping of t-cell surface markers by flow cytometry offers a detailed status of the individual's inflammatory response. the frequency of mt2 (cd4 þ cd45ra -cd62l þ cd11adim) and mt1 (cd4 þ cd45ra -cd62l -cd11abright) cells in peripheral blood, and their ratio, has been shown to differ predictably in atopics and patients with leprosy, where mt2 correlates with a th2 phenotype and mt1 with a th1 phenotype. stable asthmatics, requiring fluticasone propionate (fp) 750-1000 mg daily or equivalent, were randomized to receive, double-blinded, either seretide 1 [salmeterol and fluticasone propionate (sfc, n ¼ 16)] 50 mg/500 mg bd or fp 500 mg bd (n ¼ 17). if asthma was controlled based on lung function and symptoms at clinic visits every 6 weeks, ics dose was tapered until asthma exacerbated or 0 mg was reached. the frequency and ratio of mt2 and mt1 t cells of the patients was monitored at 6 week intervals. as treatment tapered, the frequency of mt2 cells decreased (p ¼ 0038 from first to final visit), whereas that of mt1 cells increased. the ratio of mt2/mt1 decreased (p ¼ 0049 from first to final visit). in patients receiving laba þ ics, the fall in mt2/mt1 ratio appeared to be more pronounced than in patients receiving ics alone. thus, the mt2 phenotype may be associated with stable asthma, whereas an imminent exacerbation may associate with an increase in the mt1 phenotype. laba may allow for a greater effect of fp on the mt ratio. activation of complement pathways, leading to production of c3a and c5a anaphylatoxins, has been postulated in the pathogenesis of asthma and allergic airway inflammation. the present study was undertaken to investigate the role of mannan-binding lectin (mbl), an initiator of the lectin pathway of complement, in asthma and allergic rhinitis. mbl levels and mbl-induced complement activity were determined in 19 patients of bronchial asthma with allergic rhinitis and 20 unrelated, age-matched controls of indian origin. mbl levels and activity were correlated with percent eosinophilia and percent predicted fev1 values of the patients. association of single nucleotide polymorphisms (snps) in exon 1 and intron 1 of the mbl with the disease, clinical markers, mbl levels and mbl-induced complement activity was analysed using standard statistical tools. significantly higher mbl levels and activity were observed in patients of bronchial asthma with allergic rhinitis as compared to the controls. we identified five snps, of which two, a816g in exon 1 and g1011a in intron 1 of the mbl, were novel. snp g1011a was significantly associated with the disease (p ¼ 0.0024, or ¼ 5.8696, 95% ci: 1.7316 < or < 19.8963). individuals with 'a' allele at position 1011 showed increased mbl levels, activity and disease severity. our results suggest that 'a' allele at position 1011 leading to high mbl levels and complement activity may be contributing to the severity of bronchial asthma and allergic airway inflammation. serum resistance of borrelia burgdorferi strains belonging to the b. afzelii and b. burgdorferi sensu stricto genospecies is dependent on binding of complement inhibitor factor h. we recently reported that factor h binding by b. burgdorferi is due to inducible expression of several approximately 20 kda plasmid-encoded, surface-exposed lipoproteins related to ospe (e.g. erpa, erpp and p21). in addition, a second class of factor h-binding proteins of approximately 27-35 kda has been described. the ospe-related lipoproteins are dramatically induced by b. burgdorferi during transmission from its tick vector into the mammalian host. the induction of ospe-related lipoproteins during mammalian infection may play a key a role in the borrelial evasion of the host's immune system. the goal of the present study was to define the factor h-binding regions of ospe-related proteins using mutagenesis, peptide mapping and surface plasmon resonance analysis (biacore). the combined studies revealed that the c-terminal regions of both human and mouse factor h (scrs 18-20) specifically bind to ospe-related lipoproteins. we also found fhr-1, whose c-terminal scrs 3-5 are homologous to scrs 18-20 of factor h, to bind to ospe. peptide mapping revealed five putative regions (designated i-v) in ospe that could directly interact with factor h. deleting the c-terminal 15 amino acid residues from region v of p21 abolished its ability to bind factor h. at the same time, however, synthetic peptides corresponding to the c-termini of ospe, p21 and erpp did not inhibit factor h binding to ospe. thus, the c-terminal-binding region v appears to be necessary but not sufficient for factor h binding. when a more specific mutation strategy was employed, where single amino acid residues in peptides spanning over the factor h-binding regions were mutated to alanines, we observed that lysines in the factor h-binding regions of ospe were required for factor h binding. the combined data have revealed that key lysine residues in ospe-related lipoproteins and ionic interactions are crucial for factor h interactions. furthermore, binding of ospe to the c-termini of both mouse and human factor h suggests that borrelia spirochetes utilize analogous complement resistance mechanisms in both rodents and man. in borrelia garinii strains, which in in vitro analyses have been found to be sensitive to complement killing, differences in the ospe sequences as well as in the expression of factor h-binding proteins may account for their susceptibility to serum lysis. role of yada, ail and lipopolysaccharide in serum resistance of yersinia enterocolitica serotype o:3 mannan-binding lectin (mbl), l-ficolin and h-ficolin are pattern recognition molecules of the innate immune system. we investigated the ability of these molecules to bind to different serotypes and noncapsulated variants of streptococcus pneumonia and staphylococcus aureus. we found that mbl binds to noncapsulated s. aureus strain (wood) but not any of the examined s. pneumoniae serotypes. l-ficolin binds to some capsulated s. pneumoniae serotypes (11a, 11d and 11f) as well as some capsulated s. aureus serotypes (type-1, -8, -9, -11 and -12). h-ficolin does not bind to any of the examined s. pneumoniae and s. aureus serotypes included in this study but did bind to a strain of aerococcus viridans. when bound to bacteria, mbl and h-ficolin initiated activation of complement factor c4, whereas l-ficolin did not. during this study, quantitative assays for the three proteins were developed and the concentration in 97 plasma samples were determined and the median values were estimated at 0.8 mg of mbl/ml, 3.3 mg of l-ficolin/ml and 18.4 mg of h-ficolin/ ml, respectively. the absence of early complement components (c1, c4 and c2 but not c3) is a predisposing factor for systemic lupus erythematosus (sle). recently, we demonstrated that, in c4-deficient (c4 def.) mice, igm-containing immune complexes (igm-ic) are filtered by the splenic barrier of marginal zone macrophages (mzm), resulting in an increased immune response against antigens within these igm-ic, but this could not be observed in wildtype or c3 def. mice. we hypothesized that splenic cd11b þ mzm play an important role in the induction of autoimmunity, and we therefore analysed their cytokine profile after isolation with the help of magnetic antibody cell sorting. mrna was isolated, and real-time pcr was performed with specific primers for murine ifn-g (ifn-g), interleukin-12 (il-12) and ifn-a (ifn-a). we observe a moderate increase of il-12 and ifn-g mrna in cd11b þ cells of c4 def. mice compared to wildtype cells. surprisingly, the concentration of ifn-a mrna is six times higher in c4 def. mice. preliminary results suggest that mrna in cd11b þ cells of c3 def. mice is even lower than that in wt. six hours following i.v. application of 20 mg of a abstracts 625 .................................................................................................................................................................................................. murine monoclonal igm anti-dsdna antibody, production of il-12, ifn-g and ifn-a mrna is increased in cd11b þ cells of both c4 def. and wt mice. several references described increased levels of inf-a in patients with sle. dendritic cells are discussed as a major source of ifn-a. our observation that c4-deficient, sle-susceptible mice demonstrate an increased spontaneous ifn-a production by splenic cd11b þ marginal zone macrophages could be an early sign and a trigger for the development of sle. this is supported by the fact that the absence of c3 is not a predisposing factor for sle and our observation that c3 def. animals display low levels of ifn-a mrna. 200-400 million people worldwide and represents one of the leading causes for liver cirrhosis and hepatocellular carcinoma. control over the hbv infection is achieved mainly by vaccination with hepatitis b surface antigen (hbsag). hbsag contains n-linked glycosylation side and is recognized by both mbl-a and mbl-c in a cadependent manner. hbsag-mbl complexes activate complement and may thus affect humoural immunity. to investigate the role of mbl in humoural responses to hbsag, we immununized mice that lack both mbl-a and mbl-c proteins with soluble hbsag. it has been shown that deficiencies in other complement components like c1q, c4 and c3 result in decreased antibody responses. however, mbl double ko animals mounted dramatically increased humoural responses. after priming, mbl double kos mounted hbsag-specific igm responses, which were threefold higher than wt controls. after boosting the hbsag, total igg was 10-fold higher in mbl ko than in wt control animals. similar to the response to hbsag, other glycosylated soluble antigens (e.g. invertase) induced better humoural responses in mbl double ko animals, suggesting that mbl plays an important role in a negative feedback regulation of adaptive immunity. reconstitution experiments with rmbl partially rescued the ko phenotype. we propose that the clearance of glycoprotein antigens in mbl ko is handled differently from the wt, resulting in better stimulation of humoural responses. alternatively, glycoprotein-ag-mbl-rich complexes inhibit b-cell responsiveness via putative mbl receptors. the complement system is an important part of the innate immune system. the activation of complement proceeds through three different pathways that converge in the generation of c3-activating enzyme complexes. complement activation via the lectin pathway is initiated when recognition molecules, mannan-binding lectin (mbl) or ficolin, bind to carbohydrate structures characteristic for microbial surfaces. in the circulation, mbl and ficolins are found in association with three structurally related mblassociated serine proteases (masp)-1, -2 and -3 and a small, nonenzymatic component, map19. masp-2 has been shown to elicit complement activation through the sequential proteolytic cleavage of c4 and c2 upon binding of mbl/masp-2 complexes to microbial surfaces. we have recently uncovered a polymorphism in the masp-2/map19 gene in a patient shown to be deficient in the lectin pathway of complement activation. the polymorphism results in a single amino acid substitution in the n-terminal part of the masp-2 protein. recombinant wildtype masp-2 and masp-2 containing the amino acid substitution in question was produced, and the ability to activate complement was studied. the mutation had a profound impact on masp-2 function, resulting in the lack of complement activation through the lectin pathway. elisa-based experiments showed that the mutation leads to the impairment of complement activation through influencing the binding of masp-2 to mbl or ficolins. deficiencies in the lectin pathway of complement activation have so far been accounted for only by lack of functional mbl. the mutation described above is the first defect described affecting both activation through mbl and the ficolins. .................................................................................................................................................................................................. th1, th2 and treg cell balance. dcs are present in the gut mucosa and may thus be target for modulation by gut microbes, including ingested probiotics. here, we tested the hypothesis that species of lactic acid bacteria, important members of the gut flora, differentially activate dc. a large panel of human gut-derived lactobacillus and bifidobacterium spp. was screened for dc-polarizing capacity by exposing bone marrow-derived murine dc to lethally irradiated bacteria. cytokines in culture supernatants and dc-surface maturation markers were analysed. substantial differences were found among strains in the capacity to induce interleukin-12 (il-12) and tumour necrosis factor (tnf)-a, while the differences for il-10 and il-6 were less pronounced. bifidobacteria tended to be weak il-12 and tnf-a inducers, while both strong and weak il-12 inducers were found among the strains of lactobacillus. remarkably, strains weak in il-12 induction inhibited il-12 and tnf-a production induced by an otherwise strong cytokine-inducing strain of lactobacillus casei, while il-10 production remained unaltered. selected strains were tested for induction of dc maturation markers. those lactobacilli with greatest capacity to induce il-12 were most effective in upregulating surface mhc class ii and cd86. moreover, l. casei-induced upregulation of cd86 was reduced in the presence of a weak il-12inducing l. reuteri. in conclusion, human lactobacillus and bifidobacterium spp. polarize differentially dc maturation. thus, the potential exists for th1/th2/treg-driving capacities of the gut dc to be modulated according to composition of gut flora including ingested probiotics. the intestinal micro flora is indispensable in developing and maintaining homeostasis of the gut-associated immune system. evidence indicates that lactic acid bacteria (lab), e.g. lactobacilli and bifidobacteria, have beneficial effects on the host. established health effects include increased gut maturation, antagonisms towards pathogens and immune modulation. the objective of this study is to evaluate the immunomodulating properties of a range of lab of human origin. as dendritic cells (dcs) play a pivotal role in the balance between tolerance and immunity to commensal microorganisms, in vitro-generated immature dcs serve as a suitable model for studying the immunomodulating effects of lab. human immature dcs were generated in vitro from monocytes and exposed to lethally uv-irradiated lab. the effect of various species of lab on dcs in direct contact was evaluated. furthermore, the maturation pattern of dcs separated from the bacteria by an epithelial cell layer (caco-2 cells), which should mimic the intestinal environment, was studied. cytokine secretion (il-12, il-10 and tnf-a) and upregulation of maturation surface markers on dcs (cd83 and cd86) was measured. different lab induced diverse cytokine responses. some strains were strong il-12 and tnf-a inducers and others weak. all strains induced il-10. different lab also differentially modulated expression of cd83 and cd86 on dcs. although some variation in the response to lab of dcs generated from different blood donors was observed, general differences in the effect of the various lab was revealed. experiments with the dc caco-2 coculture system are ongoing. different species of lab differentially affect dc maturation; this suggets that the gut flora plays a pivotal role in polarization of the immune response. natural killer (nk) cells are cells of the nonspecific immune system lysing altered self-cells. a noncytolytic subset of nk cells may serve a regulatory role by secreting cytokines. bacteria translocating across the gastrointestinal mucosa are presumed to gain access to nk cells, as consumption of certain lactic acid bacteria has been shown to increase in vivo nk cytotoxicity. here, we investigated how human gut flora-derived lactobacilli affect nk cells in vitro, by measuring proliferation and ifn-g production of human nk cells upon bacterial stimulation. cd3 -cd56 þ nk cells were isolated from buffy coats by negative isolation using non-nk lineage-specific antibodies and magnetic beads. nk cells were incubated with 10mg/ml uv-inactivated bacteria or 10mg/ml phytohemagglutinin (pha) for 4 days. proliferation was assessed by incorporation of radioactive thymidine into nk-cell dna. the ifn-g concentration was measured by elisa. incubation of nk cells with a lactobacillus acidophilus strain increased the proliferation of the nk cells and induced ifn-g production, both to levels comparable to pha stimulation. the proliferative response was further enhanced with autologous monocytes present, probably because cytokines, secreted by monocytes having engulfed bacteria, stimulated the nk cells. in contrast, a lactobacillus paracasei strain caused the nk cells to proliferate only in the presence of monocytes. these results demonstrate that various strains of lactobacilli have the capacity to activate nk cells in vitro, in a monocyte-dependent or -independent way. hence, the encounter of nk cells with lactic acid bacteria will affect nk-cell activation. such activation of nk cells may potentially skew an on-going or subsequent immune response towards a th1 response. lactobacilli are nonpathogenic gram-positive inhabitants of the normal human intestine known for their healthpromoting effects. in our earlier work, it is shown that human monoclonal antibody isolated from sera of a patient with waldenstrom macroglobulinaemia possess innate antibody characteristics and binds to lactic acid bacteria. according to the immune network model, immunization with this bacteria could induce the perturbations in immune system that might result in production of anti-lactobacillus antibodies, human monoclonal antibody like (ab1) and anti-idiotypic antibody (ab2). in this study, balb/c mice were immunized with two doses of bacteria lactobacillus acidophilus in complete and incomplete freund's adjuvant and phosphate-buffered saline (pbs), respectively. seven days after the last immunization, sera from immunized mice were collected and the presence of lactobacillus-specific ab1 and ab2 were determined by elisas. in the sera of immunized mice, antibodies specific to bacteria lactobacillus acidophilus were shown. the concentration of lactobacillus-specific antibodies was higher in the sera of hyperimmunized mice (mice immunized with 1 mg of igm dj) than in sera of mice immunized with 100 times lower doses of immunogen (0.01 mg per doses). moreover, ab1 and ab2 antibodies were detected in the sera of lactobacillus-hyperimmunized mice. in this study, we have shown the idiotypic network interactions in mice immunized with bacteria lactobacillus acidophilus. the normal gastrointestinal flora is crucial for the maturation of the acquired immunity via effects on antigenpresenting cells (apcs). here, we have investigated how two types of apcs, monocytes and dendritic cells (dcs), react to different bacterial strains typical of the commensal intestinal flora. purified monocytes and monocyte-derived dcs were stimulated with uv-inactivated gram-positive (lactobacillus plantarum and bifidobacterium adolescentis) and gram-negative (escherichia coli and veillonella parvula) bacterial strains. monocytes produced higher levels of il-12p70 and tnf, as detected by elisa, in response to l. plantarum than to e. coli and v. parvula. in contrast, dcs secreted high amounts of il-12p70, tnf, il-6 and il-10 in response to e. coli and v. parvula but were practically unresponsive to l. plantarum and b. adolescentis. the lack of response to the gram-positive strains correlated with a lower surface expression of toll-like reseptor 2 (tlr2) on dcs compared to monocytes. the surface expression of tlr4 on dcs was undetectable when analysed by flow cytometry, but blocking this receptor decreased the tnf production in response to v. parvula, indicating that low tlr4 expression on dcs is sufficient to mount an inflammatory response to gram-negative bacteria. ifn-g increased the expression of tlr4 on dcs and also potentiated the cytokine response to gram-negative bacteria. our results indicate that, when monocytes differentiate into dcs, their ability to respond to different commensal bacteria dramatically changes, thereby becoming unresponsive to probiotic gram-positive bacteria. these results may have important implications for the capacity of different groups of commensal bacteria to regulate mucosal and systemic immunity. probiotic bacteria, e.g. lactobacillus spp., may improve diseases such as chronic inflammatory bowel disease. we examined cytokine production and phenotypic change after in vitro stimulation of t cells from healthy volunteers using different probiotic strains. methods: t cells were cultured from colonic biopsies from eight healthy volunteers (agnholt and kaltoft, exp clin immunogenet 2001; 18:213-25) , and dendritic cells were matured from their peripheral blood mononuclear cells. t-cell cultures were stimulated with autologous bacterial sonicate or strains of lactobacillus spp., with and without the addition of dendritic cells. cytokine levels (tnf-a, ifn-g, il-10 and gm-csf) and phenotype (cd3, cd4, cd25 and cd69) were measured on day 4. results: lactobacillus spp. induced higher productions of tnf-a and il-10 than did autologous bacteria. in presence of dendritic cells, the production of all cytokines increased. however, the increases of ifn-g and tnf-a were more pronounced in wells with autologous bacteria than in wells with lactobacillus spp. the addition of dendritic cells upregulated cd25 expression without simultaneous upregulation of cd69. the upregulation was pronounced after stimulation with lactobacillus rhamnosus gg compared with autologous bacteria and other lactobacilli. discussion: in presence of dendritic cells, autologous bacteria induced inflammatory cytokines, while probiotics mainly induced regulatory cytokines. lactobacillus rhamnosus gg induced a regulatory phenotype (cd25 þ ), in part mediated by dendritic cells. future studies will address whether this shift to a cd25 þ phenotype represents a differentiation into competent regulatory t cells. in a clinical context, such cells might be used for treatment of inflammatory diseases. protein microarrays will play a key role in the postgenomic era and offer a unique possibility to perform highthroughput global proteome analysis. a chip can be printed with thousands of protein probes (e.g. antibodies), the biological sample added (e.g. a proteome) and any binding detected. we aim to develop protein microarrays based on human recombinant scfv antibody fragments for global proteome analysis. the concept of comparing proteomic maps of healthy versus diseased samples will allow diseasespecific proteins to be detected. in fact, antibody microarrays will allow us to perform comparative proteome analysis on any sample format in a species-independent manner, as long as a proteome can be isolated. however, the complexity of proteomes, containing several thousands of different proteins, is a problem. here, we have designed antibody microarrays targeting the water-soluble fraction of a proteome. to this end, an anticytokine antibody array was developed and human dendritic cells (aeactivation) was used as model system. the results showed that our antibody microarrays could be used to examine the cytokine profile in complex samples. furthermore, we have taken the first steps towards comparing our results with those of other technologies on both the protein and gene level. due to the complexity of the model proteome, we also examined the possibility to prefractionate the proteome in a simple one-step procedure (based on size) prior to the labelling step. in more detail, the sample proteome was fractionated into two fractions using membrane devices with different molecular weight cut-offs. the results showed that the fractionation considerably enhanced the assay sensitivity allowing cytokines in the pg/ml range to be readily detectable. the immunomodulatory effect of heat shock protein 70: immunization with a dna construct based on the malarial antigen fused with a fragment of hsp 70 primes for a th-1 type of response finding an appropriate adjuvant for human vaccination is crucial. heat shock proteins (hsps) act as adjuvants when coadministered with peptide antigens or given as fusion proteins. however, there is a potential risk of autoimmunity when using the complete molecules, because hsps are evolutionary conserved. to overcome this, we first evaluated the adjuvant effect against two different antigens of a less-conserved fraction of plasmodium falciparum hsp70 (pf70c) and compared it to the whole hsp70 molecule from trypanosoma cruzi (tchsp70). we found that pf70c exhibited similar adjuvant properties as the whole molecule. we later evaluated the adjuvant potential of pf70c against the malarial antigen eb200 in a chimeric dna construct. no appreciable levels of eb200-specific abstracts 629 .................................................................................................................................................................................................. antibodies were detected in mice immunized only with the dna constructs. however, dna primed the immune system, because subsequent challenge with the corresponding recombinant fusion proteins elicited a strong th-1 antibody response. in contrast, no priming effect was observed for ex vivo ifn-g production but stimulation with the hsp-chimeric fusion protein induced a stronger secretion of ifn-g in vitro than other proteins used. these results indicate that the use of hsps is promising in the design of new vaccines. high-throughput proteomics on antibody-based microarrays: the importance of probe and surface design in analogy to dna microarrays, protein microarrays offer a new distinct possibility to perform sensitive highthroughput global proteome analysis. however, the development of the protein microarray technology will place high demands upon the design of both probes and solid supports. the analysis of thousands of heterogeneous proteins on a single microarray requires the use of uniform probes, such as antibodies, directly designed for protein microarray applications. we have recently generated a human recombinant single-chain fv antibody library, genetically constructed around one framework, the ncoder-library, containing 2 â 1010 clones. single framework antibody fragments (sinfabs) selected from this library were successfully applied as probes for microarrays providing sensitive detection in the 600 attomol (mass spectrometry) and the 300 zeptomole range (fluorescence). however, the choice of framework is critical. we have shown that the selected ncoder framework displayed excellent functional on-chip stability and arrayed dehydrated probes retained their activity for several months. furthermore, we have addressed the issues of biocompatibility of the solid support and immobilization strategies for our microarray setup. an in-house-designed substrate, macroporous silicon coated with nitrocellulose (map3-nc7), displayed properties equal to, or better than, those of five commercially available supports used as reference surfaces. we have also evaluated different coupling strategies, such as adsorption, covalent coupling, diffusion and affinity coupling. using a novel affinity tag, the double-(his)6-tag, we increased the binding efficiency of sinfab-molecules to ni2 þ -coated solid supports, thereby allowing nonpurified probes to be directly applied. the mannan-binding lectin (mbl) pathway is part of the innate immune system providing a first line of defence against infections. mbl and ficolins circulate in complexes with mbl-associated serine proteases (masp-1, -2 and -3). after recognition of a microorganism by mbl, activation of the complement system occurs. masp-1 and masp-3 share five domains (making up the so-called a-chain), whereas they have unique protease domains (b-chains). before the identification of masp-3, an assay for masp was presented, based on antibodies against the a-chain of masp-1. with the new knowledge of the three masps, and the sharing of domains by masp-1 and masp-3, assays specific for the protease domains have to be constructed, if one wishes to measure the proteins individually. we present an assay for quantifying total masp-3 in plasma and serum samples. the assay is a sandwich-type assay using as catching antibody a monoclonal antibody against the common a-chain of masp-1/3 and a developing secondary antibody against the c-terminal part of the protease domain of masp-3. we have used this assay for estimating the normal concentration of the protein as well as the concentration in patients and also for characterizing by gel permeation chromatography the masp-3 protein in serum. inducible costimulator ligand (icosl) is a costimulatory molecule related to b7.1 (cd80) and b7.2 (cd86). b cells, monocytes, dendritic cells and endothelial cells express icosl. inducible costimulator (icos) interacts with icosl, and this interaction leads to signals involved in isotype switching and the development of immunological memory. hitherto, no polymorphisms of this gene have been described. the aim of this study was to reveal variation of the icosl gene in normal individuals. all eight exons, except exon 1, were sequenced with flanking introns in 10 healthy blood donors. eight single nucleotide polymorphisms (snps) and two length polymorphisms were found. one of the snps was found in the coding regions of the gene. the base involved was located in exon 3 and caused a conservative amino acid change from valine (gtt) to isoleucine (att). three individuals were heterozygous g/a for the exon polymorphism, while the remaining seven individuals were homozygous for the wildtype g/g. exon 3 encodes the immunoglobulin variable (igv)-like domain of the molecule which is situated outside the cell. this means that the amino acid could be critical for the stability of the molecule or could constitute part of the binding site for icos. the results form the basis for further experiments to find possible associations of the alleles to diseases caused by immune dysregulation. especially, the exon 3 variant is interesting and could play a role for the development of immunological diseases. besides, it would be interesting to see whether both exon 3 alleles are expressed or only the wildtype allele is functional. interferon-a/b (ifn-a/b) is increasingly used as antiviral and immunomodulatory therapies. unfortunately, bioavailability varies with ifn species and mode of administration, and all ifn species are potentially immunogenic. assays for antiviral activity (ifn) and antiviral neutralization (antibodies, nab) have been used for some time to monitor patients on ifn biologicals. these assays require laborious titrations making them unsuitable for large-scale clinical use. myxovirus a (mxa) is a resistance gtpbinding protein that is specifically induced by treatment with type 1 ifns. for example, ifn-b-induced mxa in blood leucocytes has been used as a biomarker in ifn-btreated patients with multiple sclerosis. however, the degree of specificity of mxa in this regard is unclear, and measurements of mxa protein and/or mrna are not yet suitable for routine clinical use. in an attempt to find new and better reporter genes (and, hopefully, genes and gene products with proven specificity for ifn-a and -b), microarray screenings with u133a genechips (affymetrix) were carried using human blood leucocytes and the human lung carcinoma cell line a549. we studied the simultaneous expression of 22,000 transcripts before and after exposure to human recombinant ifn-a and ifn-b and other antiviral and immunomodulatory cytokines. the results will be presented at the conference. interferon-a/b (ifn-a/b) is increasingly used as antiviral and immunomodulatory therapies. unfortunately, bioavailability varies with ifn species and mode of administration, and all ifn species are potentially immunogenic. assays for antiviral activity (ifn) and antiviral neutralization (antibodies, nab) have been used for some time to monitor patients on ifn biologicals. these assays require laborious titrations making them unsuitable for large-scale clinical use. our laboratories have therefore modified the antiviral assays for ifn bioactivity and nab, so that they are suitable for large-scale screening in specialized laboratories. the read-out is survival of a subcloned a549 cell line in the presence of an otherwise lethal amount of virus. thus, survival increases in the presence of type 1 ifn and decreases in the presence of nab against the ifn added to the cells. mxa is induced by type 1 ifn and can be used for measuring the nab activity. in another assay, the mxa level in the a549 cell line is measured. in an attempt to find a new and better reporter gene for type 1 ifn than mxa and genes specific for either ifn-a or -b, a micro array screen was carried using the u133a chip from affymetrix. the expression of 22,000 genes can be studied simultaneous with this technology. the results will be presented at the conference. in our laboratory, we have developed a database system, which we believe is of immediate interest to the general scientific community. the database represents a computerbased replacement for the laboratory notebooks used in the majority of research laboratories worldwide. in addition, the database provides an effective tool for organizing and managing laboratory information at all levels, spanning from managing and revising standard operating procedures and producing documentation of research activities to keeping track of data and conclusions. using the commercially available database toolkit software filemaker pro, we have developed a relational database solution for management of laboratory information. the system consists of a hierarchy of five interrelated databases, each pertaining to a separate type of information, namely, overall project information, information relating to individual experiment setups, documentation of daily research activity, generated data and descriptions of standard operating procedures. like other databases, each individual database consists of a number of records, each comprised of a set of fields in which information is entered. in each record, a certain field is reserved to specify the relation of the record to a record in another database at a higher level. thus, the database is essentially five databases linked by a hierarchy of one-to-many relations, organizing information in a folder-like structure. importantly, the database system allows multiple users to access and edit records simultaneously, and the data entered in one database immediately becomes accessible through the other databases. the limitations of laboratory notebooks are apparent when looking for information, which is dispersed throughout one or more notebooks, or possibly on loose sheets of paper or printouts 'somewhere'. the often complicated process of gathering laboratory data or results when writing grant applications or research papers is made considerably easier with the database system. thus, the database solution presented should be broadly attractive to researchers, irrespective of their scientific discipline. an effective sars vaccine is likely to include components that can induce specific cytotoxic t-cell (ctl) responses. the specificities of such responses are governed by hlarestricted presentation of sars-derived peptide epitopes. exact knowledge of how the immune system handles protein antigens would allow for the identification of such linear sequences directly from genomic/proteomic sequence information. the latter was recently established when a causative coronavirus (sars cov) was isolated and full-length sequenced. here, we have combined advanced bioinformatics and high-throughput immunology to perform an hla supertype, genome-wide scan for sars-specific cytotoxic t cell epitopes. the scan includes all nine human hla supertypes in total covering >99% of all major human populations. for each hla supertype, we have selected the 15 top candidates for test in biochemical-binding assays. at this time (approximately 6 months after the genome was established), we have tested the majority of the hla supertypes and identified almost 100 potential vaccine candidates. these should be further validated in sars survivors and used for vaccine formulation. we suggest that immunobioinformatics may become a fast and valuable tool in rational vaccine design. rationale: major histocompatibility complex class i (mhc i) molecules monitor the protein content of the cell by binding small derived peptides and presenting them to cytotoxic cd8 þ t cells. the goal of the human mhc project is to predict the binding strength of any given peptide/mhc complex. this prediction allows the design of peptide-based vaccines. the prediction requires representative binding data from mhc alleles from all the nine hla supertypes. here, we describe the genetic construction, protein production and purification as well as the establishment-binding assays for two recombinant mhc supertype alleles, hla-b*1501 and hla-b*5801. methods: using the quikchange multisite directed mutagenesis kit (stratagene), codon-optimized genes encoding hla-b*1501 and hla-b*5801 are created. the two mhc i molecules are fermented and purified by ion exchange chromatography, hydrophobic interaction chromatography and size exclusion chromatography. the binding (kd) of natural t-cell epitopes, as well as predicted peptide ligands, is described by radioactive immunoassays (rias) and enzyme-linked immunosorbent assays (elisas). the mhc molecules are biotinylated during expression. results: the expression of mhc i resulted in multiple disulfide bond isomers, which are separated by hydrophobic interaction chromatography and used in subsequent binding studies resulting in the determination of kd for various peptide ligands ranging from strong binders we have previously demonstrated that bioinformatics tools such as artificial neural networks (anns) are capable of performing pathogen-, genome-and hlawide predictions of peptide-hla interactions. these tools may therefore enable a fast and rational approach to epitope identification and thereby assist in the development of vaccines and immunotherapy. a crucial step in the generation of such bioinformatics tools is the selection of data representing the event in question (in casu peptide-hla interaction). this is particularly important when it is difficult and expensive to obtain data. herein, we demonstrate the importance in selecting information-rich data and we develop a computational method, query-bycommittee, which can perform a global identification of such information-rich data in an unbiased and automated manner. furthermore, we demonstrate how this method can be applied to an efficient iterative development strategy for these bioinformatics tools. methods: a large panel of binding affinities of peptides binding to hla a*0204 was measured by a radioimmunoassay (ria). this data was used to develop multiple first generation anns, which formed a virtual committee. this committee was used to screen (or 'queried') for peptides, where the anns agreed ('low-qbc'), or disagreed ('high-qbc'), on their hla-binding potential. seventeen low-qbc peptides and 17 high-qbc peptides were synthesized and tested. the high-or low-qbc data were added to the original data, and new high-or low-qbc second generation anns were developed, respectively. this procedure was repeated 40 times. the high-qbc-enriched ann performed significantly better than the low-qbc-enriched ann in 37 of the 40 tests. conclusion: these results demonstrate that high-qbcenriched networks perform better than low-qbc-enriched networks in selecting informative data for developing peptide-mhc-binding predictors. this improvement in selecting data is not due to differences in network training performance but due to the difference in information content in the high-qbc experiment and in the low-qbc experiment. finally, it should be noted that this strategy could be used in many contexts where generation of data is difficult and costly. interleukin-18 (il-18), a pro-inflammatory cytokine that is produced by both lymphoid and nonlymphoid cells, has a critical role in modulation of innate and adaptive immunity. its primary function in stimulation of ifn-g production and stimulation of nk-cell-cytotoxic activities makes this cytokine a candidate for cancer immunotherapy. in oral cavity, this cytokine is produced by oral epithelia and carcinoma cells and is related to tumour regression in nude mice bearing salivary adenocarcinoma. however, direct effects of this cytokine on oral cancer cells have not been elucidated. in this project, we investigated il-18 effect on an oral carcinoma (kb) cell line. with rt-pcr technique, kb-cell line was found to express il-18 receptors (il-18ra and il-18rb), indicating that this oral carcinoma line is a target for il-18 study. we showed that recombinant human il-18 inhibited kb-cell proliferation by 17% at concentration of 100 ng/ml (p < 0.05), whereas ldh release by these cells in treatment group and control groups was comparable, indicating that il-18 suppression of cell proliferation was not mediated by the induction of cell death. to further address this hypothesis, we found that il-18 treatment did not induce apoptotic cell death, as studied by dna laddering and tunel assays. in addition, expression pattern of cell death-controlling genes (bcl-2 and bax) was not altered by this cytokine. findings in these studies indicated that suppression of kb-cell proliferation may be attributed to control of cell cycle, growth arrest or induction of cell differentiation. the data presented in this project could provide an insight of how cancer cell directly responds to il-18, as this cytokine is an important regulator of anticancer mechanisms. aloe emodin (ae) is a naturally occurring compound with wide spectrum of biological properties, including antimicrobial, vasorelaxant, immunosuppressive and anticancer actions. this anthraquinone induces apoptosis in several tumour cell lines with special affinity to tumours of neuroectodermal origin. high amounts of nitric oxide (no) released by activated macrophages induce tumour cell death. therefore, we explored the capacity of ae to modulate no-mediated antitumour response in vitro. interestingly, while ae markedly suppressed no release from macrophages alone, it significantly potentiated no production in cocultures of macrophages and c6 cells, after 48 h of cultivation. accordingly, the viability of c6 cells cocultivated with macrophages was reduced in the presence of ae. moreover, the observed ae-imposed potentiation of no production in macrophages was closely related to macrophage culture cell density. according to these data, we proposed that no modulator capacity of ae strongly depended on intercellular contact, indicating that macrophage antitumour response was not compromised but even potentiated by ae. immunotherapy represents an attractive fourth-modality therapeutic approach, especially in the light of the shortcomings of conventional surgery, radiation and chemotherapies in the management of metastatic cancer. to this end, a large number of peptide antigens derived from taa have been applied in immunotherapeutic trials for the treatment of various malignancies, e.g. cancers of the breast, prostate and kidney, in addition to haematological cancers. in some cases the response rates have been impressive and no adverse autoimmunity have been observed. a major strategic difficulty associated with these trials relates to the choice of best-suited peptide antigens. the vast majority of the antigens described thus far is not vital for survival and growth of the tumour cells, and immunoselection of antigen-loss variants may therefore prove to be an additional obstacle for the clinical applicability of most of the known peptide epitopes. in this respect, the development of acquired antigen loss during immunotherapy has been demonstrated in several cases. obviously, the development of loss-variant tumour cells implies that these cells acquire a pronounced growth advantage and are left unaffected by further treatment. ideally, target antigens should be derived from proteins required for survival and growth of tumour cells, as antigens with these characteristics would not be inflicted by the development of loss-variant tumour cells. in this respect, several inhibitors of apoptosis proteins (iaps) are universally expressed among tumours and play an important role in tumour cell escape from apoptosis. we have characterized spontaneous t-cell reactivity against iapderived peptides in cancer patients. from the iap survivin, we have characterized peptides restricted to the class i molecules hla-a1, a2, a3, a11, b7 and b35. furthermore, we have demonstrated that survivin-specific t cells infiltrate metastatic lesions and that isolated survivinspecific ctls are capable of killing hla-matched tumour cells. survivin-derived peptides are now in clinical trial, and continued work in our lab has demonstrated that other iaps are targets for spontaneous t-cell reactivity in cancer patients. we previously reported that in mice with large progressing t-cell lymphoma tumours, dysfunctions in the antitumour ctl activity occur, associated with an accumulation of splenic arginase-producing myeloid suppressor cells (mscs). in this study, we first demonstrate that both the presence and the activation state of these msc depends on tumour evolution. while in tumour regressors hardly any arginase-producing msc can be found, both the amount and the arginase activity of this population expands from early over late progressors. this gradual induction of mscs is paralleled by an increasing suppression of ctl activity and th1, but not th2, cytokine production. upon analysing the molecular repertoire of msc in vitro, we found, besides arginase1, a well-established marker for alternatively activated myeloid cells or m2, a strong upregulation of fizz1 and ym, two additional recently identified markers for m2. further evaluation of molecular markers by microarray analysis in msc yielded genes involved in wound healing (e.g. coagulation factor xiiia), anti-inflammation (e.g. selenoprotein p), immunomodulation (e.g. pd-l2) and fat and sugar metabolism (e.g. leptin receptor). of note, many of these genes are regulated by type 2 cytokines (il-4, il-13 and il-10) and are therefore rather m2 associated. overall, our data provide new markers for msc in cancer and further establish their m2 activation state. study. only sp-a showed a significant expression in normal mucosa which was downregulated in crc. as the absolute signal level was below the noise threshold, these results have to be interpreted with caution and require confirmation by direct measurenment of the proteins. our results suggest that there is no major role for the human collectins in colorectal cancer. tetramerization is visualized by sds-page. conclusion: an effective method for the production of highly pure mhc i molecules has been applied to hla-b*1501 and hla-b*5801, and ria and elisa binding assays for those alleles have been established background: proliferation, differentiation and apoptosis are essential processes in the normal functions of the mammary epithelium. the hypothesis examined in this study is that the transcription factor bcl-6 is critically important not only for regulating b-cell growth and development but also for mammary epithelial apoptosis. methodology: twenty breast cancer cases and 31 healthy controls were used to investigate whether bcl-6 protein in involved in breast cancer (grade iii). full length bcl-6 cdna was retrovirally transduced into eph-4 cell line. we then used flow cytometry of brdurd-stained cells to investigate the cell-cycle duration of the control and transduced cell lines. tunel was used as a marker of apoptosis to find out differences in the frequencies of apoptotic cells in the control and transduced cell lines. finally, immunohistochemistry staining was performed to detect bcl-6 in breast cancer (iii). results: restoration of bcl-6 into eph-4 cells not only inhibits apoptosis but also prolongs the cell cycle and results in increased cell size and protein content. the results also indicated that the cell-cycle time of bcl-6-transduced eph-4 cells is prolonged by about 3 h, presumably as a result of the action of bcl-6 at the bcl-6 at the g1/s transition. we found differences in the frequencies of viable and apoptotic cells in cultures of the parent eph-4 cells, control-transduced eph-4 cells and bcl-6-transduced eph-4 cells. consistently, we demonstrated that bcl-6 is expressed in 90% of high grade of breast carcinoma, which is considered as the most aggressive of tumours. conclusion: together, these results suggest that bcl-6 is likely to be involved in mammary gland development and carcinogenesis. inflammatory cytokines have a critical role in modulation of both innate and adaptive immunity in response to foreign antigen. they also play an important role in anticancer immunity. for example, they can promote cell-mediated immunity against cancer cells. with their immunostimulatory effects, these cytokines are being tested for cancer treatment in the form of dna vaccine or adjuvant or therapeutic cytokines. direct effect of these cytokines on cancer cell, however, is still unclear. in this project, we investigated whether il-1( and il-18 can modulate cancer cell proliferation. we employed a simple nonradioactive proliferation (mtt) assay and detection of lactate dehydrogenase (ldh) to test the effect of these recombinant human cytokines on various cancer cell lines, including breast cancer cell line (mcf-7), oral carcinoma cell line (kb), colon cancer cell line (caco-2) and choriocarcinoma cell line (jar). cytokines used in this study had both inhibitory and stimulatory effect on cell proliferation. findings in this project could provide an insight of cancer cell response to these cytokines and this could lead to a consideration on using cytokine as immunotherapy for cancer treatment.capacity of ae to modulate nitric oxide production depended on intercellular contact donor t cells are involved in the antitumour effects observed after bmt. thus, patients receiving t-celldepleted bmt have a higher risk of leukaemia relapse compared to patients receiving nonmanipulated bmt, and patients experiencing graft-versus-host disease (gvhd) have a lower risk of disease relapse than patients who do not experience gvhd. although the importance of donor t cells for the curative action of bmt has been established, the exact mechanisms and molecules involved in this graft-versus-tumour effect remain largely unknown. in a recently initiated project, we have conducted a longitudinal study of t-cell clonotypes in patients who received peripheral blood stem cell grafts after nonmyeloablative conditioning. peripheral blood samples were obtained sequentially after transplant, and the mononuclear cells (mncs) were isolated and cryopreserved. cd8 þ t cells were isolated from the mncs by use of immunomagnetic beads or facs and analysed for the presence of clonally expanded cells by t-cell receptor clonotype mapping based on rt-pcr and denaturing gradient gel electrophoresis (dgge). using this gel-based methodology, clonally expanded t cells were monitored after transplant and compared to the clinical data of the patients. the preliminary results demonstrates the presence of clonally expanded cd8 þ t cells at all time points analysed. furthermore, a number of clonotypes persisted for more than 6 months, and other clonotypes emerged during this period. the appearance of newly emerged clonotypes which coincided with clinical gvhd could indicate a role for these t cells in the pathogenesis of gvhd. background: deficiency of the mannan-binding lectin (mbl) pathway of innate immunity leads to increased susceptibility to infections. in patients with colorectal cancer, postoperative infection is associated with poor prognosis. the aim of the present study was to evaluate (1) the relation between the mbl pathway and postoperative infectious complications and survival of patients resected for colorectal cancer and (2) the role of mbl as acute phase reactant compared to crp. methods: preoperative mbl concentration, mbl/mblassociated serine protease (masp) activity and crp were determined in serum from 611 patients and 150 healthy controls. the patients were observed for 8 years. postoperative infections, recurrence and survival were recorded. results: the mbl pathway components were increased in the patients (p < 0.0001) compared to healthy controls. low mbl levels were predictive of pneumonia (p ¼ 0.01), and pneumonia (n ¼ 87) was associated with poor survival (p ¼ 0.003, hr ¼ 1.5, 95% ci 1.1-1.9). mbl and mbl/ masp activity could not predict postoperative overall infections. mbl showed no correlation (spearman's r ¼ 0.02, 95% ci à0.06-0.10) with crp. conclusions: low preoperative mbl levels are predictive of pneumonia, which is associated with poorer survival. mbl concentration and mbl/masp activity was not predictive of other postoperative infections or long-term prognosis. mbl apparently is not a surrogate measure of crp. department of surgery, university hospital of erlangen, erlangen, germany. e-mail: michael.siassi@rzmail.uni-erlangen.de introduction: the human collectins, mannan-binding lectin (mbl), surfactant protein-a (sp-a) and surfactantprotein-d (sp-d) play a central role in the innate immune system. immunological responses to malignant transformation of epithelial cells gained increasing interest recently. a former study could demonstrate binding of mbl to certain colorectal carcinoma (crc) cell lines in vitro. we therefore examined the expression of human collectins in normal colon mucosa and in colorectal carcinomas. materials and methods: colon samples from 20 crc patients and 10 normal mucosa samples were collected immediately after surgery. the tissue was microdissected and rna isolated (qiagen, rneasy-kit). gene expression profiles were analysed using gene-chips (affymetrix, hg-u133). we analysed the data for the expression of mbl, its associated serine proteases mannan-binding lectinassociated serine protease 1/2 (masp 1/2), sp-a and sp-d. the signal intensity of the genes of interest was compared using the mann-whitney u-test. results: the expression of human collectins in normal human colon mucosa was generally low. only the expression of sp-a and masp-2 reached the noise threshold of 250 signals. these genes were significantly downregulated in crc specimens. the expression of the other proteins showed no difference in normal mucosa and crc. conclusion: as demonstrated before, the expression of human collectins in normal colon was low in this being the first lymph node to receive drainage from the tumour area, the sentinel node offers a unique possibility to obtain tumour-reactive lymphocytes. we investigated antitumour immune responses in sentinel nodes from patients with bladder cancer, by assaying tumour-specific proliferation and tcr vb repertoires. during tumour surgery, sentinel lymph nodes were identified by peritumoural injection of blue dye. fresh specimens of tumour, sentinel and nonsentinel lymph nodes were obtained, and single-cell suspensions were prepared. cells were assayed for reactivity against autologous tumour extract in [ 3 h]-thymidine incorporation assays and characterized by flow cytometry. parallel analyses of the expression of vb gene families were performed with padlock probes, linear oligonucleotides which upon target recognition can be converted to circular molecules by a ligase. probes were reacted with cdna prepared from magnetically separated cd4 þ cells, and the tcr repertoire was determined by hybridizing the products to oligonucleotide microarrays. dose-dependent proliferation in response to tumour extract could be detected in sentinel lymph nodes. common clonal expansions were detected among tumourinfiltrating lymphocytes and in sentinel lymph nodes. nonsentinel lymph nodes displayed a divergent tcr vb repertoire. these results indicate an ongoing immune response against tumour antigens in sentinel nodes, draining urinary bladder cancer. identification of sentinel lymph nodes makes it possible to obtain tumour-reactive lymphocytes for use in adoptive immunotherapy. key: cord-011436-ud35mf5l authors: li, yingying; zhao, ling; luo, zhaochen; zhang, yachun; lv, lei; zhao, jianqing; sui, baokun; huang, fei; cui, min; fu, zhen f.; zhou, ming title: interferon-λ attenuates rabies virus infection by inducing interferon-stimulated genes and alleviating neurological inflammation date: 2020-04-06 journal: viruses doi: 10.3390/v12040405 sha: doc_id: 11436 cord_uid: ud35mf5l rabies, caused by rabies virus (rabv), is a fatal neurological disease that still causes more than 59,000 human deaths each year. type iii interferon ifn-λs are cytokines with type i ifn-like antiviral activities. although ifn-λ can restrict the infection for some viruses, especially intestinal viruses, the inhibitory effect against rabv infection remains undefined. in this study, the function of type iii ifn against rabv infection was investigated. initially, we found that ifn-λ2 and ifn-λ3 could inhibit rabv replication in cells. to characterize the role of ifn-λ in rabv infection in a mouse model, recombinant rabvs expressing murine ifn-λ2 or ifn-λ3, termed as rb2c-ifnλ2 or rb2c-ifnλ3, respectively, were constructed and rescued. it was found that expression of ifn-λ could reduce the pathogenicity of rabv and limit viral spread in the brains by different infection routes. furthermore, expression of ifn-λ could induce the activation of the jak-stat pathway, resulting in the production of interferon-stimulated genes (isgs). it was also found that rrabvs expressing ifn-λ could reduce the production of inflammatory cytokines in primary astrocytes and microgila cells, restrict the opening of the blood-brain barrier (bbb), and prevent excessive infiltration of inflammatory cells into the brain, which could be responsible for the neuronal damage caused by rabv. consistently, ifn-λ was found to maintain the integrity of tight junction (tj) protein zo-1 of bbb to alleviate neuroinflammation in a transwell model. our study underscores the role of ifn-λ in inhibiting rabv infection, which potentiates ifn-λ as a possible therapeutic agent for the treatment of rabv infection. rabies causes acute incurable encephalitis and is responsible for more than 59,000 human deaths each year, thus posing a severe threat to public health [1] . rabies virus (rabv) has a non-segmented, recombinant rabv strain b2c was generated from cvs-b2c, which was attenuated from the challenge virus standard (cvs-24) in baby hamster kidney (bhk-21) cells [23] . the cloned cell line bsr cells were derived from bhk-21 cells [24] , which were cultured in dulbecco's modified eagle's medium (dmem) (gibco, grand island, ny, usa) containing 10% fetal bovine serum (fbs) (gibco, grand island, ny, usa) and 1% antibiotics (penicillin and streptomycin) (beyotime, wuhan, china). mouse brain capillary endothelial cell line b.end3 cells (atcc-crl-2299) were cultured in dmem containing 10% fbs and 1% antibiotics (penicillin and streptomycin). mouse neuroblastoma (na) cells [25] were maintained in rpmi 1640 medium (mediatech, herndon, va, usa) containing 10% fbs and 1% antibiotics (penicillin and streptomycin). african green monkey kidney cells (vero, atcc-ccl-81) were cultured in dmem containing 10% fbs and 2% antibiotics (penicillin and streptomycin). hek-293t cells (atcc-crl-3216) were maintained in rpmi1640 medium supplemented with 10% fbs and 1% antibiotics (penicillin and streptomycin). na cells or vero cells were seeded into 12-well plates and cultured for 24 h, and then infected with b2c at a multiplicity of infection (moi) of 0.01. at 24 hpi, the cells were treated with 10 or 1000 ng/ml of ifn-λ2 or ifn-λ3 (bd biosciences, san jose, ca, usa) by addition into the culture medium. at indicated time points after treatment, virus titers in the supernatant were measured. primary mixed glial cell cultures were established as described previously [26] . briefly, brain tissues from 2-day-old balb/c mice were dissociated by repeated pipetting and then passed through a 75-nm nylon mesh (corning, ny, usa). the cells were washed once in cold pbs and cultured in dmem (with high glucose) supplemented with 10% fbs and 1% penicillin-streptomycin. the medium was changed on days 3, 5, and 7. on day 10, the flasks were shaken at 260 rpm for 2 h to remove any non-adherent cells (mainly microglia). the remaining adherent astrocytes were detached with trypsin-edta and then plated again for further experiments. the purity of the isolated astrocytes for further studies was greater than 95%, which was examined by immunohistochemistry using the anti-gfap antibody. primary microglia cells were prepared from cerebral cortices of 2-day-old balb/c mice as described previously [26] . briefly, brain tissues were collected from the mice, and the cortex was dissected and minced in pbs containing 0.25% trypsin for digestion at 37 • c for 30 min with a shake every 5 min. after digestion, the dmem supplemented with 5% fbs and dnase i were added and incubated for 5 min. then the isolated cells were resuspended with dmem supplemented with 10% fbs and the cell mass were removed by 40 µm cell sieve filtration. finally, the isolated primary microglia cells were incubated at 37 • c for 7-9 days and change the culture medium with fresh dmem containing 10% fbs at day 5. the purity of the isolated primary microglia for further studies was greater than 90%, which was examined by staining the cells with anti-iba1 antibody using ifa. the rrabv vector pb2c was constructed by inserting the genome of cvs-b2c into the mammalian expression vector pcdna3.1 as described previously [27] . a transcription unit containing bsiwi and nhei restriction sites was inserted between the g-and l-coding sequences by deleting the pseudogene. the rb2c-ifnλ2 and rb2c-ifnλ3 cdna clones were generated from pb2c as previously described [28] . briefly, the pb2c vector was digested with bsiwi and nhei (neb, ipswich, ma) between the g and l genes. murine ifnλ-2/3 cdnas were prepared by rt-pcr amplification using template rna isolated from vsv-infected mouse lung tissues [29] . the ifnλ-2 and ifnλ-3 genes were then inserted into pb2c, generating pb2c-ifnλ2 and pb2c-ifnλ3, respectively. pcr primers are listed in table 1 . the full length infectious clones (pb2c-ifnλ2 and pb2c-ifnλ3) and four helper plasmids (expressing genes n, p, g, and l from the parent virus b2c) were separately transfected into bsr cells using superfect transfection reagent (qiagen, valencia, ca, usa) according to procedures described in previous studies [30] . after incubating for 4 days, the culture medium was harvested and then examined for the presence of rescued rrabvs using fitc-conjugated anti-rabv n antibodies, and the specific fluorescence would be observed under an olympus ix51 fluorescence microscope if the virus is successfully rescued. table 1 . primers for construction and rescue of recombinant rabies viruses (rabvs) expressing murine ifn-λ. sequence (5 -3 ) bsiwi and nhei sites are underlined. fluorescence morphologies were determined using a direct fluorescent antibody assay as previously described [30] . na cells were infected at a low moi (0.01) with different rrabvs, overlaid with semisolid medium containing 1% agar, and incubated at 34 • c for 48 h. the agar was removed and the adhesive cells were stained with fitc-labeled rabv n-specific antibody. twenty fluorescent foci were examined to calculate the number of infected cells per fluorescent focus by using image j software [31] . virus titers were determined using a direct fluorescent antibody assay as previously described [27] . briefly, a series of 10-fold dilutions of the virus were prepared and used to inoculate bsr cells in 96-well microplates. the inoculations were performed in quadruplicate and then incubated at 37 • c for 48 h. after incubation, the cells were fixed with 80% ice-cold acetone and stained for 1 h with fitc-conjugated rabv n protein-specific antibodies. antigen-positive foci were observed under an olympus ix51 fluorescence microscope, and virus titers were calculated and presented as focus-forming units/ml (ffu/ml). elisa was performed to quantify the amount of ifnλ-2/3 in na cell culture supernatants. the assays were performed using commercially available mouse ifnλ-2/3 elisa kits (raybiotech, atlanta, ga, usa), following the manufacturer's instructions. the samples (tissues or cells) were collected on ice and homogenized in trizol (invitrogen). total rna was isolated and used for qrt-pcr. briefly, complementary dna (cdna) was prepared with 1 µg rna as template using a first-strand cdna synthesis kit (toyobo). the thermocycler conditions were used for cdna synthesis (10 min at 25 • c, 30 min at 55 • c, and 5 min at 85 • c). each qpcr was conducted in duplicate with approximately 100 ng dnase-treated rna and 5 nm primer pairs, using a one-step sybr green qrt-pcr mix kit (toyobo). primers are listed in table 2 . the following conditions describe real-time pcr amplification (60 s at 94 • c; 15 s at 94 • c, 15 s at 60 • c, and 45 s at 72 • c for 40 cycles; 60 s at 72 • c), and the cycle threshold (ct) values were recorded. a standard curve was generated from serially diluted plasmids carrying a rabv n gene and the copy numbers of viral messenger rna of rabv n gene (mrna) and viral genome rna (vrna) were normalized to 1 µg of total rna [28] . to quantify the level of vrna, the primer vrna-f was used for reverse transcription, while the primer n mrna-r was used for reverse transcription of n-mrna quantification. the ct value was inversely correlated with the mrna concentration, and each ct unit represented a twofold change in the mrna concentration. the mrna levels of chemokines/cytokines and tj proteins were normalized to β-actin mrna levels. the results were expressed as fold change relative to mrna levels detected in mock-infected controls. table 2 . primers for qrt-pcr. sequence (5 -3 ) cell pellets were lysed in ice-cold ripa lysis buffer containing protease inhibitor cocktail (composed of a proprietary mix of aebsf, aprotinin, bestatin, e64, leupeptin, and pepstatin a to promote broad spectrum protection against endogenous proteases). the mixture was homogenized and centrifuged at 10,000× g for 10 min at 4 • c. after centrifugation, insoluble material was removed, and total protein concentration in the supernatant was measured using a bca protein assay kit (beyotime, wuhan, china). each sample was subjected to polyacrylamide gel electrophoresis, transferred onto a nitrocellulose membrane (bio-rad, richmond, ca, usa), and blocked for 1 h at 37 • c with 3% bovine serum albumin (bsa) in tris-buffered saline with 0.05% tween-20 (tbst). membranes were then incubated overnight with primary antibodies. after extensive washing with tbst, the membranes were incubated with secondary antibodies. antibody binding was visualized using enhanced chemiluminescence reagents (beyotime). bands were quantified using imagej (nih, bethesda, md, usa). the values represent the relative immunoreactivity of each protein, normalized to the respective loading control. five-week-old female balb/c mice (n = 10) were inoculated under isoflurane anesthesia. the groups received the following treatments: (a) inoculated intramuscularly (i.m.) with a 100 µl volume of 6 × 10 5 ffu; (b) infected intradermally (i.d.) in both ears with a dose of 1.5 × 10 5 ffu in 30 ul dmem; (c) inoculated intranasally (i.n.) in 20 µl of a solution containing 150 ffu of b2c, rb2c-ifnλ2, or rb2c-ifnλ3 or mock-infected with dmem. body weight loss, clinical signs, and survivor numbers were recorded daily for 21 days. the animals were scored for clinical signs as follows: 0, normal mouse; 1, disorder movement; 2, ruffled fur; 3, trembling and shaking; 4, paralysis; 5, dead. all animals were humanely euthanized at the end of the experiment. 293t cells (2×10 5 cells per well) were seeded into 24-well plates. the cells were transfected with 50 ng of luciferase reporter plasmids (a gift of prof. xiao shaobo from huazhong agricultural university) [32] , together with pcaggs-ifnλ or pcaggs, using lipofectamine 2000 (thermo scientific, shanghai, china). in parallel, 20 ng of prl-tk renilla luciferase reporter plasmid (promega, madison, wi, usa) was transfected to normalize transfection efficiency. twenty-four hours after transfection, the cells were infected with b2c, rb2c-ifnλ2, and rb2c-ifnλ3 for 12 h. luciferase activity in total cell lysates was measured using a dual-specific luciferase reporter assay system (promega, madison, wi, usa). primary astrocytes were mock infected or infected with rrabvs at a moi of 5. cell supernatants were collected at 48 hpi. inflammatory cytokines (il-1β, il-6, il-17a, ifn-γ, kc, tnf-α, and vegf) were quantified in the mock-and rabv-infected cell supernatants using a quantibody mouse cytokine array 1 kit (raybiotech, norcross, ga, usa), according to the manufacturer's protocol. the array was scanned using a genepix 4000b (molecular devices, axon instruments, silicon valley, ca, usa). data were collected using the genepix pro application at a photomultiplier tube (pmt) gain ranging from 540 to 790. a gain of 590 generated the optimal standard curve, and the results of this scan were analyzed using q-analyzer for qam-cyt-1 (raybiotech). a transendothelial permeability assay was conducted as previously described with minor modifications [33] . b.end3 cells were cultured on transwell filters (pore size 0.4 µm) until reaching 100% confluence. after treatments, fitc-dextran-10000 (10 kda; sigma-aldrich, st. louis, mo, usa) was applied apically at 1 mg/ml for 30 min. samples were then removed from the lower chamber for fluorescence measurements with a fluorimeter (excitation, 492 nm; emission, 520 nm). animals were anesthetized with ether and were perfused by intracardiac injection of phosphate-buffered saline (pbs) as described previously [18] . mouse brains were fixed with 4% paraformaldehyde for 24 h at 4 • c and then washed with pbs. brain tissues were harvested and embedded in paraffin for coronal sections. to detect rabv, nonspecific binding was blocked with 10% goat serum, and then sections were incubated with dapi and fitc-conjugated antibodies against the rabv n protein. to detect inflammatory cells, harvested sections were incubated with primary antibodies against cd45 at the concentrations indicated in the manufacturer's guidelines, and then incubated with biotinylated secondary antibodies. the sections were observed under an olympus ix51 fluorescence microscope. for immunofluorescence, b.end3 cells were seeded on coverslips. after forming a confluent monolayer, they were suspended in medium containing supernatants from infected astrocytes. the cells were subsequently fixed with 4% paraformaldehyde (pfa), permeabilized with 0.1% triton x-100, and then incubated with rabbit anti-zo-1 polyclonal antibodies. finally, they were incubated with secondary antibody conjugated with alexa fluor 488, and with dapi for nuclear counterstaining. cells were imaged using a laser confocal microscope (leica, germany). bbb permeability was determined by measuring sodium fluorescein uptake as described previously [16, 34] . briefly, 100 µl of 100 mg/ml sodium fluorescein was injected intraperitoneally into each mouse. peripheral blood was collected after circulation for 10 min, and pbs-perfused brains were then harvested. the recovered serum was mixed with an equal volume of 10% trichloroacetic acid (tca) and then centrifuged for 10 min. the volume of the supernatant was adjusted to 150 µl with 5 m naoh and 7.5% tca. homogenized brain samples in cold 7.5% tca were centrifuged for 10 min at 10,000× g to remove debris. the supernatant was adjusted to 150 µl with addition of 5 m naoh. fluorescence of serum and brain homogenate samples was measured using a spectrophotometer (biotek instruments, vt, usa) with excitation at 485 nm and emission at 530 nm. the amount of sodium fluorescein taken up into brain tissues is calculated as (µg of fluorescence cerebrum, cerebellum, or brain stem/mg of tissue)/(µg of fluorescence sera/ml of blood) to normalize values for blood levels of the dye at the time of tissue collection. data are expressed as fold differences between the amount of tracer in tissues from virus-infected mice and the amount in tissues from the uninfected control. all data were analyzed using graphpad prism 8 (graphpad software, lnc., san diego, ca, usa). for the percent survival tests, kaplan-meier survival curves were analyzed using the log rank test. for the other data, an unpaired two-tailed t-test was used to determine whether differences were statistically significant. data were representative of two independent experiments. for all results, the following notations are used to indicate significant differences between groups: *, p < 0.05; **, p < 0.01; ***, p < 0.001. to test whether ifn-λ restricts rabv replication in vitro, na or vero cells were infected with b2c strain, and recombinant mouse ifn-λ2 or ifn-λ3 at 10 ng/ml and 1000 ng/ml were added to treat the rabv infected cells at 24 h post infection (hpi), and the virus titers, the levels of mrna of rabv n gene (n mrna) and viral rna (vrna) were measured at 24 and 48 h after the treatment. in na cells, as shown in figure 1a -c, the addition of ifn-λ2 or ifn-λ3 reduced the virus titers and the levels of n mrna and vrna at both time points. notably, at 48 h after treatment with 1000 ng/ml of ifn-λ2 or ifn-λ3, more than 10-fold decreases of virus titers were observed compared with mock-treated cells. in the vero cells, an ifn-α/β independent cell line, the addition of ifn-λ2 or ifn-λ3 reduced the virus titers and the levels of n mrna and vrna at 24 h post treatment as shown in figure 1d -f. at 48 h post treatment, no significant difference was observed in the virus titers and the levels of n mrna and vrna among all. these results suggest that ifn-λ2 and ifn-λ3 inhibits rabv replication in the two cell lines used. to further characterize the role of ifn-λ in rabv infection in the mouse model, recombinant rabvs (rrabvs) expressing murine ifn-λ2 or ifn-λ3, designated as rb2c-ifnλ2 and rb2c-ifnλ3 respectively, were constructed as shown in figure 2a , and rescued as described previously [27] . the insertion of both genes into the rabv genome was verified by rt-pcr and sequencing (data not shown). to detect whether the rrabvs could express ifn-λ2 or ifn-λ3, na cells were infected with the rrabvs, and ifn-λ2 or ifn-λ3 were determined by elisa. as shown in figure 2b , elisa results indicate that ifn-λ2 and ifn-λ3 were well expressed in rb2c-ifnλ2 and rb2c-ifnλ3 infected cells, respectively, in a dose-dependent manner, whereas those expressed in b2c did not. moreover, viral growth curves on bsr, na, and vero cells were depicted. as shown in figure 2c -e, compared with parent virus b2c, more than 10-fold losses of viral titers were observed in rb2c-ifnλ2 or rb2c-ifnλ3 infected bsr or na cells at 72 and 96 hpi, and in rb2c-ifnλ2 or rb2c-ifnλ3 infected vero cells at all tested time points. additionally, consistent with the results of the growth curve, western blot experiments detecting rabv n protein showed that expression of ifn-λ2 or ifn-λ3 by the rrabvs reduced rabv n protein levels in infected na cells at 48 hpi ( figure 2f) . furthermore, the effects of expression of ifn-λ2 or ifn-λ3 on virus spread were measured by observing the fluorescence morphologies of rrabv-infected cells and counting the number of rabv-positive cells using fluorescence microscopy. as a result, the fluorescence foci of cells infected with rb2c-ifnλ2 or rb2c-ifnλ3 were significantly smaller than those infected with b2c ( figure 2g,h) , indicating that expression of ifn-λ suppresses the cell-to-cell spread of rabv. taken together, these results suggest that expression of ifn-λ2 or ifn-λ3 inhibits rabv replication and spread in infected cells. 3) . the following notations were used to indicate significant differences between groups: *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001. to further investigate whether ifn-λ restricts rabv infection in vivo, groups of five-week-old female balb/c mice were mock-infected with dmem or inoculated with b2c, rb2c-ifnλ2, or rb2c-ifnλ3 by different routes. the body weight losses of mice infected with rb2c-ifnλ2 or rb2c-ifnλ3 by the three routes (i.m., i.d., or i.n.) were lower than those infected with b2c, as shown in figure 3a ,d,g. similarly, the clinical scores of mice infected with rb2c-ifnλ2 or rb2c-ifnλ3 by either route were lower than those infected with b2c ( figure 3b,e,h) . consistently, significantly higher percentage of survivor ratios were observed in rb2c-ifnλ2 or rb2c-ifnλ3 infected mice than b2c infected mice ( figure 3c ,f,i). it is worth noting that the most striking enhancement on survival rates among the three infection routes were observed in i.n. route-that 100% and 80% of mice i.n. infected with rb2c-ifnλ3 (p = 0.0005) and rb2c-ifnλ2 (p = 0.0126), respectively, survived, while 80% of b2c i.n. infected mice died of rabies within 17 days ( figure 3i ). all the mice exhibited rabv-related symptoms and death were confirmed by rt-pcr from brain tissues. the following notations were used to indicate significant differences between groups: *, p < 0.05; **, p < 0.01; ***, p < 0.001. to investigate the viral load in the brains of infected mice, balb/c mice were i.n. infected with 150 ffu of rrabvs, or mock-infected with dmem, and viral burdens were analyzed in different parts of the brain by qrt-pcr at 6, 9, 12, and 15 days post infection (dpi). viral copy numbers were extrapolated by quantitating mrnas corresponding to the rabv n gene. as expected, viral copy numbers in the olfactory bulb, cerebrum, cerebellum, and brain stem of rb2c-ifnλ2 or rb2c-ifnλ3 infected mice were significantly lower than those infected with b2c ( figure 4a ). additionally, viral antigen (rabv n protein) in different brain tissues was also detected using immunofluorescence staining. consistent with the qrt-pcr results, almost no (or under detection limit) viral antigens were observed in all the parts of the brains of rb2c-ifnλ2 or rb2c-ifnλ3 infected mice, while an obviously positive fluorescent signal was observed in all parts of the brains of detected mice that were infected with b2c ( figure 4b ). all the above data demonstrate that ifnλ significantly reduces the pathogenicity of rabv in mice. . ifn-λ restricts rabv replication in the mouse brain. five-week-old female balb/c mice were inoculated i.n. with 150 ffu of b2c, rb2c-ifnλ2, rb2c-ifnλ3, or dmem alone. (a) n mrna copy number was measured in mouse olfactory bulb, cerebrum, cerebellum, and brain stem at 6, 9, 12, and 15 dpi by qrt-pcr (n = 5). (b) mouse brain sections (n = 3) were stained with fitc-labeled rabv n-specific antibody to detect the distribution of viral antigen in the central nervous system (cns) parenchyma (shown in green); nuclei stained with 4',6-diamidino-2-phenylindole (dapi) are shown in blue. representative images were acquired at 40× magnification (scale bar = 100 µm). error bars represent the se. the following notations were used to indicate significant differences between groups: *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001. as a typical neurotropic virus, rabv mainly infects neurons in the brain. therefore, to define the signaling pathway by which ifn-λ restricts rabv infection, a luciferase reporter assay was carried out in na cells as described in the methods section. the results suggest that expression of ifn-λ2 or ifn-λ3 could activate ifnα4, ifnβ, and isg54-isre, but down-regulated the expression of nuclear factor κb (nf-κb) ( figure 5a) . furthermore, the mrna levels of ifn-α, ifn-β, stat1, interferon-induced protein with tetratricopeptide repeats 2 (ifit2), and ifn-inducible gtpase 1 (iigp1) in different rrabv infected na cells were detected by qrt-pcr. significantly higher levels of ifn-α4, ifn-α5, ifn-β, stat1, ifit2, and iigp1 were detected in the cells infected with rb2c-ifnλ2 or rb2c-ifnλ3 than those in cells infected with b2c. meanwhile, the mrna levels of nf-κb (p65) and tnf-α were significantly decreased in na cells infected with rb2c-ifnλ2 or rb2c-ifnλ3 compared with those infected with b2c ( figure 5b) . additionally, expression of isgs (ifit2 and iigp1) and activation of jak-stat pathway, phosphorylation of stat1, were also measured by western blotting assay at 24 hpi, and high levels of phosphorylated stat1, ifit2 and iigp1 were detected in rb2c-ifnλ2 or rb2c-ifnλ3 infected cell. these data demonstrate that ifn-λ enhances the expression of isgs via activating jak-stat pathway, and thus inhibits rabv replication. figure 5 . ifn-λ activates stat1/2 and enhances the production of isgs. (a) na cells were transfected with plasmids encoding ifn-α4, ifn-β, isg54-isre or nf-κb firefly luciferase reporter, respectively. after 24 h, cells were left uninfected or were infected for 12 h with different rrabvs. luciferase assays were performed to analyze the promoter activity of isre, ifn-α4, ifn-β and nf-κb, respectively. luciferase reporter activity was expressed as fold change that normalized to renilla luciferase activity. (b) na cells were infected with b2c, rb2c-ifnλ2, or rb2c-ifnλ3 at a moi of 1. at 24 hpi, total rna was isolated and ifn-α4, ifn-α5, ifn-β, stat1, ifit2, iigp1, nf-κb (p65), and tnf-α mrna levels were analyzed by qrt-pcr. (c) protein levels of stat1, ifit1, ifit2, iigp1, and β-actin in different rrabv-infected na cells were measured by western blot at 24 hpi. error bars represent the se (n = 3). the following notations were used to indicate significant differences between groups: *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001. as is known, astrocytes and microglia cells play an important role in innate immunity and neuroinflammation in the cns. hence, primary astrocytes and microglia cells were isolated as described in the materials and methods section and infected with different rrabvs at a moi of 5, and the production of several inflammatory cytokines was measured in the infected astrocytes and microglia cells. viral titers in the supernatants of astrocytes or microglia cells incubated with b2c, rb2c-ifnλ2, or rb2c-ifnλ3 were nearly equal ( figure 6a,b) . the supernatants of astrocytes were then applied to a protein array to quantify the production of specific cytokines, and the mrna levels of gm-csf, il-1β, il-6, kc, tnf-α, and vegf were quantified in microglia cells by qrt-pcr. as shown in figure 6c , pro-inflammatory cytokines, including tnf-α, il-6, il-17a, il-1β, chemokine (c-x-c motif) ligand 1 (cxcl1/kc), and vascular endothelial growth factor (vegf, which is related to bbb opening), were significantly lower in astrocytes infected with rb2c-ifnλ2 or rb2c-ifnλ3 than those infected with b2c. similarly, the mrna levels of gm-csf, il-1β, il-6, kc, tnf-α, and vegf were significantly decreased in microglia cells infected with rb2c-ifnλ2 or rb2c-ifnλ3 than those infected with b2c ( figure 6d ). together, these results suggest that ifn-λ represses the production of inflammatory cytokines induced by rabv infection. figure 6 . ifn-λ reduces the production of inflammatory cytokines in primary astrocytes and microglia cells. astrocytes and microglia cells isolated from 2-day-old suckling mice were infected with b2c, rb2c-ifnλ2, or rb2c-ifnλ3 at a moi of 5, and cell supernatants and lysates were collected at 48 hpi for astrocytes and microglia cells, respectively, for the determination of indicated cytokines. the rrabv titers were measured using a focus-forming assay and are expressed as ffu/ml on astrocytes (a) and microglia cells (b). concentrations of the indicated cytokines in astrocyte supernatants were measured using a cytokine array (c) and microglia cells lysates were measured by qrt-pcr (d). error bars represent the se (n = 3). the following notations were used to indicate significant differences between groups: *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001. it is possible that the expression of ifn-λ in the cns can subdue the neuroinflammatory response and block the elevation of bbb permeability due to the low expression level of inflammatory cytokines and vegf in rb2c-ifnλ2 or rb2c-ifnλ3 infected astrocytes. meanwhile, the enhancement of bbb permeability that associated with rabv infection is caused by cytokines and the infiltration of inflammatory cells, as demonstrated in a previous study [16] . to test this hypothesis, six-week-old female balb/c mice were i.c. infected with rrabvs. to exclude the effect of different virus loads on the regulation of bbb permeability and neuroinflammation, the brains of mice infected by different rrabvs were harvested to analyze viral burden by qrt-pcr. at 3, 6, and 9 dpi, mrna levels of rabv n were nearly equal in different brain regions of mice infected by b2c, rb2c-ifnλ2, or rb2c-ifnλ3 ( figure 7a ). sodium fluorescein was injected into the mice via the tail vein for measurement of bbb permeability. at 3 and 9 dpi, no significant difference in the amount of sodium fluorescein uptake was detected among the three groups. at 6 dpi, leakage of sodium fluorescein from the peripheral circulation into the cerebrum, cerebellum, and brain stem in b2c-infected mice was significantly higher than those in mice infected with rb2c-ifnλ2 or rb2c-ifnλ3 ( figure 7b ). moreover, the brain sections were then stained with anti-cd45 antibody to observe and quantify the infiltration of cd45 + cells induced by different rrabvs infection. less positive signal was observed in different parts of brain from mice infected with rb2c-ifnλ2 or rb2c-ifnλ3 than those from mice infected with b2c ( figure 7c ). significantly more cd45 + lymphocytes were found in the cerebral cortex, hippocampus, hypothalamus, cerebellum, and brain stem from the mice infected with b2c than those infected with rb2c-ifnλ2 or rb2c-ifnλ3 at 6 dpi ( figure 7d ). these results indicate that ifn-λ declines the bbb permeability to prevent excessive infiltration of inflammatory cells into the cns during rabv infection. figure 7 . ifn-λ decreases bbb permeability and alleviates neurologic inflammation in the mouse brain. six-week-old female balb/c mice were inoculated i.c. with 100 ffu of rb2c, rb2c-ifnλ2, rb2c-ifnλ3, or dmem (mock). (a) at 3, 6, and 9 dpi, mice (n = 5) were euthanized, and the brains were harvested, and viral loads were determined by qrt-pcr. (b) the change in bbb permeability was assessed by measuring the amount of sodium fluorescein uptake in the cerebrum, cerebellum and brain stem using a spectrophotometer after intraperitoneal administration at 3, 6, and 9 dpi (n = 5). (c) for the assessment of neurologic inflammation, six-week-old female balb/c mice were inoculated i.c. with 100 ffu of b2c, rb2c-ifnλ2, rb2c-ifnλ3, or dmem (mock). at 6 dpi, mice brains were collected and embedded in paraffin. the sections were then stained with anti-cd45 antibody to quantify inflammation. the scale bars represent 100 µm (n = 3). (d) cd45 + lymphocytes from at least three different randomly selected areas of each brain region were counted. error bars represent the se. the following notations were used to indicate significant differences between groups: *, p < 0.05; **, p < 0.01. to explore the mechanism by which ifn-λ reduces bbb permeability, a transwell model was performed. mouse brain capillary endothelial (b.end3) cells were cultured in the upper chamber of a transwell insert, with supporting astrocytes in the lower chamber. the b.end3 cells were then treated for 24 h with extracts from the supernatants of astrocytes infected with different rrabvs, and the expression of the tj proteins zo-1 and occludin were then analyzed by qrt-pcr and western blot. both mrna ( figure 8a ) and protein expression ( figure 8b ) of zo-1 in cells treated with supernatants from b2c-infected astrocytes was significantly lower than that those incubated with the supernatants from rb2c-ifnλ2 or rb2c-ifnλ3 infected astrocytes. additionally, supernatant-treated cells were also stained with anti-zo-1 polyclonal antibodies and imaged by a laser confocal microscope to assess the integrity of zo-1. the rb2c-infected astrocytes supernatants caused dissociation of zo-1, while those treated with rb2c-ifnλ2 or rb2c-ifnλ3 infected astrocytes supernatants reduced the effect ( figure 8c,d) . to further evaluate the integrity of the endothelial monolayer, fitc-dextran 10,000 was added to the upper chamber of a transwell insert and the fluorescence in the lower chamber was monitored. as shown in figure 8e , the leakage of fitc-dextran 10,000 was significantly lower when treated with supernatants from rb2c-ifnλ2 or rb2c-ifnλ3 infected astrocytes than that treated with b2c infected astrocytes supernatant. these data suggest that ifn-λ maintains the integrity of tj proteins, resulting in the decrease in the bbb permeability during rabv infection. end3 cells. mouse brain capillary endothelial (b.end3) cells were co-cultured for 24 h with extracts from supernatants of astrocytes that had been mock-infected with dmem or infected with b2c, rb2c-ifnλ2, or rb2c-ifnλ3 at a moi of 5. expression of zo-1 and occludin was detected by qrt-pcr (a) and western blot (b). the integrity of tj protein zo-1 was measured by confocal microscopy (c). relative mean fluorescence intensity for the regions of interest (roi) was determined using image j (d). b.end3 cells were cultured on transwell inserts and treated with extracts from supernatants of astrocytes that had been mock infected with dmem or infected with b2c, rb2c-ifnλ2, or rb2c-ifnλ3 at a moi of 5. at 24 hpi, fitc-dextran-10000 was added into the upper insert of the transwell and then the permeation of fitc-dextran-10000 into the lower chamber was measured using a fluorimeter (excitation, 492 nm; emission, 520 nm) (e). error bars represent the se (n = 3). the following notations were used to indicate significant differences between groups: *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001. ifn-λ signaling through the ifnlr1 receptor on intestinal epithelial cells (iecs) induces antiviral effectors such as isgs via stat1/stat2/irf9-mediated transcription, thereby boosting defenses against intestinal viruses such as rotavirus, reovirus, and norovirus [35] [36] [37] [38] . the neurotropic west nile virus (wnv) is inhibited in the cns by ifn-λ through a mechanism that modulates endothelial cell tight junction integrity [21] . in our study, we found that ifn-λ curtails the replication of another neurotropic virus, rabv, in cells. moreover, ifn-λ also reduces rabv pathogenicity in infected mice by different infection routes, especially i.n. route. ifn-λ has been reported to be particularly important for innate pathogen defense at mucosal barriers [39] , which provides a possible explanation for a better survivor rate in rrabv-expressing ifn-λ-infected mice by i.n. route. in addition, ifn-λ is expressed in a tissue-specific manner and its receptor ifnlr1 exhibits a much more tissue-specific expression pattern, with a preference for epithelial cells [10, 40] . during intranasal challenge with rabv, the expression of ifnlr1 by nasal epithelial cells is necessary for the responsiveness of the tissue to ifn-λ and the suppression of viral replication in nasal mucosa. therefore, ifn-λ has evolved as a specific factor that can prevent the invasion of some neurotropic viruses through nasal epithelial cells. the ability of ifn-λ to induce isg expression in a targeted set of epithelial cells also suggests that ifn-λ promotes a focused antiviral or immunomodulatory response [41, 42] . the main antiviral functions of ifn-λ have been linked to the activation of the stat1/2 signaling pathway [43, 44] . activation of stat1/2 is required for optimal transcription of isgs, which establish antiviral defenses. ifn-α/β is also engaged in antiviral processes. cooperation between ifn-α/β and ifn-λ further increases stat1/2-activation. in our study, isre activity was upregulated by the expression of ifn-λ during rabv infection, indicating that ifn-λ plays a positive role in activating isre, which is involved in the downstream production of isgs [45] . isgs such as ifit2 and iigp1 were reported to restrict rabv replication [13, 46] . the two isgs were elevated after the infection of rrabvs expressing ifn-λ in our study, indicating that ifn-λ could activate the stat1/2 signaling pathway during rabv infection, resulting in the downstream production of isgs to restrict rabv infection. encephalitis induced by lab-attenuated rabv is characterized by obvious cns inflammation [18, 47, 48] . it has previously been reported that rabv induced inflammation in microglia cells mainly through p38 and nf-κb pathways [49] . the results of luciferase reporter assay in our study implied that ifn-λ expression could suitably suppress the activation of nf-κb. subsequently, production of pro-inflammation cytokines was remarkably decreased, as expected. consistent with these observations, one previous study showed that excessive expression of ifn-λ caused by iav infection, whereas the degradation of iκb and the activation of nf-κb was significantly decreased in the infected cells. in contrast, disruption of ifn-λ signaling pathway resulted in the activation of nf-κb [50] . on the other hand, ifn-λ that is induced in dcs and macrophages does not augment proinflammatory cytokine production during viral infection [51] . type i ifn provides antiviral resistance but also induces proinflammatory responses essential for countering infection [52] . previous study demonstrated that ifn-λ could upregulate suppressor of cytokine signaling 1 (socs1) and socs3, which reduce the devastating effects of excessive inflammation [53] . consistently, in our study, it was found that ifn-λ could decrease the nf-κb activation and further alleviate inflammation via suppression of neutrophil infiltration and proinflammatory cytokines such as tnf-α, il-17a, il-1α, il-1β, il-6, il-12, and vegf during rabv infection, all of which contribute to bbb breakdown. the role of ifn-λ in nf-κb activation and inflammation production in the context of rabv infection appears to be important, but the detailed mechanism of this process remains to be fully explored. although ifn-λ decreases expression of proinflammatory cytokines, it does not require the activation of inflammation to modulate bbb permeability [51] . a previous study demonstrated that stat1/2 signaling or protein synthesis is not required for endothelial barrier tightening [21] . indeed, in our study, ifn-λ signaling was found to maintain the expression of tj protein zo-1 and protect its integrity, which tightens the endothelial barrier. the tightened barrier also prevents rabv transit across epithelial surfaces, such as the nasal mucosa barrier, which could be another explanation for the strongest attenuation of rabv pathogenicity after i.n. infection. it was reported that approximately 6 kb of dna at gene loci for ifnl2 and ifnl3 have nucleotide sequence identity greater than 97% for the whole genomic region in both human and mouse, indicating that the function of ifn-λ2 and ifn-λ3 could be very similar [54] . consistently, the results in affecting rabv replication and pathogenicity are similar between rb2c-ifnλ2 and rb2c-ifnλ3 in our study. additionally, non-murine cell line vero cells, which are supposed to be an ifn-α/β independent cell line, were also used in our study to exclude the possible involvement of type i ifn during the ifn-λ inhibiting rabv replication. it has been reported that both mouse ifn-λ2 and ifn-λ3 were capable of up-regulating mhc class i antigen expression in several human cell lines and induced antiviral protection in mouse b16 cells or human ht29 cells infected with vsv [55] . these data indicate that the mouse ifn-λ could potentially be functional in cells derived from species other than mouse. in conclusion, our data indicate that ifn-λ, either ifn-λ2 or ifn-λ3, can restrict rabv infection by inducing isgs, limiting blood-brain barrier permeability to decrease the neuroinflammation, and thus attenuating the pathogenicity of rabv. current status of rabies and prospects for elimination rabies virus receptors dsrna and the innate antiviral immune response beyond viral interferon regulatory factors: immune evasion strategies rabies virus infection induces type i interferon production in an ips-1 dependent manner while dendritic cell activation relies on ifnar signaling 5 -triphosphate rna is the ligand for rig-i interferons: cell signalling, immune modulation, antiviral response and virus countermeasures identification of genes differentially regulated by interferon alpha, beta, or gamma using oligonucleotide arrays interferon-lambda cures persistent murine norovirus infection in the absence of adaptive immunity lambda interferon (ifn-lambda), a type iii ifn, is induced by viruses and ifns and displays potent antiviral activity against select virus infections in vivo critical role of k1685 and k1829 in the large protein of rabies virus in viral pathogenicity and immune evasion lab-attenuated rabies virus causes abortive infection and induces cytokine expression in astrocytes by activating mitochondrial antiviral-signaling protein signaling pathway ifit2 is a restriction factor in rabies virus pathogenicity the roles of chemokines in rabies virus infection: overexpression may not always be beneficial neuronal apoptosis does not play an important role in human rabies encephalitis enhancement of blood-brain barrier permeability and reduction of tight junction protein expression are modulated by chemokines/cytokines induced by rabies virus infection comparison of immune responses to attenuated rabies virus and street virus in mouse brain role of chemokines in the enhancement of bbb permeability and inflammatory infiltration after rabies virus infection viral pathogen-associated molecular patterns regulate blood-brain barrier integrity via competing innate cytokine signals virus infections in the nervous system interferon-lambda restricts west nile virus neuroinvasion by tightening the blood-brain barrier viral disruption of the blood-brain barrier rabies virus quasispecies: implications for pathogenesis persistent infection of bhk21/wi-2 cells with rubella virus and characterization of rubella variants rabies serogroup viruses in neuroblastoma cells: propagation, "autointerference", and apparently random back-mutation of attenuated viruses to the virulent state cell-type-specific type i interferon antagonism influences organ tropism of murine coronavirus rabies virus glycoprotein is an important determinant for the induction of innate immune responses and the pathogenic mechanisms expression of mip-1 (ccl3) by a recombinant rabies virus enhances its immunogenicity by inducing innate immunity and recruiting dendritic cells and b cells using type iii interferon to improve the efficacy of vaccine and oncolytic viral vectors the ectodomain of rabies virus glycoprotein determines dendritic cell activation dna double-strand breaks induced byfractionated neutron beam irradiation for boron neutron capture therapy porcine deltacoronavirus nsp5 antagonizes type i interferon signaling by cleaving stat2 endothelial japanese encephalitis virus infection enhances migration and adhesion of leukocytes to brain microvascular endothelia via mek-dependent expression of icam1 and the cinc and rantes chemokines expression of neuronal cxcl10 induced by rabies virus infection initiates infiltration of inflammatory cells, production of chemokines and cytokines, and enhancement of blood-brain barrier permeability interferon-lambda and interleukin 22 act synergistically for the induction of interferon-stimulated genes and control of rotavirus infection distinct roles of type i and type iii interferons in intestinal immunity to homologous and heterologous rotavirus infections leukocyte-derived ifn-alpha/beta and epithelial ifn-lambda constitute a compartmentalized mucosal defense system that restricts enteric virus infections expression of ifnlr1 on intestinal epithelial cells is critical to the antiviral effects of interferon lambda against norovirus and reovirus interferon-lambda orchestrates innate and adaptive mucosal immune responses ifn-lambda (ifn-lambda) is expressed in a tissue-dependent fashion and primarily acts on epithelial cells in vivo interferon-lambda in the context of viral infections: production, response and therapeutic implications a randomized phase 2b study of peginterferon lambda-1a for the treatment of chronic hcv infection role of the interleukin (il)-28 receptor tyrosine residues for antiviral and antiproliferative activity of il-29/interferon-lambda 1: similarities with type i interferon signaling interferon-lambda: a new addition to an old family identification of a member of the interferon regulatory factor family that binds to the interferon-stimulated response element and activates expression of interferon-induced genes interferon-inducible gtpase: a novel viral response protein involved in rabies virus infection glycoprotein-mediated induction of apoptosis limits the spread of attenuated rabies viruses in the central nervous system of mice attenuated rabies virus activates, while pathogenic rabies virus evades, the host innate immune responses in the central nervous system rabies virus-induced activation of mitogen-activated protein kinase and nf-kappab signaling pathways regulates expression of cxc and cc chemokine ligands in microglia suppression of interferon lambda signaling by socs-1 results in their excessive production during influenza virus infection interferon-lambda mediates non-redundant front-line antiviral protection against influenza virus infection without compromising host fitness pathogenic potential of interferon alphabeta in acute influenza infection intracellular protein therapy with socs3 inhibits inflammation and apoptosis the role of genomic data in the discovery, annotation and evolutionary interpretation of the interferon-lambda family characterization of the mouse ifn-lambda ligand-receptor system: ifn-lambdas exhibit antitumor activity against b16 melanoma we thank shuaipeng he (laboratory animal center, huazhong agricultural university) for excellent mice management. the authors declare no conflict of interest. the funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results. key: cord-048489-ajafw966 authors: bozza, fernando a; cruz, oswaldo g; zagne, sonia mo; azeredo, elzinandes l; nogueira, rita mr; assis, edson f; bozza, patricia t; kubelka, claire f title: multiplex cytokine profile from dengue patients: mip-1beta and ifn-gamma as predictive factors for severity date: 2008-06-25 journal: bmc infect dis doi: 10.1186/1471-2334-8-86 sha: doc_id: 48489 cord_uid: ajafw966 background: dengue virus pathogenesis is not yet fully understood and the identification of patients at high risk for developing severe disease forms is still a great challenge in dengue patient care. during the present study, we evaluated prospectively the potential of cytokines present in plasma from patients with dengue in stratifying disease severity. methods: seventeen-cytokine multiplex fluorescent microbead immunoassay was used for the simultaneous detection in 59 dengue patients. glm models using bimodal or gaussian family were determined in order to associate cytokines with clinical manifestations and laboratory diagnosis. results: il-1β, ifn-γ, il-4, il-6, il-13, il-7 and gm-csf were significantly increased in patients with severe clinical manifestations (severe dengue) when compared to mild disease forms (mild dengue). in contrast, increased mip-1β levels were observed in patients with mild dengue. mip-1β was also associated with cd56+nk cell circulating rates. il-1β, il-8, tnf-α and mcp-1 were associated with marked thrombocytopenia. increased mcp-1 and gm-csf levels correlated with hypotension. moreover, mip-1β and ifn-γ were independently associated with both dengue severity and disease outcome. conclusion: our data demonstrated that the use of a multiple cytokine assay platform was suitable for identifying distinct cytokine profiles associated with the dengue clinical manifestations and severity. mip-β is indicated for the first time as a good prognostic marker in contrast to ifn-γ that was associated with disease severity. during the last decades dengue became the most important arthropod-borne emerging viral disease in tropical countries [1] . it is estimated that about 2.5% notified cases are classified as dengue haemorrhagic fever (dhf) and about 2.5-20% of dhf cases are lethal [2] [3] [4] . in the last two decades, latin america saw a dramatic increase in frequency and in geographic extension of dengue fever. specifically, the situation in brazil has worsened during the last decade since the introduction of the dengue-3 serotype. in the past years brazil had dengue outbreaks with at least 1 million cases (2001) (2002) and within the last 18 months 900 thousand cases were reported [5] . in addition, severe disease forms are occurring with increased frequency and mortality rates. dengue pathogenesis is not completely understood, and the main determinants of the development of severe forms are not yet well established. increase in capillary permeability associated with endothelial activation and haemorrhagic phenomena are landmarks of severe clinical manifestations, strongly suggesting an alteration in immunoregulation [6] . cytokines are proteins secreted during innate and adaptive immunological responses, acting as inflammatory mediators or modulatory molecules during several haemorrhagic fevers [7] . clinical studies support a key role for cytokines in the dhf pathogenesis [8] [9] [10] [11] [12] [13] . during dengue virus infections, cytokines are involved in the disease onset and homeostatic regulation. specifically, tnf-α, il-1β and il-6 have been associated with both coagulation (f1+2 and tatc) and fibrinolysis (t-pa, papc, and d-dimmer) activation markers [14] . this activation is more striking in patients with severe clinical manifestations, although it can be found at lower degrees in patients with mild disease [15, 16] . despite the fact that cytokine network and their multiple regulatory pathways are highly complex and not fully elucidated during dengue fever, these molecules seem to represent interesting markers for patient stratification or prognosis. an emerging interest has appeared in order to define biomarkers that may have pathophysiological roles during disease and that may be used as future therapeutic targets. new technologies have been developed in order to detect multiple biomarkers within a single and small blood sample. such approaches may lead to the development of specific marker panels for dengue fever. accordingly, cytokine patterns have been indicated as serum biomarkers during infectious diseases such as hepatitis c [17] , ards [18] and sepsis [19] . in this study, we prospectively evaluated the potential use of plasma cytokine concentrations for severity stratifica-tion of patients with dengue, using a 17 cytokine-multiplex assay. among tested cytokines, we were able to recognize ten significantly altered circulating factors and to characterise cytokine patterns related to determined clinical manifestations and disease severity. the ethics committee of the oswaldo cruz foundation approved this study protocol and written informed consent was obtained from all patients or their guardians prior to blood collection. we included prospectively 59 dengue-infected patients ( a detailed history and physical examination was performed to detect hemorrhagic manifestations (positive tourniquet test for capillary fragility, skin haemorrhages, epistaxis, gingival, gastrointestinal, or urinary tract haemorrhage), signs of plasma leakage (pleural or pericardial effusion, ascites), signs of circulatory failure (cold extremities, cyanosis, hypotension, tachycardia, shock), and hepatomegaly. in addition to the suggestive clinical diagnosis, all patients had the dengue virus infection confirmed either by antidengue enzyme-linked immunoabsorbent assay (elisa)-igm, serotype specific reverse transcription-polymerase chain reaction (rt-pcr) or by virus isolation [20] [21] [22] . dengue immune response was considered as primary or secondary by igg elisa according to previously established criteria [23] . as previously reported [24] [25] [26] , we also were often unable to characterize the severe disease forms based on who criteria [3]. in nicaragua, harris et al. [24, 27] described four key severe clinical manifestations associated with dengue -shock, plasma leakage, marked thrombocytopenia or internal haemorrhage -that do not fit dhf/dss classification as single parameters. according to these criteria, we considered: • severe dengue -dengue confirmed cases plus severe thrombocytopenia (<50,000 platelets/mm 3 ) and/or hypotension (postural hypotension with decrease in systolic arterial pressure in 20 mm hg in supine position or systolic arterial pressure < 90 mm hg) and/or plasma leakage (either haemoconcentration fluctuation of packed cell volume ≥ 20% during illness course and recovery or clinical signs of plasma leakage, such as pleural effusion) and/or severe haemorrhagic manifestations. • mild dengue -dengue confirmed cases in absence of severe thrombocytopenia, hypotension, plasma leakage signs or haemorrhagic manifestations. blood samples were collected from a peripheral vein and kept on ice. plasma was collected by centrifugation at 800 g for 15 min at 4°c, aliquoted, and stored at -70°c until the analysis day. a multiplex biometric immunoassay, containing fluorescent dyed microspheres conjugated with a monoclonal antibody specific for a target protein, was used for cytokine measurement according to the manufacturer's instructions (bio-plex human cytokine assay; bio-rad inc., hercules, ca, usa). cytokines measured were: il-1β, il-2, il-4, il-5, il-6, il-7, cxcl8 (il-8), il-10, il-12 (p70), il-13, il-17, granulocyte colony stimulating factor (g-csf), granulocyte-monocyte colony stimulating factor (gm-csf), monocyte chemoattractive protein (mcp-1/ccl2), macrophage inflammatory protein (mip-1β/ccl4), and tnf-α. briefly, 20 μl plasma samples were diluted 1:4 and incubated with antibodycoupled beads. complexes were washed, then incubated with biotinylated detection antibody and, finally, with streptavidin-phycoerythrin prior to assessing cytokine concentration titres. concentrated human recombinant cytokine was provided by the vendor (bio-rad laboratories). a range of 1.95-32,000 pg/ml recombinant cytokines was used to establish standard curves and to maximize the sensitivity and the assay dynamic range. cytokine levels were determined using a multiplex array reader from luminex™ instrumentation system (bio-plex workstation from bio-rad laboratories). the analyte concentration was calculated using software provided by the manufacturer (bio-plex manager software). liquid nitrogen cryopreserved peripheral blood mononuclear leukocytes were isolated by histopaque-1077 (sigma chemical co., saint louis, mo, usa) from 35 out of 59 dengue patients. cells were stained for cd56 surface marker using anti-cd56-cy5 (igg1, clone b159) from pharmingen (san diego, ca, usa) and positive cells were detected by flow cytometry as described before [22] using facscalibur (becton-dickinson). events (10,000-20,000) were acquired and analyses were carried out with flowjo (treestar, version 4.3) software. the nonparametric mann-whitney u test was used to evaluate differences between cytokine ratios from severe and mild dengue patients. glm models were used to evaluate factors independently associated with quantitative variables. analysis of factors independently associated with dengue severity and other clinical manifestations was performed with glm with logistic regression or gaussian family. results from the logistic regressions are given as odds ratio (or). the confidence interval (ci) was established at 95%. alternatively, for a glm gaussian family t values were recorded. a probability value of p<0.05 was considered to be significant. the statistical programs r [28] and prism 4 (graph-pad software, san diego, ca, usa) were employed. the fisher's exact test was applied to determine the significance of positive samples from patients when comparing different virus serotypes or sequential infections. correlation between platelet counts and cytokine production in blood samples was estimated by spearman's correlation. from the 59 patients included, 39 were classified as severe dengue and 20 as mild dengue. detailed demographic, clinical, and laboratorial data from dengue patients are summarized in table 1 . blood collection was performed between 3 and 10 days after disease onset. in order to avoid effects due to differences in the blood collection time, we compared mild and severe dengue patient groups using mann-whitney u test, which showed no differences in the disease onset time at the moment of sample collection [see additional file 1]. the original data used to perform this analysis is shown at figure 1 . patients with mild and severe dengue were investigated for prior incidence of infection, detected by serologic immune response (igg antibodies for denv). patients with severe dengue (42%; 14 out 33) were more likely to be experiencing a secondary dengue virus infection than patients with mild dengue (28%; 6 out 20), although no statistical significance was found in fisher's exact test (p = 0.3989). among 22 patients with denv-1, 5 were classified as secondary infection, whereas among 35 patients with denv-3, 12 were classified as secondary infection (p = 0.3912). dengue fever is characterised by a high fever phase and an abrupt drop in body temperature that has been called defervescence phase. characteristically the disease outcome is defined during this phase, when patients can either recover rapidly or progress to a severe life-threatening stage. cytokines and immunoactivation markers such as ifn-γ, il-2, soluble cd8 and receptors for tnf-α [12, 29] are associated with the defervescence phase and with disease severity. ifn-α levels are higher in dhf than in df during defervescence [30] . during the febrile phase significant increase in cytokine circulating levels was detected including il-4, il-6, il-10, mcp-1 and mip-1β levels, which were maintained also elevated in defervescence (data not shown, analysed by non parametric kruskal-wallis test and dunn's multiple comparison test when compared with controls, p < 0.05); no significantly altered febrile levels were found when compared to defervescence. during the febrile cytokine levels in plasma from patients with mild and severe dengue figure 1 cytokine levels in plasma from patients with mild and severe dengue. box-and-whiskers graph. the box extends from the 25 th to the 75 th percentile and the line at the middle is the median. the error bars, or whiskers extend down to the lowest value and up to the highest. factors were sorted according to their functional groups. mann-whitney u test was used to evaluate differences between cytokine concentration from severe and mild dengue patients. * p < 0.05, ** p < 0.01 and ** p < 0.001. phase il-7 was significantly higher than in defervescence. il-1β, il-13, ifn-γ were significantly increased during defervescence as compared to control samples. significant levels of il-5, il-12, and il-17 were not detected during dengue disease in our patients. il-2 was detected both in healthy individuals and in dengue patients but no difference between these two groups was detected [see additional file 2]. we studied the cytokine profile from brazilian patients in order to compare severe and mild dengue cases during the acute phase of the disease. figure 1 shows data from patients with regard to their plasma cytokine contents, which were sorted in four groups according to their reported function. we observed that cytokine concentrations of il-1β, ifn-γ, il-4, il-6, il-7, il-13 and gm-csf were significantly increased during severe dengue as compared to mild dengue, while mip-1β levels are higher in mild dengue. mip-1β and ifn-γ were independent variables associated disease outcome as determined by a logistic regression model (table 2 and figure 2 ). while mip-1β was increased during mild dengue with odds ratio (or) of 0.181 and confidence interval (ci) 0.045-0.72, ifn-γ was associated with severity with or of 1.138 (ci, 1.0541-1.245). to assess relationships between cytokine levels and several clinical manifestations, the patient cohort with severe dengue was divided into distinct subgroups: those with hypotension, thrombocytopenia (≤50.000 counts/mm 3 ) and/or haemorrhagic manifestations. a logistic regression model was used for binomial response subgroups and glm models using gaussian family were employed for subgroups with continuous response in order to determine cytokine profiles. il-1β was associated with marked thrombocytopenia with or = 1.058 (ci, 1.012-1.106) in dengue patients. tnf-α was inversely related to thrombocytopenia with or = 0.978 (ci, 0.962-0.995) (table 2, figure 2 ). considering platelet counts as a continuous variable for statistical analysis with a gaussian family, it was possible to determine that il-8 (p = 0.0434) and mcp-1 (p = 0.0146) levels are inversely related to their counts, displaying therefore an association with thrombocytopenia, while mip-1β (p = 0.0114) confirms its association with higher counts -normal or tending to normal (table 3) . gm-csf (or = 1.004; ci, 1.001-1.007) was related with hypotension, whereas il-1β had a negative predictive table 2 and figure 2 . natural killer (nk) cells have been earlier related to mild cases of dengue [22] . forty-eight pbmc samples from thirty-five patients had their cd56+ rates determined by flow cytometry and a good correlation was observed with cytokines detected in plasma as independent factors in predicting severity table 2 . their respective mip-1β plasmatic levels (r = 0.4668; p = 0.0008). considering that different cytokines act in the immunological network as stimulating/up regulating factors and also in a feedback loop as down regulators, the cytokine balance might play a role in the immune response outcome. therefore we calculated mip-1β/ifn-γ ratios for every patient and compared those from mild dengue with those from severe dengue. ratio averages ± sem were respectively 3032 ± 514 and 864 ± 240 (p = 0.0003; mann whitney u test), confirming our earlier data that these cytokines are acting as opposing factors. the different models built here using clinical manifestations as independent variables each exhibit specific cytokine profiles. the cytokine profile identified in patients with dengue may represent a valuable tool for the characterisation of immunological response patterns and may assist the identification of patient groups at risk for developing severe disease. in the present study, the use of a multiplex analysis for cytokine plasma detection in patients with dengue could identify cytokine profiles associated with the disease severity. early identification and management of severe dengue disease are essential to prevent death. it has been increasingly recognized that the inflammatory response and deregulated cytokine production play key roles in the development of severe clinical manifestations [31] . however, cytokine profiles associated with dengue evolution and prognosis are not well established. new technologies for cytokine quantification were developed including the multiplex immunofluorescent bead array analysis system, allowing multiple biomarkers to be tested simultaneously in a small volume from one single plasma aliquot. recently, this methodology has been used for cytokine profile evaluation during several infectious diseases including viral infections [17, 32, 33] and sepsis [19] , among others. we were able to identify models for cytokine circulation during dengue acute phase that may vary with clinical manifestations. mip-1β was for the first time associated with a good prognostic and was identified in the different disease models presented here. mip-1β has been earlier detected after dengue virus cell stimuli in vitro [34, 35] but preliminary studies in vivo did not report their role in severity. in accordance with a protective role for mip-1β, changes in mip-1β levels were significantly correlated with decreases in viral titre after hepatitis c treatment [17] . in addition, mip-1β was up regulated in acute infection in chimpanzees only when viral clearance took place, but not in those animals which failed to eradicate the virus [36] . mip-1β is produced by human monocytes and dendritic cells upon different stimuli [37] as well as by activated nk cells [38] and lymphocytes [39, 40] . activated nk cells release granzyme a, which displays cytolytic functions and mip-1β is chemoattractant for nk cells, recruiting them to inflammatory sites. nk cells have been associated with mild dengue [22] . here a good correlation between mip-1β plasma levels and nk cells was observed, reinforcing the relevance of these pathways and strongly suggesting their role in dengue protective mechanisms. an early and efficient virus clearance by direct or indirect nk functions is likely controlling virus replication, restricting intense immunological activation and the dengue immunopathology and therefore favouring a mild dengue disease. in previous studies, tnf-α has been reported to be associated with severity, mainly during dhf in brazilian patients [11, 13, 41] . in the present study, however, this cytokine was not strongly associated with severity. indeed, other authors also found inconsistency or no difference in tnf-α levels in severe vs. mild disease forms [10, 42] . we may hypothesize that differences in dengue virus serotypes or in host immune response such as different tnf-α genetic polymorphisms may explain the disease outcome. in our study from 2001 (braga et al., 2001) , patients were dengue-2 infected, while in the present study, patients were dengue-1 and -3 infected. a recent report [43] describes non-significant tnf-α serum levels in adult patients and suggests that the discrepancy may have been caused by a transient tnf-α peak which was not detected at a later time point. in the present study we observed an association of ifn-γ with disease severity. indeed, increased ifn-γ concentrations have been detected in dengue patients in a variety of studies [29, [44] [45] [46] [47] . dhf induced by dengue-3 was associated with higher viremia early in illness and earlier peak plasma ifn-γ levels; maximum plasma viremia levels correlated with the degree of plasma leakage and thrombocytopenia [45] . however, in a previous study from our group we failed to observe association of ifn-γ with disease severity [44] , probably due to the small number of severe patients analyzed or to the dengue-1 incidence. ifn-γ is produced during a t-lymphocyte helper response type 1 and may reflect cd8+ t cell activation with production of inflammatory cytokines. high levels of ifn-γ were observed in patients with dengue from asian and latin america and were associated with severity [9] . ifn-γ produced by t cells may also activate mononuclear phagocytes (monocytes and dendritic cells), which would produce factors such as tnf-α, tissue factor, and plateletactivating factor, among other mediators. these factors may all participate in platelet and endothelial cell activation, leading to platelet consumption, increase in endothelial permeability, hypotension and ultimately to shock. ifn-γ has also been associated with secondary heterologous dengue virus infections [47, 48] inducing a strong antigenically cross-reactive inflammatory response, probably inefficient in terms of antibody and t-cell specific response. indeed, we observed earlier in several patients a cd8+t cell activation with hla-dr+ subset increase that was associated with severity [9] . gm-csf acts at early differentiation processes at myeloid progenitors or resting monocytes [49] . an additional stimulus may be required to activate monocytes or dendritic cells in order to produce proinflammatory cytokines [50] . gm-csf was associated with hypotension as well as mcp-1, likely acting both in concert, contrasting with mip-1β, once more associated with good prognostics. mcp-1 was earlier detected in dhf patients [51] but for the first time we reported clinical and laboratory findings associated with severity. il-8 and mcp-1, here associated with thrombocytopenia, are chemokines and may contribute to platelet activation, either by their chemoattractant properties or by their effect on endothelial permeability. both factors were detected in patients with dhf [51, 52] . these cytokines are produced by monocytes after various activation stimuli, such as virus infection, and increase the endothelial permeability by disrupting tight junctions among cells [53] . despite the fact that our study could identify cytokines with good accuracy for the stratification and/or prognosis of dengue, it has potential limitations. here we identified cytokines related to dengue severity, but the small sample size represents a shortcoming regarding the generalization of our results. in addition, only one time point was used for the measurement of cytokines, not allowing further insights provided by sequential measurements. moreover, classification of disease severity has been a matter of debate, especially for adult patients' management and classification. indeed, the who criteria for dhf has failed to identify severe disease, including fatal cases, in adult latin america population [24, 54] (s.m.o. zagne, r.m.o. nogueira, unpublished) and clearly do not satisfy the stratification of our studied population. accordingly, in the present study severe disease forms were classified following other proposed criteria [24] . while a direct correlation of cytokine concentrations and the pathophysiology of severe dengue is tempting, we believe that the full burden of disease severity cannot be attributed to a single cytokine. cytokines may be increased simply as one of the several steps in the network loops without necessarily playing a direct harmful role and most likely more than one factor may be involved, including others not tested here such as il-18, tgf-β, and mif among others [9] . we can suggest a mechanism explaining our cytokine models for dengue fever (figure 3) . mip-1β would be associated with a protective pathway for its chemoattractive and activating effect on nk cells, which in turn are efficient cells in early virus clearance, by their antiviral cytokine production and cytotoxic activity against infected target cells. ifn-γ has a deleterious effect for the host regarding its action in activating t cells for virus anti-genic cross-reactive response and monocyte/dendritic cell activation. mononuclear monocytes are activated by ifnγ and gm-csf among other cytokines and in turn produce several factors such as il-1β and mcp-1 that may act on vascular permeability leading to plasma leakage and haemoconcentration. as suggested by other authors, it is likely that viral replication in antigen presenting cells, cytokine liberation and circulation, and t cell activation may not be a linear process [55] , but in fact a complex interaction network, with positive and negative feedbacks, where viral clearance and pathologic events take place, such as increased vascular permeability and circulatory collapse, and their balance may determine the disease outcome. our study demonstrated the plasma cytokine profile in dengue fever from a brazilian population detected by a multiplex bead immunoassay. mip-β is indicated for the first time as a good prognostic marker and in contrast to ifn-γ that was associated with the disease severity. both cytokines can discriminate mild from severe cases. moreover, we show here for the first time that during the dengue course different cytokine profiles may be present and vary according to determined clinical manifestations. the cytokine profiles identified herein by bead array multiplex system may favour an early identification of patients with the worst prognosis and may contribute to the establishment of more directed therapeutic procedures than the present ones. hypothetic mechanism to explain cytokine models during dengue fever figure 3 hypothetic mechanism to explain cytokine models during dengue fever. mip-1β is associated with a good prognostic and ifn-γ has a predictive value for severity. gm-csf, mcp-1, il-1β, il-6, il-8, il-12, il-13 are also playing important roles during dengue pathogenesis (see text for detailed description). epidemic dengue/dengue hemorrhagic fever as a public health, social and economic problem in the 21st century world health organization: dengue hemorrahagic fever diagnosis treatment and control secretaria de vigilância em saúde-dengue -informe epidemiológico da dengue immunopathological mechanisms in dengue and dengue hemorrhagic fever viral hemorrhagic fevers role of t cells, cytokines and antibody in dengue fever and dengue haemorrhagic fever activated peripheral lymphocytes with increased expression of cell adhesion molecules and cytotoxic markers are associated with dengue fever disease pathophysiologic and prognostic role of cytokines in dengue hemorrhagic fever serum levels of tumor necrosis factor-alpha (tnf-alpha), interleukin-6 (il-6), and interleukin-1 beta (il-1 beta) in dengue-infected patients early immune activation in acute dengue illness is related to development of plasma leakage and disease severity detection of circulant tumor necrosis factoralpha, soluble tumor necrosis factor p75 and interferongamma in brazilian patients with dengue fever and dengue hemorrhagic fever the role of cytokines in activation of coagulation and fibrinolysis in dengue shock syndrome systemic host inflammatory and coagulation response in the dengue virus primo-infection dengue hemorrhagic fever in infants: a study of clinical and cytokine profiles multiplex cytokine profiling of initial therapeutic response in patients with chronic hepatitis c virus infection the clinical significance of serum and bronchoalveolar lavage inflammatory cytokines in patients at risk for acute respiratory distress syndrome cytokine profiles as markers of disease severity in sepsis: a multiplex analysis diagnosis of dengue by using reverse transcriptase-polymerase chain reaction schatzmayr hg: dengue epidemic in the state of rio de janeiro, brazil: virological and epidemiological aspects nk cells, displaying early activation, cytotoxicity and adhesion molecules, are associated with mild dengue disease evaluation of an igg enzyme-linked immunosorbent assay for dengue diagnosis clinical, epidemiologic, and virologic features of dengue in the 1998 epidemic in nicaragua clinical diagnosis and assessment of severity of confirmed dengue infections in vietnamese children: is the world health organization classification system helpful? dengue in travelers assessment of the world health organization scheme for classification of dengue severity in nicaragua r: a language and environment for statistical computing. r foundation for statistical computing activation of t lymphocytes in dengue virus infections. high levels of soluble interleukin 2 receptor, soluble cd4, soluble cd8, interleukin 2, and interferon-gamma in sera of children with dengue high levels of interferon alpha in the sera of children with dengue virus infection recent advances in deciphering viral and host determinants of dengue virus replication and pathogenesis multiplex analysis of cytokines in the blood of cynomolgus macaques naturally infected with ebola virus (reston serotype) hpv-16 l1 vlp vaccine elicits a broad-spectrum of cytokine responses in whole blood activation of terminally differentiated human monocytes/macrophages by dengue virus: productive infection, hierarchical production of innate cytokines and chemokines, and the synergistic effect of lipopolysaccharide mip-1 alpha and mip-1 beta induction by dengue virus intrahepatic gene expression during chronic hepatitis c virus infection in chimpanzees macrophage inflammatory protein-1 coordinate expression of cytokines and chemokines by nk cells during murine cytomegalovirus infection activation of nk cells by cc chemokines. chemotaxis, ca2+ mobilization, and enzyme release alpha and beta chemokines induce nk cell migration and enhance nk-mediated cytolysis elevated tumour necrosis factor in dengue fever and dengue haemorrhagic fever plasma levels of tumour necrosis factor alpha and transforming growth factor beta-1 in children with dengue 2 virus infection in french polynesia correlation of serum levels of macrophage migration inhibitory factor with disease severity and clinical outcome in dengue patients characterisation of lymphocyte response and cytokine patterns in patients with dengue fever differing influences of virus burden and immune activation on disease severity in secondary dengue-3 virus infections th(2) immune response in patients with dengue during defervescence: preliminary evidence altered cytokine responses of dengue-specific cd4+ t cells to heterologous serotypes dengue-specific t cell responses in peripheral blood mononuclear cells obtained prior to secondary dengue virus infections in thai schoolchildren alternative activation of macrophages effects of granulocyte-monocyte colony-stimulating factor (gm-csf) on expression of adhesion molecules and production of cytokines in blood monocytes and ovarian cancer-associated macrophages mcp-1, a highly expressed chemokine in dengue haemorrhagic fever/dengue shock syndrome patients, may cause permeability change, possibly through reduced tight junctions of vascular endothelium cells il8 release, tight junction and cytoskeleton dynamic reorganization conducive to permeability increase are induced by dengue virus infection of microvascular endothelial monolayers predictors of spontaneous bleeding in dengue dengue and dengue hemorrhagic fever, brazil of cascades and perfect storms: the immunopathogenesis of dengue haemorrhagic fever-dengue shock syndrome (dhf/dss) this work was supported by fundação oswaldo cruz (pdtsp-dengue), decict/conselho de desenvolvimento científico e tecnológico (cnpq), fundação de amparo à pesquisa do estado do rio de janeiro (faperj). the authors thank the program for technological development in tools for health-pdtis-fiocruz for use of its luminex facilities. we acknowledge in memoriam dr. jussara p. nascimento for her constant encouragement, dr. marcelo a. pinto for suggestions and dr. andrea schwager for the manuscript revision. the authors declare that they have no competing interests. fab and ogc contributed equally to the study. fab contributed to the study conception and design, carried out clinical studies, helped in data analysis and in drafting the manuscript. ogc performed data and statistical analysis. smoz carried out the clinical studies. efa and carried out the luminex immunoassays. ela collected and stored samples and patient data and helped in the luminex immunoassays. rmrn was responsible for the confirmatory diagnostics. ptb conceived the study and design and helped to draft the manuscript. cfk conceived the study and design, participated in data and statistical analysis and drafted the manuscript. all authors read and approved the final manuscript. the pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2334/8/86/prepub key: cord-001964-iy6qzq58 authors: muñoz-gonzález, sara; pérez-simó, marta; colom-cadena, andreu; cabezón, oscar; bohórquez, josé alejandro; rosell, rosa; pérez, lester josué; marco, ignasi; lavín, santiago; domingo, mariano; ganges, llilianne title: classical swine fever virus vs. classical swine fever virus: the superinfection exclusion phenomenon in experimentally infected wild boar date: 2016-02-26 journal: plos one doi: 10.1371/journal.pone.0149469 sha: doc_id: 1964 cord_uid: iy6qzq58 two groups with three wild boars each were used: group a (animals 1 to 3) served as the control, and group b (animals 4 to 6) was postnatally persistently infected with the cat01 strain of csfv (primary virus). the animals, six weeks old and clinically healthy, were inoculated with the virulent strain margarita (secondary virus). for exclusive detection of the margarita strain, a specific qrt-pcr assay was designed, which proved not to have cross-reactivity with the cat01 strain. the wild boars persistently infected with csfv were protected from superinfection by the virulent csfv margarita strain, as evidenced by the absence of clinical signs and the absence of margarita rna detection in serum, swabs and tissue samples. additionally, in pbmcs, a well-known target for csfv viral replication, only the primary infecting virus rna (cat01 strain) could be detected, even after the isolation in st cells, demonstrating sie at the tissue level in vivo. furthermore, the data analysis of the margarita qrt-pcr, by means of calculated δct values, supported that pbmcs from persistently infected animals were substantially protected from superinfection after in vitro inoculation with the margarita virus strain, while this virus was able to infect naive pbmcs efficiently. in parallel, ifn-α values were undetectable in the sera from animals in group b after inoculation with the csfv margarita strain. furthermore, these animals were unable to elicit adaptive humoral (no e2-specific or neutralising antibodies) or cellular immune responses (in terms of ifn-γ-producing cells) after inoculation with the second virus. finally, a sequence analysis could not detect csfv margarita rna in the samples tested from group b. our results suggested that the sie phenomenon might be involved in the evolution and phylogeny of the virus, as well as in csfv control by vaccination. to the best of our knowledge, this study was one of the first showing efficient suppression of superinfection in animals, especially in the absence of ifn-α, which might be associated with the lack of innate immune mechanisms. members of the pestivirus genus, within the flaviviridae family, account for a variety of diseases in farm animals, the most economically important of which are bovine viral diarrhoea virus (bvdv) and classical swine fever virus (csfv). classical swine fever virus (csfv) is the etiological agent of a highly contagious viral disease of swine affecting domestic pigs and wild boars [1] , which has caused major losses in stock farming [2, 3] . csfv is composed of a lipid envelope, a capsid and a single plus-strand rna genome carrying a single, large open reading frame (orf) flanked by two untranslated regions (utrs). the orf encodes a polyprotein of approximately 3900 amino acids, which are processed by cellular and viral proteases in the four structural proteins-c, e rns , e1, e2-and in the 8 non-structural proteins-n pro , p7, ns2, ns3, ns4a, ns4b, ns5a, and ns5b [4] . recently, it was proved that csfv can generate postnatal persistence by infecting both newborn piglets and wild boars with either low-and/or moderate-virulence strains, respectively. over the six weeks after postnatal infection, most of the infected animals remained clinically healthy, despite persistent high virus titres in the blood, organs and body secretions. importantly, these animals were unable to mount any detectable humoral or cellular immune responses. at necropsy, the most prominent gross pathological lesion was severe thymus atrophy. four weeks after infection, pbmcs from persistently infected seronegative piglets were unresponsive to both specific csfv and non-specific pha stimulation in terms of ifn-γ-producing cells. these results suggested the development of an immunosuppression state in these postnatally persistently infected pigs [5, 6] . in addition, it was shown that six-week-old, persistently csfv-infected pigs were unable to elicit specific immune responses following vaccination with a csfv lapinised c-strain vaccine (hclv) [7] . interestingly, the rna of the vaccinal c-strain was undetectable by specific rt-pcr [8] in any of the samples analysed after vaccination, including blood, nasal and rectal swabs, or organs throughout the experiment, suggesting a phenomenon of homologous interference, also known as superinfection exclusion (sie), between the high viral load generated by the primary persistent infection and the csfv vaccine strain. the sie phenomenon, defined as the ability of a primary virus infection to interfere with a secondary infection by the same or a closely related virus, has been described in a broad range of virus-host systems, including bacteria, plants, and animals, and in important pathogens of humans, such as rubella virus, human immunodeficiency virus (hiv), and hepatitis c virus (hcv), among others [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] . from an evolutionary standpoint, sie might be a conservative strategy, reducing the likelihood of recombination events between related strains [17, 21, 22] , thus determining the stability of viral sequences within the same cell. from a practical standpoint, sie has significant implications for the treatment or prevention of viral infections. in this regard, cross-protection of crops by purposeful infection with milder virus isolates is a widely accepted practice, and it is viewed as an effective and economical antiviral management strategy [23] . additionally, transplantation of hcv-infected liver grafts has been suggested as a treatment for already infected patients, given that the transplantation of a healthy organ would lead to rapid damage to the newly transplanted liver by the virus of the recipient patient [15, 24] . previous studies conducted in cell cultures with bvdv demonstrated that cells acutely infected with this virus were protected from a second infection by a homologous bvdv strain [17] . additionally, it was shown that csfv is generally noncytopathic, and it readily establishes persistent infections in cell culture. nevertheless, when persistently infected cultures were serially passaged more than 100 times, spontaneous generation of cytopathogenic (cp) csfv variants could occur. the few surviving cells of the cytopathic effect (cpe), although still infected, were also protected from the cpe after superinfection with cp csfv [25] . both studies supported the ability of pestiviruses to generate sie in cell cultures. thus, along with the availability of a persistent infection model of csfv, in the present study, we sought to assess sie against a highly virulent csfv strain at the organism level in six-week-old wild boars, rendered persistently csfv-infected at birth. our results showed that sie could occur at the systemic level in csfv-infected swine. pk-15 cells (atcc ccl 33) and sk6 cells [26] were cultured in dulbecco's modified eagle medium (dmem), supplemented with 10% foetal bovine serum (fbs), pestivirus-free, at 37°c in 5% co 2 . the cells were infected with 0.1 tcid 50 /cell in 2% fbs, and the virus was harvested 48 h later. additionally, st cells (atcc crl 1746) were cultured in dmem, supplemented with l-glutamine (2%) and 10% foetal bovine serum (fbs), pestivirus-free at 37°c in 5% co 2. peroxidase-linked assay (pla) [27] was used for viral titration following the statistical methods described by reed and muench [28] . the catalonia 01 (cat01) strain used in this study was isolated from the spanish csf epizootic in 2000-2001 [29] . this isolate belongs to the csfv 2.3 genogroup [30] . the course of infection by this strain was found to be mild [29, 31] . finally, the virulent margarita strain, which belongs to the csfv 1.4 genogroup [29, 32, 33] , was used. to elucidate the capacity of csfv to generate sie, two groups (a and b), with three male, sixweek-old wild boars in each, were used. these animals were acquired from gestion cinegetica integral sl farm (segovia, spain) and were housed in the experimental isolation facilities in the biosecurity level 3 laboratory of the centre de recerca en sanitat animal (cresa); they were fed a conventional piglet starter diet and pellets until the end of the trial (startrite 100, kwikstart, and prestarter; sca iberica s.a., zaragoza, spain) and were handled according to previous studies conducted in cresa [6] . group a (animals 1 to 3) was used as controls, and they tested pestivirus-free at the beginning of the study. the second group (group b), housed in an independent isolation unit at the bsl-3 facility of cresa, (animals 4 to 6), were postnatally persistently csfv-infected animals. these animals, which had been intranasally infected in the first 24 h after birth with the csfv cat01 strain, were viraemic and apparently healthy at six weeks old, although being immunosuppressed, they lacked csfv-specific cellular and humoral responses [5, 6] . both groups had an average weight of 6 kg per animal. after a five-day acclimation period, all of the animals were experimentally infected by i.m. injection in the neck [33] [34] [35] with 10 5 tcid 50 csfv margarita strain. in previous studies, this viral dose caused acute csf and often induced death at 10-15 days post-infection (dpi) [36] . sera and nasal and rectal swabs were collected at 0, 3, 7, 10 and 13 dpi. blood samples for the isolation of pbmcs were obtained at day 0 and at the time of euthanasia. a trained veterinarian recorded the clinical signs daily in a blinded manner [36] . the clinical signs compatible with csfv infection were anorexia, fever, conjunctivitis, diarrhoea, constipation, cyanosis of the skin, abdominal petechiae, dyspnoea, tremors, locomotive disturbances, reluctant walking, swaying movement of the hindquarters, posterior paresis, convulsions from mild to severe and prostration. particular stress was placed upon the registration of nervous symptoms [29, 33, 34, 36] . the clinical status of the animals was scored from 0 to 6 [29, 33, 34, 36] as follows: 0: no signs; 1: mild pyrexia; 2: pyrexia plus mild clinical signs; 3: mild-to-moderate clinical signs; 4: moderate clinical signs; 5: moderate-to-severe clinical signs; and 6: death. for ethical reasons, the animals were euthanised when the clinical score reached 5, when exhibiting a fall of the hindquarters, when there was inability to drink or feed, when prostration occurred or when exhibiting moderate nervous disorders. after euthanasia, an exhaustive necropsy was conducted, in which the presence of pathological symptoms in different organs and tissues was evaluated. surviving wild boars were euthanised at 13 dpi, and urine and tissues (spleen, liver, intestine, mesenteric lymph node, prescapular lymph node, bone marrow, medulla oblongata, lung, kidney, thymus and tonsil) were obtained at necropsy. euthanasia was performed according to european directive 2010/63/eu, using a pentobarbital overdose of 60-100 mg/kg administered via the anterior vena cava. the animal care and procedures were in accordance with the guidelines of the good experimental practices (gep), under the supervision of the ethical and animal welfare committee of the autonomous university of barcelona (uab), and they were approved under number 8804, according to the existing national and european regulations. additionally, the biosafety level of the viruses used in this study was stated as biosecurity level 3, as approved by the biosafety committee of the uab, with registration assignment ar-296-15. fifteen representative sequences of the three csfv genogroups were retrieved from genbank and aligned using bioedit [37] . two primers and probes were designed for specific detection of the margarita strain sequence (1.4 csfv genogroup) by targeting the 5´end of the e2 gene, as follows: forward primer (2333-2356), 5´-aagattacgaccacaatttacaac-3´; reverse primer (2411-2431), 5´-tcc tactgaccacattaagcg-3´and probe (2369-2389), 5´-ccatcaaggctatctgcacgg-3´. the nucleotide positions were based on the genome sequence of the margarita strain (genbank accession number aj704817). the probe was labelled with 6-fam at the 5´end and with bhq1 at the 3´end. the primers and probe were purified by reverse phase hplc. the one-step rt-pcr protocol was undertaken using the commercially available taqman1 one-step rt-pcr master mix reagents kit (applied biosystems roche). the real-time rt-pcr assay was optimised using a total volume of 25 μl. real-time qrt-pcr was performed using an applied biosystems1 7500 fast real-time pcr system. the temperature profile was 30 min at 50°c (reverse transcription), 15 min at 95°c (inactivation reverse transcriptase/activation taq polymerase), followed by 42 cycles of 15 s at 94°c (denaturation), 30 s at 57°c (annealing) and 30 s at 68°c (elongation). identical temperature profiles were used for all of the real-time rt-pcr runs, and fluorescence values were collected during the annealing step. twenty csfv rna preparations strains were used to determine the specificity and sensitivity of the assay (table 1) [30, 38] . to exclude the possibility of presence of csfv cat01 strain rna interfering with the assay sensitivity for the csfv margarita strain rna detection, mixtures from serial rna dilutions from both viral strains were analysed. in addition, mixtures from rna serum samples of group b (prior to the margarita strain inoculation), with samples from group a at 7 days post-infection with the margarita strain, were analysed. initial sample volume of 150 μl to obtain a final volume of 50 μl of rna, which was stored at -80°c. the presence of csfv rna in the serum and in nasal and rectal swabs, as well as in tissue samples, was analysed by a generic csfv qrt-pcr [39] . this test was used in our laboratory for inter-laboratory comparisons of csfv diagnoses, organised by the eu reference laboratory. positive results were considered for threshold cycle values (ct) equal to or less than 42. samples in which fluorescence was undetectable were considered negative. additionally, the qrt-pcr specific for the margarita strain, designed in this work (described above), was used to distinguish those samples infected with the margarita strain. serum samples were tested with neutralisation peroxidase-linked assay (npla) [40] , and the titres were expressed as the reciprocal dilution of serum that neutralised 100 tcid 50 of the cat01 or margarita strain in 50% of the culture replicates. the detection of e2-specific antibodies was performed using a commercial elisa kit (idexx); the samples were considered positive when the blocking percentage was 40%, following the manufacturer's recommendations. anti-ifn-α monoclonal antibodies (k9 and k17) and ifn-α recombinant protein (pbl biomedical laboratories, piscataway, new jersey, usa) were used in elisa to detect ifn-α in serum samples at 0, 3, 7 and 10 dpi [34, [41] [42] [43] . the cut-off value of the assay was calculated as the average of the optical density of negative controls (blank and negative sera before csfv infection) plus three standard deviations. cytokine concentrations in serum were determined using a regression line built with the optical densities of the cytokine standards used in the tests. elispot assay to detect csfv-specific ifn-γ cells was performed as previously described [34] , using pbmcs that were obtained at day 0 and at the time of euthanasia. briefly, plates (costar 3590, corning) were coated overnight with 5 μg/ml capture antibody (p2g10, pharmigen). detection was performed using a biotinylated antibody (p2c11, pharmigen). a total of 5x10 5 pbmcs/well were plated in triplicate at 0.1 multiplicity of infection (moi) of the cat01 and margarita csfv strains. moreover, the same samples were incubated in the presence of phytohaemagglutinin (pha) (10 μg/ml). the controls were incubated in the presence of mock-stimulated wells. the numbers of spots in the media for mock-stimulated wells were considered to constitute the baseline for the calculation of antigen-specific frequencies of ifn-γ-producing cells. samples from animal 1 (group a: margarita acutely infected wild boar; 10 dpi), animal 5 (group b: cat01 persistently infected wild boar and superinfected with csfv margarita strain; 13 dpi), and a pestivirus-free wild boar (animal 1 before infection), were used to assess sie in pbmcs (fig 1) . the pbmcs were isolated from whole blood by centrifugation on ficoll gradients (histopaque-1077; sigma). the number and viability of the pbmcs were determined by staining with trypan blue [33] . a total of 4x10 5 pbmcs/well from each animal were plated in quintuplicate at 37°c in 96-well plates with: (i) vehicle; (ii) the cat01 strain at a 0. was analysed by generic csfv qrt-pcr [39] and for the specific margarita strain by qrt-pcr detection assay (see above, section 2.3). for virus isolation, an established cell line sensitive for specific csfv proliferation, st cells, were cultured at 37°c in 96-well plates in triplicate in the presence of each of the collected cell suspensions. after 72 h, the supernatants were removed, and the collected st cells were washed twice and resuspended in 200 μl of sterile pbs. after two cycles of freeze-thaw at -80°c, the presence of csfv rna in the st cell samples was analysed by qrt-pcr for csfv [39] and the margarita strain (see above). in parallel, a st plate similarly inoculated with cell suspensions was used for confirmation by pla [27] . a delta ct (δct) for margarita strain rna detection was calculated as the differences between (i) the margarita ct value detected from the isolation of st from groups a or b and (ii) the ct value in st inoculated with margarita-infected naïve pbmc extract, being δct = ct (a) -ct (b) . the whole protocol was repeated twice, in st and also in sk6 cells using pbmcs from animals 1 (group a), 4 and 5 (group b) and cells from the naïve animal (number 1, group a), collected before margarita infection. the e2-gene fragment reported by lowings et al. [44] was amplified by end point rt-pcr [45] in sera, tonsil, lung and spleen from animals 1, 3 (group a), 4 and 5 (group b), collected at necropsy. additionally, the viral inoculums used in the experimental infections (cat01 and margarita strains) were evaluated. the amplification products were checked by electrophoresis on 2% agarose gel and were directly cleaned with a wizard1 pcr preps dna purification system (promega, madison, wisconsin, usa). sequencing reactions were conducted under bigdye tm terminator-cycling conditions using an abi 3130xl. forward and reverse sequences obtained from each amplicon were assembled using the contig express application in vector nti software, version 11 (invitrogen). the sequences from the e2-gene fragment obtained were aligned to analyse the sequence found in each sample. of the 20 csfv rna strains analysed, the assay detected only the csfv rna from the margarita strain (1.4 genogroup), while the other 19 csfv rna extractions were negative (table 1 ). this result indicated that the newly developed assay was highly specific for the detection of the csfv margarita strain, and there was no cross-reactivity with the other tested csfv strains from genogroup 2 (including the cat01 strain). the specificity of the assay was based primarily on mismatches in the probe-binding region but also to some extent on mismatches in primer-binding regions. the sensitivity of the assay was evaluated by testing 10-fold dilutions of the margarita strain rna. the analytical sensitivity was estimated to be as high as 0.4 tcid 50 . the assay had a reaction coefficient (r 2 ) of 0.994 (data not shown). positive results were considered for threshold cycle values (ct) equal to or less than 38. finally, the presence of cat01 rna strain in the sample containing the margarita strain rna did not affect the assay sensitivity (data not shown). animals persistently infected with the cat01 strain and inoculated with the virulent margarita strain (group b) showed neither clinical signs of disease nor fever at any time throughout the study, maintaining good health status (fig 2) . in contrast, animals from group a, infected with the margarita strain, presented mild clinical signs at 2 dpi that progressed to moderate within 48-72 h. at 7 dpi, animal 2 showed a clinical score value of 4; however, it was found dead at 8 dpi, with lesions of haemorrhagic diathesis. animals 1 and 3 progressed to dyspnoea, weight loss, swaying movement of the hindquarters, posterior paresis and high fever until 10 dpi, when euthanasia was performed. margarita rna was undetected in the sera from animals in group b, except for animal 4 at 13 dpi with a high ct value (ct 36.84), considered a low rna viral load (36, 39) (fig 3) . additionally, csfv margarita strain rna could not be detected in any of the nasal or rectal swabs collected from group b (data not shown). furthermore, in group b, csfv margarita rna was found only in the liver of animal 4 and also in the spleen of animals 4 and 5, with a low rna viral load. in contrast, all wild boars from group b (csfv persistently infected with cat01 strain) maintained during the whole trial a high and constant csfv rna load in serum, swabs and organs, when examined by generic csfv q-rt-pcr (table 2 ). in contrast, both qrt-pcrs (generic and specific for margarita strain) were positive in organs and samples collected from animals in group a ( table 2 ). the ct values were positive by the csfv generic qrt-pcr [39] , in both serum and swab samples, from 3 dpi onwards. ct values for the specific margarita assay were similar to those obtained by the csfv generic qrt-pcr. to evaluate the induction of csfv-specific antibodies, serum samples were analysed at different times after csfv margarita strain infection. the absence of antibody response, in terms of e2-specific antibodies and neutralising antibody titres, was found in both csfv acutely and persistently superinfected groups during the entire experiment (data not shown). previously, it was shown that csfv pi animals were unable to elicit an innate immune response, in terms of ifn-α production, against a csfv life-attenuated vaccine [7] . however, we wondered whether superinfection with a csfv virulent strain would trigger detectable levels of ifn-α in the csfv-superinfected wild boars (group b), given that ifn-α has been largely related to disease severity, as a hallmark of csfv acute infection [34, 46] . in the present work, we observed that progression of disease in group a was correlated with an increase in the levels of endogenous ifn-α after infection, as measured by elisa, with values that reached more than 240 u/ml in two of three animals at 7 dpi and 10 dpi (data not shown). in contrast, ifn-α was undetectable in all of the serum samples analysed both before (day 0) and after margarita inoculation of csfv catalonia persistently infected pigs (group b) (data not shown). pbmcs from all of the animals were analysed for virus-specific and non-specific ifn-γ responses by elispot assay at 0 and 13 dpi post-margarita strain inoculation. very few ifnγ-producing cells were found upon csfv and pha stimulation of pbmcs from all 3 of the csfv-superinfected animals (group b). these results supported our previous results showing that postnatal infection of piglets with csfv could result in virus persistence due to a lack of b-and t-cell responses (data not shown). it is well known that white blood cells, including the pbmcs, are targets for csfv replication [47, 48] . consequently, to examine whether the pbmcs collected from the csfv-superinfected animals (group b) and the acutely infected animals (group a), were permissive (or not) to csfv superinfection, we assayed in vitro inoculation of such samples, with either cat01 or margarita csfv strains. similarly, pbmc samples were mock-infected. additionally, pbmcs from a naïve animal were used as controls. as was expected, csfv-specific margarita rna was detected in the pbmcs from animals developing the csf acute disease (group a) in both mock and margarita-infected samples. furthermore, pbmcs from group b in vitro inoculated with margarita were also positive for csfv-specific margarita rna detection, but with a high ct value correlated with a lower rna load (table 3) . otherwise, pbmcs from group b in vitro mock-infected were negative for csfv-specific margarita rna detection (table 3) . following these findings, to decipher whether the detected rna load in group b might correspond to rna traces from the inocula or to the infecting virus, the previously analysed pbmc extracts were inoculated into a st cell line. consistently, the detected rna load notably increased in st after inoculation with the extract from margarita in vitro inoculated-naïve pbmcs; the obtained 7.76δ ct positive value confirmed the infectivity of the virus recovered from the pbmc samples. in contrast, margarita rna in group b in vitro mock-infected pbmcs remained undetectable even after st inoculation. furthermore, margarita rna load detection in group b in vitro margarita-infected pbmc samples decreased after inoculation of st cells, corresponding to higher ct values than those previously detected directly from pbmc extracts. remarkably, an 11.6 δct value was found in the st cells with margarita in vitro inoculated pbmcs from group b, relative to the value obtained in the st cell extracts from margaritainoculated naïve pbmcs (table 3 ). the whole protocol was repeated twice for animals 1 (group a), 4 and 5 (group b) in both sk6 and st cells, supporting the results with similar ct values (data not shown). similarly, the cells' positive infection was confirmed by pla testing, although this test cannot differentiate between cat01 and margarita csfv strains. to detect the presence of csfv rna of both viral strains (cat01 and/or margarita) in the sera, tonsil, and spleen of animals 1 and 3 (group a) and 4 and 5 (group b), the e2-gene fragment reported by lowings et al. [44] as a phylogenetic marker was amplified by end point rt-pcr [45] . in all of the samples analysed from animals that developed the csf acute form (group a), the sequence corresponding to the margarita strain (aj704817) used as the inoculum was detected. furthermore, the samples analysed from superinfected animals (group b: csfv catalonia 01 persistently infected inoculated with csfv margarita strain) only showed the sequence corresponding to the cat01 strain [30] (fig 4) . despite its significance, the mechanisms of mutual exclusion by viral variants are far from being completely understood, and the actual knowledge is basically derived from studies at the cellular level in established cell lines [14, 16, 19, 49] . very few reports have demonstrated the phenomenon of sie at the organism level, and, to our knowledge, these models have been limited to plant viruses, west nile virus (wnv) in mosquitoes, and peking duck hepatitis b virus (dhbv) [50] [51] [52] . in addition, it has not yet been demonstrated in a mammalian host at the systemic level. previous works have reported the capability of csfv to generate postnatally persistent infection in both domestic pigs and wild boars [5, 6] . subsequently, it was also shown that postnatally persistently infected pigs were unable to elicit a specific immune response to a csfv live attenuated vaccine and that the viral vaccine rna was undetectable in any of the samples analysed [7] . against this background, we assessed the capacity of csfv to generate sie in csfv persistently infected swine. for that purpose, csfv persistently infected wild boars were inoculated with a csfv strain that induce acute disease with a higher replication rate [29, 36] . because pestiviruses are immunologically and genetically closely related, accurate serological characterisation of csfv isolates is impeded by the extensive cross-reactions observed among pestivirus members and the limited availability of mabs capable of differentiating among different csfv isolates [27, 45, 53] . to differentiate the csfv margarita strain rna from the csfv cat01 strain rna in the samples from the present study, a specific qrt-pcr for margarita strain rna detection was developed. thus, alongside the model of infection with the margarita strain, the qrt-pcr assay developed allowed for clear discernment of whether there was actually a blockage that prevented susceptibility to infection by the second virus in both the absence of clinical signs and the absence of molecular detection of the superinfecting virus. notwithstanding the high infection rate of the cat01 strain in persistently infected animals from group b (primary virus infection), good health status was maintained after inoculation with the margarita csfv virulent strain (secondary infection) in the absence of viral detection in sera throughout the study, except in one animal at 13 dpi with a low margarita strain rna load (animal 4). despite the important role that neutralising antibodies play in csfv protection [29, 54] , complete absence of neutralising antibodies response was found after margarita strain infection in these animals. similarly, absence of an ifn-γ-producing cell response against csfv or pha was also observed. considering the role played by ifn-γ in the control of csfv infection [34, 55] and the lack of responsiveness to ifn-γ-producing cells after pha stimulation, the csfv-superinfected animals maintained a immunosuppression state similar to that previously described in postnatal persistent infection [5, 6] . previous work has proved how the failure to induce optimal levels of the humoral and cellular responses after csfv infection promoted the spread of the virus and its relationship with disease progression [29, 54] . in this regard, the implications of the cellular and neutralising antibody response in clinical protection against the acute form in the csfv-superinfected animals from this study are excluded. furthermore, no superinfecting virus excretion was detected in any of the animals from group b, whilst the high viral load generated by the strain that induced the persistent infection (cat01 strain or primary infection) was maintained until the end of the trial, supporting our previous results [7] . in contrast, the csfv margarita strain generated the acute form of the disease in animals from group a, with high margarita rna loads in all of the samples analysed. in addition, the failure of the humoral response in the pigs that developed acute csf was previously described [29] . in addition to the adaptive immune response, the innate immune response to the virus, as measured by type i ifn-α in the serum, also seemed to be impaired, in terms of ifn-α detection because ifn-α values were undetectable in the sera from postnatally persistently infected wild boars after csfv margarita strain inoculation. at the same time, the progression of the acute disease in group a was correlated with an increase in levels of endogenous ifn-α, as has been previously described [29, 46, 56, 57] . the absence of an ifn-α response in the cat01 persistently infected animals after margarita strain inoculation (secondary infection) probably was due to the almost complete lack of margarita strain replication in these animals. otherwise, specific csfv-blockade phenomena for ifn-α might be occurring. efficient viral strategies to escape the type i ifn-induced antiviral mechanisms have been described within pestivirus. in this regard, the viral rna triggers ifn synthesis, and the viral rnase e rns inhibits ifn expression induced by extracellular viral rna [58] . in addition, the viral protein n pro suppresses type i ifn (ifn-α/β) induction by mediating proteasomal degradation of ifn regulatory factor 3 (irf-3) [58] [59] [60] . for instance, in persistent infection, bvdv maintains "self-tolerance" by avoiding the induction of ifn, without compromising the ifn action against unrelated viruses ("nonself") [58] . in the case of csfv-infected pigs, it has been recently demonstrated that functional n pro significantly reduced local ifn-α mrna expression responses at local sites of virus replication [61] . these highly selective "self" models of evasion of the interferon defence system might be key elements in the success of persistent infections and could promote, in addition, the generation of sie phenomena. previous reports have suggested that the availability of mammalian models for sie in vivo is hampered by the interferon response generated against the infecting virus in these species [11, 24] . it is noteworthy that csfv postnatally persistently infected swine have shown an immunosuppression state comprising a reduction in interferon responses (types i and ii) [5] [6] [7] . this immunological status might promote the maintenance of a high and constant csfv load, as already described, preventing second viral entry [5] . nevertheless, further studies would be needed to clarify the molecular mechanisms involved in this phenomenon. at 13 dpi, low levels of margarita rna were detected only in some collected tissues from persistently and superinfected wild boars (group b), principally in animal 4, in which margarita rna was detected from the spleen and liver, as well as in the serum. however, the level of margarita rna detection was approximately fifteen times less than the acutely infected animals from group a ( table 2 ). the margarita rna levels found in the superinfected animals might be correlated with the low margarita strain viral loads in some macrophages in these tissues [62] . in contrast, despite pbmcs being a well-known target for csfv viral replication [62] , after in vitro assay, the presence of csfv margarita rna could not be detected in either the pbmcs or st cell extracts from group b. additionally, the in vitro superinfection of isolated pbmcs failed when they were derived from persistently infected piglets but were clearly positive for assays with cells from naïve animals, as demonstrated by means of calculated δct values, supporting that pbmcs from persistently infected animals were substantially protected from superinfection after in vitro inoculation with the margarita virus strain. these results suggest that sie still occurs at the tissue level (table 3 ). in contrast, the margarita strain rna could not be detected after the sequence analyses of the samples from persistently infected margarita-inoculated animals (group b) nor even in the tonsil, one of the main targets for csfv replication [3, 63] . nevertheless, next-generation sequence analyses would be of great interest to analyse these samples in detail, emphasising the spleen and liver tissues that were also positive for rna margarita strain detection after superinfection. altogether, although it is a very complex mechanism, if compared with the acutely infected group a, these results showed that a phenomenon of csfv sie occurred at the systemic level. nevertheless, the colonisation of a multi-cellular host is a complex process during which the viral load can dramatically change in different organs and at different stages of the infection, and not all of the potential target cells are infected in persistently infected animals despite the high viral load generated by the cat01 csfv strain in persistently infected animals [5, 6] . illustrative examples include some of the works performed to demonstrate sie at the cellular level because some cells uninfected by one viral primary infection are subsequently infected by the second viral infection [11, 50] . in contrast, the implications of other mechanisms in the host cannot be excluded, and it remains unclear whether the observed phenomenon is really due to a blockage at the level of infection of cells. this was precisely in the case of a citrus tristeza virus (ctv) sie model, wherein a ctv protein (p33) was required to mediate sie at the organism level but that did not appear to be implicated in exclusion at the cellular level [50] . overall, our results suggested efficient suppression of viral superinfection in a mammalian host, especially in the absence of ifn-α, indicating a lack of innate immune mechanisms. considering the role of this phenomenon from an evolutionary standpoint, their implications within an epidemic situation might be relevant to the evolution and phylogeny of csfv. although this phenomenon must be studied in greater depth, the possible outcome for the generation of new csfv strains circulating in an endemic situation and the impact on disease control, including vaccination with live attenuated vaccines, cannot be underrated. virus taxonomy eighth report of the international committee on taxonomy of viruses annu rev anim biosci recent advances in the development of recombinant vaccines against classical swine fever virus: cellular responses also play a role in protection molecular biology of pestiviruses. animal viruses: molecular biology postnatal persistent infection with classical swine fever virus and its immunological implications post-natal persistent infection with classical swine fever virus in wild boar: a strategy for viral maintenance? transbound emerg dis efficacy of a live attenuated vaccine in classical swine fever virus postnatally persistently infected pigs a generic real-time taqman assay for specific detection of lapinized chinese vaccines against classical swine fever biochemical and immunological aspects of the exclusion of lambda by superinfection with t4 a coat-independent superinfection exclusion rapidly imposed in nicotiana benthamiana cells by tobacco mosaic virus is not prevented by depletion of the movement protein superinfection exclusion is an active virus-controlled function that requires a specific viral protein the multiplicity of cellular infection changes depending on the route of cell infection in a plant virus superinfection exclusion of alphaviruses in three mosquito cell lines persistently infected with sindbis virus a novel mode of poxvirus superinfection exclusion that prevents fusion of the lipid bilayers of viral and cellular membranes hepatitis c virus superinfection of liver grafts: a detailed analysis of early exclusion of non-dominant virus strains rubella virus-induced superinfection exclusion studied in cells with persisting replicons dual mechanisms of pestiviral superinfection exclusion at entry and rna replication superinfection exclusion by vesicular stomatitis virus superinfection exclusion in cells infected with hepatitis c virus the nef protein of human immunodeficiency virus establishes superinfection immunity by a dual strategy to downregulate cell-surface ccr5 and cd4 sequence variability of borna disease virus: resistance to superinfection may contribute to high genome stability in persistently infected cells influenza a virus neuraminidase limits viral superinfection natural resistance mechanisms of plants to viruses evasion of superinfection exclusion and elimination of primary viral rna by an adapted strain of hepatitis c virus porcine cells persistently infected with classical swine fever virus protected from pestivirus-induced cytopathic effect establishment, viral susceptibility and biological characteristics of a swine kidney cell line sk-6 production of monoclonal antibodies against swine fever virus and their use in laboratory diagnosis a simple method of estimating fifty per cent endpoints the impact of csfv on the immune response to control infection positive selection pressure on the b/c domains of the e2-gene of classical swine fever virus in endemic areas under c-strain vaccination descriptive epidemiology of the outbreak of classical swine fever in catalonia (spain), 2001/02. vet rec [internet origin and evolution of viruses causing classical swine fever in cuba a dna vaccine expressing the e2 protein of classical swine fever virus elicits t cell responses that can prime for rapid antibody production and confer total protection upon viral challenge interferon-gamma induction correlates with protection by dna vaccine expressing e2 glycoprotein against classical swine fever virus infection in domestic pigs characterization of c-strain "riems" tav-epitope escape variants obtained through selective antibody pressure in cell culture partial protection against classical swine fever virus elicited by dendrimeric vaccine-candidate peptides in domestic pigs bioedit: a user-friendly biological sequence alignment program for windows 95/98/nt development and validation of a novel sybr green real-time rt-pcr assay for the detection of classical swine fever virus evaluated on different real-time pcr platforms validation of a real-time rt-pcr assay for sensitive and specific detection of classical swine fever the neutralizing peroxidase-linked assay for detection of antibody against swine fever virus a sensitive immunoassay for porcine interferon-alpha enrichment of coronavirus-induced interferon-producing blood leukocytes increases the interferon yield per cell: a study with pig leukocytes type-a cpg oligonucleotides activate exclusively porcine natural interferon-producing cells to secrete interferon-alpha, tumour necrosis factor-alpha and interleukin-12 classical swine fever virus diversity and evolution molecular epidemiology of classical swine fever in cuba high ifn-alpha responses associated with depletion of lymphocytes and natural ifn-producing cells during classical swine fever. j interferon cytokine res comparison of methods for improved rna extraction from blood for early detection of classical swine fever virus by real-time reverse transcription polymerase chain reaction. j vet diagnostic investig diagnostic methods for detection of classical swine fever virusstatus quo and new developments cellular mechanisms in the superinfection exclusion of vesicular stomatitis virus a viral protein mediates superinfection exclusion at the whole-organism level but is not required for exclusion at the cellular level superinfection exclusion in duck hepatitis b virus infection is mediated by the large surface antigen a positively selected mutation in the wnv 2k peptide confers resistance to superinfection exclusion in vivo a multiplex rt-pcr assay for the rapid and differential diagnosis of classical swine fever and other pestivirus infections. vet microbiol vaccinology of classical swine fever: from lab to field characterisation of vaccineinduced, broadly cross-reactive ifn-γ secreting t cell responses that correlate with rapid protection against classical swine fever virus lymphocyte apoptosis during classical swine fever: implication of activation-induced cell death acute induction of cell deathrelated ifn stimulated genes (isg) differentiates highly from moderately virulent csfv strains bvdv: a pestivirus inducing tolerance of the innate immune response n pro of classical swine fever virus prevents type i interferon-mediated priming of conventional dendritic cells for enhanced interferon-α response classical swine fever virus npro interacts with interferon regulatory factor 3 and induces its proteasomal degradation npro of classical swine fever virus contributes to pathogenicity in pigs by preventing type i interferon induction at local replication sites classical swine fever virus marker vaccine strain cp7_e2alf: shedding and dissemination studies in boars classical swine fever virus strain "c". how long is it detectable after oral vaccination? j vet med b infect dis vet public health we thank valentí rosell, iván cordón and david solanes for their help in the animal facilities. key: cord-003614-63rzzos3 authors: klotz, daniela; gerhauser, ingo title: interferon-stimulated genes—mediators of the innate immune response during canine distemper virus infection date: 2019-04-01 journal: int j mol sci doi: 10.3390/ijms20071620 sha: doc_id: 3614 cord_uid: 63rzzos3 the demyelinating canine distemper virus (cdv)-leukoencephalitis represents a translational animal model for multiple sclerosis. the present study investigated the expression of type i interferon (ifn-i) pathway members in cdv-induced cerebellar lesions to gain an insight into their role in lesion development. gene expression of 110 manually selected genes in acute, subacute and chronic lesions was analyzed using pre-existing microarray data. interferon regulatory factor (irf) 3, irf7, signal transducer and activator of transcription (stat) 1, stat2, mx protein, protein kinase r (pkr), 2′-5′-oligoadenylate synthetase (oas) 1 and interferon-stimulated gene (isg) 15 expression were also evaluated using immunohistochemistry. cellular origin of stat1, stat2, mx and pkr were determined using immunofluorescence. cdv infection caused an increased expression of the antiviral effector proteins mx, pkr, oas1 and isg15, which probably contributed to a restricted viral replication, particularly in neurons and oligodendrocytes. this increase might be partly mediated by irf-dependent pathways due to the lack of changes in ifn-i levels and absence of stat2 in astrocytes. nevertheless, activated microglia/macrophages showed a strong expression of stat1, stat2 and mx proteins in later stages of the disease, indicating a strong activation of the ifn-i signaling cascade, which might be involved in the aggravation of bystander demyelination. canine distemper virus (cdv) is an enveloped, single-stranded, negative-sense rna virus [1, 2] , which can infect terrestrial and aquatic carnivores via the oropharyngeal route [3, 4] . the clinical course of the disease depends on the cdv strain as well as the immune status and age of the infected host [2, 4] . cdv-infected animals can either trigger a strong humoral and cellular immune response between the 8th and 14th day after infection and recover or, in the case of a weak immune response, die or develop a persistent infection [4, 5] . clinically, canine distemper is predominantly characterized by catarrhal respiratory and enteric disease signs, but a systemic form affecting also the central nervous system (cns) and unusual manifestations including the so-called "hard pad" disease can occur [4] . the clinical disease of canine distemper is similar in pathogenesis and symptoms to measles virus infection in humans [4] . neurologic manifestations of canine distemper can be caused by gray and/or white matter disease [4] [5] [6] [7] . the rarer polioencephalitis is divided into "old dog encephalitis", "post-vaccination distemper encephalitis" and "inclusion body polioencephalitis" [6, 7] . however, the nervous form usually manifests as leukoencephalitis within the cerebellum [5] . the lesions can be the original microarray analysis performed by ulrich et al. (2014) revealed 780 differentially expressed probe sets and the dominating change was an up-regulation of genes related to the innate and the humoral immune response [47] . of 110 manually selected genes of the ifn-i pathway, 57% (63/110) were up-regulated in a relevant manner (significant fold change > 1.5) at least in one group of cdv-induced lesions, and gene expression usually peaked in the subacute stage ( figure 1 , table 1, table s1 ). toll-like receptor (tlr) 2, tlr3 and tlr7 (fold change up to 4.52, 8.00 and 2.83) were moderately increased, whereas pkr and mda5 were highly up-regulated (fold change up to 15.62 and 33.76) . only a mild increase in irf3, irf5 and nuclear factor of kappa light polypeptide gene enhancer in b-cells (nfkb) 1 gene expression (fold change < 2) was found, whereas irf1 and irf7 were strongly induced (fold change up to 30.13 and 113.54) . no transcriptional changes were found in ifn-i gene expression and ifnar genes were only mildly increased (fold change < 2) in the subacute stage of cdv-infection. similarly, most signal transducers including jak1, socs1 and tyk2 did not show significant transcriptional changes. nevertheless, stat1, stat2 and irf9 were moderately up-regulated (fold change up to 19.19, 4.57 and 11.44) . the classical isgs were strongly induced by cdv-infection with particularly high levels for isg15, ifi44l, isg56, oas2 and mx2 (fold change up to 928. 35, 419.93, 179 .05, 173.57 and 147.59). additionally, several mhc class i and class ii genes were highly up-regulated (fold change up to 135. 55 and 30.97 ). an overview of fold changes for selected interferon-dependent genes is given in figure 1 , table 1 and table s1 . the original microarray analysis performed by ulrich et al. (2014) revealed 780 differentially expressed probe sets and the dominating change was an up-regulation of genes related to the innate and the humoral immune response [47] . of 110 manually selected genes of the ifn-i pathway, 57% (63/110) were up-regulated in a relevant manner (significant fold change > 1.5) at least in one group of cdv-induced lesions, and gene expression usually peaked in the subacute stage ( figure 1 , table 1 , table s1 ). toll-like receptor (tlr) 2, tlr3 and tlr7 (fold change up to 4.52, 8.00 and 2.83) were moderately increased, whereas pkr and mda5 were highly up-regulated (fold change up to 15.62 and 33.76) . only a mild increase in irf3, irf5 and nuclear factor of kappa light polypeptide gene enhancer in b-cells (nfkb) 1 gene expression (fold change < 2) was found, whereas irf1 and irf7 were strongly induced (fold change up to 30.13 and 113.54) . no transcriptional changes were found in ifn-i gene expression and ifnar genes were only mildly increased (fold change < 2) in the subacute stage of cdv-infection. similarly, most signal transducers including jak1, socs1 and tyk2 did not show significant transcriptional changes. nevertheless, stat1, stat2 and irf9 were moderately up-regulated (fold change up to 19.19, 4.57 and 11.44 55 and 30.97 ). an overview of fold changes for selected interferon-dependent genes is given (nfκb, irf1, irf3, irf5, irf7) . these transcription factors translocate into the nucleus and induce ifn-i expression. right side: receptor binding of ifn-i activates the jak-stat signaling pathway leading to the formation of stat1/stat2 heterodimers in the cytoplasm. these heterodimers form a complex with irf9, termed interferon-stimulated gene factor 3 (isgf3). in the nucleus, isgf3 induces the transcription of interferon-stimulated genes including isg15, mx1, mx2, pkr, irf7, oas1, oas2 and oasl. irf7 also acts in a positive feedback loop to stimulate ifn-i expression. columns behind the gene symbols display fold changes in three different stages of cdv-infection (acute; subacute; chronic). fold changes are shown on a logarithmic scale. (nfκb, irf1, irf3, irf5, irf7) . these transcription factors translocate into the nucleus and induce ifn-i expression. right side: receptor binding of ifn-i activates the jak-stat signaling pathway leading to the formation of stat1/stat2 heterodimers in the cytoplasm. these heterodimers form a complex with irf9, termed interferon-stimulated gene factor 3 (isgf3). in the nucleus, isgf3 induces the transcription of interferon-stimulated genes including isg15, mx1, mx2, pkr, irf7, oas1, oas2 and oasl. irf7 also acts in a positive feedback loop to stimulate ifn-i expression. columns behind the gene symbols display fold changes in three different stages of cdv-infection (acute; subacute; chronic). fold changes are shown on a logarithmic scale. lesions (130) found in cerebellar tissue of 15 cdv-infected dogs were analyzed using he-and lfb-stainings and cdv immunohistochemistry. in 16 lesions, cdv antigen changes but not histological ones were detected (group 2). twenty-six acute lesions were characterized by white matter vacuolation and absence of demyelination (group 3). forty-eight subacute lesions showed demyelination in the lfb-staining but no inflammatory cell infiltration in the he-staining (group 4). forty chronic lesions demonstrated advanced demyelination, perivascular cuffing and lymphocytes in the parenchyma (group 5). in addition, 53 areas were randomly selected in the cerebellar white matter of six control dogs for evaluation (group 1). representative pictures of the investigated lesions are shown in figure in general, immunohistochemical analysis detected an increase in the protein expression of all investigated ifn-i pathway members in cdv-induced white matter lesions (figures 3 and 4 , table 2, figure s1 ). in general, immunohistochemical analysis detected an increase in the protein expression of all investigated ifn-i pathway members in cdv-induced white matter lesions (figures 3 and 4 , table 2, figure s1 ). a statistically significant up-regulation of irf3 protein expression was only found in group 2 lesions compared to controls (p < 0.001). endothelial cells, neurons and glial cells were positive for irf3 in all investigated groups. in addition, perivascular lymphocytes present in group 5 expressed irf3. irf7 protein was not expressed in perivascular lymphocytes but in glial cells and neurons in infected and control groups. there was a prominent staining in the granular cell layer and a slight staining of purkinje cells. the irf7 protein expression was significantly up-regulated in acute (p = 0.006), subacute (p < 0.001) and chronic lesions (p < 0.001) and continuously increased during lesion progression. stat1 protein expression was increased at all stages of cdv-induced leukoencephalitis (group 2: p = 0.003; groups 3-5: p < 0.001). in cdv-infected cerebellar tissue, stat1 proteins were mainly detected in glial cells, whereas only a few neurons and endothelial cells expressed stat1 and perivascular cells were mainly negative. in control tissue, only single glial cells, as well as some neurons and endothelial cells, slightly expressed stat1. stat2 protein expression was significantly elevated only in chronic lesions (group 5: p = 0.02). perivascular lymphocytes were negative for stat2, whereas neurons, especially purkinje cells and glia cells, expressed stat2 with a strong intensity in cdv-infected dogs and a weak intensity in control dogs. isg15, mx and pkr proteins were significantly increased at all investigated stages of cdv-induced leukoencephalitis (p < 0.001). perivascular lymphocytes expressed mx and pkr proteins but not isg15. mx proteins were also detected in endothelial cells, neurons and glial cells of cdv-infected dogs. in control tissue, only some neurons and endothelial cells showed a mild mx expression. furthermore, glial cells and neurons, especially purkinje cells and some endothelial cells, were positive for pkr in cdv-infected dogs. in control animals, pkr proteins were only detected in neurons, especially purkinje cells. isg15 proteins were detected in neurons, endothelial cells and glial cells of cdv-infected dogs, whereas control animals only showed a weak isg15 protein expression in neurons and endothelial cells. a significant up-regulation of oas proteins was found in acute (p = 0.008), subacute (p < 0.001) and chronic lesions (p = 0.01) but not in cdv-infected tissue without histological lesions (group 2; p = 0.09). in cdv-infected dogs, oas proteins were detected in perivascular lymphocytes, endothelial cells, glial cells and neurons. in control animals, only neurons and endothelial cells expressed oas proteins. control dogs; group 2 are foci with cdv antigen expression but without histological lesions; group 3 are acute cdv lesions; group 4 are subacute foci without inflammation; group 5 are chronic cdv lesions with inflammation. shown are the percentages of the immunopositive area using box plots in a logarithmic scale and significant differences between the cdv-infected groups 2-5 and the control group 1 based on kruskal-wallis-tests and dunn's multiple comparison tests. * p < 0.05; ** p < 0.01; *** p < 0.001. ++ ++ ++ ++ +++ + +++ ++ + +++ + ++ endothelial cells +++ +++ (+) (+) + (+) ++ (+) ++ (+) + + (+) spearman's rank correlation coefficients demonstrated a moderate correlation between cdv antigen and stat1, isg15, mx and pkr protein expression (table 3) . cdv antigen correlated only weakly with irf3 and stat2 and did not correlate with irf7 protein expression. irf3 also correlated weakly with stat1, oas and pkr. moreover, there was a weak to moderate correlation between stat1, isg15, mx, oas and pkr, a moderate correlation between irf7 and isg15, and a strong correlation between isg15 and mx (table 3) . double staining with cellular markers revealed a stat1 expression in nogoa + oligodendrocytes, gfap + astrocytes and iba1 + microglia/macrophages, which was limited to histological lesions. stat2 was detected in nogoa + oligodendrocytes located in white matter lesions and also in normal-appearing white matter. stat2 was also found in activated iba1 + microglia/macrophages (gitter cells), which were present in subacute and chronic white matter lesions. mx was expressed by intralesional gfap + astrocytes, nogoa + oligodendrocytes and iba1 + microglia/macrophages. pkr was also found in intralesional gfap + astrocytes and some nogoa + oligodendrocytes but not in iba1 + microglia/macrophages ( figure 5 ). immunofluorescence double-staining was also used to determine the infection status of cells expressing stat1, stat2 and mx. a low number of cdv-infected cells expressed stat1 and mx. however, cdv-infected cells expressing stat2 were nearly absent ( figure 6 ). the present study gives an overview of the protein expression of selected ifn-i pathway members and compares it with existing microarray data. previous studies often investigated isg expression in cell cultures, whereas studies on the protein expression of isgs in a naturally occurring animal disease are rare [48] [49] [50] [51] [52] . irf3 plays, together with the closely related irf7, an important role in the ifn-i signaling cascade and the control of viral infections [53] . ysebrant de lendonck et al. (2014) reported that irf3 is constitutively expressed in most tissues and cell types [54] . similarly, in the present study, irf3 was expressed in most cell types present in cdv-induced cerebellar lesions as well as in control tissue (endothelial cells, neurons, glial cells and perivascular inflammatory cells). this strong constitutive expression of irf3 might also explain that the present microarray analysis and immunohistochemical investigation did not detect changes in the gene or protein expression of this transcription factor in acute, subacute or chronic cerebellar lesions. nonetheless, there was a significant increase in irf3 protein expression in cdv-infected areas lacking histological lesions (group 2) compared to controls demonstrating an early activation of ifn-i signaling in the disease process. irf3 interacts with other transcription factors, coactivators and repressors including other irfs and nf-κb and thereby regulates t cell differentiation into th1, th2 and th17 cells as well as induces apoptosis during viral infections [54] . thus, irf3 most likely modulates adaptive immune responses at all stages of cdv-induced cerebellar lesions due to its action as a signaling platform, which irf3 can fulfill without changes in expression. unlike irf3, irf7 is reported to be expressed only at low levels in most cell types and has to be continuously produced due to its short half-life time of 0.5-1 h [55, 56] . nevertheless, irf7 protein was present in neurons of the granular layer and individual glial cells, even in control cerebellum, showing low constitutive expression of this transcription factor in the canine cns. oasl1knockout mice studies showed that oasl1 inhibits irf7 translation and thereby negatively the present study gives an overview of the protein expression of selected ifn-i pathway members and compares it with existing microarray data. previous studies often investigated isg expression in cell cultures, whereas studies on the protein expression of isgs in a naturally occurring animal disease are rare [48] [49] [50] [51] [52] . irf3 plays, together with the closely related irf7, an important role in the ifn-i signaling cascade and the control of viral infections [53] . ysebrant de lendonck et al. (2014) reported that irf3 is constitutively expressed in most tissues and cell types [54] . similarly, in the present study, irf3 was expressed in most cell types present in cdv-induced cerebellar lesions as well as in control tissue (endothelial cells, neurons, glial cells and perivascular inflammatory cells). this strong constitutive expression of irf3 might also explain that the present microarray analysis and immunohistochemical investigation did not detect changes in the gene or protein expression of this transcription factor in acute, subacute or chronic cerebellar lesions. nonetheless, there was a significant increase in irf3 protein expression in cdv-infected areas lacking histological lesions (group 2) compared to controls demonstrating an early activation of ifn-i signaling in the disease process. irf3 interacts with other transcription factors, coactivators and repressors including other irfs and nf-κb and thereby regulates t cell differentiation into th1, th2 and th17 cells as well as induces apoptosis during viral infections [54] . thus, irf3 most likely modulates adaptive immune responses at all stages of cdv-induced cerebellar lesions due to its action as a signaling platform, which irf3 can fulfill without changes in expression. unlike irf3, irf7 is reported to be expressed only at low levels in most cell types and has to be continuously produced due to its short half-life time of 0.5-1 h [55, 56] . nevertheless, irf7 protein was present in neurons of the granular layer and individual glial cells, even in control cerebellum, showing low constitutive expression of this transcription factor in the canine cns. oasl1-knockout mice studies showed that oasl1 inhibits irf7 translation and thereby negatively regulates ifn-i production during acute and chronic viral infections [57, 58] . the present study detected a strong increase in oasl1 mrna transcripts in acute, subacute and chronic lesions, which might affect irf7 expression. nonetheless, there was a strong up-regulation in irf7 gene and protein expression in cerebellar lesions, which was most pronounced at later stages of cdv-induced leukoencephalitis. this continuous increase in irf7 expression is probably caused by the positive feedback mechanism of irf7 and indicates that the ifn-i signaling pathway contributes to lesion progression [56, [59] [60] [61] . stat1 proteins can form stat1/stat2 heterodimers and stat1/stat1 homodimers to induce transcription of isgs and pro-inflammatory cytokines after ifn-i and ifnγ stimulation, respectively [40] . svitek et al. (2014) demonstrated that cdv has to block stat2 signaling for immune evasion in ferrets, whereas inhibition of stat1 does not contribute to cdv virulence [45] . the present study demonstrated a strong increase in stat1 mrna transcripts and proteins in the cdv-infected cerebellum (groups 2-5), whereas control animals expressed only minimal amounts of stat1. stat2 proteins were constitutively expressed in cdv-infected and control tissue and only weakly up-regulated in a chronic lesion, but microarray analysis revealed a moderate increase in stat2 mrna transcripts also in acute and subacute lesions. however, the antibody used to detect stat1 only binds to phosphorylated and thus activated proteins (p84/p91), whereas the anti-stat2 antibody recognizes activated and non-activated proteins. this difference explains the detection of less prominent changes in stat2 compared to stat1 protein expression using immunohistochemistry. interestingly, both transcription factors were found in neurons, oligodendrocytes and activated microglia/macrophages, whereas astrocytes, endothelial cells and inflammatory cells only expressed stat1 but not stat2. this observation suggests that the canonical ifn-i pathway with stat1/stat2 heterodimer formation only plays a minor role in the latter cells and non-canonical jak-stat signaling via stat1/stat1 homodimers or other pathways might mediate isg expression [40] . moreover, the present analysis of the previously published microarray did not detect an increase in ifn-i expression in the cdv-infected cerebellum, which is most likely caused by the non-structural v protein of cdv interfering with mda5-dependent virus detection [45] . consequently, the ifn-i response and the down-stream jak-stat signaling cascade are blocked during canine distemper encephalitis. nevertheless, the present study demonstrated a strong increase in isg gene and protein expression, which seems to be mediated by ifn-i-independent pathways. similarly, a recent microarray study of mice infected by theiler's murine encephalomyelitis virus (tmev) described a strong isg expression in demyelinating spinal cord lesions despite low ifn-i levels [62] . isg15 expression can directly be induced by irf1 (formerly called isgf2), which is a transcription factor involved in anti-viral and anti-bacterial immune responses, t cell and nk cell differentiation, mhc class i and ii expression and induction of apoptosis [55, 63, 64] . correspondingly, the present canine study demonstrated a strong up-regulation of irf1 and several mhc genes at all stages of cdv-induced cerebellar lesions. dla-88 was particularly increased in acute and subacute distemper lesions (fold change up to 135,55). most importantly, this highly polymorphic mhc class i protein can present peptides derived from cdv hemagglutinin, large polymerase, matrix, nucleocapsid, and v proteins [65, 66] . irf7 is also described as inducing isg transcription in the absence of ifn-i [67] . consequently, irf1-and irf7-dependent pathways presumably mediate a major part of isg expression in the cdv-infected cerebellum, counteracting common viral strategies, which modulate or inhibit the ifn-i response [67, 68] . isg15 showed the strongest increase of all investigated genes, with a fold change of almost 1000 in subacute lesions. in addition, a weak isg15 immunoreactivity was detected in neurons and endothelial cells of uninfected canine cerebella, confirming previous descriptions of a low constitutive isg15 expression [69] . isg15 represents an ubiquitin-like protein, which binds to over 150 cellular proteins involved in immune regulation, including members of the ifn-i, nf-κb and jnk pathways [69, 70] . this process known as isgylation can activate or inhibit the respective signaling cascades in a species-specific manner [71] . murine isg15 targets rig1, which reduces type i interferon promoter activity and nf-κb responses [72] . in contrast, human isg15 can bind to irf3 preventing its degradation by the proteasome and thereby increasing the ifn-i response [73] . nevertheless, humans born with inactivating isg15 mutations are highly susceptible to mycobacterial and autoinflammatory diseases but not to viruses, this underlining the complexity of isg15-mediated effects [71, 74] . the susceptibility of isg15-deficient patients to environmental mycobacteria is probably related to the cytokine-like functions of secreted isg15, which acts in synergy with il12 to induce ifnγ production by t cells or nk cells, thus enhancing the antimycobacterial activity of macrophages [70, 71, 75, 76] . only a few studies have investigated the functions of isg15 in dogs, indicating an antiviral activity of this protein in canine cells, which might be mediated by binding isg15 to viral proteins interfering with viral replication [17, 69, 70, 77] . ifnγ can be found in the csf of dogs with chronic but not early lesions [78] [79] [80] . consequently, highly up-regulated isg15 levels in cdv-induced cerebellar lesions most likely contribute to viral elimination and might modulate the canine immune system. pkr can be activated by double-stranded rna and isgylation, which results in autophosphorylation and subsequently in phosphorylation of the eukaryotic translation initiation factor 2 alpha (eif2α), thereby down-regulating cellular and viral protein synthesis [81] [82] [83] [84] . in addition, the activation of this constitutively expressed virus sensor protein can induce apoptosis, thus ensuring the eradication of infected cells from the tissue [85, 86] . the present study demonstrated a strong increase in the expression of phosphorylated, hence activated pkr in the cdv-infected cerebellum. this protein was detected in neurons, intralesional astrocytes and oligodendrocytes as well as perivascular lymphocytes but not in microglia/macrophages. similarly, tmev-infected mice showed a strong expression of activated pkr proteins in spinal cord white matter lesions [62] . nevertheless, these proteins were predominantly expressed by intralesional gitter cells and to a lesser extent by perivascular immune cells, oligodendrocytes and neurons but not by astrocytes in tmev-infected mice. similarly, in mice suffering from experimental allergic encephalomyelitis, another mouse model of human ms, activated pkr proteins were found in oligodendrocytes and neurons as well as in intralesional t cells and macrophages [87] . these observations suggest a so far undescribed species-specific difference in pkr expression and/or activation of murine and canine astrocytes and microglia/macrophages. similar to pkr, oas proteins are constitutively expressed in many tissues (spleen, lung, liver, thymus, small intestine, brain) and bind double-stranded rna resulting in their activation [88] [89] [90] . activated oas proteins then induce the formation of 2 -5 -oligoadenylates from atp molecules, which trigger the ubiquitously expressed endoribonuclease rnase l [91] . this enzyme can degrade cellular and viral rna and thereby inhibit viral protein synthesis [91] [92] [93] . the production of small rna cleavage products by rnase l again initiates ifn-i production, creating a positive feedback mechanism in antiviral defense [93, 94] . rnase l has also been described as a potent inducer of apoptosis in fibroblasts [95] . in addition, kristiansen et al. (2010) suggested that oas proteins produced by virus-infected cells can be released into the extracellular space, where it acts as an rnase l-independent paracrine antiviral agent to protect neighboring cells from infection [96] . the present study revealed a strong increase in oas gene and protein expression in acute, subacute and chronic cerebellar lesions, which most likely contributes to the restriction of viral replication. oas proteins were detected in neurons and endothelial cells as well as in intralesional glial cells and perivascular inflammatory cells. previous studies demonstrated that the oas-rnase l pathway is particularly important in murine macrophages and inhibits the replication of picornaviridae including tmev [93, [97] [98] [99] [100] [101] . interestingly, the oas activity in canine serum is 10-to 100-fold higher than in cats, rabbits and guinea pigs, indicating a prominent role of oas proteins in the canine humoral immune system [46] . however, the exact role of rnase l-dependent and independent functions of oas proteins in the pathogenesis of cdv-induced leukoencephalitis remains to be determined. the present study showed a significant increase in the expression of mx proteins during all stages of canine distemper leukoencephalitis, which was even found in cdv-infected areas without histological lesions. the presence of mx proteins in neurons and endothelial cells of the normal canine brain demonstrates a low but constitutive expression of this antiviral protein, whose expression was described to be strictly dependent on ifn-i [102] . similarly, porter et al. (2006) found a strong mx expression in astrocytes, macrophages/microglia and neurons in cdv-infected brain tissue [44] . endothelial cells were also described to be immunopositive for mx by porter et al. (2006) , but this staining was interpreted as non-specific [44] . however, strong expression of mx proteins in canine distemper lesions most likely impedes viral replication and, similar to increased isg15, pkr and oas levels, contributes to the typical decrease in viral antigen observed during the progression of the disease [2, 4, 103] . moreover, the low number of cdv-infected cells expressing stat1, stat2 and mx indicates that the expression of these type i ifn pathway members is not directly induced by virus infection. the strong expression of stat1, stat2 and downstream isg proteins in oligodendrocytes and neurons is probably related to their well-known restricted cdv infection, which is characterized by the presence of cdv nucleic acid sequences and lack of viral antigen [4] . in contrast, astrocytes did not express stat2 proteins, which favors viral replication in these cells due to the blocking of the canonical jak-stat pathway. the cell-type specific differences in the activation of the ifn-i pathway might also explain the fact that up to 95% of all cdv-infected cells within acute lesions are astrocytes [104] , whereas oligodendrocytes hardly express cdv protein [105] . however, the restriction of virus replication in neurons and oligodendrocytes does not prevent axonal pathology and massive down-regulation of myelin gene expression (oligodendrocyte dystrophy) [105, 106] . both processes contribute to the initiation of the demyelination process, whereas later stages of demyelination are mainly mediated by so-called bystander mechanisms [2, 4, 47, 107] . this bystander demyelination results from the activation of macrophages/microglia, which is characterized by an increased phagocytic activity, oxygen radical production and expression of mhc class ii molecules and the release of myelin damaging enzymes such as matrix-metalloproteinases (mmps) [2, 4, [108] [109] [110] . the progressive development of demyelination is driven by a strong continuous expression of il1, il6, il8, il12 and tnfα promoting innate and th1-biased immune responses [2, 4, 80, [111] [112] [113] . interestingly, recent studies demonstrated that the ifn-i pathway is critically involved in the differentiation and activation of immune cells as well as the expression of mmps and pro-inflammatory cytokines [55, [114] [115] [116] [117] [118] [119] [120] . ifn-i can modulate the phenotype of microglia, regulate phagocytosis and affect blood-brain barrier integrity [115] . irf1 is required for nk cell development and differentiation of cd8 + t cells promotes the differentiation of th1 cells via transcriptional control of il12p40, inhibits tnfα-induced mmp9 expression and contributes to the activation of the nlrp3 inflammasome [55, 114, 116, 117] . irf3 stimulates the transcription of il6, il8 and mmps through interaction with c-jun and the ap-1 promoter site [118] [119] [120] . irf7 regulates the expression of il6 through stabilization of il6 mrna and induces the transcription of cxcl10, a chemokine for macrophages, t cells and nk cells [118] . moreover, studies of monogenic autoinflammatory cns diseases demonstrated that they can be caused by an uncontrolled up-regulation of ifn-i signaling and jak inhibition may be a successful therapeutic strategy in patients with these type i interferonopathies [121] [122] [123] . consequently, the strong activation of the ifn-i signaling pathway in infiltrating immune and resident cns cells including activated microglia/macrophages most likely contributes to immunopathological mechanisms aggravating cdv-triggered demyelination. this study was conducted in accordance with the german animal welfare act. all animals used in this study were dead at the time of submission for necropsy in order to investigate the causes of death and disease. consequently, the authors confirm that this study does not contain data obtained from animal experiments and no animals were infected or sacrificed for the purpose of this retrospective pathological case-control study. moreover, all dog owners provided written consent for the dogs' tissues to be collected and used for research purposes. paraffin-embedded cerebellar tissue of 15 naturally cdv-infected dogs and six control dogs without any cns lesion was collected from the archive of the department of pathology of the veterinary university of hannover, germany and used for histological analysis and immunostainings. anamnestic data of the investigated dogs are summarized in table s2 . serial two-micrometer paraffin sections were stained with hematoxylin and eosin (he) or luxol fast blue (lfb) or used for immunohistochemistry. the lesions in the cdv-infected dogs were categorized with he-and lfb-staining as previously described [47, 106, 124] . briefly, randomly selected areas in the cerebellar white matter of dogs without any lesions were used as controls (group 1). white matter lesions of cdv-infected dogs were divided into four groups. group 2 ("antigen without lesion") included areas with antigen expression but with no lesions in he-or lfb-stainings. group 3 lesions ("acute") were characterized by vacuolation in the he staining and absence of demyelination in the lfb-staining. group 4 ("subacute") included lesions with demyelination but with no inflammatory infiltrates in the he-staining. group 5 ("chronic") contained demyelinated lesions with variable numbers of inflammatory cells in the perivascular area or diffusely distributed ones in the parenchyma. an miame-compliant expression raw data set of a previously published microarray study upon cdv-induced demyelinating leukoencephalitis performed on genechip canine genome 2.0 arrays (affymetrix inc., santa clara, usa) was accessed via the arrayexpress database (accession number: e-mexp-3917; http://www.ebi.ac.uk/arrayexpress) [47] . rna used for the original microarray study was isolated from frozen cerebellar specimens of 12 control dogs and 14 cdv-infected dogs, which were grouped using a classification system similar to the present study (control; acute; subacute; chronic) [47] . fold changes in this data set were calculated as the ratio of the inverse-transformed arithmetic means of the log2-transformed expression values between the respective groups. downregulations are given as negative reciprocal values [47] . in order to perform a bottom-up analysis of transcriptional changes in cdv-induced lesions, 110 candidate genes including 12 pattern recognition receptors (prrs), ten ifn regulatory factors, eight type i/ii ifns, five type i/ii/iii receptors, 17 signal transducers, 46 ifn-dependent antiviral effectors and 12 mhc class i/ii genes were manually extracted from peer-reviewed published literature (table s1) [12, 30, 62, 125, 126] . if necessary, murine and human genes were converted into orthologous canine gene symbols using the madgene web tool (http: //cardioserve.nantes.inserm.fr/madtools/madgene/) [127] . the expression values of these genes were evaluated for significant differences between the respective groups employing independent pairwise mann-whitney u tests (see statistical analysis). for immunohistochemistry, sections were dewaxed, rehydrated and incubated in ethanol with 0.5% hydrogen peroxide for 30 min to block endogenous peroxidase. except for stat1, sections were pretreated by boiling them in 10 mm sodium citrate buffer (ph 6) for 20 min in a microwave oven (800 w). sections were blocked with phosphate-buffered saline (pbs) containing 20% goat serum for 30 min at room temperature and incubated with rabbit polyclonal anti-phosphorylated stat1 (p84/p91, sc-346, santa cruz biotechnology inc., santa cruz, ca, usa, 1:400), anti-oas (sc-98424, santa cruz, 1:600) and anti-isg15 (sc-50366, santa cruz, 1:600), rabbit monoclonal anti-irf3 (ab68481, abcam inc., cambridge, ma, usa, 1:2000), anti-irf7 (ab109255, abcam, 1:8000) and anti-phosphorylated pkr (phospho t446, ab32036, abcam, 1:600) as well as mouse monoclonal anti-cdv (d110; kindly provided by prof. dr. a. zurbriggen, university of bern, bern, switzerland, 1:100), anti-stat2 (sc-1668, santa cruz, 1:100) and anti-mx (m143, kindly provided by prof. dr. haller and pd. dr. kochs, university medical center freiburg, freiburg, germany, 1:1000) antibodies overnight at 4 • c. sections incubated with normal rabbit serum (r4505, sigma-aldrich chemie gmbh, munich, germany) or igg-containing ascites of balb/c mice instead of primary antibodies served as negative controls. biotinylated goat-anti-rabbit igg (ba-1000) or goat-anti-mouse igg (ba-9200), diluted 1:200 (vector laboratories inc., burlingame, ca, usa), were used as secondary antibodies. immunolabeling was visualized by using the avidin-biotin-peroxidase complex method (pk6100, vector laboratories) and the chromogen 3,3 -diaminobenzidine-tetrahydrochloride with 0.05% hydrogen peroxide. finally, sections were slightly counterstained with mayer's hematoxylin. every immunohistochemically stained slide was photographed with a digital microscope (hs all-in-one fluorescence microscope bz-9000 generation ii, biorevo, keyence deutschland gmbh, neu-isenburg, germany) and afterwards analyzed using analysis software (analysis 3.1 software package, soft imaging system gmbh, münster, germany). percentage of immunopositive areas are shown as box-and-whisker plots with median, minimum and maximum in a logarithmic scale. to determine the cellular origin of selected members of the jak-stat signaling pathway (stat1, stat2) and downstream isgs (mx and pkr), immunofluorescence double-labeling was performed using markers for oligodendrocytes (nogoa), astrocytes (gfap) and microglia/macrophages (iba1). moreover, double-staining was used to investigate the infection status of cells expressing stat1, stat2 and mx. immunofluorescence was performed on selected sections of subacute and chronic lesions due to the strong expression of the investigated markers in the microarray analysis and immunohistochemical evaluation. dewaxing, rehydration, pretreatment and blocking of non-specific binding with 20% goat serum or 20% horse serum was performed as described for immunohistochemistry without blocking the endogenous peroxidase. sections were co-incubated with antibodies directed against phosphorylated stat1 (sc-346; 1:100), stat2 (sc-1668; 1:50), mx (m143; 1:100) or phosphorylated pkr (ab32036; 1:50) and antibodies directed against gfap (goat polyclonal, ab53554, abcam, 1:200 or rabbit polyclonal, dakocytomation gmbh, hamburg, germany, 1:1000), nogoa (c-4, mouse monoclonal, sc-271878, santa cruz, 1:200 or rabbit polyclonal, millipore/chemicon, catalog-no. ab5664p, 1:500), iba1 (mouse monoclonal, ab15690, abcam, 1:300 or rabbit polyclonal, thermofisher scientific, schwerte, germany, catalog-no. pa5-27436, 1:500) or cdv (d110, mouse monoclonal, 1:100 or #25, kindly provided by c. örvell, karolinska university hospital, stockholm, sweden, rabbit polyclonal, 1:2000) overnight at 4 • c. negative controls were included in accordance with the immunohistochemical investigation. subsequently, sections were incubated with cy2-and cy3-conjugated secondary antibodies (goat anti-rabbit, 111-165-144; goat anti-mouse, 115-545-003 or 115-165-166; donkey anti-goat, 705-165-147; jackson immunoresearch europe ltd., cambridge, uk; goat anti-rabbit, a-11034; invitrogen/thermo fisher; donkey anti-rabbit, ab150073; abcam; 1:200) for 45 min at room temperature in the dark. nuclei were visualized by 0.01% bisbenzimide (h33258, sigma-aldrich) and sections were mounted with mounting medium (shandon immu-mount, thermofisher scientific, catalog-no. 9990402, usa). sections were photographed with a digital microscope (hs all-in-one fluorescence microscope bz-9000 generation ii, biorevo, keyence deutschland gmbh, neu-isenburg, germany). microarray data were analyzed with non-parametric mann-whitney u tests (prism 6, graphpad software ltd., la jolla, ca, usa), comparing each group of cdv-infected dogs with controls. immunohistochemically data were analyzed using kruskal-wallis tests and dunn's multiple comparison tests comparing groups 2-5 (cdv-infected) with group 1 (control). spearman's rank coefficients were calculated to evaluate the correlation of cdv antigen and isg protein expression. statistical significance was designated as p < 0.05. the ifn-i pathway is activated in the early stages of cdv-induced leukoencephalitis, leading to the expression of various isgs, which restrict viral replication particularly, in neurons and oligodendrocytes. in contrast, the lack of stat2 expression in astrocytes and subsequent suppression of canonical jak-stat signaling might explain their high infection rate in cerebellar lesions. the up-regulation of isgs seems to be partly mediated by irf1-and irf7-dependent pathways counteracting the inhibition of the ifn-i response by cdv. however, the strong activation of the ifn-i signaling cascade, especially in activated microglia/macrophages, probably contributes to immune-mediated mechanisms of demyelination in later stages of the disease. these results encourage further research to elucidate the regulation of ifn-i signaling and its contribution to demyelinating cns diseases in dogs and humans in order to develop novel treatment strategies targeting specific ifn-i pathway members. the authors declare no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. matrix genes of measles virus and canine distemper virus: cloning, nucleotide sequences, and deduced amino acid sequences new aspects of the pathogenesis of canine distemper leukoencephalitis demyelination in canine distemper virus infection: a review pathogenesis and immunopathology of systemic and nervous canine distemper virale infektionen von welpen und junghunden unter berücksichtigung der staupevirusinfektion abundant expression of viral nucleoprotein mrna and restricted translation of the corresponding viral protein in inclusion body polioencephalitis of canine distemper restricted virus protein translation in canine distemper virus inclusion body polioencephalitis demyelination in experimental canine distemper virus infection: immunological, pathologic, and immunohistological studies the pathogenesis of canine distemper virus induced demyelination aspects of canine distemper virus and measles virus encephalomyelitis class ii cytokine receptors and their ligands: key antiviral and inflammatory modulators interferon-stimulated genes and their antiviral effector functions role of early cytokines, including alpha and beta interferons (ifn-alpha/beta), in innate and adaptive immune responses to viral infections gerhauser, i. type i interferons in the pathogenesis and treatment of canine diseases interferon-γ: gene and protein structure, transcription regulation, and actions interferon-lambda: a new addition to an old family functional characterization of canine interferon-lambda antiviral and antiproliferative effects of canine interferon-lambda1 ifn-lambdas mediate antiviral protection through a distinct class ii cytokine receptor complex integrative genomic analyses on interferon-lambdas and their roles in cancer prediction independent comparison of interferon trial study, g. every-other-day interferon beta-1b versus once-weekly interferon beta-1a for multiple sclerosis: results of a 2-year prospective randomised multicentre study (incomin) pathology in practice. canine distemper virus disease in a dog pattern recognition receptors and inflammation pattern recognition receptors and the innate immune response to viral infection innate immune recognition: mechanisms and pathways innate immunity: the virtues of a nonclonal system of recognition mechanisms and pathways of innate immune activation and regulation in health and cancer toll-like receptor signalling rig-i like receptors and their signaling crosstalk in the regulation of antiviral immunity interferon-inducible antiviral effectors intrinsic cellular defenses against virus infection by antiviral type i interferon the jak-stat pathway at twenty the molecular details of cytokine signaling via the jak/stat pathway the interferon receptors interplay between janus kinase/signal transducer and activator of transcription signaling activated by type i interferons and viral antagonism interferon-induced nuclear factors that bind a shared promoter element correlate with positive and negative transcriptional control transcriptional responses to polypeptide ligands: the jak-stat pathway identification of new type i interferon-stimulated genes and investigation of their involvement in ifn-beta activation interferon regulated gene (irg) expression-signature in a mouse model of chikungunya virus neurovirulence canonical and non-canonical aspects of jak-stat signaling: lessons from interferons for cytokine responses stat2 mediates innate immunity to dengue virus in the absence of stat1 via the type i interferon receptor in vitro and in vivo properties of the virus causing natural canine distemper encephalitis interferon in cerebrospinal fluid. a marker for viral persistence of canine distemper encephalomyelitis immunohistochemical evaluation of mx protein expression in canine encephalitides morbillivirus control of the interferon response: relevance of stat2 and mda5 but not stat1 for canine distemper virus virulence in ferrets high level activity of 2', 5'-oligoadenylate synthetase in dog serum transcriptional changes in canine distemper virus-induced demyelinating leukoencephalitis favor a biphasic mode of demyelination fluorescence-activated cell sorting-based analysis reveals an asymmetric induction of interferon-stimulated genes in response to seasonal influenza a virus myxovirus resistance protein a inhibits hepatitis c virus replication through jak-stat pathway activation differential expression of genes related to innate immune responses in ex vivo spinal cord and cerebellar slice cultures infected with west nile virus interferon-stimulated gene 15 (isg15) conjugates proteins in dermatomyositis muscle with perifascicular atrophy hepatitis c virus induces interferon-lambda and interferon-stimulated genes in primary liver cultures type i interferon [corrected] gene induction by the interferon regulatory factor family of transcription factors interferon regulatory factor 3 in adaptive immune responses irf family of transcription factors as regulators of host defense the irf family transcription factors at the interface of innate and adaptive immune responses oasl1 inhibits translation of the type i interferon-regulating transcription factor irf7 negative regulation of type i ifn expression by oasl1 permits chronic viral infection and cd8(+) t-cell exhaustion master regulators of signalling by toll-like receptors and cytosolic pattern-recognition receptors structural insights into interferon regulatory factor activation differential viral induction of distinct interferon-alpha genes by positive feedback through interferon regulatory factor-7 interferon-stimulated genes-essential antiviral effectors implicated in resistance to theiler's virus-induced demyelinating disease constitutive expression of an isgf2/irf1 transgene leads to interferon-independent activation of interferon-inducible genes and resistance to virus infection irf1 up-regulates isg15 gene expression in dsrna stimulation or csfv infection by targeting nucleotides-487 to -325 in the 5' flanking region the canine mhc class ia allele dla-88*508:01 presents diverse self-and canine distemper virus-origin peptides of varying length that have a conserved binding motif polymorphism analysis of four canine mhc class i genes transcription factor redundancy ensures induction of the antiviral state viral evasion strategies in type i ifn signaling-a summary of recent developments isgylation-a key to lock the cell gates for preventing the spread of threats beyond isglylation: functions of free intracellular and extracellular isg15 isg15: in sickness and in health negative feedback regulation of rig-i-mediated antiviral signaling by interferon-induced isg15 conjugation positive regulation of interferon regulatory factor 3 activation by herc5 via isg15 modification isg15 deficiency and increased viral resistance in humans but not mice mycobacterial disease and impaired ifn-gamma immunity in humans with inherited isg15 deficiency a human 15-kda ifn-induced protein induces the secretion of ifn-gamma identification and function analysis of canine stimulator of interferon gene (sting) increase of pro-inflammatory cytokine expression in non-demyelinating early cerebral lesions in nervous canine distemper dominating interleukin-10 mrna expression induction in cerebrospinal fluid cells of dogs with natural canine distemper virus induced demyelinating and non-demyelinating cns lesions increased expression of pro-inflammatory cytokines and lack of up-regulation of anti-inflammatory cytokines in early distemper cns lesions protein kinase pkr and rna adenosine deaminase adar1: new roles for old players as modulators of the interferon response the impact of pkr activation: from neurodegeneration to cancer activation of double-stranded rna-activated protein kinase (pkr) by interferon-stimulated gene 15 (isg15) modification down-regulates protein translation the double-stranded rna-dependent protein kinase pkr: structure and function a double-stranded rna-activated protein kinase-dependent pathway mediating stress-induced apoptosis essential role for the dsrna-dependent protein kinase pkr in innate immunity to viral infection immunohistochemical localization of phosphorylated protein kinase r and phosphorylated eukaryotic initiation factor-2 alpha in the central nervous system of sjl mice with experimental allergic encephalomyelitis on the discovery of interferon-inducible, double-stranded rna activated enzymes: the 2'-5'oligoadenylate synthetases and the protein kinase pkr. cytokine growth factor rev continuous production of interferon in normal mice: effect of anti-interferon globulin, sex, age, strain and environment on the levels of 2-5a synthetase and p67k kinase expression of interferon-induced genes in different tissues of mice fascination with 2-5a-dependent rnase: a unique enzyme that functions in interferon action activation of p38 mitogen-activated protein kinase and c-jun nh(2)-terminal kinase by double-stranded rna and encephalomyocarditis virus: involvement of rnase l, protein kinase r, and alternative pathways inhibition of the oas/rnase l pathway by viruses small self-rna generated by rnase l amplifies antiviral innate immunity the 2-5a system in viral infection and apoptosis extracellular 2'-5' oligoadenylate synthetase stimulates rnase l-independent antiviral activity: a novel mechanism of virus-induced innate immunity evasion of antiviral innate immunity by theiler's virus l* protein through direct inhibition of rnase l cell-type-specific activation of the oligoadenylate synthetase-rnase l pathway by a murine coronavirus a full-length murine 2-5a synthetase cdna transfected in nih-3t3 cells impairs emcv but not vsv replication constitutive expression of (2'-5') oligo a synthetase confers resistance to picornavirus infection constitutive expression of a 2',5'-oligoadenylate synthetase cdna results in increased antiviral activity and growth suppression interferon-induced mx proteins in antiviral host defense restricted expression of viral surface proteins in canine distemper encephalitis astrocytic infection in canine distemper virus-induced demyelination oligodendroglial pathology in canine distemper axonal pathology and loss precede demyelination and accompany chronic lesions in a spontaneously occurring animal model of multiple sclerosis the neurobiology of canine distemper virus infection up-regulation of mrna for matrix metalloproteinases-9 and -14 in advanced lesions of demyelinating canine distemper leukoencephalitis phase-dependent expression of matrix metalloproteinases and their inhibitors in demyelinating canine distemper encephalitis repertoire of microglial and macrophage responses after spinal cord injury interleukin-1beta, -6, -12 and tumor necrosis factor-alpha expression in brains of dogs with canine distemper virus infection early t cell response in the central nervous system in canine distemper virus infection identification of cd4+ and cd8+ t cell subsets and b cells in the brain of dogs with spontaneous acute, subacute-, and chronic-demyelinating distemper encephalitis irf1 is a transcriptional regulator of zbp1 promoting nlrp3 inflammasome activation and cell death during influenza virus infection microglial interferon signaling and white matter interferons inhibit tumor necrosis factor-alpha-mediated matrix metalloproteinase-9 activation via interferon regulatory factor-1 binding competition with nf-kappa b regulation of immunity and oncogenesis by the irf transcription factor family targeting interferon regulatory factors to inhibit activation of the type i ifn response: implications for treatment of autoimmune disorders synoviocyte innate immune responses: ii. pivotal role of ifn regulatory factor 3 poly(i:c) induces expressions of mmp-1, -2, and -3 through various signaling pathways including irf3 in human skin fibroblasts therapeutic approaches to type i interferonopathies the type i interferonopathies type i interferon-mediated monogenic autoinflammation: the type i interferonopathies, a conceptual overview up-regulation of major histocompatibility complex class ii antigen expression in the central nervous system of dogs with spontaneous canine distemper virus encephalitis the yin and yang of viruses and interferons interferon-stimulated genes: roles in viral pathogenesis madgene: retrieval and processing of gene identifier lists for the analysis of heterogeneous microarray datasets the authors wish to thank bettina buck, caroline schütz, kerstin schöne and petra grünig for their excellent technical assistance. furthermore, we would like to thank a. zurbriggen (university of bern, switzerland) and c. örvell (karolinska university hospital, stockholm, sweden) for providing the antibodies against cdv and o. haller and g. kochs (university medical center freiburg, germany) for providing the antibody against mx. key: cord-002248-92pzqj35 authors: terasaki, kaori; ramirez, sydney i.; makino, shinji title: mechanistic insight into the host transcription inhibition function of rift valley fever virus nss and its importance in virulence date: 2016-10-06 journal: plos negl trop dis doi: 10.1371/journal.pntd.0005047 sha: doc_id: 2248 cord_uid: 92pzqj35 rift valley fever virus (rvfv), a member of the genus phlebovirus within the family bunyaviridae, causes periodic outbreaks in livestocks and humans in countries of the african continent and middle east. rvfv nss protein, a nonstructural protein, is a major virulence factor that exhibits several important biological properties. these include suppression of general transcription, inhibition of ifn-β promoter induction and degradation of double-stranded rna-dependent protein kinase r. although each of these biological functions of nss are considered important for countering the antiviral response in the host, the individual contributions of these functions towards rvfv virulence remains unclear. to examine this, we generated two rvfv mp-12 strain-derived mutant viruses. each carried mutations in nss that specifically targeted its general transcription inhibition function without affecting its ability to degrade pkr and inhibit ifn-β promoter induction, through its interaction with sin3-associated protein 30, a part of the repressor complex at the ifn-β promoter. using these mutant viruses, we have dissected the transcription inhibition function of nss and examined its importance in rvfv virulence. both nss mutant viruses exhibited a differentially impaired ability to inhibit host transcription when compared with mp-12. it has been reported that nss suppresses general transcription by interfering with the formation of the transcription factor iih complex, through the degradation of the p62 subunit and sequestration of the p44 subunit. our study results lead us to suggest that the ability of nss to induce p62 degradation is the major contributor to its general transcription inhibition property, whereas its interaction with p44 may not play a significant role in this function. importantly, rvfv mp-12-nss mutant viruses with an impaired general transcription inhibition function showed a reduced cytotoxicity in cell culture and attenuated virulence in young mice, compared with its parental virus mp-12, highlighting the contribution of nss-mediated general transcription inhibition towards rvfv virulence. rift valley fever virus (rvfv), a member of the genus phlebovirus within the family bunyaviridae, causes periodic outbreaks in livestocks and humans in countries of the african continent and middle east. rvfv nss protein, a nonstructural protein, is a major virulence factor that exhibits several important biological properties. these include suppression of general transcription, inhibition of ifn-β promoter induction and degradation of doublestranded rna-dependent protein kinase r. although each of these biological functions of nss are considered important for countering the antiviral response in the host, the individual contributions of these functions towards rvfv virulence remains unclear. to examine this, we generated two rvfv mp-12 strain-derived mutant viruses. each carried mutations in nss that specifically targeted its general transcription inhibition function without affecting its ability to degrade pkr and inhibit ifn-β promoter induction, through its interaction with sin3-associated protein 30, a part of the repressor complex at the ifn-β promoter. using these mutant viruses, we have dissected the transcription inhibition function of nss and examined its importance in rvfv virulence. both nss mutant viruses exhibited a differentially impaired ability to inhibit host transcription when compared with mp-12. it has been reported that nss suppresses general transcription by interfering with the formation of the transcription factor iih complex, through the degradation of the p62 subunit and sequestration of the p44 subunit. our study results lead us to suggest that the ability of nss to induce p62 degradation is the major contributor to its general transcription inhibition property, whereas its interaction with p44 may not play a significant role in this function. importantly, rvfv mp-12-nss mutant viruses with an impaired general transcription inhibition function showed a reduced cytotoxicity in cell culture and attenuated virulence in young mice, rift valley fever virus (rvfv) is the pathogen causing rift valley fever, which affects both humans and domestic ruminants, primarily in countries of the african continent and middle east. the virus is an arbovirus and circulates between mosquito vectors and ruminants in endemic areas. rvfv causes high mortality rates in young ruminants and a high rate of abortions in pregnant ruminants [1] . humans are infected with the virus either by mosquito bite or by direct contact with materials of infected animals. the majority of patients show influenzalike symptoms but few develop hemorrhagic fever, neurological symptoms, and ocular disease [2] . due to its major impact on public health, rvfv is classified as a category a priority pathogen by the national institute of allergy and infectious diseases. currently there is no approved vaccine available for humans and animals in non-endemic areas. rvfv belongs to the family bunyaviridae, genus phlebovirus. rvfv is an enveloped virus and carries 3 segmented rna genomes, the l, m and s segments, which are of negative or ambisense polarity. the l segment encodes l protein, a viral rna-dependent rna polymerase. m rna encodes 78kda protein, nsm protein, gn protein and gc protein, the latter two of which are major envelope glycoproteins and generated by co-translational cleavage of precursor gn/gc polyprotein. 78kda protein is dispensable for virus replication [3] , whereas it plays important roles in virus dissemination in mosquitoes [4, 5] . nsm is a viral anti-apoptotic protein [6, 7] and also is important for efficient virus replication in macrophage cell lines [5] . s rna expresses a nucleocapsid (n) protein and a nonstructural protein nss by using an ambisense coding strategy. the n protein encapsidates the viral rna and forms a ribonucleocapsid complex with l protein [8] . rvfv nss protein is a phosphoprotein with an apparent molecular weight of 31 kda and is localized in both the cytoplasm and nucleus [9] . in the nucleus, nss forms filament-like structures by self-dimerization through its c-terminal domain [10] . rvfv nss protein is a major virus virulence factor and has various important biological functions, which are important for countering the host antiviral response. one of the nss functions is suppression of ifn-β mrna transcription. nss binds to sin3-associated protein 30 (sap30), a subunit of a corepressor complex, and maintains ifn-β promoter in a transcriptionally silent state, leading to suppression of ifn-β mrna transcription [11] . in addition to the specific inhibition of ifn-β transcription, nss suppresses general transcription; it has been proposed that nss exerts suppression of general transcription by interacting with subunits of transcription factor ii h (tfiih) complex, p44 and p62 [12, 13] . a recent study showed that the nss-mediated general transcription inhibition contributes to the inhibition of ifn-β mrna transcription [14] . although nss-mediated transcription suppression has been considered to be important in suppressing the host antiviral response, the exact effects of the nss-mediated general transcription inhibition on virus virulence have not been defined. nss promotes the degradation of doublestranded rna-dependent protein kinase r (pkr), an antiviral ifn-stimulated gene product, through a proteasome pathway to prevent phosphorylation of eif2-α triggered by rvfv infection [15] [16] [17] [18] . furthermore, nss contributes to cellular stress responses such as an increase in reactive oxygen species, activation of dna damage signaling, cell cycle arrest, and activation of the p53 signaling pathway [19] [20] [21] [22] . although how nss induces the cellular stresses remains largely unknown, these stress responses may contribute to rvfv-induced cell death. to elucidate the mechanisms of the different functions of nss, it would be of great value to characterize a series of nss mutants, each of which specifically lacks one of these nss functions but retains other functions. however, introducing any short in-frame deletion in the rvfv nss resulted in loss of all functions [23] , suggesting to us that the nss protein structure is important for its biological activity. hence, it has been challenging to generate nss mutants that lack a specific biological function but retain its other functions. the lack of these nss mutants has prevented a detailed mechanistic analysis of each biological function of the nss. in this study, we generated two rvfv mutants, each carrying mutations in nss, that specifically targeted its general transcription inhibition function, to delineate the mechanism of its inhibition by nss. furthermore, we examined the importance of the nss-mediated inhibition of general transcription on virus-induced cytotoxicity and tested its role in rvfv virulence by using a young mouse model. all mouse studies were performed in facilities accredited by the association for assessment and accreditation of laboratory animal care in accordance with the recommendations in the guide for the care and use of laboratory animals (institute of laboratory animal resources, national research council, national academy of sciences, 1996). the animal protocol (protocol number, 1105023a) was approved by the institutional animal care and use committee of the university of texas medical branch. a standard recombinant pcr method, in which pprot7-s encoding antiviral-sense s rna [24] served as a template, was used to generate pprot7-s carrying mutations in nss coding region. the nss coding region of pprot7-s was amplified by using primers carrying mfe i or a not i site. after digestion with mfe i and not i, the pcr fragment was cloned into eco ri and the not i site of a pcaggs plasmid, resulting in the plasmids expressing nss protein. for expression of human fbxo3 isoform 1 (fbxo3/1) [14] , intracellular rnas of mrc-5 cells were subjected to cdna synthesis, and the fbxo3/1 gene was amplified with primers carrying mfe i or the not i site. after addition of an n-terminal v5 tag, the pcr product was cloned into ecor i and the not i site of pcaggs. for expression of human sap30, pcr product encoding human sap30 with n-terminal v5 tag was cloned into ecor i and the xho i site of pcaggs. all of the constructs were confirmed by sequencing. bsr-t7/5 cells which stably express t7 rna polymerase [25] were maintained in glasgow's minimal essential medium (mem) (lonza) containing 10% fetal bovine serum (fbs), 10% tryptose phosphate broth, mem amino acids solution and geneticin (1 mg/ml). vero e6 cells were maintained in dulbecco's modified mem (gibco) containing 5% fbs. mrc-5 cells were maintained in eagle's mem (emem) (gibco) containing 10% fbs, mem non-essential amino acids solution (gibco), and 1% sodium pyruvate (sigma). hela cells were maintained in emem containing 10% fbs. mp-12 is a highly attenuated rvfv strain obtained after 12 serial passages of an rvfv zh548 strain in the presence of 5-fluorouracil [26] . a recombinant mp-12 strain and other mp-12-derived mutants were rescued from cdnas as described previously [24] , except that bsr-t7/5 cells were used in place of bhk/t7-9 cells. titers of the rescued viruses were determined by using a plaque assay [24] . for the virus carrying r16h/ m250k mutations in nss, passage 0 (p0) virus obtained from plasmid-transfected bsr-t7/5 cells were serially diluted and inoculated into veroe6 cells. the highest titer of p1 virus was selected and used for the study. anti-pkr rabbit polyclonal antibody, anti-flag tag mouse monoclonal antibody, anti-flag tag rabbit monoclonal antibody, and anti-v5 tag rabbit monoclonal antibody were purchased from cell signaling technology. anti-gtf2h1 (p62) mouse monoclonal antibody, anti-gtf2h2 (p44) mouse polyclonal antibody, and anti-xpd mouse monoclonal antibody were purchased from abcam. anti-tfiih p44 (n-17) goat polyclonal antibody, anti-β-actin goat polyclonal antibody and horseradish peroxidase (hrp)-labeled anti-mouse, anti-goat, and anti-rabbit secondary antibodies were purchased from santa cruz biotechnology. anti-gst-n rabbit polyclonal antibody was generated by inoculating a rabbit with gst-n fusion protein (the entire n protein was fused with the c terminus of gst protein) followed by affinity purification of the serum [3] . anti-mp-12 mouse serum was provided by dr. robert b. tesh at the university of texas medical branch. the monoclonal antibody h2k k d k (h2k), which is against major histocompatibility complex class i antigen, was obtained from dr. paul gottlieb at the university of texas at austin. cells were washed with phosphate-buffered saline (pbs) and suspended in sds polyacrylamide gel electrophoresis (sds-page) sample buffer. samples were boiled for 3-5 min and subjected to sds-page. proteins were electroblotted onto polyvinylidene difluoride membranes (immune blot: bio rad). after blocking with skim milk, the membranes were incubated with the primary antibody for 1 h at room temperature and with the secondary antibody for 1 h at room temperature. the proteins on the membrane were detected by using an ecl western blotting detection reagent (ge healthcare life sciences) or ecl plus western blotting substrate (pierce). vero e6 cells were infected with either mp-12 or its mutants at a multiplicity of infection (m.o. i.) of 3. at 16 h post infection (p.i.), the culture media was replaced with methionine/cysteinefree medium. after starvation for 30 min, the infected cells were labeled with 100 μci/ml of 35 s-methionine/cysteine (1,000 ci/mmol; mp biomedicals) for 1 h. the radiolabeled cells were suspended in 2x sds-page sample buffer, resolved by sds-page and visualized by coomassie blue staining or autoradiography. the click-it rna alexa fluor 488 imaging kit was purchased from thermo fisher scientific. vero e6 cells were infected with recombinant viruses at an m.o.i. of 3 and incubated with 1 mm of 5-ethynyl uridine (5eu) for 1 h at 16 h p.i. cellular rna was stained with alexa fluor, 488-coupled azide for 30 min by following the manufacturer's protocol. for a negative control, mock-infected cells were treated with 5 μg/ml of actinomycin d (actd) for 30 min prior to 5eu treatment and for 1 h during 5eu treatment. after the click reaction, rvfv n protein was stained with anti-gst-n rabbit polyclonal antibody followed by alexa fluor 594 conjugated anti-rabbit antibody. images were captured on a zeiss axiophot 2 fluorescence microscope with a 40x magnification lens and processed with the imagej software [27] . for flow cytometry analysis, the infected cells were detached from the dish by accumax (innovative cell technologies) after the 5eu treatment and suspended in culture media. after washing with pbs containing 1% bovine serum albumin (bsa), cells were fixed with 2% formaldehyde/pbs for 30 min at room temperature and blocked with blocking buffer (pbs containing 0.2% saponin and 1% bsa) for 15 min on ice. cellular rna was stained by alexa fluor 488-coupled azide for 30 min in the presence of 0.5% saponin and 1% bsa. after washing with the blocking buffer, rvfv n protein was stained by anti-gst-n rabbit polyclonal antibody for 30 min on ice followed by alexa fluor 594 conjugated anti-rabbit antibody. the cells were washed with pbs containing 1% bsa, passed through a cell strainer (bd falcon), and analyzed on an lsrii fortessa (bd biosciences). single cells were gated based on their forward scatter and side scatter profile. more than 30,000 count of the gated single cells were analyzed for each experiment. confluent vero e6 cells grown in 96-well plates were either mock infected or with mutant virus at an m.o.i. of 3. cell viability was determined by using viral toxglo (promega), which measures cellular atp. at various times p.i., atp detection reagent was added to each well. after incubation for 10 min, luminescence was measured by spectramax m5e (molecular devices). total rnas were extracted by using trizol reagent (invitrogen) and subjected to northern blot analysis as described previously [28] . strand-specific digoxigenin-labeled rna probes and a digoxigenin (dig) system (roche) were used to detect rna. the 564-nucleotide-long, 293 cell-derived, and dig-labeled ifn-β riboprobe [29] was used for ifn-β mrna detection. cells cultured on chamber slides were fixed in 4% paraformaldehyde for 15 min. the fixed cells were permeabilized with 0.2% tritonx-100 for 15 min and blocked with 1% bsa in pbs for 30 min. after the blocking, the cells were stained with primary antibody diluted with the blocking solution, followed by incubation with alexa fluor 488 or 594-conjugated secondary antibodies (molecular probes). images were captured by a zeiss axiophot 2 fluorescence microscopy and processed with imagej software [27] . to detect the interaction between nss and p44, we infected hela cells with mp-12-nss-flag, which expresses nss with the c-terminal flag tag or its mutant viruses at an m.o.i. of 3. at 8 h p.i., the cells were lysed in lysis buffer [50 mm tris-hcl, ph 7.6, 0.1% np-40, 150 mm nacl, 1 mm edta, protease inhibitors (sigma), and 100 u/ml benzonase (sigma)]. after 3 cycles of freeze-thaw, the cell lysate was cleared by centrifugation at 4°c and 100,000 x g for 1 h and incubated with dynabeads protein g (life technologies) conjugated with anti-gtf2h2 (p44) mouse monoclonal antibody or h2k antibody according to the manufacturer's protocol. precipitates were washed with the lysis buffer with 0.1% tween 20 and analyzed by western blotting using the anti-p44 goat polyclonal, and anti-p44 mouse monoclonal and anti-flag mouse monoclonal antibodies. to detect the interaction between nss and sap30, we transfected hela cells with pcaggs-v5-sap30 which encodes human sap30 carrying an n-terminus v5 tag by using fugene hd transfection reagent (promega). at 16 h post transfection, the cells were infected with mp-12-nss-flag or its mutant viruses. the cells were lysed with lysis buffer (50 mm tris-hcl, ph 7.5, 100 mm nacl, 1% np-40, 1 mm edta, and protease inhibitor cocktail) [11] at 8 h p.i. the cell lysate was cleared by centrifuge at 21,000 x g and 4°c for 15 min and incubated with anti-v5-tag mab-magnetic beads (mbl international) according to the manufacturer's protocol. precipitates were washed with the lysis buffer containing 450 mm nacl and analyzed by western blotting by using anti-v5 tag rabbit monoclonal and anti-flag mouse monoclonal antibodies. for detection of interaction between nss and p62, hela cells were infected with mp-12-nss-flag or its mutant viruses at an m.o.i. of 3. at 5 h p.i., the cells were lysed in lysis buffer [20 mm tris-hcl, ph 7.6, 0.1% tritonx-100, 150 mm nacl, 1 mm edta, protease inhibitors (sigma), and 100 u/ml benzonase (sigma)] [13] . after 3 cycles of freeze-thaw, the cell lysate was cleared by centrifugation at 4°c and 100,000 x g for 1 h and incubated with dynabeads protein g conjugated with anti-gtf2h1 (p62) mouse monoclonal antibody according to the manufacturer's protocol. precipitates were analyzed by western blotting using the anti-p62 mouse monoclonal and anti-flag mouse monoclonal antibodies. to detect the interaction between nss and fbxo, we transfected hela cells with pcaggs-v5-fbxo3/1, which encodes fbxo3/1 carrying an n-terminus v5 tag, by using fugene hd transfection reagent. at 16 h post transfection, the cells were infected with mp-12-nss-flag or its mutant viruses and lysed with lysis buffer (50 mm tris-hcl, ph 7.5, 0.2% np-40, 5% glycerol, 100 mm nacl, 1.5 mm mgcl 2 , and protease inhibitor cocktail) [14] at 5 h p.i. the cell lysate was cleared by centrifugation at 21,000 x g and 4°c for 15 min and incubated with anti-v5-tag mab-magnetic beads (mbl international) according to the manufacturer's protocol. precipitates were washed with the lysis buffer and analyzed by western blotting by using the anti-v5 tag rabbit monoclonal and anti-flag mouse monoclonal antibodies. hela cells prepared in 12-well plates were transfected with 0.25 μg of the pcaggs-based plasmid encoding either nss or mutant nss or an empty vector along with 0.1 μg of prl-sv40 (promega) which expresses renilla luciferase under control of an sv40 promotor. renilla luciferase activities in the transfected cells were measured by using a renilla luciferase assay system (promega) at 24h post transfection. eighteen-day-old cd-1 mice were intraperitoneally inoculated with 10 4 pfu of mp-12, mp-12-m250k or mp-12-r16h/m250k or with hank's balanced salt solution (hbss). the animals were observed for survival for 22 days post inoculation. we previously generated and characterized an rvfv mp-12 strain-derived mutant virus, which is deficient for efficient virus genome co-packaging due to a large deletion in the 5' untranslated region of m rna segment [30] . to obtain virus variants, carrying mutations that can compensate for this deficiency, we serially passaged this mutant virus in type i ifn-incompetent vero e6 cells; the virus inoculum, used for each passage, was diluted 10 times and the released viruses were harvested at 4 days p.i. the titer of the mutant virus progressively increased with each passage; at passage 18, the virus titer was 2.9 x 10 6 pfu/ml, which was 2 logs higher than the initial titer of 2.0 x 10 4 pfu/ml prior to the serial passage. we isolated 34 plaque-cloned viruses from passage level 18, and 28 had a large deletion in the nss gene in the s segment, whereas 6 plaque-cloned viruses retained the full-length nss gene. we determined the full genome sequence of 5 clones that retained the full-length nss gene and found that all of the isolated viruses had several mutations in the l, m, and s segments. table 1 shows mutations found in the nss genes of 5 plaque-cloned viruses and the uncloned passage 18 virus. all five plaque-cloned viruses and the passage 18 virus had an amino-acid substitution at position 250, including m250k or m250t mutations. two plaque-cloned viruses and the passage 18 virus had a d100g substitution. other mutations found in plaque-cloned viruses were r16h and a75e. although k202n mutation was found in the uncloned passage 18 virus sample, this mutation was absent in the plaque-cloned viruses. to test whether the mutations affect nss functions, we generated mp-12-based mutant viruses, each carrying r16h, r16h+m250k (nss-r16h/m250k), a75e, d100g, d100g+m250t, m250k (nss-m250k), or m250t mutations in the nss, by using a reverse genetics system [24] . accumulations of nss protein in veroe6 cells infected with mp-12 carrying the d100g mutation or the d100g+m250t mutations were significantly lower than those in mp-12 infected cells (fig 1) , indicating that the d100g mutation affected efficient nss accumulation. due to the poor accumulation of nss, we excluded the mp-12 mutant carrying the d100g mutation and that carrying the d100g+m250t mutation from subsequent analysis. in cells infected with the other mutants, levels of nss protein accumulation were similar to that in mp-12 infected cells. in this study, we refer to mp-12 nss as wild type nss (wt nss). to evaluate host transcriptional shut-off activities of these nss mutants, levels of rna synthesis in virus-infected veroe6 cells were measured by labeling with 5eu from 16 h to 17 h p.i. mock-infected cells alone, as well as those treated with actd, and mp-12-infected cells and cells infected with mp-12δnss, which lacks the nss gene [24] , served as controls. 5eu-labeled rna, which was detected as a fluorescence signal, was mainly observed in the nucleus but not in the cytoplasm, indicating that viral rna synthesis, which occurs exclusively in the cytoplasm, was not very active at 16h post infection. as shown in fig 2a, strong signals of fluorescent-labeled rna were observed in the nucleus of mock-infected cells and mp-12δnssinfected cells, demonstrating active host rna synthesis. in contrast, actd-treated cells showed very weak fluorescent signals, demonstrating actd-mediated host transcriptional shut-off. mp-12-infected cells showed weaker fluorescent signals in the nucleus, compared with those in mp-12δnss-infected cells, demonstrating an inhibition of host transcription by nss. fluorescent intensity observed in cells infected with virus carrying mutation m250t, r16h or a75e in nss was similar to that in mp-12-infected cells, suggesting that host transcription was still inhibited by these nss mutants. in contrast, cells infected with mp-12 carrying nss-r16h/ we next used flow cytometry analysis to quantitatively measure the levels of 5eu-labeled rnas in mp-12-m250k-infected cells and in mp-12-r16h/m250k-infected cells. we used the same controls as described above. fig 2b shows the results using density plots, wherein 5eu incorporation and n protein levels are shown on the x and y axes, respectively. in mockinfected and actd-treated samples, most of the cells were found in quadrant 3 (q3) and q4, respectively, demonstrating active host transcription in the former, but not in the latter ( fig 2b-a and 2b-b) . in all infected samples, 86.8-93.4% of cells were rvfv n protein positive (q1+q2), demonstrating that most of the cells were infected. the level of 5eu-labeled rna in mp-12δnss-infected cells was similar to that in mock-infected cells, demonstrating similar rna synthesis activity in these two samples (fig 2b-a and 2b-f ). mp-12-infected cells showed lower levels of 5eu-labeled rna compared with those in mp-12δnss infected cells, demonstrating its lower rna transcription activity (fig 2b-c and 2b-f ). mp-12-r16h/m250kinfected and mp-12δnss-infected cells showed similar density plot patterns, suggesting to us that host transcription inhibition did not occur in mp-12-r16h/m250k-infected cells (fig 2be and 2b-f). mp-12-m250k-infected cells showed lower levels of 5eu-labeled rna compared with those in mp-12δnss-infected cells (fig 2b-d and 2b-f) . however, the percentage of mp-12-m250k-infected cells with low transcription activity was 43.0% (q1/q1+q2), while in the mp-12 samples, it was 56.6%. these results suggested that mp-12-m250k replication suppressed host general transcription, and yet the inhibitory activity of mp-12-m250k was weaker than that of mp-12. we next examined the effect of differences in the transcription inhibitory activities of the mutant viruses on global protein synthesis by incorporation of 35 s-methionine/cysteine into newly synthesizing proteins in mock-infected cells and in cells infected with mp-12, mp-12δnss, mp-12-m250k, or mp-12-r16h/m250k (fig 2c) . consistent with our previous study [24] , mp-12 replication, but not mp-12δnss replication, suppressed host protein synthesis. mp-12-r16h/m250k replication did not inhibit host protein synthesis, while mp-12-m250k replication moderately inhibited host protein synthesis. taken together, these analyses showed that nss-r16h/m250k lost the general transcription suppression activity, leading to efficient host protein synthesis in infected cells, while nss-m250k exerted inefficient general transcription suppression activity, causing moderate levels of host protein synthesis inhibition in infected cells. analysis of replication kinetics of mp-12, mp-12δnss, mp-12-r16h/m250k and mp-12-m250k showed that all viruses replicated efficiently with similar replication kinetics in vero e6 cells (fig 3a) . mp-12 formed clear plaques in vero e6 cells, whereas mp-12-r16h/m250k and mp-12-m250k formed turbid-type plaques like mp-12δnss (fig 3b) , indicating that the r16h/ m250k mutation and the m250k mutation in nss affected virus-induced cytotoxicity. cell viability assays showed that mp-12 replication caused a 90% reduction in cell viability as compared with mock-infected cells at 72 h p.i. (fig 3c) , whereas mp-12δnss-infected cells and mock-infected cells showed similar cell viabilities throughout the course of the experiments. mp-12-r16h/m250k-infected cells and mp-12-m250k-infected cells showed an 11% and 39% decrease in cell viability, respectively, as compared with that in mock-infected cells at 72 h p.i., demonstrating that both of the mutant viruses were less cytotoxic than mp-12. mp-12, mp-12-m250k and mp-12-r16h/m250k had similar replication kinetics, regardless of their differential ability to induce cytotoxicity (fig 3) , implying that virus-induced cytotoxicity did not have a significant impact on rvfv replication kinetics in vero e6 cells. because others reported that mp-12 replication induces nss-dependent p53 stabilization, which contributes to virus-induced cell death [21] , we next examined stabilization of p53 in cells infected with mp-12 and other mutant viruses (fig 3d) . amounts of p53 protein significantly increased in mp-12-infected cells, but not in mp-12δnss-infected cells, confirming the nss-dependent p53 stabilization in the infected cells. accumulation of p53 also occurred in mp-12-m250k-infected cells, whereas the accumulation levels were lower than those in nss interacts with pkr and triggers degradation of the pkr by a proteasome pathway [15] [16] [17] [18] . to investigate whether the nss mutants retain the ability for pkr degradation, we compared the total amount of pkr in virus-infected cells. similar levels of reduction in the amounts of pkr occurred in cells infected with mp-12, mp-12-m250k or mp-12-r16h/250k, suggesting that nss-r16h/m250k and nss-m250k promoted pkr degradation as efficiently as wt nss (fig 4) . le may et al. proposed that interaction of nss with sap30 interferes with the recruitment of co-activator protein cbp at activation sites on the ifn-β promotor, leading to the inhibition of ifn-β mrna transcription [11] . to investigate the ability of mp-12-r16h/m250k or mp-12-m250k to suppress ifn-β mrna transcription, we examined the levels of ifn-β mrna in mrc-5 cells infected with mp-12, mp-12-δnss, mp-12-m250k or mp-12-r16h/250k at various times p.i. (fig 5a) . mp-12δnss replication induced robust ifn-β mrna transcription, whereas ifn-β mrna was not detectable in mp-12-or mp-12-m250k-infected cells. in contrast, low levels of ifn-β mrna accumulation occurred in mp-12-r16h/m250k-infected cells, demonstrating that the nss-r16h/m250k was unable to efficiently inhibit ifn-β mrna transcription. mp-12-m250k replicated as efficiently as did mp-12 in mrc-5 cells, whereas the virus titer of mp-12δnss was significantly lower than that of mp-12 at 72 h p.i. (fig 5b) , demonstrating the efficient inhibition of ifn-β production in the former, but not in the latter. although mp-12-r16h/m250k replicated better than mp-12δnss, it replicated less efficiently mp-12-r16h/m250k-flag). cells, transiently expressing a v5-tagged sap30, were infected with these viruses, and the co-localization of the mutated nss protein with sap30 was examined by fluorescence microscopy analysis. like wt nss, both nss mutants formed filament-like structures in the nucleus (fig 5c) . the expressed sap30 was mainly observed in the nucleus and was co-localized with wt nss and both mutated nss in the filaments. we also performed co-immunoprecipitation analysis to examine nss-sap30 interaction. as a negative control, v5-tagged venus was expressed in place of v5-tagged sap30. anti-v5 antibody co-precipitated wt nss and both nss mutants along with v5-tagged sap30, while the amounts of nss-m250k that were co-immunoprecipitated with sap30 were lower than those of wt nss ( fig 5d) . anti-v5 antibody did not co-precipitate wt nss along with v5-tagged venus (fig 5d) . these data demonstrate that both nss mutants bound to sap30 in infected cells. taken together, these data indicated that nss-r16h/m250k was unable to completely block ifn-β transcription despite its ability to bind to sap30. both nss mutants retained some nss functions, including degradation of pkr and sap30 binding, and yet these nss mutants and wt nss showed different levels of general transcriptional suppression activities in virus-infected cells. transcriptional suppression activities of the mutated nss were also examined in cells transiently expressing nss along with renilla luciferase from co-transfected plasmids. luciferase activity was strongly inhibited in the cells coexpressing wt nss and renilla luciferase (fig 6a) . consistent with the data obtained from infected cells (fig 2) , nss-r16h/m250k expression did not inhibit luciferase activity, whereas nss-m250k expression moderately inhibited the luciferase activity. western blot analysis of nuclear and cytoplasmic fractions from virus-infected cells showed that like wt nss, nss-r16h/m250 and nss-m250k were also distributed in both the nucleus and the cytoplasm (s1 fig) , demonstrating that these mutations did not affect the subcellular localization of nss. these data prompted us to further examine the interplay between these nss mutants and a form of host transcription machinery, tfiih, as other studies have shown an association of tfiih with nss in the nss-induced host transcription shut-off. rvfv nss binds to p44, a subunit of tfiih, and interferes with the formation of the tfiih complex by inhibiting the subsequent interaction of p44 and xpd [12] . the reduction in the abundance of xpd and p44 also occurs upon rvfv infection, although the mechanisms that govern the reduction of these proteins are unclear [12] . in addition, nss binds to both p62, a tfiih subunit, and fbxo3, a component of e3 ubiquitin ligase, which leads to p62 degradation [13, 14] . we first examined the abundance of tfiih components, including p44, p62 and xpd, in infected cells. substantial reduction of p62 abundance and moderate reductions in the abundances of p44 and xpd occurred in cells infected with mp-12, or mp-12-m250k at 16 h p.i. (fig 6b, left panels) . in contrast, there was no substantial reduction in the abundance of these tfiih components in cells infected with mp-12δns or mp-12-r16h/m250k. the same results were obtained when viruses carrying flag-tagged nss were used. to exclude the possibility that different transcription suppression activities of these viruses affected the results, we repeated the experiments in the presence of actd and obtained similar results (fig 6b, right panels) . next, we tested the interaction of mutated nss with p44 and p62. cells infected with mp-12 or its mutants, all of which carried flag-tagged nss, were subjected to co-immunoprecipitation analysis using anti-p44 antibody (fig 6c) . anti-p44 antibody co-immunoprecipitated wt nss and both mutant nss along with p44, whereas control anti-h2k antibody precipitated neither p44 nor nss. these data demonstrated that both nss mutants bound to p44. to examine the interaction of p62 and nss, cells were infected with the viruses as described above, and cell extracts were prepared at 5 h p.i., when p62 was still detectable in mp-12-nss-flag-infected cells (fig 6d) . anti-p62 antibody, but not anti-h2k antibody, co-immunoprecipitated wt nss and both mutant nss along with p62, demonstrating binding of the mutant nss proteins to p62. we noted that the amounts of nss-m250k that were co-immunoprecipitated with p44 and with p62 were lower than those of wt nss, implying that the binding efficiencies of nss-m250k for p44 and for p62 were lower than those for wt nss. fbxo3 is an interactor of rvfv nss that is engaged in the degradation of p62; nss interacts with the full-length fbxo3 protein (fbxo3/1) as well as with a shorter splice variant of the fbxo3 that lacks the c-terminal acidic domain and poly(r) region [14] . because nss-r16h/m250k did not induce p62 degradation regardless of its ability to bind to p62 (fig 6d) , we suspected that nss-r16h/m250k would not interact with fbox3. to test this possibility, cells transiently expressing the v5-tagged fbxo3/1 were mock infected or infected with mp-12-nss-flag, mp-12-m250k-flag, or mp-12-r16/m250k-flag. at 5 h p.i., cell extracts were prepared and subjected to co-immunoprecipitation analysis by using anti-v5 antibody ( fig 6e) . all of the nss proteins, including nss-r16/m250k, were co-immunoprecipitated with fbxo3/1. table 2 summarizes interactions of the nss mutants with p44, p62 and fbxo3. nss is a major virulence factor of rvfv [31] . the nss-mediated inhibition of type i ifn production is thought to contribute to the virulence of the virus, yet it remains unclear whether the general transcription suppression function of nss contributes to this virulence. we examined the importance of the nss-mediated transcription inhibition in rvfv virulence by using a young mouse model. although mp-12 is known as an attenuated strain, the intraperitoneal inoculation of 10 4 pfu of mp-12 into 18-day-old cd1 mice resulted in the death of 55% of the mice within 13 days p.i. (fig 7) . under the same experimental conditions, none of the mp-12-r16h/m250k-inoculated mice and 20% of the mp-12-m250k-inoculated mice died. no obvious clinical signs, including neurological symptoms, were observed. these data demonstrated that the mp-12-r16h/m250k lacked virulence, and mp-12-m250k was less virulent than mp-12 in this young mouse model. in this study, we demonstrated that the m250k and r16h/m250k mutations in nss differentially reduced its inhibitory activity on host transcription without affecting its ability to inhibit ifn-β transcription, through its interaction with sap30, and induced pkr degradation. nss-r16h/m250k, which completely lacked the activity to inhibit general transcription, correspondingly lost the ability to promote the degradation of p62. unexpectedly, nss-r16h/ m250k was able to interact with fbox3 and p62 (fig 6) . these data possibly suggest that the fbxo3-nss-p62 interaction may not be sufficient to trigger p62 degradation. nss-m250k exhibited a partially reduced activity to inhibit general transcription. the results of co-immunoprecipitation assays showed that a reduced amount of nss-m250k co-precipitated with sap30, p62 and p44, compared with that in wt nss (figs 5 and 6 ). however, the slightly impaired ability of nss-m250k to inhibit general transcription could not be solely attributed to the lower binding efficiency of nss-m250k to p44 because nss-m250k efficiently suppressed ifn-β transcription and induced p62 degradation, despite its lower efficiency of binding to sap30 and p62. moreover, nss-r16h/m250k, which lacked transcription suppression activity, efficiently interacted with p44 ( fig 6) . these data bring into question the importance of nss-p44 interaction for its host transcriptional shut-off function. one possibility is that the nss-p44 interaction may only make a modest contribution towards its transcription inhibition activity and possibly could have additional, as yet unidentified, biological function(s). a second possibility is that only wt nss, but not the mutated nss, is able to interfere with the formation of an active tfiih complex. nss competes with xpd for binding to p44, resulting in inhibition of the tfiih complex formation [12] . although both nss-m250k and nss-r16h/m250k retained the ability to bind to p44, it is possible that the binding of these mutated nss to p44 did not exclude the binding of xpd. the r16h single mutation did not affect the ability of nss to inhibit transcription (fig 2) . although the m250k mutation is in close proximity to the ωxav motif located at the c-terminal region of nss, which is essential for p62 degradation [32] , mp-12-m250k still retained the ability to degrade p62 (fig 6) . however, the r16h/m250k double mutant lost the ability to induce p62 degradation. these results imply that the combined mutations, r16h and m250k, induced an unfavorable structural alteration in nss, which abolished its function to degrade p62. mp-12-r16h/m250k replication induced low levels of ifn-β mrna (fig 5) , indicating that nss-r16h/m250k was not able to block ifn-β mrna synthesis as efficiently as wt nss despite its ability to interact with sap30 (fig 5) . it has been reported that nss-induced p62 degradation contributes to the inhibition of ifn-β production [14] . accordingly, our data suggested that mp-12-r16h/m250k was unable to completely block ifn-β mrna synthesis due to a lack of ability to promote the degradation of p62 (fig 6) . although mp-12-r16h/m250k replication induced ifn-β mrna synthesis, the level of the ifn-β mrna was significantly lower than that in mp-12δnss-infected cells (fig 5) , suggesting the importance of interaction of nss with sap30 for ifn-β inhibition. taken together, the data shown here and those of others [11, 14] strongly imply that nss-sap30 interaction and nss-induced general host transcriptional suppression function are both necessary for efficient inhibition of ifn-β mrna transcription. mp-12δnss or mp-12 encoding a reporter gene in place of the nss gene causes less prominent cytopathic effects than does mp-12, demonstrating the contribution of the nss towards the induction of cytotoxicity [24] . mp-12 replication induces nss-dependent p53 stabilization, which contributes to virus-induced cell death [21] , and yet it was unclear which function of the nss contributed to the induction of the p53-mediated cytotoxicity. as shown in fig 3, mp-12-infected cells showed the lowest cell viability, followed in order by mp-12-m250k, which moderately suppressed host general transcription, and mp-12-r16h/m250k, which did not suppress host transcription. hence, there was a correlation between the strength of nss-mediated, host transcriptional shut-off activity and cell viability in the infected cells (table 3) . likewise, accumulation of p53 was the highest in mp-12-infected cells, followed in order by that in mp-12-m250k-infected cells and mp-12-r16h/m250k-infected cells, suggesting to us that p53 stabilization also correlates with the transcription inhibition activities of the different nss mutants. these results indicate that the nss-mediated host transcriptional shut-off triggered p53 stabilization, leading to p53-mediated cell death. although mp-12 is an attenuated rvfv strain, 55% of 18-day-old cd1 mice died after intraperitoneal inoculation with 10 4 pfu within 13 days p.i. (fig 7) . as the immune system is not yet fully developed in young mice, it likely failed to prevent systemic infection by mp-12. consistent with this notion, intraperitoneal inoculation of mp-12 into severe combined immune deficiency mice also caused 100% mortality [33] . mp-12-r16h/m250k carrying nss that lacks the host transcription inhibition function was completely attenuated in 18-day-old cd1 mice (fig 7) . mp-12-m250k carrying nss with an impaired ability to inhibit transcription also exhibited reduced virulence when compared to that in its parental virus mp-12. these data highlight the role of the host transcription inhibition function of nss in rvfv virulence. type i ifn has been shown to play a critical role in protecting the host from rvfv-induced disease in animal models [34] . notably, both the nss mutants, carrying either the m250k single mutation or the r16h/m250k double mutation, retained the ability to bind to sap30, the factor that is targeted by nss to inhibit ifn-β mrna transcription. however, the induction of ifn-β mrna synthesis was not completely blocked in mp-12-r16h/m250k-infected mrc-5 cells, most probably due to the lack of inhibition of host transcription in these cells expressing the mutated nss. these data suggest the possibility that the inability of mp-12-r16h/m250k to completely block the production of ifn-β in infected mice could have contributed towards its attenuation. in addition, the production of other antiviral and/or proinflammatory cytokines could have also contributed towards the attenuated phenotype of both of these mutant viruses, carrying nss with an impaired ability to block host transcription. it is also possible that the lower levels of virus-induced cytocidal effects might have contributed to the lower virulence of these nss mutant viruses, as both mutant viruses caused less severe cytopathic effects and cytotoxicity than did mp-12 in cultured cells (fig 3) . we believe that experiments using rvfv mutants, carrying the m250k single mutation and the r16h/m250k double mutation, in animal models would yield valuable information about the role of nss-mediated host transcription inhibition in regulating host cytokine responses and its impact on the pathogenesis of rvfv. mp-12 is an attenuated live vaccine candidate, but it still harbors residual virulence in a young mouse model. although further studies are required to test the immunogenicity and protective efficacy of mp-12-m250k and mp-12-r16h/m250k, there is a potential for developing these mp-12-derived nss mutant viruses as safer live attenuated rvfv vaccine candidates. we found that the serial passage of an mp-12-derived mutant virus having a large deletion in the 5' untranslated region of m rna segment [30] in vero e6 cells resulted in accumulation of variant viruses that were able to replicate better than the original mutant virus. most of viruses in passage 18 had a large internal deletion in the nss gene, which may mean their nss proteins are biologically inactive. others have reported that rvfv carrying large deletions in the nss gene start accumulating from the 15 th serial passage in bhk cells of the rvfv p strain, which is defective in ifn-α/β signaling [35] . in our experiment, the large deletions in the nss gene were detected after the 5 th serial passage of the virus. although the cell lines used in the studies were different, the deletion of the nss gene as early as after 5 passages implied that there was a selective pressure to remove the nss gene from the virus genome during the passaging of the mutant virus. the full-length nss gene of the uncloned passage 18 viruses had m250k, m250t, k202n and d100g mutations (table 1 ). in the cells infected with mp-12 carrying nss with a d100g mutation, the accumulation of nss was poor (fig 1) , indicating that the d100g mutation affects the efficient accumulation of nss. as two out of the five plaque-cloned viruses (clones 3 and 4, table 1 ) also had the d100g mutation in nss, this possibly affected its accumulation in infected cells. the remaining three clones carried nss with a m250k or r16h/m250k mutation. our results showed that these mutations in nss partially or completely abolished its host transcription inhibition function without affecting its ability to interact with sap30 to inhibit ifn-β mrna synthesis. these data implied that the host transcription function of nss was unfavorable for the mutant virus, carrying a deletion in the 5' utr, to replicate well in veroe6 cells. mp-12-m250k and mp-12-r16h/m250k replication induced low cytotoxicity due to its lower inhibitory activity on host transcription (figs 2 and 3) . we suspect that the lack of nss-induced host transcription suppression created a favorable cellular environment for the mutant virus, thereby allowing the emergence and accumulation of mutant viruses that lacked this function. further studies are required to delineate the importance of nss-mediated host transcription inhibition in the virus life cycle. with mp-12-nss-flag or its mutants, at an m.o.i. of 2 and at 6 h p.i., the cells were lysed using the lysis buffer (10 mm tris-hcl, ph 7.5, 10 mm kcl, 1.5 mm mgcl 2 , 0.5% triton x-100, protease inhibitor cocktail) followed by incubation on ice for 10 min. after centrifugation at 2,000 x g for 2 min, the resulting supernatant was collected as the cytoplasmic fraction (c). the pellet from this centrifugation was washed once with the lysis buffer (without triton x-100), suspended in 1x sds sample buffer and denoted as the nuclear fraction (n). the subcellular fractions were analyzed by western blotting using anti-flag, anti-hsp90 and anti-lamin a antibodies. (tif) breaking the chain: rift valley fever virus control via livestock vaccination the pathogenesis of rift valley fever nsm and 78-kilodalton proteins of rift valley fever virus are nonessential for viral replication in cell culture deletion of the nsm virulence gene of rift valley fever virus inhibits virus replication in and dissemination from the midgut of aedes aegypti mosquitoes the rift valley fever accessory proteins nsm and p78/nsm-gn are distinct determinants of virus propagation in vertebrate and invertebrate hosts nsm protein of rift valley fever virus suppresses virusinduced apoptosis the c-terminal region of rift valley fever virus nsm protein targets the protein to the mitochondrial outer membrane and exerts antiapoptotic function recent advances in the molecular and cellular biology of bunyaviruses the rift valley fever virus nonstructural protein nss is phosphorylated at serine residues located in casein kinase ii consensus motifs in the carboxy-terminus the carboxy-terminal acidic domain of rift valley fever virus nss protein is essential for the formation of filamentous structures but not for the nuclear localization of the protein a sap30 complex inhibits ifnbeta expression in rift valley fever virus infected cells tfiih transcription factor, a target for the rift valley hemorrhagic fever virus nss protein of rift valley fever virus promotes posttranslational downregulation of the tfiih subunit p62 virulence factor nss of rift valley fever virus recruits the f-box protein fbxo3 to degrade subunit p62 of general transcription factor tfiih rift valley fever virus nss protein promotes post-transcriptional downregulation of protein kinase pkr and inhibits eif2alpha phosphorylation nss protein of rift valley fever virus induces the specific degradation of the double-stranded rna-dependent protein kinase protein kinase r degradation is essential for rift valley fever virus infection and is regulated by skp1-cul1-f-box (scf) fbxw11-nss e3 ligase nss virulence factor of rift valley fever virus engages the f-box proteins fbxw11 and beta-trcp1 to degrade the antiviral protein kinase pkr reactive oxygen species activate nfkappab (p65) and p53 and induce apoptosis in rvfv infected liver cells induction of dna damage signaling upon rift valley fever virus infection results in cell cycle arrest and increased viral replication p53 activation following rift valley fever virus infection contributes to cell death and viral production nonstructural nss protein of rift valley fever virus interacts with pericentromeric dna sequences of the host cell, inducing chromosome cohesion and segregation defects functional analysis of rift valley fever virus nss encoding a partial truncation rescue of infectious rift valley fever virus entirely from cdna, analysis of virus lacking the nss gene, and expression of a foreign gene generation of bovine respiratory syncytial virus (brsv) from cdna: brsv ns2 is not essential for virus replication in tissue culture, and the human rsv leader region acts as a functional brsv genome promoter mutagen-directed attenuation of rift valley fever virus as a method for vaccine development nih image to imagej: 25 years of image analysis rift valley fever virus nonstructural protein nss promotes viral rna replication and transcription in a minigenome system severe acute respiratory syndrome coronavirus nsp1 suppresses host gene expression, including that of type i interferon, in infected cells mechanism of tripartite rna genome packaging in rift valley fever virus characterization of clone 13, a naturally attenuated avirulent isolate of rift valley fever virus, which is altered in the small segment a ωxav motif in the rift valley fever virus nss protein is essential for degrading p62, forming nuclear filaments and virulence recombinant rift valley fever vaccines induce protective levels of antibody in baboons and resistance to lethal challenge in mice interplay between the virus and host in rift valley fever pathogenesis host alternation is necessary to maintain the genome stability of rift valley fever virus we thank robert tesh for anti-mp12 antibody; paul gottlieb for the monoclonal antibody h2k k d k ; mark griffin (flow cytometry and cell sorting core, the university of texas medical branch) for support with the flow cytometry analyses; and krishna narayanan for critical reading of the manuscript. conceptualization: kt sm. key: cord-005953-5z89yeb6 authors: nan title: abstracts des 114. internistenkongresses 2008 date: 2008 journal: med klin (munich) doi: 10.1007/s00063-008-1026-y sha: doc_id: 5953 cord_uid: 5z89yeb6 nan so far, platelets were regarded static cells without further movement once they were adherent to fibrinogen or other extracellular matrices. migration of adherent platelets has not been discussed by the scientific audience so far. methods and results: 2-dimensional gel-electrophoresis of platelets revealed that fibrinogen-adherent platelets synthesize or modify multiple proteins. most of these proteins include cytoskeletal proteins and proteins of the migration apparatus. we concluded that adherent platelets might be able to migrate on the surface of an extracellular matrix or endothelium. to demonstrate platelet migration we exposed fibrinogen-coated coverslips with adherent platelets to continuous flow under high shear rates as in the arterial vascular system. all non-adherent platelets were removed from the experiment. under continuous flow we observed shear stress induced mechanotaxis of human platelets some of which started to actively migrate along the direction of flow. migrating platelets were still tightly adherent to the extracellular matrix and would have otherwise been swept away by the continuous flow. we found, that these migrating platelets underwent cytoskeletal rearrangement and formed pseudopodia. to support that our observation is an active cellular process, we blocked cellular signal transduction on pi3-kinase level with ly294002/wortmannin. after exposure to ly294002/wortmannin, we could no longer observe platelet migration under flow conditions. we subsequently examined chemokine-directed migration of platelets inlcuding sdf1, rantes and ena-78. we created a chemokine source constantly releasing chemokines and thereby generating a gradient. as for the sdf1 (stromal cell derived factor-1; 10μg/ml) source we observed that about 30% of platelets adherent around this artificial source started to move towards the sdf-1 source. platelets were actively migrating for several hours in this in-vitro setting. during migration platelets polarized and formed pseudopodia with modification of several proteins of the migratory aparatus under sdf1 exposure. when the sdf-1 source was removed, platelets migrated without a specific direction. also with blockade of the cxcr4 receptor, the specific receptor for sdf-1, directed platelet migration could no longer be observed. we are currently performing inhibitory experiments on different levels of cell signalling to characterize the migratory apparatus of platelets. the role of other cytokines associated with cell migration including rantes and ena-78 will be investigated. we apply immunofluorescence, western-blot, 2-dimensional gel electrophoresis and mass spectrometry to differentiate the migratory apparatus of migrating cells. conclusion: platelets that adhere to fibrinogen are not irreversibly fixed on matrix surface. they are able to move into a specific direction guided by shear stress induced mechanotaxis or directed along a chemokine gradient . by these mechanisms, platelets might be able to migrate into a platelet clot to further stabilize the hemostatic plug. moreover, platelet migration may be a physiological mechanism to precisely cover denuded vascular lesions and further play a role in the recruitment of platelets to the site of vascular lesions or inflammation. influence of genetic variation in four obesity candidate genes on body mass index and metabolism j. aberle 1 , p. peitsmeyer 1 , n. a. beck 1 , f. u. beil 1 1 3. medizinische klinik, hamburg objective: it is believed that is in most cases obesity derives from an obesogenic environment on the basis of a genetic predisposition. a large number of genes and genetic variations have been associated with obesity. research methods and procedures: in a cohort of 1134 caucasion men and women with a body mass index (bmi) of 25 kg/m² or more we investigated four candidate genes at baseline and following a 3 months low fat caloric restriction diet by polymerase chain restriction and restriction digestion: the 1359 g/a variant of the cannabinoid receptor 1 (cb1), the p129t polymorphism in fatty acid amide hydrolase (faah), the l7p variation in neuropeptide y (npy) and the -420c>g polymorphism in resistin. results: comparing groups according to genotype for each gene separately revealed a significantly greater reduction of body weight and bmi in carriers of at least on mutant allele in cb1 as well as a greater reduction in triglycerides in resistin mutant allele carriers. we furthermore investigated gene-to-gene interaction: adding faah mutant alleles to carriers of at least one mutant allele in cb1, or resistin significantly influenced triglycerides, total-or ldl-cholesterol levels respectively. combination of npy n-allele with either faah t-allele or cb1 a-allele as well resulted in an increase of triglyceride levels. discussion: our results support the hypothesis of obesity as a complex genetic disorder with several genes involved in the development of an obese phenotype and obesity related comorbidities. rt-pcr, die konzentrationen sezernierter proteine mittels immunoassay quantifiziert. ergebnisse: die adiponektinstimulation von rasf resultierte in veränderungen der genexpression sowohl auf mrna-als auch proteinebene. unter diesen regulierten genen waren proinflammatorische zytokine ( schlussfolgerung: adiponektin nimmt einfluss auf rasf, indem u.a. die expression und sekretion von proinflammatorischen und prodestruktiven/matrixabbauenden mediatoren modifiziert werden. diese erkenntnisse könnten dabei helfen, die pathophysiologischen mechanismen in der ra besser zu verstehen und potentielle zielmoleküle zur therapeutischen intervention zu identifizieren. in dieser globalen studie wurde der nutzen einer verlängerten thromboseprophylaxe nach totalem hüftgelenkersatz untersucht. es wurde die kurzzeit-thromboseprophylaxe mit enoxaparin mit einer verlängerten thromboseprophylaxe von bis zu 5 wochen mit einem neuen, oralen, direkten faktor-xa. bei studienbeginn und nach 5 tagen wurde die wundfläche vermessen und eine 3 mm große biopsie aus der wunde entnommen, aus der mit hilfe der real-time pcr (taqman ® ) die mmp-mrna bestimmt wurde. die wundflüssigkeit wurde mit hilfe der gaze täglich gesammelt und mittels zymographie aufgearbeitet. ergebnisse: die expression von mmp-9mrna zeigte keine veränderungen in beiden gruppen. die mmp-2 und -9-konzentrationen in der wundflüssigkeit unterschieden sich nicht signifikant in beiden gruppen zu beginn der studie. allerdings wurde eine signifikante reduktion der aktiven form von mmp-2 zwischen dem 1. und 5. behandlungstag in der pi-gruppe verzeichnet (p=0,043). die ergebnisse von mmp-2pro zeigten in der therapiegruppe ebenfalls eine sinkende tendenz (25% reduktion zu 27% anstieg in der kontrollgruppe) erreichten aber nicht das signifikanzniveau. außerdem zeigte sich eine reduktion der wundgröße in der pi-gruppe von 24% im vergleich zur kontrollgruppe (p=0,006). schlussfolgerung: es traten keine veränderung der mmp-mrna-expression unter pi-therapie auf. allerdings fanden wir eine signifikante reduktion der biologisch aktiven form von mmp-2 in der wundflüssigkeit. die lokale behandlung mit einem pi beeinflusst also primär nicht die expression von proteasen, sondern scheint die wundflüssigkeit so zu modulieren, dass eine geringere aktivität von mmps auf der wundoberfläche zu finden ist und durch eine geringere proteolytische aktivität eine verbesserte wundheilung eintritt. einleitung einer antiretroviralen therapie in zwei ländlichen regionen in rwanda (n=11), fibrose in mehreren regionen (n=9). nur die gruppe, die baseline keine fibrose aufwies zeigte unter dreijähriger ert eine normalisierung der wanddicke (von 13,1± 0,9 auf 11,8 ± 1,0 mm; p<0,032) und eine weitgehende funktionelle normalisierung der regionalen herzfunktion (strain rate radial: von 2,35 ± 0,16 s-1 auf 2,95 ± 0,39 s-1, p=0,017; vergleichskollektiv 2,7 ± 0,3 s-1). die anderen beiden gruppen zeigten zwar in hinblick auf die wandstärken einen positiven effekt, hinsichtlich der herzfunktion konnten sie jedoch bei bereits deutlich erniedrigten funktionswerten zum baseline-zeitpunkt lediglich stabilisiert werden (reduktion der wandstärke nach 3 jahren ert: gruppe wenig fibrose= 10 mm; gruppe viel fibrose= 12 mm) schlussfolgerung: die enzymersatztherapie ist eine effektive langzeitbehandlung bei patienten mit fabry kardiomyopathie. hierbei fällt insbesondere auf, dass eine rechtzeitige therapie, bevor sich eine myokardiale fibrose entwickelt hat, indiziert ist. klinikgestützte poststationäre mobile gesundheitsservices; "hospital-to home®" -im poststationären versorgungsmanagement chirurgischer patienten adult-onset still´s disease (aosd) represents a rare systemic inflammatory disorder with an estimated incidence between 1 and 2 cases per million inhabitants in western europe. due to this small incidence only few data on diagnosis and therapy are available. the etiology of aosd remains elusive. currently the hypothesis of an exacerbated immune response based on dysregulation of cytokine mediated signalling cascades is favoured among scientists. according to resent results the proinflammatoy cytokine interleukin 1 (il-1) seems to play a central role in pathogenesis of the more common juvenile as well as adult-onset still´s disease. in view of that, we report about a fast and sustained response of a 44year old woman with aosd under first-line treatment with the il-1 receptor antagonist anakinra. the fast mode of action compared to conventional dmards and tnf inhibitors paired with an acceptable safety profile had prompted us to initiate a primary il-1 receptor blocking therapy. the observed rapid clinical response (cessation of fever within 12 hours) and normalization of highly elevated acute phase laboratory parameters within a few days encourage a first line use of anakinra in aosd-patients. longer observation periods and larger series of patients will be needed to answer open questions like optimal length of anakinra-treatment, long-term outcome and complications. cardiovascular complications in patients with diabetes mellitus are associated with increased oxidative stress in vascular tissue. a key event for no synthase (nos) uncoupling involves downregulation of bh4 synthesizing enzyme gtpcyclohydrolase i (gch-i). we here investigated the effects of at1-receptor block ade with chronic telmisartan therapy on gch-i expression, nos uncoupling and endothelial function in streptozotocin (stz)-induced diabetes mellitus (type i). diabetes was induced by single i.v. injection of stz (60mg/kg, 7 weeks) in male wistar rats (250g). telmisartan (25mg/kg/d) therapy did not improve blood glucose and body weight. aorta from diabetic animals had vascular dysfunction as revealed by isometric tension studies. ros produced by nadph oxidase, mitochondria and xanthine oxidase were increased in the diabetic group. nadph oxidase subunits mrna and protein expression was increased in response to stz. importantly, the expression of the gch-i and enos activating ser1177 phosphorylation was decreased by stz. therapy with telmisartan normalized all these parameters almost completely. the results of the present studies demonstrate for the first time that at1 receptor treatment of diabetic animals with telmisartan is able to prevent downregulation of the important bh4 synthesizing enzyme gch-i, which prevents enos uncoupling and an activation of cardiovascular superoxide sources. der einfluss der sauren sphingomyelinase auf die expression von matrix-metalloproteinase-1 in intestinalen epithelzellen und fibroblasten background: the calcineurin (cn)/nf-at signaling cascade takes a crucial role during t-cell activation and the development of myocardial hypertrophy. in addition to nfat, the phosphatase cn is also translocated into the nucleus. for nuclear import, specific carrier proteins, so-called importins, recognize nuclear localization sequences (nlss) in the sequence of the respective target proteins. we developed a synthetic import blocking peptide (ibp) which is identical to the nls sequence of cn. ibp prevented the interaction between cn and its importin and subsequently inhibited nuclear import of cn. methods and results: according to immunohistochemical studies both the subcellular localization of cn, the inhibitory effect of ibp and also the specificity of the blocking peptide were demonstrated. while the inhibitory effect within the t-cell activation is analyzed by [3h]-thymidine incorporation (7135 ± 2,503 vs. 2957 ± 1161 [%]), the impact on angiotensin ii stimulated cardiomyocytes was examined on the transcriptional (nfat reporter assay; 145 ± 10 vs. 108 ± 5 [%]) and on the translational level ([3h]-leucine incorporation; 254 ± 27 vs. 88 ± 49 [%]). by using ibp, the expression of hypertrophy markers, e.g. myosin heavy chain β, was suppressed, the cell size was not increased. interestingly, phosphatase activity was not effected (cna assay, biomol). the translocation of diverse transcription factors, like nfat, nfκb or erk2, was not effected, which illustrated the specifity of ibp. the results could be confirmed in a pilot animal study for heart failure. discussion: ibp prevents the nuclear translocation of calcineurin both in t-cells and in cardiomyocytes. the inhibitory activity is specific for calcineurin und does not affect the cn phosphatase activity. ibp has also an effect in vivo, where it has been demonstrated to suppress the development of myocardial hypertrophy in an animal model. in summary, ibp suppresses t-cell activation und prevents the development of myocardial hypertrophy. proteasome inhibitors suppress angiogenesis by altering endothelial vegfr-2 expression the ubiquitin-proteasome system is the major pathway for intracellular protein degradation in eukaryotic cells that controls a wide range of cellular regulatory proteins, including transcription factors and cell cycle regulatory proteins. recent evidence established the importance of the proteasome also in tumor development, showing anti-tumor and anti-angiogenic actions by selective inhibitors in vivo. as signaling via the vegfr2 pathway is critical for angiogenic responses to occur, we explored whether anti-angiogenic effects via proteasome inhibition were mediated in part through diminished endothelial vegfr2 expression. our studies show that different proteasome inhibitors (mg132, alln and lactacystin) all blocked vegfr2 expression in a time-and concentration-dependent manner, which was paralleled by respective inhibition of capillary like structure formation and endothelial cell migration. in contrast, tie-2 or vegfr-1 expression was not significantly affected by proteasome inhibitor treatment. the suppressive effects on vegfr2 expression were neither conveyed by increased shedding nor by shortened protein half-life, suggesting that transcriptional mechanisms accounted for the observed effects. in line with this conclusion, proteasome inhibition significantly suppressed vegfr2 mrna accumulation. in addition, inhibitor treatment considerably decreased transcriptional activity of 5'-deletional vegfr2 promoter gene constructs. proteasome inhibition-mediated repression was conveyed by a gc-rich region, harboring one consensus sp1 binding site. subsequent emsa analyses demonstrated diminished constitutive sp1-dependent dna binding in response to proteasome inhibition. hence, vegfr-2 expression may constitute a critical molecular target of proteasome inhibitors that mediates their anti-angiogenic effects in vivo. loss of ifn-γ inducibility of the mhc class ii antigen processing pathway in head and neck cancer: evidence for posttranscriptional as well as epigenetic regulation abnormalities of the major histocompatibility complex (mhc) antigens by tumor cells impair the cellular immune response and promote tumor evasion from immune surveillance. so far, studies analyzing the mhc class ii expression levels in hnc are limited. therefore, we investigated the constitutive and interferon (ifn)-γ-regulated expression profiles of mhc class ii apm in various hnc cell lines and also analyzed the mhc class ii expression in hnc lesions. hnc cell lines analyzed in vitro lacked constitutive mhc class ii surface expression. despite the ifn-γ-mediated induction at the mrna level, six out of ten cell lines did not show any relevant mhc class ii surface expression. this phenomenon might be attributed to a posttranscriptional dysregulation of specific mhc class ii apm components. one cell line displayed a loss of ifn-γ induced ciita-expression that corresponded to impaired mhc class ii surface expression, which could be linked to hypermethylation of the ifn-γ-responsive ciita-promoter iv. in vivo, immunohistochemistry analyses of 35 patients revealed that about 86% of hnc tissues exhibit a negative or only marginally positive staining, whereas 14% displayed a heterogenous or highly positive mhc class ii surface expression. there was no statistical correlation between tumor differentiation and the mhc class ii expression in hnc lesions. taken together these results suggest a high frequency of mhc class ii abnormalities in hnc in vitro and in vivo, which could occur at different steps of the antigen processing pathway. this information may have a significant impact on practical and clinical aspects of tumor immunotherapeutic strategies. ciita versus ifn-γ induced mhc class ii expression in head and neck cancer cells effective and safe reduction of blood pressue by the combination of amlodipine 5/valsartan 160 mg in patients with hypertension and metabolic risk factors not controlled by amlodipine 5 mg or felodipine 5 mga subanalysis of the express-m trial introduction: atrial fibrillation (af) is the most common cardiac arrhythmia and frequently occurs in patients with coronary heart disease. prophylactic oral anticoagulation (oac) is indicated in patients with af and at intermediate or high risk for thrombembolic events. however, an anticoagulant regimen has not been standardized for patients with af after coronary stent implantation, which may require additional dual antiplatelet therapy. methods: we identified retrospectively 159 patients with af who underwent pci in our department from 1999-2004. we compared baseline variables and incidence of a combined endpoint (stroke, mi, death, bleeding) in patients receiving clopidogrel and aspirin (n=103, group 1), versus patients receiving the combination of clopidogrel and aspirin with low molecular weight heparin (lmwh) (n=42, group 2), versus patients receiving the combination of clopidogrel and aspirin with oac (n=14, group 3) at discharge. results: the groups were not relevantly different regarding classical risk factors, however, patients discharged with triple therapy including oac seemed to be at (30) higher risk: patients in group 3 had decreased left ventricular ejection fraction and increased inflammatory state as measured by plasma fibrinogen. in a median follow up of 1,4 years (range from 2 months to 5.7 years) 2 severe bleeding events, 4 myocardial infarctions, 13 strokes, and 9 cardiovascular deaths occurred. kaplan meyer analyses showed that the occurrence of severe events was not significantly different (p=0.433). conclusion: in our analyses no treatment regimen seemed to be superior. however, in the follow-up period incidence of myocardial infarctions and strokes were high in patients with solely clopidogrel and aspirin treatment, and incidence of cardiovascular deaths was high in patients receiving the combination of clopidogrel and aspirin with lmwh. the number of patients receiving the combination of clopidogrel and aspirin with oac (triple therapy) was low, however, only one severe event was found in the follow-up period in this group. the current data underline the importance for prospective trials investigating the optimal anticoagulant-/antiplatelet treatment in patients with af after coronary stent implantation. valvuloplastie der kalzifizierten aortenstenose: neue erfolge durch weiterentwicklung der herkömmlichen methode background: we have shown that assessment of cardiac output (co) by the inert gas rebreathing method provides reliable measurements as compared to magnetic resonance tomography (mrt) in patients with sinus rhythm. during atrial fibrillation, however, co measurement based on the determination of stroke volume may be impaired by heart rate variation, potentially leading to divergent results. the present study therefore aimed to compare co measurements by inert gas rebreathing to mrt in patients with atrial fibrillation. methods: in a prospective case-control study, we included 30 consecutive patients with atrial fibrillation undergoing cardiac mrt, and 30 control patients pair-matched for gender, co by mrt, height and weight from a collective of 250 patients with sinus rhythm undergoing mrt. the co of reclining patients was determined by inert gas rebreathing with dinitric oxide and sulfur hexafluoride (innocor, innovision, odense dk) immediately before or after the mrt. the data determined by mrt for co served as reference value comparing the methods by means of bland-altman analysis. the study collective covered a wide range of patients (18 to 79 years, median 67, 21 men in the atrial fibrillation group vs. 23 to 78, median 66 in the sinus rhythm group). bland-altman analysis showed a good correspondence of the two methods for co with an average deviation of 0.2±1.2 l/min in the atrial fibrillation group vs. 0.3±1.1 l/min in the sinus rhythm group. no statistically significant difference could be found between the two groups (p = 0.48, mann-whitney-test). conclusion: co measurements by mrt and by the rebreathing method with dinitric oxide and sulfur hexafluoride provide closely related results even in the presence of atrial fibrillation. the rebreathing method therefore allows an easy and reliable non-invasive determination of the co in this patient collective. the importance of the method for diagnosing and treating cardiac diseases remains to be assessed by further studies. when analyzing the plasma lipid profile for each polymorphism, we found the following differences in subjects with gallstones: significantly higher triglyceride and cholesterol levels in carriers of the common abcb11 allele and significantly lower hdl-cholesterol concentrations in carriers of the common rs31653 allele. however, differences were also detected in controls with regard to cholesterol and total hdl-cholesterol concentrations. our results do not support a link between common abcb4 and abcb11 polymorphisms, lithogenic dyslipidemia and gallstone risk. evaluation der prävalenz schlafbezogener atemstörungen bei patienten mit vorhofflimmern und unauffälliger linksventrikulärer pumpfunktion mittels kardiorespiratorischer polygraphie hintergrund: in erregbaren herzzellen sind spannungsgesteuerte natriumkanäle für die auslösung und weiterleitung von aktionspotenzialen verantwortlich. natriumkanäle bestehen aus einer die pore bildenden α-untereinheit und zusätzlichen β-untereinheiten. verschiedene isoformen der α-untereinheiten haben ganz bestimmte entwicklungs-und lokalisationsmuster im herzmuskel, und sie besitzen spezifische pharmakologische und funktionelle eigenschaften. das kugelfischgift, tetrodotoxin (ttx), ein spezifischer natriumkanalblocker, inhibiert konzentrationsabhängig verschiedene α-isoformen. die kardiale isoform nav1.5 wird durch mikromolekulare konzentrationen blockiert (ttxresistent), während neuronale ttx-sensitive isoformen bereits durch konzentrationen im nanomolaren bereich inhibiert werden. die α-untereinheiten sind mit zusätzlichen β-untereinheiten assoziiert. methoden und resultate: humanes vorhofmyokard wurde mittels der patchclamp-technik, kontraktilitätsmessungen, immunhistochemie und western blot untersucht. wir konnten zeigen, dass nicht nur kardiale ttx-resistente (ttxr) natriumkanäle am menschlichen herzen vorkommen, sondern ebenfalls neuronale ttx-sensitive (ttxs) isoformen exprimiert werden. ttxs-natriumkanäle tragen zu 15% zum gesamtnatriumstrom bei, während die restlichen 85% des stroms von ttxr-kanälen übernommen werden. funktionelle einschränkungen durch die blockade von neuronalen natriumkanälen konnten in kontraktilitätsmessungen ebenfalls nachgewiesen werden. immunhistochemische experimente bestätigten isoformenspezifische lokalisation der α-untereinheiten nav1.1 bis 1.6 sowie aller bekannten β-untereinheiten (1-4). schlussfolgerung: im humanen herzen kommen neben kardialen auch neuronale natriumkanäle vor. sie tragen funktionell 15% zum gesamtnatriumstrom im menschlichen herzen bei und haben ebenfalls einen einfluss auf die kontraktilität. p44/p42-mapk-aktivierung in hypokontraktilen gefäßen zirrhotischer ratten: geänderte mechanismen der aktivierung jedoch gleichzeitig die g-protein abhängige aktivierung der p44/p42 mitogenactivated protein kinase (mapk). die g-protein abhängige p44/p42 aktivierung durch den angiotensin-ii typ1 rezeptor (at1r) erfolgt dagegen über protein-kinase c. methoden: leberzirrhose bei ratten wurde durch gallengangsligatur (bdl) induziert. isolierte aorten wurden in vitro mit bzw. ohne angiotensin-ii und verschiedenen inhibitoren inkubiert. protein-expressionen und -phosphorylierungen wurden über western-blot analyse untersucht. die interaktion zwischen dem at1r und b-arrestin 2 wurde in co-immunopräzipitations-experimenten untersucht. ergebnisse: trotz ausbleibender angiotensin-ii induzierter aktivierung der g-protein abhängigen kontraktilen signalwege in aorten von bdl ratten kommt es in aorten zirrhotischer und nicht-zirrhotischer ratten zu einer angiotensin-ii stimulierten aktivierung der p44/p42 mapk. diese angiotensin-ii stimulierte erk-aktivierung ist in aorten von bdl ratten, nicht jedoch der nicht-zirrhotischen kontrollen, resistent gegenüber hemmung der protein-kinase c und a. gleichzeitig führte die stimulation mit angiotensin-ii zur bindung von b-arrestin 2 an den at1r, die bei bdl ratten verstärkt ist. diskussion: bei leberzirrhose kommt es in extrahepatischen gefäßen zu änderungen in der kopplung von vasokonstriktor-rezeptoren an ihre signalwege. anstatt g-protein abhängiger signalwege unter normalen umständen (z.b. kontraktions-kaskaden) werden bei leberzirrhose b-arrestin 2-abhängige signalwege aktiviert. the prediabetic and diabetic in vivo modification of circulating low density lipoproteins decreases their potential to stimulate adrenal steroidogenesis context: biochemical modification of low density lipoprotein (ldl) has been implicated in the pathogenesis of impaired glucose tolerance (igt) and type 2 diabetes mellitus (dm2) and its related micro-and macrovascular disease. objective: since ldl serves as a major cholesterol source for adrenal steroidogenesis we investigated whether oxidative and glycoxidative modifications of ldl isolated from igt and dm2 subjects influence aldosterone and cortisol release from human adrenocortical cells. in a cross-sectional study ldl were isolated from 30 subjects with normal glucose tolerance (ngt), 30 subjects with igt, and 26 patients with dm2. individual oxidation and glycoxidation characteristics of ldl apolipoprotein b-100 were assessed by gas chromatography-mass spectrometry (gc-ms) analysis. human adrenocortical cells (nci-h295r) were incubated for 24h with 100 μg/ml of isolated ldl and after removal of supernatants the cells were further stimulated for next 24h with angiotensin ii. aldosterone and cortisol release were then quantified in the supernatants after 24h and 48h, respectively. results: circulating ldl from igt and dm2 subjects were substantially more oxidized and glycoxidized than the ldl samples from ngt subjects. each of the five measured oxidation/glycoxidation markers was significantly positively associated with glycemic control, measured as hba1c. ldl stimulated aldosterone and cortisol release from adrenocortical cells. however, hormone secretion was inversely related to the degree of ldl modification. conclusion: prediabetic and diabetic biochemical ldl modifications may promote impaired adrenocortical aldosterone and cortisol synthesis. die modulation der interaktion von nephrin und β-arrestin2 durch protein-kinase c alpha steuert die nephrin-vermittelte endozytose und signaltransduktion zielsetzung: hepatische sternzellen (hscs) sind die primären fibrogenen zellen in der fibrotischen leber. selektive apoptose von hscs stellt einen neuen antifibrotischen therapieansatz dar. wir haben kürzlich gezeigt, dass das endocannabinoid 2-arachidonoylglycerol (2-ag) die proliferation von hscs blockieren sowie apoptose induzieren kann. hepatozyten sind jedoch gegenüber diesen mechanismen resistent. der exakte molekulare mechanismus dieser selektiven zelltod-induktion in hscs ist noch nicht geklärt. ziel dieser arbeit war es, eine mögliche rolle von cox-2 im differentiellen zelltod-verhalten primärer hscs und hepatozyten durch 2-ag-metabolisierung in pro-apoptotischen prostaglandin-d2-glycerolester (pgd2-ge) zu überprüfen. methoden: primäre hepatozyten und hscs wurden durch collagenaseperfusion aus lebern gesunder ratten isoliert. 2-ag-oder pgd2-ge-induzierte apoptose wurde mittels ldh-assay und western blot für caspase 3-und parp-cleavage analysiert. bildung von reaktiven sauerstoffspezies (ros) wurde durch dcfda-fluoreszenz ermittelt. cox-2-expression wurde mittels western blot bestimmt. ergebnisse: hscs zeigten eine signifikante cox-2-proteinexpression, wohingegen hepatocyten keine wesentliche cox-2-expression aufwiesen. behandlung von primären in kultur aktivierten hscs mit 2-ag induzierte einen dosisabhängigen zelltod (70% nach 24h bei 10μm), begleitet von parp-und caspase 3-cleavage. pgd2-ge induzierte in primären hscs ebenfalls dosisabhän-gig apoptose. dagegen verursachte 2-ag in primären hepatozyten sogar bis zu konzentrationen von 100μm keinen zelltod, pgd2-ge jedoch führte ab konzentrationen von 25μm zum zelltod. 2-ag und pgd2-ge führten zur bildung von ros in hscs, was auf gemeinsame ros-abhängige apoptose-signalwege hinweist. schlussfolgerung: hscs und hepatozyten weisen einen signifikanten unterschied in der expression von cox-2 auf, was eine unterschiedliche metabolisierung des endocannabinoids 2-ag bedingt. die produktion von pgd2-ge durch cox-2 in hscs trägt möglicherweise zur disparaten empfindlichkeit von hepatozyten und hscs gegenüber 2-ag-induzierter apoptose bei. die rolle des hypoxie-induzierbaren-faktors 1a in der strahlentherapieresistenz von humanen adenokarzinomzellen der lunge (a549) zielsetzung: hypoxie hat einen entscheidenden einfluss auf die chemo-und strahlentherapieresistenz von soliden tumoren und somit auf die prognose von tumorerkrankungen. die hypoxie-induzierbaren-faktoren (hif-1a und hif-2a) sind transkriptionsfaktoren, die über spezifische zielgenaktivierung adaptive prozesse in den hypoxischen tumorarealen steuern. in dieser studie wurde die strahlensensibilität der adenokarzinomzelllinie der lunge (a549) unter dem einfluss von hif-1 und hif-2 untersucht. methoden: im ersten schritt wurde die auswirkung von bestrahlung 6 gy (1x, 4x, 10x) auf die a549 zellen untersucht (zellzählung, kolonie-assay, immunzytofluoreszenz: caspase-3, kerngrößen, multinuklei). unter normoxischen und hypoxischen bedingungen wurde die hif-1a und hif-2a expression mittels western blot und pcr mit und ohne radiatio untersucht. die strahlentherapieresistenz unter inhibition von hif (sirna) wurde in vitro (kolonie-assay, zellzählung) und in vivo (tumor growth delay assay) untersucht. ergebnisse: bestrahlung mit 6 gy führte zu einer reduktion der proliferation von a549 zellen ohne beeinflussung der apoptose. gleichzeitig änderte sich die kernmorphologie mit einem anstieg der kerngröße und vermehrten multinuklei unter fraktionierter bestrahlung. dies konnte auf einen anstieg der "seneszenten" zellen zurückgeführt werden. ein direkter einfluss einmaliger radiatio auf die expression von hif-1 oder hif-2 wurde nicht beobachtet. allerdings konnte über die inhibition von hif-1a mittels sirna eine erhöhung der strahlensensibilität in den a549 zellen in vitro erreicht werden. dies wurde in vivo mittels tumor growth delay assay bestätigt. schlussfolgerung: die aktivierung von hif-1a in hypoxischen tumorarealen hat einen entscheidenden einfluss auf die sensibilität von a549 zellen gegenüber bestrahlung. conditional overexpression of neuronal nitric oxide synthase is cardioprotective in ischemia-reperfusion introduction: we previously demonstrated that conditional overexpression of the neuronal nitric oxide synthase (nnos, nos1) inhibited l-type ca 2+ -channels. we now hypothesize that nnos overexpression has an impact on myocardial contractility and acts cardioprotective after ischemia-reperfusion. we assessed cardiac function in the newly established transgenic mouse model with conditional, myocardial nnos overexpression. nos-activity (22 ± 1.5 vs. 29 ± 1μm/sec, n=18, p<0.05) was significantly enhanced after nnos overexpression. co-immunoprecipitation experiments indicated interaction of nnos with sr ca 2+ atpase and additionally with l-type ca 2+ -channels in nnos overexpressing animals. ica,l (reduction of 40±6 rel.%, n=12, p<0.05) as well as intracellular ca 2+ -transients and fractional shortening in cardiomyocytes were clearly impaired in nnos overexpressing mice (3.0 ± 0.4f/f0 vs. 2.2 ± 0.2f/f0, n=13, p<0.005 and 7.7 ± 1.3% vs. 3.8 ± 0.5%, n=13, p<0.05). in vivo examinations of the nnos overexpressing mice showed a decrease of +dp/dtmax (reduction for 52 ± 17%, n=12, p<0.05) as well as a reduced ejection fraction (43±5% vs. 63±9%, n=12, p<0.05). ischemia-reperfusion experiments showed a cardioprotective effect of nnos overexpression (30 min post-ischemia, lvdp 20±6 in non-induced animals vs. 60±11 mmhg in nnos overexpressing animals, n=6, p<0.05). discussion: in summary, we demonstrated that under basline conditions, conditional transgenic overexpression of nnos resulted in a mild reduction of myocardial contractility, mainly due to inhibition of the l-type ca 2+ -channel. in contrast, under pathophysiological conditions (i.e. ischemia-reperfusion) nnos overexpression acts cardioprotective. these effects might be caused by a reduction of myocardial ca 2+ -overload after reperfusion. hintergrund und ziele: die pathogenese der erhöhten apoptose in der hepatitis c virus (hcv)-infizierten leber ist weitestgehend unbekannt. für zahlreiche hcv proteine ist eine modulation von zellulären apoptosemechanismen beschrieben worden. trail, der tnf-related apoptosis-inducing ligand, wurde kürzlich als zytotoxisch für hcv-infizierte hepatozyten beschrieben. um die zugrunde liegenden molekularen mechanismen einer potentiellen rolle von trail bei der hcv-induzierten hepatitis und der viralen clearance zu studieren, untersuchten wir die effekte der hcv replikation auf die trail-induzierte apoptose in humanen hepatomazellen. methoden: huh7.5 zellen wurden mit dem in zellkultur infektiösen hcv stamm jfh-1 sowie den mutanten jfh-1/δe1e2, jfh-1/gnd und sgr-jfh-1 hcv rna transfiziert bzw. infiziert. apoptose wurde durch bestimmung von parp, caspase-8 und -9 spaltprodukten sowie mittels tunel-reaktion untersucht. ergebnisse: transfektion von huh7.5 zellen mit replikations-kompetenter jfh-1, nicht jedoch mit replikations-defizienter jfh-1/gnd rna, führte zu einer apoptose von huh7.5 zellen, nachgewiesen mittels parp spaltung und positiver tunel reaktion. die verstärkte trail apoptose war in mit jfh-1/δe1e2 und sgr-jfh-1 subgenomischer hcv rna transfizierten huh7.5 zellen ähnlich stark wie beim vollständigen jfh-1 virus, was auf eine unabhängigkeit der apoptose-sensibilisierung von strukturproteinen sowie die notwendigkeit der viralen replikation hindeutet. untersuchungen zur signaltransduktion der jfh-1-bedingten apoptose-sensibilisierung zeigen eine deutliche abhängigkeit von caspase-9 aktivierung sowie eine jfh-1-induzierte suppression von bcl-2 und bcl-xl. dies deutet auf eine zentrale bedeutung des mitochondrialen signaltransduktionsweges bei der jfh-1-induzierten apoptose hin. schlussfolgerung: unsere daten zeigen eine apoptosesensibilisierung von huh7.5 hepatomazellen durch hcv replikation, welche unabhängig von hcv strukturproteinen und wahrscheinlich mitochondrial beding ist. diese ergebnisse geben wichtige hinweise für die pathogenese der hepatitis c und die mechanismen der viralen clearance. bier, jedoch nicht ethanol stimuliert in vitro die enzymsekretion der pankreasazinuszelllinie ar4-2j und frisch isolierter pankreasazinuszellen der ratte a. gerloff 1 , m. v. singer 1 , p. feick 1 1 ii. medizinische klinik, universitätsklinikum mannheim, mannheim hintergrund: da der pankreasazinuszelle in der frühen phase der entwicklung der alkoholischen pankreatitis eine wichtige rolle zugesprochen wird, wurde die wirkung von alkohol (ethanol) auf die funktion dieser zelle bereits seit jahren (44) intensiv untersucht. allerdings wird alkohol meist in form von schmackhaften getränken konsumiert, die viele nichtalkoholische inhaltsstoffe enthalten, welche ebenfalls einen pathophysiologischen einfluss auf die funktion des pankreas haben können. ziel: vergleich der wirkung von bier und adäquaten ethanollösungen auf die proteinsekretion und signaltransduktion der pankreasazinuszelllinie ar4-2j (ratte) und frisch isolierter ratten-azinuszellen. methoden: ar4-2j-zellen wurden für 72h mit dexamethason differenziert und frisch isolierte azinuszellen durch collagenase-verdau des pankreas von spraque-dawley-ratten gewonnen. nach inkubation der zellen für 60 min. wurde die amylasefreisetzung als maß der proteinsekretion mit hilfe eines kommerziellen testkits bestimmt. ergebnisse: die inkubation von ar4-2j-zellen mit bier (1-10% (v/v)) bewirkte eine dosisabhängige stimulation der basalen amylasesekretion. reiner ethanol in vergleichbarer konzentration wie im bier hatte keinen effekt auf die amylasefreisetzung. die bestimmung von ldh nach 24h inkubation der ar4-2j-zellen zeigte, dass die bierinduzierte amylasefreisetzung nicht auf einer membranschädigung beruhte. die verwendung selektiver inhibitoren und des ca-indikators fura-2/am ergab, dass die bierinduzierte amylasesekretion vorwiegend durch die aktivierung von phospholipase c und der anschließenden kalzium-freisetzung aus intrazellulären speichern vermittelt wird. der stimulatorische effekt von bier auf die proteinsekretion war in frisch isolierten azinuszellen reproduzierbar. schlussfolgerung: unsere daten zeigen, dass der effekt von bier auf die sekretion von pankreasazinuszellen auf nichtalkoholische inhaltsstoffe zurückzuführen ist. diese inhaltsstoffe müssen bei der untersuchung von alkoholinduzierten pathologischen und funktionellen veränderungen berücksichtigt werden. pharmakokinetik und pharmakodynamik einer fckw-freien, fixen kombination aus beclometason-dipropionat und formoterol (-1,46, 0,59 und -2,45 mm hg) erreichte keine statistische signifikanz. bei den mit liraglutid behandelten patienten zeigte sich im vergleich zu placebo eine senkung der triglyceride (adjustiert) von -19% (0,65 mg, p=0,03), -15% (1,25 mg, p=0,09) und -22% (1,90 mg, p=0,01) . es gab keine klinisch relevanten und konsistenten unterschiede bezüglich der gesamtcholesterin-, hdl-, ldl-, apob-, il-6-, tnf-α-, leptin-und adiponectin-konzentrationen zwischen den liraglutid-behandlungsgruppen und placebo, eine signifikante reduktion des ldl-cholesterins wurde unter placebo im vergleich zu den aktiven behandlungs-gruppen beobachtet, das galt aber nicht für apob, bei dem lediglich in der 1,25 mg behandlungsgruppe ein unterschied zu verzeichnen war. es konnte ein ausgeprägter effekt von liraglutid auf die pai-1-konzentrationen mit reduktionen von -14% (0,65 mg, p=0,29), -29% (1,25 mg, p=0,02) bzw. -25% (1,90 mg, p=0,05) gegenüber placebo beobachtet werden. das galt auch für die bnp-konzentrationen mit dosisabhängigen senkungen von -26% (p=0,1), -30% (p=0,05) und -38% (p=0,01) gegenüber placebo für die entsprechenden liraglutid-behandlungsgruppen. die beobachteten dosisabhängigen senkungen des crp gegenüber placebo (-3%, -12% und -20%) erreichten keine statistische signifikanz. schlussfolgerung: die behandlung mit liraglutid war mit einer blutdrucksenkung und einer abnahme der triglycerid-, pai-1-und bnp-konzentrationen verbunden, die beobachteten effekte müssen in langzeitstudien bestätigt werden und bedürfen weiterer untersuchungen. objectives: the majority of treated hypertensive patients do not achieve target blood pressure levels <140/90 mmhg. one key reason is inadequate adherence with the prescribed drug regimen. dosing regimens are either not executed as prescribed (non compliance) or patients stop taking the medication (non persistence). it has been demonstrated that drug adherence with angiotensin receptor antagonists like valsartan is superior in comparison to other drug classes. the present study was designed to evaluate whether drug adherence could be further improved by the use of supportive tools. design and methods: 28 centers were randomized to provide pharmacological treatment with or without a set of supportive measures (e.g. structured physicianpatient interaction, printed information about hypertension, reminder stickers, pill box with alarm, home blood pressure measurement). 202 newly diagnosed patients or patients with stage 1 hypertension (blood pressure at baseline 149.8±6.2/93.9±4.4 mmhg) who had not been treated for at least 1 year were included in this trial. all patients entered the 34-week treatment phase with valsartan 160 mg. titration to valsartan 160 mg/hctz 12.5 mg was allowed if necessary. drug adherence was assessed by electronic monitors (medication event monitoring system -mems). results: patients treated with a valsartan-based therapy in combination with supportive measures demonstrated lower rates of treatment discontinuation. per-sistence at the end of the 8 months observation period was 88% in the control patients and 96% in patients receiving supportive measures. in addition, better execution of the dosing regimen (compliance) could be observed in patients receiving supportive measures but this effect tended to fade with time. bp control improved more in patients with the supportive measures. conclusions: drug adherence can be improved with the use of supportive measures. in this study, while the effect on compliance decreased over time, the effect of chosen supportive measures on persistence seems to be long lasting. randomized, double blind parallel group study to evaluate the reduction of the urinary albumin/ creatinine ratio by valsartan plus lisinopril versus lisinopril or valsartan alone in hypertensive patients with microalbuminuria -the valeria trial objectives: microalbuminuria is known as an independent predictor for stroke, myocardial infarction and death and has a higher prevalence in hypertensive subjects than in the general population. intensified inhibition of the renin-angiotensin-aldosterone system seems to be an efficient treatment option and might be achieved by dual blockage with arbs and aceis. it was the aim of the study to compare the efficiency and safety of a combination therapy comprising valsartan and lisinopril with valsartan and lisinopril monotherapy in patients with hypertension and microalbuminuria. methods: this was a randomized, double blind parallel group study. patients with hypertension (mean sitting diastolic blood pressure = 85 mmhg and < 110 mmhg) and microalbumiuria (urinary albumin/creatinine ratio (uacr) = 2.5 mg/mmol and = 25.0 mg/mmol for men and uacr = 3.5 mg/mmol and = 35.0 mg/mmol for women in the first morning urine sample on at least two of three visits) were eligible for participation. after a wash-out/placebo-run-in phase of max. 3 weeks, patient were randomized to treatment (1:1:1) with either lisinopril 10-40 mg (lis), valsartan 80-320 mg (val) or the combination of valsartan/linisopril 80/10-320/20 mg (com) for 30 weeks. results: median uacr at baseline in the com, val, and lis group was 11.6 mg/mmol, 7.5 mg/mmol, and 8.8 mg/mmol, respectively. at baseline, systolic/ diastolic bp for com, val, and lis was 150.4/90.1 mmhg, 153.1/91.9 mmhg, and 153.0/90.6 mmhg. treatment with com, val, and lis resulted in an uacr reduction of 64%, 53%, and 41% (median) after 30 weeks of treatment. the reduction achieved with com was significantly greater than with lis (p=0.036). normalization of microalbuminuria (uacr < 2.5 mg/mmol for men and < 3.5 mg for women) with com, val, and lis was achieved in 38%, 31% and 17% of the patients (p=0.0344 for com vs. lis). blood pressure differences between the groups were not statistically significant. all treatments were well tolerated. conclusion: in patients with hypertension and microalbuminuria the combination of valsartan and lisinopril provided a significantly better reduction of uacr and doubled the rate of patients with normalized uacr compared to lisinopril alone. analyse der mikrozirkulation der oberen extremitäten unter ldl-apherese the baseline ppg of the rebleeder (group b) was 24.43 +-2.63 mmhg before tips and 12,57 +-2.82 mmhg after tips insertion, giving an overall ppg reduction of 48,52%. 28 patients who underwent tips for variceal bleeding never had a bleeding episode again after tips (group a) . the baseline ppg of these patients was 21.8 +-3.79 mmhg before tips and 10,0 +-2,71 mmhg after tips, giving an overall ppg reduction of 54,15% statistical analysis showed that the ppg difference between 12,57 mmhg in rebleeder (group b) and 10mmhg in non-rebleeder (group a) was statistically significant (p-value < 0.05). patients with refractory ascites never suffered from an episode of bleeding after tips independently from their initial ppg reduction. conclusion: patients who underwent tips for recurrent variceal bleeding do have a significant higher risk for rebleeding with an initial ppg reduction to 12,57 mmhg whereas those patients in which the ppg reduction to 10 mmhg can be achieved a rebleeding episode is unlikely. we conclude that a ppg reduction to at least 10mmhg should be aimed to prevent further bleeding episodes. these findings differ from previous reports in which ppg of less than 12 mmhg is thought to prevent rebleeding. ergebnisse: tgr männchen jedoch nicht tgr-weibchen entwickeln eine albuminurie (tgr-c: 129 mg/24h) und glomerulosklerose, die durch flutamid zu >90% unterdrückt wird (tgr-fl: 6 mg/24h). testosterongabe induziert in tgr-weibchen jedoch nicht in wt-weibchen albuminurie (tgr-ovt: 87 mg/24h, wt-ovt: 1.7 mg/24h) und glomerulosklerose (schadensindex-tgr-ovt: 152, wt-ovt: 5,3). ovariektomie induziert keine albuminurie. testosteron stimuliert in tgr-und wt-weibchen das renale wachstum und die glomeruläre angiotensinogenexpression, das glomeruläre wachstum wird jedoch nur in tgr-weibchen erhöht. androgenrezeptoren werden im glomerulus geschlechtsunabhängig exprimiert. schlussfolgerung: at1 rezeptorüberexpression in podozyten transgener ratten induziert albuminurie und glomerulosklerose. testosteron ist ein entscheidender kofaktor, der möglicherweise über eine direkte stimulation der glomerulären angii bildung wirkt. hohe prävalenz der ass-non-responder bei acvb-patienten in den multiplen testanalysen hintergrund und zielsetzung: in der therapie der hiv-infektion sind wechselwirkungen und unverträglichkeit der mittlerweile zahlreich zur verfügung stehenden antiretoviralen medikamente ein grosses problem. mehr als 50% der antiretroviralen medikamente und begleitenden antiinfektiva gegen opportunistische infektionen weisen positiv geladene reste (kationen) auf. die ausscheidung organischer kationen über die leber und niere erfolgt u.a. über die organischen kationentransporter (oct) 1 und 2, die eine breite substratspezifität aufweisen. ziel der studie war die charakterisierung kationischer antiretroviraler medikamente und antiinfektiva als inhibitoren und substrate von oct 1/ 2. methoden: hek-293 zellen wurden stabil transfiziert mit humanem oct 1 und 2 kloniert im eukaryotischen expressionsvektor pcdna3. die ic50-werte wurden durch messungen der spezifischen aufnahme von 3h-gelabeltem 1-methyl-4-phenylpyridinium (mpp+) bestimmt. die quantitative bestimmung der spezifischen substrate erfolgte mittels liquid chromatography electrospray-ionizationtandem mass spectrometry (lc-esi-ms/ms). km und vmax wurden mit der michaelis menten gleichung bestimmt. ergebnisse: pentamidine (ic50(hoct1) = 0.4 (1) μmol/l (mittelwert (standardfehler); (oct2) 3.8(1)), nelfinavir (7(1); 33(11)), ritonavir (14(2); 51(22)), lamivudine (17(3); 13(3)), trimethoprim (20 (3); 131(12)), zalcitabine (24(5); 25 (7)), saquinavir (37 (7); 105(29)) und indinavir (37(6); 275(57)) zeigten eine signifikante hemmung von oct1/2. lamivudine und zalcitabine sind substrate von oct1/2 (lamivudine: oct1(vmax=2.0(0.2) nmol/mg/min; km=249(51) μmol/l) oct2 (vmax=1.1(0.2); km=248(86)) zalcitabine: oct1(vmax=1.0(0.1); km= 242(56); oct2(vmax=0.6(0.1); km=232 (13) ergebnisse: der endogene ligand s1p, der selektive s1p1-rezeptor agonist sew 2871 und der unselektive s1p-rezeptor agonist fty720 transaktivieren den ebenfalls bereits als vermittler protektiver intrazellulärer signalwege beschriebenen egf-rezeptor. damit assoziert ist die aktivierung von akt in ratten-kardiomyozyten. im rattenmyokard verbessert bei applikation beginnend mit reperfusion fty720, nicht jedoch der selektive s1p1 rezeptor agonist sew2871 die erholung der mechanischen funktion signifikant. in humanen myokardpräparaten zeigt fty720 ex vivo einen analogen effekt bei applikation unter reperfusion. zusammenfassung: stimulation von s1p1 und s1p3-rezeptoren induziert die aktivierung von akt in kardiomyozyten. eine verbesserung der erholung der mechanischen funktion lässt sich nur über stimulation des s1p3 rezeptors unter reperfusion erzielen. s1p-rezeptoren scheinen daher ein klinisch interessanter angriffspunkt für eine behandlung im sinne einer pharmakologischen postkonditionierung. systemic effects of glucose content in pd fluids on life span and neuronal function in c. elegans background: glucose content in peritoneal dialysis fluids has a decisive role in the formation of glucose degradation products (gdps) during sterilization and is thus associated with the degradation of peritoneal membrane integrity. absorption of gdps from the peritoneal cavity in peritoneal dialysis patients results in rising plasma age levels. one precursor in age formation is methylglyoxal, which is metabolized by glyoxalase-1. to further investigate systemic effects of glucose content in pd fluids on life span and neuronal function, wild type and glyoxalase-1 overexpressing c. elegans were used as model system. homologies to 70% of all human genes are preserved in c. elegans. thus, this model system allows not only performance of life span assays, but also analysis of neuronal function. methods: c. elegans were cultivated under normal conditions and in the presence of 1.5% and 4% glucose derived from peritoneal dialysis fluids to perform life span assays. simultaneously locomotive patterns as parameter of neuronal function and neuronal gfp expression were evaluated for each group. results: wild type c. elegans showed a significant reduction of life span of 65% under the influence of glucose 1,5% and of 77% using 4% glucose. in contrast, glyoxalase-1 overexpression led to an increased resistance towards glucose expo-sure with no significant reduction of life span under 1.5% or 4% glucose. additionally, with glucose exposure an impairment of neuronal function was observed in wild type c. elegans displaying severe changes in locomotive patterns, which were not present in transgenic c. elegans. neuronal gfp expression in wild type c. elegans declined significantly with glucose exposure without a dose dependant effect. conclusion:. glucose derived from pd fluids has a significant impact on life span and neuronal impairment in c. elegans. whether potential systemic toxicity and neurotoxicity in pd patients can be attributed to pd fluids has to be further evaluated. common pathways in endogenous major depression and depression during ifn-α therapy for chronic hepatitis c background: a major complication of combination therapy with pegylated interferon-alpha (ifn-α) and ribavirin in patients with chronic hepatitis c is the induction of depressive side effects. methods: to elucidate the underlying pathophysiological mechanisms, a total of 50 caucasian patients with histologically proven chronic hepatitis c were treated with standard combination therapy consisting of pegylated ifn-α2a and ribavirin. the transcriptional profile 12h before and 12h after the first injection of ifn-α was analysed using human genomic microarrays (affymetrix, hg u133a) and quantitative real-time rt-pcr. class prediction analysis was performed to identify genes which are differentially regulated in patients with or without ifninduced depression. furthermore, pbmc from 21 patients hospitalized for se vere major depression and 11 controls were cultivated in vitro with pegylated ifn-α2a to validate the data in this cohort. results: 11/50 hcv patients (22%) developed clinically relevant ifn-induced depression. using class prediction analysis, the development of depression could be predicted with 91% accuracy by 16 genes including dynlt1, gch1, tor1b, disc1, mef2a and st3gal5 that were previously described to be relevant for recurrent major depression or neuronal development in the brain. in accordance with this data, increased endogenous ifn-production and selective hyper-responsiveness of these genes to ifn stimulation were observed in hcvnegative patients with severe major depression. conclusions: these data suggest that selective hyper-responsiveness to exogenous (ifn therapy) or endogenous (endogenous depression) type i ifns may lead to the development of depressive symptoms. these data could lead to the discovery of novel therapeutic approaches to treat ifn-induced and major endogenous depression. renal toxicity of glucose degradation products in peritoneal dialysis purpose (zielsetzung): in peritoneal dialysis (pd) residual renal function contributes to improved patient survival and quality of life. glucose degradation products (gdp) impair not only the peritoneal membrane, but also appear in the systemic circulation with the potential for organ toxicity. here we show that in a model of advanced renal failure, gdp affect the structure and function of the remnant kidney. sprague-dawley rats were randomly assigned to a twostage subtotal nephrectomized (snx) or sham operation and were left untreated for 3 weeks. the snx+gdp group received chemically defined gdp intravenously for 4 weeks; the snx and the sham-operated rats remained without gdp. the complete follow-up for all groups was 7 weeks post-operatively. we analyzed renal damage using a semiquantitative score for glomerulosclerosis and tubulointerstitial damage as well as for immunohistochemical analyses. the snx+gdp rats developed significantly more albuminuria (12.3 ± 4.96 mg/24 h vs. snx 5.68 ± 2.77 mg/24 h; p ≤ 0.05) and showed a significantly higher score of glomerulosclerosis index (2.18 ± 0.33 vs. 1.82 ± 0.23; p ≤0.01) and tubulointerstitial damage index (2.17 ± 0.29 vs. 1.78 ± 0.22; p ≤ 0.01). in the snx+gdp group the expression of carboxymethyllysin and methylglyoxal was significantly higher in the tubulointerstitium and the glomeruli compared to the snx rats. caspase 3 staining and tunel assay were more pronounced in the tubulointerstitium and the glomeruli of the snx+gdp group. in snx+gdp animals, the expression of the slit diaphragm protein nephrin, was significantly lower compared to snx and sham operated animals. in subtotally nephrectomized rats, administration of gdp increased albuminuria, indices of glomerular and tubulointerstitial damage significantly, specifically with a perturbation of podocytes. it is likely that gdp-free pd solutions maintain and stabilize residual renal function in pd. non-parenchymal liver cells (wu et al., hepatology, in press ). therefore, the aim of this study was to investigate whether hbv has the ability to suppress tlr-induced antiviral responses in parenchymal and non-parenchymal liver cells. we have isolated murine kc by counterflow elutriation as well as murine lsec by anti-lsec microbeads. in addition, primary murine hepatocytes were isolated by a two-step perfusion method. npcs and hepatocytes were cultivated in the presence or absence of hbsag, hbeag, hbv virions or supernatants from hbv-producing hbv-met cells, and were stimulated by tlr 1-9 ligands. supernatants were collected and tested for antiviral cytokines by viral protection assay or co-cultured with differentiated hbv-met cells. primary hepatocytes, undifferentiated hbv-met cells and highly differentiated hbv-met cells were stimulated by tlr 1-9 ligands. results: pretreatment of hepatocytes and npcs with hbv-met cell supernatants (contain hbsag, hbeag and virions), hbsag, hbeag and hbv virions almost completely abrogated tlr 3 and tlr 4 induced antiviral activity which correlated with suppressed isgs induction by poly i:c and lps on the transcriptional level. tlr 1-9 stimulation had no direct antiviral effect on hbv-met cells in contrast to primary hepatocytes. in addition, expression of pro-inflammatory cytokines (tnf-a, il-6) after tlr -3, -4, -7, -8 and -9 stimulation were significantly reduced in highly differentiated hbv-met cells compared with undifferentiated hbv-met cells. our data indicate that integrated hbv can downregulate the tlr-mediated activation of hepatocytes. furthermore, hbv can almost completely abrogate the antiviral activity of hepatocytes and npcs induced by tlr 3 and tlr 4 stimulation. this has major implications for the interaction between hbv and the immune system. schimäre im pankreas: ein glukagon-produzierendes neuroendokrines karzinom assoziiert mit hypoglykämien conclusions: dbe-ercp is an alternative method for diagnostic as well as therapeutic interventions in the biliary as well pancreatic system in the operated patient. however, it should be limited to selected patients, e.g. with contraindications for ptc, as it is a time consuming as well as a cost intensive procedure. undifferenziertes embryonales sarkom der leber -eine seltene primäre leberneoplasie beim erwachsenen patients with metabolic syndrome (ms) and type 2 diabetes are at increased risk for coronary artery disease. lipoprotein metabolism is characterized by elevated triglycerides (tg), low hdl cholesterol (hdl-c) and a predominance of atherogenic dense ldl (dldl). this is also denoted as atherogenic lipoprotein phenotype (alp). methods: multicenter, randomized, open-label cross-over study investigating the effect of combination therapy of fluvastatin/fenofibrate (80/200 mg) (ff) on the ldl-subfraction distribution compared to combination therapy of simvastatin/ ezetimibe (20/10 mg) (se) in patients with ms and type 2 diabetes. at baseline and after 6 weeks of combination therapies ldl-subfractions were measured by endpoint gradient ultracentrifugation. results: 75 patients were randomized, 69 completed the study. blood lipid samples of 56 patients could be analysed. 38 out of 56 patients (68%) showed a profile dominated by dldl. in these patients, tg were elevated and hdl-c was lower compared to non dldl patients. in all patients reduction of total and ldl cholesterol by se was stronger than by ff. the increase of hdl-c was stronger with se in the non dldl group, whereas in the dldl group there was no difference between treatments. in non dldl patients there was no difference with regard to tg reduction and no effect on calculated ldl-radius. however, in the dldl group ff was more efficient in reducing tg (p=0.020 between treatments) and se reduced ldl radius even further, whereas ff increased ldl radius to larger ldl particles (p<0.001 between treatments). conclusions: simvastatin/ezetimibe combination is more efficient in reducing total and ldl cholesterol. this is also true for hdl-c increase in patients without dldl. however, in ms patients with dldl fluvastatin/fenofibrate were more efficient in reducing tg and increasing ldl radius. overall, the number of patients with aes and myalgia was 15. 1% hintergrund: bnp und nt-probnp sind molekulare biomarker, welche ebenso für die diagnostik und prognose der chronischen herzinsuffizienz genutzt werden können wie für die optimierung einer medikamentösen therapie und das langzeit-management dieser erkrankung. erstmalig untersuchten wir den einfluss eines strukturierten, multi-modalen, stationären interventionsprogramms -wie es in deutschland in vielen kardiovaskulären rehabilitationskliniken angeboten wird -auf den zeitlichen verlauf der nt-probnp-werte während und sechs monate nach einer solchen intervention. methodik: diese studie wurde in sechs deutschen kardiovaskulären rehabilitationszentren durchgeführt mit folgenden vier interventionsmodalitäten: (1) optimierung der medikamentösen therapie, (2) erziehung zu einem und durchführung eines krankheits-bezogenen ausdauertrainings-programms, (3) intensive information über art und verlauf der erkrankung und erziehung zu einem adäquaten ernährungsverhalten, und (4) teilnahme an den physischen und psychischen entspannungsübungen. zur steigerung der motivation und dokumentation wurden speziell für diese studie entwickelte patienten-tagebücher ausgegeben, welche den gesamten sechsmonatigen studienverlauf abdeckten. nt-probnp-analysen erfolgten am anfang (r1), während (r2) und am ende der stationären rehabilitationsperiode (r3), ebenso -auf ambulanter basis -nach drei monaten (r4) und sechs monaten r5) nach entlassung. zum vergleich wurde eine gleichaltrige kontrollgruppe von patienten herangezogen, welche nicht an einem solchen intensiven programm teilnahmen, sondern in einer herzinsuffizienz-ambulanz medizinisch betreut wurden. hypothese: wir erwarteten einen positiven effekt unseres interventions-programms auf die nt-probnp-spiegel während des stationären aufenthaltes, und entweder eine konstanz dieser werte nach der entlassung oder -auf grund von mangelhafter compliance -sogar einen wiedereinstieg der werte bis zu sechs monaten nach entlassung. ergebnisse: komplette nt-probnp-werte lagen für 60 patienten vor, welche an einer chronischen herzinsuffizienz im stadium nyha ii -iii litten, eine klinisch relevante dekompensation erlitten hatten und eine lvef = 35% aufwiesen: der medianwert der nt-probnp-spiegel betrug 1985 pg/ml am anfang (r2), war geringfügig aber nicht signifikant reduziert auf 1644 (r2) und 1755 pg/dl (r3) während des stationären aufenthaltes, fiel jedoch signifikant und klinisch relevant auf 978 pg/ml nach drei monaten (r4; p<0,01) und auf 719 pg/ml nach sechs monaten ab (r5; p<0,01). der medianwert der kontrollgruppe änderte sich nicht während der sechsmonatigen beobachtungsperiode (1164 pg/ml und 1107 pg/ml; ns). schlussfolgerungen: im gegensatz zu unserer hypothese hat eine modernes, strukturiertes, stationäres rehabilitationsprogramm keinen kurzzeit-effekt auf die nt-probnp-spiegel innerhalb von wochen, sondern vielmehr einen signifikanten und größenordnungsmäßig klinisch relevanten langzeit-effekt innerhalb von monaten. dies könnte in der tatsache begründet liegen, dass die molekularen und strukturellen umbauprozesse des linken ventrikels im sinne eines umgekehrten "left ventricular remodelling" entsprechend zeit benötigen. da die nt-probnp-spiegel mit der prognose der erkrankung assoziiert sind, kann indirekt geschlossen werden, dass unsere stationäre rehabilitationsstrategie langfristig positive effekte auf hospitalisierungsrate und überlebensprognose hat. cancer fatigue und gestörte ruhe/aktivitätsregulation bei rezidivfreien mammakarzinom-patientinnen, ergebnisse einer prospektiven studie r=0, 358, p=0, 015) . vergleichbare beziehungen bestanden zwischen dem pap und ahi (r=0,39, p=0,002), dem ai (r=0,39, p=0,002) und dem zai (r=0,40, p=0,016). das hzv war bei patienten mit csa (1,93±0,5 l/ min/m 2 ) signifikant niedriger als bei osa (2,55±1,0 l/min/m 2 ) oder patienten ohne sas (2,22±0,4 l/min/m 2 ). das hzv korrelierte negativ mit dem zai (r= -0,344, p=0,008), dem ai (r= -0,31, p=0,02) und dem ahi (r= -0,21, p<0,05). bei pat mit einer osa waren solche beziehungen nicht nachweisbar. die vorliegenden befunde unterstützen die these, wonach das auftreten einer zsa ein parameter für die schwere einer hi sein kann und erhöhte pa-und pcw-drücke über pulmonal-vagale j-rezeptoren zur hyperventilation und damit entstehung und unterhaltung einer zsa beitragen können. der quotient aus serum-natrium/ urin-natrium-konzentrationen zu den serum-kalium/ urin-kalium-konzentrationen (sus:pup) im screening auf einen primären aldosteronismus hintergrund: das herzzeitvolumen (hzv) ist ein wichtiger parameter in der diagnostik und therapie kardialer erkrankungen. die aktuellen standardmethoden zur bestimmung des hzv sind jedoch entweder invasiv (rechtsherzkatheter, picco) oder technisch aufwändig bzw. teuer (magnetresonanztomographie, mrt). die bisherigen nicht-invasiven methoden zur messung des hzv mittels rückatmung von kohlendioxid oder thorakaler bioimpedanz sind zwar einfach durchzuführen, weisen aber methodisch bedingte ungenauigkeiten auf. ziel der vorliegenden prospektiven studie war es daher, eine neue methode zur bestimmung des hzv mittels cw-doppler ultraschall (cwd) zu evaluieren. methodik: bei 32 konsekutiven patienten wurde unmittelbar vor oder nach einer kardialen mrt das herzzeitvolumen (hzv) mittels cwd im liegen bestimmt (uscom, uscom ltd, sydney australia). als referenzwerte dienten die in der mrt bestimmten werte, die mit dem arithmetischen mittel aus zwei aufeinanderfolgenden cwd messungen verglichen wurden. der statistische vergleich der methoden erfolgte mittels bland-altman-analyse. ergebnisse: das patientenkollektiv bestand aus 20 männern (alter 17-80 jahre, median 51 jahre) und 12 frauen (alter 27-78 jahre, median 64 jahre). insgesamt konnte bei 29 von 32 konsekutiven patienten im untersuchungszeitraum das hzv und sv mittels cwd bestimmt werden, bei 3 patienten (9%) konnte kein ausreichendes ultraschallsignal empfangen werden. das hzv mittels mrt lag bei 5,2 +/-1,1 l/min (mittelwert +/-sd, minimum 3,0 l/min, maximum 7,5 l/min), das hzv mittels cwd bei 4,7 +/-1,1 l/min (minimum 2,8 l/min, maximum 8,0 l/min). die bland-altman-analyse ergab eine gute übereinstimmung der beiden methoden für das hzv von 0,5 +/-1,0 l/min (mittlere abweichung +/-sd) und für die bestimmung des schlagvolumens von 6,9 +/-16 ml. die methode zeigte eine gute reproduzierbarkeit mit einer mittleren abweichung von 0,1 +/-0,5 l/min für das hzv und 0,3 +/-7,7 ml für das sv. schlussfolgerung: der cw-doppler ultraschall erlaubt eine zuverlässige nichtinvasive bestimmung des hzv mit guter reproduzierbarkeit. der zukünftige stellenwert der methode in der diagnostik und therapiesteuerung muss durch weitere untersuchungen belegt werden. nichtinvasive bestimmung des herzzeitvolumens mittels inertgas-rückatmung und cw-doppler-ultraschall -sind die ergebnisse vergleichbar? bei 4 patienten (10%) war die messung mittels cwd nicht möglich, bei 2 patienten (5%) konnte die igr-messung nicht durchgeführt werden. das hzv mittels igr lag bei 4,9+/-1,1 l/min (mittelwert+/-sd, 2,7 bis 7,6 l/min) und bei 4,6+/-1,2 l/min (2,5 bis 8,0 l/min) mittels cwd. die bland-altman-analyse ergab eine gute übereinstimmung für das hzv von 0,4+/-1,1 l/min (mittlere abweichung+/-sd) und für das sv von 2+/-18 ml. es zeigte sich eine gute reproduzierbarkeit mit einer mittleren abweichung von 0,2+/-0,6 l/min für das hzv mittels igr bzw. 0,1+/-0,5 l/min mittels cwd. schlussfolgerung: die bestimmung des hzv und sv mittels igr und cwd ergab eine gute übereinstimmung und reproduzierbarkeit, so dass die methoden als vergleichbar einzuschätzen sind. zur identifikation des optimalen patientenkollektives für jede der beiden methoden sind weitere untersuchungen zur erarbeitung eines score-systems geplant. validierung einer einfach anzuwendenden neuartigen oszillometrischen methode zur bestimmung der pulswellen-geschwindigkeit obesity in humans is mainly due to high-fat intake and associated with an increased incidence of glomerular injury that is associated with low-grade inflammation and increased production of reactive oxygen species (ros). lauric acid (c12:0), a major constituent of coconut oil, has anti-inflammatory activities, however its effects on pro-inflammatory gene expression in the kidney during obesity are unknown. the aim of the study was to quantify and compare renal cortical gene expression of pro-and antioxidant enzymes and icam-1 in c57bl/6j mice fed with standard chow diet (sd), or diets rich in animal fat (afd) or vegetable fat (lauric acid, vfd) for 15 weeks. changes in body weight and glucose tolerance (ip ggt) were also determined. both high-fat diets caused weight gain and glucose intolerance to a similar degree (n.s., afd vs. vfd, both p<0.001 vs. sd). expression levels of proinflammatory genes (no synthase 2, nos2; catalytic subunit of the phagocytic nadph oxidase, nox2; intracellular adhesion molecule-1, icam-1) were more than 30-fold lower compared to antioxidant genes (gluthatione peroxidase, gpx and cu/zn superoxide dismutase, sod1) and the regulatory subunit of the nadph oxidase complex p22phox. only vfd-treatment reduced renal expression of p22phox. treatment with afd but not with vfd increased mrna expression of nos2, nox2, and icam-1. vfd, but not afd treatment was associated with upregulation of the antioxidant genes sod1 and gpx. these data suggest that fat diets rich in lauric acid, in contrast to diets containing animal fats, have distinct and possibly beneficial effects on expression of enzymes regulating cellular redox state in the kidney. contractility in the carotid artery obesity is a risk factor for diseases such as diabetes mellitus and atherosclerosis and has been associated with increased carotid artery intima thickness in obese patients. leptin has been implicated in formation of reactive oxygen species (ros), and functional leptin deficiency or impairment of leptin receptor activity result in obesity. endothelin-1 (et-1), ros and reduced no bioactivity are involved in vascular changes in obesity and diabetes. the aim of this study was to investigate the role of ros in et-1 mediated vasoreactivity in the carotid artery of obese leptin-deficient ob/ob and lean c57bl/6j wild-type mice. rings were preincubated for 30 minutes with the no synthase inhibitor, l-nitro-argininemethylester (l-name, 300 μmol/l) to block etb receptor-mediated no release. rings were exposed to et-1 (0.01-300 nmol/l) in the presence or absence of the ros antioxidant epck-1 (a combination of vitamin c and vitamin e, 0.1 mg/ml). endothelium-dependent relaxation to acetylcholine (ach) was in-vestigated in the presence of non-specific cyclooxygenase (cox) inhibitiors. maximal et-1-mediated contraction in control mice was similar to obese mice. antioxidant treatment with epc-k1 reduced et-1-induced contractions in control (from 44.7 ± 6.7% to 22.7 ± 3.8%, p<0.05), but not in obese mice (55.8 ± 11.9% vs. 49.2 ± 8.9%, n.s.) endothelium-dependent relaxation to ach remained unchanged in both groups. in conclusion, these results indicate that ros contribute to et-1-induced contractions in the carotid artery of healthy mice, an effect that disappears during obesity. leptin may modulate contraction to et-1 in the carotid artery by mechanisms that are independent of ros. high dietary intake is a major cause of obesity, a risk factor for the development of hypertension, diabetes, and atherosclerosis. the aim of this study was to investigate whether high dietary fat intake-induced vascular changes are reversible upon dietary fat restriction. body weight, glucose tolerance and vascular reactivity were measured in c57bl/6j mice, which were fed either with standard chow or with high-fat diet (hfd, 58% kcal from fat) for 6 months or with hfd for 6 months followed by fat restriction for 4 months. vascular reactivity was analyzed in the carotid artery in response to the α-adrenergic receptor agonist phenylephrine (pe; 3x10 -8 m), and to acetylcholine (ach; 10 -10 -10 -5 m) in the presence or absence of ng-nitro-l-arginine methyl ester (l-name; 3x10 -4 m), a nitric oxide (no) synthase inhibitor. high-fat diet increased body weight and impaired glucose tolerance (p<0.05 vs. control). vascular contractions in response to pe (3x10 -8 m) and ach (10 -7 m) were increased both in the absence or presence of no (p<0.05 vs. control), while the endothelium-dependent relaxation was unaffected. in contrast, dietary fat restriction normalized the body weight similar to control animals and also the relative insulin resistance (p<0.05 vs. hfd). in parallel, vascular contraction to ach was attenuated in the absence of l-name (p<0.05 vs. hfd). unexpectedly, dietary fat restriction did not reverse pe-and ach-mediated contraction in the absence of no induced by hfd. in conclusion, intake of high dietary fat has a "memory" effect on vascular reactivity, which cannot completely be reversed even upon dietary fat restriction. this may contribute to an increased risk of vascular injury seen in patients even after normalization of body weight and insulin resistance. die phagozytotische aufnahme von hcv-infizierten apoptose-körperchen durch hsc führt zur induktion der fibrogenese a minority of neuroendocrine tumors (net) present functional syndromes by uncontrolled secretion of peptide hormones or messengers. peptide receptor radiotherapy (prrt) and transcatheter arterial chemoembolisation (tace) have demonstrated efficacy as monotherapy in the treatment of functional nets. prrt has a slower and longer acting mechanism of action whereas tace can be immediately effective. however, there are no reports that both therapies have been applied in combination. we treated three consecutive patients with severe functional syndromes caused by hepatic metastasis of nets by sequential prrt and tace. the first patient (female 57 years) suffered from a pancreatic net metastasized into the liver. severe hypercalcemia with levels of 3.9 mmol/l developed leading to fatique and somnolence despite medical treatment.the patient received one course of 40 mci y-90 dota-tate prrt and 11 days later, tace was performed at the right liver lobe due to persistent hypercalcemia. after 5 days, calcium levels were normalized and the patients was able to walk with support. after demission, the patients died of progressive disease 6 weeks later. the second patient (male, 58 years) presented with somnolence and hypoglycaemia down to 1 mmol/l by disseminated insulinoma of the pancreas. the patient received two courses of prrt and three coursed of tace involving both liver lobes. he tolerated the combined treatment well and is off intravenous glucose since the first tace. the third patient (male, 62 years) presented with severe hypoglycaemia due to hyperinsulinemia caused by disseminated net of unknown origin with prominent liver metastases in both liver lobes. initially, the patient was treated by tace followed by 2 courses of prrt with 177-lu. the treatment was well tolerated, the patient is off intravenous glucose. a second tace was intended but was not be performed due to remission of the liver metastases. initial experience with combined treatment of prrt and tace demonstrated that hepatic failure did not evolve in our patients and that the treatment was well tolerated. however, prognosis remains poor due to disseminated and progressive disease. die zahl zirkulierender endothelialer progenitorzellen steigt mit zunehmendem schweregrad der peripheren arteriellen verschlusskrankheit these data indicate that the key mirna-processing enzymes dicer and drosha and consequently the overall expression of mirnas are downregulated during neointima formation in vivo. moreover, the augmented proliferative-and attenuated apoptotic response observed following knock down of drosha and dicer in vsmc implicate an important role of these enzymes and of mirnas for vsmc function during the development of vascular proliferative disease. (76) hochaktives endokrines organ. das fettgewebe produziert nämlich zahlreiche adipokinine, welche möglicherweise einen autokrinen und parakrinen einfluss auf den fettstoffwechsel, sowie eine endokrine wirkung auf andere organe haben. die entdeckung solcher faktoren eröffnet die frage, ob die adipokinine die gesuchte verbindung zwischen adipositas und den damit assoziierten erkrankungen sein könnten. im diesen zusammenhang haben wir untersucht, ob das fettgewebe die herzfunktion direkt mittels sekretion von kardioaktiven faktoren beeinflusst. wir isolierten deshalb adipozyten aus humanem fettgewebe und untersuchten anschließend den effekt ihrer sekretionsprodukte auf kardiomyozyten in zellkultur sowie mittels langendorff system auf isoliert-perfundierte rattenherzen. wie wir feststellen konnten, hatten die sekretionsprodukte der adipozyten die kontraktion der kardiomyozyten durch eine verminderung des intrazellullären kalziumstroms stark gehemmt. durch erhitzen oder trypsin-behandlung wurden die kardiodepressiven faktoren inaktiv. mittels filtration nach molekulargewicht konnten die kardiodepressiven faktoren zwischen 10 und 30kd isolierten werden. auf ähnliche weise führte die perfusion von isoliertem herz mit adipozytenfaktoren zu einer starken reduktion der kraftentwicklung sowie zu einer reduktion des koronarflusses durch eine kontraktion der koronararterien. zusammenfassend, die ermittelten daten lassen auf einen bislang unbekannten, negativen einfluss des fettgewebes auf die herzkontraktion schließen. dies geschieht einerseits direkt durch die verminderung des intrazellullären kalziumstroms in die kardiomyozyten, andererseits indirekt durch die reduktion des koronarflusses. diese daten liefern somit eine neue erklärung für den zusammenhang zwischen adipositas und herzinsuffizienz. a comparison of serum fractalkine in patients with coronary heart disease, insulin dependent diabetes, and healthy controls background: atherosclerosis is a multifactorial process that involves inflammation of the vessel wall arising from interactions of leukocytes with vascular endothelial and other local cells. these interactions are regulated by cytokines, including chemokines, as well as adhesion molecules. the chemokine fractalkine (fkn) and its receptor, cx3cr1, have emerged as interesting intermediaries in atherosclerosis. fkn is a unique chemokine because it exists in soluble and membrane-anchored forms. membrane-bound fkn contributes to adhesion of cx3cr1-expressing leukocytes, thus providing a novel pathway for leukocyte activation. soluble fkn has leukocyte chemotactic activity. therefore, serum fractalkine levels may indicate the inflammatory situation in chd patients. we investigated the serum concentrations of fkn in 50 non-diabetic patients with coronary heart disease (chd), and 50 insulin dependent diabetics (iddm) in patients attending for rehabilitation in the curschmann-clinic, timmendorfer strand. serum fkn was also determined in 50 healthy controls. fkn was analyzed by elisa. results: soluble fkn was slightly, but not significantly increased in patients with chd in comparison to healthy controls (mean ± standard deviation: 722±884 pg/ml in chd-patients versus 693±748 pg/ml in controls, p=0.833). surprisingly, soluble fkn was decreased in insulin dependent diabetics (mean±sd 430±257 pg/ml thus, iddm versus controls p=0.058, and iddm versus chd p=0.034). conclusion: previous studies described that soluble fkn is increased in chd patients compared with healthy controls and that membrane-bound fkn was increased in human carotid arteries and animal models. the decreased soluble fkn in insulin dependent diabetics compared with chd-patients and controls in this study may partly be explained by the quiescent disease state predominant in our rehabilitation patients. hintergrund: neben der jeweiligen bilddokumentation und dem deskriptiven befund des interventionalisten exsistieren keine objektiven daten zur veränderung der hämodynamik während peripheren arteriellen gefässinterventionen und dem unmittelbaren hämodynamischen erfolg nach der intervention. versuche der etablierung eines monitorings durch dopplerultraschall scheiterten in der vergangenheit offensichtlich u.a. an der aufwändigen fixierung der dopplersonden. methode: wir verwenden zur kodomo die doppler x-plore sonde (7,5 mhz, firma medi-stim, deisenhofen), welche durch einen saugnapf mit ultraschallgeldepot über einer zuvor dopplersonographisch detektierten fussarterie fixiert wird. es können so während der intervention kontinuierliche dopplersignale dokumentiert werden. ergebnisse: phänomene die mittels kodomo bei infrainguinalen eingriffen periinterventionell erfasst werden können sind: verbesserung des dopplerflusses postinterventionell, artefakte durch kontrastmittel, periphere embolien, veränderungen des dopplersignals während der angioplastie. beispielhaft wird ein fall mit filiformer superficialisstenose links demonstriert (abb. 1). schlussfolgerung: kodomo gibt dem interventionalisten über die bildgebung hinausgehende hämodynamische zusatzinformationen und ist ein verfahren zur unmittelbaren dokumentation des hämodynamischen erfolges der interventionellen therapie. es spart in konsequenter anwendung möglicherweise kontrast-mittel, durchleuchtungs-und untersuchungszeit. durch den artefiziellen gefäßverschluss zum zeitpunkt der angioplastie kann der kollateralfluss der zielläsion zukünftig gemessen werden. einfluss von shunt-verkalkungen auf das überleben von hämodialysepatienten introduction: two-dimensional strain (2ds) is a novel method to measure strain from standard two-dimensional echocardiographic images. strain imaging has been proposed as a sensitive tool for the assessment of the left ventricular myocardial function. the aim of our study was to characterize global and regional function abnormalities using this technique in patients (pts.) with constrictive pericarditis (cp) and restrictive cardiomyopathy (rcm). methods: we studied 52 consecutive patients (pts.) with heart failure of either proven pericardial (cp) or myocardial origin (rcm; biopsy proven cardiac amy-loidosis). global longitudinal strain (gls) and regional peak systolic strain (pss) was assessed by 2ds in the apical four-chamber-view using a dedicated software package (vivid 7, ge healthcare). all pts. underwent a complete echocardiographic and hemodynamic assessment. results: out of the 52 pts. (mean age: 60±12 years) 29 had cp, and 23 rcm. the thickness of the interventricular septum (ivsd) was significant increased in pts. with rcm (17±5 vs. 9±1 mm). mean gls was -11.2±3.9% in the cp-group and -7.7±2.9% in the rcmgroup (p<0.01). pts. with rcm showed a significant decreased longitudinal pss in the septal segments (basal: -5.7±4.6% vs. -17.5±6.3%, p<0.01, mid: -8.8±3.9% vs. -18.5±4.7%, p<0.01; apical: -11.5±5.7% vs. -16.2±6.7%; p<0.05) and a decreased pss in the lateral segments (basal: -8.8±5.2% vs. -12.4±6.6%, n.s.; mid: -6.9±3.4% vs. -7.7±6.3%, n.s.; apical: -6.4±3.5% vs. -10.0± 6.9%, n.s.). conclusion: two-dimensional strain is a simple and rapid method to measure gls and pss. this technique might be used as new helpful tool for the differentiation between pts. with rcm and cp, and for the detection of early regional myocardial dysfunction before the onset of congestive heart failure (chf) in patients with cardiac amyloidosis. nichtinvasive koronare plaquedifferenzierung: mehrschicht-computertomographie validiert mit intravaskulärem ultraschall über die klassischen risiken hinaus sind auch lokale faktoren an der initiation und progression von atherosklerotischen ablagerungen beteiligt. diese faktoren beinhalten multiple variablen wie wechselwirkungen zwischen gewebe und flüssigkeiten, wandspannung und flussgeschwindigkeit . die direkte messung dieser faktoren ist in-vivo nur im tierversuch möglich. entsprechende parameter können jedoch über den einsatz moderner bildgebungstechniken (cardio-cta) unter verwendung von flusssimulationen (computational fluid dynamics=cfd) berechnet werden. das ziel dieser studie war daher 1) die durchführbarkeit der in-vivo cfd-berechnung an humanen koronararterien zu demonstrieren und 2) die ergebnisse mit radiofrequenzmessungen im intravaskulären ultraschall (ivus) zu korrelieren. methoden und ergebnisse: zehn patienten mit suspektem koronarem befund wurden prospektiv in die studie einbezogen. diese erhielten innerhalb von vier wochen eine ct-gestützte koronarangiographie (dual source 64 slice ct) und eine invasive koronarangiographie. bei diesen patienten wurde die intravaskuläre ultraschalluntersuchung in allen drei hauptstammgefäßen durchgeführt, die erhebung der radiofrequenzdaten geschah simultan. mit hilfe der axialen cta-schnitte wurde ein gittermodell der jeweiligen gefäße erstellt. dieses konnte genutzt werde um die gewebeflüssigkeitinteraktionen, flussgeschwindigkeit und wandspannungsverhältnisse zu simulieren. die berechnung der cfd-parameter konnte in 24/30 herzkranzarterien erfolgreich durchgeführt werden. siebzehn koronararterien wurden zusätzlich durch ivus beurteilt. die wandspannung der gefäße korrelierte umgekehrt zu vorhandenen durch ivus ermittelte plaques(p<0,05). ein zusammenhang zwischen den erhobenen cfd-daten und den radiofrequenzdaten der gewebe konnte bisher nicht nachgewiesen werden. zusammenfassung: die ergebnisse dieser studie demonstriert die möglichkeit gewebeflüssigkeitsinteraktionen in menschlichen koronararterien, mit hilfe von modernen computertomographischer bildgebung, nichtinvasiv abzuschätzen. die bedeutung dieser zusätzlichen informationen muss prospektiv weiter untersucht werden. welchen einfluss hat die myokardiale fibrose und funktion bei patienten mit hochgradiger aortenklappenstenose auf den klinischen langzeitverlauf nach aortenklappenersatzoperation? ziel dieser studie ist es, die entwicklung des linksventrikulären remodellings sowie der regionalen myokardialen funktion bei patienten mit hochgradiger aortenklappenstenose sowohl vor als auch im verlauf nach aortenklappenersatzoperation zu untersuchen. methodik: bei 65 patienten mit hochgradiger aortenklappenstenose wurde sowohl präoperativ als auch 9 monate postoperativ eine konventionelle echokardiographie und eine strain-rate-imaging studie mit bestimmung der longitudinalen systolischen spitzen-strain-rate durchgeführt. zur beurteilung der myokardialen fibrose wurde eine magnetresonanz tomographie (mrt) mittels gadolinium late enhancement technik durchgeführt und eine biopsie aus dem linksventrikulären septum intraoperativ entnommen. ergebnisse: die patienten wurden entsprechend der nyha-klasse 9 monate nach der operation in drei gruppen aufgeteilt. gruppe1 (gutes outcome=nyha-klasse i; n=22), gruppe 2 (mittleres outcome, nyha-klasse ii; n=24), gruppe 3 (=schlechtes outcome, nyha -klasse iii bzw. iv; n=19). präoperativ zeigten sich bei allen drei gruppen eine vergleichbare ejektions-fraktion (gruppe 1=55±13%; gruppe 2=51±13%; gruppe 3=51±13%). wohingegen bei der longitudinalen strain rate signifikant höhere werte in gruppe 1 als in gruppe 3 gemessen werden konnten (gruppe 1=1,32±0,2 s -1 ; gruppe 2=0,98±0,2 s -1 ; gruppe 3=0,58±0,2 s -1 ). interessanterweise stieg die longitudinale strain rate erst mit der abnahme der wandstärken nach 9 monaten an. außerdem konnte ein signifikant höherer grad an interstitieller fibrose mittels biopsie-score (gruppe 1=0,3±0,5; gruppe 2=0,5±0,8; gruppe 3=1,6±0,5) sowie auch ein häufigeres late enhancement in der mrt bei patienten mit schlechtem klinischem outcome nachgewiesen werden (segmente mit fibrose: gruppe 1=0,6±0,4; gruppe 2=0,9±0,3; gruppe 3=1,9±0,3). zusammenfassung: diese daten lassen vermuten, dass bei patienten mit hochgradiger aortenklappenstenose sowohl eine myokardiale fibrose als auch eine reduzierte regionale myokardiale funktion präoperativ entscheidene hinweise auf den klinischen langzeit-verlauf nach aortenklappenersatzoperation geben können. indikationen und limitationen der ultraschall-elastographie bei patienten mit erkrankungen des pankreas vor dem hintergrund immer wirksamerer, aber auch sehr kostenintensiver behandlungsoptionen einerseits und dem bewusstsein der früh einsetzenden strukturellen schädigung im arthritischen prozess ("window of opportunity") andererseits, ist ein monitoring des therapieansprechens bei patienten mit rheumatoider arthritis von besonderer bedeutung. eine exakte darstellung des entzündlichen substrates gelingt besonders mit der sonographie, wodurch therapieversager frühzeitig identifiziert werden können. die arthrosonographie erlaubt eine sehr gute differenzierung zwischen exsudativen und proliferativen synovialisveränderungen sowie die beurteilung der perfusion durch die doppler-verfahren. voraussetzung für eine sonographische beurteilung des verlaufes des arthritischen prozesses ist eine quantifizierung der entzündungsaktivität. in anlehnung an den von scheel und kollegen publizierten synovitisscore, wurden patienten mit aktiver rheumatoider arthritis, die durch eine biologikatherapie (tnf-inhibitoren, rituximab, abatacept) in klinische remission gebracht werden konnten (das28: <2,6), standardisiert sonographisch nachuntersucht. bei der mehrheit der ra-patienten konnte trotz klinischer remission durch die sonographischen nachuntersuchungen 3 und 6 monate nach initiierung der biologikatherapie eine persistierende entzündungaktivität nachgewiesen werden. während eine deutliche therapiebedingte abnahme der hyperperfusion zu beobachten war, konnte nur eine geringgradige abnahme der synovialen proliferationen und ergussbildung festgestellt werden. diese ergebnisse zeigen, dass bei der mehrzahl der ra-patienten trotz klinischer remission eine sonographisch nachweisbare snovitis persistiert, was möglicherweise die ursache für den radiologischen krankheitsprogress bei einem teil dieser patienten darstellt. zielsetzung/aims: granulocyte-colony stimulating factor (g-csf) was shown to improve cardiac function after myocardial infarction (mi). recently, we histologically demonstrated enhanced arteriogenesis and reduced infarct size by g-csf treatment after mi in mice. in this study, we non-invasively, repetitively, and quantitatively investigated g-csf effects on perfusion by a pinhole single-photon emission computed tomography (spect) system after mi in mice. methoden/methods: mi was induced by lad ligation in wt mice (c57bl/6j). g-csf (100 μg/kg; n=5) or saline (n=4) was daily injected for 5 days. extent of left ventricular (lv) perfusion defects was determined with a [99mtc]-sestamibi triple headed pinhole spect system at 6 days (baseline) and 30 days after lad occlusion. polar maps were normalized by mean of a standardized reference region of interest (roi) in the septal region (100%). best threshold value for identifying infarcted areas was determined by comparing perfusion defects with the histological infarct sizes in these animals. defect size was indicated as% of lv myocardium. primary endpoint was change of defect size from baseline to 30 days after mi. ergebnisse/results: best threshold for identifying infarcted areas compared to histology was less than 65% of the septal reference roi. mean infarct size was similar in saline (20,55% ± 0,90) and g-csf group (19,16% ± 1,10) at baseline. at day 30 after mi, a slight difference (p=0,05) was found between saline (20,24 ± 0,84) and g-csf (16,54% ± 1,24) groups. however, change of defect size was significantly different (p=0,02) between g-csf (-2,63% ± 0,76) and control animals (-0,31 ± 0,14) . schlussfolgerung/conclusions: this is the first study, demonstrating the suitability of triple headed pinhole spect system for repetitive infarct size measurement in mice, offering a new non-invasive imaging technique for cardiovascular therapy monitoring. in this context, we showed that g-csf administration significantly reduces lv perfusion defects indicating positive effects on ventricular remodelling after mi in mice. endoscopy in patients with acute leukemia after intensive chemotherapy conclusions: endoscopy can be performed relatively safely in patients who received myelosuppressive chemotherapy. the procedure may induce fever and bacteremia. the percentage of patients in whom endoscopic hemostasis was applicable and effective was low. we recommend conservative treatment first. endoscopy should be reserved for patients who are unstable or refractory. impact of pregnancy on prevalence of goitre and nodular thyroid disease in women living in a region of borderline iodine deficiency purpose: an interplay of genetic, epigenetic and environmental factors contributes to thyroid disease. in a cross-sectional study we aimed to determine the actual influence of parity on goitre and nodular thyroid disease (ntd) in women living in a region with borderline iodine deficiency. methods: thyroid ultrasonography (7.5mhz; merck thyromobil) was performed by the same investigator in 731 women living in thuringia and saxony. furthermore, age and bmi were documented and all women were asked about the number of previous pregnancies, family history of thyroid disease and past or present smoking. results: goitre prevalence was 17.6%. solitary thyroid nodules were detected in 21.2%, multiple nodules in 24.6% of the study population. age was positively correlated with goitre prevalence and ntd (due to multiple but not solitary nodules). no association was found between parity and goitre prevalence. in contrast, a significant increase in both, solitary (22.2%) and multinodular thyroid disease (23.7%) was observed in women with at least one pregnancy compared to nullipara (11.9% and 16.9%, respectively). bmi in women with goitre (27.3kg/ m2) was significantly higher than in women without (24.8kg/m2). in addition, a significant correlation was detected between bmi and presence of multinodular disease (26.5kg/m2). 24,5% of women with goitre reported a positive family history for thyroid disease, as opposed to 15% of women with normal size thyroid gland. neither goitre nor ntd were associated with a present or past history of smoking. conclusion: ntd and/or goitre are present in up to 50% of woman in an area with borderline iodine deficiency. besides age, bmi and family history, parity is positively correlated with presence of ntd, whereas smoking was not associated with goitre/ntd. defizite in der medizinischen versorgung von patienten mit nebennierenkarzinom in deutschland 3 1 kardiologie, universität heidelberg, heidelberg; 2 koordinierungszentrum für klinische studien leipzig, universität leipzig, leipzig; 3 kardiologie, universität würzburg, würzburg; 4 institut für herzkreislaufforschung, dortmund; 5 kardiologie, universität marburg, marburg; 6 kardiologie, berlin; 7 institut für frauenspezifische gesundheitsforschung, deutsches herzzentrum berlin, berlin; 8 kardiologie, universität göttingen, göttingen; 9 kardiologie, essen einleitung: ältere patienten und frauen sind in großen klinischen studien zur chronischen herzinsuffizienz regelhaft unterrepräsentiert. die vorliegende arbeit analysiert daher die geschlechts-und altersabhängige leitlinien-ädhärenz im rahmen der erhebungen des kompetenznetzwerkes herzinsuffizienz. methodik: wir evaluierten die datensätze aller patienten mit systolischer herzinsuffizienz (lvef echokardiographisch ≤45%) die zwischen 01/2004 und 06/2007 in das kompetenznetzwerk herzinsuffizienz eingeschlossen wurden: n=2333; mittleres alter 62,9±13,5 jahre; 24,8% frauen. die nyha-angepasste, leitliniengerechte verschreibung der pharmakotherapie (=adhärenz) wurde über die einnahmehäufigkeit der herzinsuffizienzmedikation unter berücksichtigung von kontraindikationen erfasst. der guideline-adherence-indicator (gai) wurde für betablocker, ace-hemmer/at1-blocker und aldosteron-rezeptorblocker berechnet (=gai-3), sowie für die zusätzliche indikation für diuretikum und glykosid (=gai-5). ergebnisse: der gai-3 betrug in den nyha klassen i/ii/iii/iv 94/92/88/85% und der gai-5 94/91/72/72% (p für trend jeweils <0.001). die therapie-adhärenz (gai-3 und gai-5) war bei männern höher als bei frauen (p=0.048 und p<0.001). in der multivariablen ordinalen regression waren zwar alter (or pro dekade 0, 91, p=0, 017; und or 0, 79, p<0, 001) und nyha stadium iii-iv (or 0,59 und 0,13, p jeweils <0,001), nicht jedoch das geschlecht (p=0,24 und p=0,53) prädiktoren des gai-3 und gai-5. zusammenfassung: aktuelle daten des kompetenznetz herzinsuffizienz zeigen bei der therapie der chronischen herzinsuffizienz im vergleich zu früheren studien in deutschland hinsichtlich der verordneten substanzklassen eine klinisch hoch relevante zunahme der leitlinien-adhärenz. in zukünftigen studien wird zu klären sein, ob auch leitliniengerechte dosierungen in der klinischen praxis sinnvoll umsetzbar sind. effekt von nt-probnp purpose: the endothelial specific angiopoietin (ang)-tie ligand-receptor system has a key role in regulating vascular integrity and quiescence. the antagonistic ligands ang-1 and ang-2 control the expression of endothelial adhesion molecules and intercellular tight junctions. the role of ang-2 in anca-associated vasculitis (aav) has not been investigated yet. methods: we measured serum ang-2 in 20 healthy controls and 40 patients with aav (15 patients with active aav at initial presentation and during follow-up, 10 patients in stable long-term remission, 10 patients prior to systemic relapse, and 5 patients with active "limited" wg (ent). the disease activity was monitored by the birmingham vasculitis activity score (bvas) and the enumeration of circulating endothelial cells (cecs). for statistical analysis we used one-way anova with dunn´s correction, friedmann and wilcoxon tests, spearman´s rho test and linear regression. results: ang-2 was elevated in active aav (mean 6.1 ± 3.5 ng/ml sem) compared to controls (1.2 ± 1.0 p<0.05). most notably, ang-2 was also elevated in archival serum samples of patients in long-term remission just before systemic vasculitic relapse (2.9 ± 2.2 ng/ml p<0.05). in contrast, ang-2 was normal in patients with stable remission of aav (1.7 ± 0.7 ng/ml and "limited" granulomatous ent disease (1.3 ± 0.5 ng/ml). ang-2 was elevated at initial presentation (6.1 ± 3.5 ng/ml sem) and declined rapidly after immunosuppessive therapy at 1 month (2.4 ± 1.4 ng/ml), 2-3 months (2.7 ± 2.3 ng/ml) and 6 months (1.4 ± 1.0 ng/ml). linear regression analysis demonstrated a strong association of ang-2 with bvas (rs2 = 0.49 p<0.0001) and cecs and (rs 2 = 0.48 p<0.001). these results indicate that ang-2 might be a useful early marker to discriminate systemic vasculitic activity in aav from limited granulomatous ent disease and remission. moreover, ang-2 might also be a mediator of endothelial inflammation and detachment. further investigation of the ang/tie-system in vasculitis is crucial since pharmacologic inhibitors of ang-2 will become available shortly. warfarin-induzierte kalzifikation in mäuseneine neues modell zur untersuchung der interaktion von verkalkungsinhibitoren aim of the study: although hypercholesterolemia, a predominant risk factor of coronary heart disease, is related to cholesterol metabolism, the association between cholesterol metabolism and coronary heart disease is not well known. therefore, this study investigates effects of cholesterol metabolism on coronary heart disease and family history of cardiovascular diseases. methods: in addition to conventional coronary risk factors (age, sex, bmi, arterial hypertension, diabetes mellitus, smoking, family history of cardiovascular diseases, plasma cholesterol) campesterol and sitosterol (indicators of cholesterol absorption) and lathosterol (indicator of cholesterol synthesis) were determined in 40 consecutive men and women with severe aortic stenosis. on coronary angiograms prior to aortic valve replacement extend of coronary heart disease was determined (0, 1, 2, 3-vessel disease). furthermore, a semiquantitative score of the extend of coronary atherosclerotic lesions was determined. results: in patients with a positive history of cardiovascular diseases the ratio of campesterol to lathosterol was significantly increased (p<0.005). there was a significant increase in campesterol to lathosterol ratio in plasma with increasing extend of coronary heart disease (0, 1, 2, 3-vessel disease; p<0.05). furthermore, there was a positive correlation of coronary vessel score with the ratio of campesterol to lathosterol in plasma (r=0.52; p<0.001) and in aortic valve cusps (r=0.33; p<0.03), indicating that enhanced absorption and reduced synthesis is related to the extend of coronary heart disease. logistic regression analysis revealed that of all coronary risk factors tested the ratio of campesterol to lathosterol was the sole, significant predictor of coronary heart disease in this subset of patients (p<0.01). the results of this study suggest that in patients with severe aortic stenosis elevated ratios of campesterol to lathosterol are directly related to concomitant coronary heart disease and may serve as a predictor for coronary heart disease in humans. bei postmenopausalen frauen und bei männern mit rheumatoider arthritis und glukokortikoid-therapie besteht zu über 80 prozent die indikation für eine bisphosphonat-therapie nach dvo-leitlinie hintergrund: ein geringes ansprechen auf clopidogrel nach koronarer stentimplantation ist assoziiert mit einem erhöhten risiko für das auftreten von stentthrombosen. die cytochrom p450 (cyp) isoenzyme cyp2c19 and cyp3a4/5 sind bei der bioaktivierung von clopidogrel beteiligt. das ziel der vorliegenden studie ist es, den zusammenhang zwischen dem ansprechen auf clopidogrel und genetischer varianten der cyp isoenzyme bei patienten mit symptomatischer koronarer herzerkrankung zu untersuchen. methoden und ergebnisse: genotypisierungen für cyp2c19 (*2, *3, *17), cyp3a4*1b und cyp3a5*3 polymorphismen wurden bei konsekutiv eingeschlossenen patienten (n=237), die einer koronare stentimplantation aufgrund einer symptomatischen koronaren herzerkrankung erhielten, durchgeführt. die adenosin diphosphat (adp)-induzierte thrombozytenaggregation wurde frühestens 6 stunden nach erstgabe von 600 mg clopidogrel gemesen. träger der cyp2c19*2 variante zeigten eine signifikant höhere residuelle thrombozytenaggregation (rta) (chi-quadrat 29; p<0.0001). die übrigen untersuchten polymorphismen zeigten keinen signifikanten einfluß auf die rta. auf der grundlage des predict-score zur vorhersage einer erhöhten rta wurden der cyp2c19*2 genotyp und zuvor identifizierte nicht-genetische einflussvariablen (alter, akutes koronarsyndrom, typ 2 diabetes, reduzierte linksventrikuläre funktion, und niereninsuffizienz) untersucht. die logistische regressionsanalyse ergab eine signifikante korrelation der nicht-genetischen risikofaktoren (chi-quadrat 5,3; p=0,021) und des cyp2c19*2 polymorphismus (chi-quadrat 21,3; p<0,0001) mit einer erhöhten rta, und einen kumulative assoziation zwischen rta und der kombination aus genetischen und nicht-genetischen faktoren (chi-quadrat 25,85; p <0,0001). diskussion: träger von zumindest einem cyp2c19*2 allel haben ein ungefähr 4-faches risiko, eine erhöhte rta zu entwickeln. genotypisierung der funktionsverlust-variante cyp2c19*2 könnte zu einer verbesserten prädiktion des ansprechens auf die antithrombozytäre therapie mit clopidogrel beitragen. the shape of the glucose curve during an oral glucose tolerance test correlates with changes in glucose tolerance m. reimann 1 , j. li 1 , s. bornstein 1 , j. schulze 2 , p. schwarz 1 1 medical clinic iii, endocrinology, medical faculty carl gustav carus, technical university dresden, dresden; 2 sächsische landesärztekammer, dresden introduction and objectives: the shape of the glucose curve during an oral glucose tolerance test (ogtt) can be categorized in monophasic and biphasic. the latter has been associated with normal glucose tolerance. the aim of the study was to explore the association between the shape of the glucose curve and changes of glucose tolerance. research design and methods: ogtt data from 615 subjects with different stages of glucose tolerance were analyzed at baseline and three years follow up. the shape of the glucose curve at baseline was classified as monophasic, biphasic and unclassified. the shape index was calculated as the difference between glucose at 120 min and at 90 min and treated as continuous variable in correlation analyses. in the biphasic group, there was a significantly higher proportion of subjects with normal glucose tolerance and a lower proportion of subjects with impaired glucose tolerance and type 2 diabetes. subjects with a biphasic shape had a significant lower bmi and a better profile of carbohydrate metabolism. the shape index correlated significantly with changes of plasma glucose at baseline and at 120 min and insulinauc after adjustment for glucose tolerance state. the prevalence ratio for disease regression was significantly higher in subjects with biphasic glucose curve shape. the shape index gives additional information regarding the stage of glucose tolerance beyond the who classification. a biphasic shape is associated with improvements in plasma glucose and may predict regression of disease irrespective of glucose tolerance. die residuelle aggregation unter dualer thrombozytenhemmung beeinflusst die inzidenz schwerer kardiovaskulärer ereignisse unabhängig vom einsatz medikamentenbeschichteter stents kritisch kranke patienten weisen häufig eine charakteristische konstellation des thyreotropen regelkreises auf, die als non-thyroidal-illness-syndrom (ntis) bezeichnet wird und mit einer erhöhten morbidität und mortalität verbunden ist. trotz langjähriger forschung zu details dieses komplexes sind wichtige fragen noch immer offen. so ist bislang noch unbekannt, inwiefern vorbestehende schilddrüsenerkrankungen zur entstehung und ausprägung eines ntis beitragen. im rahmen der prospektiven aqua-fontis-studie untersuchten wir daher 164 patienten, die auf einer internistischen, einer chirurgischen und einer herzchirurgischen intensivstation des universitätsklinikums bergmannsheil versorgt wurden, bezüglich des auftretens von autoantikörpern gegen thyreoglobulin (tgak), schilddrüsen-peroxidase (tpo-ak) oder tsh-rezeptoren (trak). die antikörpertiter, die wir 24 stunden nach aufnahme auf die intensivstation bestimmten, korrelierten wir mit parametern der schilddrüsenhomöostase, der dauer des stationären aufenthaltes und dem überleben der patienten. über 98% der untersuchten patienten wiesen negative oder intermediäre tgakoder tpo-ak-titer auf, weniger als ein prozent zeigten positive antikörpertiter. ausnahmslos alle untersuchten patienten waren trak-negativ. die höhe sämtlicher antikörpertiter korrelierte weder mit dem auftreten einer thyreotropen insuffizienz noch mit der dauer des stationären aufenthaltes oder der intensivbehandlung und auch nicht mit dem überleben der betroffenen patienten. auch in der subgruppe der überlebenden patienten korrelierten die antikörpertiter nicht mit der dauer der behandlung. es kann daher festgestellt werden, dass bei kritisch kranken personen schilddrüsenautoantikörper mit annähernd der gleichen prävalenz wie bei einer normalpopulation auftreten. darüber hinaus haben autoimmunthyreopathien keinen stellenwert in der prognose von patienten mit ntis. typ-2-diabetes mittels ekg identification of markers for prediction of the clinical course remains a major challenge in the management of diabetic nephropathy. we established a proteomics approach for identification of diabetic nephropathy related biomarkers in urine. we used seldi-tof mass spectrometry and sax2 protein arrays to compare protein profiles from urine of four defined patient groups. samples from patients with type 2 diabetes (dm) (n = 45) without nephropathy and without microalbuminuria (dm-wnp), dm patients with macro-or microalbuminuria (dm-np) (n = 38), patients with proteinuria due to non-diabetic renal disease (n = 34), and healthy controls (n = 45) were analysed. anionic exchange, reversephase fractionation, gel electrophoresis, and mass spectrometry were used to isolate and identify proteins with high discriminatory power. a protein with m/z 6188 (p <0.0000004) was strongly released in the urine of healthy controls, patients with proteinuria due to non-diabetic disease, and dm-wnp in contrast to dm-np-patients. a m/z 14766 protein (p <0.00008) was selectively excreted in the urine of dm-np patients, whereas the protein with m/z 11774 (p <0.000004) was significantly excreted by patients with proteinuria and dm-np. the m/z 11774 and m/z 14766 mass peaks were identified as beta-2-microglobulin and uba52, an ubiquitin ribosomal fusion protein respectively. the protein with m/z 6188 was identified as a processed form of ubiquitin. moreover the ubiquitin degradation assay confirmed the potential role of a urinary protease whose absence was specific for diabetic nephropathy. the release of high amounts of uba52 in urine of dm-np patients could serve as a diagnostic marker. the identification of the protease and longitudinal studies in larger patient groups will determine the usefulness of the short form of ubiquitin as a marker for predicting the clinical course and the potential role of the protease in the pathophysiology of diabetic renal involvement. rolle von freien fettsäuren und fettsäuretransportproteinexpression in der apoptosevermittelten pathogenese der fettlebererkrankung extravascular lung water index (elwi) has been demonstrated to predict mortality and to correlate to pao2/fio2-ratio and compliance in patients with sepsis and ards. however, with an increasing number of obese patients, there is the question which body weight should be used for indexation of extravascular lung charité -universitätsmedizin berlin, campus virchow-klinikum, berlin; 2 koordinierungszentrum für klinische studien würzburg therefore it was the aim, to investigate the correlation of elwi to pao2/ fio2-ratio and oxygenation index (mean airway pressure* 100/pao2) using different weight parameters for indexation. methods: in 25 patients of a medical icu with a body mass index >25kg/m2, 260 measurements of extravascular lung water were performed using the picco system elwi correlated to the functional parameters with high significance (p<0.001) independently of the the index used: correlation to pao2/fio2-ratio conclusions: 1.) in obese patients, extravascular lung water and its indices adjusted to abw, pbw, ibw and adpw significantly (p<0.001) correlate to pao2/ fio2-ratio and oxygenation index. 2.) the highest correlation to pao2/fio2-ratio was found using adbw unsere fallsammlung zeigt den facettenreichtum seltener pilzinfektionen. weitere zentren sind eingeladen, ihre fälle unter www.fungiscope.net zu registrieren ps47, ps60, ps99 ps3, ps15, ps16 ps31, ps42, ps46 ps254, ps255, ps258, ps259 ps237 trinkmann, frederik . . ps95, ps174 ps58 van den elsen antithrombozytäre effekte von ace-hemmern und at1-blockern: modifizierte ex-vivo-plättchenaggregation bei 418 kardiovaskulären patienten a. viktor 1 , i. tuleta 2 , m. steinmetz 2 , g. bauriedel 3 , g. nickenig 2 , d. skowasch 2 1 abteilung für kardiologie und pulmologie, zentrum für innere medizin, stuttgart; 2 innere medizin (kardiologie/pneumologie), universitätsklinikum bonn, medizinische klinik ii, bonn; 3 innere medizin (kardiologie), klinikum meiningen, medizinische klinik i, meiningen hintergrund: ace-hemmer und at1-rezeptorblocker (arbs) sind eckpfeiler in der therapie kardiovaskulärer patienten. in mehreren randomisierten und plazebo-kontrollierten studien konnte eine signifikante verminderung von kardiovaskulärer mortalität, myokardinfarkt-und schlaganfallrate nachgewiesen werden. vor diesem hintergrund untersucht diese studie mögliche antithrombozytäre effekte von ace-hemmern und arbs; vergleichskollektive sind patienten mit ass/clopidogrel und unbehandelte patienten. methoden: proben von insgesamt 418 kardiovaskulären patienten wurden mittels vollblutaggregometrie analysiert. dabei war die agonisten-induzierte plättchenaggregation (adp, kollagen) durch die zunahme der impedanz (ohm) quantifiziert. die daten wurden mit vorliegen bzw. fehlen der medikation korreliert. ergebnisse: als zentraler befund war die plättchenaggregation bei studienteilnehmern mit ace-hemmern, arbs, ass und clopidogrel vermindert. die kollagen-induzierte plättchenaggregation wurde unter ace-hemmern (23,1%, p<0,001) und arbs (33,4%, p<0,001) signifikant reduziert; unter therapie ass (35,6%, p<0,001) bzw. ass/clopidogrel (26,9%, p=0,234) nahm sie ebenfalls ab. nach adp-induktion war die plättchenaggregation unter therapie mit ace-hemmern (36,6%, p=0,003) und arbs (38,7%, p=0,011) signifikant reduziert; unter ass (38,7%, p=0,007) und ass/clopidogrel (82,8%, p=0,011) zeigte sich ebenfalls eine reduktion. eine zusätzliche verlaufsbeobachtung nach neueinstellung mit dem wirkstoff valsartan zeigte nach 28 tagen eine signifikant reduzierte kollagen-induzierte plättchenaggregation (36%, p=0,006). eine in vitro-messreihe mit der rohsubstanz valsartan konnte für äquivalente therapeutische dosen keine signifikante beeinflussung zeigen. folgerung: die therapie mit arbs und ace-hemmern führt zu einer signifikanten verminderung der plättchenaggregation ex vivo. das antithrombotische potential der arbs und ace-hemmer könnte für die reduktion der konsekutiven thrombosen und der progression atherosklerotischer organleiden mitverantwortlich sein und so die reduktion kardiovaskulärer endpunkte zumindest miterklären. kombination einer dendritischen zellvakzine mit gemcitabin im murinen pankreaskarzinom:charakterisierung der immunantwort und wirksamkeit m. dauer with or without the tlr-9 ligand cpg before (prophylactic) or after (therapeutic) tumor induction. cd8+ t cell responses were analyzed in peripheral blood. antigen uptake, activation and cytokine production of dendritic cells (dc) in lymph nodes was analyzed by flow cytometry. data: vaccine draining lymph nodes increased in size and cellularity. the iscom vaccine was effectively taken up by dc, resulting in upregulation of activation markers, production of il-12 and potent t cell stimulation. key: cord-023419-lnmc6vv5 authors: steinhauer, c.; wingren, c.; borrebaeck, c. a. k. title: high‐throughput proteomics on antibody‐based microarrays: the importance of probe and surface design date: 2008-06-28 journal: scand j immunol doi: 10.1111/j.0300-9475.2004.01423ax.x sha: doc_id: 23419 cord_uid: lnmc6vv5 in analogy to dna microarrays, protein microarrays offer a new distinct possibility to perform sensitive high‐throughput global proteome analysis. however, the development of the protein microarray technology will place high demands upon the design of both probes and solid supports. the analysis of thousands of heterogeneous proteins on a single microarray requires the use of uniform probes, such as antibodies, directly designed for protein microarray applications. we have recently generated a human recombinant single‐chain fv antibody library, genetically constructed around one framework, the ncoder‐library, containing 2 × 1010 clones. single framework antibody fragments (sinfabs) selected from this library were successfully applied as probes for microarrays providing sensitive detection in the 600 attomol (mass spectrometry) and the 300 zeptomole range (fluorescence). however, the choice of framework is critical. we have shown that the selected ncoder framework displayed excellent functional on‐chip stability and arrayed dehydrated probes retained their activity for several months. furthermore, we have addressed the issues of biocompatibility of the solid support and immobilization strategies for our microarray setup. an in‐house‐designed substrate, macroporous silicon coated with nitrocellulose (map3‐nc7), displayed properties equal to, or better than, those of five commercially available supports used as reference surfaces. we have also evaluated different coupling strategies, such as adsorption, covalent coupling, diffusion and affinity coupling. using a novel affinity tag, the double‐(his)6‐tag, we increased the binding efficiency of sinfab‐molecules to ni2(+)‐coated solid supports, thereby allowing nonpurified probes to be directly applied. the nuclear receptor heterodimers of liver x receptors (lxrs) are recently identified as key transcriptional regulators of genes involved in lipid homeostasis and inflammation. lxrs and their ligands are negative regulators of macrophage inflammatory gene expression. multiple sclerosis (ms), a demyelinating disease of the central nervous system of unknown cause, is characterized by recurrent inflammation involving macrophages and their inflammatory mediators. sweden belongs to the countries with a high ms incidence. in italy, incidence is lower, with an exception for sardinia where the incidence is even higher than that in sweden. subjects from sardinia are ethnically more homogeneous and differ from swedes, also regarding genetic background and environment. we studied lxrs and their related molecules of blood mononuclear cells (mncs) from female patients with untreated relapsing-remitting ms from sassari, sardinia and stockholm, sweden. sex-and age-matched healthy controls (hcs) were from both areas. mrna expression was evaluated by real-time pcr. lxr-a was lower (p < 0.05) in ms (mean ae sem: 3.1 ae 0.2; n ¼ 37) compared to hc (3.6 ae 0.1; n ¼ 37). lxr-a was lower in ms from stockholm (2.6 ae 0.2; n ¼ 22) compared to corresponding hc (3.4 ae 0.1; n ¼ 22; p < 0.01) and compared to ms (3.8 ae 0.2; n ¼ 15; p < 0.001) and hc (4 ae 0.2; n ¼ 15; p < 0.001) from sardinia. ms patients from stockholm, but not from sassari, also expressed lower (p < 0.05) lxr-b (à4.1 ae 0.4) compared to corresponding hc (à2.9 ae 0.3). ms from stockholm was associated with higher abca-1 (6.1 ae 0.4 versus 5.0 ae 0.3; p < 0.05) and higher estrogen receptor-b-cx (2.4 ae 0.4 versus 0.8 ae 0.4; p < 0.01) compared to corresponding hc. the hc from sassari had higher androgen receptor (2.9 ae 0.2) compared to ms from sassari (1.4 ae 0.3; p < 0.01), ms (1.3 ae 0.4; p < 0.01) and hc from stockholm (1.2 ae 0.3; p < 0.01). ms from sassari had lower cyclooxygenase-1 compared to corresponding hc (5.1 ae 0.4 versus 6.6 ae 0.3; p < 0.01) and lower prostaglandin-e (à0.03 ae 0.5) compared to the hc (1.4 ae 0.5; p < 0.05) and ms (2.7 ae 0.4; p < 0.05) and hc from stockholm (1.9 ae 0.4, p < 0.001). our findings identify lxrs and their related molecules as being involved in ms from stockholm but not from sassari, while sex hormone receptors seem to be involved in ms in sassari. multiple sclerosis: ifn-b induces cd123 + bdca2 -dendritic cells that produce il-6 and il-10 and have no enhanced type i interferon production y. m. huang, 1 s. adikari, 1 u. båve, 2 a. sanna 1,3 & g. alm 4 dc antigens (bdca) and investigate their ability to produce type i ifn in response to virus stimulation. we show that ifn-b induces development of cd123 þ dc from human blood monocytes, which coexpress bdca4 þ but are negative for bdca2 -, a specific marker for plasmacytoid dc. such ifn-b-modulated dc produce large amounts of il-6 and il-10, but no il-12p40 and have no enhanced ifn-b and ifn-b production. the findings indicate that ifn-bmodulated dc represent a myeloid dc subset with diminished cd11c, bdca-1 and cd1a expression, having potent th2-promoting function but lacking antiviral capacity. the association of psoriasis with throat infections by streptococcus pyogenes suggests a potential antigenic target for the t cells that are known to infiltrate dermis and epidermis of psoriatic skin. streptococcal m protein shares an extensive sequence homology with human epidermal keratins. keratins 16 (k16) and 17 (k17) are mostly absent from uninvolved skin but are upregulated in psoriatic lesions. there is increasing evidence that cd8 þ t cells play an important effector role in psoriasis and m proteinprimed t cells may recognize these shared epitopes in skin via molecular mimicry. to identify candidate epitopes, peptides with sequences from k17 were selected on the basis of predicted binding to hla-cw6 and sequence similarities with m6 protein. matched peptides from the sequence of m6 protein and a set of peptides with poor predicted binding were also selected. cw6 þ individuals with psoriasis and cw6 þ healthy controls, having a family history of psoriasis, were recruited. pbmcs were incubated with the peptide antigens. t-cell activation in the cd4 þ , cd8 þ and later the skin-homing cutaneous lymphocyteassociated antigen (cla)-expressing subset of cd8 þ t cells was evaluated by cd69 expression and intracellular ifn-g accumulation using flow cytometry. we demonstrate that cw6 þ psoriasis patients had significant cd8 þ t-cell ifn-g responses to peptides from k17 and m6 protein selected on the basis of sequence homology and predicted hla-cw*0602 binding. these responses were about 10 times more frequent in the skin-homing cutaneous lymphocyte-associated antigen-expressing (cla þ ) subset of cd8 þ t cells. cd4 þ t cells showed only borderline responses. cd8 þ t cells from cw6 þ nonpsoriatic individuals responded to some m6 peptides but very rarely to k17 peptides, and this also applied to the cla þ cd8 þ subset. these findings indicate that psoriatic individuals have cd8 þ t cells that recognize keratin self-antigens and that epitopes shared by streptococcal m protein and human keratin may be targets for the cd8 þ t cells that infiltrate psoriatic skin lesions. autoantibodies directed against citrulline-containing proteins have an impressive specificity of nearly 100% in ra patients and a suggestive involvement in the pathogenesis. the targeted epitopes are generated by a post-translational modification catalysed by the calcium-dependent enzyme peptidyl arginine deaminase that converts the positively charged arginine to polar but uncharged citrullin. the aim of this study was to analyse the presence of citrulline in the joints at different time points of collagen-induced arthritis in da rats by immunohistochemistry and to investigate how immunogenicity and arthritogenicity was affected by citrullination of rat serum albumin (rsa) and collagen type ii (cii). our results indicate that citrulline could be detected in joints of arthritic animals, first appearance at the onset of disease and increasing as disease progressed into a chronic state. unimmunized animals or time points before clinical signs of arthritis were negative. by morphology, we state that some infiltrating macrophages as well as the cartilage surface stain positive for citrulline, while the major source of citrullinated proteins appears to be fibrin depositions. a specific cit-rsa t-cell response was observed in animals challenged by citrullinated rsa, no response was recorded when rsa was used as a stimulus. the igg analysis reveals not only a response towards the modified protein but also cross-reactivity to native rsa. no t-cell or b-cell response was noted in animals injected with unmodified rsa. cit-cii induced a disease with higher incidence and earlier onset than did the native counterpart. we conclude that, in contrast to the human disease, citrulline does not seem to appear before clinical signs. as inflammation proceeds, citrulline is detected specifically in the joints. all other organs investigated were negative. we also conclude that citrullination of a protein can break tolerance and increase its arthritogenic properties. ectopic germinal centers (gcs) can be detected in the salivary glands of approximately 1/5 of patients with sjögren's syndrome (ss) and appear in both primary and secondary ss. previously, ectopic gc have been associated with increased local autoantibody production. the aim of this study was to determine whether gc in primary sjögren's syndrome (pss) defines a distinct seroimmunological phenotype. retrospectively, a material of 130 haematoxylin and eosin-stained paraffin-embedded tissue sections of minor salivary gland tissue from patients with pss was morphologically screened for the presence of ectopic gc. gc-like lesions were detected in 33/130 (25%) of the pss patients. seventy-two pss patients lacking these structures (gc-) were randomly selected for comparison. focus score was significantly increased in the gc þ patients compared to the gcpatients (p ¼ 0.035). in the gc þ group, 54.5% of the patients presented with anti-ro/ssa compared to 43.7% in the gcgroup. anti-la/ssb was detected in 31.3% of the gc þ patients compared to 25.7% of the gcpatients. sixty-one percentage of gc þ patients presented with increased levels of igg, a nonsignificant difference when compared to 39.4% in the gcpatients (p ¼ 0.089). levels of rf, ana, ena, igm and iga were similar in both patient groups, as were esr and crp. in conclusion, patients with ectopic gc have a higher focus score and more often present with autoantibodies and increased levels of igg compared to pss patients with regular focal infiltration (gc -). our findings may indicate a certain seroimmunological phenotype and warrant for further prospective studies. association between mannose-binding lectin and vascular complications in type 1 diabetes complement activation and inflammation have been suggested in the pathogenesis of diabetic vascular lesions. we investigated serum mannose-binding lectin (mbl) levels and polymorphisms in the mbl gene in type 1 diabetic (t1dm) patients with and without diabetic nephropathy and associated macrovascular complications. polymorphisms in the mbl gene and serum mbl levels were determined in 199 t1dm patients with overt nephropathy and 192 t1dm patients with persistent normoalbuminuria matched for age, sex and duration of diabetes as well as in 100 healthy control subjects. the frequencies of high and low expression mbl genotypes were similar in patients with t1dm and healthy controls. high mbl genotypes were significantly more frequent in diabetic patients with nephropathy than in the normoalbuminuric group, and the risk of having nephropathy, given a high mbl genotype, assessed by odds ratio was 1.52 (1.02-2.27), p ¼ 0.04. median serum mbl concentrations were significantly higher in patients with nephropathy than in patients with normoalbuminuria [2306 mg/l (iqr 753-4867 mg/l) versus 1491 mg/l (iqr 577-2944), p ¼ 0.0003], and even when comparing patients with identical genotypes, serum mbl levels were higher in the nephropathy group than in the normoalbuminuric group. patients with a history of cardiovascular disease had significantly elevated mbl levels independently of nephropathy status [3178 mg/l (iqr 636-5231 mg/l) versus 1741 mg/l (iqr 656-3149 mg/l), p ¼ 0.02]. the differences in mbl levels between patients with and without vascular complications were driven primarily by pronounced differences among carriers of high mbl genotypes (p < 0.0001). our findings suggest that mbl may be involved in the pathogenesis of microvascular and macrovascular complications in type 1 diabetes and that determination of mbl status might be used to identify patients at increased risk of developing these complications. neuroimmunology unit, center for molecular medicine, karolinska institutet, stockholm, sweden. e-mail: judit.wefer@cmm.ki.se dna vaccine coding for the encephalitogenic peptide mog 91-108 protects lew.1av1 from subsequent development of experimental autoimmune encephalomyelitis (eae). protection is associated with a type 1 immune response and is dependent on the presence of cpg dna motifs. the mechanisms underlying the observed reduction of eae development in protected rats have not been fully clarified. we investigated immunological characteristics of lymphocytes after dna vaccinaton and subsequent eae induction. we confirm that protection was not associated with suppression of t1 cells, as transcription of the novel molecule rat t-cell immunoglobulin-and mucindomain-containing molecule (tim-3), reported to be exclusively expressed on differentiated t1 cells, was not altered by dna vaccination. we did not note any clonal deletion upon tolerization, but detected an antigen-specific lymphocyte population upregulating ifng upon recall stimulation 3 weeks after protective dna vaccination. in protected rats, we observed (1) no alterations in antigenspecific th2 or th3 responses, (2) reduced mhc ii expression on splenocytes early after eae induction, (3) antigen-specific upregulation of ifnb upon recall stimulation and (4) reduced il-12rb2 on lymphocytes. we thus demonstrate an association of the protective effect of dna vaccination with expression of ifnb. we are currently investigating the cellular mechanisms behind this ifnb-mediated protection. multiple sclerosis (ms) is an autoimmune condition characterized by degeneration of nerve fibre myelin sheets. a candidate autoantigen, myelin basic protein (mbp), has especially attracted attention. the presence of anti-mbp antibodies is a predictor of definite ms, but their role in the pathogenesis remains obscure. t cells have long been known to play a pivotal role in the pathogenesis of ms. recently, an important role for b cells as autoantigen-presenting cells has been demonstrated in other autoimmune diseases, including rheumatoid arthritis and diabetes. the uptake of mbp by b cells and the presentation of mbp-derive peptides to t helper (th) cells by b cells may be promoted by the formation of complement (c) activating immune complexes (ics) between mbp and natural autoantibodies in healthy individuals and disease-associated anti-mbp antibodies in ms patients, respectively. we have investigated the formation of mbp-containing ic, the binding of mbp to b cells, the mbp-elicited induction of th-cell and b-cell proliferation and the cytokine production in peripheral blood mononuclear cells (pbmcs) from healthy donors grown in the presence of intact or c-inactivated serum from healthy donors or patients with ms. while mbp did not induce measurable proliferation of b cells nor cd4 þ t cells, we observed the production of tnf-a, ifn-g and il-10 by pbmc in response to incubation with mbp in the presence of sera from healthy controls as well as sera from ms patients. by contrast, no production of il-2, il-4 and il-5 was detected. we are currently investigating the capability of ms sera to promote the formation of mbp-containing ic and thereby enhance the cytokine responses, by virtue of elevated anti-mbp contents. the phagolysosomally localized acid sphingomyelinase (asmase) activated by proinflammatory cytokines such as tnf and ifn-g generates the signalling molecule ceramide which in turn results in the activation of proteases like cathepsin d. these characteristics of asmase suggest a possible role of this molecule in the phagocytotic uptake and phagosomal degradation processes of antigens or in antigen presentation. we show here that asmase -/mice fail to eliminate the noncytopathic lymphocytic choriomeningitis (lcm) virus as rapidly as littermate wildtype mice. investigation of the immune response revealed a reduced expansion of cd8 þ t cells. the secretion of ifn-g in response to contact with target cells as well as the cytolytic activity of virus-specific cd8 þ t cells was severely impaired. additionally, both phases of the lcm virus-specific dth response, mediated by cd8 þ and cd4 þ t cells consecutively, were diminished in asmase -/mice. however, the secondary memory response of virus-specific ctl was not altered, and the 614 abstracts ................................................................................................................................................................................................. virus was effectively controlled for at least 3 months by asmase -/mice. in conclusion, the results of this study suggest an involvement of the asmase in the activation, expansion or maturation of virus-specific cd8 þ t cells during the acute infection of mice with the lcm virus. novel markers for alternative activation of macrophages: macrophage galactose-type c-type lectins 1 and 2 in parallel with the th1/th2 dichotomy, macrophages are capable of developing into functionally and molecularly distinct subpopulations, due to differences in, for example cytokine environment and pathological conditions. while the best-studied, classically activated macrophage is induced by type i stimuli such as ifn-g, a type ii cytokine environment antagonizes the classical activation of macrophages and is capable of alternatively activating macrophages. however, molecular markers associated with these type ii cytokine-dependent, alternatively activated macrophages remain scarce. besides the earlier documented markers macrophage mannose receptor and arginase 1, we recently demonstrated that murine alternatively activated macrophages are characterized by increased expression of fizz1 and ym. we now report that expression of the two members of the mouse macrophage galactose-type c-type lectin gene family, termed mmgl1 and mmgl2, is induced in diverse populations of alternatively activated macrophages, including peritoneal macrophages elicited during infection with the protozoan trypanosoma brucei or the helminth taenia crassiceps, and alveolar macrophages elicited in a mouse model of allergic asthma. we also demonstrate that, in vitro, interleukin-4 and interleukin-13 upregulate mmgl1 and mmgl2 expression and that, in vivo, induction of mmgl1 and mmgl2 is dependent on interleukin-4 receptor signalling. moreover, we show that regulation of mgl expression is similar in human monocytes and monocyte-derived macrophages. hence, macrophage galactose-type c-type lectins represent novel markers for both murine and human alternatively activated macrophages; thus, paving the way for further characterization of the phenotype of macrophages occurring in th2 conditions. background: human parvovirus b19 (b19) is a ubiquitous pathogen, normally causing a mild self-limiting disease, but also capable of causing both significant pathology and long-term persistence. the small size and stability of the virus makes it suitable for mapping of the full breath and the kinetics of the cellular immune responses following acute viral infection. methods: five patients with acute primary b19 infection were included in the study and followed consecutively for up to 200 weeks. cellular immune responses were mapped by ifng enzyme-linked immunospot to overlapping peptides spanning the whole b19 genome. results: in all five acutely infected patients, we were able to monitor the kinetics of a strong specific cellular immune reaction. responses peaked at levels of 850-1850 sfc/ million pbmcs, roughly corresponding to 0.3-0.6% b19specific cd8 þ cells circulating in peripheral blood at 10-80 weeks post-infection. the responses in individual patients were directed to three or four different peptide pools, and the specificity was confined to the same cd8 epitopes present in the pools throughout the follow-up period. the majority of responses were directed to the virus nonstructural protein, only two patients showed any response to the capsid proteins, elicited by the same epitope in both cases. conclusion: the cellular immune responses to acute b19 infection are surprisingly narrow in distribution and remain at high levels for up to 80 weeks post-infection. the initial epitope specificity is maintained, and the majority of responses target the virus nonstructural protein, which is not included in vaccine preparations, evaluated against the infection. the relationship between malnutrition and malaria is controversial. on one hand, malaria may cause malnutrition, while on the other, malnutrition itself may modulate susceptibility to the disease. we investigated the association between plasmodium falciparum malaria and malnutrition in a cohort of children living on the coast of kenya. the study involved longitudinal follow-up for clinical malaria episodes and anthropometric measurements at four cross-sectional surveys. we used poisson regression analysis to investigate the association between malaria and nutritional status. compared to baseline (children with a waz or haz score of !à2), the crude incidence rate ratios (irrs) for malaria in children with low haz or waz scores (<à2) during the period prior to assessment were 1.17 (95% ci 0.91-1.50; 0 ¼ 0.21) and 0.94 (0.71-1.25; 0.67), respectively, suggesting no association between malaria and the subsequent development of pem. however, we found that age was acting as an effect modifier in the association between malaria and malnutrition. the irr for malaria in children 0-2 years old who were subsequently characterized as wasted was 1.65 (1.10-2.20; p ¼ 0.01), and a significant overall relationship between malaria and low-haz was found on regression analysis when adjusting for the interaction with age (irr 1.89; 1.01-3.53; p < 0.05). although children living on the coast of kenya continue to suffer clinical episodes of uncomplicated malaria throughout their first decade, the association between malaria and malnutrition appears to be limited to the first 2 years of life. a. astrinidou-vakaloudi, 1 s. xytsas, 1 i. diamanti, 1 h. ioannidis 2 & p. pangidis 2 1 microbiology department of general hospital of thessaloniki 'agios pavlos', thessaloniki, greece, and 2 nefrology, 2 nd ika hospital of thessaloniki, thessaloniki, greece. e-mail: stasa@hol.gr aim: renal dysfunction may influence the colonization of gastric mucosa by urea-splitting bacteria such as helicobacter pylori, by increasing urea concentrations in the gastric juice. our aim was to investigate the prevalence of h. pylori in patients with end-stage renal disease (esrd), receiving long-term haemodialysis treatment. methods: this study included 40 sera from patients with esrd (29 male and 11 female) undergoing periodic haemodialysis; mean time of treatment was 42.6 months. using elisa technique, we investigated the presence of igg and iga antibodies against h. pylori as well as igg caga (antibodies specific for caga(þ) strains of h. pylori). sera from 40 healthy blood donors were used as a control group. results: h. pylori igg antibodies were detected in 32 out of 40 (80%) patients in the dialysis group, while 31/40 (77.5%) tested positive for iga. igg caga antibodies were present in 13 out of 40 (32.5%). prevalence of h. pylori igg, iga and caga igg antibodies in the control group was 33, 7 and 15%, respectively. conclusions: although international data suggest that prevalence of h. pylori infection is the same in esrd patients as in healthy individuals, in our study that seems not to be the case. the higher blood and gastric juice urea levels may be a risk factor (among many others), but more studies are required in order to understand the relation of h. pylori infection in this group of patients. flanders interuniversity institute for biotechnology, department of molecular and cellular interactions, free university of brussels, brussels, and 2 pasteur institute of brussels, mycobacterial immunology, brussels, belgium. e-mail: tgartner@vub.ac.be immunity against tuberculosis (tb), caused by mycobacterium tuberculosis, depends largely on activation and maintenance of strong cell-mediated immune responses involving both cd4 þ and cd8 þ t cells and the ability to respond with th1-type cytokines, particularly ifn-g. recent studies suggested that bcg, the only licensed vaccine against m. tuberculosis, may fail to induce t-cell responses in the lung mucosa and may therefore not protect against pulmonary tb. a decrease in tb mortality may be achieved by enhancing immunity in the lung. the present study evaluated the induction of antigen-specific immunity in the lung by intranasal (i.n.) delivery of the lipoprotein i (opri) from pseudomonas aeruginosa. opri has shown to be a toll-like receptor 2/4 agonist that, when given subcutaneously, induces type-1 immune responses against heterologous antigens. here, a fusion of opri to ag85a of mtb (opri-ag85a) was used as a subunit vaccine in homologous prime-boost immunizations. in addition, opri-ag85a was combined with an ag85a-encoding dna vaccine (ag85a dna) or with bcg in heterologous prime-boost vaccinations. intranasal and parenteral delivery with opri-ag85a elicited comparable t-cell responses in the spleen; in addition, i.n. delivery elicited specific t-cell responses in the lung lymph nodes (llns). intramuscular delivery of ag85a dna induced significant systemic th1 immune responses. intranasal boosting with opri-ag85a enhanced this response and in addition induced an antigen-specific ifn-g response in lln. opri may therefore be an efficient adjuvant for mucosal boosting. we continue to evaluate the protection induced by opri-based prime-boost vaccinations against pulmonary tb. results on the immunogenicity and protection against intravenous mtb h37rv infection will be presented. toll-like receptors (tlrs) are pattern recognition receptors of the innate immune system, which recognize molecular structures on pathogens or cellular stress-associated molecules. tlr-ligand interactions trigger activation of inflammatory signal transduction and expression of genes involved in host defense. in this study, we have examined the requirement for different tlr adaptor molecules in virus-induced chemokine expression and are currently trying to identify the tlr involved. we have found that both a herpesvirus [herpes simplex virus (hsv)] and a paramyxovirus (sendai virus) require a functional genome to induce expression or proinflammatory chemokines in human and murine monocytic cell lines. for both viruses, this is independent of the tlr adaptor molecules trif and mal. however, overexpression of the vaccinia virus-encoded inhibitor of tlr-signalling a52r or dominant-negative myd88 totally inhibited hsv-induced rantes expression but only partially prevented sendai virus from inducing this chemokine. this suggests that hsv-induced rantes expression occurs via a tlr pathways, whereas sendai virus utilizes both tlr-dependent and -independent pathways to stimulate expression of rantes. we are currently trying to identify the tlrs involved. data from these studies will also be presented at the meeting. 2 0 -5 0 -oligoadenylate synthetases are interferon-induced, double-stranded rna-activated antiviral enzymes which are the only proteins known to catalyse 2 0 -specific nucleotidyl transfer. this first crystal structure of a 2 0 -5 0oligoadenylate synthetase reveals a structural conservation with the 3 0 -specific poly(a) polymerase that, coupled with structure-guided mutagenesis, supports a conserved catalytic mechanism for the 2 0 -and 3 0 -specific nucleotidyl transferases. comparison with structures of other superfamily members indicates that the donor substrates are bound by conserved active site features while the acceptor substrates are oriented by nonconserved regions. the 2 0 -5 0oligoadenylate synthetases are activated by viral doublestranded rna in infected cells and initiate a cellular response by synthesizing 2 0 -5 0 -oligoadenylates, that in turn activate rnase l. this crystal structure suggests that activation involves a domain-domain shift and identifies a putative dsrna activation site that is probed by mutagenesis. we demonstrated that this site is required both for the binding of dsrna and for the subsequent activation of oas. this rna-binding site is different from known rna-binding site; rather than forming a defined three-dimensional domain, it is located at the interface of the two major domains in oas. this novel architecture ensures that the dsrna helix can make simultaneously contact with both domains of oas and ensure the subsequent structural rearrangement leading to the activation of oas. our work provides structural insight into cellular recognition of double-stranded rna of viral origin and identifies a novel rna-binding motif. bacteria-specific iga antibodies are efficient opsonins for neutrophils and mononuclear phagocytes, provided that the phagocytes express the fca receptor (cd89). expression of cd89 can be stimulated by inflammatory cytokines, activated complement factors and certain microbial components. in one study, unstimulated phagocytes were able to ingest iga antibody-treated pneumococci, but only in the presence of complement, which was found to be activated by the iga antibodies along the alternative pathway. pneumococci produce iga1 protease that cleaves human iga1, but not iga2, molecules in the hinge region. this leaves iga1 as faba (monovalent) deprived of fca which contains the docking site for cd89. iga1 is the vastly predominant subclass of iga in the upper airways and circulation of humans. aims: to examine the effects of iga1 protease activity and complement on phagocytosis of iga antibody-coated pneumococci by an unstimulated human phagocytic cell line (hl60). materials and methods: iga1 and iga2 monoclonal antibodies to serotype 4 pneumococcal capsular polysaccharide (ps) were generated by heterohybridoma technique involving b cells from human vaccinees. isogenic serotype 4 pneumococci with and without iga1 protease activity, respectively, were obtained after inactivation of the iga gene of the tigr4 strain. opsonophagocytosis was quantitated using the assay described by romero-steiner et al. based on enumeration of surviving bacteria by culture. the integrity of iga molecules was examined by western blotting. results: both iga1 and iga2 antibody to type-4 polysaccharide-induced phagocytosis of iga1 protease-deficient type-4 pneumococci equally well in the absence as in the presence of complement. iga1 antibody to type-4 polysaccharide displayed a fourfold higher opsonophagocytosis titer against iga1 protease deficient compared to homologous wildtype target bacteria. a similar effect of iga1 protease activity of the target bacteria was not observed in a parallel experiment where iga2 antibody to type-4 polysaccharide served as opsonin. iga1 antibody extracted from iga1 protease-producing target bacteria was almost exclusively in the form of faba. conversely, iga1 from protease-deficient bacteria and iga2 from both types of bacteria were intact. conclusions: these results indicate that the iga1 protease activity of s. neumoniae may help the bacteria escape iga1 antibody-mediated opsonophagocytosis. besides, in these experiments, iga-mediated opsonophagocytosis was independent of complement. vitamins e and c have been found to increase the cellular and humeral immunity of pigs. vitamin e deficiency has also been found to predispose pigs to different diseases, e. coli infection is one among them. after weaning, the vitamin e status of pigs often decreases to a critical low level. in this experiment, we studied whether vitamin c supplementation would be a possible feeding strategy to optimize the immune status of weaners. the interaction between vitamin e and c is interesting due to the reported sparing action on vitamin e or synergism between these to vitamins. piglets were weaned at day 28 of age from sows fed increasing dietary vitamin e during lactation, and piglets were during the following 3 weeks fed either a control diet or this diet supplemented with 500 mg stay-c per kg. blood sampling was obtained weekly from day 28 and until day 49 of age. on the same days, one piglet per dietary treatment was killed and alveolar macrophages (am) were harvested. vitamin c supplementation increased the concentration of igm in serum of piglets throughout the weaning period. although the vitamin e concentration in am decreased with increasing age of the piglets, the concentration was numerically higher in piglets of sows fed the high dietary level of vitamin e. however, vitamin c supplementation tended to increase the total am concentration of vitamin e after weaning and increased the proportion of the biologically most active isomer of vitamin e [rrr-(a-tocopherol)] in the am. the eicosanoid synthesis by am was not influenced by the vitamin c supplementation, but the synthesis of leukotriene b4 was decreased 2 weeks after weaning compared to other days of am harvesting. in conclusion, dietary vitamin c supplementation improved the immune responses of piglets after weaning. a whole blood stimulation assay with escherichia coli (o111:b4) endotoxin was established to measure the capacity of dairy cows to produce the proinflammatory cytokine tumour necrosis factor-a (tnf-a) ex vivo. initially, a time-and dose-dependent study was carried out to find the optimal stimulation conditions for the tnf-a response. the tnf-a response peaked between 3 and 4 h at 38.5 c. a dose in the range of 5-10 g of e. coli lipopolysaccharide (lps)/ml whole blood was found to give the maximum tnf-a response. thirty-eight danish-holstein dairy cows were investigated for their tnf-a responsiveness ex vivo in the periparturient period. heparin-stabilized blood samples were collected seven times over a period of 4 months (weeks à3, à1, 2, 3, 5, 9 and 13 around calving) and stimulated with 5 g/ml of e. coli lps. indeed, fluctuations in the tnf-a responsiveness occurred over time. moreover, the mean tnf-a responsiveness of 38 cows was found to be significantly increased (p < 0.001) in the weeks close to calving. however, in the more stabile physiological periods, some cows had a consistently low tnf-a response, whereas others had high a tnf-a response. we are currently investigating whether high and low tnf-a responders to e. coli lps also exist in dairy cows in vivo. moreover, the importance of tnf-a responsiveness ex vivo to dairy cows' susceptibility and clinical response to experimental e. coli infections in the udder is being investigated. coelomic cytolytic factor (ccf) is a 42 kda invertebrate pattern recognition molecule isolated from the coelomic fluid of the earthworm eisenia foetida (oligochaeta, annelida). ccf displays a number of similarities with the mammalian cytokine tumour necrosis factor-a (tnfa) as a result of a shared n,n 0 -diacetylchitobiose lectin-like domain. however, these similarities are solely functional and are not based on any (dna or amino acid) sequence homology, thus suggesting a form of convergent evolution. in particular, the lectin-like domain of tnf-a has been shown to induce membrane depolarization in various mammalian cell types, through interactions with endogenous amiloride-sensitive ion channels. this nonreceptor-mediated activity of tnf-a has been reported to be involved in the resorption of oedema. likewise, the lectin-like domain of ccf also induces membrane depolarization in mammalian cells. here, we show that ccf appears to be able to induce oedema resorption in an alveolar epithelial cell line through its lectin-like domain. this lectin-like domain of ccf interacts (directly or indirectly) with endogenous sodium and/or chloride channels, and not potassium channels, on mammalian cells. additionally, we suggest that the jnk/sapk and erk1/2 pathways are involved in ccf-induced macrophage activation. these results further establish the functional analogy between an invertebrate pattern recognition molecule and a mammalian cytokine and, from a more applied point of view, suggest the possibility of utilizing ccf in the treatment of oedema. release of svegf and svegfr1 from white blood cells and platelets during surgery and stimulation with bacterial antigens introduction: the influence of surgery on release of soluble vascular endothelial growth factor (svegf) and the soluble vascular endothelial growth factor inhibitory receptor 1 (svegfr1) is unknown. we studied the effect of major and minor surgery on potential variations in svegf and svegfr1 concentrations in vivo and on bacterial antigen-induced release of svegf and svegfr1 from whole blood in vitro. methods: sixty-one patients with abdominal diseases undergoing five different surgical procedures were included. blood samples were drawn from anaesthetized patients before and after the operation. white blood cells and platelets were counted, and plasma svegf and svegfr1 was determined by an elisa method. whole blood from each blood sample was stimulated in vitro with bacteria-derived antigens (lps or protein-a) and svegf and svegfr1 levels were subsequently determined in the supernatants. stimulation with isotonic saline served as control assay. neither svegf or svegfr1 in plasma changed during surgery. in vitro stimulation of blood samples with bacteria-derived antigens resulted in a significant increase in svegf (p < 0.0001) and a less pronounced but still significant increase in svegfr1. release of svegf due to stimulation was significantly higher after the operation (nonsignificant), whereas svegfr1 release remained largely unchanged after surgery. correlation between bacterial antigen-induced release of svegf and neutrophile cell count was highly significant (p < 0.0001). there was no correlation between svegf and platelet cell count, and bacterial antigen-induced svegfr1 release did not correlate with counts of neutrophils and platelets. conclusions: plasma svegf and svegfr1 concentrations did not change during surgery. in vitro bacterial stimulation led to increased release of svegf and svegfr1, which was not significantly amplified during surgery and which may be related to number of circulating neutrophils. natural killer cell functions and subsets after in vitro stimulation with il-2 and il-12, with special emphasis on intracellular ifn-g and nk-cell cytotoxicity r. nyboe, 1,2 t. rix, 1,2 j. krog, 1,2 e. tønnesen 1 & m. hokland 2 1 department of anaesthesiology and intensive care, aarhus university hospital, and 2 institute of medical microbiology, and immunology, university of aarhus, aarhus, denmark. e-mail: rnsr@studmed.au.dk materials and methods: isolated cryopreserved human peripheral blood mononuclear cells (pbmcs) were stimulated with il-2 and il-12. this stimulation has previously been shown to activate nk cells. cell cytotoxicity was measured by flow cytometry after incubation with k562 cells. this method was compared to the current standard 51cr release assay. cells were treated with bfa to accumulate ifn-g, stained for surface markers, permeabilized and stained for intracellular ifn-g. flow cytometry was then performed to measure intracellular ifn-g production in pbmc, especially in nk cells. results: we have demonstrated that stimulation with il-2 and il-12 is effective in increasing the number of ifn-gpositive cells. there is a distinct difference between the cd3-cd56dim and the cd3-cd56bright subsets, with a much greater proportion of ifn-g-positive cells in the cd3-cd56bright subset. the effects of stimulation with il-2 and il-12 on cytotoxicity will be presented, as will the relation between ifn-g production and cytotoxicity. in addition, we will present results of these assays applied to porcine cells. discussion: in combination, these tests will address nk cell function by combining cytotoxicity with ifn-g production in nk cell subsets. the results will demonstrate whether this could serve as a useful tool in describing nkcell function, which could be of value in clinical and experimental settings. culture of regulatory t-cell lines from bronchial mucosa t lymphocytes play a major role in many immune responses. in the last decade, special focus has been on the function of th1 and th2 effector cells. now the importance of regulatory cd4 þ cd25 þ t cells in maintenance of the immunological homeostasis emerges. sarcoidosis is a multisystem granulomatous disorder often affecting the lungs. the typical sarcoid granulomas consists of epitheloid cells, macrophages and lymphocytes, mainly cd4 þ t cells of th1 phenotype. we have cultured t cells from bronchial biopsies of patients with sarcoidosis as well as from controls in high levels of interleukin 2 (il-2) and il-4 and demonstrate spontaneously arising cd4 þ cd25 þ populations and high concentrations of il-10 in these cultures. the main difference between cultures of sarcoid origin compared to controls is a very much higher concentration of the inflammatory cytokines il-6 and tnf-a in cultures of sarcoid origin. the effects of hyperbaric exposure on human peripheral blood mononuclear cells, with special emphasis on natural killer cell cytotoxicity and subsets materials and methods: as an experimental physiological stress model, we examined the effects of hyperbaric exposure on peripheral blood mononuclear cells (pbmcs) obtained from venous blood drawn from eight divers during a simulated heliox saturation dive. eight persons working in normobar atmosphere outside the pressurized chamber served as control donors. the spontaneous cytotoxicity of the pbmcs was estimated in a 4 h 51cr-release assay using k562 as nk-sensitive target cells. the pbmcs were characterized, using 4-colour flow cytometry, with special emphasis on the nk-cell subsets. the data were statistically analysed using a multivariate regression model (stata 8.2). p values <0.05 was considered statistically significant. results: the estimated cytotoxicity increased significantly in both the group of divers and control donors during the dive (pdivers < 0.01 and pcontrols < 0.01). although the cytotoxicity increased relatively more (p < 0.01) in the group of divers compared to the group of control donors between day 1 and 2. discussion: the increased cytotoxicity of pbmc estimated in the group of divers indicate that parts of the cellular immune system are affected during the extreme physiological conditions induced during the initial phase of the presented experimental hyperbaric setup. the increase in cytotoxicity observed in the group of control donors could hypothetically reflect the stress level in persons working outside the pressurized chamber during the dive. the interaction with adhesion molecules expressed by vascular endothelium is the first step in lymphocyte infiltration into tissues. at both cutaneous and mucosal sites interleukin-10 (il-10), il-12 and transforming growth factor (tgf)-b are important regulators of chronic inflammatory disease, where cutaneous lymphocyteassociated antigen (cla) and ae integrin (cd103) may be expressed. unlike cla, cd103 is not believed to play a role in tissue-specific homing but may help to retain t cells within epithelial layers. we have previously shown that il-12 alone can together with an unknown cofactor increase the expression of cla. stimulation with streptococcal pyrogenic exotoxin c (spec) increased the expression of cd103 by cd8 þ but not cd4 þ t cells. while il-12 increased superantigen-stimulated expression of cla, this cytokine strongly inhibited the cd103 expres-sion, and a combination of il-12 and tgf-b completely abrogated the induced cd103 expression. conversely, il-10 suppressed cla but increased cd103 expression. these findings indicate that, in addition to suppressing the development of th1-mediated inflammatory responses, il-10 may also inhibit the migration of cd8 þ t cells into the skin while il-12 promotes such migration. thus, the expression of cla and cd103 may be antagonistically regulated by il-10 and il-12, and the balance between these cytokines could influence the t-cell migration of inflammatory cells into epithelial tissues. during contact sensitivity reaction, immune cells proliferate. in order to study the histological picture of these proliferation phases, we used a mouse model of contact sensitivity in the oral mucosa and on skin. we also used bromodeoxyuridin (brdu, an analogue to thymidin) that is incorporated into the nucleus during cell replication. the hapten oxazolone (oxa) was used to sensitize and elicit the oral mucosa and/or the ear skin. mice were killed at various times after elicitation, and unsensitized animals were also exposed to the hapten as controls. brdu (25 mg/ kg animal) was injected i.p. 2 h before the kill. specimens from the oral mucosa, ear skin and submandibular and auricular lymph nodes were cut and fixed in 4% paraformaldehyde. they were then treated with acid and biotinylated anti-brdu antibody and developed using abc-kit and dab. the analyses were performed using a leica light microscope and the computer program analysis. in the oral mucosa, the frequency of proliferating cells were increasing during the observation period, 4-24 h after elicitation, regardless of site of sensitization. the proliferating cells were found mainly in the basal cell layer of the epithelium. similar patterns were found in ear skin. the regional lymph nodes demonstrated a few scattered proliferating cells 4 h after elicitation. after 24 h, these cells were found frequently in the whole lymph node. control animals exhibited considerable less proliferating cells at all times. we conclude that most proliferating cells were found 24 h after elicitation locally at the hapten-exposed sites (the oral mucosa or the ear skin) as well as in the regional lymph nodes. the endogenous nucleoside adenosine is released in excess during inflammation or other metabolic stress and is generally known to deliver tissue protective anti-inflammatory effects. adenosine acts via four adenosine receptors of which the a2a receptor is the predominant form in t cells. adenosine levels are elevated in asthmatic lung, and adenosine can directly induce mast cell degranulation and bronchoconstriction in these patients. instead, the role of anti-inflammatory mechanisms of adenosine on t cells in asthma is unclear. aim: to study the a2a receptor expression in peripheral blood cd4 þ t cells in asthmatic and healthy individuals using flow cytometric and quantitative real-time pcr methods. results: unstimulated cd4 þ cells of asthmatic patients expressed significantly lower levels (p < 0.001) of a2a receptor in protein level (mean percentage of cells positive ae sem: 76.8 ae 1.2, n ¼ 6) compared to healthy individuals (90.4% ae 1.9, n ¼ 4). double staining for cd69 expression showed that stimulation of cd4 þ cells decreased a2a expression in both groups but indicated that the detected lower levels of a2a in unstimulated cells of asthmatics was not due to preactivation in these patients. surprisingly, a2a mrna expression in unstimulated cd4 þ cells was significantly higher (p < 0.05) in asthmatics (n ¼ 28) compared to healthy controls (n ¼ 7). the expression did not correlate with serum total ige levels. conclusions: asthmatic individuals express less a2a adenosine receptor on their peripheral cd4 þ t cells. the higher mrna levels instead may point to a negative feedback regulation in the receptor expression. the role of possibly decreased adenosine-mediated anti-inflammatory effects in asthma pathogenesis require further studies on this t-cell mediated disease. the chronic inflammatory skin disease atopic eczema (ae) affects almost 15% of the population in many countries today. the pathogenesis of ae is not fully understood. a combination of genetic predisposition and environmental factors like microorganisms seems to contribute to the symptoms. the yeast malassezia sympodialis is part of our normal skin micro flora but can act as an allergen and elicit specific ige and t-cell reactivity in patients with ae. recently, we identified a novel major m. sympodialis allergen, designated mala s 11 (22.4 kda), with sequence similarity to the mitochondrial enzyme manganese superoxide dismutase (mnsod). interestingly, mala s 11 has a high degree of homology to human mnsod. the aim of this study was to examine the effects of recombinant mala s 11 on antigen-presenting dendritic cells. monocytederived dendritic cells (mddcs) from healthy blood donors were cultured with or without mala s 11 for different time periods. it was found that the maturation marker cd83 and the costimulatory molecules cd80 and cd86 were upregulated on the mddcs exposed to mala s 11 for 24 h, as demonstrated by flow cytometry. furthermore, coculture of mddcs with mala s 11 for 9 h induced an increased production of the inflammatory cytokines il-6 (200-fold), tnf-a (100-fold) and il-8 (sixfold), as detected by the cytometric bead array (cba) analysis. our results suggest that mala s 11 affects the immune response through dc maturation and production of inflammatory cytokines. the potential cross-reactivity with human mnsod needs to be explored and the exact role of mala s 11 in the pathogenesis of ae assessed in clinical studies involving skin prick and atopy patch tests. allergen-specific immunotherapy (sit) is commonly conducted with allergen extracts adsorbed to aluminium hydroxide (alum). drawbacks linked to the use of alum, such as the formation of granuloma at the site of injection, have led to suggestions of novel allergen carriers. an alternative carrier is 2 mm carbohydrate-based particles (cbps). in mouse, allergen-coupled cbps have been demonstrated to skew the allergen-specific immune response towards a th1-like activity (grönlund et al. immunology, 2002) . we here coupled the recombinant major cat allergen fel d 1 to cbps (cbp-fel d 1) by cyanogen-bromide activation, resulting in covalent binding. the effect of cbp-fel d 1 on monocyte-derived dendritic cells (mddcs) from healthy human blood donors was studied. we found that the majority of the cd1a þ mddcs were capable of taking up fitc-labelled cbp-fel d 1, as demonstrated by flow cytometry and confocal laser scanning microscopy. furthermore, incubation with cbp-fel d 1 resulted in an upregulation of the costimulatory molecule cd86 on the mddcs, which was not observed with fel d 1 or cbps alone. finally, cbp-fel d 1 induced a fivefold increase in the release of the pro-inflammatory cytokine tumour necrosis factor (tnf)-a and a fourfold increase in the release of the chemokine interleukin-8 from mddcs. taken together, the effects cbps possess make them interesting as novel allergen carriers for sit. the cysteine protease der p1 from dust mite of the genus dermatophagoides pteronyssinus is a major type i allergen. about 80% of house dust mite (hdm) allergic individuals are reactive to this protease in standard assays for detection of ige. a curative treatment for atopic allergy is immunotherapy (it) with hdm extracts which are complex mixtures occasionally resulting in anaphylactic reactions. novozymes focuses on developing a recombinant variant of der p1 which exhibit lowered risk of ige-mediated allergic reactions, while maintaining its ability to trigger proper th-cell responses. this may provide a safer alternative for specific it of hdm allergy. a secreted recombinant form of pro-der p 1 expressed by saccharamyces cerevisiae was obtained by fusion of the pro-enzyme to a fungal signal peptide. the n-glycosylation site of der p1 was mutated resulting in a deglycosylated pro-enzyme with a molecular mass of 35 kda. protein purification procedure was developed to obtain nearly pure der p1 protein followed by determination of concentration by active-site-titration with the cysteine protease inhibitor e64. the deglycosylated recombinant pro-der p 1 revealed immunologic similarity to the native der p 1 molecule when compared in basophile histamine release, ige-binding assays and t-cell proliferation assays. by in silico epitope mapping of a modelled 3-dimensional structure of der p1, five putative igg and ige epitopes were predicted. by protein engineering, the predicted epitopes were removed one by one in der p1 and screening for hypoallergenic variants was performed. combining inhaled long-acting b-2 agonist (laba) and inhaled corticosteroid (ics) seems to offer asthma control at a lower dose of ics than achieved by ics alone. fine mapping of t-cell surface markers by flow cytometry offers a detailed status of the individual's inflammatory response. the frequency of mt2 (cd4 þ cd45ra -cd62l þ cd11adim) and mt1 (cd4 þ cd45ra -cd62l -cd11abright) cells in peripheral blood, and their ratio, has been shown to differ predictably in atopics and patients with leprosy, where mt2 correlates with a th2 phenotype and mt1 with a th1 phenotype. stable asthmatics, requiring fluticasone propionate (fp) 750-1000 mg daily or equivalent, were randomized to receive, double-blinded, either seretide 1 [salmeterol and fluticasone propionate (sfc, n ¼ 16)] 50 mg/500 mg bd or fp 500 mg bd (n ¼ 17). if asthma was controlled based on lung function and symptoms at clinic visits every 6 weeks, ics dose was tapered until asthma exacerbated or 0 mg was reached. the frequency and ratio of mt2 and mt1 t cells of the patients was monitored at 6 week intervals. as treatment tapered, the frequency of mt2 cells decreased (p ¼ 0038 from first to final visit), whereas that of mt1 cells increased. the ratio of mt2/mt1 decreased (p ¼ 0049 from first to final visit). in patients receiving laba þ ics, the fall in mt2/mt1 ratio appeared to be more pronounced than in patients receiving ics alone. thus, the mt2 phenotype may be associated with stable asthma, whereas an imminent exacerbation may associate with an increase in the mt1 phenotype. laba may allow for a greater effect of fp on the mt ratio. activation of complement pathways, leading to production of c3a and c5a anaphylatoxins, has been postulated in the pathogenesis of asthma and allergic airway inflammation. the present study was undertaken to investigate the role of mannan-binding lectin (mbl), an initiator of the lectin pathway of complement, in asthma and allergic rhinitis. mbl levels and mbl-induced complement activity were determined in 19 patients of bronchial asthma with allergic rhinitis and 20 unrelated, age-matched controls of indian origin. mbl levels and activity were correlated with percent eosinophilia and percent predicted fev1 values of the patients. association of single nucleotide polymorphisms (snps) in exon 1 and intron 1 of the mbl with the disease, clinical markers, mbl levels and mbl-induced complement activity was analysed using standard statistical tools. significantly higher mbl levels and activity were observed in patients of bronchial asthma with allergic rhinitis as compared to the controls. we identified five snps, of which two, a816g in exon 1 and g1011a in intron 1 of the mbl, were novel. snp g1011a was significantly associated with the disease (p ¼ 0.0024, or ¼ 5.8696, 95% ci: 1.7316 < or < 19.8963). individuals with 'a' allele at position 1011 showed increased mbl levels, activity and disease severity. our results suggest that 'a' allele at position 1011 leading to high mbl levels and complement activity may be contributing to the severity of bronchial asthma and allergic airway inflammation. serum resistance of borrelia burgdorferi strains belonging to the b. afzelii and b. burgdorferi sensu stricto genospecies is dependent on binding of complement inhibitor factor h. we recently reported that factor h binding by b. burgdorferi is due to inducible expression of several approximately 20 kda plasmid-encoded, surface-exposed lipoproteins related to ospe (e.g. erpa, erpp and p21). in addition, a second class of factor h-binding proteins of approximately 27-35 kda has been described. the ospe-related lipoproteins are dramatically induced by b. burgdorferi during transmission from its tick vector into the mammalian host. the induction of ospe-related lipoproteins during mammalian infection may play a key a role in the borrelial evasion of the host's immune system. the goal of the present study was to define the factor h-binding regions of ospe-related proteins using mutagenesis, peptide mapping and surface plasmon resonance analysis (biacore). the combined studies revealed that the c-terminal regions of both human and mouse factor h (scrs 18-20) specifically bind to ospe-related lipoproteins. we also found fhr-1, whose c-terminal scrs 3-5 are homologous to scrs 18-20 of factor h, to bind to ospe. peptide mapping revealed five putative regions (designated i-v) in ospe that could directly interact with factor h. deleting the c-terminal 15 amino acid residues from region v of p21 abolished its ability to bind factor h. at the same time, however, synthetic peptides corresponding to the c-termini of ospe, p21 and erpp did not inhibit factor h binding to ospe. thus, the c-terminal-binding region v appears to be necessary but not sufficient for factor h binding. when a more specific mutation strategy was employed, where single amino acid residues in peptides spanning over the factor h-binding regions were mutated to alanines, we observed that lysines in the factor h-binding regions of ospe were required for factor h binding. the combined data have revealed that key lysine residues in ospe-related lipoproteins and ionic interactions are crucial for factor h interactions. furthermore, binding of ospe to the c-termini of both mouse and human factor h suggests that borrelia spirochetes utilize analogous complement resistance mechanisms in both rodents and man. in borrelia garinii strains, which in in vitro analyses have been found to be sensitive to complement killing, differences in the ospe sequences as well as in the expression of factor h-binding proteins may account for their susceptibility to serum lysis. role of yada, ail and lipopolysaccharide in serum resistance of yersinia enterocolitica serotype o:3 mannan-binding lectin (mbl), l-ficolin and h-ficolin are pattern recognition molecules of the innate immune system. we investigated the ability of these molecules to bind to different serotypes and noncapsulated variants of streptococcus pneumonia and staphylococcus aureus. we found that mbl binds to noncapsulated s. aureus strain (wood) but not any of the examined s. pneumoniae serotypes. l-ficolin binds to some capsulated s. pneumoniae serotypes (11a, 11d and 11f) as well as some capsulated s. aureus serotypes (type-1, -8, -9, -11 and -12). h-ficolin does not bind to any of the examined s. pneumoniae and s. aureus serotypes included in this study but did bind to a strain of aerococcus viridans. when bound to bacteria, mbl and h-ficolin initiated activation of complement factor c4, whereas l-ficolin did not. during this study, quantitative assays for the three proteins were developed and the concentration in 97 plasma samples were determined and the median values were estimated at 0.8 mg of mbl/ml, 3.3 mg of l-ficolin/ml and 18.4 mg of h-ficolin/ ml, respectively. the absence of early complement components (c1, c4 and c2 but not c3) is a predisposing factor for systemic lupus erythematosus (sle). recently, we demonstrated that, in c4-deficient (c4 def.) mice, igm-containing immune complexes (igm-ic) are filtered by the splenic barrier of marginal zone macrophages (mzm), resulting in an increased immune response against antigens within these igm-ic, but this could not be observed in wildtype or c3 def. mice. we hypothesized that splenic cd11b þ mzm play an important role in the induction of autoimmunity, and we therefore analysed their cytokine profile after isolation with the help of magnetic antibody cell sorting. mrna was isolated, and real-time pcr was performed with specific primers for murine ifn-g (ifn-g), interleukin-12 (il-12) and ifn-a (ifn-a). we observe a moderate increase of il-12 and ifn-g mrna in cd11b þ cells of c4 def. mice compared to wildtype cells. surprisingly, the concentration of ifn-a mrna is six times higher in c4 def. mice. preliminary results suggest that mrna in cd11b þ cells of c3 def. mice is even lower than that in wt. six hours following i.v. application of 20 mg of a abstracts 625 .................................................................................................................................................................................................. murine monoclonal igm anti-dsdna antibody, production of il-12, ifn-g and ifn-a mrna is increased in cd11b þ cells of both c4 def. and wt mice. several references described increased levels of inf-a in patients with sle. dendritic cells are discussed as a major source of ifn-a. our observation that c4-deficient, sle-susceptible mice demonstrate an increased spontaneous ifn-a production by splenic cd11b þ marginal zone macrophages could be an early sign and a trigger for the development of sle. this is supported by the fact that the absence of c3 is not a predisposing factor for sle and our observation that c3 def. animals display low levels of ifn-a mrna. 200-400 million people worldwide and represents one of the leading causes for liver cirrhosis and hepatocellular carcinoma. control over the hbv infection is achieved mainly by vaccination with hepatitis b surface antigen (hbsag). hbsag contains n-linked glycosylation side and is recognized by both mbl-a and mbl-c in a cadependent manner. hbsag-mbl complexes activate complement and may thus affect humoural immunity. to investigate the role of mbl in humoural responses to hbsag, we immununized mice that lack both mbl-a and mbl-c proteins with soluble hbsag. it has been shown that deficiencies in other complement components like c1q, c4 and c3 result in decreased antibody responses. however, mbl double ko animals mounted dramatically increased humoural responses. after priming, mbl double kos mounted hbsag-specific igm responses, which were threefold higher than wt controls. after boosting the hbsag, total igg was 10-fold higher in mbl ko than in wt control animals. similar to the response to hbsag, other glycosylated soluble antigens (e.g. invertase) induced better humoural responses in mbl double ko animals, suggesting that mbl plays an important role in a negative feedback regulation of adaptive immunity. reconstitution experiments with rmbl partially rescued the ko phenotype. we propose that the clearance of glycoprotein antigens in mbl ko is handled differently from the wt, resulting in better stimulation of humoural responses. alternatively, glycoprotein-ag-mbl-rich complexes inhibit b-cell responsiveness via putative mbl receptors. the complement system is an important part of the innate immune system. the activation of complement proceeds through three different pathways that converge in the generation of c3-activating enzyme complexes. complement activation via the lectin pathway is initiated when recognition molecules, mannan-binding lectin (mbl) or ficolin, bind to carbohydrate structures characteristic for microbial surfaces. in the circulation, mbl and ficolins are found in association with three structurally related mblassociated serine proteases (masp)-1, -2 and -3 and a small, nonenzymatic component, map19. masp-2 has been shown to elicit complement activation through the sequential proteolytic cleavage of c4 and c2 upon binding of mbl/masp-2 complexes to microbial surfaces. we have recently uncovered a polymorphism in the masp-2/map19 gene in a patient shown to be deficient in the lectin pathway of complement activation. the polymorphism results in a single amino acid substitution in the n-terminal part of the masp-2 protein. recombinant wildtype masp-2 and masp-2 containing the amino acid substitution in question was produced, and the ability to activate complement was studied. the mutation had a profound impact on masp-2 function, resulting in the lack of complement activation through the lectin pathway. elisa-based experiments showed that the mutation leads to the impairment of complement activation through influencing the binding of masp-2 to mbl or ficolins. deficiencies in the lectin pathway of complement activation have so far been accounted for only by lack of functional mbl. the mutation described above is the first defect described affecting both activation through mbl and the ficolins. .................................................................................................................................................................................................. th1, th2 and treg cell balance. dcs are present in the gut mucosa and may thus be target for modulation by gut microbes, including ingested probiotics. here, we tested the hypothesis that species of lactic acid bacteria, important members of the gut flora, differentially activate dc. a large panel of human gut-derived lactobacillus and bifidobacterium spp. was screened for dc-polarizing capacity by exposing bone marrow-derived murine dc to lethally irradiated bacteria. cytokines in culture supernatants and dc-surface maturation markers were analysed. substantial differences were found among strains in the capacity to induce interleukin-12 (il-12) and tumour necrosis factor (tnf)-a, while the differences for il-10 and il-6 were less pronounced. bifidobacteria tended to be weak il-12 and tnf-a inducers, while both strong and weak il-12 inducers were found among the strains of lactobacillus. remarkably, strains weak in il-12 induction inhibited il-12 and tnf-a production induced by an otherwise strong cytokine-inducing strain of lactobacillus casei, while il-10 production remained unaltered. selected strains were tested for induction of dc maturation markers. those lactobacilli with greatest capacity to induce il-12 were most effective in upregulating surface mhc class ii and cd86. moreover, l. casei-induced upregulation of cd86 was reduced in the presence of a weak il-12inducing l. reuteri. in conclusion, human lactobacillus and bifidobacterium spp. polarize differentially dc maturation. thus, the potential exists for th1/th2/treg-driving capacities of the gut dc to be modulated according to composition of gut flora including ingested probiotics. the intestinal micro flora is indispensable in developing and maintaining homeostasis of the gut-associated immune system. evidence indicates that lactic acid bacteria (lab), e.g. lactobacilli and bifidobacteria, have beneficial effects on the host. established health effects include increased gut maturation, antagonisms towards pathogens and immune modulation. the objective of this study is to evaluate the immunomodulating properties of a range of lab of human origin. as dendritic cells (dcs) play a pivotal role in the balance between tolerance and immunity to commensal microorganisms, in vitro-generated immature dcs serve as a suitable model for studying the immunomodulating effects of lab. human immature dcs were generated in vitro from monocytes and exposed to lethally uv-irradiated lab. the effect of various species of lab on dcs in direct contact was evaluated. furthermore, the maturation pattern of dcs separated from the bacteria by an epithelial cell layer (caco-2 cells), which should mimic the intestinal environment, was studied. cytokine secretion (il-12, il-10 and tnf-a) and upregulation of maturation surface markers on dcs (cd83 and cd86) was measured. different lab induced diverse cytokine responses. some strains were strong il-12 and tnf-a inducers and others weak. all strains induced il-10. different lab also differentially modulated expression of cd83 and cd86 on dcs. although some variation in the response to lab of dcs generated from different blood donors was observed, general differences in the effect of the various lab was revealed. experiments with the dc caco-2 coculture system are ongoing. different species of lab differentially affect dc maturation; this suggets that the gut flora plays a pivotal role in polarization of the immune response. natural killer (nk) cells are cells of the nonspecific immune system lysing altered self-cells. a noncytolytic subset of nk cells may serve a regulatory role by secreting cytokines. bacteria translocating across the gastrointestinal mucosa are presumed to gain access to nk cells, as consumption of certain lactic acid bacteria has been shown to increase in vivo nk cytotoxicity. here, we investigated how human gut flora-derived lactobacilli affect nk cells in vitro, by measuring proliferation and ifn-g production of human nk cells upon bacterial stimulation. cd3 -cd56 þ nk cells were isolated from buffy coats by negative isolation using non-nk lineage-specific antibodies and magnetic beads. nk cells were incubated with 10mg/ml uv-inactivated bacteria or 10mg/ml phytohemagglutinin (pha) for 4 days. proliferation was assessed by incorporation of radioactive thymidine into nk-cell dna. the ifn-g concentration was measured by elisa. incubation of nk cells with a lactobacillus acidophilus strain increased the proliferation of the nk cells and induced ifn-g production, both to levels comparable to pha stimulation. the proliferative response was further enhanced with autologous monocytes present, probably because cytokines, secreted by monocytes having engulfed bacteria, stimulated the nk cells. in contrast, a lactobacillus paracasei strain caused the nk cells to proliferate only in the presence of monocytes. these results demonstrate that various strains of lactobacilli have the capacity to activate nk cells in vitro, in a monocyte-dependent or -independent way. hence, the encounter of nk cells with lactic acid bacteria will affect nk-cell activation. such activation of nk cells may potentially skew an on-going or subsequent immune response towards a th1 response. lactobacilli are nonpathogenic gram-positive inhabitants of the normal human intestine known for their healthpromoting effects. in our earlier work, it is shown that human monoclonal antibody isolated from sera of a patient with waldenstrom macroglobulinaemia possess innate antibody characteristics and binds to lactic acid bacteria. according to the immune network model, immunization with this bacteria could induce the perturbations in immune system that might result in production of anti-lactobacillus antibodies, human monoclonal antibody like (ab1) and anti-idiotypic antibody (ab2). in this study, balb/c mice were immunized with two doses of bacteria lactobacillus acidophilus in complete and incomplete freund's adjuvant and phosphate-buffered saline (pbs), respectively. seven days after the last immunization, sera from immunized mice were collected and the presence of lactobacillus-specific ab1 and ab2 were determined by elisas. in the sera of immunized mice, antibodies specific to bacteria lactobacillus acidophilus were shown. the concentration of lactobacillus-specific antibodies was higher in the sera of hyperimmunized mice (mice immunized with 1 mg of igm dj) than in sera of mice immunized with 100 times lower doses of immunogen (0.01 mg per doses). moreover, ab1 and ab2 antibodies were detected in the sera of lactobacillus-hyperimmunized mice. in this study, we have shown the idiotypic network interactions in mice immunized with bacteria lactobacillus acidophilus. the normal gastrointestinal flora is crucial for the maturation of the acquired immunity via effects on antigenpresenting cells (apcs). here, we have investigated how two types of apcs, monocytes and dendritic cells (dcs), react to different bacterial strains typical of the commensal intestinal flora. purified monocytes and monocyte-derived dcs were stimulated with uv-inactivated gram-positive (lactobacillus plantarum and bifidobacterium adolescentis) and gram-negative (escherichia coli and veillonella parvula) bacterial strains. monocytes produced higher levels of il-12p70 and tnf, as detected by elisa, in response to l. plantarum than to e. coli and v. parvula. in contrast, dcs secreted high amounts of il-12p70, tnf, il-6 and il-10 in response to e. coli and v. parvula but were practically unresponsive to l. plantarum and b. adolescentis. the lack of response to the gram-positive strains correlated with a lower surface expression of toll-like reseptor 2 (tlr2) on dcs compared to monocytes. the surface expression of tlr4 on dcs was undetectable when analysed by flow cytometry, but blocking this receptor decreased the tnf production in response to v. parvula, indicating that low tlr4 expression on dcs is sufficient to mount an inflammatory response to gram-negative bacteria. ifn-g increased the expression of tlr4 on dcs and also potentiated the cytokine response to gram-negative bacteria. our results indicate that, when monocytes differentiate into dcs, their ability to respond to different commensal bacteria dramatically changes, thereby becoming unresponsive to probiotic gram-positive bacteria. these results may have important implications for the capacity of different groups of commensal bacteria to regulate mucosal and systemic immunity. probiotic bacteria, e.g. lactobacillus spp., may improve diseases such as chronic inflammatory bowel disease. we examined cytokine production and phenotypic change after in vitro stimulation of t cells from healthy volunteers using different probiotic strains. methods: t cells were cultured from colonic biopsies from eight healthy volunteers (agnholt and kaltoft, exp clin immunogenet 2001; 18:213-25) , and dendritic cells were matured from their peripheral blood mononuclear cells. t-cell cultures were stimulated with autologous bacterial sonicate or strains of lactobacillus spp., with and without the addition of dendritic cells. cytokine levels (tnf-a, ifn-g, il-10 and gm-csf) and phenotype (cd3, cd4, cd25 and cd69) were measured on day 4. results: lactobacillus spp. induced higher productions of tnf-a and il-10 than did autologous bacteria. in presence of dendritic cells, the production of all cytokines increased. however, the increases of ifn-g and tnf-a were more pronounced in wells with autologous bacteria than in wells with lactobacillus spp. the addition of dendritic cells upregulated cd25 expression without simultaneous upregulation of cd69. the upregulation was pronounced after stimulation with lactobacillus rhamnosus gg compared with autologous bacteria and other lactobacilli. discussion: in presence of dendritic cells, autologous bacteria induced inflammatory cytokines, while probiotics mainly induced regulatory cytokines. lactobacillus rhamnosus gg induced a regulatory phenotype (cd25 þ ), in part mediated by dendritic cells. future studies will address whether this shift to a cd25 þ phenotype represents a differentiation into competent regulatory t cells. in a clinical context, such cells might be used for treatment of inflammatory diseases. protein microarrays will play a key role in the postgenomic era and offer a unique possibility to perform highthroughput global proteome analysis. a chip can be printed with thousands of protein probes (e.g. antibodies), the biological sample added (e.g. a proteome) and any binding detected. we aim to develop protein microarrays based on human recombinant scfv antibody fragments for global proteome analysis. the concept of comparing proteomic maps of healthy versus diseased samples will allow diseasespecific proteins to be detected. in fact, antibody microarrays will allow us to perform comparative proteome analysis on any sample format in a species-independent manner, as long as a proteome can be isolated. however, the complexity of proteomes, containing several thousands of different proteins, is a problem. here, we have designed antibody microarrays targeting the water-soluble fraction of a proteome. to this end, an anticytokine antibody array was developed and human dendritic cells (aeactivation) was used as model system. the results showed that our antibody microarrays could be used to examine the cytokine profile in complex samples. furthermore, we have taken the first steps towards comparing our results with those of other technologies on both the protein and gene level. due to the complexity of the model proteome, we also examined the possibility to prefractionate the proteome in a simple one-step procedure (based on size) prior to the labelling step. in more detail, the sample proteome was fractionated into two fractions using membrane devices with different molecular weight cut-offs. the results showed that the fractionation considerably enhanced the assay sensitivity allowing cytokines in the pg/ml range to be readily detectable. the immunomodulatory effect of heat shock protein 70: immunization with a dna construct based on the malarial antigen fused with a fragment of hsp 70 primes for a th-1 type of response finding an appropriate adjuvant for human vaccination is crucial. heat shock proteins (hsps) act as adjuvants when coadministered with peptide antigens or given as fusion proteins. however, there is a potential risk of autoimmunity when using the complete molecules, because hsps are evolutionary conserved. to overcome this, we first evaluated the adjuvant effect against two different antigens of a less-conserved fraction of plasmodium falciparum hsp70 (pf70c) and compared it to the whole hsp70 molecule from trypanosoma cruzi (tchsp70). we found that pf70c exhibited similar adjuvant properties as the whole molecule. we later evaluated the adjuvant potential of pf70c against the malarial antigen eb200 in a chimeric dna construct. no appreciable levels of eb200-specific abstracts 629 .................................................................................................................................................................................................. antibodies were detected in mice immunized only with the dna constructs. however, dna primed the immune system, because subsequent challenge with the corresponding recombinant fusion proteins elicited a strong th-1 antibody response. in contrast, no priming effect was observed for ex vivo ifn-g production but stimulation with the hsp-chimeric fusion protein induced a stronger secretion of ifn-g in vitro than other proteins used. these results indicate that the use of hsps is promising in the design of new vaccines. high-throughput proteomics on antibody-based microarrays: the importance of probe and surface design in analogy to dna microarrays, protein microarrays offer a new distinct possibility to perform sensitive highthroughput global proteome analysis. however, the development of the protein microarray technology will place high demands upon the design of both probes and solid supports. the analysis of thousands of heterogeneous proteins on a single microarray requires the use of uniform probes, such as antibodies, directly designed for protein microarray applications. we have recently generated a human recombinant single-chain fv antibody library, genetically constructed around one framework, the ncoder-library, containing 2 â 1010 clones. single framework antibody fragments (sinfabs) selected from this library were successfully applied as probes for microarrays providing sensitive detection in the 600 attomol (mass spectrometry) and the 300 zeptomole range (fluorescence). however, the choice of framework is critical. we have shown that the selected ncoder framework displayed excellent functional on-chip stability and arrayed dehydrated probes retained their activity for several months. furthermore, we have addressed the issues of biocompatibility of the solid support and immobilization strategies for our microarray setup. an in-house-designed substrate, macroporous silicon coated with nitrocellulose (map3-nc7), displayed properties equal to, or better than, those of five commercially available supports used as reference surfaces. we have also evaluated different coupling strategies, such as adsorption, covalent coupling, diffusion and affinity coupling. using a novel affinity tag, the double-(his)6-tag, we increased the binding efficiency of sinfab-molecules to ni2 þ -coated solid supports, thereby allowing nonpurified probes to be directly applied. the mannan-binding lectin (mbl) pathway is part of the innate immune system providing a first line of defence against infections. mbl and ficolins circulate in complexes with mbl-associated serine proteases (masp-1, -2 and -3). after recognition of a microorganism by mbl, activation of the complement system occurs. masp-1 and masp-3 share five domains (making up the so-called a-chain), whereas they have unique protease domains (b-chains). before the identification of masp-3, an assay for masp was presented, based on antibodies against the a-chain of masp-1. with the new knowledge of the three masps, and the sharing of domains by masp-1 and masp-3, assays specific for the protease domains have to be constructed, if one wishes to measure the proteins individually. we present an assay for quantifying total masp-3 in plasma and serum samples. the assay is a sandwich-type assay using as catching antibody a monoclonal antibody against the common a-chain of masp-1/3 and a developing secondary antibody against the c-terminal part of the protease domain of masp-3. we have used this assay for estimating the normal concentration of the protein as well as the concentration in patients and also for characterizing by gel permeation chromatography the masp-3 protein in serum. inducible costimulator ligand (icosl) is a costimulatory molecule related to b7.1 (cd80) and b7.2 (cd86). b cells, monocytes, dendritic cells and endothelial cells express icosl. inducible costimulator (icos) interacts with icosl, and this interaction leads to signals involved in isotype switching and the development of immunological memory. hitherto, no polymorphisms of this gene have been described. the aim of this study was to reveal variation of the icosl gene in normal individuals. all eight exons, except exon 1, were sequenced with flanking introns in 10 healthy blood donors. eight single nucleotide polymorphisms (snps) and two length polymorphisms were found. one of the snps was found in the coding regions of the gene. the base involved was located in exon 3 and caused a conservative amino acid change from valine (gtt) to isoleucine (att). three individuals were heterozygous g/a for the exon polymorphism, while the remaining seven individuals were homozygous for the wildtype g/g. exon 3 encodes the immunoglobulin variable (igv)-like domain of the molecule which is situated outside the cell. this means that the amino acid could be critical for the stability of the molecule or could constitute part of the binding site for icos. the results form the basis for further experiments to find possible associations of the alleles to diseases caused by immune dysregulation. especially, the exon 3 variant is interesting and could play a role for the development of immunological diseases. besides, it would be interesting to see whether both exon 3 alleles are expressed or only the wildtype allele is functional. interferon-a/b (ifn-a/b) is increasingly used as antiviral and immunomodulatory therapies. unfortunately, bioavailability varies with ifn species and mode of administration, and all ifn species are potentially immunogenic. assays for antiviral activity (ifn) and antiviral neutralization (antibodies, nab) have been used for some time to monitor patients on ifn biologicals. these assays require laborious titrations making them unsuitable for large-scale clinical use. myxovirus a (mxa) is a resistance gtpbinding protein that is specifically induced by treatment with type 1 ifns. for example, ifn-b-induced mxa in blood leucocytes has been used as a biomarker in ifn-btreated patients with multiple sclerosis. however, the degree of specificity of mxa in this regard is unclear, and measurements of mxa protein and/or mrna are not yet suitable for routine clinical use. in an attempt to find new and better reporter genes (and, hopefully, genes and gene products with proven specificity for ifn-a and -b), microarray screenings with u133a genechips (affymetrix) were carried using human blood leucocytes and the human lung carcinoma cell line a549. we studied the simultaneous expression of 22,000 transcripts before and after exposure to human recombinant ifn-a and ifn-b and other antiviral and immunomodulatory cytokines. the results will be presented at the conference. interferon-a/b (ifn-a/b) is increasingly used as antiviral and immunomodulatory therapies. unfortunately, bioavailability varies with ifn species and mode of administration, and all ifn species are potentially immunogenic. assays for antiviral activity (ifn) and antiviral neutralization (antibodies, nab) have been used for some time to monitor patients on ifn biologicals. these assays require laborious titrations making them unsuitable for large-scale clinical use. our laboratories have therefore modified the antiviral assays for ifn bioactivity and nab, so that they are suitable for large-scale screening in specialized laboratories. the read-out is survival of a subcloned a549 cell line in the presence of an otherwise lethal amount of virus. thus, survival increases in the presence of type 1 ifn and decreases in the presence of nab against the ifn added to the cells. mxa is induced by type 1 ifn and can be used for measuring the nab activity. in another assay, the mxa level in the a549 cell line is measured. in an attempt to find a new and better reporter gene for type 1 ifn than mxa and genes specific for either ifn-a or -b, a micro array screen was carried using the u133a chip from affymetrix. the expression of 22,000 genes can be studied simultaneous with this technology. the results will be presented at the conference. in our laboratory, we have developed a database system, which we believe is of immediate interest to the general scientific community. the database represents a computerbased replacement for the laboratory notebooks used in the majority of research laboratories worldwide. in addition, the database provides an effective tool for organizing and managing laboratory information at all levels, spanning from managing and revising standard operating procedures and producing documentation of research activities to keeping track of data and conclusions. using the commercially available database toolkit software filemaker pro, we have developed a relational database solution for management of laboratory information. the system consists of a hierarchy of five interrelated databases, each pertaining to a separate type of information, namely, overall project information, information relating to individual experiment setups, documentation of daily research activity, generated data and descriptions of standard operating procedures. like other databases, each individual database consists of a number of records, each comprised of a set of fields in which information is entered. in each record, a certain field is reserved to specify the relation of the record to a record in another database at a higher level. thus, the database is essentially five databases linked by a hierarchy of one-to-many relations, organizing information in a folder-like structure. importantly, the database system allows multiple users to access and edit records simultaneously, and the data entered in one database immediately becomes accessible through the other databases. the limitations of laboratory notebooks are apparent when looking for information, which is dispersed throughout one or more notebooks, or possibly on loose sheets of paper or printouts 'somewhere'. the often complicated process of gathering laboratory data or results when writing grant applications or research papers is made considerably easier with the database system. thus, the database solution presented should be broadly attractive to researchers, irrespective of their scientific discipline. an effective sars vaccine is likely to include components that can induce specific cytotoxic t-cell (ctl) responses. the specificities of such responses are governed by hlarestricted presentation of sars-derived peptide epitopes. exact knowledge of how the immune system handles protein antigens would allow for the identification of such linear sequences directly from genomic/proteomic sequence information. the latter was recently established when a causative coronavirus (sars cov) was isolated and full-length sequenced. here, we have combined advanced bioinformatics and high-throughput immunology to perform an hla supertype, genome-wide scan for sars-specific cytotoxic t cell epitopes. the scan includes all nine human hla supertypes in total covering >99% of all major human populations. for each hla supertype, we have selected the 15 top candidates for test in biochemical-binding assays. at this time (approximately 6 months after the genome was established), we have tested the majority of the hla supertypes and identified almost 100 potential vaccine candidates. these should be further validated in sars survivors and used for vaccine formulation. we suggest that immunobioinformatics may become a fast and valuable tool in rational vaccine design. rationale: major histocompatibility complex class i (mhc i) molecules monitor the protein content of the cell by binding small derived peptides and presenting them to cytotoxic cd8 þ t cells. the goal of the human mhc project is to predict the binding strength of any given peptide/mhc complex. this prediction allows the design of peptide-based vaccines. the prediction requires representative binding data from mhc alleles from all the nine hla supertypes. here, we describe the genetic construction, protein production and purification as well as the establishment-binding assays for two recombinant mhc supertype alleles, hla-b*1501 and hla-b*5801. methods: using the quikchange multisite directed mutagenesis kit (stratagene), codon-optimized genes encoding hla-b*1501 and hla-b*5801 are created. the two mhc i molecules are fermented and purified by ion exchange chromatography, hydrophobic interaction chromatography and size exclusion chromatography. the binding (kd) of natural t-cell epitopes, as well as predicted peptide ligands, is described by radioactive immunoassays (rias) and enzyme-linked immunosorbent assays (elisas). the mhc molecules are biotinylated during expression. results: the expression of mhc i resulted in multiple disulfide bond isomers, which are separated by hydrophobic interaction chromatography and used in subsequent binding studies resulting in the determination of kd for various peptide ligands ranging from strong binders we have previously demonstrated that bioinformatics tools such as artificial neural networks (anns) are capable of performing pathogen-, genome-and hlawide predictions of peptide-hla interactions. these tools may therefore enable a fast and rational approach to epitope identification and thereby assist in the development of vaccines and immunotherapy. a crucial step in the generation of such bioinformatics tools is the selection of data representing the event in question (in casu peptide-hla interaction). this is particularly important when it is difficult and expensive to obtain data. herein, we demonstrate the importance in selecting information-rich data and we develop a computational method, query-bycommittee, which can perform a global identification of such information-rich data in an unbiased and automated manner. furthermore, we demonstrate how this method can be applied to an efficient iterative development strategy for these bioinformatics tools. methods: a large panel of binding affinities of peptides binding to hla a*0204 was measured by a radioimmunoassay (ria). this data was used to develop multiple first generation anns, which formed a virtual committee. this committee was used to screen (or 'queried') for peptides, where the anns agreed ('low-qbc'), or disagreed ('high-qbc'), on their hla-binding potential. seventeen low-qbc peptides and 17 high-qbc peptides were synthesized and tested. the high-or low-qbc data were added to the original data, and new high-or low-qbc second generation anns were developed, respectively. this procedure was repeated 40 times. the high-qbc-enriched ann performed significantly better than the low-qbc-enriched ann in 37 of the 40 tests. conclusion: these results demonstrate that high-qbcenriched networks perform better than low-qbc-enriched networks in selecting informative data for developing peptide-mhc-binding predictors. this improvement in selecting data is not due to differences in network training performance but due to the difference in information content in the high-qbc experiment and in the low-qbc experiment. finally, it should be noted that this strategy could be used in many contexts where generation of data is difficult and costly. interleukin-18 (il-18), a pro-inflammatory cytokine that is produced by both lymphoid and nonlymphoid cells, has a critical role in modulation of innate and adaptive immunity. its primary function in stimulation of ifn-g production and stimulation of nk-cell-cytotoxic activities makes this cytokine a candidate for cancer immunotherapy. in oral cavity, this cytokine is produced by oral epithelia and carcinoma cells and is related to tumour regression in nude mice bearing salivary adenocarcinoma. however, direct effects of this cytokine on oral cancer cells have not been elucidated. in this project, we investigated il-18 effect on an oral carcinoma (kb) cell line. with rt-pcr technique, kb-cell line was found to express il-18 receptors (il-18ra and il-18rb), indicating that this oral carcinoma line is a target for il-18 study. we showed that recombinant human il-18 inhibited kb-cell proliferation by 17% at concentration of 100 ng/ml (p < 0.05), whereas ldh release by these cells in treatment group and control groups was comparable, indicating that il-18 suppression of cell proliferation was not mediated by the induction of cell death. to further address this hypothesis, we found that il-18 treatment did not induce apoptotic cell death, as studied by dna laddering and tunel assays. in addition, expression pattern of cell death-controlling genes (bcl-2 and bax) was not altered by this cytokine. findings in these studies indicated that suppression of kb-cell proliferation may be attributed to control of cell cycle, growth arrest or induction of cell differentiation. the data presented in this project could provide an insight of how cancer cell directly responds to il-18, as this cytokine is an important regulator of anticancer mechanisms. aloe emodin (ae) is a naturally occurring compound with wide spectrum of biological properties, including antimicrobial, vasorelaxant, immunosuppressive and anticancer actions. this anthraquinone induces apoptosis in several tumour cell lines with special affinity to tumours of neuroectodermal origin. high amounts of nitric oxide (no) released by activated macrophages induce tumour cell death. therefore, we explored the capacity of ae to modulate no-mediated antitumour response in vitro. interestingly, while ae markedly suppressed no release from macrophages alone, it significantly potentiated no production in cocultures of macrophages and c6 cells, after 48 h of cultivation. accordingly, the viability of c6 cells cocultivated with macrophages was reduced in the presence of ae. moreover, the observed ae-imposed potentiation of no production in macrophages was closely related to macrophage culture cell density. according to these data, we proposed that no modulator capacity of ae strongly depended on intercellular contact, indicating that macrophage antitumour response was not compromised but even potentiated by ae. immunotherapy represents an attractive fourth-modality therapeutic approach, especially in the light of the shortcomings of conventional surgery, radiation and chemotherapies in the management of metastatic cancer. to this end, a large number of peptide antigens derived from taa have been applied in immunotherapeutic trials for the treatment of various malignancies, e.g. cancers of the breast, prostate and kidney, in addition to haematological cancers. in some cases the response rates have been impressive and no adverse autoimmunity have been observed. a major strategic difficulty associated with these trials relates to the choice of best-suited peptide antigens. the vast majority of the antigens described thus far is not vital for survival and growth of the tumour cells, and immunoselection of antigen-loss variants may therefore prove to be an additional obstacle for the clinical applicability of most of the known peptide epitopes. in this respect, the development of acquired antigen loss during immunotherapy has been demonstrated in several cases. obviously, the development of loss-variant tumour cells implies that these cells acquire a pronounced growth advantage and are left unaffected by further treatment. ideally, target antigens should be derived from proteins required for survival and growth of tumour cells, as antigens with these characteristics would not be inflicted by the development of loss-variant tumour cells. in this respect, several inhibitors of apoptosis proteins (iaps) are universally expressed among tumours and play an important role in tumour cell escape from apoptosis. we have characterized spontaneous t-cell reactivity against iapderived peptides in cancer patients. from the iap survivin, we have characterized peptides restricted to the class i molecules hla-a1, a2, a3, a11, b7 and b35. furthermore, we have demonstrated that survivin-specific t cells infiltrate metastatic lesions and that isolated survivinspecific ctls are capable of killing hla-matched tumour cells. survivin-derived peptides are now in clinical trial, and continued work in our lab has demonstrated that other iaps are targets for spontaneous t-cell reactivity in cancer patients. we previously reported that in mice with large progressing t-cell lymphoma tumours, dysfunctions in the antitumour ctl activity occur, associated with an accumulation of splenic arginase-producing myeloid suppressor cells (mscs). in this study, we first demonstrate that both the presence and the activation state of these msc depends on tumour evolution. while in tumour regressors hardly any arginase-producing msc can be found, both the amount and the arginase activity of this population expands from early over late progressors. this gradual induction of mscs is paralleled by an increasing suppression of ctl activity and th1, but not th2, cytokine production. upon analysing the molecular repertoire of msc in vitro, we found, besides arginase1, a well-established marker for alternatively activated myeloid cells or m2, a strong upregulation of fizz1 and ym, two additional recently identified markers for m2. further evaluation of molecular markers by microarray analysis in msc yielded genes involved in wound healing (e.g. coagulation factor xiiia), anti-inflammation (e.g. selenoprotein p), immunomodulation (e.g. pd-l2) and fat and sugar metabolism (e.g. leptin receptor). of note, many of these genes are regulated by type 2 cytokines (il-4, il-13 and il-10) and are therefore rather m2 associated. overall, our data provide new markers for msc in cancer and further establish their m2 activation state. study. only sp-a showed a significant expression in normal mucosa which was downregulated in crc. as the absolute signal level was below the noise threshold, these results have to be interpreted with caution and require confirmation by direct measurenment of the proteins. our results suggest that there is no major role for the human collectins in colorectal cancer. tetramerization is visualized by sds-page. conclusion: an effective method for the production of highly pure mhc i molecules has been applied to hla-b*1501 and hla-b*5801, and ria and elisa binding assays for those alleles have been established background: proliferation, differentiation and apoptosis are essential processes in the normal functions of the mammary epithelium. the hypothesis examined in this study is that the transcription factor bcl-6 is critically important not only for regulating b-cell growth and development but also for mammary epithelial apoptosis. methodology: twenty breast cancer cases and 31 healthy controls were used to investigate whether bcl-6 protein in involved in breast cancer (grade iii). full length bcl-6 cdna was retrovirally transduced into eph-4 cell line. we then used flow cytometry of brdurd-stained cells to investigate the cell-cycle duration of the control and transduced cell lines. tunel was used as a marker of apoptosis to find out differences in the frequencies of apoptotic cells in the control and transduced cell lines. finally, immunohistochemistry staining was performed to detect bcl-6 in breast cancer (iii). results: restoration of bcl-6 into eph-4 cells not only inhibits apoptosis but also prolongs the cell cycle and results in increased cell size and protein content. the results also indicated that the cell-cycle time of bcl-6-transduced eph-4 cells is prolonged by about 3 h, presumably as a result of the action of bcl-6 at the bcl-6 at the g1/s transition. we found differences in the frequencies of viable and apoptotic cells in cultures of the parent eph-4 cells, control-transduced eph-4 cells and bcl-6-transduced eph-4 cells. consistently, we demonstrated that bcl-6 is expressed in 90% of high grade of breast carcinoma, which is considered as the most aggressive of tumours. conclusion: together, these results suggest that bcl-6 is likely to be involved in mammary gland development and carcinogenesis. inflammatory cytokines have a critical role in modulation of both innate and adaptive immunity in response to foreign antigen. they also play an important role in anticancer immunity. for example, they can promote cell-mediated immunity against cancer cells. with their immunostimulatory effects, these cytokines are being tested for cancer treatment in the form of dna vaccine or adjuvant or therapeutic cytokines. direct effect of these cytokines on cancer cell, however, is still unclear. in this project, we investigated whether il-1( and il-18 can modulate cancer cell proliferation. we employed a simple nonradioactive proliferation (mtt) assay and detection of lactate dehydrogenase (ldh) to test the effect of these recombinant human cytokines on various cancer cell lines, including breast cancer cell line (mcf-7), oral carcinoma cell line (kb), colon cancer cell line (caco-2) and choriocarcinoma cell line (jar). cytokines used in this study had both inhibitory and stimulatory effect on cell proliferation. findings in this project could provide an insight of cancer cell response to these cytokines and this could lead to a consideration on using cytokine as immunotherapy for cancer treatment.capacity of ae to modulate nitric oxide production depended on intercellular contact donor t cells are involved in the antitumour effects observed after bmt. thus, patients receiving t-celldepleted bmt have a higher risk of leukaemia relapse compared to patients receiving nonmanipulated bmt, and patients experiencing graft-versus-host disease (gvhd) have a lower risk of disease relapse than patients who do not experience gvhd. although the importance of donor t cells for the curative action of bmt has been established, the exact mechanisms and molecules involved in this graft-versus-tumour effect remain largely unknown. in a recently initiated project, we have conducted a longitudinal study of t-cell clonotypes in patients who received peripheral blood stem cell grafts after nonmyeloablative conditioning. peripheral blood samples were obtained sequentially after transplant, and the mononuclear cells (mncs) were isolated and cryopreserved. cd8 þ t cells were isolated from the mncs by use of immunomagnetic beads or facs and analysed for the presence of clonally expanded cells by t-cell receptor clonotype mapping based on rt-pcr and denaturing gradient gel electrophoresis (dgge). using this gel-based methodology, clonally expanded t cells were monitored after transplant and compared to the clinical data of the patients. the preliminary results demonstrates the presence of clonally expanded cd8 þ t cells at all time points analysed. furthermore, a number of clonotypes persisted for more than 6 months, and other clonotypes emerged during this period. the appearance of newly emerged clonotypes which coincided with clinical gvhd could indicate a role for these t cells in the pathogenesis of gvhd. background: deficiency of the mannan-binding lectin (mbl) pathway of innate immunity leads to increased susceptibility to infections. in patients with colorectal cancer, postoperative infection is associated with poor prognosis. the aim of the present study was to evaluate (1) the relation between the mbl pathway and postoperative infectious complications and survival of patients resected for colorectal cancer and (2) the role of mbl as acute phase reactant compared to crp. methods: preoperative mbl concentration, mbl/mblassociated serine protease (masp) activity and crp were determined in serum from 611 patients and 150 healthy controls. the patients were observed for 8 years. postoperative infections, recurrence and survival were recorded. results: the mbl pathway components were increased in the patients (p < 0.0001) compared to healthy controls. low mbl levels were predictive of pneumonia (p ¼ 0.01), and pneumonia (n ¼ 87) was associated with poor survival (p ¼ 0.003, hr ¼ 1.5, 95% ci 1.1-1.9). mbl and mbl/ masp activity could not predict postoperative overall infections. mbl showed no correlation (spearman's r ¼ 0.02, 95% ci à0.06-0.10) with crp. conclusions: low preoperative mbl levels are predictive of pneumonia, which is associated with poorer survival. mbl concentration and mbl/masp activity was not predictive of other postoperative infections or long-term prognosis. mbl apparently is not a surrogate measure of crp. department of surgery, university hospital of erlangen, erlangen, germany. e-mail: michael.siassi@rzmail.uni-erlangen.de introduction: the human collectins, mannan-binding lectin (mbl), surfactant protein-a (sp-a) and surfactantprotein-d (sp-d) play a central role in the innate immune system. immunological responses to malignant transformation of epithelial cells gained increasing interest recently. a former study could demonstrate binding of mbl to certain colorectal carcinoma (crc) cell lines in vitro. we therefore examined the expression of human collectins in normal colon mucosa and in colorectal carcinomas. materials and methods: colon samples from 20 crc patients and 10 normal mucosa samples were collected immediately after surgery. the tissue was microdissected and rna isolated (qiagen, rneasy-kit). gene expression profiles were analysed using gene-chips (affymetrix, hg-u133). we analysed the data for the expression of mbl, its associated serine proteases mannan-binding lectinassociated serine protease 1/2 (masp 1/2), sp-a and sp-d. the signal intensity of the genes of interest was compared using the mann-whitney u-test. results: the expression of human collectins in normal human colon mucosa was generally low. only the expression of sp-a and masp-2 reached the noise threshold of 250 signals. these genes were significantly downregulated in crc specimens. the expression of the other proteins showed no difference in normal mucosa and crc. conclusion: as demonstrated before, the expression of human collectins in normal colon was low in this being the first lymph node to receive drainage from the tumour area, the sentinel node offers a unique possibility to obtain tumour-reactive lymphocytes. we investigated antitumour immune responses in sentinel nodes from patients with bladder cancer, by assaying tumour-specific proliferation and tcr vb repertoires. during tumour surgery, sentinel lymph nodes were identified by peritumoural injection of blue dye. fresh specimens of tumour, sentinel and nonsentinel lymph nodes were obtained, and single-cell suspensions were prepared. cells were assayed for reactivity against autologous tumour extract in [ 3 h]-thymidine incorporation assays and characterized by flow cytometry. parallel analyses of the expression of vb gene families were performed with padlock probes, linear oligonucleotides which upon target recognition can be converted to circular molecules by a ligase. probes were reacted with cdna prepared from magnetically separated cd4 þ cells, and the tcr repertoire was determined by hybridizing the products to oligonucleotide microarrays. dose-dependent proliferation in response to tumour extract could be detected in sentinel lymph nodes. common clonal expansions were detected among tumourinfiltrating lymphocytes and in sentinel lymph nodes. nonsentinel lymph nodes displayed a divergent tcr vb repertoire. these results indicate an ongoing immune response against tumour antigens in sentinel nodes, draining urinary bladder cancer. identification of sentinel lymph nodes makes it possible to obtain tumour-reactive lymphocytes for use in adoptive immunotherapy. key: cord-004334-y1fcw1dj authors: kalodimou, georgia; veit, svenja; jany, sylvia; kalinke, ulrich; broder, christopher c.; sutter, gerd; volz, asisa title: a soluble version of nipah virus glycoprotein g delivered by vaccinia virus mva activates specific cd8 and cd4 t cells in mice date: 2019-12-24 journal: viruses doi: 10.3390/v12010026 sha: doc_id: 4334 cord_uid: y1fcw1dj nipah virus (niv) is an emerging zoonotic virus that is transmitted by bats to humans and to pigs, causing severe respiratory disease and often fatal encephalitis. antibodies directed against the niv-glycoprotein (g) protein are known to play a major role in clearing niv infection and in providing vaccine-induced protective immunity. more recently, t cells have been also shown to be involved in recovery from niv infection. so far, relatively little is known about the role of t cell responses and the antigenic targets of niv-g that are recognized by cd8 t cells. in this study, niv-g protein served as the target immunogen to activate niv-specific cellular immune responses. modified vaccinia virus ankara (mva), a safety-tested strain of vaccinia virus for preclinical and clinical vaccine research, was used for the generation of mva–niv-g candidate vaccines expressing different versions of recombinant niv-g. overlapping peptides covering the entire niv-g protein were used to identify major histocompatibility complex class i/ii-restricted t cell responses in type i interferon receptor-deficient (ifnar−/−) mice after vaccination with the mva–niv-g candidate vaccines. we have identified an h2-b-restricted nonamer peptide epitope with cd8 t cell antigenicity and a h2-b 15mer with cd4 t cell antigenicity in the niv-g protein. the identification of this epitope and the availability of the mva–niv-g candidate vaccines will help to evaluate niv-g-specific immune responses and the potential immune correlates of vaccine-mediated protection in the appropriate murine models of niv-g infection. of note, a soluble version of niv-g was advantageous in activating niv-g-specific cellular immune responses using these peptides. nipah virus (niv) is an emerging zoonotic pathogen of global concern that was ranked recently by the world health organization (who) as a high-priority pathogen. niv is a negative-sense, single-stranded rna virus that is a member of the genus henipavirus (family paramyxoviridae). niv was first identified during a large outbreak affecting humans and pigs in malaysia and singapore in 1999 [1] . from 2001 onwards, seasonal outbreaks are observed almost annually in bangladesh and sporadically hohenpeißenberg, germany) and had access to food and water ad libitum. all experiments were approved by the government of upper bavaria, munich germany and were performed in compliance with the german animal welfare act (55.2vet-2532.vet_02-17-93, 09.01.2017). primary chicken embryo fibroblasts (cef) were isolated from 10-day-old spf chicken embryos (valo, cuxhaven, germany) and grown in minimum essential medium (mem) (sigma-aldrich, taufkirchen, germany) supplemented with 10% heat-inactivated fetal bovine serum (fbs) (sigma-aldrich), 1% penicillin-streptomycin (sigma-aldrich), and 1% mem nonessential amino acid solution (sigma-aldrich). human hela cells (atcc ccl-2) were maintained in mem supplemented with 10% heat-inactivated fbs (sigma-aldrich) and the above antibiotics. df-1 cells (atcc crl-12203) were grown in vle dulbecco's modified eagle's medium (dmem) (merck, darmstadt, germany) supplemented with 10% heat-inactivated fbs (sigma-aldrich), 1% penicillin-streptomycin (sigma-aldrich), 1% mem nonessential amino acid solution (sigma-aldrich), and 1% hepes solution (sigma-aldrich). cells were maintained at 37 • c in a humidified 5% co 2 atmosphere. the cdna that encoded for the entire 602 amino acid sequence of the niv-g protein (nipah virus isolate ummc1, genbank accession number ay029767.1) was modified in silico by introducing silent codon alterations to remove termination signals of vaccinia virus early transcription (tttttnt) and g/c nucleotide runs. for construction of the soluble form of niv-g protein (nivsg), the cytoplasmic and transmembrane domains were deleted and an internal leader sequence and amino acid linker sequences were added as described previously [40, 48] . for vaccinia virus (vacv)-specific transcriptional regulation, we placed the nivsg gene sequences under control of the synthetic vacv early/late promoter (pmh5) [49] and used the strong natural early vacv promoter pvgf [50] [51] [52] for expression of niv-g sequences. the cdnas encoding for niv-g or nivsg including the cleavage sites for the restriction endonucleases xhol and apal were generated by dna synthesis (genewiz, leipzig, germany). cdna sequences were cloned into the mva transfer plasmid plw-73 [53] , which directs insertion of heterologous sequences to a site between the open reading frames (orf) of the essential viral genes, mva069r and mva070l. recombinant mva viruses expressing the nivsg and niv-g proteins were generated as described previously [54, 55] . to summarize, cef cells at 80-90% confluence were infected with nonrecombinant mva (clonal isolate mva f6) at a multiplicity of infection (moi) of 0.05 and transfected with vector plasmid dna containing nivsg or niv-g gene sequences using x-tremegene™ hp dna transfection reagent (roche diagnostics, penzberg, germany). after 48 h incubation, cells were harvested, and the recombinant viruses mva-nivsg and mva-niv-g were clonally isolated by screening for co-expression of the fluorescent protein marker gfp and plaque passaging several times. resultant vector virus isolates were quality controlled using standard protocols [54] . polymerase chain reaction (pcr) analysis of genomic viral dna served to confirm the genetic identity and genomic stability of the mva vector viruses. the replicative capacity of the recombinant mva-niv viruses compared with nonrecombinant mva was tested by multi-step growth experiments in cef and human hela cells. to generate vaccine preparations, recombinant mva-niv viruses were amplified in cef, purified by ultracentrifugation through 36% sucrose cushions, and reconstituted in 10 mm tris-hcl buffer, ph 9.0, to make stock preparations [54] . cef and hela cells were infected with recombinant mva-nivsg and mva-niv-g viruses at a moi of 5. cells infected with mva (moi 5) and inoculation medium alone (mock) were used as controls. cell lysates were prepared or culture supernatants were collected at various time points after infection and stored at −80 • c. cell-associated and secreted proteins were resolved by sodium dodecyl sulfate (sds)-polyacrylamide (10%) gel electrophoresis (sds-page), and proteins were transferred onto nitrocellulose membranes by wet electroblotting. to investigate the glycosylation pattern, proteins were pretreated using the protein deglycosylation mix ii kit (new england biolabs, ipswich, ma, usa) according to the manufacturer's instructions prior to sds page. membranes were blocked with blocking buffer, which consisted of pbs containing 5% non-fat dried milk powder (carl roth, karlsruhe, germany) and 0.1% v/v tween 20 (sigma-aldrich) for one hour at room temperature. membranes were then probed with the primary antibody (polyclonal mouse anti-niv-g (1:2000) or polyclonal rabbit anti-niv-g (1:5000)) diluted in blocking buffer overnight at 4 • c. membranes were washed three times with pbs containing 0.1% tween 20 (pbs/t) and probed with goat anti-mouse igg conjugated to horseradish peroxidase (hrp) (1:5000; agilent dako, glostrup, denmark) or goat anti-rabbit igg hrp (1:5000; cell signaling technology, leiden, the netherlands). membranes were washed again with pbs/t and were developed using supersignal ® west dura extended duration substrate (thermo fisher scientific, planegg, gemany). blots were visualized using a microchemi 4.2 imager (dnr bio-imaging systems, neve yamin, israel). confluent monolayers of hela cells were infected with recombinant mva-nivsg or mva-niv-g viruses at moi 0.05. controls included mva (moi 0.05) and inoculation medium alone (mock). after infection, cells were incubated for 16 h in a 37 • c incubator. cells were fixed with 4% paraformaldehyde (pfa) (sigma-aldrich) and on some occasions, permeabilized with 0.5% triton x-100 (sigma-aldrich). cells were blocked in blocking buffer containing 1% bovine serum albumin (bsa) (sigma-aldrich). cells were stained with polyclonal rabbit anti-niv-g diluted 1:10,000 in pbs containing 0.5% bsa (pbs/bsa). cells were washed with pbs and stained with the secondary antibody goat anti-mouse igg alexa fluor (af) 568 (1:1000; thermo fisher scientific) diluted in pbs/bsa buffer. fluorescence was visualized using the keyence bz-x710 microscope (keyence, osaka, japan). mice were immunized with 10 8 pfu in 50 µl vaccine buffer (10 mm tris and 140 mm nacl, ph 7.4) of recombinant mva-nivsg, recombinant mva-niv-g or mva or vaccine buffer as a mock vaccine control via the intraperitoneal or intramuscular routes. mice received either one (prime) or two (prime-boost) immunizations over a 21 day interval. for t cell analysis, mice were euthanized 8 days after immunization. blood was collected on days 0, 18, and 31. coagulated blood was centrifuged at 1300× g for 5 min in minicollect vials (greiner bio-one, alphen aan den rijn, the netherlands) in order to separate serum, which was subsequently stored at −20 • c until further use. antigen-specific igg responses induced by immunization with the vaccine candidates were analyzed by enzyme-linked immunosorbent assay (elisa) using purified soluble recombinant niv glycoprotein g expressed in mammalian hek293 cells. flat bottom 96-well elisa plates (nunc™ maxisorp™ plates, thermo scientific) were coated with 50 ng/well recombinant protein (100 µl volume) and incubated overnight at 4 • c. plates were washed three times with 200 µl/well pbs/t. plates were blocked with blocking buffer containing 1% bovine serum albumin (sigma-aldrich) and 0.15 m sucrose (sigma-aldrich) in pbs for 1 h at 37 • c. plates were then washed with pbs/t as described above. sera were serially diluted in pbs containing 1% bsa (pbs/bsa) three-fold down the plate, starting at a dilution of 1:100 (100 µl volume/well) and incubated for 1 h at 37 • c. after washing, plates were incubated with 100 µl/well goat anti-mouse igg conjugated hrp (1:2000; agilent dako, denmark) diluted in pbs/bsa for 1 h at 37 • c. plates were then washed with pbs/t as described earlier. then, 100 µl/well 3,3 , 5,5 -tetramethylbenzidine (tmb) liquid substrate system for elisa (sigma-aldrich) was added, and plates were incubated until a color change was observed. the reaction was stopped by the addition of 100 µl/well stop reagent for tmb substrate (450 nm, sigma-aldrich). the absorbance was measured on an elisa plate reader at 450 nm with a 620 nm reference wavelength. total igg titers were calculated from the inflection point of the titration curve as logarithms of the reciprocal. the protein sequence for niv-g was obtained from the uniprot database (id: q9ih62). using an in silico approach, we identified 130 individual synthetic peptides that spanned the external domain niv-g protein, starting from the third amino acid of the n-terminal side to the c terminus (amino acids 73-602). our peptide library consisted of 15mer peptides that overlapped by 11mer. all peptides were synthesized by thermo fisher scientific as crude material (<50% purity) on a 1-4 mg scale. the two-dimensional peptide pool matrix system was used for screening [56, 57] . briefly, peptides were organized into two-dimensional matrix peptide pools (h1-h10 and v1-v11) containing 11-13 peptides. for mapping of potential cd8 t cell epitopes in positive 15mer peptides, every possible sequence of peptides 8-11mer in length was determined. theoretical peptides were then analyzed for binding to the mouse major histocompatibility complex (mhc) class i allele h2-b using the syfpeithi database. the peptides of each amino acid sequence length were synthesized and tested. for identification of cd4 t cell epitopes, mhc class ii binding predictions were performed on 15mer peptides found within positive peptide pools identified by ifn-γ enzyme-linked immunospot assay (elispot). using the mhc class ii binding, t cell epitope prediction resource of the immune epitope database (iedb, https://www.iedb.org/), peptides restricted to mouse mhc class ii allele h2-iab were analyzed using "iedb recommended" prediction method [58] . the most promising candidates were then chosen for further experimental epitope prediction studies. all peptides were dissolved to a concentration of 2 mg/ml in pbs, aliquoted, and stored at −20 • c until use. t cell analysis by elispot was performed as described previously [55] . briefly, spleens were collected from mice 8 days after the final immunization. single-cell suspensions were prepared by teasing spleens through a 70 µm cell strainer (falcon ® corning, corning, ny, usa). red blood cells (rbc) were removed using red cell lysis buffer (sigma-aldrich), and cells were washed and resuspended in rpmi-10, which consisted of rpmi-1640 (sigma-aldrich) containing 10% heat-inactivated fbs (sigma-aldrich) and 1% penicillin-streptomycin (sigma-aldrich). for experiments that required cd4 and cd8 t cell purification, splenocytes were incubated with mouse cd4 and cd8 microbeads (miltenyi biotec, bergisch gladbach, germany) and processed by negative selection using the quadromacs separator (miltenyi biotec). ifn-γ-producing cells were measured by ifn-γ elispot assay using the mouse ifn-γ elispotplus kit (mabtech, stockholm, sweden) as described in the manufacturer's protocol. in summary, 2 × 10 5 splenocytes were seeded onto 96-well flat bottom plates (100 µl/well) (sarstedt, nümbrecht, germany), and 100 µl/well peptide pools, subpools, or individual peptides were added (each peptide diluted to 2 µg/ml in rpmi-10). after mixing, the splenocyte/peptide mixtures were transferred onto plates precoated with ifn-γ detection antibody and incubated for 48 h at 37 • c. nonstimulated cells were used as a mock control, and the positive controls cultures were treated with phorbol myristate acetate (pma) and ionomycin (both from sigma-aldrich) or vaccinia virus (vacv)-specific cd8 t cell epitope, b8r 20-27 (tsykfesv) [59] . after incubating, plates were processed as described in the kit manufacturer's protocol (mabtech). spots were counted and analyzed using the automated elispot plate reader (a. el. vis eli.scan and a. el. vis elispot analysis software, hannover, germany). splenocytes were diluted to 1 × 10 7 cells/ml in rpmi-10, and 100 µl/well (1 × 10 6 cells) was added onto a 96-well u-bottom plate. then, 100 µl/well peptide diluted to 16 µg/ml in rpmi-10 was added to give a final peptide concentration of 8 µg/ml. the vacv cd8 t cell epitope b8r 20-27 (final concentration of 8 µg/ml) was used as a positive control along with pma (10 ng/ml) plus ionomycin (500 ng/ml). pbs diluted in rpmi-10 was used as a mock stimulated control. after plating, cells were incubated for 2 h at 37 • c. then, 20 µl/well 10x brefeldin a, a golgi inhibitor that was prepared by diluting 1000x brefeldin a (biolegend, san diego, ca, usa) in rmpi-10, was added to give a final dilution of 1x brefeldin a. cells were then incubated for an additional 4 h at 37 • c. after incubating, plates were centrifuged (500 g for 3 min), and the supernatant was removed. samples were filtered through 50 µm nylon mesh (sefar pty ltd., huntingwood, nsw, australia) into 5 ml round bottom facs tubes (sarstedt). single-color controls were prepared for each facs analysis using onecomp ebeads™ compensation beads (ebioscience, thermo fisher scientific) for fluorophore-conjugated antibodies and cells for the viability dye zombie aqua. data acquisition was performed by macsquant vyb flow analyser (miltenyi biotec), and data was analyzed using flowjo (flowjo llc, bd life sciences, ashland, or, usa). data were analyzed using graphpad prism version 5.0 (graphpad software inc., san diego, ca, usa) and were expressed as mean ± standard error of the mean (sem). statistical analysis was performed using the unpaired, two-tailed t-test to compare two groups and one-way anova to compare three or more groups. the threshold for statistical significance was p < 0.05. to generate the recombinant mva viruses delivering niv-g antigens, we used the gene from niv malaysia (isolate ummc1, genbank accession number ay029767.1) and generated codon-optimized gene sequences encoding a full-length glycoprotein g (niv-g) or a soluble external domain g protein (nivsg). these synthetic gene sequences were placed under the transcriptional control of the vaccinia virus-specific promoters pvgf or pmh5 and introduced into the mva genome by homologous recombination targeting the intergenic site between the essential mva genes 069 and 070l. the clonal isolation of the recombinant viruses mva-nivsg and mva-niv-g was facilitated by the co-production of the green fluorescent reporter protein (gfp), as previously described [54] . the final recombinant viruses containing the niv-g gene sequences (mva-niv-g and mva-nivsg) were obtained after removal of the gfp reporter gene by intragenomic homologous recombination ( figure s1a , marker gene deletion). to verify the identity of the desired modification, we performed the standard quality control experiments as described previously [54] . pcr analysis of viral genomic dna confirmed the proper insertion of the recombinant gene sequences at the target site in the genome of mva (figure s1b-e) and the genetic characteristics and stability of the recombinant mva viruses. we assessed the growth behavior of the recombinant viruses mva-nivsg and mva-niv-g in multi-step growth analyses in human hela cells and primary cef, which are routinely used for amplification of recombinant mva in vaccine manufacturing ( figure s1f ). mva-nivsg and mva-niv-g efficiently replicated in cef and demonstrated an increase of infectivity titers that was comparable to that obtained with nonrecombinant mva. however, mva-niv-g and mva-nivsg did not productively grow in human hela cells, confirming that they had retained the characteristic replication deficiency of mva in cells of mammalian origin. these findings corroborated the expected mva phenotype and confirmed that the recombinant viruses could be handled under laboratory conditions of biosafety level 1. of note, we originally generated another recombinant mva virus using the synthetic early-late promoter pmh5 for transcriptional control of recombinant gene expression and production of the full-length niv-g protein. however, upon growth testing, this candidate virus failed to reach levels of infectious progeny in cef to be eligible for large-scale amplification as needed for vaccine production processes. our vaccine candidates, mva-nivsg and mva-niv-g, should produce recombinant niv glycoprotein g in its full-length form (niv-g) and, in parallel, the soluble form (nivsg). niv-g is a 602 amino acid long protein consisting of an n-terminal internal domain, a transmembrane domain, and a c-terminal external domain ( figure 1a ). for nivsg protein, an internal leader sequence and three amino acid linkers have replaced the internal and transmembrane domains ( figure 1a ), as described previously [19, 48] . we assessed the correct expression and studied the cellular localization of the niv-g and nivsg by immunofluorescence microscopy of mva-niv-infected cells immunolabeled with anti-niv-g antibody, followed by a fluorescently labelled secondary antibody. cell nuclei were stained with dapi (300 nm). as anticipated, we observed different patterns of green fluorescence, with varying cellular localizations depending on the mva-niv construct. green fluorescence, specific for niv-g, was identified in permeabilized and nonpermeabilized cells infected with mva-niv-g ( figure 1b ), but not in mva-infected or mock control cells. this data confirmed that the recombinant full-length niv-g protein encoded by mva-niv-g was indeed anchored on the cell surface. in contrast, the niv-g-specific staining in cells infected with mva-nivsg virus appeared to be predominantly located within the cells and was readily detected after permeabilization. as anticipated, we failed to detect nivsg in considerable amounts without permeabilization, confirming that nivsg was not expressed on the cell surface ( figure 1b) . to further investigate the synthesis of niv-g proteins after infection with mva-nivsg and mva-niv-g, respectively, total proteins from infected cef and hela cell cultures were analyzed by western blot using a niv-g-specific antibody. total cell lysates or culture supernatants obtained from cef and hela cultures infected with recombinant mva virus were separated by sds-page and immunoblotted. we specifically detected a protein with an estimated molecular mass of approximately 72-75 kda in lysates from cef cells and hela cells infected either with mva-niv-g or mva-nivsg ( figure 1c ). in the cell lysates, the glycoprotein was first detectable at 4 h post-infection. since the nivsg gene encoding sequences were modified to result in the secreted soluble version (sg), we performed additional western blot experiments to determine whether the protein still maintained glycosylation sites comparable to wild-type niv-g. lysates and supernatants obtained from cultures of df-1 cells infected with mva-nivsg were treated with enzymes that deglycosylate proteins and analyzed by western blotting. enzyme treatment resulted in a reduction in the molecular mass of recombinant nivsg protein from 70-75 kda to 58-60 kda ( figure 1d ), matching the expected size of unmodified g protein. this suggested that recombinant nivsg has retained the normal glycosylation pattern of wild-type niv-g. to assess the immunogenicity of the recombinant mva-niv-g/nivsg candidate vaccines, we vaccinated ifnar−/− mice with 10 8 pfu via the intramuscular route at days 0 and 21. serum samples were tested for niv-g-binding igg antibodies by elisa 18 days after the first immunization (prime) and 10 days after the second immunization (prime-boost) (figure 2 ). even a single application of the mva-niv-g vaccines induced abundant levels of niv-g-specific igg antibodies in the mice. after booster immunization, all vaccinated animals produced even higher levels of circulating niv-g-specific antibodies, with the antibody titers increasing by approximately ten-fold. currently, there is only limited information available on niv-specific t cell immunity. in order to characterize the niv-g-specific t cell response induced by our candidate vaccines, we first aimed to identify niv-g polyprotein-specific t cell epitopes. ifnar−/− mice on a c57bl/6 background (mhc i = h2-db/h2-kb (h2-b) and mhc ii = h2-iab) were immunized twice with the mva-nivsg candidate vaccine via the intraperitoneal (i.p.) route, and splenocytes were prepared 8 days after the final inoculation, cd4 and cd8 t cells were purified and restimulated with overlapping 15mer peptides corresponding to the niv-g protein. we pooled overlapping peptides using a twodimensional peptide matrix system ( figure 3a ) and screened for their ability to induce ifn-γ by elispot. ifn-γ production above background levels was observed after stimulation with 2 out of the 21 peptide pools tested ( figure 3b ). cd8 t cells from mice immunized with mva-nivsg, but not the mva and mock groups, showed elevated numbers of ifn-γ spot-forming counts (sfc) after stimulation with peptide pools v1 and h1 ( figure 3b ). in the next step, the peptides within the v1 and h1 peptide pools were used to characterize the t cell epitope specificities in more detail. for this, pools v1 (v1a and v1b) and h1 (h1a and h1b) were subdivided into subpools containing 5-7 peptides each. since two 15mer peptides were shared between the pools, #1 (g73-87 = ytrstdnqavikdal) and #2 (g77-91 = tdnqavikdalqgiq), these peptides were also tested separately. for this experiment, we used an identical immunization protocol and splenic cd8 t cell purification procedure. cd8 t cells from these mice were restimulated with the above subpools and individual peptides #1 and #2. stimulation with the subpools h1a and v1a resulted in ifn-γ production above background levels in the mva-nivsg group with ifn-γ sfc mean ± sem values of 455 ± 99 and 271 ± 74 ifn-γ sfc/10 6 splenocytes, respectively ( figure 1c ). subpools h1b and v1b, however, did not stimulate cd8 t cells in the mva-nivsg group. stimulation of cd8 t cells with individual peptides #1 and #2, which were present in subpools h1a and v1a, resulted in different outcomes. ifn-γ production above the background was observed in cultures stimulated with #1 (mean = 349 ± 87 sfc/10 6 cells), but not #2, in the mva-nivsg group. these data suggested that the 15mer peptide #1 contained peptide sequences that stimulated niv-g-specific cd8 t cells. niv-g or niv-sg currently, there is only limited information available on niv-specific t cell immunity. in order to characterize the niv-g-specific t cell response induced by our candidate vaccines, we first aimed to identify niv-g polyprotein-specific t cell epitopes. ifnar−/− mice on a c57bl/6 background (mhc i = h2-db/h2-kb (h2-b) and mhc ii = h2-iab) were immunized twice with the mva-nivsg candidate vaccine via the intraperitoneal (i.p.) route, and splenocytes were prepared 8 days after the final inoculation, cd4 and cd8 t cells were purified and restimulated with overlapping 15mer peptides corresponding to the niv-g protein. we pooled overlapping peptides using a two-dimensional peptide matrix system ( figure 3a ) and screened for their ability to induce ifn-γ by elispot. ifn-γ production above background levels was observed after stimulation with 2 out of the 21 peptide pools tested ( figure 3b ). cd8 t cells from mice immunized with mva-nivsg, but not the mva and mock groups, showed elevated numbers of ifn-γ spot-forming counts (sfc) after stimulation with peptide pools v1 and h1 ( figure 3b ). in the next step, the peptides within the v1 and h1 peptide pools were used to characterize the t cell epitope specificities in more detail. for this, pools v1 (v1a and v1b) and h1 (h1a and h1b) were subdivided into subpools containing 5-7 peptides each. since two 15mer peptides were shared between the pools, #1 (g 73-87 = ytrstdnqavikdal) and #2 (g 77-91 = tdnqavikdalqgiq), these peptides were also tested separately. for this experiment, we used an identical immunization protocol and splenic cd8 t cell purification procedure. cd8 t cells from these mice were restimulated with the above subpools and individual peptides #1 and #2. stimulation with the subpools h1a and v1a resulted in ifn-γ production above background levels in the mva-nivsg group with ifn-γ sfc mean ± sem values of 455 ± 99 and 271 ± 74 ifn-γ sfc/10 6 splenocytes, respectively ( figure 1c ). subpools h1b and v1b, however, did not stimulate cd8 t cells in the mva-nivsg group. stimulation of cd8 t cells with individual peptides #1 and #2, which were present in subpools h1a and v1a, resulted in different outcomes. ifn-γ production above the background was observed in cultures stimulated with #1 (mean = 349 ± 87 sfc/10 6 cells), but not #2, in the mva-nivsg group. these data suggested that the 15mer peptide #1 contained peptide sequences that stimulated niv-g-specific cd8 t cells. graphs shows ifn-γ sfc (spot-forming counts) of stimulated cd8 t cells. differences between groups were analyzed by one-way anova. asterisks represent statistically significant overall differences for a specific peptide subpool or individual peptide. * p < 0.05. to map the potential cd8 t cell epitope within the niv-g protein in more detail, we dissected the 15mer peptide #1 into every possible 8-11mer sequence ( figure 4a ). the sequences were then analyzed for h2-b binding computationally using the syfpeithi database, and the four best of each amino acid length were chosen (table 1) . to test these peptides, we immunized ifnar−/− mice twice with mva-nivsg via the i.p. route and analyzed total splenocyte activation by elispot assay. of the sixteen 8-11mer overlapping peptides tested, eight resulted in the stimulation of measurable ifn-γ in the mva-nivsg group ( figure 4b ). the positive peptides included three 11mer in length (11.1, 11.2, and 11.3), two 10mer in length (10.2 and 10.3), two 9mer in length (9.3 and 9.4), and one 8mer in length (8.4) ( figure 4b ). the mean ifn-γ sfc value was lower for cells stimulated with peptide 8.4 relative to peptide 9.3. the mean values for the mva-nivsg group were 645 ± 112 ifn-γ sfc/10 6 cells for peptide 9.3 and 380 ± 85 ifn-γ sfc/10 6 for peptide 8.4. comparative analysis of the positive peptide sequences demonstrated that the sequence of peptide 9.3 (rstdnqavi) was present in all positive 10-11mer peptides (table 1 ). in addition, the sequence of peptide 8.4 (stdnqavi) was present in the eight positive peptides. to characterize the induction of ifn-γ sfc by these peptides in more detail, ifnar−/− mice were vaccinated twice via the i.m. route, a commonly used route for human vaccinations, and in vitro stimulated splenocytes were analyzed by elispot assay. for this experiment, we chose the most promising peptides of each amino acid sequence length (peptides 8.4, 9.3, 10.2, 10.3, 11.1, and 11.2). the six peptides tested significantly stimulated the activation of ifn-γ in the mva-nivsg group relative to the mva and pbs controls ( figure 4c ). mean sfc values, however, did not show statistically significant variations between each individual peptide. importantly, the mean sfc value for peptide 9.3 was again nonsignificantly elevated relative to peptide 8.4 (mean ± sem = 663 ± 213 and 305 ± 148 ifn-γ sfc/10 6 cells respectively). consequently, we chose peptide 9.3 (rstdnqavi) (mean = 906 ± 109 ifn-γ sfc/10 6 cells) as a potential h2-b-restricted epitope candidate of niv-g ( table 2 ). the alignment of peptide 9.3 to the full sequence of niv-g is shown in figure s3 . (11.1, 11.2, 10.2, 10.3, 9.3, 8.4) . differences between groups were analyzed by one-way anova. asterisks represent statistically significant overall differences for a specific peptide. * p < 0.05, ** p < 0.01. 1 peptide chosen as the most promising h2-b-restricted peptide of niv-g; 2 peptide chosen as the most promising h2-iab-restricted peptide of niv-g. rows in bold indicate promising niv-g-specific t cell epitopes. to comparatively evaluate the activation of niv-g-specific immunity after i.m. vaccination with mva-nivsg and mva-niv-g using a standard dose of 10 8 pfu, we stimulated splenocytes with peptide 9.3 ( table 2 ) and analyzed cytokine production by ifn-γ elispot assay and ifn-γ plus tnf-α ics. comparisons between the two candidate vaccines showed that the mean sfc values were significantly higher for the mva-nivsg group relative to the mva-niv-g group ( figure 5a and figure s2a ). the means of the mva-nivsg group were 914 ± 221 ifn-γ sfc/10 6 cells, and the means of the mva-niv-g group were 410 ± 68 ifn-γ sfc/10 6 cells ( figure 3c ). ifn-γ ics data showed that both the frequency and absolute number of ifn-γ+ cd8 t cells were significantly higher than the control background levels ( figure 5b ). our peptides did not induce ifn-γ production by cd4 t cells ( figure s2b ), indicating that they indeed stimulated antigen-specific cd8 t cells only. when we compared our two candidate vaccines, we found that the percentage and absolute number of ifn-γ+ cd8 t cells was significantly higher in the mva-nivsg group. the mean frequencies of ifn-γ+ cd8 t cells were 0.84% ± 14% for the mva-nivsg group and 0.33% ± 0.04% for the mva-niv-g group. mean absolute number of ifn-γ+ cd8 t cells were in the range of 2040 ± 380 cells/10 6 splenocytes and 978 ± 112 cells/10 6 splenocytes for the mva-nivsg and mva-niv-g groups, respectively. taken together, our elispot and ifn-γ ics data indicate that mva-nivsg activates higher numbers of peptide-specific cd8 t cells than mva-niv-g. when we analyzed for coproduction of ifn-γ and tnf-α, we found that the majority of niv-g peptide-specific cd8 t cells from mva-nivsg and mva-nivg immunized mice were double positive for the cytokines (ifn-γ + tnf-α+) ( figure s4a-c) . after showing that prime-boost immunization with our two vaccine candidates generated robust niv-g-specific cd8 t cell responses, we tested their immunogenicity after a single vaccination. ifnar−/− mice were vaccinated once with mva-nivsg, mva-niv-g, mva, or mock via the i.m. route and analyzed by elispot and ifn-γ + tnf-α ics as described before. our elispot results overall showed elevated sfc counts relative to the background levels of our mva and mock controls ( figure 5c ). statistically significant differences between the mva-nivsg and mva-niv-g were detected, with mean sfc values of 704 ± 67 ifn-γ sfc/10 6 cells and 233 ± 46 ifn-γ sfc/10 6 for them, respectively. facs analysis showed that the frequency and absolute number of ifn-γ+ cd8 t cells was higher in the experimental groups relative to the mva and mock controls ( figure 5d ). the mean percentage of ifn-γ + cd8 t cells was 0.14% ± 0.049% for the mva-niv-g groups, whereas for the mva-nivsg group, the mean percentage range for the peptides was 0.65% ± 0.21%. while mva-niv-g prime immunization induced a low frequency of ifn-γ+ cells, the mva-nivsg group showed ifn-γ+tnf-α+ secreting t cells after a single vaccination ( figure s4d-f) , although percentages were lower than what was observed after prime-boost immunization ( figure s4a-c) . this indicates that prime immunization with mva-nivsg was sufficient to generate a sizeable population of polyfunctional antigen-specific cd8 t cells. table 2) and measured by elispot assay and ifn-γ ics plus facs analysis. (a,b) antigen-specific cd8 t cell response induced by prime-boost immunization. (a) ifn-γ sfc for stimulated splenocytes measured by elispot assay. (b) ifn-γ production by stimulated splenic cd8 t cell measured by ics and facs analysis. graphs show frequency and absolute number (per 10 6 splenocytes) of antigen-specific ifn-γ+ cd8 t cells. (c,d) antigen-specific cd8 t cell response induced by prime immunization. (c) ifn-γ sfc for stimulated splenocytes measured by elispot assay. (d) frequency and absolute number (per 10 6 splenocytes) of antigen-specific cd8 t cells measured by ifn-γ ics plus facs analysis. differences between individual groups were analyzed by one-way anova and tukey post-hoc test. asterisks represent statistically significant differences between two groups. * p < 0.05, ** p < 0.01, *** p < 0.001. initially, we also analyzed purified cd4 t cell cultures from mva-nivsg immunized mice for their ability to stimulate ifn-γ by elispot using a two-dimensional pooled-peptide matrix system ( figure 3a , section 3.4.1). in contrast to cd8 t cell-enriched splenocytes, the ifn-γ sfc signals in these cultures were lower (figures 3b and 6a) . in order to determine definitively which peptide pools were above background levels, we selected a cut off value of 20 ifn-γ sfc/10 6 cells ( figure 6a , grey line). mean ifn-γ sfc values above background were observed in 8 out of the 21 pools (pools h3, h4, v2, v3, v5, v6, v9, and v11) ( figure 6a ). in order to identify potential cd4 t cell epitopes of h2-iab, we performed a computational analysis of the 15mer peptides in the five most positive peptide pools measured by elispot assay (pools h3, h4, v3, v6, and v9). using mhc ii binding predictions obtained from the iedb online resource, we found two promising peptides. these peptides were #49 (lfmtnvwtppnpntv) and #50 (nvwtppnpntvyhcs) ( table 2 ). next, we used ifn-γ ics to identify directly antigen-specific cd4 t cells after stimulation with these peptides. for this, splenocytes from mice that had been vaccinated with mva-niv-g or mva-nivsg were stimulated with peptides #49 and #50 and analyzed by flow cytometry. a small population of peptide-specific ifn-γ+ cd4 t cells was observed in the mva-nivsg and mva-niv-g groups ( figure 6b ). due to low frequencies of ifn-γ-producing cells, we chose a cut off value of 0.1% to differentiate between positive signals and background. overall, cd4 t cells from the mva-nivsg group had a frequency of ifn-γ+ above the cut off relative to mva-niv-g group ( figure 6c ). the mean percentage of ifn-γ+ cd4 t cells for the two peptides was 0.09-0.17% and 0.04-0.07% for mva-nivsg and mva-niv-g, respectively. moreover, the frequency and absolute number of ifn-γ-producing cd4 t cells in peptide #50 stimulated cultures was significantly higher in the mva-nivsg group when compared with the mva-niv-g group (mean = 290 ± 65 and 140 ± 46 cells/10 6 splenocytes respectively). for peptide #49, mva-nivsg vaccinated mice showed a significantly elevated frequency of ifn-γ+ cd4 t cells only. in conclusion, our data indicate that peptide #50 (nvwtppnpntvyhcs) is a promising h2-iab-restricted cd4 t cell epitope candidate of niv-g. the alignment of the peptide to the full amino acid sequence of wild-type niv-g is shown in figure s3 . the continuous threat of suddenly emerging niv outbreaks, particularly in bangladesh and india, demonstrate the need for countermeasure approaches ready to use in an immediate public health response. at present, there are no licensed niv vaccines for use in humans available. the existence of a niv candidate vaccine should significantly reduce the risk of infection and transmission of the virus in the case of an outbreak scenario. there are some experimental niv vaccines that have already been tested in different preclinical animal models. the major focus of these approaches was to evaluate the immunogenicity and efficacy in the context of niv challenge infection. in those studies, efficacy has been mostly associated with the generation of niv-specific antibodies, and immune monitoring is mainly relying on the detection of virus-neutralizing antibodies [31, 60, 61] . however, there is relatively little known about the induction and the relevance of niv-specific cellular immune responses. in that context, the availability of appropriate tools to investigate the role of t cells in niv-specific immunity is an important prerequisite in the development of new vaccines and therapeutics. thus, it will be indispensable to monitor in animal models the contribution of virus-specific t cells to protective immunity but also to potential niv antigen-specific immune pathology. here, we identified a major histocompatibility complex (mhc) haplotype h2-b-restricted peptide epitope in the niv-g protein by stimulating t cells from mva−nivsg vaccinated ifnar−/− mice with a two-dimensional (2d) matrix pool of overlapping peptides. ifnar−/− mice have been already established as a valuable preclinical animal model for niv infection with a ld 50 of 8 × 10 3 pfu after intraperitoneal challenge infection [15] . moreover, in previous studies, we have already successfully demonstrated that interferon type i receptor knockout mice (ifnar−/−) [47] can be readily used as animal models to study the immunogenicity and protective capacity of mva immunization [62, 63] . here, we wished to specifically assess the ability of mva-delivered niv-g antigen to induce the activation of cellular immune responses in mice. in general, the envelope g protein is known as the well-conserved attachment glycoprotein for both hev and niv. in a previous study, a recombinant adeno-associated virus vaccine expressing a full-length niv-g protein protected hamsters in an niv infection model [38] . in another approach, a soluble version of hev-g has been engineered and showed an even more advantageous efficacy when tested in different preclinical animal models [42, 43, 64] . using hevsg, a monoclonal antibody m102.4 was derived and has already been successfully tested as a therapeutic approach in humans. to further enhance henipavirus g protein-induced immunogenicity, we also designed and tested a soluble version of the niv-g protein (nivsg) similar to the hevsg antigen used for the generation of m102.4. to comparatively evaluate the immunogenicity of the nivsg protein, we also generated an mva expressing full-length g. the recombinant viruses mva-niv-g and mva-nivsg produced stable amounts of niv-g antigen upon in vitro infection of human cells, indicating the unimpaired expression of the target gene under transcriptional control of the synthetic vaccinia virus-specific early-late promoter pmh5 or the strong natural early promoter pvgf. in the case of mva-nivsg, removal of the transmembrane domain and cytoplasmic tail resulted in the secretion of the niv-g from mva-infected cells and accumulation also in the supernatant of cell cultures, as demonstrated in western blot analysis and immunostaining. a similar result was obtained with hevsg, as expressed by conventional recombinant vacv [41] . in contrast, the full-length mva-produced niv-g protein was not released in the supernatant, indicating the stable presentation on the cell surface through the transmembrane domains. another important aspect of proper protein expression, folding and conformational stability is influenced by the n-glycans. recent studies indicated that niv-g n-glycans reduce fusion efficiency because removal of some n-glycans caused cell-cell hyperfusogenicity and increased viral entry [65] . glycosidase treatment of full-length mva-produced niv-g resulted in a polypeptide of 58 kda, corresponding to the molecular mass predicted from the niv g gene encoding sequences. the glycosidase treatment of the nivsg also indicated the presence of all the n-glycans sites within the soluble version of the glycoprotein. a first in vivo evaluation in mice revealed that treatment with the mva-niv-g and mva-nivsg candidate vaccines resulted in the induction of similar levels of g-binding serum antibodies, confirming the immunogenicity of both antigen versions [66] . in that context, the presence of binding antibodies seems to play a substantial role in the blocking of niv entry, since the mechanism of niv neutralization is complex and involves more antigenic sites than those required for simple receptor binding [67] . however, more recent studies in different animal models suggest that cellular immune responses are also involved in mediating protection against niv infection [38, 68] . this observation is further supported by studies in a pig model for niv infection showing substantial activation of cd8 t cells after oral infection with niv [45] . in line with these preclinical data, humans surviving niv infection [2] showed significant levels of proliferating (ki-67+) cd8 t cells, indicating the presence of acute effector cells. these data emphasize that in addition to the humoral immune responses, t cells could be associated with recovery from niv infection. in a more recent study, stroh and coworkers confirmed the activation of niv-specific cd8 t cells in mice after vaccination with niv-like particles. these data further highlight that t cells may play a critical role in niv infection [68] . another hypothesis is that niv-specific t cells could be involved in potential immunopathologies. in this context, data from infections with other neurotropic viruses, for example, west nile virus, demonstrated that antigen-specific t cells can open the blood brain barrier and contribute to virus infections of the brain [69] [70] [71] [72] [73] . thus, to allow for more detailed studies characterizing t cells in niv-associated immunity or pathogenesis, it is essential to identify niv-g peptide epitopes allowing for the specific mhc-restricted antigen presentation and the activation of niv-specific t cells. we identified a nonamer epitope niv-g-9.3 [75] [76] [77] [78] [79] [80] [81] [82] [83] (rstdnqavi). analysis of this peptide sequence showed that niv-g 9.3 75-83 could be functionally conserved in hendra virus, but not cedar virus (another recognized henipavirus), g antigens ( figure s3 ). structural and functional analyses reveal promiscuous and species-specific use of ephrin receptors by cedar virus [74] . this potential epitope will support a more detailed experimental characterization of t cells induced by niv infection in the mouse model and their contribution for pathogenesis and protection. in this study, we found that mva-nivsg vaccination induced significantly higher amounts of niv-g epitope-specific cd8 t cells compared with the mva-niv-g candidate vaccine. this was a somewhat surprising observation as the immunizations with both antigens had elicited very comparable levels of g-specific antibodies. it is tempting to speculate that nivsg, as a soluble antigen, can trigger enhanced t cell responses because it is available to two different pathways of antigen presentation. on the one hand, nivsg as intracellularly synthetized antigen is endogenously processed and presented via mhc-i on the cell surface to activate cd8 t cells. in addition, the nivsg is secreted in high amounts from the mva-nivsg-infected cells, and such extracellular antigen can efficiently fuel the cross-presentation pathway and thereby induce elevated cd8 and cd4 t cell immune responses [75, 76] . interestingly, the mva-nivsg candidate vaccine also proved to rapidly induce niv-g epitope-specific cd8 t cells after single-dose application. these data are of relevance, since the epidemiology of the more recent niv outbreaks demonstrated that a potential vaccine candidate should rapidly protect. importantly, an h2b-restricted epitope has been identified in ifnar−/− mice, which serve as an established small animal model for niv infection [15] . in addition, we showed the induction of niv-g-specific cd4 t cells upon prime-boost immunization in the ifnar−/− with the mva-niv vaccines and using peptides for in vitro stimulation, as identified by using mhc ii binding predictions obtained from the iedb online resource (www.iedb.org). this data goes well along with the hypothesis that cd4 t cell responses are also significantly elevated upon infection [77] . again, mva-nivsg vaccination results in more efficient activation of cd4 t cell responses. further experiments will be needed to characterize the contribution of niv g-specific cd4 t cells for niv infection in more detail. taken together, our findings showed the activation of niv-g-specific t cells in ifnar−/− mice following vaccination with mva-based candidate vaccines. we confirmed the identification of potential h2-b-restricted niv-g cd8 and cd4 t cell peptide epitopes. in this study we also demonstrated that an mva-nivsg candidate vaccine may have superior immunogenicity, resulting in niv-specific antibodies and t cells in ifnar−/− mice. these data emphasize the promise of future studies in this animal model further evaluating the role of niv-specific t cells activated by the g-9.3 [75] [76] [77] [78] [79] [80] [81] [82] [83] and g-50 269-283 peptide epitopes, in both vaccine-induced protection and potential contribution to niv-induced pathologies. supplementary materials: the following are available online at http://www.mdpi.com/1999-4915/12/1/26/s1, figure s1 : generation and characterization of recombinant mva-nivsg and mva-niv-g, figure s2 : ifn-γ production by cd8 and cd4 t cells stimulated by the niv-g peptide 9.3, figure s3 : amino acid alignment of h2-b-and h2-iab-restricted peptides, figure s4 : quality of activated cd8 t cells from mice immunized with recombinant mva expressing niv-g. author contributions: g.k., s.v., c.c.b., g.s. and a.v. conceived and designed the experiments; g.k., s.v., s.j. and a.v. performed the experiments; g.k., s.v., u.k., c.c.b., g.s. and a.v. analyzed the data; g.k., g.s. and a.v. wrote the paper. all authors have read and agreed to the published version of the manuscript. nipah virus: a recently emergent deadly paramyxovirus adaptive immune responses in humans during nipah virus acute and convalescent phases of infection nipah virus infection henipavirus infection of the central nervous system clinical presentation of nipah virus infection in bangladesh emerging trends of nipah virus: a review long-term neurological and functional outcome in nipah virus infection relapsed and late-onset nipah encephalitis neuropsychiatric sequelae of nipah virus encephalitis henipaviruses: an updated review focusing on the pteropid reservoir and features of transmission changing resource landscapes and spillover of henipaviruses pathology of acute henipavirus infection in humans and animals animal challenge models of henipavirus infection and pathogenesis henipavirus infections: lessons from animal models date palm sap linked to nipah virus outbreak in bangladesh transmission of nipah virus-14 years of investigations in bangladesh a functional henipavirus envelope glycoprotein pseudotyped lentivirus assay system ephrin-b2 ligand is a functional receptor for hendra virus and nipah virus ephrinb2 is the entry receptor for nipah virus, an emergent deadly paramyxovirus two key residues in ephrinb3 are critical for its use as an alternative receptor for nipah virus quantitative analysis of nipah virus proteins released as virus-like particles reveals central role for the matrix protein functional studies of host-specific ephrin-b ligands as henipavirus receptors potent neutralization of hendra and nipah viruses by human monoclonal antibodies therapeutic treatment of nipah virus infection in nonhuman primates with a neutralizing human monoclonal antibody pathogenic differences between nipah virus bangladesh and malaysia strains in primates: implications for antibody therapy. sci a neutralizing human monoclonal antibody protects against lethal disease in a new ferret model of acute nipah virus infection nipah virus: vaccination and passive protection studies in a hamster model recombinant nipah virus vaccines protect pigs against challenge single injection recombinant vesicular stomatitis virus vaccines protect ferrets against lethal nipah virus disease single-dose live-attenuated nipah virus vaccines confer complete protection by eliciting antibodies directed against surface glycoproteins single-dose replication-defective vsv-based nipah virus vaccines provide protection from lethal challenge in syrian hamsters single-dose live-attenuated vesicular stomatitis virus-based vaccine protects african green monkeys from nipah virus disease peri-exposure protection against nipah virus disease using a single-dose recombinant vesicular stomatitis virus-based vaccine rabies-based vaccine induces potent immune responses against nipah virus recombinant measles virus vaccine expressing the nipah virus glycoprotein protects against lethal nipah virus challenge a single-dose chadox1-vectored vaccine provides complete protection against nipah bangladesh and malaysia in syrian golden hamsters protection against henipavirus infection by use of recombinant adeno-associated virus-vector vaccines hendra virus and nipah virus animal vaccines receptor binding, fusion inhibition, and induction of cross-reactive neutralizing antibodies by a soluble g glycoprotein of hendra virus site occupancy and glycan compositional analysis of two soluble recombinant forms of the attachment glycoprotein of hendra virus a recombinant subunit vaccine formulation protects against lethal nipah virus challenge in cats vaccination of ferrets with a recombinant g glycoprotein subunit vaccine provides protection against nipah virus disease for over 12 months a hendra virus g glycoprotein subunit vaccine protects african green monkeys from nipah virus challenge protection against henipaviruses in swine requires both, cell-mediated and humoral immune response chapter five-modified vaccinia virus ankara: history, value in basic research, and current perspectives for vaccine development functional role of type i and type ii interferons in antiviral defense biochemical, conformational, and immunogenic analysis of soluble trimeric forms of henipavirus fusion glycoproteins development of a replication-deficient recombinant vaccinia virus vaccine effective against parainfluenza virus 3 infection in an animal model kinetic analysis of a complete poxvirus transcriptome reveals an immediate-early class of genes simultaneous high-resolution analysis of vaccinia virus and host cell transcriptomes by deep rna sequencing myristoylation increases the cd8+t-cell response to a gfp prototype antigen delivered by modified vaccinia virus ankara elucidating and minimizing the loss by recombinant vaccinia virus of human immunodeficiency virus gene expression resulting from spontaneous mutations and positive selection easy and efficient protocols for working with recombinant vaccinia virus mva cells responding to the middle east respiratory syndrome coronavirus nucleocapsid protein delivered by vaccinia virus mva in mice pooled-peptide epitope mapping strategies are efficient and highly sensitive: an evaluation of methods for identifying human t cell epitope specificities in large-scale hiv vaccine efficacy trials iedb-ar: immune epitope database-analysis resource in 2019 identification of poxvirus cd8+ t cell determinants to enable rational design and characterization of smallpox vaccines a vlp-based vaccine provides complete protection against nipah virus challenge following multiple-dose or single-dose vaccination schedules in a hamster model hendra virus vaccine, a one health approach to protecting horse, human, and environmental health rapid expansion of cd8+ t cells in wild-type and type i interferon receptor-deficient mice correlates with protection after low-dose emergency immunization with modified vaccinia virus ankara critical role of perforin-dependent cd8+ t cell immunity for rapid protective vaccination in a murine model for human smallpox a recombinant hendra virus g glycoprotein-based subunit vaccine protects ferrets from lethal hendra virus challenge n-glycans on the nipah virus attachment glycoprotein modulate fusion and viral entry as they protect against antibody neutralization antibodies to henipavirus or henipa-like viruses in domestic pigs in ghana neutralization assays for differential henipavirus serology using bio-plex protein array systems henipavirus-like particles induce a cd8 t cell response in c57bl/6 mice pathways exploited by flaviviruses to counteract the blood-brain barrier and invade the central nervous system toll-like receptor 3 mediates west nile virus entry into the brain causing lethal encephalitis west nile virus-induced disruption of the blood-brain barrier in mice is characterized by the degradation of the junctional complex proteins and increase in multiple matrix metalloproteinases nile virus: crossing the blood-brain barrier cd8+ t cells mediate recovery and immunopathology in west nile virus encephalitis structural and functional analyses reveal promiscuous and species specific use of ephrin receptors by cedar virus cross-priming of cytotoxic t cells dictates antigen requisites for modified vaccinia virus ankara vector vaccines distinct pathways of antigen uptake and intracellular routing in cd4 and cd8 t cell activation pteropid bats are confirmed as the reservoir hosts of henipaviruses: a comprehensive experimental study of virus transmission the authors declare no conflict of interest. the funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results. key: cord-000164-094d0rn6 authors: suthar, mehul s.; ma, daphne y.; thomas, sunil; lund, jennifer m.; zhang, nu; daffis, stephane; rudensky, alexander y.; bevan, michael j.; clark, edward a.; kaja, murali-krishna; diamond, michael s.; gale, michael title: ips-1 is essential for the control of west nile virus infection and immunity date: 2010-02-05 journal: plos pathog doi: 10.1371/journal.ppat.1000757 sha: doc_id: 164 cord_uid: 094d0rn6 the innate immune response is essential for controlling west nile virus (wnv) infection but how this response is propagated and regulates adaptive immunity in vivo are not defined. herein, we show that ips-1, the central adaptor protein to rig-i-like receptor (rlr) signaling, is essential for triggering of innate immunity and for effective development and regulation of adaptive immunity against pathogenic wnv. ips-1(−/−) mice exhibited increased susceptibility to wnv infection marked by enhanced viral replication and dissemination with early viral entry into the cns. infection of cultured bone-marrow (bm) derived dendritic cells (dcs), macrophages (macs), and primary cortical neurons showed that the ips-1-dependent rlr signaling was essential for triggering ifn defenses and controlling virus replication in these key target cells of infection. intriguingly, infected ips-1(−/−) mice displayed uncontrolled inflammation that included elevated systemic type i ifn, proinflammatory cytokine and chemokine responses, increased numbers of inflammatory dcs, enhanced humoral responses marked by complete loss of virus neutralization activity, and increased numbers of virus-specific cd8+ t cells and non-specific immune cell proliferation in the periphery and in the cns. this uncontrolled inflammatory response was associated with a lack of regulatory t cell expansion that normally occurs during acute wnv infection. thus, the enhanced inflammatory response in the absence of ips-1 was coupled with a failure to protect against wnv infection. our data define an innate/adaptive immune interface mediated through ips-1-dependent rlr signaling that regulates the quantity, quality, and balance of the immune response to wnv infection. west nile virus (wnv) is a neurotropic flavivirus and is an emerging public health threat. infection with wnv now constitutes the leading cause of mosquito-borne and epidemic encephalitis in humans in the united states [1] . wnv is enveloped and contains a single strand positive sense rna genome of approximately 11 kb in length that encodes three structural (c, prm/m, and e) and seven non-structural proteins (ns1, ns2a, ns2b, ns3, ns4a, ns4b, and ns5). it cycles enzootically between birds and culex mosquitoes, with humans infected as dead-end hosts. wnv infection has been modeled in inbred mice wherein infection and pathogenesis recapitulate many of the features of human infection (reviewed in [2] ). following subcutaneous inoculation, wnv replicates in dendritic cells (dcs) at the portal of entry and in the draining lymph node. a primary viremia develops and virus spreads to visceral organs including the spleen, where further amplification occurs, leading to central nervous system (cns) dissemination and encephalitis. in humans, wnv causes an acute febrile illness that can progress to severe and sometimes lethal neuroinvasive disease, especially in the elderly and immunocompromised [3] . however, healthy young adults are also afflicted with severe neurological disease [4, 5, 6] , indicating that virulence can occur independently of immune deficiencies or aging. intracellular innate immune defenses and the actions of type i interferon (ifn) provide a first-line of defense against virus infection and are essential for the control of wnv replication, dissemination, and neurovirulence [7] . innate antiviral immune defenses are triggered through the recognition of conserved pathogen associated molecular pattern (pamp) motifs within viral products by intracellular pathogen recognition receptor (prr) proteins in infected cells. prr signaling directs downstream activation of latent transcription factors, including nf-kb, interferon regulatory factor (irf)-3 and irf-7, in a cell type-specific manner to induce antiviral response programs that include expression of proinflammatory cytokines, chemokines, type i ifn, and interferon stimulated genes (isgs) [7, 8, 9, 10] . the isg products induced through autocrine and paracrine actions of ifn confer antiviral activity by limiting virus replication and cell-to-cell virus spread. modulation of ifn signaling has been identified as a virulence feature of pathogenic strains of wnv [11, 12] . the rlrs, retinoic acid inducible gene-i (rig-i) and melanoma differentiation antigen 5 (mda5) [13, 14, 15, 16] , are prrs that play critical roles in triggering immune defenses against rna virus infection, including wnv. rig-i and mda5 are cytosolic rna helicases that contain an amino terminal tandem caspase activation and recruitment domain (card). upon engaging rna substrates, the rlrs undergo a conformational change and bind to the mitochondrial associated protein, interferon promoter stimulator-1 (ips-1) through a card-card interaction, leading to ips-1-dependent signaling of ifn production and expression of immune response genes [17, 18] . rlr signaling and ips-1 function have an essential role in triggering ifn defenses during wnv infection of mouse embryo fibroblasts (mefs) and human cell lines in vitro. cells lacking either rig-i or mda5 were attenuated in their ability to generate an effective innate immune response to infection, whereas cells lacking both rig-i and mda5 or those deficient in ips-1 alone were unable to respond to infection with wnv and related flaviviruses [19, 20, 21, 22] . recent studies examined the role of another class of pattern recognition receptors, toll like receptor (tlr)3 and tlr7, and show that these receptors are also important prrs of wnv infection, as they play a role in signaling ifn production and an inflammatory response upon viral ligand recognition [23, 24, 25] . tlr3 has been shown to contribute to both enhancement and protection of cns inflammation and neurovirulence of wnv in vivo [23, 24] , while tlr7-dependent signaling was shown to be essential for directing proper immune cell homing to sites of wnv infection during the adaptive immune response in vivo [25] . type i ifn, a major product of prr signaling, has been shown to link innate and adaptive immune responses. however, the specific prr pathways that mediate this during acute wnv infection have not been delineated nor has the rlr pathway been evaluated in this context. the quantity and quality of the innate and adaptive immune responses after infection must be carefully regulated to avoid aberrant inflammation and immunopathogenesis. regulatory t (t reg ) cells and inflammatory dendritic cell (dc) subsets regulate inflammation during acute virus infection through t cell suppression and by modulating the trafficking and inflammatory cytokine production of immune cells into infected tissues [26, 27, 28] . thus, the level of local and peripheral t reg cells, and the composition of local dc subsets that develop during wnv infection may determine immune control and wnv disease. here, we assessed the role of rlr signaling and ips-1 in wnv infection and immunity. our studies define ips-1 as an essential modulator of immunity in vivo and demonstrate that ips-1-dependent signaling orchestrates an innate/adaptive immune interface that regulates immune responses to effectively control wnv infection. wnv infection of primary embryonic fibroblasts recovered from rig-i 2/2 mice revealed that rig-i was important in eliciting innate antiviral immune defenses early during infection, whereas mda5 was important for enhancing and sustaining this response [21] . we further evaluated wnv infection of rig-i 2/2 or mda5 2/2 mice and confirmed that rig-i serves a dominant role among the rlrs for the acute induction of innate immune defenses and protection against wnv infection in vivo (data not shown). since the rlrs signal innate defenses through the ips-1 adaptor protein [29] , we also examined the role of ips-1 in protection against wnv infection upon a sub-lethal virus challenge of wild type and ips-1 2/2 mice. ips-1 2/2 mice were highly susceptible to wnv infection and exhibited 100% mortality with an average survival time (ast) of 7.3 days as compared to wild type mice (38.5% mortality with an ast of 13.2 days; p,0.0001; fig 1a) . thus, rig-i and ips-1-dependent signaling are essential for protection against wnv infection. to define the role of ips-1 in controlling wnv in vivo, wild type and ips-1 2/2 mice were infected subcutaneously (s.c.) with 100 pfu of wn-tx and viral burden within peripheral tissues and the cns was measured over time post-infection (pi). ips-1 2/2 mice exhibited increased viremia compared to wild type mice (45.7 fold enhancement at day 1 pi, p,0.05) throughout the course of infection (fig 1b) . similarly, viral loads in the spleen were elevated in the infected ips-1 2/2 mice (fig 1c) . wnv infection of ips-1 2/2 mice displayed an expanded tissue tropism as infectious virus was found in the kidneys, a tissue that is not normally permissive to infection in wild type mice ( fig 1d) . wnv is typically detected in the cns of wild type mice after s.c. challenge between 4 and 8 days pi [2] . consistent with this time course, infected wild type mice exhibited detectable viral loads (average viral titer of 10 1.8 pfu/gram of tissue) in the brain by day 6 p.i., although virus was not detected in the spinal cord (fig 1e and f ). in contrast, wnv spread to the brain ( fig 1e) and spinal cord of ips-1 2/2 mice (fig 1f) by day 2 pi, with viral loads rising through day 6 pi. together these results indicate that ips-1, likely west nile virus (wnv) is a mosquito-transmitted rna virus that has emerged in the western hemisphere and is now the leading cause of arboviral encephalitis in the united states. however, the virus/host interface that controls wnv pathogenesis is not well understood. previous studies have established that the innate immune response and interferon (ifn) defenses are essential for controlling virus replication and dissemination. in this study, we assessed the importance of the rig-i like receptor (rlr) signaling pathway in wnv pathogenesis through analysis of mice lacking ips-1, the central adaptor molecule of rlr signaling. our studies revealed that ips-1 is essential for protection against wnv infection and that it regulates processes that control virus replication and triggering of innate immune defenses. we found that ips-1 plays an important role in establishing adaptive immunity through an innate/adaptive interface that elicits effective antibody responses and controls the expansion of regulatory t cells. thus, rlrs are essential for pathogen recognition of wnv infection and their signaling programs help orchestrate immune response maturation, regulation of inflammation, and immune homeostasis that define the outcome of wnv infection. through rlr signaling of innate immune defenses, limits wnv replication, viremia, and peripheral spread, and is essential for the control of viral invasion of the cns. myeloid cells, including tissue and lymphoid dc and macrophages (mw), are among the first cells to encounter wnv during infection and thus function to restrict the spread of virus to distant tissues and the cns [2] . to define the role of ips-1 in controlling virus replication and innate immunity in myeloid cells, we analyzed wnv infection and host responses in primary bone marrow-derived dc and mw recovered from wild type and ips-1 2/2 mice. dc and mw were infected at an moi of 1.0 (relative to viral plaque assay quantification of bhk-21 cells; see methods) and evaluated for virus replication, ifn induction, and innate immune triggering of isg expression (fig 2) . ips-1 2/2 dcs sustained significantly higher wnv replication at 36 and 48 hours pi compared to wild type infected cells (fig 2a) . wnv infection of wild type dcs induced ifn-b secretion but this response was completely abolished in ips-1 2/2 dcs (fig 2b) . the lack of ifn-b induction in ips-1 2/2 dcs correlated with a lack of isg expression including rig-i, mda5, and stat-1 ( fig 2c) . in addition, expression of isg54 and isg49, which are direct irf-3 target genes [30, 31] , were not induced during wnv infection of ips-1 2/2 dcs (fig 2c) . moreover, isg56, another irf-3 target gene [31] , was induced late during infection and to lower levels as compared to isg54 and isg49 in wild type, infected dcs. wnv infection of ips-1 2/2 mw resulted in significantly higher virus replication between 24 and 48 hours pi as compared to infected wild type cells ( fig 2d) . whereas wild type infected mw expressed ifn-b, this response was completely abolished in ips-1 2/2 mw (fig 2f) . we also observed a differential expression of isgs and irf-3target genes within wnv-infected mw. rig-i, mda5, and stat-1 were not induced in ips-1 2/2 mw, whereas, isg56, isg49, and pkr were expressed at reduced levels and with delayed kinetics. these data establish that ips-1-dependent rlr signaling is the major innate immune signaling pathway that controls virus replication in conventional dcs and mw. the rlr signaling pathway triggers the innate immune response to wnv infection in primary cortical neurons neurons represent the target cell of wnv infection in the cns and their death after infection is a key factor in pathogenesis and neurological sequelae [32, 33] . to define the role of rlr signaling in restricting virus replication in neurons, primary cortical neurons were generated from wild type and ips-1 2/2 mice. cells were infected at an moi of 1.0 with wn-tx and virus yield, ifn-b induction, and isg expression were evaluated. in the absence of ips-1, wnv replicated faster and to higher levels resulting in a 2.2 and 4.2-fold (p,0.05) increase in viral production at 24 hrs and 48 pi, respectively as compared to infected wild type neuronal cells ( fig 3a) . this relatively modest virologic effect in neurons compared to that observed in ips-1 2/2 dc and mw was expected, as ifn-a or -b pre-treatment only inhibits wnv infection in cortical neurons to a maximum of 5 to 8-fold [12] , suggesting that the ifn response is comparably less potent in neurons. ifn-b expression was induced to lower levels in ips-1 2/2 neurons compared to wild type infected neurons at 24 (10-fold, p,0.05) and 36 hours pi (5-fold, p,0.05) despite the higher levels of virus replication (fig 3a and 3b) . expression of isgs, (including rig-i and mda5) and irf-3 target genes (including isg56 and isg49) followed this pattern and were dependent on ips-1 for rapid and high level expression ( fig 3c) . the presence of ifn-b and isg transcripts in ips-1 2/2 cells at 48 hrs pi is consistent with the finding that tlr3 has an independent and subordinate role in triggering innate immune responses in cortical neurons at later time points after wnv infection [23] . these results demonstrate that the rlr signaling pathway controls virus replication and induces innate immune responses against wnv infection in cortical neurons. neuronal destruction and cns inflammation are enhanced in wnv infected ips-1 2/2 mice to determine the role of the rlr pathway in protection of neurons against wnv pathogenesis in vivo, we conducted histological analysis of brain tissue from wild type and ips-1 2/2 mice infected with wn-tx ( fig 4a) . analysis of brain sections from infected wild type mice revealed little or no inflammation or neuronal damage, with sparse and focal cell infiltrates restricted to the hippocampus and cerebral cortex on day 6 pi. by day 10 pi (a timepoint in wild type mice in which peak virus replication in the cns occurs [34] ) cellular infiltrates were present in the parenchyma and neuronal destruction was observed throughout the cortex and hippocampus. in contrast, brain sections from infected ips-1 2/2 mice recovered on day 6 pi displayed extensive injury to neurons in the cortex and granular neurons of the hippocampus. damaged neurons appeared pyknotic with vacuolation, degeneration and cell dropout. somewhat surprisingly, we observed extensive inflammation in the brains from infected ips-1 2/2 mice within the cortex, hippocampus, and cerebellum (data not shown) displaying prominent perivascular and parenchymal immune cell infiltrates. to evaluate the composition and antigen-specificity of the inflammatory cells within the brains of wnv-infected mice, lymphocytes were isolated from infected brains on day 6 pi and were characterized from pools (n = 5) of wild type and ips-1 2/2 infected mice. brains from ips-1 2/2 infected mice showed an 2.9fold increase in the total number of immune cells as compared to wild type infected mice (fig 4b) , and this was associated with an increase in absolute numbers of infiltrating cd4+ and cd8+ t cells ( fig 4c) . among the brain cd8+ t cells isolated from ips-1 2/2 mice, there was a remarkable 27-fold increase in the number of antigen-specific cells that expressed ifn-c after treatment with an immundominant ns4b peptide ( fig 4d) [35, 36] . analysis of microglia/mw cells, based on relative surface expression of cd45 and cd11b [37] , revealed increased numbers of microglial cells (cd45+ lo /cd11b+) and infiltrating macrophages (cd45+ hi / cd11b+) within the brains of infected ips-1 2/2 mice when compared to wild type mice (fig 4e) . similar findings were observed in the spinal cords from infected ips-1 2/2 mice (data not shown). combined with the histological analysis, these results demonstrate that in the absence of ips-1, wnv infection induces a strong inflammatory response in the cns. while this response is likely associated with increased viral loads, the failure of this increased inflammatory response to elicit protection or control cns pathology, in the absence of ips-1, suggests a role for the rlr signaling pathway as a regulatory program that controls the quality of the inflammatory response to wnv infection. to further characterize how ips-1 modulates the inflammatory response to wnv infection, we measured levels of systemic type i ifn, proinflammatory cytokines, and chemokines present in the serum of wnv-infected mice at 1 and 4 days pi. paradoxically, a trend towards more rapid induction and increased levels of type i ifn were observed in the serum of ips-1 2/2 mice compared to wild type mice (fig 5a) . we note that in this case type i ifn was detected and quantified through a mouse-specific type i ifn bioassay, which does not differentiate between the ifn-a or -b species. this result is consistent with our recent studies showing that serum type i ifn levels accumulate during wnv infection in an irf-7-dependent but irf-3-independent manner [8, 9] . in this case ifn-a species are likely accumulating through a tlr7dependent signaling pathway involving plasmacytoid dcs, which do not require the rlr pathway for ifn production [38] . more recently, town et al. assessed the role of tlr7 and myd88 2/2 during wnv infection and found that mice lacking myd88 produced elevated levels of systemic ifn during wnv infection [25] . thus, during wnv infection systemic ifn is regulated through rlr-dependent and independent processes. correspondingly, when compared to wild type mice, ips-1 2/2 infected animals, which show higher viremia (fig 1b) produced increased serum levels of proinflammatory cytokines and chemokines in response to wnv infection. elevated levels of systemic il-6, tnfa, cxcl10, and ifn-c were observed at 1 and/or 4 days pi in ips-1 2/2 mice (fig 5b) . serum cytokine levels were also compared between uninfected wild type and ips-1 2/2 mice and showed no differences in basal cytokine expression (data not shown). these results demonstrate that in the absence of ips-1, greater proinflammatory cytokine and chemokine responses are induced during wnv infection. altered wnv-specific antibody profiles in ips-1 2/2 mice wnv-specific antibody responses are essential for suppressing viremia and virus dissemination and limiting lethal wnv infection [39, 40] . to determine if a deficiency in ips-1 modulated the quality and quantity of the humoral immune response, we characterized the antibody profile in sera during wnv infection. in wild type mice, neutralizing virus-specific igm antibodies are typically detectable by day 4 pi with wnv and production of neutralizing virus-specific igg antibodies follow between days 6 and 8 pi [40] . a time course analysis in wild type and ips-1 2/2 infected mice showed that between 4 and 6 days pi, wnv-infected ips-1 2/2 mice exhibited significantly higher levels of virus-specific igm, igg, and igg subclasses as compared to infected wild type mice ( table 1) . wnv-specific igg1 antibodies were detected at low levels on day 6 pi in sera from wild type and ips-1 2/2 mice. additionally, we observed a ,72.9-fold increase in wnv-specific igg2a levels in infected ips-1 2/2 as compared to wild type mice on day 6 pi and ,2.2-fold increase on day 8 pi. assessment of the virus-specific antibody responses through a prnt assay revealed that neutralization titers in sera from wild type mice increased dramatically between 6 and 8 days pi. sera from ips-1 2/2 infected mice exhibited a modest increase in neutralization titer to 1:1280, despite having much higher levels of virusspecific antibodies. this difference translated into a serum neutralization index that was ,39-fold lower on day 6 pi in the infected ips-1 2/2 mice compared to wild type mice. these results demonstrate that the humoral responses in wnv-infected ips-1 2/2 mice are distinct from responses in wild type infected mice. thus, rlr signaling and ips-1 actions likely contribute to regulatory processes that govern the levels, igg class switching, and neutralizing capacity of antibodies generated in response to wnv infection. to characterize the immune parameters associating with the dysregulated inflammatory and humoral responses in wnv infected ips-1 2/2 mice, we analyzed the immune cell composition in draining lymph node and spleen tissues. wild type and ips-1 2/2 mice were challenged with diluent alone or with wn-tx, and draining popliteal lymph node (dln) and the spleen were harvested at 1 and 6 days pi, respectively. analysis of the popliteal dln provides insight into how ips-1 modulates the inflammatory response immediately after infection whereas assessment of the spleen elucidates characteristics of the adaptive immune response prior to the infection endpoint. immune cells were isolated from the popliteal dln and were characterized by flow cytometry (fig 6) . analysis of cd8a dc subsets, which are phenotypically the major antigen presenting cells within lymphoid tissues and are implicated in eliciting virus-specific cd8 t cell in response to acute wnv infection [41] , showed that infected wild type and ips-1 2/2 mice exhibited similar increases in the numbers of cd8a + and cd8a 2 dcs compared to mock-infected mice (fig 6a, b) . however, a significant increase (,3-fold; p,0.05) of a proinflammatory dc subset, characterized as cd11c + cd11b hi ly6c + , was observed within the popliteal dlns of ips-1 2/2 infected mice (fig 6c) . this dc subset is monocytederived and typically recruited to sites of acute inflammation where they propagate the inflammatory response [42] . we found that these dc subsets were not significantly expanded and showed no differences in their recruitment to the dln in either wild type or ips-1 2/2 infected mice at 12 hours pi (data now shown). thus, as early as 24 hours pi, an elevated cellular inflammatory response is initiated in the ips-1 2/2 mice. in contrast, similar increases in plasmacytoid dcs were observed within infected wild type and ips-1 2/2 infected mice (fig 6d) , demonstrating that an absence of ips-1 did not directly affect expansion and/or recruitment of this dc subset. within the popliteal dlns, mock-infected ips-1 2/2 mice compared to wild type mice generally showed elevated numbers of b cells, cd4+ t cells (p,0.05), and cd8+ t cells (fig 6e, f, and g) . we further analyzed the lymphocyte composition of the spleen on day 6 after wnv infection (fig 7) . gross pathologic analysis revealed that wnv infection of ips-1 2/2 mice results in massive splenomegaly whereas infection of wild type mice induces only a slight increase in spleen size (fig 7a) . cell analysis revealed increased numbers of total lymphocytes in the spleens of infected ips-1 2/2 mice as compared to wild type mice (fig 7b) . regulatory t (t reg ) cells have recently been shown to contribute to the dampening of inflammation and adaptive immune responses during acute virus infections [26, 43, 44] . moreover, a reduction in the number of circulating t reg in mice leads to enhanced lethality after wnv infection [45] . a time course analysis of t regs in wild type mice revealed that wnv infection results in a significant increase in the numbers of foxp3+ t cells as compared to mock-infected mice beginning on day 4 and peaking by day 6 pi (fig 7c) , indicating the expansion of t regs during acute wnv infection. despite their marked increase in viral load, the infected ips-1 2/2 mice did not display an increase in the numbers of foxp3+ t cells at any timepoint analyzed. thus, ips-1 signaling directly or indirectly impacts t reg proliferation and does so independently of viral load. we also observed that spleens from infected ips-1 2/2 mice exhibited significantly increased numbers of cd8+ t cells in comparison to those from infected wild type mice, whereas the expansion of splenic cd4+ t cells in wild type and ips-1 2/2 mice were not different (fig 7d) . the spleens from wnv-infected ips-1 2/2 mice showed significantly higher numbers (3.9-fold; p,0.05) of wnv-specific cd8+ t cells producing ifnc. to further define the phenotype associated with wnv-induced splenomegaly in ips-1 2/2 mice, we also assessed the numbers of nk cells and neutrophils. spleens from infected ips-1 2/2 mice contained greater numbers of nk cells (cd4 2 cd8 2 nk1.1 + cells), nkt cells (cd4+/cd8+/nk1.1+ cells) and neutrophils (cd11b + gr1 + cells) (fig 7e) . although wnvinfected wild type mice infected displayed slight increases in the absolute numbers of these specific cell types, a deficiency of ips-1 resulted in a more marked enhancement of these immune cell populations. in this study, we establish a major role for rlr signaling in protection from wnv pathogenesis, and demonstrate that ips-1 is critical for the control of wnv infection in vivo. our studies indicate that ips-1-dependent rlr signaling functions to establish balanced, effective, and protective innate and adaptive immune responses, and that ips-1 links adaptive immune regulation with the innate immunity triggered by rlr signaling during wnv infection. in the absence of ips-1 in vitro, innate immune defense programs of myeloid dcs and macrophages were ablated or severely attenuated. moreover, in vivo analysis of infected ips-1 2/ 2 mice showed altered igg and igm antibody responses with diminished virus neutralization activity. the inflammatory response to wnv infection is regulated by ips-1-dependent processes such that a deficiency of ips-1 resulted in elevated proinflammatory cytokine and chemokines and increased numbers of inflammatory dcs, antigen-specific t cells, natural killer cells, and neutrophils in lymphoid organs, and activated macrophage/ microglial cells within the cns. the dysregulated inflammatory response to wnv infection in ips-1 2/2 mice was associated with a reduction in the numbers of t reg cells and their failure to expand during acute infection. these observations demonstrate the critical role of ips-1 in mediating rlr signaling of innate antiviral immunity against wnv infection, and reveal novel features of ips-1 function in regulating immune homeostasis, inflammation, and adaptive immunity to infection. although infection of primary dcs, macrophages, and neuronal cells failed to induce type i ifn upon wnv infection, wnvinfected ips-1 2/2 mice showed enhanced systemic type i ifn responses. this finding agrees with previous studies that indicate both ips-1-dependent and -independent pathways contribute to the systemic type i ifn production in vivo [8, 9, 23, 25] . most importantly, the enhanced tissue tropism and rapid viral entry into the cns observed in the ips-1 2/2 mice is not affected by the elevated systemic ifn responses. this suggests that local tissuespecific and intracellular responses triggered by rlr-dependent signaling are more essential for reducing viral burden and dissemination. one possible explanation is that a distinct set of rlr-responsive genes function to control virus replication at the site of infection. this could explain, in part, the elevated levels of virus replication, enhanced tissue tropism and cell-to-cell spread in mice or cells deficient in irf3 or irf-7, each of which are downstream transcription factors of rlr signaling [8, 9, 10] . additionally, it is likely that pdcs, which are specialized dendritic cells for producing systemic type i ifn during a viral infection [46] , are likely contributing to the ifn responses observed during wnv infection. studies by silva et al. have shown that wnv triggers ifn induction in pdcs through a replication-independent manner [47] . interestingly, within the dln, we observed similar expansion of pdcs between wild type and ips-1 2/2 infected mice, yet at the same timepoint (24 hours pi), elevated systemic type i ifn responses were observed in ips-1 2/2 mice. this suggests two possibilities: 1) splenic pdcs or circulating pdcs are likely responding to the high levels virus in the serum from the ips-1 2/2 infected mice to produce ifn at 24 hours pi and/or 2) various other cell types that express tlr3 and/or tlr7 are responding to wnv infection and contributing to systemic ifn responses. taken together, these studies indicate that rlr signaling and the actions of irf-3/7 are important in triggering ifn production and isg expression to limit wnv replication and spread, and that tlr signaling may impart additional, rlrindependent defenses that regulate immunity against wnv infection. the production of and response to type-i ifn is a major linkage point between innate and adaptive immunity, as ifn-a and ifn-b sustain b cell activation and differentiation [48, 49, 50] , expand antigen-specific cd8+ t cells [51, 52] , cd4+ t cells [53] , and activation of nk cells [54] . one of the most intriguing aspects of this study was the global alteration of the immune response elicited in the ips-1 2/2 mice, indicating that rlr signaling couples innate immunity with regulation of the adaptive immune response. infection of ips-1 2/2 mice exhibited increased igm and igg wnv-specific antibodies, enhanced wnv-specific cd8+t cell response, and increased expansion of neutrophils, nk cells and nk-t cells. one trivial explanation for these differences is that there is an increased antigen load in the absence of ips-1 and, as a result, enhanced virus-specific (e.g. cd8+ t cells, igg and igm antibodies) and nonspecific (e.g. neutrophils, nk cells) responses. however, there are several key findings from this study that argue against these responses simply being attributed to higher antigen load: (1) in the absence of ips-1, the cd4 and cd8 t cells, which are protective against wnv infection [34, 35, 36, 55, 56, 57, 58] , were significantly enhanced in the peripheral and cns compartments but failed protect against infection. one explanation for this observation is that the expansion and migration of cd4 and cd8 t cells to different tissues was itself uncontrolled, resulting in t cell-mediated pathology rather than t cell-mediated protection. (2) while the quantity of virus-specific igm and igg antibody responses were greatly enhanced in the absence of ips-1, there was a marked reduction in antibody quality in terms of neutralization capacity. in contrast deficiencies in tlr3 or myd88 (data not shown) did not alter virus-specific antibody responses and neutralization capacities. collectively, these findings suggest that rlr-dependent signaling coordinates effective antibody responses during wnv infection through as yet undefined pathway. (3) while systemic ifn responses provide a link between innate and adaptive immune responses, our studies suggest that the prr signaling pathways (rlr-dependent vsindependent) and levels of ifn production in combination with production other proinflammatory cytokines or chemokines regulate the quantity and quality of the immune response during virus infection. thus, in the absence of ips-1 signaling, infected conventional dcs or mw, two integral cell types in establishing adaptive immunity, likely do not produce ifn or the normal array and level of proinflammatory cytokines/ chemokines. instead, ifn and other mediators may be strictly produced by infected or bystander cells during wnv infection, occurring with altered kinetics and magnitude, through tlr-dependent pathways, such as tlr3 and/or tlr7 [23, 25] . (4) in the absence of ips-1, the enhanced expansion of ly6c+ ''inflammatory'' dcs failed to limit early wnv replication and dissemination. this inflammatory dc subset migrates to sites of infection, secretes pro-inflammatory cytokines, and promotes cd8+ t cell expansion during a secondary virus infection, suggesting it sustains the effector t cell response [59] . our data indicate that ly6c+ dc recruitment and/or expansion is governed by ips-1-dependent events of rlr signaling. thus, the aberrant recruitment/expansion of these inflammatory dcs may contribute to immunopathogenesis and limit development of an effective immune response to control wnv virus infection. (5) the lack of t reg expansion during wnv infection correlated with altered ifn levels, increased proinflammatory cytokines and chemokine levels, and an increased number and distribution of antigen-specific cd8+ t cells. these observations implicate an indirect or direct role for ips-1 in regulating t reg levels during wnv infection, and provide evidence that links a lack of t reg expansion to immune dysregulation. while their importance in autoimmunity is established [60] , recent studies have implicated an integral role for t regs in controlling inflammation and adaptive immune responses during acute virus infections [26, 43, 44] . during acute infection t regs function to locally contain and control the immune response with the dual outcome of suppressing viral dissemination while decreasing the likelihood of immune-mediated pathology. in support of this model, infection studies with herpes simplex virus (hsv) and mouse hepatitis virus (mhv), two well established models of viral encephalitis, have demonstrated the importance of t regs in limiting proinflammatory cytokine and chemokine responses to protect the cns and enhance survival [26, 43] . recent work also implicates t regs in the control of wnv pathogenesis, wherein peripheral expansion of t regs was associated with asymptomatic infection among wnv-infected blood donors but reduced t reg levels associated with wnv disease [45] . furthermore, these studies revealed that the conditional depletion of t reg cells in mice results in enhancement of wnv virulence and expansion of antigen-specific cd8 t cells. interestingly, from our studies, type i ifn does not appear to be the major contributor to t reg expansion during wnv infection, as t regs failed to expand in the ips-1 2/2 infected mice despite their elevated levels of systemic type ifn. these observations suggest that rlr signaling through ips-1 provides essential signals that directly or indirectly impart the expansion of t regs during wnv infection. we propose that ips-1 coordinates an innate/adaptive immune interface wherein ips-1-signaling after rlr engagement regulates the quantity, quality, and balance of the subsequent immune response. the integrity of the innate/adaptive immune interface is central to the eliminating virus but also restricting immunopathogenesis and inflammation during infection. rlr signaling is essential for triggering the innate immune response to rna viruses that cause human disease, including the influenza viruses, respiratory syncytial virus and other paramyxoviruses, picornaviruses, reoviruses, flaviviruses, and hepatitis c virus [14, 19, 22] . thus, in addition to wnv, ips-1-dependent rlr signaling will likely have a broad impact for the control of inflammation, immune response quality, and viral disease. bhk21 and l929 cells were maintained in dulbecco's modified eagle medium (dmem) supplemented with 10% fetal bovine serum (fbs), 2mm l-glutamine, 1 mm sodium pyruvate, antibiotic-antimycotic solution, and 16 nonessential amino acids (complete dmem). wnv strain tx 2002-hc (wn-tx) was isolated by as previously described [11] . working stocks of wn-tx were generated by a single round of amplification on vero-e6 (ccl-81; atcc) cells, and supernatants were collected, aliquoted, and stored at 280uc. virus stocks were titered by a standard plaque assay on bhk21 cells as previously described [40] . ips-1 2/2 (c57bl/66129sv/ev) and their wild type littermate control mice have been published [38, 61] and were obtained as a generous gift from dr. s. akira (osaka university, osaka, japan). mice were genotyped and bred under pathogen-free conditions in the animal facility at the university of washington. experiments were performed with approval from the university of washington institutional animal care and use committee. the methods for mice use and care were performed in accordance with the university of washington institutional animal care and use committee guidelines. age-matched six to twelve week old mice were inoculated subcutaneously (s.c.) in the left rear footpad with 100 pfu of wn-tx in a 10 ml inoculum diluted in hanks balanced salt solution (hbss) supplemented with 1% heatinactivated fbs. mice were monitored daily for morbidity and mortality. for in vivo virus replication studies, infected mice were euthanized, bled, and perfused with 20 ml of phosphate-buffered saline (pbs). whole brain, spinal cord, kidney, and spleen were removed, weighed, homogenized in 500ul of pbs, and titered by plaque assay. bone-marrow derived dc and mw were generated as described previously [9] . briefly, bone marrow cells from wild type and congenic deficient mice were isolated and cultured for 7 days in either rpmi-1640 supplemented with granulocyte-macrophagecolony stimulating factor, and interleukin-4 (peprotech) to generate myeloid dc or in dmem supplemented with macrophage colony stimulating factor (peprotech) to generate mw. on day 7, dc or mw were infected with wn-tx at an moi of 1.0 and at 12, 24, 36, and 48 hours post-infection (hpi), supernatants were collected for titration of viral burden by plaque assay on bhk21 cells and levels of ifn-b (described below). cells were collected in parallel for western blot analysis. cortical neurons were isolated from 15-day-old embryonic mice and cultured as described previously [62] . on day 6 of culture, neurons were infected with wn-tx at an moi of 1.0 and at 12, 24, 36, and 48 hpi, supernatants were collected for virus titration by plaque assay on bhk21 cells and cells were collected for rna analysis by rt-qpcr (described below). cells were lysed in modified ripa buffer (10mm tris [ph 7.5], 150mm nacl, 0.5% sodium deoxycholate, and 1% triton x-100) supplemented with protease inhibitor cocktail (sigma) and phosphatase inhibitor cocktail ii (calbiochem). protein extracts (25 mg) were analyzed by immunoblotting as described previously [11] . the following primary antibodies were used to probe blots: mouse anti-wnv from the center for disease control; rabbit anti-isg56, rabbit anti-isg54, rabbit anti-isg49, kindly provided by dr. g. sen; mouse anti-pkr from santa cruz; rabbit anti-rig-i and rabbit anti-mda5 from ibl; mouse anti-tubulin from sigma; and rabbit anti-stat-1 from cell signaling. secondary antibodies included peroxidase-conjugated goat anti-rabbit, goat anti-mouse, donkey anti-rabbit, and donkey anti-mouse were from jackson immunoresearch. for analysis of viremia, serum was separated (bd microtainer tube sst) and rna was extracted as previously described [8] . wnv rna copy number was measured by rt-quantitative pcr (rt-qpcr) as previously described [63] . for cultured cells, total rna was extracted using the rneasy kit (qiagen), dnase treated (ambion) and evaluated for isg49, isg56, ifn-b, rig-i, and mda5 rna expression by one-step sybr green rt-qpcr. specific primer sets for isg-49, isg-56, rig-i, and ifn-b have been described previously [30, 64] . primer sets for mda5 are: 59-gtggtcgagccagagctgat and 39-tgtctcatg-ttcgataactcctgaa. ifn-a and -b were measured in sera using a biological assay as previously described [65] . briefly, l929 cells were seeded at 3610 4 cells/well in a 96 well plate one day prior to the addition of interferon standards or experimental samples. mouse sera (diluted 1:10 in l929 media) were treated with uv light for 20 minutes to eliminate residual virus. duplicate sera samples were then added to the 96-well plates in two-fold dilutions along with a murine ifnb standard. the following day, emcv challenge virus was added to the cells in 50 ml/well at an moi of 5.0. twenty-four hours later, cytopathic effect was measured by a blinded scorer and ifn levels in the sera was calculated based on the ifn standard. ifn-b in cell culture supernatants was analyzed using mouse-specific elisa kits from pbl biomedical laboratories according to the manufacturer's protocol. wnv-specific igm, total igg, igg1, and igg2a levels were determined by an elisa using purified recombinant e protein as previously described [55] . the neutralization titer of serum antibody was determined by using a previously described plaque reduction neutralization assay [40] . briefly, sera samples from mock or wn-tx infected mice were diluted in dmem followed by incubation at 56uc for 30 minutes to inactivate virus and complement factors. sera were further diluted in two-fold increments and incubated with 100 pfu of wn-tx at 37uc for 1 hour. standard plaque assays were performed on bhk21 cells and the dilution at which 50% of plaques were neutralized was determined by comparing the number of plaques formed from wnv-infected sera samples to mock infected sera samples. cytokine/chemokine analysis wnv infected sera were analyzed for the presence and levels of tnf-a, ifn-c, cxcl10 (ip-10), and il-6 by a mouse-specific cytokine/chemokine milliplex elisa (millipore). mock-infected or wnv-infected mice were exsanguinated and perfused with pbs, 4% paraformaldehyde, ph 7.3. brains were embedded in paraffin and 10-mm sections were prepared and stained with hematoxylin and eosin (h&e) by the uw histology pathology laboratory. sections were analyzed using a nikon eclipse e600 microscope (uw keck microscope facility). draining lymph nodes from mice were isolated and digested with collagenase (roche) and type i dnase in serum-free rpmi media at 37uc for 40 minutes with mechanical disruption. cells were then incubated with rpmi media containing 10% fbs with edta and hepes for 10 minutes at room temperature, pelleted, and resuspended in pbs containing 2% fbs and 0.1% sodium azide (facs staining buffer). splenocytes were isolated, washed, and re-suspended in rpmi 1640 containing 10% fbs before in vitro stimulation. cells were washed twice before facs staining. for isolation of cns immune cells, mice were euthanized and perfused extensively with pbs to remove residual intravascular leukocytes. brains and spinal cords from 5 mice per experimental group were isolated and pooled. tissues were minced in rpmi media, triturated, and digested with liberase (roche) and type i dnase in serum-free rpmi media at 37uc for 45 min. immune cells were isolated after gradient centrifugation from a 37/70% percoll interface and washed twice with facs staining buffer. immune cells were stained with antibodies specific to cd11c, cd11b, b220, cd3, cd25, cd4, cd8, nk1.1, gr-1, siglec h, and cd45 (all reagents from ebiosciences). intracellular foxp3 staining was performed as previously described [26] . intracellular ifn-c staining was performed on splenocytes and cns immune cells as previous described [35, 36] . briefly, lymphocytes were stimulated with 1 mg/ml of the wnv ns4b peptide (ssvwnat-tai) for 4 h at 37uc. cells were washed and stained for cell surface markers followed by permeabilization-fixation using the cytofix-cytoperm kit (bd-pharmingen) and stained with a pacific-blue conjugated ifn-c antibody (ebiosciences) at 4uc for 30 min, washed and analyzed by flow cytometry. flow cytometry was performed on a bd lsrii machine using bd facsdiva software. cell analysis was performed on flowjo (v.8.7.2) software. for in vitro studies and immune cell analysis an unpaired student t-test was used to determine statistical differences. for in vivo viral burden analysis, mann-whitney analysis was used to determine statistical differences. kaplan-meier survival curves were analyzed by the log-rank test. a p-value #0.05 was considered significant. all data were analyzed using prism software (graphpad prism5). west nile virus activity-united states pathogensis of west nile virus infection: a balance between virulence, innate and adaptive immunity, and viral evasion virology, pathology, and clinical manifestations of west nile virus disease west nile virus meningoencephalitis in an immunocompetent adolescent severe west nile virus disease in healthy adults emerging viruses in transplantation: there is more to infection after transplant than cmv and ebv evasion and disruption of innate immune signalling by hepatitis c and west nile viruses cell-specific irf-3 responses protect against west nile virus infection by interferondependent and -independent mechanisms interferon regulatory factor irf-7 induces the antiviral alpha interferon response and protects against lethal west nile virus infection the host response to west nile virus infection limits viral spread through the activation of the interferon regulatory factor 3 pathway resistance to alpha/beta interferon is a determinant of west nile virus replication fitness and virulence alpha/beta interferon protects against lethal west nile virus infection by restricting cellular tropism and enhancing neuronal survival mda-5: an interferon-inducible putative rna helicase with double-stranded rnadependent atpase activity and melanoma growth-suppressive properties regulating intracellular anti-viral defense and permissiveness to hepatitis c virus rna replication through a cellular rna helicase, rig-i the rna helicase rig-i has an essential function in double-stranded rnainduced innate antiviral responses shared and unique functions of the dexd/h-box helicases rig-i, mda5, and lgp2 in antiviral innate immunity lengthdependent recognition of double-stranded ribonucleic acids by retinoic acidinducible gene-i and melanoma differentiation-associated gene 5 regulation of innate antiviral defenses through a shared repressor domain in rig-i and lgp2 distinct rig-i and mda5 signaling by rna viruses in innate immunity west nile virus evades activation of interferon regulatory factor 3 through rig-i-dependent and -independent pathways without antagonizing host defense signaling establishment and maintenance of the innate antiviral response to west nile virus involves both rig-i and mda5 signaling through ips-1 differential roles of mda5 and rig-i helicases in the recognition of rna viruses toll-like receptor 3 has a protective role against west nile virus infection toll-like receptor 3 mediates west nile virus entry into the brain causing lethal encephalitis toll-like receptor 7 mitigates lethal west nile encephalitis via interleukin 23-dependent immune cell infiltration and homing coordination of early protective immunity to viral infection by regulatory t cells natural regulatory t cells in infectious disease monocyte-derived dendritic cells in innate and adaptive immunity card games between virus and host get a new player novel characteristics of the function and induction of murine p56 family proteins transcriptional profiling of interferon regulatory factor 3 target genes: direct involvement in the regulation of interferon-stimulated genes infection and injury of neurons by west nile encephalitis virus west nile virus infection in the golden hamster (mesocricetus auratus): a model for west nile encephalitis role of cd8+ t cells in control of west nile virus infection protective capacity and epitope specificity of cd8(+) t cells responding to lethal west nile virus infection antigenspecific cytotoxic t lymphocytes protect against lethal west nile virus encephalitis replicon particles of venezuelan equine encephalitis virus as a reductionist murine model for encephalitis cell type-specific involvement of rig-i in antiviral response a critical role for induced igm in the protection against west nile virus infection b cells and antibody play critical roles in the immediate defense of disseminated infection by west nile encephalitis virus batf3 deficiency reveals a critical role for cd8alpha+ dendritic cells in cytotoxic t cell immunity in vivo induction of immune responses to pathogens by conventional dendritic cells role of regulatory t cells in coronavirus-induced acute encephalitis regulatory t cells promote early influx of cd8+ t cells in the lungs of respiratory syncytial virus-infected mice and diminish immunodominance disparities tregs control the development of symptomatic west nile virus infection in humans and mice production of type i interferons: plasmacytoid dendritic cells and beyond differential activation of human monocyte-derived and plasmacytoid dendritic cells by west nile virus generated in different host cells early b-cell activation after west nile virus infection requires alpha/beta interferon but not antigen receptor signaling type i ifn receptor signals directly stimulate local b cells early following influenza virus infection early type i interferon-mediated signals on b cells specifically enhance antiviral humoral responses type i interferons act directly on cd8 t cells to allow clonal expansion and memory formation in response to viral infection innate inflammatory signals induced by various pathogens differentially dictate the ifn-i dependence of cd8 t cells for clonal expansion and memory formation cutting edge: the direct action of type i ifn on cd4 t cells is critical for sustaining clonal expansion in response to a viral but not a bacterial infection the reciprocal interaction of nk cells with plasmacytoid or myeloid dendritic cells profoundly affects innate resistance functions cd8+ t cells require perforin to clear west nile virus from infected neurons cd8+ t cells mediate recovery and immunopathology in west nile virus encephalitis west nile virus-specific cd4 t cells exhibit direct antiviral cytokine secretion and cytotoxicity and are sufficient for antiviral protection cd4+ t-cell responses are required for clearance of west nile virus from the central nervous system dendritic cell-induced memory t cell activation in nonlymphoid tissues tregs are regulated by cytokines: implications for autoimmunity essential role of ips-1 in innate immune responses against rna viruses pkr and rnase l contribute to protection against lethal west nile virus infection by controlling early viral spread in the periphery and replication in neurons detection of west nile virus lineages 1 and 2 by real-time pcr innate immunity induced by composition-dependent rig-i recognition of hepatitis c virus rna differential induction of type i interferon responses in myeloid dendritic cells by mosquito and mammalian-cell-derived alphaviruses we thank kristy szetter and jessica briley for technical help and robert immormino for generation of purified wnv e protein for elisa. key: cord-003261-fz8ucwwm authors: freundt, eric c.; drappier, melissa; michiels, thomas title: innate immune detection of cardioviruses and viral disruption of interferon signaling date: 2018-10-12 journal: front microbiol doi: 10.3389/fmicb.2018.02448 sha: doc_id: 3261 cord_uid: fz8ucwwm cardioviruses are members of the picornaviridae family and infect a variety of mammals, from mice to humans. replication of cardioviruses produces double stranded rna that is detected by helicases in the rig-i-like receptor family and leads to a signaling cascade to produce type i interferon. like other viruses within picornaviridae, however, cardioviruses have evolved several mechanisms to inhibit interferon production. in this review, we summarize recent findings that have uncovered several proteins enabling efficient detection of cardiovirus dsrna and discuss which cell types may be most important for interferon production in vivo. additionally, we describe how cardiovirus proteins l, 3c and l(∗) disrupt interferon production and antagonize the antiviral activity of interferon effector molecules. picornaviridae is an important family of single-stranded, positive-polarity rna viruses that includes >30 genera with over 75 species (zell et al., 2017) . within picornaviridae, the genus cardiovirus includes encephalomyocarditis virus (emcv), theiler's murine encephalomyelitis virus (tmev) and saffold viruses (safv). although emcv has been described as a potential zoonotic agent, safvs are the only cardioviruses known to regularly infect humans, with the vast majority of people showing evidence of infection (zoll et al., 2009; carocci and bakkali-kassimi, 2012) . emcv has been found to infect over 30 host species and contains one serotype, mengo virus, which was isolated in 1948 in the mengo district of uganda (dick et al., 1948) . tmev was discovered in 1937 by max theiler and is found in wild mice and rats worldwide. tmev can cause different diseases, depending on the virus strain and host genetics, ranging from fatal encephalitis to a chronic demyelinating disease that has served as a model for multiple sclerosis (brahic et al., 2005) . the genome of cardioviruses is approximately 7.8-8.5 kb and contains 5 and 3 untranslated regions (figure 1) . translation of the genome gives rise to a polyprotein that is cleaved by the 3c protease, leading to the production of 12 proteins. two additional proteins, l * and 2b * , are expressed from alternate open reading frames. l * is only expressed by tmev and is important for infection of macrophages, persistence of the virus in mice and inhibiting rnase l (van eyll and michiels, 2000; sorgeloos et al., 2013) , 2b * results from a frameshifting mechanism conserved in cardioviruses that regulates the ratio of structural and non-structural proteins translated over time. protein 2b * itself is only thought to be important for replication of emcv, as mutants that abolish its expression had a small plaque phenotype. 2b * in tmev and safv is unlikely to act as a protein as it is predicted to encode a peptide of 14 amino acids (loughran et al., 2011) . in this review, we focus on the ways in which cardioviruses trigger the innate immune response and the efficient mechanisms that they have evolved to suppress these signaling pathways. we consider which cell types may be most important for production of interferon (ifn) in vivo, and also describe how cardioviruses disrupt the functions of interferon effectors. double-stranded rna (dsrna) is a necessary product of picornavirus replication, as positive-stranded genome is copied to produce a negative-stranded, full-length template, which is in turn used to produce additional genomes. dsrna is recognized by several sensor proteins within the cell and triggers a signal transduction pathway that results in transcription of the type i ifn (ifn-α/β) genes, as well as ifn-λ. in the endosome, dsrna is detected by toll-like receptor 3 (tlr3), which signals through the adaptor protein trif to activate ifn regulatory factor 3 (irf-3) and nuclear factor kappa b (nf-κb) (yamamoto et al., 2003) . cytoplasmic dsrna is detected by the rig-i-like receptor (rlr) family of proteins, which includes rig-i (retinoic acid-induced gene i) and mda5 (melanoma differentiation-associated gene 5). upon recognition of dsrna, these proteins undergo a conformational change that exposes n-terminal caspase activation and recruitment domains (cards). the rlrs are then capable of stimulating the mitochondrial antiviral signaling (mavs) protein, also known as ips-1, cardif, and visa, which in turn activates tank-binding kinase-1 (tbk1), inducible i-κb kinase (ikk-ε) and irf-3, which then translocates to the nucleus to facilitate transcription of ifn genes (reviewed in gebhardt et al., 2017) . although rig-i and mda5 both detect dsrna within the cytosol, their functions are non-redundant. rig-i recognizes relatively short dsrna species (<1 kb) with 5 ppp or 5 pp, which are produced in certain virus infections (hornung et al., 2006; pichlmair et al., 2006) . in contrast, mda5 recognizes long dsrna, which is present during picornavirus replication (kato et al., 2008; pichlmair et al., 2009) . thus, while rig-i is activated during infection with flaviviruses, paramyxoviruses, influenza, and others, mda5 is responsible for detection of picornaviruses (gitlin et al., 2006; kato et al., 2006; pichlmair et al., 2009; wang et al., 2010; feng et al., 2012) . the importance of mda5 for control of cardioviruses was demonstrated in mda5-deficient mice, which failed to control emcv infection and did not efficiently produce ifn (gitlin et al., 2006; kato et al., 2006) . in addition to rig-like helicases described above, which activate the mavs pathway, another ifn-inducible rna helicase, moloney leukemia virus 10 homolog (mov10) was reported to enhance ifn induction (cuevas et al., 2016) . mov10 expression in hek293 cells restricted emcv replication. interestingly, mov10 acts through irf-3 activation, in a rlr and mavs-independent way and signals require ikk-ε but not tbk1. such mavs-independent pathways are likely not critical for global ifn production in emcv infected mice, given the major impact of mda5 or mavs deficiency in mice, but they may be important in specific cell types or in conditions where the other pathways may be less potent. laboratory of genetics and physiology 2 (lgp2), also known as dhx58, is also a member of the rlr family but lacks a card domain (yoneyama et al., 2005) . given its structural similarity and lack of a card domain, lgp2 was initially thought to negatively regulate dsrna recognition by rig-i, as its overexpression limited ifn induction by sendai virus and newcastle disease virus (rothenfusser et al., 2005) . however, although the negative effect of lgp2 on rig-i remained controversial, later studies have established that lgp2 acts as a co-activator of mda5. mice deficient for lgp2 were impaired in responding to rna ligands for mda5 or to emcv infection (venkataraman et al., 2007; satoh et al., 2010) . lgp2 was also shown to increase the rate of mda5 interaction with rna and downstream signaling by facilitating the formation of numerous, shorter mda5 filaments (bruns et al., 2014) . thus, it appears that lgp2 can act as both a positive and negative regulator of rlr signaling, with the outcome likely dependent on the concentration of lgp2 (bruns and horvath, 2015) . however, recombinant mda5 was directly activated as measured by an atp hydrolysis assay by the replicative form of dsrna coxsackievirus b3, showing that lgp2 is not essential for activation of mda5 in vitro by dsrna (feng et al., 2012) . both lgp2 and mda5 are important for detecting cardiovirus replication. mefs deficient for either protein produce lower amounts of ifn-β when infected with emcv (deddouche et al., 2014) . intriguingly, lgp2 may enhance activation of mda5 during emcv infection by binding to rna complementary to the leader (l) gene and forming a complex with mda5. this rna sequence from l was also shown to be a potent activator of mda5 in the absence of virus infection (deddouche et al., 2014) . although dsrna can bind and activate recombinant mda5 in the absence of other proteins, it is likely that additional partners are required for efficient activation of mda5 in vivo. for example, mda5 is phosphorylated in resting cells, and requires dephosporylation by pp1α/γ (wies et al., 2013; takashima et al., 2015) . additional proteins that participate in recognition of cardiovirus dsrna have recently been described, including a study showing that tar rna binding protein (trbp) interacts with lgp2 in a yeast two-hybrid screen (komuro et al., 2016) . lgp2 was found to interact with trbp in co-immunoprecipitation experiments and depletion of trbp by sirna reduced interferon production induced by tmev and emcv. moreover, trbp enhanced ifn induction by tmev and emcv when overexpressed. depletion of trbp did not affect induction of ifn by sendai virus, which is recognized by rig-i. this study establishes that trbp participates in detection of cardiovirus dsrna by lgp2/mda5 but the mechanism and its importance in vivo remain to be elucidated. as trbp is a component of the rnai machinery (chendrimada et al., 2005; haase et al., 2005) , other molecules involved in rnai were assessed for their role in dsrna detection and protein activator of pkr (pact) was found to also participate in activation of ifn signaling by cardioviruses. when pact was depleted by sirna, interferon production was decreased during infection of both tmev and emcv. overexpression of pact also increased ifn activation when lgp2 was co-expressed with mda5. intriguingly, single-stranded tmev genome enhanced the association of lgp2 and pact, which suggests that a secondary structure in the tmev genome might facilitate this interaction (miyamoto and komuro, 2017) . in a separate report, pact was shown to be required for induction of ifn by emcv but not sendai virus. this study also demonstrated that pact and mda5 were recruited to dsrna (poly(i:c)) but not single stranded rna, and that pact expression increased the amount of mda5 oligomerization (lui et al., 2017) . both trbp and pact have also been reported to bind to double stranded rna-dependent protein kinase (pkr), and pact can bind rig-i (park et al., 1994; patel and sen, 1998; kok et al., 2011) . at the present time, it is not clear if these interactions are important for mediating recognition of cardioviruses. yet another partner in detecting dsrna in cardiovirus infection was recently uncovered. a cdna screen to identify genes involved in regulating ifn signaling revealed that dhx29 expression increased transcription of an ifn-β reporter plasmid in response to high molecular weight (hmw) poly(i:c) (zhu et al., 2018) . depletion of dhx29 resulted in decreased phosphorylation of tbk1 and irf-3 in response to hmw poly(i:c) and emcv as well as decreased production of ifn-β. the authors also show that dhx29 binds to mda5 but not mavs or rig-i, and binding could only be detected when mda5 was activated by emcv or hmw poly(i:c). mechanistically, this study demonstrated that dhx29 mediated rna binding of mda5 by interacting with mda5 through its n-terminus and rna through its dexd and helicase domains, and that dhx29 promotes formation of mda5 filaments, which are required for activation (zhu et al., 2018) . dhx29 was also independently described to interact with rig-i (sugimoto et al., 2014) , although it may be of greater importance for activation of mda5 (zhu et al., 2018) . together, these studies show that multiple proteins facilitate recognition of dsrna by mda5 during cardiovirus infection (figure 2 ). thus far, these proteins include lgp2, dhx29, pact and trbp. additional helicases are also likely to participate in rlr-dsrna complex formation but their activities remain to be clarified (oshiumi et al., 2016) . moreover, recent discoveries have identified a novel role for pkr in recognition of dsrna and activation of mda5, which will be discussed below. as the number of proteins mediating mda5 activation grows, so does the number of questions about how this pathway functions. for example, what role does each of the proteins play and how do they work together mechanistically to activate mda5? do they play non-redundant roles, or do they function in the same way but in different cell types? resolving these questions will allow for deeper understanding of the first line of immune defense against rna viruses. upon recognition of dsrna, pkr controls virus infection by phosphorylating eukaryotic initiation factor 2 (eif2α), which inhibits translation (farrell et al., 1977) . in this way, an infected cell can suppress production of viral proteins. phosphorylation of eif2α also leads to stress granule formation and induces autophagy, and both pathways are commonly observed during virus infection (paul and munz, 2016; poblete-duran et al., 2016) . not surprisingly, many viruses have evolved strategies to inhibit the activation or function of pkr (reviewed in garcia et al., 2007) , as evidenced by the fact that pkr-deficiency did not modify the survival time of emcv infected mice (yang et al., 1995) . in addition to its role in inhibiting translation, recent evidence has emerged to show that pkr participates in production of ifn. for example, pkr appears to be important for nuclear translocation of irf-3 following mda5 activation (pham et al., 2016) . pkr was found to bind to mda5 and this interaction was not disrupted by nuclease treatment, indicating that binding does not depend on the presence of rna. in pkr-deficient cells, emcv infection failed to induce irf-3 translocation to the nucleus. moreover, a constitutively active mutant of pkr induced ifn through mavs. the effect of pkr on induction of ifn required its catalytic activity but did not depend on phosphorylation of eif2α (pham et al., 2016) . in a separate report, pkr was shown to be important for normal processing of ifn-β mrna, suggesting that pkr may function at multiple points in the ifn pathway (schulz et al., 2010) . additionally, activation of pkr by hmw poly(i:c) was shown to be inhibited in cells depleted of mda5 and mavs, suggesting that mavs influences activation of pkr. moreover, mavs and pkr were found to interact in co-ip experiments, and the interaction depended on the card domain in mavs and the dsrna binding domain of pkr (zhang et al., 2014) . together, these studies clearly indicate a role for pkr in mda5-dependent ifn induction, although several mechanisms may be involved, which require clarification. the role of pkr in ifn production after cardiovirus infection remains to be resolved. in one study that examined mengo virus infection, knockdown of pkr led to decreased induction of ifn-β in hela cells, suggesting that pkr may play a role in the mda5 pathway during cardiovirus infection (langereis et al., 2013) . this observation fits with the model proposed by onomoto et al. (2012) , which was based on influenza virus infection and suggests that pkr triggers the formation of "antiviral stress granules" that serve as a recruitment platform for dsrna and rig-like helicases, thereby enhancing ifn production. pkr is, however, not essential for ifn production in cardiovirus-infected cells because mengo virus possessing a deletion in the zinc-finger of l, which abrogates its functions as an interferon antagonist, was found to induce interferon in the cells lacking pkr and rnase l (feng et al., 2012) . cardioviruses may also inhibit pkr through activity of the l protein, as stress granule formation was prevented by the l protein of mengo, tmev, and safv-2 during virus infection (borghese and michiels, 2011) . however, direct inhibition of pkr by l remains to be demonstrated. during infection, viral dsrna can also be released into the extracellular milieu, either non-specifically during lysis of infected cells or possibly intentionally by exocytosis to trigger innate immunity by uninfected cells. dsrna can then be endocytosed by neighboring cells and infiltrating immune cells and lead to ifn production. tlr3 recognizes dsrna in endosomes and signals through trif to activate irf-3 and nf-κb. the importance of tlr3 in context of cardiovirus infection may depend on the model of infection. for example, mice deficient for myd88 or tlr3 were not significantly more susceptible than wild-type mice to emcv infection . in a separate report, however, tlr3-deficient mice had higher viral loads in the liver and heart and were more susceptible to infection (hardarson et al., 2007) . also, a study that evaluated the role of tlr3 in controlling a strain of emcv with tropism for β cells of the pancreas found that tlr3 protected mice from a fatal infection and that tlr3-deficent mice produced less ifnβ early in infection (15 and 18 h post-infection). however, this deficiency was transient and mice lacking tlr3 produced levels of ifn-β equivalent to wild-type animals at 24 h post-infection. in the same experiments, the authors demonstrated that mice lacking mda5 succumbed to the infection more rapidly than tlr3-deficient mice and produced less ifn-β at 24 h postinfection (mccartney et al., 2011) . finally, a recent report using intracerebral inoculation of the gdvii strain of tmev evaluated the importance of these molecules for control of virus replication and induction of ifn. trif-/-, myd88-/-, and mice lacking both trif and myd88 showed wild-type levels of ifn induction, while mavs-deficient animals were slightly but significantly impaired. when trif, myd88 and mavs were all depleted, however, mice were unable to induce ifn. only mice lacking myd88 and trif, or mice lacking myd88, trif and mavs showed increased titers of gdvii in the brain (pfefferkorn et al., 2016) . thus, both tlr3 and rlrs contribute to controlling virus replication in vivo, although the relative importance of these pathways may depend on the virus and route of inoculation. surprisingly, mda5 is also responsible for the vast majority of ifn produced from extracellular dsrna in vivo. mice deficient for mda5 produced substantially less ifn when administered polyi:c, whereas tlr3-deficient mice responded like wild-type (gitlin et al., 2006) . these results raise the question of how dsrna taken up through endocytosis could gain access to the cytoplasm. the mechanism by which dsrna could be internalized and then access the cytoplasmic rlrs has been unresolved until a recent discovery identified sidt2, the mammalian ortholog of the sid-1 dsrna transporter in caenorhabditis elegans, as a transporter of dsrna from the endosome to the cytoplasm. sidt2 was shown to be important for mediating detection of dsrna in the context of emcv infection in vivo. mice that were deficient for sidt2 failed to control replication, produced less ifn-β, and succumbed to infection (nguyen et al., 2017) . these data suggest that a crucial pathway for innate signaling in emcv infection is release of viral rna into the extracellular milieu, endocytosis, and subsequent transfer of viral rna to the cytoplasm to access mda5. two proteins encoded by cardioviruses were shown to counteract ifn production in infected cells: protease 3c, which is responsible for the processing of the virus-encoded polyprotein, and the leader protein (l), which corresponds to the n-terminal peptide of the polyprotein. 3c is a cysteine proteinase with a trypsin-like serine protease fold, responsible for most cleavages occuring during the maturation of the viral polyprotein (pelham, 1978) . like 3c proteases of other picornaviruses that were shown to target critical factors involved in ifn induction such as rig-i (barral et al., 2009) , emcv 3c was reported to cleave traf family member-associated nf-kb activator (tank) in infected cells, thus disrupting the complex involving tbk1, ikke and irf3 and limiting type i ifn production (huang et al., 2017) . likewise, emcv 3c was shown to target mov10, an rna helicase that acts in a mavs-independent way, as a possible innate immune evasion mechanism (cuevas et al., 2016 ; figure 3 ). l is a small, multifunctional protein of 67-76 amino acids expressed by all cardioviruses (figure 1 ). l contains an n-terminal zinc finger motif (cys-his-cys-cys), an acidic domain, a serine/threonine rich domain, and a c-terminal theilo domain, which is present in safv and tmev but absent in emcv. l was shown to be dispensable for replication of tmev in cell culture but its loss inhibits spread in cells that have a functional interferon response, like l929 cells, and also impairs the viral persistence in vivo (van pesch et al., 2001) . l deletions in figure 3 | interferon antagonism by the cardiovirus 3c protease. the 3c protease is responsible for cleavage of the cardiovirus polyprotein produced from translation of the genome. in addition, the cardiovirus 3c protease cleaves host proteins, such as tank and mov10, to prevent the cell from producing ifn. frontiers in microbiology | www.frontiersin.org emcv prevent the virus from shutting off host protein synthesis and enable interferon production (zoll et al., 1996 (zoll et al., , 2002 . mengo virus containing a mutation in the zinc finger of l failed to inhibit ifn synthesis and its replication was inhibited during low moi infections in vitro. in mice lacking the ifn α/β receptor, the mutant virus behaved as wild-type, but in wild-type mice, replication of the l mutant virus was impaired and it failed to cause disease, demonstrating that activity of l is important for pathogenesis in vivo (hato et al., 2007) . mutations in the zinc finger domain or the theilo domain of tmev or safv l inhibit its ability to antagonize interferon signaling (ricour et al., 2009) . a critical step in production of ifn following detection of viral replication by mda5 and other molecules is nuclear translocation of irf-3 and nf-κb. these proteins enable transcription of the ifn genes to produce mrna, which must then be exported from the nucleus for translation. since picornaviruses do not replicate within the nucleus, many viruses within this family disrupt nucleocytoplasmic trafficking, which results in inhibition of ifn production and translocation of nuclear proteins to the cytosol to benefit viral replication (reviewed in yarbrough et al., 2014; flather and semler, 2015) . the mechanism of how l interferes with ifn production may be due to its abilities to disrupt nucleocytoplasmic trafficking, activation of irf-3, and assembly of stress granules in infected cells (figure 4) . each of these activities will be explored below. the l protein of cardioviruses perturbs the function of the nuclear pore complex (npc) (porter et al., 2006) . in mammals, the npc consists of approximately 30 different proteins, called nucleoporins (nups) and enables transit across the nuclear membrane (gorlich and kutay, 1999; wente and rout, 2010) . while small molecules and ions are able to diffuse through the npc, molecules larger than approximately 20-40 kda require active transport, which is regulated by transport receptors called karyopherins (yarbrough et al., 2014) . transport into the nucleus requires a short amino acid motif, called a nuclear localization sequence (nls) that can interact with either the α or β subtypes of karyopherins, depending on the sequence of the protein's nls. binding and dissociation of nls-containing proteins by karyopherins is also regulated by small gtpase ran. in the cytosol, ran is bound to gdp and can bind cargo proteins. once in the nucleus, however, ran is converted to the gtp bound form by the ran guanine nucleotide exchange factor (rangef) and dissociates from cargo. export then requires a nuclear export sequence (nes) that binds to karyopherins bound to rangtp, and dissociation of this complex occurs in the cytoplasm when a ran gtpase-activating protein (rangap) hydrolyzes gtp to gdp. in this way, the rangdp/gtp gradient regulates directional transport into and out of the nucleus. localization of l to the nucleus depends on expression of 2a, which contains a nls in its c-terminus (groppo et al., 2011) . upon nuclear localization, l interacts with ran with high affinity and 2a is displaced as the binding sites for 2a and ran partially overlap (petty et al., 2014) . l from emcv, tmev, and safv induce hyper-phosphorylation of nups including nup62 and nup98 (ricour et al., 2009; ciomperlik et al., 2015) , likely by recruiting and activating a kinase, which may be facilitated by l binding of exportins crm1 and cas (ciomperlik et al., 2016) . chemical inhibition of erk and p38 was able to block l-mediated hyper-phosphorylation of nups (porter et al., 2010) . additionally, l of emcv is phosphorylated by casein kinase 2 (ck2) and this phosphorylation is required for nup phosphorylation, although ck2 did not phosphorylate l of safv or tmev . it is possible, although it remains to be shown, that these kinases also play a role in inhibition of nucleocytoplasmic trafficking by l of tmev and safv. in addition to its role in disrupting nucleocytoplasmic trafficking, tmev and mengo l prevent production of type i ifn in infected cells by interfering with irf-3 dimerization and tmev l also prevents export of mrna from the nucleus (delhaye et al., 2004; ricour et al., 2009) . for both tmev and mengo virus, dimerization of irf-3 was impaired despite the protein having been phosphorylated. inactivation of irf-3 occurs despite reports that it accumulates in the nucleus of infected cells (delhaye et al., 2004) . these data suggest that dsrna is detected in cardiovirus infected cells leading to activation of mavs and downstream kinases, but that irf-3 is unable to induce ifn transcription. stress granules can form in cells during virus infection and often result from inhibition of translation following phosphorylation of eif2α by pkr (white and lloyd, 2012) . the l protein of mengo, tmev, and safv-2 inhibits stress granule formation during infection and ectopic expression of l was able to prevent thapsigargin-and arsenite-induced stress granules (borghese and michiels, 2011) . stress granules formed during infection with viruses containing deletions in the zinc-finger domain or a mutation in the theilo domain of l, indicating that these motifs are also important for inhibition of stress granules (borghese and michiels, 2011) . while l inhibits nucleocytoplasmic trafficking, stress granule formation, and possibly pkr activation, it has not been possible to uncouple these events using l mutants. when one function of l is disrupted, all functions are simultaneously impaired. therefore, it remains possible that l inhibits ifn production by blocking pkr activation, by interfering with irf-3 dimerization or nucleocytoplasmic trafficking, or through a combination of these mechanisms (figure 4) . the importance of these antiviral pathways in controlling infection is underscored by the variety of mechanisms that viruses have evolved to prevent their activity. for example, the l protein of foot-and-mouth disease virus (fmdv), a picornavirus in the genus aphthovirus, has proteolytic activity whereas the l protein of cardioviruses does not. despite the major differences in these proteins, they all still function to inhibit induction of ifn. fmdv l pro can cleave eif4g (devaney et al., 1988; kirchweger et al., 1994; guarne et al., 1998) and therefore reduce translation of cellular mrnas, and can also perturb ifn transcription by cleaving nf-κb (de los santos et al., 2006 . however, in the once in the nucleus, l interacts with high affinity with ran gtpase, thus displacing 2a. the l-ran complex would activate a kinase and trigger nucleoporin hyper-phosphorylation, thereby leading to nuclear pore complex dismantling and to nucleocytoplasmic trafficking perturbation. on the other hand, l may inhibit pkr, thus preventing translation arrest through eif2α phosphorylation and therefore block assembly of stress granules. inhibition of ifn gene transcription by l may result from irf-3 trafficking perturbation and/or from the absence of pkr-enhanced dsrna detection by mda5. context of a chimeric mengo virus infection, fmdv l pro was less effective at inhibiting ifn induction in vitro and in vivo (hato et al., 2010) . similar convergent evolution is apparent when considering the 2a protein of picornaviruses. whereas 2a functions as a protease for most picornaviruses and cleaves mediators of type i interferon signaling, this activity is not present in cardioviruses. nevertheless, l still targets some of these same molecules for inactivation (agol and gmyl, 2010) . additionally, both 2a of enteroviruses and l of cardioviruses inhibit stress granule assembly (yang et al., 2018) . the functions of l appear to be sufficiently important to the virus so that it maintains high levels of l expression throughout infection. emcv and tmev undergo a frameshift during translation later in infection by 2a binding to a stem-loop structure in the genome (napthine et al., 2017) . this frameshift decreases expression of non-structural proteins 2bc-3abcd by 74-82% (finch et al., 2015) . a follow up study using metabolic labeling estimated the frameshifting to be 46-76% efficient (ling and firth, 2017) . this mechanism may allow for cardioviruses, and perhaps other picornaviruses, to increase the translation of structural proteins later in infection. due to its position in the genome, however, l expression would remain high throughout infection despite it not having a structural role for virus assembly. thus, it may be important for cardioviruses to express sufficient levels of l to counteract the immune response throughout the replication cycle. with effective ways to inhibit the production of interferon during infection, control of cardioviruses likely depends on nearby uninfected cells to produce interferon. intriguingly, these pathways seem to also depend on mda5, although tlr3 may also be important in certain cell types such as plasmacytoid dendritic cells (hornung et al., 2006) . as discussed, recent data suggest a model where viral dsrna is released, endocytosed, and then the rna is translocated to the cytosol where it is detected by mda5. in the cns, astrocytes appear to be the primary producers of ifn-β for several neurotropic viruses that preferentially infect neurons, such as tmev and la crosse virus (kallfass et al., 2012; pfefferkorn et al., 2016) . using transgenic mice that expressed firefly luciferase under the control of the ifn-β promoter restricted to different cell types, the authors were able to determine that 73% of ifn-β production during a neurotropic tmev infection was from astrocytes, whereas only 1% was from neurons, which are the primary target of infection. in mice lacking mavs, ifn-β production was slightly but significantly reduced, suggesting that the rlr pathway is active during infection but may not be the only pathway activated by tmev in astrocytes. intriguingly, mice deficient for myd88 and trif did not show a significant decrease in ifn-β induction, although induction of ifn-β was completely abrogated in mice deficient for mavs, myd88 and trif. therefore, it appears that both rlr and tlr signaling are important for ifn-β production after tmev infection of the cns. astrocytes were also shown to be primary producers of ifn during infection with rabies virus and vesicular stomatitis virus. in the case of rabies virus, astrocytes are stimulated to produce ifn by an abortive infection. that the virus is unable to replicate fully may prevent expression of viral interferon antagonists and allow for robust production of ifn. how viral replication is prevented in these cells will be important to uncover and may lead to novel insights about viral control in vivo. it is likely that abortive infection of astrocytes occurs during infection by tmev. however, this remains to be demonstrated and viral rna may well be encountered by other means. mda5 is critical for induction of ifn against cardioviruses in the periphery as well. ex vivo, cells such as macrophages, conventional dendritic cells and fibroblasts depend on mavs for production of ifn in response to dsrna (sun et al., 2006) . similarly, mda5 was shown to be essential in these cells for type i ifn production after emcv infection, in contrast to pdcs which induce ifn production in a tlr-dependent fashion (gitlin et al., 2006; kato et al., 2006) . after emcv infection of mice, some ifn is produced through tlrs, likely by pdcs, but most ifn was produced by mda5 activation (gitlin et al., 2006; kato et al., 2006) . levels of ifn-i were strongly decreased in the serum of mda5-deficient mice infected by emcv. whereas mda5 expression strongly influenced survival in response to infection, the effect of myd88 depletion had a modest effect and loss of trif or rig-i did not affect survival . thus, mda5 is essential for controlling emcv infection in the periphery. interferon secreted by infected cells binds to its receptor on surrounding cells, activating a signaling cascade that leads to expression of hundreds of interferon-stimulated genes (isgs). two of these isgs, pkr and oligoadenylate synthetases (oas) are part of the best-characterized interferon effector pathways. as described earlier, pkr is likely antagonized by the l protein, as l inhibits pkr-induced stress granule assembly. moreover, a recent study reported increased sumo3 conjugation figure 5 | inhibition of rnase l activation by l * tmev l * binds rnase l ankyrin repeats 1 and 2 (numbered) through a direct protein-protein interaction, thereby preventing association of 2-5a with rnase l monomers and the consequent dimerization and activation of the enzyme. of pkr in emcv-infected cells, which dampens pkr activation and promotes caspase-dependent pkr degradation (maarifi et al., 2018) . oligoadenylate synthetases are enzymes responsible for rnase l activation. cardioviruses have evolved two strategies to interfere with the oas-rnase l pathway. in an infected cell, oas are activated by dsrna and produce 2 -5 oligoadenylates (2-5a). binding of two 2-5a molecules to the ankyrin domain of the latent endoribonuclease rnase l triggers its dimerization and activation (figure 5) . active rnase l then cleaves viral and cellular ssrna leading to decreased viral replication and ultimately to apoptosis of the cell. interestingly, rna fragments generated by rnase l can amplify ifn production in a rig-i, mda5 and mavs-dependent way (malathi et al., 2007) . rnase l targets both viral and cellular mrna but is also predicted to cleave the genome of ssrna viruses, as reported for emcv (li et al., 1998) . in addition to 2 -phosphodiesterases and phosphatases that tightly regulate the system by degrading 2-5a within minutes of their synthesis, rnase l activity can be negatively regulated by the rnase l inhibitor (rli/abce) (bisbal et al., 1995) . rli/abce expression is induced by emcv and correlates with rnase l inhibition (martinand et al., 1999) . accordingly, overexpression of rli/abce inhibited the action of ifn against emcv (bisbal et al., 1995) . rnase l inhibition by emcv-induced rli is, however, partial as rnase l antiviral activity against emcv was demonstrated in vitro using dominant negative rnase l and oas1 overexpression (chebath et al., 1987; zhou et al., 1998) and in vivo, in rnase l-deficient mice, which presented increased emcv infection and mortality compared to wild-type mice (zhou et al., 1997) . the l * protein of tmev was found to potently inhibit rnase l through a direct protein-protein interaction (sorgeloos et al., 2013) . mechanistically, l * binds to rnase l ankyrin repeats 1 and 2, thereby preventing 2-5a binding to the enzyme and further activation steps (drappier et al., 2018; figure 5 ). in wild-type macrophages, replication of l * -mutant was significantly impaired as compared to that of the wild-type virus (sorgeloos et al., 2013) . in contrast, l * -mutant and wildtype viruses replicated to the same level in rnase l-deficient primary peritoneal macrophages. moreover, l * was shown to be active in vivo in the context of mhv chimeric viruses; l * could substitute for another viral rnase l inhibitor, namely the ns2 phosphodiesterase of mhv, in the liver of infected mice (drappier et al., 2018) . the fact that the virus devotes one of its proteins to rnase l antagonism highlights the importance of this antiviral pathway against tmev. interestingly rnase l inhibition by l * is highly species-specific; l * of a mouse tmev strain inhibits mouse rnase l but not its orthologs from other species including rat (sorgeloos et al., 2013; drappier et al., 2018) . accordingly, l * of a rat tmev strain inhibits rat but not mouse rnase l. theiler's murine encephalomyelitis virus is the only cardiovirus expressing l * , and thus the only cardiovirus known to directly inhibit rnase l. this could stem from its tropism for macrophages, which are the main tmev target during the chronic phase of infection and in which the oas-rnase l system is particularly active (zhao et al., 2012) . however, macrophages were reported to play important roles in emcv pathogenesis, including for viral replication and dissemination in piglets (papaioannou et al., 2003) and as reservoir cells for emcv persistence in rats (psalla et al., 2006) . since emcv is sensitive to rnase l activity, it is possible that another emcv protein will have developed some rnase l antagonistic activity, which might be identified using the appropriate host-pathogen context. interactions between safvs and rnase l have yet to be described, but it is also likely that these viruses have evolved ways of inhibiting this pathway. given the many ways that picornaviruses inhibit interferon production and signaling in infected cells, it is not surprising that the most important producers of ifn would be uninfected or abortively infected cells. indeed, cardioviruses efficiently block ifn in infected cells but loss of mda5 in mice causes them to be more susceptible to virus infection. these data indicate that detection of cytoplasmic dsrna by mda5 occurs in cells that are not productively infected (gitlin et al., 2006) and the recent finding that sidt2 mediates this process opens many new exciting areas of research (nguyen et al., 2017) . which molecules might be important for release of viral rna? is there a role for exosomes in this process? how might these pathways be stimulated pharmacologically? preventing release of viral rna and subsequent detection by uninfected cells may represent selective pressure favoring non-lytic release. given the exquisite genetic malleability in response to natural selection displayed by viruses, it is likely that viruses will have evolved mechanisms of inhibiting detection of viral rna by uninfected cells, perhaps by restricting dsrna release or by secreting proteins that inhibit rna transport into uninfected cells. it will be exciting to see how discoveries unfold in this area of research. several recent studies involving cardioviruses have revealed a more complicated picture regarding initial detection of replicating rna and induction of ifn. while it is clear that the helicases lgp2, dhx29, pact and trbp work in concert with mda5 for detection of dsrna, it remains to be determined how these molecules coordinate and interact and whether they function in a cell-type specific manner. it will also be important to resolve the way in which pkr functions to activate ifn signaling. future studies in this area will likely have broad relevance for innate detection of viruses. finally, the mechanisms by which l disrupts nucleocytoplasmic trafficking, stress granule formation, and interferon production clearly require further clarification. mutational analysis of l has revealed that these activities are tightly coupled, suggesting that the l interacts with protein(s) that can serve as a common node in each of these pathways. as ifn signaling and stress granules are important for a variety of viral pathogens, answers to these questions may provide broadly relevant insight into host-pathogen interactions. ef, md, and tm wrote the manuscript and approved its final version. ef was supported by a david delo research grant. md was supported by action de recherches concertée (arc). research in the tm lab was supported by the belgian fund for medical research (frsm, pdr # t.0185.14) and by eos joint program of fonds de la recherche scientifique -fnrs and fonds wetenschapelijk onderzoek -vlaanderen -fwo (eos id: 30981113). viral security proteins: counteracting host defences rig-i is cleaved during picornavirus infection encephalomyocarditis virus leader is phosphorylated by ck2 and syk as a requirement for subsequent phosphorylation of cellular nucleoporins cloning and characterization of a rnase l inhibitor. a new component of the interferon-regulated 2-5a pathway the leader protein of cardioviruses inhibits stress granule assembly the genetics of the persistent infection and demyelinating disease caused by theiler's virus lgp2 synergy with mda5 in rlrmediated rna recognition and antiviral signaling the innate immune sensor lgp2 activates antiviral signaling by regulating mda5-rna interaction and filament assembly the encephalomyocarditis virus constitutive expression of (2'-5') oligo a synthetase confers resistance to picornavirus infection trbp recruits the dicer complex to ago2 for microrna processing and gene silencing three cardiovirus leader proteins equivalently inhibit four different nucleocytoplasmic trafficking pathways cardiovirus leader proteins bind exportins: implications for virus replication and nucleocytoplasmic trafficking inhibition mov10 provides antiviral activity against rna viruses by enhancing rig-i-mavs-independent ifn induction the leader proteinase of foot-and-mouth disease virus inhibits the induction of beta interferon mrna and blocks the host innate immune response degradation of nuclear factor kappa b during foot-and-mouth disease virus infection a conserved domain in the leader proteinase of foot-and-mouth disease virus is required for proper subcellular localization and function identification of an lgp2-associated mda5 agonist in picornavirus-infected cells the leader protein of theiler's virus interferes with nucleocytoplasmic trafficking of cellular proteins leader protein of foot-and-mouth disease virus is required for cleavage of the p220 component of the cap-binding protein complex mengo encephalomyelitis; a hitherto unknown virus affecting man a novel mechanism of rnase l inhibition: theiler's virus l * protein prevents 2-5a from binding to rnase l phosphorylation of initiation factor elf-2 and the control of reticulocyte protein synthesis mda5 detects the double-stranded rna replicative form in picornavirus-infected cells characterization of ribosomal frameshifting in theiler's murine encephalomyelitis virus picornaviruses and nuclear functions: targeting a cellular compartment distinct from the replication site of a positivestrand rna virus the dsrna protein kinase pkr: virus and cell control discrimination of self and non-self ribonucleic acids essential role of mda-5 in type i ifn responses to polyriboinosinic:polyribocytidylic acid and encephalomyocarditis picornavirus transport between the cell nucleus and the cytoplasm mutational analysis of the emcv 2a protein identifies a nuclear localization signal and an eif4e binding site structure of the foot-and-mouth disease virus leader protease: a papainlike fold adapted for self-processing and eif4g recognition trbp, a regulator of cellular pkr and hiv-1 virus expression, interacts with dicer and functions in rna silencing toll-like receptor 3 is an essential component of the innate stress response in virus-induced cardiac injury the mengovirus leader protein blocks interferon-alpha/beta gene transcription and inhibits activation of interferon regulatory factor 3 differential ifn-alpha/beta production suppressing capacities of the leader proteins of mengovirus and foot-and-mouth disease virus 5'-triphosphate rna is the ligand for rig-i encephalomyocarditis virus 3c protease attenuates type i interferon production through disrupting the tank-tbk1-ikkepsilon-irf3 complex visualizing production of beta interferon by astrocytes and microglia in brain of la crosse virus-infected mice length-dependent recognition of double-stranded ribonucleic acids by retinoic acid-inducible gene-i and melanoma differentiation-associated gene 5 differential roles of mda5 and rig-i helicases in the recognition of rna viruses foot-and-mouth disease virus leader proteinase: purification of the lb form and determination of its cleavage site on eif-4 gamma the double-stranded rna-binding protein pact functions as a cellular activator of rig-i to facilitate innate antiviral response the tar-rna binding protein is required for immunoresponses triggered by cardiovirus infection mda5 localizes to stress granules, but this localization is not required for the induction of type i interferon rnase l mediates the antiviral effect of interferon through a selective reduction in viral rna during encephalomyocarditis virus infection an analysis by metabolic labelling of the encephalomyocarditis virus ribosomal frameshifting efficiency and stimulators ribosomal frameshifting into an overlapping gene in the 2b-encoding region of the cardiovirus genome pact facilitates rna-induced activation of mda5 by promoting mda5 oligomerization differential effects of sumo1 and sumo3 on pkr activation and stability small self-rna generated by rnase l amplifies antiviral innate immunity rnase l inhibitor is induced during human immunodeficiency virus type 1 infection and down regulates the 2-5a/rnase l pathway in human t cells rna sensor-induced type i ifn prevents diabetes caused by a beta cell-tropic virus in mice pact is required for mda5-mediated immunoresponses triggered by cardiovirus infection via interaction with lgp2 protein-directed ribosomal frameshifting temporally regulates gene expression sidt2 transports extracellular dsrna into the cytoplasm for innate immune recognition critical role of an antiviral stress granule containing rig-i and pkr in viral detection and innate immunity accessory factors of cytoplasmic viral rna sensors required for antiviral innate immune response pathogenesis of encephalomyocarditis virus (emcv) infection in piglets during the viraemia phase: a histopathological, immunohistochemical and virological study tar rna-binding protein is an inhibitor of the interferon-induced protein kinase pkr pact, a protein activator of the interferoninduced protein kinase, pkr autophagy and mammalian viruses: roles in immune response, viral replication, and beyond translation of encephalomyocarditis virus rna in vitro yields an active proteolytic processing enzyme binding interactions between the encephalomyocarditis virus leader and protein 2a abortively infected astrocytes appear to represent the main source of interferon beta in the virus-infected brain pkr transduces mda5-dependent signals for type i ifn induction rig-i-mediated antiviral responses to single-stranded rna bearing 5'-phosphates activation of mda5 requires higher-order rna structures generated during virus infection who regulates whom? an overview of rna granules and viral infections a picornavirus protein interacts with ran-gtpase and disrupts nucleocytoplasmic transport nucleoporin phosphorylation triggered by the encephalomyocarditis virus leader protein is mediated by mitogen-activated protein kinases pathogenesis of experimental encephalomyocarditis: a histopathological, immunohistochemical and virological study in rats inhibition of mrna export and dimerization of interferon regulatory factor 3 by theiler's virus leader protein the rna helicase lgp2 inhibits tlrindependent sensing of viral replication by retinoic acid-inducible gene-i lgp2 is a positive regulator of rig-i-and mda5-mediated antiviral responses protein kinase r contributes to immunity against specific viruses by regulating interferon mrna integrity evasion of antiviral innate immunity by theiler's virus l * protein through direct inhibition of rnase l helicase proteins dhx29 and rig-i cosense cytosolic nucleic acids in the human airway system the specific and essential role of mavs in antiviral innate immune responses riok3-mediated phosphorylation of mda5 interferes with its assembly and attenuates the innate immune response influence of the theiler's virus l * protein on macrophage infection, viral persistence, and neurovirulence the leader protein of theiler's virus inhibits immediate-early alpha/beta interferon production loss of dexd/h box rna helicase lgp2 manifests disparate antiviral responses mda5 and mavs mediate type i interferon responses to coxsackie b virus the nuclear pore complex and nuclear transport regulation of stress granules in virus systems dephosphorylation of the rna sensors rig-i and mda5 by the phosphatase pp1 is essential for innate immune signaling role of adaptor trif in the myd88-independent toll-like receptor signaling pathway picornavirus 2a protease regulates stress granule formation to facilitate viral translation deficient signaling in mice devoid of double-stranded rna-dependent protein kinase viral subversion of nucleocytoplasmic trafficking shared and unique functions of the dexd/h-box helicases rig-i, mda5, and lgp2 in antiviral innate immunity ictv virus taxonomy profile: picornaviridae ips-1 plays an essential role in dsrna-induced stress granule formation by interacting with pkr and promoting its activation antagonism of the interferon-induced oas-rnase l pathway by murine coronavirus ns2 protein is required for virus replication and liver pathology interferon action and apoptosis are defective in mice devoid of 2' ,5'-oligoadenylate-dependent rnase l impact of rnase l overexpression on viral and cellular growth and death dhx29 functions as an rna co-sensor for mda5-mediated emcv-specific antiviral immunity saffold virus, a human theiler'slike cardiovirus, is ubiquitous and causes infection early in life mengovirus leader is involved in the inhibition of host cell protein synthesis the mengovirus leader protein suppresses alpha/beta interferon production by inhibition of the iron/ferritin-mediated activation of nf-kappa b key: cord-003239-nph2ezii authors: zhu, zixiang; du, xiaoli; li, pengfei; zhang, xiangle; yang, fan; cao, weijun; tian, hong; zhang, keshan; liu, xiangtao; zheng, haixue title: early growth response gene-1 suppresses foot-and-mouth disease virus replication by enhancing type i interferon pathway signal transduction date: 2018-09-27 journal: front microbiol doi: 10.3389/fmicb.2018.02326 sha: doc_id: 3239 cord_uid: nph2ezii early growth response gene-1 (egr1) is a multifunctional transcription factor that is implicated in viral infection. in this study, we observed that foot-and-mouth disease virus (fmdv) infection significantly triggered egr1 expression. overexpression of egr1 suppressed fmdv replication in porcine cells, and knockdown of egr1 considerably promoted fmdv replication. a previously reported fmdv mutant virus (with two amino acids mutations in sap domain) that displays a strong type i interferon (ifn) induction activity was used in this study. we found that sap mutant fmdv infection induced a higher expression of egr1 than wildtype fmdv infection, and also triggered higher ifn-β and ifn-stimulated genes (isgs) expression than wildtype fmdv infection. this implied a link between egr1 and type i ifn signaling. further study showed that overexpression of egr1 resulted in sendai virus (sev)-induced ifn-stimulated response element (isre) and nf-κb promoter activation. in addition, the sev-induced isgs expression was impaired in egr1 knockdown cells. egr1 upregulation promoted type i ifn signaling activation and suppressed fmdv and seneca valley virus replication. suppression of the transcriptional activity of egr1 did not affect its antiviral effect against fmdv. this study reveals a new mechanism evolved by egr1 to enhance type i ifn signaling and suppress fmdv replication. foot-and-mouth disease virus (fmdv) is a non-enveloped virus with positive-sense and singlestranded rna genome. the viral genome is approximately 8.5 kb nucleotides in length, including a single large open reading frame that encodes a polyprotein. the polyprotein is subsequently processed by viral proteases during protein synthesis, generating several intermediates and 12 mature proteins (sobrino and domingo, 2001; grubman and baxt, 2004) . during co-evolution with the hosts, these viral proteins have acquired many functions to counteract host antiviral responses, cause immunosuppression, and promote viral replication and infection (mason et al., 2003; rodriguez pulido and saiz, 2017) . therefore, fmdv causes an acute vesicular disease of infected animals, which is called foot-and-mouth disease (fmd). fmd is a highly contagious disease that can lead to significant economic losses to the local livestock industry (rweyemamu et al., 2008a; paton and taylor, 2011; zai-xin, 2015; bouguedour and ripani, 2016) . the understanding of host-fmdv interaction as well as the involved mechanism contributes to the planning of new strategies for fmd prevention (domingo et al., 2005; rweyemamu et al., 2008b; rodriguez pulido and saiz, 2017) . accordingly, many researches on host responses in fmdv-infected cells have to be investigated. early growth response gene-1 (egr1), also designated zif268, is a host transcriptional regulator that expresses rapidly after a number of stimuli like oxygen deprivation, growth factors, cytokines, shear stress and injury (brand et al., 1992; khachigian et al., 1997; nishi et al., 2002; jo et al., 2011; li et al., 2013) . egr1 is involved in diverse biologic functions and a broad variety of host signal transduction cascades that mediates cell growth, survival, differentiation, apoptosis and proliferation (pagel and deindl, 2011; papanikolaou et al., 2014) . different pathways have been identified that participate in egr1 induction and then regulates several biological behaviors. such as, the ras homologue gene family (rho) genes are involved in cell cycle progression, and the rho/rho-kinase pathway has been shown to regulate egr1 expression (barrientos et al., 2007; pagel and deindl, 2011) . as a zinc-finger dna-binding protein, egr1 also regulates expression of diverse gene families by binding to promoter sequences of target genes (papanikolaou et al., 2014) . therefore, egr1 is involved in activation of signal transduction of many pathways. several studies indicate that egr1 is linked to viral infection and immune response. egr1 modulates pro-apoptotic pathway and promotes venezuelan equine encephalitis virus (veev) replication (baer et al., 2016) . knockdown of egr1 in rhabdomyosarcoma cells decreases enterovirus 71 (ev71) replication (song et al., 2015) . it seems that egr1 might play a positive role in these viruses replication. however, egr1 also appears critical for the initiation of immune response in b cells and t cells. egr1 plays roles in regulation of the expression of sever cytokines including interleukin-2, cd44, icam-1 and tumor necrosis factor genes (skerka et al., 1995; mcmahon and monroe, 1996; shin et al., 2009; cubero and nieto, 2012) . an sap domain [scaffold-attachment factor (saf)-a/b, apoptotic chromatin-condensation inducer in the nucleus (acinus) and pias (protein inhibitor of activated signal transducer and activator of transcription) domain] previously was identified within the fmdv l pro by de los santos et al. (2009) . mutation of l pro sap domain promotes type i ifn signaling activation and decreases virus growth. in addition, animals inoculated with the fmdv sap mutant display strong neutralizing antibody response and t cell response comparing with infection with wildtype fmdv (de los santos et al., 2009; diaz-san segundo et al., 2012) . in this study, a robust egr1 upregulation was observed in both wildtype and sap mutant fmdv-infected cells comparing with the mock-infected cells by viewing the protein abundance of egr1. therefore, we investigated the correlation between fmdv infection and egr1, and determined the antiviral role of egr1 against fmdv. sap mutant fmdv infection induced a higher expression of egr1 than wildtype fmdv infection. sap mutant fmdv infection also triggered higher ifn-β and ifn-stimulated genes (isgs) expression than wildtype fmdv infection. we also found that overexpression of egr1 enhanced sendai virus (sev)-induced interferon (ifn)-stimulated response element (isre) activation. sev-induced isgs expression was impaired in egr1 knockdown cells, which may serve as a link between upregulation of egr1 and type i ifn signaling. further study showed that egr1 enhanced tbk1 phosphorylation during fmdv infection. it indicated that egr1 upregulation promoted type i ifn signaling activation by enhancing tbk1 phosphorylation and resulted in decreased fmdv replication. this study reveals a link between egr1 and innate immune response during fmdv infection. porcine kidney pk-15 cells, human embryonic kidney 293t cells (hek293t) cells described previously were maintained in dulbecco's modified eagle's medium supplemented with 10% heat-inactivated fetal bovine serum, 100 u/ml penicillin, and 100 µg/ml streptomycin sulfate. all the cells were cultured at 37 • c under 5% co2. sendai virus (sev), a model rna virus widely used to activate type i ifn signaling in cells, was kindly provide by hongbing shu's laboratory (wuhan university, china) (zhou et al., 2014; . fmdv strain o/by/cha/2010 (genbank number: jn998085) described previously was used for virus infection (zheng et al., 2012) . commercial antibodies used in this study include an anti-egr1 mouse monoclonal antibody (abcam, cambridge, ma, united states), anti-tbk1 rabbit antibody from cell signaling technology (cst) inc. (beverly, ma, united states), anti-phospho-tbk1 rabbit antibody (cst), anti-c-myc mouse antibody (santa cruz biotechnology, santa cruz, ca, united states) and anti-β-actin mouse antibody (santa cruz biotechnology). anti-ifn-β and anti-ifn-α antibodies (5000 nu/ml, pbl biomedical laboratories) and igg isotype antibodies were used in the type i ifn-blocking experiments as previously described (trottier et al., 2009) . anti-fmdv vp1 protein polyclonal antibody was previously produced in our laboratory . transfection reagents include opti-mem medium and the lipofectamine 2000 that were purchased from invitrogen. poly (i:c) was purchased from invivogen. ifn-β was purchased from pbl biomedical laboratories. the full-length porcine egr1 cdna fragment was cloned into a pcdna tm 3.1/myc-his(-)a vector (invitrogen) to construct a myc-tagged egr1 eukaryotic expressing plasmid (myc-egr1, including a c-terminal myc tag). the constructed plasmid was analyzed and verified by dna sequencing. a series of plasmids expressing ha-tagged type i ifn pathway-related proteins [including mda5, rig-i(card), visa, tbk1, irf3 and irf7], and the ifn-β promoter luciferase reporter plasmids and control plasmid renilla luciferase prl-tk were kindly provided by hongbing shu's laboratory (zhou et al., 2014; . the plasmids were transfected into cells using opti-mem medium and the lipofectamine 2000 (invitrogen) reagent according to the manufacture's protocol. trizol reagent (invitrogen) was used to extract cellular or viral rna following the instruction of the protocol. the firststrand cdna was synthesized by reverse transcription reaction with the extracted rnas as templates. reverse transcription was performed with m-mlv reverse transcriptase (invitrogen) and random hexamer primers (takara) according to the manufacturer's recommendations. the quantification of the cdna was performed by qpcr. the relative amounts of the synthesized cdna was determined as an indicator of the target transcripts. qpcr was carried out using sybr premix ex taq (takara) on a quantstudio 5 real-time pcr instrument (applied biosystems) according to the manufacturer's instructions. the glyceraldehyde-3-phosphate dehydrogenase (gapdh) gene was used for normalization in qpcr analysis. relative transcript levels were calculated using 2 − ct method as described previously . all the primers used in this study were listed in table 1 . for western blotting, the cells were collected at the indicated time points and. the lysed cell extracts were resolved by 10% sds-page and transferred onto a nitrocellulose membrane (pall). the nitrocellulose membrane was then blocked with 10% skim milk powder in tbst (20 mm tris, 55 mm nacl, 0.1% tween 20) overnight at 4 • c. the membrane was incubated with primary and secondary antibodies as described previously (zhu et al., 2013) . the membrane was washed 3 × 5 min, before being protein abundance analysis. the antibody-antigen complexes were visualized using enhanced chemiluminescence detection reagents (thermo). small interfering rna (sirna) was used to knockdown egr1 protein expression. sirna fragments were chemically synthesized by genepharma company (china). the sequences of the sirnas used in this study include: 5 -ccaugga caacuacccuaatt-3 (egr sirna-353), 5 -gccuaguga gcaugaccaatt-3 (egr sirna-749), and 5 -gcuguca ccaacuccuucatt-3 (egr sirna-1819). a non-targeting sirna (nc sirna) was used as a negative control. sirna fragments were transfected into cells using lipofectamine 2000 as described previously . forty-eight hours after sirna transfection, cells were used for further experiments. to examine the effect of sirna on egr1 expression, egr1 mrna and protein abundance were measured by qpcr and western blotting respectively. hek293t cells seeded on 24-well plates were co-transfected with 100 ng luciferase reporter plasmid with 10 ng internal control renilla luciferase reporter plasmid (to normalize for transfection efficiency) prl-tk (promega), together with the indicated plasmids and/or empty vector controls using lipofectamine 2000 according to the manufacture's instruction. to make the cells receive the same amounts of total plasmids, the empty vector plasmids were used in all transfection experiments. as for sevmediated type i ifn signaling pathway activation, the cells were mock-infected or infected with sev (100hau/ml) for 16 h; and the dual luciferase assays were then performed according to the promega dual-luciferase reporter assay system protocol. the relative luciferase activity was expressed as arbitrary units by normalizing firefly to renilla luciferase activity. as for type i ifn pathway adaptor molecules-induced ifn-element activation assay, the hek293t cells were co-transfected with the reporter plasmids with the indicated plasmid or vector plasmid for 24 h; and the luciferase activities were measured. all experiments were performed at least in triplicate. the measured values are represented as mean ± sd from three independent experiments. the statistical significance analyses were performed using the student's t-test. data considered significant when * p < 0.05, and highly significant when * * p < 0.01. pk-15 cells were infected by equal amounts of wildtype or sap mutant fmdv for 12 h as previously described (zhu et al., 2015) . the expression levels of egr1 and viral vp1 protein were detected by western blotting. we observed that egr1 protein level is significantly upregulated both in wildtype and sap mutant fmdv-infected cells at 12 h postinfection (hpi) ( figure 1a) . therefore, the correlation between fmdv infection and egr1 was further investigated. the dynamics of egr1 in fmdv-infected cells were determined. transcripts of egr1 were considerably upregulated after fmdv infection and reached to the highest level at 8 hpi. no significant changes were observed in mock-infected cells ( figure 1b) . egr1 protein expression was also gradually upregulated as the infection progressed ( figure 1c ). this indicates that fmdv infection triggers upregulation of egr1. to investigate whether egr1 is an ifn inducible gene, hek293t and pk-15 cells were incubated with ifn-β to induce the expression of ifn inducible genes. the expression of two ifn inducible genes isg15 and isg54 was highly induced by incubation of ifn-β. however, the expression of egr1 was not changed by treatment of ifn-β ( figure 1d ). this indicated that egr1 expression was not induced by ifnβ treatment; however, fmdv infection could induce egr1 expression. to investigate the potential role of egr1 during fmdv infection, we evaluated the viral replication level in egr1 overexpressed cells. pk-15 cells were transfected with different doses of egr1 expressing plasmids, the cells were incubated with equal amounts of fmdv (0.5 moi) at 24 h post-transfection (hpt). the viral protein and viral rna expression level was measured at 12 hpi. overexpression of egr1 significantly suppressed both the viral protein expression and viral rna replication. the viral titers in both vector and egr1 plasmids (2 µg) transfected cells were measured and compared, which showed that fmdv yields were also decreased by overexpression of egr1 (figure 2a) . to further confirm the antiviral role of egr1 during fmdv infection, the sirnas that target egr1 were designed and evaluated. pk-15 cells were transfected with the nc sirna or egr1 sirna for 48 h, the interference efficacy of the sirnas was determined by qpcr analysis. the egr1 sirna-1819 showed the highest efficacy and was used for egr1 knockdown assay ( figure 2b) . pk-15 cells were transfected with egr1 sirna-1819, the cells were infected with fmdv at 48 hpt and incubated for another 12 or 16 h. the expression of egr1 and fmdv vp1 protein was detected using western blotting. knockdown of egr1 considerably increased vp1 protein expression during fmdv infection (figure 2c) . the relative fold-change in abundance of fmdv vp1 protein in fmdv-infected nc sirna or egr1 sirna cells was determined by densitometric analysis and normalized to β-actin, which confirmed that knockdown of egr1 enhanced fmdv vp1 protein expression (figure 2c , right panel). viral rna detection also suggested that knockdown of egr1 promoted viral replication ( figure 2d) . the viral titers were subsequently measured at 16 hpi, which showed that knockdown of egr1 significantly promoted fmdv propagation ( figure 2d , right panel). these results suggest the antiviral role of egr1 against fmdv. both wildtype and sap mutant fmdv infection resulted in egr1 upregulation, however, sap mutant fmdv resulted in a higher upregulation of egr1 ( figure 1a) . previous study indicates sap mutant fmdv infection induces higher expression of ifn-β and isgs than wildtype fmdv infection (de los santos et al., 2009) . we also investigated the expression state of ifn-β and isgs (isg15 and mx1) in the cells infected by wildtype or sap mutant fmdv. at 12 hpi, there was ∼3-fold difference in ifn-β transcripts for sap mutant fmdv-infected cells relative to wildtype fmdv-infected cells ( figure 3a) . a similar pattern was observed for isg15 and mx1, varying from 2-to 4-fold higher for sap mutant fmdv compared to wildtype fmdv ( figure 3a) . these results were similar to the previous results reported by de los santos et al. (2009) . this showed that both egr1 expression and type i ifn signaling were enhanced in sap mutant fmdvinfected cells. this implied a link between egr1 and type i ifn pathway. to identify the role of egr1 on type i ifn signaling, egr1 is overexpressed in hek293t cells, and the sev that is routinely used to induce type i ifns in cell culture was used to activate type i ifn signaling. overexpression of egr1 significantly promoted sev-induced type i ifn signaling, showing a dosedependent manner (figure 3b) . the expression of ifn-β, isg15 and mx1 in egr1 overexpressed cells were subsequently evaluated. the results showed that overexpression of egr1 considerably promoted sev-induced ifn-β, isg15 and mx1 expression ( figure 3c) . the effect of egr1 on sev-induced nf-κb activation was also evaluated by dual luciferase reporter assay, which also showed that egr1 positively enhanced nf-κb-mediated transcriptional activity ( figure 3d) . the role of egr1 on poly (i:c)-induced type i ifn signaling was further evaluated. overexpression of egr1 significantly promoted poly (i:c)-induced type i ifn signaling ( figure 3e) . the expression of poly (i:c)-induced isgs was also measured. the results figure 1 | state of egr1 in fmdv-infected cells. (a) pk-15 cells were incubated with equal amounts of sap mutant fmdv or wildtype fmdv for 12 h, the abundance of egr1 and viral vp1 protein was detected. (b) pk-15 cells were infected with wildtype fmdv or mock-infected for 0, 2, 4, 8, 12, or 16 h. the transcripts of egr1 and viral rna were detected by qpcr. (c) pk-15 cells were infected with wildtype fmdv for 0, 2, 4, 8, 12, or 16 h. the expression levels of egr1 and vp1 protein were detected by western blotting. (d) hek293t or pk-15 cells were mock-treated or incubated with ifn-β at a concentration of 10 ng/ml for 12 h. the expression levels of isg15, isg54 and egr1 was measured by qpcr. showed that overexpression of egr1 considerably promoted poly (i:c)-induced isg15 and isg54 expression ( figure 3f ). sevinduced isgs expression levels in egr1 knockdown cells were also analyzed. the sirna interference efficacy was also verified in hek293t cells ( figure 3g) . egr1 was knocked down by transfection of sirna, and the cells were infected by sev at 48 hpt and incubated for 16 h. the expression of ifn-β, isg15 and mx1 were measured. the transcript levels of ifn-β, isg15 and mx1 remarkably decreased in egr1 knockdown cells comparing with that in nc sirna cell ( figure 3h) . these results suggested a positive regulatory role of egr1 on type i ifn signaling. the type i ifn blocking antibody experiments were also performed. anti-ifn-β and anti-ifn-α antibodies (5000 nu/ml) was used to block type i ifn signaling in pk-15 cells, the egr1overexpressed cells were treated with ifn antibodies for 1 h and then infected with fmdv. the viral yields were measured at 12 hpi. type i ifn blocking antibodies obviously abrogated the inhibitory effects of egr1 on fmdv propagation ( figure 3i) . these results suggested that egr1 suppressed fmdv replication by enhancing type i ifn signaling. we also evaluated the antiviral role of egr1 against another picornavirus seneca valley virus (svv) which showed a close relationship with fmdv, and we found that upregulation of egr1 also enhanced isgs expression (isg15 and isg54) and suppressed svv replication ( figure 3j) (b) pk-15 cells were transfected with nc (negative control) or sirna (egr1 sirna-353, egr1 sirna-749 or egr1 sirna-1819) for 48 h. the egr1 mrna levels were measured by qpcr. (c) schematic diagram of the strategy in egr1 knockdown experiment and investigation of the viral replication state in egr1 knockdown cells. pk-15 cells were transfected with nc sirna or egr1 sirna-1819 for 48 h. the cells were infected with fmdv for 0, 12, or 16 h. viral protein abundance was measured by western blotting. relative fold-change in abundance of vp1 protein was determined by densitometric analysis using quantity one software (bio-rad) and normalized to β-actin. (d) viral rna levels in fmdv-infected nc sirna or egr1 sirna-1819 cells at 0, 12, and 16 hpi were measured by qpcr. viral yields in fmdv-infected nc sirna or egr1 sirna-1819 cells at 16 hpi were measured by tcid 50 assay. * p < 0.05 was considered as statistically significant and * * p < 0.01 was considered as highly significant. i ifn signaling pathway. to screen the potential proteins that were targeted by egr1, hek293t cells were co-transfected with the myc-vector or myc-tagged egr1 plasmids and the indicated plasmids expressing rig-i, rig-i(card) (the card domain of rig-i), mda5(helicase) (the helicase domain of mda5), visa, tbk1, irf3 and irf7, together with isre luciferase reporter plasmid and the internal control plasmid prl-tk. luciferase activity was measured at 24 h after transfection. overexpression of adaptor proteins rig-i, rig-i(card), mda5), visa, tbk1, irf3 or irf7 all activated the isre luciferase reporter system, and overexpression of mda5(helicase) did not activate the isre luciferase reporter system (figure 4) . tbk1 or its upstream proteins (rig-i, mda5 and visa) mediated type i ifn signaling was significantly enhanced by overexpression of egr1. however, overexpression of egr1 did not promote irf3 and irf7 mediated type i ifn signaling (figure 4) . irf3 and irf7 are the downstream proteins of tbk1. therefore, we speculated that tbk1 or its upstream molecules (rig-i, mda5 and visa) were the target/targets of egr1 to enhance type i ifn signal transduction. to investigate whether egr1 interacted with the adaptors of type i ifn pathway, the coimmunoprecipitation assay was performed by co-transfection of the myc-egr1 plasmids and the plasmids expressing various ha-tagged adaptors of type i ifn pathway. the transfectants were immunoprecipitated with anti-ha antibodies and subjected to western blotting analysis. no interaction was observed between egr1 and the adaptors (figure 5a) . egr1 enhanced the activation of isre luciferase reporter stimulated by tbk1 and its upstream molecules. tbk1 might be a key adaptor to enhance type i ifn signaling. the influence of egr1 on tbk1 expression and tbk1 phosphorylation levels were evaluated in fmdv-infected cells. pk-15 cells were transfected with 2 µg of myc-egr1 or its empty vector plasmids. the cells were infected with equal amounts of fmdv at 24 hpt and collected at 0, 6, 12, or 18 hpi. overexpression of egr1 had no influence on tbk1 expression during fmdv infection. however, it significantly promoted tbk1 phosphorylation levels after fmdv infection comparing with that in the empty vector transfected cells (figure 5b) . the vp1 was used as an indicator of viral replication. overexpression of egr1 also resulted in decreased vp1 abundance ( figure 5b ). this confirmed that upregulation of egr1 enhanced type i ifn signaling and suppressed fmdv replication. as a transcription factor, the transcriptional activity is significantly involved in the regulatory function of egr1. the transcripts of ifn-β, isg15 and mx1 were detected by qpcr. (d) hek293t cells were transfected with vector or myc-egr1 plasmids together with nf-κb luciferase reporter plasmid and prl-tk. the transfected cells were infected with sev, and the luciferase activity was measured by dual luciferase assay. (e) hek293t cells were transfected with vector plasmids or myc-egr1 plasmids and solvent control or poly (i:c) together with isre luciferase reporter plasmid and prl-tk. the luciferase activity was measured by dual luciferase assay. (f) hek293t cells were co-transfected with vector plasmids or myc-egr1 plasmids and solvent control or poly (i:c), the expression of isg15 and isg54 expression was measured by qpcr. (g) hek293t cells were transfected with nc or egr1 sirna-1819 for 48 h. the egr1 transcripts were measured by qpcr. (h) hek293t cells were transfected with nc sirna or egr1 sirna-1819 for 48 h. the cells were then mock-infected or infected with sev for 16 h. the transcripts of ifn-β, isg15 and mx1 were detected by qpcr. (i) pk-15 cells were transfected with equal amounts of vector or egr1 expressing plasmids for 24 h, the egr1-transfected cell were mock-treated or treated with ifn antibodies for 1 h and then infected with fmdv. the viral yields were measured by tcid 50 assay at 12 hpi. (j) pk-15 cells were transfected with vector plasmids or myc-egr1 plasmids for 24 h. the transfected cells were infected with svv for 12 h. the viral rna, isg15 and isg54 expression levels were detected by qpcr. * p < 0.05 was considered as statistically significant and * * p < 0.01 was considered as highly significant. to investigate whether the transcriptional activity is related to the antiviral function of egr1, znegr1, a previous reported dominant-negative mutant of egr1 that lacks a transcriptional function, was used as an inhibitor of the transcriptional activity of egr1 (levkovitz and baraban, 2001) . the myc-tagged znegr1 expressing plasmid was constructed ( figure 6a) . pk-15 cells were transfected with vector, myc-egr1 plasmids or cotransfected with myc-egr1 and myc-znegr1 figure 4 | the target of egr1 in type i ifn pathway activation. hek293t cells were co-transfected with myc-egr1 or empty vector plasmids and the constructs expressing rig-i, rig-i(card), mda5, visa, tbk1, irf3 or irf7, together with isre luciferase reporter plasmid and the internal control plasmid prl-tk. dual luciferase activity was determined at 24 hpt. * * p < 0.01 was considered as highly significant. plasmids and subjected to fmdv infection. overexpression of egr1 suppressed fmdv replication, and egr1-mediated antiviral effect was not blocked by cotransfection with znegr1 ( figure 6b) . we further evaluated the localization of egr1 in mock-or fmdv-infected cells, and we found fmdv infection did not change the localization of egr1 compared with that in the mock-infected cells ( figure 6c) . these data suggest that the transcriptional activity is not involved in the antiviral function of egr1. egr1, as a multifunctional transcription factor, plays regulatory roles in a variety of cellular responses. in addition, egr1 shows an anti-tumor function. overexpression of egr1 decreases tumorigenesis in nude mice and various of human tumor cell lines (huang et al., 1994 (huang et al., , 1995 . induction of tgfβ1 and p53 may lead to the tumor suppressor property of egr1 (baron et al., 2006) . p53, as a tumor suppressor, has also been implicated in other functions that play important roles in disease and health (fuhrman et al., 2009 ). p53-dependent antiviral defense has been widely reported (takaoka et al., 2003; shin-ya et al., 2005; muñoz-fontela et al., 2008) . such as, p53 serves as an antiviral protein during influenza a virus infection by enhancing host innate and adaptive immune responses (muñoz-fontela et al., 2011) . egr1 directly induces the transcription of p53 (liu et al., 2004) , whether egr1 is also involved in host antiviral responses remains unknown. in this study, we determined that egr1 revealed an antiviral function against fmdv, which indicated that egr1 is implicated in host antiviral response. egr1 can be upregulated upon viral infection by epstein-barr virus, mouse hepatitis virus (mhv), veev, ev71, rabies viruses and japanese encephalitis virus infections (saha and rangarajan, 2003; cai et al., 2006; kim et al., 2013; song et al., 2015; baer et al., 2016) . however, egr1 expression is related to viral pathogenesis during veev, mhv and ev71 replication. all these studies were performed using mouse or human cells, and most of these viruses can cause central nervous system (cns) diseases. in this study we investigated the function of porcine egr1 and showed the antiviral role of porcine egr1 against fmdv. fmdv infection does not cause any cns disease. whether the difference of species or tissue tropism resulted in the different role of egr1 in different virus infections remain unknown. however, egr1 has been suggested to participate in ifn-γ-stat1 pathway in t cells (shin et al., 2009) . t-bet is a th1-specific transcription factor that is directly involved in t cells differentiation (djuretic et al., 2007) . egr1 regulates t-bet expression by binding to the promoter of t-bet and induces t-bet transcription (shin et al., 2009) . t-bet plays a vital role in innate immunity, and lacking of t-bet expression increases host susceptibility to inflammatory disease (garrett et al., 2007) . this implies a regulatory role of egr1 in innate immunity. besides, overexpression of egr1 downregulates nfκb inhibitor (kim et al., 2013) , which also implies a potential role of egr1 in innate immunity. in this study, we determined that egr1 is implicated in innate immunity during fmdv infection. a higher egr1 expression was observed in sap mutant fmdv-infected cells comparing with that in the wildtype fmdv-infected cells. it has been determined that sap mutant fmdv infection resulted in stronger type i ifn signaling than wildtype fmdv infection (de los santos et al., 2009) . we found that sap mutant fmdv infection triggered higher expression of ifn-β and isgs than wildtype fmdv infection. this result was similar as the result reported by de los santos et al. (2009) previously. whether the higher expression of egr1 correlated with the higher expression of ifn-β and isgs was therefore investigated. overexpression of egr1 significantly activated type i ifn signaling and ifn-β and isgs expression. knockdown of egr1 considerably impaired sev-induced ifn-β and isgs expression. type i ifn blocking antibodies obviously abrogated the inhibitory effects of egr1 on fmdv propagation. this suggested egr1 is implicated in type i ifn pathway activation. a link between egr1 and type i ifn pathway was reported for the first time. further investigation of egr1-mediated enhancive effect showed that egr1 promoted activation of the type i ifn signaling during fmdv infection. overexpression of egr1 upregulated tbk1 phosphorylation during fmdv infection. tbk1 phosphorylation enhanced type i ifn signaling and strengthened antiviral activity which subsequently suppressed fmdv replication. egr1 is a transcription factor; however, it does not induce tbk1 expression. the interaction between egr1 and various adaptors of type i ifn signaling pathway was not observed by performing coimmunoprecipitation assay. the role of the transcriptional activity of egr1 for its antiviral function against fmdv was also evaluated. suppression of the transcriptional activity of egr1 did not affect its antiviral effect. egr1 might enhance type i ifn signaling independent of its transcriptional activity. how does egr1 promote tbk1 phosphorylation is not clear. several phosphatases have been identified as regulator of phosphorylation of tbk1 (gabhann et al., 2010; zhao, 2013) . the regulation of egr1 on these phosphatases should be studied in future, and the detailed mechanism of egr1 to promote tbk1 phosphorylation should be further investigated. in addition, the effect of egr1 on the adaption of other upstream molecules of tbk1 should also be exploited. in summary, we present the first investigation of egr1 in regulation of type i ifn signaling during fmdv infection. we determined that egr1 showed an antiviral function against fmdv. egr1 promoted activation of the type i ifn signaling during fmdv infection and resulted in the decreased replication of fmdv. egr1 suppressed fmdv replication independent of its transcriptional activity. these findings identify an important role of egr1 in enhancement of type i ifn signaling during fmdv infection. however, the exact mechanism for egr1 to promote type i ifn signaling should be investigated in future to gain deeper understanding of egr1-mediated functions. venezuelan equine encephalitis virus induces apoptosis through the unfolded protein response activation of egr1 the transcription factor egr1 is a direct regulator of multiple tumor suppressors including tgfβ1, pten, p53 and fibronectin: egr1 is a potential target of gene therapy for prostate cancer two novel members of the ablim protein family, ablim-2 and -3, associate with stars and directly bind f-actin review of the foot and mouth disease situation in north africa and the risk of introducing the disease into proto-oncogene expression in porcine myocardium subjected to ischemia and reperfusion induction of transcription factor egr-1 gene expression in astrocytoma cells by murine coronavirus infection arachidonic acid stimulates tnfα production in kupffer cells via a reactive oxygen species-perk1/2-egr1-dependent mechanism a conserved domain in the leader proteinase of footand-mouth disease virus is required for proper subcellular localization and function inoculation of swine with foot-and-mouth disease sap-mutant virus induces early protection against disease transcription factors t-bet and runx3 cooperate to activate ifng and silence il4 in t helper type 1 cells foot-and-mouth disease virus evolution: exploring pathways towards virus extinction nucleolar proteins suppress caenorhabditis elegans innate immunity by inhibiting p53/cep-1 absence of ship-1 results in constitutive phosphorylation of tank-binding kinase 1 and enhanced tlr3-dependent ifn-beta production communicable ulcerative colitis induced by t-bet deficiency in the innate immune system foot-and-mouth disease suppression of v-sis-dependent transformation by the transcription factor, egr-1 egr-1 negatively regulates human tumor cell growth via the dna-binding domain a compound isolated from rumex japonicus induces early growth response gene-i expression egr-1 is activated in endothelial cells exposed to fluid shear stress and interacts with a novel shear-stress-response element in the pdgf a-chain promoter epsteinâ-barr virus latent membrane protein 1 increases genomic instability through egr-1-mediated upregulation of activation-induced cytidine deaminase in b-cell lymphoma a dominant negative inhibitor of the egr family of transcription regulatory factors suppresses cerebellar granule cell apoptosis by blocking c-jun activation the vp3 structural protein of foot-and-mouth disease virus inhibits the ifn-β signaling pathway esterase d enhances type i interferon signal transduction to suppress foot-and-mouth disease virus replication crucial role for early growth response-1 in the transcriptional regulation of mir-20b in breast cancer physical interaction between p53 and primary response gene egr-1 molecular basis of pathogenesis of fmdv the role of early growth response gene 1 (egr-1) in regulation of the immune response transcriptional role of p53 in interferonmediated antiviral immunity p53 serves as a host antiviral factor that enhances innate and adaptive immune responses to influenza a virus early growth response-1 gene mediates up-regulation of epidermal growth factor receptor expression during hypoxia early growth response 1-a transcription factor in the crossfire of signal transduction cascades a systems approach identifies co-signaling molecules of early growth response 1 transcription factor in immobilization stress developing vaccines against foot-and-mouth disease and some other exotic viral diseases of livestock molecular mechanisms of foot-andmouth disease virus targeting the host antiviral response epidemiological patterns of foot-and-mouth disease worldwide planning for the progressive control of foot-and-mouth disease worldwide common host genes are activated in mouse brain by japanese encephalitis and rabies viruses t-bet expression is regulated by egr1-mediated signaling in activated t cells intracellular interferon triggers jak/stat signaling cascade and induces p53-dependent antiviral protection a regulatory element in the human interleukin 2 gene promoter is a binding site for the zinc finger proteins sp1 and egr-1 foot-and-mouth disease in europe. fmd is economically the most important disease of farm animals. its re-emergence in europe is likely to have consequences that go beyond severe alterations of livestock production and trade early growth response-1 facilitates enterovirus 71 replication by direct binding to the viral genome rna integration of interferon-|α|/|β| signalling to p53 responses in tumour suppression and antiviral defence retinoids inhibit measles virus through a type i ifn-dependent bystander effect progress and prospect of the technologies to control foot-andmouth disease and its pathogen characteristics worldwide negative regulation of tbk1-mediated antiviral immunity genetic characterization of a new pandemic southeast asia topotype strain of serotype o foot-and-mouth disease virus isolated in china during the erassociated protein zdhhc1 is a positive regulator of dna virus-triggered, mita/sting-dependent innate immune signaling nonstructural protein 1 of influenza a virus interacts with human guanylate-binding protein 1 to antagonize antiviral activity foot-andmouth disease virus viroporin 2b antagonizes rig-i mediated antiviral effects by inhibition of its protein expression comparative proteomic analysis of wild-type and sap domain mutant foot-and-mouth disease virus-infected porcine cells identifies the ubiquitin-activating enzyme ube1 required for virus replication zz, xd, and hz conceived the study and wrote the manuscript. zz, xd, pl, xz, fy, and wc performed the experiments. ht, kz, and xl collected the data, analyzed the data, and revised the manuscript. the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © 2018 zhu, du, li, zhang, yang, cao, tian, zhang, liu and zheng. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord-017785-zwnkrs23 authors: baker, michelle l.; schountz, tony title: mammalia: chiroptera: immunology of bats date: 2018-03-10 journal: advances in comparative immunology doi: 10.1007/978-3-319-76768-0_23 sha: doc_id: 17785 cord_uid: zwnkrs23 bats are a large and diverse group comprising approximately 20% of all living mammalian species. they are the only mammals capable of powered flight and have many unique characteristics, including long lifespans, echolocation, and hibernation, and play key roles in insect control, pollination, and seed dispersal. the role of bats as natural reservoirs of a variety of high-profile viruses that are highly pathogenic in other susceptible species yet cause no clinical disease in bats has led to a resurgence of interest in their immune systems. equally compelling is the urgency to understand the immune mechanisms responsible for the susceptibility of bats to the fungus responsible for white syndrome, which threatens to wipe out a number of species of north american bats. in this chapter we review the current knowledge in the field of bat immunology, focusing on recent highlights and the need for further investigations in this area. bats (order chiroptera) are a diverse group of nocturnal mammals comprising approximately 20% of all mammalian taxa and consisting of more than 1300 species across 21 families (simmons 2005) . phylogenetic analysis places bats within the superorder laurasiatheria, sister to carnivores (e.g., cats, dogs), ungulates (e.g., horses, cows), and cetaceans (e.g., dolphins) ( fig. 1) (tsagkogeorga et al. 2013) . bats are believed to have diverged from other eutherian mammals approximately 88 million years ago (mya) (lei and dong 2016) . the traditional classification system divided bats into two suborders: microchiroptera (microbats) and megachiroptera (megabats). microbats are defined by their smaller size (4-16 cm), the use of echolocation, and the use of hibernation during the winter for many species. megabats consist of the flying foxes (also called fruit bats) and are larger nonecholocating bats (up to 1.6 kg with wingspans of 1.7 m) belonging to the pteropodidae family. however, more recent phylogenetic analyses based on molecular data have led to a reclassification of bats into the suborders yinpterochiroptera and yangochiroptera. the yinpterochiroptera suborder includes the nonecholocating pteropodidae family (flying foxes) and the echolocating rhinolophoidea family, while the yangochiroptera suborder consists of the remaining echolocating microbats (teeling et al. 2005 (teeling et al. , 2016 . the two suborders of bats are estimated to have diverged approximately 63 mya (lei and dong 2016) . although the new classification has strong statistical support, it remains controversial as it suggests that laryngeal echolocation evolved twice in chiroptera, once in yangochiroptera and once in the rhinolophoids (teeling et al. 2000) . of all the mammals, bats are the most ecologically diverse. they are the only mammals that have evolved powered flight and have adapted to a variety of environments across all continents with the exception of the polar regions. their diets are equally diverse, including fruits, pollen, insects, small vertebrates, and even blood, and they play important roles in the ecosystem through seed dispersal, pollination, and insect control. bats have longer lifespans relative to other mammals, typically living 3.5 times longer than mammals of similar size (austad 2010) . maternal investment is generally high, with most species giving birth to a single pup per year (from tsagkogeorga et al. 2013 with permission) and pups averaging approximately 23% of maternal body weight at birth (barclay and harder 2003) . curiously, despite their longer lifespans, there is anecdotal evidence that bats are resistant to tumors . the characteristic that has drawn the most attention in recent decades is their role as natural reservoirs for a variety of viruses that are highly pathogenic in other species yet rarely cause clinical disease in bats. this characteristic in particular has led to renewed interest in the immune systems of bats. bats are highly gregarious mammals, with most species living in high-density colonies, providing ideal environments for transmission and maintenance of pathogens within populations. combined with their frequent movement between roosts, transmission of viruses, bacteria, parasites, and fungi could potentially occur readily between individuals and populations, resulting in a situation of constant pathogen exposure. approximately 200 viruses have been detected across different bat species, and many of the viruses identified in bats are highly pathogenic in other species, including humans (moratelli and calisher 2015) ; however, they likely host many more (anthony et al. 2013) . examples include high-profile viruses such as the severe acute respiratory syndrome coronavirus (sars-cov), marburg virus, and hendra and nipah paramyxoviruses. these viruses occasionally spill over to other susceptible hosts, causing severe disease and mortality yet causing no disease in bats. the long coevolutionary history of bats and viruses has likely resulted in the establishment of a state of equilibrium, allowing both the viruses and their host to coexist in a disease-free state typical of natural reservoirs. bats also host a variety of other pathogens, including parasites, bacteria, and fungi. unlike viral infections, there are examples of these pathogens causing disease among bats. the fungus that causes white nose syndrome (wns), pseudogymnoascus destructans, has resulted in mass mortalities among a number of north american microbat populations, with some species now threatened with extinction (blehert et al. 2009 ). evidence for lower fungal loads consistent with the development of resistance to the fungus have been observed in some bat populations, providing hope that selection on immune genes may lead to the development of resistance or tolerance mechanisms (langwig et al. 2017) . however, it is unlikely that this will occur rapidly enough for many affected populations. several bacterial infections, including tick-borne spirochaete bacteria, borrelia spp., and some enteric bacteria, have also been associated with pathology in bats (reviewed in brook and dobson, 2015) . brooks and dobson (2015) presented evidence that bats may have evolved mechanisms to eliminate intracellular pathogens such as viruses at the expense of their ability to eliminate extracellular pathogens (bacteria, parasites, and fungi) and hypothesize that mitochondrial adaptations may play a role. in light of the increasing emergence of infectious diseases and the impacts of pathogens such as wns, deciphering the immune systems of bats has never been more critical and offers potential for identifying novel antiviral therapies and approaches to the conservation of bats threatened by diseases such as wns. fortunately, progress in the area of bat immunology is rapidly advancing as new groups enter the field and advances in technology provide opportunities for more rapid discovery. several reviews that have appeared over the last 5 years have described the various aspects of the immune systems of bats butler et al. 2014; schountz 2014; baker and zhou 2015; schountz et al. 2017) . in this chapter we provide a broad overview, with a focus on recent highlights in bat immunology and areas for future research. although few studies have examined the histology of bat lymphoid tissues, from an anatomical perspective, bats appear to have the majority of primary and secondary lymphoid organs present in other mammals, including thymus, bone marrow, spleen, and lymph nodes (papenfuss et al. 2012; zhou et al. 2016b) . bone marrow has been isolated from long bones, including humerus and radius, and from the ribs but appears to be absent in the distal wing bones (papadimitriou et al. 1996; zhou et al. 2016b) . notably absent, at least in the species that have been examined to date, are peyer's patches, which are generally located in the submucosa and lamina propria of the small intestine. no peyer's patches were present in the horseshoe bat, rhinolophus hildebrandtii, or the common pipistrelle bat, pipistrellus pipistrellus (strobel et al. 2015; makanya and john 1994) . the submucosa of the intestine of the horseshoe bat was devoid of lymphoid tissue, with the exception of a few aggregations of lymphoid nodules in the rectal submucosa (makanya and john 1994) . a range of immune cell types also appear to be present in bats. morphological characteristics have been used to identify lymphocytes, neutrophils, eosinophils, basophils, and macrophages in the brazilian free-tailed bat, tadarida brasilensis (turmelle et al. 2010a) . macrophages and t-and b-cell populations have also been identified in the indian flying fox, pteropus giganteus, based on cellular adherence and scanning electron microscopy (sarkar and chakravarty 1991) . more recently, the phenotype, morphology, and function of dendritic cells and macrophages have been characterized from bone marrow from the black flying fox, pteropus alecto (zhou et al. 2016b) . cells resembling follicular dendritic cells (fdcs) have also been described in the indian flying fox (sarkar and chakravarty 1991) . unlike dendritic cells that originate in the bone marrow, fdcs are of mesenchymal origin and are found in primary and secondary lymphoid follicles in b-cell areas of lymphoid tissue. fdcs are essential for high-affinity antibody production and for the development of b-cell memory. they also have the ability to maintain intact antigen for extended periods (van nierop and de groot 2002; heesters et al. 2014) . whether they play the same role in bats remains to be determined but presents an interesting possibility for the maintenance of persistent viral infections. the lack of species-specific reagents has often been a hindrance to comparative immunologists. however, bat immunology made a resurgence in an age of rapid advances in species-independent approaches such as next-generation sequencing, proteomics, and gene editing technologies such as crispr/cas9. rnaseq studies on tissues and cells from a variety of different species of bats have provided evidence that bats have nearly all of the major components of the immune system that are present in other mammals, including receptors and molecules associated with innate and adaptive immunity and micrornas (papenfuss et al. 2012; shaw et al. 2012; cowled et al. 2014) . rnaseq data from virus-infected bat cells and wnsinfected bat tissues have also offered insights into the genes associated with hostpathogen responses (wynne et al. 2014 (wynne et al. , 2017 field et al. 2015) . to date, partial genome sequences of 14 bat species are available in the ncbi database, providing valuable insights into the evolution of immune genes and essential sequence information for the design of primers and the development of reagents essential for studies of the immune responses of bats. the bat1k project, which aims to sequence the genomes of the approximately 1300 species of bats, will no doubt provide a valuable resource for comparing the immune repertoire of different species of bats (teeling et al. 2018) . the genomes of bats are condensed compared to other mammals, ranging from 1.6-3.54 gb. smaller genome sizes in both bats and birds have been hypothesized to be associated with the metabolic requirements of flight (kapusta et al. 2017 ). a number of genomic regions associated with immunity have been analyzed in detail, in particular in the black flying fox (p. alecto), using a combination of wholegenome data and additional sequencing. these include regions associated with innate, for example, type i interferon (ifn), and adaptive immunity, for example, major histocompatibility class i (mhc-i) and mhc-ii. consistent with the smaller size of the genomes of bats, these regions are also condensed and contain fewer genes compared with the corresponding region from other mammals (ng et al. , 2017 zhou et al. 2016a) . for example, the type i ifn locus of the black flying fox is highly condensed and contains fewer ifn genes than any other species sequenced to date (fig. 2) . the description of the genomes of two divergent bat species, the australian black flying fox (p. alecto) and david's myotis (mytois davidii), provided the first glimpse into unique genetic signatures within immune pathways of bats, lending support to the idea of inadvertent changes in the immune system associated with the evolution of flight (zhang et al. 2013 ). these include changes in the genes associated with dna response/dna repair pathways that are tightly linked with innate immune pathways (fig. 3) . the dna damage sensor, dna-dependent protein kinase catalytic subunit (dna-pkcs), which is also part of the cytoplasmic microbial nucleic acid sensing complex, was among the genes that have undergone selection in bats (ferguson et al. 2012) . accelerated evolution of innate immune genes including nuclear factor-kb (nf-kb) family member rel, ifnar1, toll-like receptor 7 (tlr7), ifn stimulated gene 15 (isg15), interleukin-18 (il-18), and nucleotidebinding oligomerization domain-like receptor (nlr) family, pyrin domain containing 3 (nlrp3) were also observed in the genomes of the two bats, an observation that may be a consequence of the coevolution of bats with viruses (zhang et al. 2013) . notably absent from the black flying fox and david's myotis genomes is the pyrin and hin domain (pyhin) gene family, which are involved in the recognition of foreign dna (zhang et al. 2013 ). this finding was recently confirmed in eight additional bat species across both suborders (ahn et al. 2016 ). the family member, absent in melanoma 2 (aim2), is a cytosolic dna sensor and also part of the inflammasome complex that results in the activation of inflammatory cytokines, including il1β and il18. a second component of the inflammasome, nlrp3, has undergone positive selection in the black flying fox and david's myotis, consistent with the possibility that the formation of inflammasomes is impaired in bats, which may in turn dampen the inflammatory response against pathogens (zhang et al. 2013) . the absence of a number of natural killer (nk) cell receptors from bat rnaseq and genome data sets is also striking (papenfuss et al. 2012; shaw et al. 2012; zhang et al. 2013) . genes that encode mammalian nk cell receptors are located within the leukocyte receptor complex (lrc) and the natural killer complex (nkc) of the genome. the two families have undergone convergent evolution to bind mhc-i molecules for the control of nk cell function. genes within the lrc encode immunoglobulin (ig)-like genes, including the killer cell ig-like receptors (kir), leukocyte ig-like receptors (lilrs), and leukocyte-associated ig-like receptors (lairs). those within the nkc encode lectin-like receptors, including the ly49 c-type lectin family. the composition of the lrc and nkc varies considerably among species. while most species have expanded either their lrc or nkc gene families, there are exceptions to this rule. in humans and nonhuman primates, the main nk cell receptors are encoded in the lrc and belong to the ig superfamily. rodents and horses have only expanded their ly49 c-type lectin family of nk receptors (kelley et al. 2005) . in contrast, cattle appear to have diversified nk genes within both the nkc and lrc regions, whereas domestic dogs and four species of marine carnivores contain single copies of kir and ly49 genes (hammond et al. 2009; schwartz et al. 2017) . in bats, kirs and ly49-like receptors appear to be absent from transcriptome and genome data sets from the black flying fox, and only a single pseudogene of ly49 was identified in the genome of david's myotis bat (papenfuss et al. 2012; zhang et al. 2013) . two kirs have been identified in the genome of the big brown bat, eptesicus fuscus, but whether they are functional remains to be determined (guethlein et al. 2015) . overall, evidence to date is consistent with the contraction of both kir and ly49 families of receptors in bats. other nk cell coreceptors have been identified in bat genome and rnaseq data sets, hinting at some level of nk cell function in bats. these include the presence of cd94 and nkg2c, which form heterodimers to generate inhibitory signals. the more divergent nkg2d, which binds mhc-i chain-related genes, mica/b, and the ul16 binding proteins (ulbps) in humans (kelley et al. 2005) , was also detected. coreceptors, including cd16, cd56, and cd244, were also transcribed in the black flying fox (papenfuss et al. 2012) . the failure to identify a number of nk cell receptors in several bat species supports the hypothesis that bats may have atypical nk cell responses or use different subsets of receptors. the availability of rnaseq and genomic data has also accelerated the characterization of a variety of immune genes and provided opportunities to examine transcription in various tissues and cells. molecular information exists for a variety of mammalian cytokines that have been described in bats including interleukins (il2, il4, il6, il10, il12), cytokines (tnfα, tgfβ), and ifns (types i, ii, and iii) (iha et al. 2009; he et al. 2010 he et al. , 2014 kepler et al. 2010; zhou et al. 2011a zhou et al. , 2016a janardhana et al. 2012; loria-cervera et al. 2014) . detailed descriptions of pattern recognition receptors, tlrs, and rig-i like helicases have also been reported (iha et al. 2010; cowled et al. 2011 cowled et al. , 2012 although only a few studies have examined the nature of ig genes in bats, a few unusual characteristics have already emerged that have been extensively reviewed elsewhere (butler et al. 2014) . the constant regions of bat igs appear to correspond to the canonical structure and repertoire found in other eutherian mammals. bats transcribe igm, igd, iga, ige, and multiple subclasses of igg (baker et al. 2010; butler et al. 2011; wynne et al. 2013) , although some species do not have ighδ genes and others have only a single ighγ gene gerrard et al. 2017) . studies of the heavy chain variable (vh) region repertoires of black flying foxes and little brown bats (myotis lucifugus) suggest bats may have the greatest number of vh gene segments among mammals (baker et al. 2010; bratsch et al. 2011) . furthermore, evidence from little brown bats indicates that bats may depend more on combinatorial diversity and less on somatic hypermutation . the antigen-binding region of black flying fox vh genes contains amino acids typically associated with lower antigen avidity but greater specificity (baker et al. 2010) . this, combined with the lack of evidence for somatic hypermutation, is consistent with the possibility that highly specific vh segments are encoded in the genomes of bats because of the long coevolutionary history of bats and viruses. the availability of cell lines from a range of different bat species has provided opportunities to study several aspects of the immune response of bat cells in vitro. this has been particularly useful for studying host-virus interactions. ifn responses of bat cells and cell lines following stimulation with viruses and synthetic tlr ligands, including polyinosinic:polycytidylic acid (polyi:c) and bacterial lipopolysaccharide (lps), have demonstrated that ifn production pathways are functional in bat cells and supernatant from stimulated cells has antiviral activity (stewart et al. 1969; omatsu et al. 2008; crameri et al. 2009; kepler et al. 2010; zhou et al. 2011b ). significantly, ifnα and ifn signaling molecules, such as ifn regulatory factor 7 (irf7), are constitutively expressed in unstimulated pteropid bat tissues and cells, consistent with the possibility that the innate immune systems of bats are at higher states of activation than other mammals, presumably allowing bats to rapidly respond to microbial infection (zhou et al. , 2016a . the constitutive expression of ifnα has been described in two species of pteropid bats (p. alecto and cynopterus brachyotis) and is a first for any species. curiously, fetal and kidney cell lines from a third pteropid bat species, the egyptian rousette bat (rousettus aegyptiacus), have low constitutive expression of ifnα, indicating that high baseline levels of ifnα may not be a feature of all bat species (kuzmin et al. 2017) . the downstream signaling events triggered by ifn result in the induction of hundreds of ifn-stimulated genes (isgs), which are responsible for the antiviral state induced by ifns. the profile of isgs in unstimulated bat cells and the kinetics of isg induction following stimulation with ifn also differs from other species. unstimulated cells from the black flying fox have higher levels of isgs compared to human cells. the isg profile of bat cells consists predominantly of a subset not associated with the acute inflammatory responses that often accompany elevated ifn activity (cheon et al. 2013; zhou et al. 2016a ). stimulation of cells from the black flying fox with ifnα also leads to the induction of novel subsets of isgs, including ribonuclease l (rnasel), that are not known to be induced by ifn to other species and the isg response is elevated for a shorter period of time in bat compared to human cell lines (de la cruz-rivera et al. 2017; zhang et al. 2017) ; rnasel is also elevated in bats that die from experimental tacaribe virus infection (gerrard et al. 2017) . overall, these studies point to differences in the regulation and profile of bat isgs as being central to the ability of bats to tolerate constitutive ifnα expression without pathology. consistent with the nature of the isg response, additional evidence is also accumulating for differences in the activation of other components of the inflammatory immune response in bats. comparison of the inflammatory cytokine production of polyi:c-stimulated cell lines from big brown bats (e. fuscus) and humans have demonstrated that the induction of high levels of proinflammatory cytokines, tnfα and il8, occurs in human but not in bat cells (banerjee et al. 2017 ). this result again demonstrates that bats may regulate their immune response more tightly compared to other species. experimental infections of bat cells and cell lines have also provided insight into the antiviral response of bats, revealing differences in the responses to different viruses and between cell types. infection of black flying fox splenocytes with the bat paramyxovirus, tioman virus, resulted in the downregulation of type i ifns and the upregulation of type iii ifns, indicating that type iii ifns may play an important role in the ability of bats to coexist with viruses (zhou et al. 2011a ). in contrast, henipavirus infection antagonizes type i and type iii ifn production and signaling in black flying fox cells but only ifn production in human cells (virtue et al. 2011a, b) . the difference in the behavior of bat ifns upon tioman and henipavirus infection may reflect different ifn production mechanisms in splenocytes, which are professional immune cells, and cloned bat cells, which are predominantly fibroblastlike (crameri et al. 2009 ). infection of cells from the black flying fox with henipavirus and the egyptian rousette bat with ebola or marburg results in the induction of ifnβ, but curiously no increase in ifnα has been observed, at least at the time points examined in these studies (zhou et al. 2016a; kuzmin et al. 2017) . as described earlier, p. alecto has high constitutive ifnα, which may account for its low induction, but this does not appear to be the case for the rousette bat. both marburg and ebola viruses, but particularly marburg, induced a potent innate immune response in rousette cells, which was generally stronger than that in human cells. the timing of induction of ifns and isgs in ebola-virus-infected cells was also delayed compared to cells infected with marburg virus (kuzmin et al. 2017) . the natural reservoir for marburg virus is known to be the rousette bat, but the reservoir for ebola is unknown and believed to be another bat species. the differences in host response of rousette bat cells to the two filoviruses may therefore reflect adaptations associated with the role of this species as a natural reservoir for marburg but not ebola. although isg responses have also been examined following viral infections in vitro, their ability to restrict viral replication has only been examined for a few isgs (de la cruz et al. 2017; zhou et al. 2013 ). the best-characterized isgs include myxovirus resistance (mx) genes and 20-50-oligoadenylate synthetase 1 (oas1). mx proteins are large gtpases that were initially described as inhibitors of influenza viruses and act by detecting viral replication and then trapping viral components. the oas1 proteins are activated by dsrna leading to the activation of rnase l, which then degrades both cellular and viral rna (sadler and williams 2008) . mx1 and oas1 from the black flying fox have been demonstrated to be highly upregulated by pteropine orthoreovirus nb (prv1nb) virus infection, an orthoreovirus carried by pteropid bats . furthermore, bat mx1 proteins from pteropidae, phyllostomidae, and vespertilionidae demonstrate antiviral activity against ebola and bat influenza-like viruses. however, thogoto virus, a tick-transmitted orthomyxovirus that is not known to infect bats, was not inhibited by bat mx1 despite the ability of human mx1 to inhibit thogoto virus replication. evidence for positive selection in two variable and surface-exposed regions of bat mx1 genes were hypothesized to explain some of the species-specific antiviral activities of these proteins (fuchs et al. 2017 ). however, antiviral activity of black flying fox rnasel has been demonstrated against the yellow fever flavivirus, which is carried by mosquitoes, consistent with differences in specificity among different bat isgs (de la cruz et al. 2017 ). cell-mediated immune (cmi) responses are controlled by cd8 + cytotoxic and cd4 + helper t-lymphocyte populations and result in the killing of virus-infected cells or activation of the antibody and cytokine response. fewer studies have examined cmi in bats. the single type ii ifn, ifnγ, is produced by black flying fox bat cells stimulated with mitogens such as phytohaemagglutinin (pha) and cona, and recombinant bat ifnγ has antiviral activity against semliki forest virus and hev in vitro . at least in vitro, ifnγ from the black flying fox appears to have activity similar to that of ifnγ from other mammals, consistent with its role in the cmi response. curiously, in rousette bat cell lines, ifnγ is induced following infection with marburg virus but not following infection with ebola virus, indicating there may be differences in the cmi response induced by these two closely related viruses (kuzmin et al. 2017) . a number of earlier studies have described the in vitro responses of pteropid bats and microbats to t-cell mitogens and mixed lymphocyte responses in pteropid bats (mcmurray and thomas 1979; chakraborty and chakravarty 1983; chakravarty and paul 1987; paul and chakravarty 1987) . although these studies have been relatively crude due to the absence of specific reagents, they have all reported delayed responses compared with those of conventional laboratory animals. the presence of regulatory t cells was implicated in the delay in mitogenic responses of b cells in bats . whether these cells are involved in the delay in t-cell-mediated immune responses observed in bats remains to be determined. more recent studies have used proteomics to functionally characterize black flying fox mhc-i molecules and identify endogenous and viral peptide ligands. peptides derived from bat mhc-i molecules display a relatively broad length distribution, consistent with earlier observations based on sequence information demonstrating relatively large peptide binding grooves in the bat class i molecules wynne et al. 2016) . furthermore, an unusual preference for a c-terminal proline residue was identified in endogenous and hendra virus (hev)-derived peptides presented by bat mhc-i molecules, consistent with the possibility that differences in antigen presentation or processing may exist in bats ). bats are capable of mounting antibody responses to viruses and model antigens, and the appearance of antibodies appears to follow the same succession as that of other mammals with the early appearance of igm followed by igg (hatten et al. 1968 (hatten et al. , 1970 chakraborty and chakravarty 1983; wellehan jr et al. 2009 ). although all of the ig isotypes have been detected at the mrna level in a variety of bat tissues, iga protein appears to be present at surprisingly low levels in tissues and secretions from the black flying fox, which may have implications for its role in mucosal immunity in bats ). there are also differences in the time course, quantity, and duration of antibody responses, and questions exist over the protective nature of antibodies in bats (hatten et al. 1968; mcmurray et al. 1982; chakraborty and chakravarty 1984; davis et al. 2007; wellehan jr et al. 2009; turmelle et al. 2010b) . responses to antigens such as ϕx174 bacteriophage and sheep red blood cells have demonstrated that the generation of neutralizing antibodies is delayed in the big brown bat, the pteropid bat, and the indian flying fox (pteropus giganteus) (hatten et al. 1968; chakraborty and chakravarty 1984) . isotype switching from igm to igg also appears to be delayed in the big brown bat (hatten et al. 1968 ). despite genetic evidence for limited somatic hypermutation in the little brown bat, an increase in antibody affinity as measured by the ability of antibodies to dissociate from ϕx174 increased following secondary immunization in the big brown bat (hatten et al. 1970) . measures of cmi in bats have been crude relative to studies in other species and are limited to studies demonstrating t-cell-mediated inflammation to protein antigens such as purified protein derivative (ppd), pha, and bovine serum albumin (bsa). such skin sensitivity tests in two bat species, the common vampire bat (desmodus rotundus) and seba's short-tailed bat (carollia perspicillata), immunized with ppd or bsa revealed delayed responses in both species compared to similar reactions in mice (mcmurray and thomas 1979) . lack of inflammatory responses have also been reported in most indian flying foxes subjected to skin sensitivity tests using the contact allergen 2-4 dinitrofluorobenzene (chakraborty and chakravarty 1983) . unlike conventional laboratory animals, few "clean" captive colonies of bats exist, and experimental infections often rely on the use of wild caught individuals, which represent a mixed population of unknown age, susceptibility, and prior viral exposure. experimental infections have been performed on a number of species of bats using rabies virus, australian bat lyssavirus (ablv), marburg, hev, nipah virus (niv), japanese b encephalitis (je) virus, and tacaribe virus (tcrv) (williamson et al. 1998 (williamson et al. , 1999 almeida et al. 2005; davis et al. 2007; middleton et al. 2007; turmelle et al. 2010b; halpin et al. 2011; cogswell-hawkinson et al. 2012; paweska et al. 2012) . although the only immune parameter measured during these studies has been antibody responses, these experiments have provided valuable information on the kinetics of viral infection, the timing and duration of antibody responses and the nature of protective immunity following reinfection. with the exception of rabies virus, ablv and tcrv, bats generally show no clinical signs of disease following infection. neutralizing antibodies to a variety of viruses have been detected in wild caught bats, demonstrating they are capable of mounting an antibody response to viruses (halpin et al. 2000; lau et al. 2005; leroy et al. 2005) . the transfer of maternal antibody to pups occurs in bats, and the decline of maternal antibodies has been examined in captive black flying, variable flying foxes (pteropus hypomelanus), and straw-colored fruit bats (eidolon helvum) baker et al. 2014 ). however, whether bats transfer maternal antibody both pre-and postpartum and the isotypes involved is unknown. the interpretation of antibody responses in bats is extremely challenging, and, as described earlier, the nature of antibody responses in bats often differs both qualitatively and quantitatively compared to other species. rabies and ablv are among the only viruses known to result in clinical disease in naturally infected and experimentally infected bats. however, not all bats develop disease, and the mechanisms responsible for differences in disease outcome between individuals are not understood. evidence from experimental infections has demonstrated that even the development of neutralizing antibodies does not always provide protection from reexposure. for example, a group of wild caught bats (12 big brown bats, e. fuscus, and 12 mexican free tailed bats, tadarida brasiliensis) challenged by oral-nasal inoculation with rabies virus all developed antirabies neutralizing antibodies within 3 months. rechallenge by intramuscular inoculation 6 months later resulted in an amnestic response in 21 animals, including 9 that developed clinical rabies (davis et al. 2007 ). low seroconversion rates have also been reported in big brown bats inoculated with rabies by intramuscular challenge with only 15 of 43 inoculated animals developing antibodies. this study also reported clinical disease following secondary or tertiary infections in bats that had seroconverted following primary inoculation (turmelle et al. 2010b) . similarly, almeida et al. (2005) described the intramuscular challenge of 40 vampire bats (d. rotundus) with rabies virus, of which 30 bats survived. once again, there was no correlation between the level of neutralizing antibody and survival. many bats that developed low or undetectable antibodies, as well as those with high antibody titers, survived infection. infection of gray-headed flying foxes, pteropus poliocephalus, with rabies or ablv results in similar rates of mortality and seroconversion. mccoll et al. (2002) reported clinical signs of disease in three of ten ablv-infected and two of four rabies-infected gray-headed flying foxes, none of which seroconverted prior to euthanasia. five of the ablv-infected survivors seroconverted by 23 dpi, with titers waning by 50 dpi. one of the rabies-infected survivors also seroconverted, but not until 70 dpi (mccoll et al. 2002) . these studies indicate that antibodies may not provide protection and support a role for other components of the immune response in those animals that survive infection. unlike rabies and ablv infections, clinical disease has not been reported in any bat species either naturally or experimentally infected with a variety of other bat-borne viruses, including hev, niv, marburg, ebola, and je viruses. however, similar to rabies infection, the role of the antibody response in providing protection remains unclear, and many animals survive infection but fail to seroconvert. the henipaviruses hev and niv are carried by pteropid bats. in australia, hev antibodies have been identified in all four species of australian flying foxes (p. alecto, p. poliocephalus, p. scapulatus, and p. conspicillatus) . niv antibodies have been identified in bats from southeast asia and africa. in malaysia, two pteropid species, small flying foxes (p. hypomelanus) and malayan flying foxes (p. vampyrus), are considered to be the reservoir hosts . a number of experimental infections of pteropid bat species have been performed to understand the nature of viral infection in the natural reservoir of these viruses. niv infection of 11 gray-headed flying foxes by subcutaneous injection resulted in the production of neutralizing antibody in all individuals inoculated, but in a separate study, only 4 of 8 malayan flying foxes that were infected by the oral-nasal route produced a neutralizing antibody response (middleton et al. 2007; halpin et al. 2011) . both subcutaneous and oral-nasal routes of infection have also been used for hev inoculation of pteropid bats. neutralizing antibody responses were detected in 10 of 20 black flying foxes inoculated oral-nasally with hev (halpin et al. 2011) . similarly, in gray flying foxes challenged with hev, neutralizing antibodies were detected in two of four bats inoculated by subcutaneous injection and three of the four bats inoculated by the oral-nasal route, with none of the bats displaying clinical signs of disease (williamson et al. 1998) . a study of four gray-headed flying foxes in late gestation infected subcutaneously with hev also described the presence of neutralizing antibodies in all four bats, and no abnormalities were observed in the fetuses or adults at necropsy (williamson et al. 1999) . in other mammals, pregnancy results in a bias in the immune response toward humoral immunity and away from cmi, which could be harmful to a fetus (szekeres-bartho 2002) . whether the nature of the maternal immune response facilitates greater production of antibody in infected bats during pregnancy remains to be investigated. a natural reservoir of marburg virus are the egyptian rousette bats (r. aegyptiacus) (towner et al. 2009) , and a number of experiments have been performed to study the nature of viral transmission and infection in this species (paweska et al. 2012; schuh et al. 2017a, b) . marburg virus is capable of horizontal transmission between inoculated and naïve r. aegyptiacus. all inoculated bats seroconverted, with igg antibodies peaking between 14-28 dpi. marburg virus antibody titers in both inoculated and in contact bats declined within 1 month following attainment of peak levels and were undetectable after 2 months (schuh et al. 2017a) . a subsequent study revealed that bats rechallenged with marburg virus 17-24 months following primary experimental infection developed virus-specific secondary antibody, indicative of the development of long-term protective immunity (schuh et al. 2017b) . clearly, additional work is needed to understand the antibody responses of bats and the nature of antibody-mediated protection against various viruses. given what we have learned about innate immunity, particularly in pteropid bats, it is possible that innate immune mechanisms, such as ifn, reduce viral replication to low levels, delaying the generation and magnitude of an antibody response. evidence for a highly diverse germline repertoire of antibodies and the absence of somatic hypermutation could indicate that bats have evolved a repertoire of antibodies that are highly pathogen specific. such antibodies may provide some level of early protection without reaching the higher titers observed in other species. although no studies have examined the cmi responses of bats to viral infections, the generation of an ifnγ reagent for pteropid bats has been described and will assist in future studies to examine cmi in bats ). wns is caused by a cold-loving (pyrophilic) and keratinophilic fungus (p. destructans) first identified in north american bats in 2006 that infects the epidermis and dermis of the muzzle, ears, and wings. since its discovery, it has been detected in six species of north american bats, and infected populations have undergone a decline of up to 90%, with several species threatened with regional extinction within the next decade. p. destructans infects bats during hibernation, causing them to arouse early, leading to depletion of energy reserves and ultimately leading to a severe inflammatory response and resulting histopathology. the fungus is widely distributed in north america and europe and has recently been found in asia (hoyt et al. 2016) . although naturally infected european bats also develop histopathological lesions in response to p. destructans, no mass mortality is observed in european or asian bats (zukal et al. 2016) . similar to the situation with viruses, the long coevolutionary relationship of european and asian bats with p. destructans has presumably led to an equilibrium between the host and pathogen. in the longer term, this may also evolve in north american bats, and evidence of some level of resistance has been reported in some populations (langwig et al. 2017) . however, the rate of mortality among some bat species is too high to ignore. understanding the hostpathogen relationship and the genes and pathways associated with disease tolerance and resistance will be important for identifying viable treatments and assessing the immune responses of bats to drugs or vaccines. earlier reports describing the immune response of bats during hibernation indicate that, like other hibernating mammals, their immune responses are suppressed during torpor when they are initially infected with p. destructans. for example, hibernating e. fuscus bats maintained at 8 °c fail to generate antibodies in response to infection with je virus (sulkin et al. 1966 ). in addition, activation plasma complement against bacteria (escherichia coli, staphylococcus aureus) and fungi (candida albicans) is lower in hibernating little brown bats compared to nonhibernating bats (moore et al. 2011) . several studies have now begun to examine the host response of bats to p. destructans to determine the level of immune activation that occurs during torpor and after arousal. p. destructans begins to colonize bat skin during hibernation, yet visible signs of inflammation are characteristically absent in torpid animals, and neutrophils and macrophages are absent from sites of pathogen invasion in hibernating bats with wns. in little brown bats, overt skin damage does not occur until 2-3 weeks after bats have emerged from hibernation with intense neutrophilic inflammation associated with invasive p. destructans infection (meteyer et al. 2012) . studies of bats from wns-affected and unaffected sites have also demonstrated significantly higher circulating leukocyte counts in wns-affected bats with elevated body temperatures (above 20 °c). the latter is consistent with the mobilization of cells associated with arousal from torpor and euthermia (moore et al. 2013) . the absence of neutrophil and t-cell infiltration has been confirmed through rnaseq analysis of wns-infected little brown bat wing tissues during hibernation (field et al. 2015) . despite the absence of neutrophil invasion, increases in gene expression for inflammatory cytokines have been detected in wing tissues from hibernating wns infected bats compared to hibernating bats not affected by wns. these include il1β, il6, il17c, il20, il23a, il24, and g-csf and chemokines, such as ccl2 and ccl20. hibernating little brown bats exhibiting visible fungal infections elevated levels of transcripts for proinflammatory cytokines, il23 and tnfα, the anti-inflammatory cytokine il10, and the antimicrobial peptide cathelicidin in lung tissue compared to hibernating uninfected bats (rapin et al. 2014) . overall, these studies are consistent with the induction of an innate antifungal response in wns-infected bats prior to emergence from hibernation followed by infiltration of immune cells and, presumably, activation of adaptive immune responses following arousal. overactivation of the immune response following arousal from torpor, combined with a depletion of energy reserves, appears to be the main cause of mortality. in contrast to p. destructans, bats are known to carry other fungal pathogens, such as histoplasma capsulatum, without disease. h. capsulatum is a pathogenic fungus that causes pulmonary and systemic infections in humans. bats are considered to be the main reservoir of this fungus, and it is commonly found in bat guano (taylor et al. 2005) . although bats are susceptible to infection, mortality is rare in bats that are inoculated intranasally, which is the most likely route of natural infection. higher mortality rates are observed in bats inoculated intraperitoneally (mcmurray and greer 1979; greer and mcmurray 1981) . great fruit eating bats (artibeus lituratus) respond to infection with the generation of complement fixing antibodies by 3 weeks and precipitating antibodies by 5 weeks post infection (mcmurray and greer 1979) . natural infection rarely results in disease, indicating that, similarly to the situation with most viruses, bats have likely evolved mechanisms to control infection, at least under conditions where they are infected under nontorpid conditions. the field of bat immunology is very much in its infancy, and significant opportunities exist for future research. thanks to advances in technology, such as wholegenome sequencing and rnaseq, considerable progress has been made, in particular with regard to our understanding of the immune system of the black flying fox, p. alecto. however, as bats are a highly diverse group of mammals that have evolved independently for a long period of time, it is possible that different immune mechanisms exist between the two suborders and across species. there is likely much more to be learned from comparative studies across different bat species. comparative genomics of bats have provided important clues to the adaptations that may allow bats to coexist with viruses in the absence of disease. these include evidence for positive selection on a variety of immune genes and differences in the repertoires of nk cell receptors. additional genomic data, including long read assemblies, will be required to resolve highly repetitive regions such as the lrc and nkc to confirm the absence of important receptor families and to resolve other repetitive regions of the bat immunome. a number of genomic regions also remain largely unexplored, partly owing to their repetitive nature. these include b-and t-cell receptor (bcr and tcr) regions. examining the repertoire and diversity of these regions will provide opportunities to examine their functional activities and importance. for example, no information exists on the repertoire of tcrs in bats and the relative importance and roles of αβ and γδ t cells. observations from genomic data sets pave the way to further addressing the role of different components of the immune system in the responses of bats to infection. the mechanisms involved in tcr and bcr diversification also remain unknown. the roles of terminal deoxynucleotidyl transferase (tdt), recombination activating gene (rag), and activation-induced cytidine deaminase (aid) on recombination, somatic hypermutation, gene conversion, and class switching remain to be explored. a number of important differences in the innate immune system have also been identified in bats that are at odds with the responses in humans and other species. in particular, the constitutive activation of ifnα in the black flying fox is striking. in other mammals, constitutive ifn expression can have implications for inflammation and autoimmunity. identifying the mechanisms responsible for the ability of bats to tolerate high levels of ifn in the absence of inflammation has significant potential for identifying novel therapeutics to treat viral diseases in humans and other species. to this end, functional characterization of the different subsets of isgs already identified in both unstimulated and stimulated cells would provide valuable insights into the mechanisms responsible for the control of viral infection in the absence of inflammation. additionally, dissection of the signaling pathways responsible for the control of ifn response will contribute to our understanding of differences in the regulation of ifn in bats compared to other species. as described earlier, a number of functional differences have been identified in the immune system of bats compared to other species. these include the nature of cell-mediated and antibody responses of bats. to advance our understanding of the nature of these responses, appropriate bat-specific reagents will be required. some commercially available human and mouse antibodies generated against highly conserved intracellular proteins are cross reactive with bat proteins and have already proven useful (zhou et al. 2016b) . a handful of bat-specific antibodies have also been generated wynne et al. 2013) . additional reagents will be necessary to advance the field, including monoclonal antibodies for use in flow cytometry, immunohistochemistry, and elisas, to dissect the roles of different cell types, including b and t cells, dendritic cells, and macrophages. reagents will also be required to examine the responses of various cytokines to examine proinflammatory and anti-inflammatory pathways for comparison to other species and to answer specific questions, including the confirmation of cytokine expression at the protein level (e.g., ifnα to confirm its constitutive expression). recombinant cytokines and growth factors will also be important for examining the responses of cells to cytokine stimulation and the expansion of specific subsets of antigenspecific lymphocytes. lastly, the development of closed breeding colonies of bats will be essential in progressing research into immunity in bats, overcoming the issues associated with wild caught individuals of unknown age and history of infection. renewed interest in bat immunology emerged following the identification of bats as reservoirs for a number of viruses, including sars-cov and ebola, that are highly pathogenic in other species. prior to the emergence of these viruses, few studies had examined any aspect of bat immunology. a number of important observations have already been made through studies of the immune systems of bats, with evidence for adaptations not observed in any other species. significant progress has now been made in the identification of genes and pathways associated with immunity, and one of the recurring themes that is emerging with regard to viral infections is the ability of bats to control inflammatory responses. regulation of the immune system is likely an important mechanism for preventing pathology associated with infection. however, bats are an extraordinarily diverse group of mammals, and the adaptions identified to date may not apply across all bat species. in contrast to the apparent regulation of the immune response during viral infections, uncontrolled inflammatory responses due to infection with pathogens such as wns clearly demonstrate that bats are capable of overactivating their immune system, causing immunopathology. as described earlier, there are still gaps in our understanding of the immune systems of bats, and significant opportunities exist. studies of bat immunology provide opportunities to identify novel mechanisms that could be applied to redirecting the immune system of other species to prevent disease and to the conservation of bats affected by pathogens such as wns. unique loss of the pyhin gene family in bats amongst mammals: implications for inflammasome sensing experimental rabies infection in haematophagous bats desmodus rotundus a strategy to estimate unknown viral diversity in mammals methusaleh's zoo: how nature provides us with clues for extending human health span bat immunology immunoglobulin heavy chain diversity in pteropid bats: evidence for a diverse and highly specific antigen binding repertoire antiviral immune responses of bats: a review viral antibody dynamics in a chiropteran host lack of inflammatory gene expression in bats: a unique role for a transcription repressor studying immunity to zoonotic diseases in the natural host-keeping it real bat white-nose syndrome: an emerging fungal pathogen? the little brown bat, m. lucifugus, displays a highly diverse vh, dh and jh repertoire but little evidence of somatic hypermutation bats as 'special' reservoirs for emerging zoonotic pathogens the two suborders of chiropterans have the canonical heavy-chain immunoglobulin (ig) gene repertoire of eutherian mammals the immunoglobulin genes of bats dichotomy of lymphocyte population and cell mediated immune responses in a fruit bat, pteropus giganteus antibody-mediated immune response in the bat, pteropus giganteus analysis of suppressor factor in delayed immune responses of a bat, pteropus giganteus ifn[beta]-dependent increases in stat1, stat2, and irf9 mediate resistance to viruses and dna damage tacaribe virus causes fatal infection of an ostensible host, the jamaican fruit bat molecular characterisation of toll-like receptors in the black flying fox pteropus alecto molecular characterisation of rigilike helicases in the black flying fox, pteropus alecto characterisation of novel micror-nas in the black flying fox (pteropus alecto) by deep sequencing ) establishment, immortalisation and characterisation of pteropid bat cell lines effects of aerosolized rabies virus exposure on bats and mice the ifn response in bat cells consists of canonical and non-canonical isgs with unique temporal expression kinetics duration of maternal antibodies against canine distemper virus and hendra virus in pteropid bats dna-pk is a dna sensor for irf-3-dependent innate immunity the natural history of hendra and nipah viruses the white-nose syndrome transcriptome: activation of anti-fungal host responses in wing tissue of hibernating little brown myotis evolution and antiviral specificities of interferon-induced mx proteins of bats against ebola, influenza, and other rna viruses transcriptomic signatures of tacaribe virus-infected jamaican fruit bats pathogenesis of experimental histoplasmosis in the bat, artibeus lituratus co-evolution of mhc class i and variable nk cell receptors in placental mammals isolation of hendra virus from pteropid bats: a natural reservoir of hendra virus pteropid bats are confirmed as the reservoir hosts of henipaviruses: a comprehensive experimental study of virus transmission evolution and survival of marine carnivores did not require a diversity of killer cell ig-like receptors or ly49 nk cell receptors immune response in chiroptera to bacteriophage øx174 studies on the immune capabilities of chiroptera positive selection of the bat interferon alpha gene family anti-lyssaviral activity of interferon κ and ω from the serotine bat, eptesicus serotinus follicular dendritic cells: dynamic antigen libraries widespread bat white-nose syndrome fungus, northeastern china molecular cloning and sequencing of the cdnas encoding the bat interleukin (il)-2, il-4, il-6, il-10, il-12p40, and tumor necrosis factor-alpha molecular cloning and expression analysis of bat toll-like receptors 3, 7 and 9 cloning, expression and antiviral activity of ifnγ from the australian fruit bat, pteropus alecto dynamics of genome size evolution in birds and mammals comparative genomics of natural killer cell receptor gene clusters chiropteran types i and ii interferon genes inferred from genome sequencing traces by a statistical gene-family assembler innate immune responses of bat and human cells to filoviruses: commonalities and distinctions resistance in persisting bat populations after white-nose syndrome invasion severe acute respiratory syndrome coronavirus-like virus in chinese horseshoe bats phylogenomic analyses of bat subordinal relationships based on transcriptome data fruit bats as reservoirs of ebola virus cloning and sequence analysis of peromyscus yucatanicus (rodentia) th1 (il-12p35, ifn-γ and tnf) and th2 (il-4, il-10 and tgf-β) cytokines the morphology of the intestine of the insectivorous horseshoe bat (rhinolophus hildebrandti, peters): a scanning electron and light microscopic study pathogenesis studies with australian bat lyssavirus in grey-headed flying foxes (pteropus poliocephalus) immune responses in bats following intranasal infection with histoplasma capsulatum cell-mediated immunity in two species of bats role of immunoglobulin classes in experimental histoplasmosis in bats pathology in euthermic bats with white nose syndrome suggests a natural manifestation of immune reconstitution inflammatory syndrome experimental nipah virus infection in pteropid bats (pteropus poliocephalus) specific alterations in complement protein activity of little brown myotis (myotis lucifugus) hibernating in whitenose syndrome affected sites hibernating little brown myotis (myotis lucifugus) show variable immunological responses to white-nose syndrome bats and zoonotic viruses: can we confidently link bats with emerging deadly viruses? evolution and comparative analysis of the bat mhc-i region insights into the ancestral organisation of the mammalian mhc class ii region from the genome of the pteropid bat, pteropus alecto induction and sequencing of rousette bat interferon α and β genes ontogenetic and anatomic variation in mineralization of the wing skeleton of the mexican free-tailed bat, tadarida brasiliensis the immune gene repertoire of an important viral reservoir, the australian black flying fox phytohaemagglutinin mediated activation of bat (pteropus giganteus) lymphocytes virological and serological findings in <italic>rousettus aegyp-tiacus</italic> experimentally inoculated with vero cells-adapted hogan strain of marburg virus activation of innate immune-response genes in little brown bats (myotis lucifugus) infected with the fungus pseudogymnoascus destructans interferon-inducible antiviral effectors analysis of immunocompetent cells in the bat, pteropus giganteus: isolation and scanning electron microscopic characterization immunology of bats and their viruses: challenges and opportunities immunological control of viral infections in bats and the emergence of viruses highly pathogenic to humans modelling filovirus maintenance in nature by experimental transmission of marburg virus between egyptian rousette bats egyptian rousette bats maintain long-term protective immunity against marburg virus infection despite diminished antibody levels the evolution of the natural killer complex; a comparison between mammals using new high-quality genome assemblies and targeted annotation transcriptome sequencing and annotation for the jamaican fruit bat (artibeus jamaicensi) mammal species of the world: a taxonomic and geographic reference persistent infection in bats and bat cell cultures with japanese encephalitis virus histological and histochemical analysis of the gastrointestinal tract of the common pipistrelle bat (pipistrellus pipistrellus) studies of arthropod-borne virus infections in chiroptera immunological relationship between the mother and the fetus geographical distribution of genetic polymorphism of the pathogen histoplasma capsulatum isolated from infected bats, captured in a central zone of mexico molecular evidence regarding the origin of echolocation and flight in bats a molecular phylogeny for bats illuminates biogeography and the fossil record phylogeny, genes, and hearing: implications for the evolution of echolocation in bats bat biology, genomes, and the bat1k project: to generate chromosome-level genomes for all living bat species isolation of genetically diverse marburg viruses from egyptian fruit bats phylogenomic analyses elucidate the evolutionary relationships of bats histological assessment of cellular immune response to the phytohemagglutinin skin test in brazilian free-tailed bats (tadarida brasiliensis) host immunity to repeated rabies virus infection in big brown bats human follicular dendritic cells: function, origin and development interferon production and signaling pathways are antagonized during henipavirus infection of fruit bat cell lines interferon signaling remains functional during henipavirus infection of human cell lines mass extinctions, biodiversity and mitochondrial function: are bats 'special' as reservoirs for emerging viruses? detection of specific antibody responses to vaccination in variable flying foxes (pteropus hypomelanus) transmission studies of hendra virus (equine morbilli-virus) in fruit bats, horses and cats experimental hendra virus infection in pregnant guinea-pigs and fruit bats purification and characterisation of immunoglobulins from the australian black flying fox (pteropus alecto) using anti-fab affinity chromatography reveals the low abundance of iga proteomics informed by transcriptomics reveals hendra virus sensitizes bat cells to trail mediated apoptosis characterization of the antigen processing machinery and endogenous peptide presentation of a bat mhc class i molecule comparative transcriptomics highlights the role of the ap1 transcription factor in the host response to ebolavirus nipah virus infection in bats (order chiroptera) in peninsular malaysia comparative analysis of bat genomes provides insight into the evolution of flight and immunity ifnar2-dependent gene expression profile induced by ifn-α in pteropus alecto bat cells and impact of ifnar2 knockout on virus infection type iii ifns in pteropid bats: differential expression patterns provide evidence for distinct roles in antiviral immunity type iii ifn receptor expression and functional characterisation in the pteropid bat, pteropus alecto bat mx1 and oas1, but not pkr are highly induced by bat interferon and viral infection irf7 in the australian black flying fox, pteropus alecto: evidence for a unique expression pattern and functional conservation contraction of the type i ifn locus and unusual constitutive expression of ifn-α in bats unlocking bat immunology: establishment of pteropus alecto bone marrow-derived dendritic cells and macrophages white-nose syndrome without borders: pseudogymnoascus destructans infection tolerated in europe and palearctic asia but not in key: cord-000478-88wo4xen authors: gowen, brian b.; ennis, jane; russell, andrew; sefing, eric j.; wong, min-hui; turner, jeffrey title: use of recombinant adenovirus vectored consensus ifn-α to avert severe arenavirus infection date: 2011-10-24 journal: plos one doi: 10.1371/journal.pone.0026072 sha: doc_id: 478 cord_uid: 88wo4xen several arenaviruses can cause viral hemorrhagic fever, a severe disease with case-fatality rates in hospitalized individuals ranging from 15-30%. because of limited prophylaxis and treatment options, new medical countermeasures are needed for these viruses classified by the national institutes of allergy and infectious diseases (niaid) as top priority biodefense category a pathogens. recombinant consensus interferon alpha (cifn-α) is a licensed protein with broad clinical appeal. however, while cifn-α has great therapeutic value, its utility for biodefense applications is hindered by its short in vivo half-life, mode and frequency of administration, and costly production. to address these limitations, we describe the use of def201, a replication-deficient adenovirus vector that drives the expression of cifn-α, for preand post-exposure prophylaxis of acute arenaviral infection modeled in hamsters. intranasal administration of def201 24 h prior to challenge with pichindé virus (picv) was highly effective at protecting animals from mortality and preventing viral replication and liver-associated disease. a significant protective effect was still observed with a single dosing of def201 given two weeks prior to picv challenge. def201 was also efficacious when administered as a treatment 24 to 48 h post-virus exposure. the protective effect of def201 was largely attributed to the expression of cifn-α, as dosing with a control empty vector adenovirus did not protect hamsters from lethal picv challenge. effective countermeasures that are highly stable, easily administered, and elicit long lasting protective immunity are much needed for arena and other viral infections. the def201 technology has the potential to address all of these issues and may serve as a broad-spectrum antiviral to enhance host defense against a number of viral pathogens. the arenaviridae family of viruses has several members that can cause viral hemorrhagic fever, an acute, often-fatal, viral syndrome characterized by intense fever, malaise, and less frequently, bleeding and neurologic manifestations. case fatality rates of hospitalized patients suffering from arenaviral hemorrhagic fever (ahf) range from 15-30% [1, 2, 3, 4] . arenaviruses known to cause ahf include junín, machupo, guanarito, sabiá, and chapare in the south american continent, and lassa and lujo in west and southern africa, respectively. primary transmission of the arenaviruses from respective rodent reservoir hosts to humans occurs via exposure to contaminated excreta [5] . person-to-person transmission can occur through contact with blood or other body fluids during the care and management of infected individuals [1, 6] . notably, these viruses are considered a threat to national security and are classified as highest priority pathogens by the niaid [7] . at present, the treatment of ahf is limited to ribavirin and immune plasma [8, 9] . the latter has only been proven to be effective in treating cases of argentine hemorrhagic fever (junín virus infection) within 8 days of disease onset. off-label usage of ribavirin has been shown to be effective in treating lassa fever when therapy was initiated within 6 days of the development of clinical symptoms. however, there are toxicities associated with ribavirin therapy at dosages required for efficacious use, which may contribute to the observed poor patient compliance in completing prescribed treatment regimens [10, 11] . very limited case data using ribavirin to treat other ahfs supports the use of emergency protocols [1, 12, 13] , however the utility of ribavirin therapy remains to be seen. interferon alpha (ifn-a) is an effective part of the host innate immune response, which can be manufactured as a recombinant human protein with broad clinical appeal [14] . consensus (c)ifna, also known as ifn alfacon-1 and infergen, is a licensed, second generation ifn-a engineered to contain the most frequently occurring amino acids among the nonallelic ifn-a subtypes. previously, we have demonstrated that cifn-a can be used effectively alone, or in combination with ribavirin, to treat pichindé virus (picv) infection in hamsters [15, 16] , an experimental model of acute arenaviral disease [17] . however, while cifn-a has clinical value, its usefulness is hindered by its short half-life and cost to manufacture. there is an initial distributive half-life of 7 minutes and a beta half-life of 2 to 5 hours [14] . the rapid systemic clearance requires frequent dosing to achieve desired therapeutic levels. consequently, treatment can result in well-documented toxicities which include headache, depression, hair loss, fever, and malaise. in order to combat the rapid degradation, pegylated forms of recombinant ifn-a have been introduced with half-lives that are on the order of days instead of hours, thus reducing the number of injections to once per week [18] . however, the cost to manufacture peg-ifn-a is exceedingly high, and the pegylation process has been shown to reduce the activity of ifn-a, thereby further increasing the production costs. to circumvent the fast decay of cifn-a, a replicationincompetent, recombinant adenovirus type 5 (rad5) gene delivery platform was designed to drive constitutive expression of the cifna gene from transduced nasal epithelial target cells. this rad5 cifn-a virus, called def201, was first developed in mice and recently shown to be active against yellow fever virus (yfv) infection in hamsters [19, 20] . the intranasal (i.n.) inoculation used in the yfv study prevents the host immune system from recognizing the ad5 vector, thereby bypassing any possible preexisting immunity [21] . in the present study, we evaluated the use of def201 administered i.n. for the prevention and treatment of picv infection in hamsters. all animal procedures complied with usda guidelines and were conducted at the aaalac-accredited laboratory animal research center at utah state university under protocol 1229, approved by the utah state university institutional animal care and use committee. female golden syrian hamsters were obtained from charles river laboratories (wilmington, ma) and acclimated for a minimum of 6 days prior to experimentation. they were fed standard hamster chow and tap water ad libitum. animals were approximately 7-9 weeks old at the time of virus challenge. picv, strain an 4763, was provided by dr. david gangemi (clemson university, clemson, south carolina). the virus was passaged once through hamsters. virus stocks were prepared from pooled livers harvested from infected hamsters. virus dilutions were made in minimal essential medium (mem), and infectious inoculum was given bilaterally in two intraperitoneal (i.p.) injections of 0.1 ml each. the recombinant adenovirus vectored cifn-a (rad5-huifn-a; def201) and the rad5 empty vector (rad ev) control virus were provided by defyrus, inc. (toronto, on, canada) at a concentration of 6610 9 and 2610 11 plaque-forming units (pfu)/ml, respectively. both viruses were prepared in pbs for i.n. instillation in a 200 ml volume. virus titers were assayed using an infectious cell culture assay as previously described [22] . briefly, a specific volume of liver or spleen homogenate or serum was serially diluted and added to triplicate wells of vero (african green monkey kidney; american type culture collection, manassas, va) cell monolayers in 96well microplates. the viral cytopathic effect (cpe) was determined 7 to 8 days post-virus inoculation, and the 50% endpoints were calculated as described [23] . the assay detection ranges were 2.8 to 9.5 log 10 50% cell culture infectious doses (ccid 50 )/g of liver or spleen and 1.8 to 8.5 log 10 ccid 50 /ml of serum. in samples presenting with undetectable liver or spleen virus, a value of ,2.8 was assigned (,1.8 for serum). conversely, in cases wherein virus exceeded the detection range, a value of.9.5 (.8.5 for serum) was assigned. for statistical analysis, values of 2.8 or 9.5 log 10 (1.8 or 8.5 for serum) were assigned as needed for samples with undetectable or saturated virus levels, respectively. detection of alt in serum is an indirect method for evaluating liver disease. serum alt levels were measured using the alt (sgpt) reagent set purchased from pointe scientific, inc. (lincoln park, mi) per the manufacturer's recommendations. the reagent volumes were adjusted for analysis on 96-well microplates. experimental design def201 dose range titration experiment. hamsters were weighed on the morning prior to the day of infection and grouped (n = 15 for drug treatment groups, 26 for the placebo group) so that the average hamster weight per group across the entire experiment varied by less than 5 grams. varying pfu amounts of def201, the rad ev control virus, or saline placebo treatments were administered in a single i.n. dose 24 h prior to challenge with ,5 pfu of picv. five animals from each group were sacrificed on day 7 of infection. serum was collected for assaying alt activity, and virus titers were determined for liver, spleen, and serum samples as described above. the remaining 10 animals (21 for the placebo group) were observed 21 days for mortality and weighed individually every 3 days starting on day 0. sham-infected normal controls (n = 3) were included for comparison. extended pre-exposure prophylaxis experiment. the design was similar to the def201 titration experiment with the following differences. hamsters were weighed on the morning of initial pretreatment (day 214 relative to the infection) and grouped (n = 15 per group). groups were treated once i.n. with 10 8 pfu of def201, rad ev control virus, or saline placebo. treatments were given 14 or 7 days prior to challenge with ,5 pfu of picv. animals were observed for 28 days post-challenge for mortality. post-exposure prophylaxis experiment. the design was similar to the def201 pre-exposure prophylaxis experiment with the following differences. single dose i.n. treatments with 10 8 pfu of def201 or rad ev were administered 24 h prior to, or 6, 24, or 48 h after challenge with ,5 pfu of picv. on day 28 postinfection, the surviving animals (including 6 naïve sham-infected controls) were re-challenged. morbidity and mortality were observed out to 58 days after the initial challenge. kaplan-meier survival plots and all statistical evaluations were done using prism (graphpad software, ca). the log-rank test was used for survival analysis. for analyzing differences in viral titers, alt levels, and weight change, a one-way analysis of variance (anova) with newman-keuls post test or the kruskal-wallis (two-tailed) test with the dunn's post test was performed based on gaussian distribution of the data. in the initial trial, hamsters were treated with 10 6 to 10 8 pfu of def201 one day prior to challenge with a lethal dose of picv. pretreatment with the highest dose of 10 8 pfu of def201 resulted in 100% survival, and 10 7 and 10 6 pfu doses also significantly protected 90% and 60% of hamsters, respectively, from mortality ( figure 1a) . moreover, the hamster that succumbed in the 10 7 group, survived 19 days. importantly, only one out of ten hamsters treated with 10 8 pfu of the control rad ev virus survived the infection; however, there did appear to be a slight delay in the time to death in the hamsters that received the control virus treatment. the weights of the hamsters were measured every 3 days to assess weight gain over the course of the experiment as a marker of well being ( figure 1b) . notably, from day 3 to day 6, a time before weight loss due to illness from picv infection would have been expected, hamster weights decreased as the dose of def201 increased. this would suggest that the higher treatment doses may have resulted in some loss of appetite, probably due to mild illness due to expression of consensus ifn since no overt effects were noticeable when handling the animals. the hamsters that received the 10 6 pfu dose of def201 gained weight through day 6 similarly to the animals treated with saline placebo and the normal controls (sham-infected, untreated) ( figure 1b) . the high-dose of rad ev control virus also resulted in a slight reduction in weight compared to the controls, suggesting that the immune response to the adenoviral vector alone may have caused some malaise in the animals. there was no elevation in serum alt levels on day 7 of infection in samples collected from parallel treated and infected hamsters receiving def201 (figure 2a ). eighty percent of the rad ev group and 100% of the pbs placebo group had elevated levels of alt, reflective of liver disease. interestingly, the 10 7 and 10 8 pfu def201 groups presented with little to no day-7 virus burden in the serum, liver, or spleen, while the 10 6 group developed viral titers that were comparable to the rad ev and placebo controls ( figure 2b-d) . a delay in the development of liver disease in the 10 6 pfu def201treated animals may explain the reduced alt levels. alternatively, saturation of liver virus titers in the low-dose def201, rad ev, and placebo groups may have masked a substantial difference between the former and the viral vector and vehicle control groups. we next evaluated the prophylactic window of protection against picv infection using the 10 8 pfu dose of def201. animals were treated one or two weeks prior to challenge with a lethal dose of picv. consistent with the trend observed in initial dose titration study, hamsters treated with the 10 8 pfu dose of def201 had significantly reduced weights compared to those that received the rad ev and placebo control treatments ( figure 3a) . nevertheless, the pretreatment with def201 seven days before infection was highly protective (90% survival rate; figure 3b ). notably, the single hamster that failed to survive the challenge succumbed on day 5, which was several days before the mean time to death measured in both the placebo and rad ev groups. an autopsy to determine the cause of death was not performed. in hamsters treated two-weeks prior to picv challenge, def201 significantly reduced mortality (50% survival) and extended the time of death in the animals that succumbed ( figure 3c ). in contrast, uniform lethality was seen with animals that received the rad ev and placebo treatments. of the 5 surviving animals pre-treated with def201, one was anorexic at the conclusion of the study on day 28 post-infection. this was reflected by a 27% weight loss compared to the animals starting weight. it is possible that this hamster, which appeared ill and lethargic, was not able to completely prevent the infection. it was unclear whether it would have ultimately recovered if the observation period had been extended. on both the 7-day ( figure 4a , c, e, g) and 14-day ( figure 4b , d, f, h) pretreatments, def201 significantly reduced day-7 viral loads and liver disease (alt) compared to the controls. the absence of elevated alt levels in the def201-treated hamsters may be explained by the 2-3 log 10 reduction in liver virus burden ( figure 4e , f) and a delay in the development of liver disease. although tissue titers were slightly lower when def201 was given 7 days prior to challenge compared to the 14 day pretreatment, this was not evident with serum viral burden. because most animals had measurable replicating picv ( figure 4c-h) , it is likely that survivors would have been immunized and protected from subsequent challenge. this may not be the case with hamsters treated with def201 24 h prior to challenge since most had no detectable virus titers in spleen, liver, or serum on day 7 of picv infection ( figure 2b-d) . having observed dramatic protection when administered up to 2 weeks prior to challenge, a final experiment was conducted to determine the therapeutic value of def201 in the hamster picv infection model. when def201 was administered 6 or 24 h after challenge, highly significant protection was observed ( figure 5 ). efficacy waned when def201 treatment was delayed to 48 h postinfection. as anticipated, the treatment given 24 pre-challenge verified previous activity, with all animals surviving challenge. interestingly, there was higher than expected survival with the control rad ev treatments initiated 24 and 48 h post-challenge, suggestive of a slight antiviral effect as the time of treatment was further delayed ( figure 5) . the surviving hamsters from this experiment were re-challenged with picv to assess the ability of def201 to enhance longer-term protection via acquired immunity. with the exception of 4 animals in the 24 h def201 pretreatment group, and a single animal in the rad ev 48 h group, all animals that were challenged with picv on day 0 of the experiment survived a second challenge figure 3 . def201 extended pre-exposure prophylaxis protects hamsters from lethal picv challenge. animals were treated i.n. with a single dose 10 8 pfu of def201, the rad ev control virus, or pbs placebo 7 or 14 days prior to picv infection. animal weights were measured two weeks prior to, and at the time of, picv challenge. the effect of 7-day and 14-day pretreatments on a) weight change over the two-week period prior to picv challenge and the extended picv prophylaxis efficacy data for the b) 7-day and c) 14-day pretreatments are shown. ***p,0.001 compared to respective placebo-treated animals. b p,0.01, c p,0.001 compared to respective rad ev-treated animals. doi:10.1371/journal.pone.0026072.g003 on day 28 ( figure 5 ). all six naïve animals that were initially shaminfected succumbed as expected. in animals that were sacrificed on day 7 relative to the first infection, reductions in alt and viral titers were most evident in the groups that received def201 within 24 h of infection ( figure 6) . notably, in the animals treated with the control rad ev, there was an interesting trend that developed with the 6 h post-infection group having the greatest alt levels and viral titers, followed by the 24, 48, and 224 h groups. this trend may suggest a low-level immune stimulation in the hamsters relative to the time at which the rad ev was given. the resulting lack of measurable viral replication in the 224 h def201 group ( figure 6b-d) is likely insufficient to elicit immunological memory. it is unclear as to why one of the first infection survivors from the 48 h rad ev group ultimately succumbed to the second infection. in the present study, our findings demonstrate that expression of cifn-a following a single i.n. administration of def201 offers a strong protective effect in hamsters against challenge with picv that included limiting liver disease and inducing an antiviral state that inhibited systemic and tissue viral replication. the lack of significant antiviral activity elicited by the rad ev control virus suggests that the enhanced antiviral response produced by def201 is largely due to the expression of the cifn-a gene. the weak stimulatory effect seen in 1 of the 3 experiments was not surprising considering the number of host systems that play a role in sensing the adenovirus vector [24] ; however, the effect was short-lived. in contrast, the enhancement of the host antiviral defenses by def201 was long-lasting with a 14-day pre-picv challenge prophylactic window. moreover, def201 was effective when given 1-2 days post-picv infection. these data also suggest that sufficient viral replication may be necessary to elicit an adaptive immune response that confers lasting protective immunity, as, for the most part, only re-challenged animals from the 24 h def201 pretreatment group succumbed to a second challenge with a lethal picv inoculum. presumably, the robust innate immunity and antiviral state induced by the def201 pretreatment rapidly controlled the ,5 pfu challenge dose obviating the development of the adaptive immune response and immunological memory. the pathogenic arenaviruses have evolved strategies to suppress and evade the host immune response [25, 26, 27, 28] , resulting in uncontrolled replication and broad dissemination. however, they appear to be unable to block the induction of ifn stimulated genes via exogenous type i ifn [29] , which may, in part, explain the success of def201 and cifn-a treatments [15] . also essential to the success of def201 was early intervention prior to significant viral replication and engagement of innate immune suppressive functions. indeed, early induction of a strong type i ifn response is associated with favorable disease outcome in nonhuman primates challenged with lassa virus [30] . early post-exposure prophylaxis was also required with exogenous cifn-a protein administered by the i.p. route [15, 16] . with the multiple strategies that arena and other pathogenic viruses have in place to subdue the ifn-mediated host antiviral response [31] , the utility of def201, recombinant ifn proteins, and ifn inducing agents will depend upon the nature of the ifn pathway blockade and require early administration to be effective post-exposure. notably, with daily cifn-a protein injections of up to 40 mg/kg, significant protection was observed; however, survival rates did not exceed 80% in those studies employing the same picv hamster model system and virus stock [15, 16] . in contrast, def201 consistently elicited greater protection (90-100%). the improved efficacy observed with def201 may be explained by a combination of factors that includes constitutive expression of fully glycosylated protein and reduced animal stress levels by avoiding daily injections for 7-10 days. we hypothesize that with the appropriate dose of def201, therapeutic levels of consensus ifn-a can be maintained, effectively eliminating the daily bolus effect produced by i.p. injections. in addition, because cifn-a is produced in genetically engineered escherichia coli, the native glycosylation pattern is lost. conceivably, enhanced immunotherapeutic activity results from fully glycosylated cifn-a expressed from cells transduced with def201. previous studies in mice with a related def201 virus expressing mouse ifn-a (mdef201) have shown the utility of adenovirusbased system to counter viral infections [20, 32, 33] . more recently, in a different hamster model of viral hemorrhagic fever, several of us reported on efficacy of def201 in mitigating yfv infection and disease [19] . yfv infection appears to be more sensitive to the effects of def201, as a lower dose was able to provide complete protection. taken together with the results of the present study, the experimental animal data support the broad use of def201 for extended pre-exposure and early post-exposure prophylaxis applications. further investigations using advanced arenavirus models based on challenge of nonhuman primates with pathogenic arenaviruses [17] are needed to better evaluate the potential of def201 to prevent severe disease in humans. nonhuman primate models should allow the full spectrum of cifn-a activity not possible in hamsters or guinea pigs. the familiarity of the fda with adenovirus gene delivery technology and approved cifn-a protein support the development of def201 for clinical use. an important step in the development process is the safety/toxicology testing in rodents, which is presently underway. in our studies, the highest dose of 10 8 pfu of def201 administered by the i.n. route appeared to be well-tolerated in hamsters despite evidence of weight loss. they did not appear visibly ill, but clearly the treatment was having some effect that possibly led to reduced food and water consumption consistent with mild toxicity seen with ifn-a therapy. the i.n. delivery route is designed to circumvent pre-existing immunity to adenovirus type 5 in humans [21, 34] , and may limit systemic inflammation that could occur by parenteral administration of large numbers of adenovirus particles. ultimately, the production of a shelf-stable, powdered formulation of def201 for easy i.n. administration and long-term storage would be ideal for stock-piling in the event of the need for mass distribution due to intentional release or (re)emerging disease outbreaks of arena or other viral etiology. nosocomial outbreak of novel arenavirus infection, southern africa treatment of argentine hemorrhagic fever new opportunities for field research on the pathogenesis and treatment of lassa fever venezuelan hemorrhagic fever: clinical and epidemiological studies of 165 cases arenaviridae: the viruses and their replication the counter-bioterrorism research agenda of the national institute of allergy and infectious diseases (niaid) for cdc category a agents efficacy of immune plasma in treatment of argentine haemorrhagic fever and association between treatment and a late neurological syndrome lassa fever. effective therapy with ribavirin review of the literature and proposed guidelines for the use of oral ribavirin as postexposure prophylaxis for lassa fever ribavirin for lassa fever postexposure prophylaxis brief report: treatment of a laboratory-acquired sabia virus infection animals were treated as described in figure 5 and sacrificed on day 7 of infection for analysis of serum (a) alt and (b) virus titer, and (c) liver and (d) spleen viral titers. *p,0.05, ***p,0.001 compared to rad ev-treated animals treatment of bolivian hemorrhagic fever with intravenous ribavirin interferon-alpha as an immunotherapeutic protein interferon alfacon-1 protects hamsters from lethal pichinde virus infection combinatorial ribavirin and interferon alfacon-1 therapy of acute arenaviral disease in hamsters animal models of highly pathogenic rna viral infections: hemorrhagic fever viruses enhanced circulating half-life and antitumor activity of a site-specific pegylated interferonalpha protein therapeutic treatment of yellow fever virus with an adenovirus-vectored interferon, def201, in a hamster model pre-and post-exposure protection against western equine encephalitis virus after single inoculation with adenovirus vector expressing interferon alpha nasal delivery of an adenovirus-based vaccine bypasses pre-existing immunity to the vaccine carrier and improves the immune response in mice vitro and in vivo activities of t-705 against arenavirus and bunyavirus infections a simple method of estimating fifty percent endpoints recognition of virus infection and innate host responses to viral gene therapy vectors human macrophages, but not dendritic cells, are activated and produce alpha/beta interferons in response to mopeia virus infection differential inhibition of type i interferon induction by arenavirus nucleoproteins short double-stranded rnas with an overhanging 59 ppp-nucleotide, as found in arenavirus genomes, act as rig-i decoys z proteins of new world arenaviruses bind rig-i and interfere with type i interferon induction inhibition of the type i interferon response by the nucleoprotein of the prototypic arenavirus lymphocytic choriomeningitis virus early and strong immune responses are associated with control of viral replication and recovery in lassa virus-infected cynomolgus monkeys type 1 interferons and the virus-host relationship: a lesson in detente singledose intranasal administration with mdef201 (adenovirus vectored mouse interferon-alpha) confers protection from mortality in a lethal sars-cov balb/c mouse model alpha interferon as an adenovirus-vectored vaccine adjuvant and antiviral in venezuelan equine encephalitis virus infection comparative seroprevalence and immunogenicity of six rare serotype recombinant adenovirus vaccine vectors from subgroups b and d key: cord-005393-rhji4io9 authors: popko, brian; corbin, joshua g.; baerwald, kristine d.; dupree, jeffrey; garcia, annie m. title: the effects of interferon-γ on the central nervous system date: 1997 journal: mol neurobiol doi: 10.1007/bf02740619 sha: doc_id: 5393 cord_uid: rhji4io9 interferon-gamma (ifn-γ) is a pleotropic cytokine released by t-lymphocytes and natural killer cells. normally, these cells do not traverse the blood-brain barrier at appreciable levels and, as such, ifn-γ is generally undetectable within the central nervous system (cns). nevertheless, in response to cns infections, as well as during certain disorders in which the cns is affected, t-cell traffic across the blood-brain barrier increases considerably, thereby exposing neuronal and glial cells to the potent effects of ifn-γ. a large portion of this article is devoted to the substantial circumstantial and experimental evidence that suggests that ifn-γ plays an important role in the pathogenesis of the demyelinating disorder multiple sclerosis (ms) and its animal model experimental allergic encephalomyelitis (eae). moreover, the biochemical and physiological effects of ifn-γ are discussed in the context of the potential consequences of such activities on the developing and mature nervous systems. interferon-gamma (ifn-?) was originally discovered by wheelock (1965) as an activity that interfered with viral replication. tlymphocytes and natural killer cells are the only cells known to be capable of expressing this cytokine (reviewed in trinchieri and perussia, 1985) . because under normal condi-under these conditions, neuronal and glial cells that normally do not encounter ifn-',v are exposed to the potent, pleotropic effects of this cytokine. there is considerable evidence that suggests that the effects of ifn-?~ are important in a variety of cns disorders (reviewed in ransohoff and benveniste, 1996) . in this article, we discuss various molecular, biochemical, cellular, and physiological properties of ifn-~, and its evoked response. moreover, the potential role this cytokine plays in cns disorders is discussed. a considerable amount of information has been learned concerning the molecular biology and biochemistry of ifn-~, since the human and mouse cdnas were cloned over a decade ago (gray et al., 1982; goeddel, 1982, 1983) . the human and mouse proteins are 143 and 134 amino acids in length, respectively, and are encoded for by four exons that reside on 6 kb of dna. interestingly; the human and mouse proteins share only limited homology, approx 40% at the amino acid level, and do not bind to the other's receptor to an appreciable extent (reviewed by farrar and schreiber, 1993) . both proteins contain two glycosylation sites (gray and goeddel, 1982; ealick et al., 1991) , although glycosylation is not essential for ifn-'i activity (kelker et al., 1983) . one factor that may play a role in regulating the biological activity of ifn-y is message stability, since the ifn-y mrna contains an au rich sequence in the 3'-untranslated region that has been shown to reduce [he mr_na halfqife of other cytokines dramatically (shaw and kamen, 1986) . biologically active ifn-~/from both human and mouse is a noncovalently bound homodimer that contains two receptor binding sites . the ifn-1 receptor is a multimeric receptor complex composed of a ligand binding subunit (a-chain) (aguet et al., 1988 ) and a transmembrane, accessory factor (~-chain) (soh et al., 1994; hemmi et al., 1994) . the c~-chain is a ubiq-uitously expressed, glycosylated, cell-surface protein that binds ifn-~ , with high affinity (reviewed by farrar and schreiber, 1993) . the extent of glycosylation is variable depending on cell type and accounts for the range of sizes of the a-chain, between 80 and 95 kda. on binding, ifn-7 induces (r-chain dimerization, which is believed to be critical to the initiation of the signal transduction cascade (greenlund et al., 1993; fountoulakis et al., 1992) . this feature of the ifn-7 receptor has been exploited to generate mutant forms that confer ifn-3, unresponsiveness to cells in a dominant manner (dighe et al., 1993 (dighe et al., , 1995 . the crystal structure of the complex between ifn-y and the a-chain of its receptor has recently been reported (walter et al., 1995) . the ~-chain of the receptor, which associates with the or-chain in an ifn-,f-dependent manner, is essential for the induction of the ifn-7 signaling cascade (bach et al., 1996) . recently, considerable progress has been made in the elucidation of the ifn-,~-induced signal transduction pathway (sadowski et al., 1993; darnell et al., 1994; shuai, 1994; vilcek and oliveira, 1994; david, 1995; schindler, 1995) . the janus kinases jak1 and jak2 become phosphorylated following binding of ifn-7 to its receptor, with which the jak kinases associate (muller et al., 1993; wafting et al., 1993) . the jak1 kinase appears to associate with the a-chain, and the jak2 kinase associates with the [3-chain (sakatsume et al., 1995) . following activation, the jak kinases phosphorytate the transcriptional factor known as signal transducer and activator of transcription (stat-i~z), w~hich binds to a specific dna element referred to as ~,-activation site (gas) . stat-la binding results in the rapid transcriptional induction of genes containing the gas element. for example, the genes encoding the gaunylate binding protein and the igg fc receptor are induced in this way: other genes that are stimulated by ifn-~/, such as the class i and class ii molecules of the major histocompatibility complex (mhc), require a longer period (several hours) before transcriptional induction is detected. these genes lack the gas element and are likely activated through ifn-?-induced intermediates (benveniste and benos, 1995) . (summarized in table 1) ifn-? plays a critical role in the regulation of the immune response (young and hardy, 1995) . for this reason, it is often referred to as immune interferon. ifn-y facilitates the stimulation of the ~1 subpopulation of t-lymphocytes, which control cell-mediated immunity and which, in fact, express ifn-y; whereas it impedes the stimulation and activity of the th2 subpopulation of t-cells, :which express il-4 and are involved in regulating humoral immu-ni~ (gajewski et al., 1989; swain et al., 1991; paul and seder, 1994; seder and paul, 1994; reiner and seder, 1995) . cytotoxic t-cells and natural killer cells are also activated by ifn-7 (trinchieri and perussia, 1985) . ifn-? also plays a role in regulating antibody production, promoting an igg2a response through a direct effect on b-cells (snapper and paul, 1987) . importantly, ifn-? is a potent stimulator of expression of the antigen-presenting components of the immune system. mhc class i and ii molecules are dramatically upregulated in the presence of ifn-'y on a variety of cell types (reviewed in vilcek et al., 1985; benveniste and benos, 1995) . ifn-? is also a powerful effector molecule of the inflammatory response (reviewed in schreiber and celada, 1985) . ifnq,-stimulated macrophages release a number of inflammatory cytokines, including tumor necrosis factor-alpha (tnf-a), il-1, il-6, and il-8. in addition, ifn-? increases the microbicidal activities of macrophages through the induction of the synthesis of hydrogen peroxide and nitric oxide . ifn-? is also a potent stimulator of the expression of the fc receptors for igg immunoglobins on macrophages (erbe et al., 1990) . all of these actions of ifn-? on macrophages serve to stimulate the cytocidal activity of these cells. multiple sclerosis (ms) is the most common human demyelinating disease that affects the cns. although the etiology of ms has not been established, it is widely believed that immunological mechanisms are involved (reviewed in martin et al., 1992; raine, 1994a,b) . re cns is immunologically distinct owing to the presence of the blood~brain barrier (crone, 1986) , which inhibits entry of both immune cells and humoral factors. in ms patients, this barrier is disrupted (martin et al., 1992) allowing increased access of blood components (cellular and noncellular) to the cns. the enhanced permeability of the blood-brain barrier is thought to be a contributing factor in immunemediated demyelinating disorders (reviewed in poser, 1987 poser, , 1993 . importantly, there is an increased infiltration of t-lymphocytes (cd4+ and cd8+) into the cns of ms patients (martin et al., 1992; utz and mcfarland, 1994) , with cd4+ (t-helper) t-cells being predominant at the sites of active demyellnation (booss et al., 1983; traugott et al., 1983) . t-cell responses to a variety of myelin antigens, including myelin basic protein (mbp) and proteolipid protein (plp), have been identified in ms patients (reviewed in miller et al., 1995) . experimental allergic encephalomyelitis (eae) is the primary animal model used in the study of ms (reviewed in alvord et al., 1984; zamvil and steinman, 1990; martin and mcfarland, 1995) . eae can be induced in a variety of laboratory animal species by immunization with either cns tissue, myelin, or volume i4, 1997 myelin components. as demonstrated through passive transfer studies, eae is a t-cell (cd4+, thl) mediated disease model (zamvil and steinman, 1990; voskuhi et al., 1993) . a relapsing/remitting form of eae is induced at high incidence in sjl mice immunized with an encephalitogenic epitope of plp (tuohy et al., 1988a (tuohy et al., ,b, 1989 (tuohy et al., , 1992 mcrae et al., 1992) . this model of eae exhibits many of the clinical, pathological, and immunological feat~ares of ms. the t-cells that infiltrate into the cns in ms and eae are activated and produce ifn-i, which is believed to play a role in the pathogenesis of irnmune-rnediated demyelinating disorders (reviewed in hartung et al., 1992; olsson, 1992; panitch, 1992 ). there is considerable circumstantial evidence that suggests that ifn-~, is a key mediator of inflammation in ms and eae. many of the pathological events observed in ms and eae, such as increased mhc expression, macrophage activation, increased expression of leukocyte adhesion molecules on blood-brain barrier endothelial cells (olsson, 1992) , and reactive gliosis (balasingam et al., 1994) , are consistent with the known effects of ifn-~,. ifn-7 is detected in active ms lesions (traugott and lebon, 1988) , and increased numbers of ifn-~,-secreting tlymphocytes are present in the cerebrospinal fluid of ms patients (olsson et al., 1990) . beck et al. (198ff) reported a cor~iation between the onset of clinical symptoms of ms and mitogenstimulated ifn-y production. moreover, eae can be transferred to naive animals using ifn-~,-secreting tqymphocytes isolated from animals experiencing eae (zamvil and stelnman, 1990) , and a significant increase in the level of ifn-?, mrna is observed in the cns of animals with severe eae compared to animals with mild eae (renno et al., 1994) . perhaps the best direct evidence for the deleterious effects of ifn-y in ms came inadvertently from a small clinical trial in the early 1980s. of 18 ms patients receiving ifn-~/, seven experienced exacerbations during the 4-wk treatment period, which was significantly higher than the pretreatment exacerbation rate (panitch et at., 1987; panitch, 1992) . not only did this study clearly eliminate ifn-~/as a potential therapeutic agent for ms, but it also demonstrated that ifn-~, activity can lead to a wo~,ens of tlne disease course, suggesting a role for this cytokine in normal disease progression. there are, however, contradictory experimental data concerning the role of ifn-~, in eae. the administration of antibodies to ifn-~, e~anced the severity of eae in mildly susceptible mice (billiau et al., 1988; voorthuis et al., 1990; duong et al., 1992) , and resulted in eae susceptibility in resistant strains (duong et al., 1994) . moreover, treatment of sjl/j mice, which are very sensitive to eae, with ifn-~, results in enhanced survival (billiau et al., 1988) . nevertheless, the protective effect of ifn-~, is not observed when eae is passively transferred. voorthuis et al. (1990) reported that intraventricular administration of ifn-~/ into eae-induced rats resulted in complete suppression of clinical signs, and willenborg et al. (1995) , using a viral delivery system, suggested that ifn-y had no effect on disease. recently, ferber et al. (1996) examined the eae susceptibility of b10.pl mice that contained an experimentally inactivated ifn-~, gene (dalton et al., 1993) . b10.pl mice are normally very susceptible to mbp-induced eae, and the ifn-?, ka~ockout mice appear equally sensitive to disease induction. although morphological data were not presented, this work dearly demonstrates that ifn-~, is not essential for eae induction, at least not in b10.pl mice. to understand better the effects of ifn-u on cns cells in vivo, in the absence of other t-cellderived factors, a variety of approaches have been used to deliver this cytokine to the cns. the direct injection of ifn-~, into various animal models has been shown to upregulate the volume 14, 1997 expression of both mhc class i and class ii glycoproteins on various neuronal celt types that do not normally express these proteins at detectable levels. wong et al. (1984) demonstrated a dramatic increase in the expression of the mhc class i glycoprotein in astrocytes, neurons, oligodendrocytes and microglia following the delivery of ifn-y to the cns of 2-dold mice. direct injection of ifn-y into the cns has also been demonstrated to increase the expression of mhc class i on rat endothelial and ependymal cells and class ii on microglia, ependymal, and perivascular cells (sethna and lampson, 1991) . continuous iv infusion of ifn-~f into lewis rats for a period of 3 d induced mhc class ii expression on microglia, ependymal and endothelial cells of large blood vessels (steiniger and van der meide, 1988) . these findings support the earlier work of momburg et at. (1986) who reported that the iv infusion of ifn-y into mice resulted in class ii expression in many organs, including the brain, where "round cells in the vicinity of meningeal blood vessel," presumably microglia, were class ii immunoreactive. ifn-y has also been suggested to play a role in the breakdown of the blood-brain barrier and the recruitment of inflammatory cells into the cns. seth_ha and lampson (1991) reported that a single injection of ifn-y into the rat basal ganglia induced the infiltration of cd4+ t-cells into the perivascular space and monocytes/ macrophages, and presumptive natural killer cells into the brain parenchyma. these authors suggested that such cellular recruitment may be facilitated by ifn-y-induced changes in the endothelial cells, resulting in an alteration in the permeability of the blood-brain barrier. moreover, simmons and willenborg (1990) , using direct injection of ifn-~. into the spinal cord of rats, reported an inflammatory response similar in pattern to that observed in early eae with the accumulation of inflammatory cells in the meninges and around spinal cord veins also suggestive of a compromised blood-brain barrier. tjuvajev et ai. (1995) reported an ifn-y-induced breakdown of the blood-brain barrier in an in vivo brain tumor model, and huynh and dorovini-zis (1993) demonstrated that brain endothelial cells undergo dramatic morphological, functional, and permeability changes in response to ifn-y, suggesting that this cytokine alters bloodbrain barrier function. steffen et al. (1994) demonstrated increased expression of vascular cell adhesion molecules (vcam) and intercellular adhesion molecule-1 (icam-1) on the brain endothelium of sjl mice experiencing eae, and antibodies to these molecules inhibited binding of peripheral lymphocytes to inflamed brains. furthermore, mccarron et al. (1993) showed that ifn-y significantly increased the adhesion of mgp-specific encephatitogenic t-ceils to mouse cerebrovascular endothelial cells, and that this effect correlated with the induction of icam-1 expression by the endothelial cells. the studies discussed above consistently indicate a role for ifn-y in the inducement of expression of mhc and cell adhesion molecules, and the recruitment of inflammatory cells; however, the infusion of ifn-y into the cns has also been implicated in a variety of other processes. for example, yong et al. (1991) reported that the direct administration of ifn-~/ into the adult mouse brain following corticectomy resulted in an increase in trauma-initiated gliosis. brosnan et al. (1989) , using the visual pathway of the rabbit, demonstrated that the injection of ifn-y significantly reduced neural conduction and induced subtle axonal pathology. in addition, the simultaneous injection of ifn-7 and a myelin/oligodendrocyte glycoprotein antibody significantly delayed cervical somatosensory evoked potentials, and caused massive demyelination in the spinal cord (vass et al., 1992) . vass et al. (1992) also reported an ifn-y-induced increase in mhc expression, which accompanied spinal cord demyelination. in complement with various delivery systems previously discussed, we have used a transgenic approach to examine the effects of ifn-y on the developing nervous system, (corbin et al., 1996) . transgenic mice were generated in which the expression of ifnq was volume 14, i997 placed under the transcriptional control of the mbp gene (mbp/ifn-y transgenic animals). transgenic mice generated with tl-ds construct have a shaking/shivering phenotype that is similar to that observed in naturally occurring mouse models of hypomyelination (e.g., sb_iverer, jimpy, quaking), and these transgenic animals have dramatically less cns myelin than control animals. reactive gliosis and increased macrophage/microglia f4/80 immunostaining were also observed. additionally, mhc class i and class ii mrna levels were increased in the cns of mbp/ifn-y transgenic mice, and the increase in mhc class i mrna expression was detected in both white and gray matter regions. surprisingly, cerebelfar granule cell migration was also disrupted in these animals. these results strongly support the hypothesis that ifn-~, is a key effector molecule in irnmtme-mediated demyelinating disorders, and suggest that the presence of this cytokine may also disrupt the cytoarchitecture of the developing nervous system. the mechanism by which ifn-7 exerts its action within the cns remains unresolved. one idea concerning the potential cellular site of action of ifn-y in cns demyelinating disorders is that ifn-1 activates macrophages/ microglial cells, which in turn mediate cytotoxic effects on oligodendr~ytes (fig. 1) . tnf-(z, which has previously been shown to be toxic to oligodendrocytes in vitro, is expressed by ifn-y-activated macrophages/microglia (selmaj et al., 1991) . more recent in vitro experiments suggest that the microglial cell toxicity to otigodendrocytes is mediated through cellsurface expression of tnf-cz and the production of nitric oxide (merrill et al., 1993) . nitric oxide is very labile, such that to elicit their cytotoxic effects, the activated microglia need to be in intimate contact with the target oligodendrocytes. oligodendrocytes undergo necrotic cell death following exposure to nitric oxide (mitrovic et al., 1995) . in support of the hypoth~ esis that nitric oxide is important in the pathogenesis of immune-mediated demyelinating diseases, it has been shown that nitric oxide synthase mrna levels are increased in mice experiencing eae (koprowski et al., 1993) , and nadph-diaphorase histochemical staining, a marker for nitric oxide production, as well as nitric oxide synthase rnrna levels were found to be elevated in the brains of ms patients (b6 et al., 1994) . ifn-y may also have a direct deleterious effect on oligodendrocytes (fig. i) , which have been shown to express ifn-7 receptors (torres et al., 1995) . we have examined the effects of ifn-7 on oligodendrocytes using the moch-1 cell line, which was derived from an oligodendroglioma that developed in a transgenic line of mice and expresses a variety of features of oligodendrocytes (hayes et al., 1992; li et al, 1995) . when moch-1 cells are cultured in medium containing low concentrations of volume 14, 1997 serum (1% fetal bovine serum [fbs]) they extend multiple, thin, branched processes and have phase-bright cell bodies, which are typi ~ cal features of cultured primary oligodendrocytes (reviewed in popko et al., 1994) . furthermore, these cells are immunoreactive against antibodies to galactocerebroside, a glycolipid marker of mature oligodendrocytes, and abundantly express myelin protein genes (hayes et al., 1992) . mochq cells cultured in 1% fbs or chemically defined medium do not express detectable levels of the astrocyte marker glial fibrillary acidic protein (gfap). when moch-1 cells are cultured in 1% fbs medium containing ifn-7. however, they transform to flat, astrocyte-like cells with enlarged cell bodies and appear more adhesive ~ in addition to these morphological changes, ifn-1 stimulates the expression of abundant levels of the astrocyte marker gfap, as well as slightly elevated levels of mbp and plp mrna. ifn-7 also induces an enormous increase of mhc class i expression, as well as a comparatively modest increase of mhc class ii mrna expression in mochq cells. the morphological and molecular changes mc)ch-1 cells undergo in response to ifn-7 suggest that ifn-7 may induce a direct effect on oligodendrocytes, or a subpopulafion of these cells, in vivo . recent studies further support the hypothesis that ifn-y has a direct, deleterious effect on oligodendrocytes. using a morphological assay, oligodendrocytes in vitro were shown to undergo apoptotic cell death at very high frequency following exposure to relatively low concentrations of ifn-7 (vartanian et al., 1995) . the addition of rnicroglia to the culture did not significantly increase the frequency of otigodendrocyte death, further supporting a direct effect of the cytokine. interestingly, vartanian et ai. (1995) also detected apoptotic oligodendrocytes, along with ifn-7, in the advancing margin of active ms plaques, but not in the chronic portion of the plaque or in adjacent unaffected white matter. agresti et al. (1996) have also observed deleterious effects of ifn-y on cultured oligodendrocytes. in the absence of cell death, they observed a decrease in the ability of oligodendrocyte progenitors to divide and differentiate, as well as changes in myelin protein gene mrnas and a general metabolic depression. these effects were shown to be fully reversible following cytokine withdrawal (agresti et al., 1996) . the harmful effects of ifn-y on myelination might be mediated through the induction of mhc expression in oligodendrocytes, which normally do not express these molecules at an appreciable level. in support of this possibility, transgenic mice in which mhc class i expression has been targeted to oligodendrocytes are severely hypomyelinated (turnley et al., 1991b; yoshioka et al, 1991; turnley and morahan, 1995) . this hypomyelh~ation occurs in the absence of immune celt infiltration, suggesting that mhc class i expression in oligodendrocytes is sufficient to disrupt myelination. recently; power et al. (1996) examined these transgenic animals in more detail and demonstrated that cell-surface expression of mhc class i in oligodendrocytes of these mice was minimal moreover, immunocytochemistry for f~2-microglobulin, which is required for cellsurface expression of mhc class i, revealed that expression of ~2-microglobulin was undetectable in oligodendrocytes and limited to microglia in lhe cns. power et al. (1996) suggest that the accumulation of mhc class i protein m the cytoplasm of the oligodendrocytes, owing to the absence of f~2-microglobulin, may interfere with normal myelin protein trafticking, leading to the observed hypomyelination in these mice. in vitro, oligodendrocytes at all stages of differentiation can be induced by ifn-7 to express cell-surface mhc class i (suzumura et al., 1986; turnley et al., 1991a; massa et al., 1993) . nevertheless, it is unclear what the consequences of oligodendroglial expression of mhc class i are. as mentioned previously, cd8+ t-cells are present in the cns of ms patients (martin et al., 1992; utz and mcfarland, 1994) . it is, however, unknown whether oligodendrocytes in vivo can present antigen to these cells. in contrast, many investigators have searched for, but failed to demonstrate, ifn-7mediated induction of mhc class ii on oligodendrocytes in culture (wong et al., 1984; suzumura et al., 1986; turnley et al., 1991a; satoh et al., 1991b) , suggesting that oligodendrocytes are refractory to mhc class ii induction. calder et al. (1988) demonstrated that oligodendrocyte precursors (o-2a progenitor cells) can be induced to express mhc class ii by ifn-~/, but that maturation into oligodendrocytes leads to loss of inducibility. nevertheless, bergsteinsdottir et al. (1992) were able to induce mhc class ii expression in a subset of mature oligodendrocytes in culture with ifn-7 in the presence of the synthetic glucocorticoid dexamethasone. although gtucocorticoids are normal cns constituents (birmingham et al., 1984; kumar et al., 1989) , the in vivo significance of these in vitro observations is unclear. another activity of ifn-t that might be relevant to the pathogenesis of immune-mediated demyelinating disorders concerns the ability of this cytokine to stimulate the expression of mat-shock (stress) proteins. these are ubiquitously expressed proteins that are believed to play a role as chaperones in normal protein synthesis and degradation (reviewed in jindal, 1996) . the expression of these proteins increases following cell stress, and this response is thought to be protective. heat-shock proteins are highly conse~rved across divergent species and are very irnmunogenic. it has been demonstrated that there is increased heat shock protein expression in ms and eae lesions, and importantly, that there is an immune response to [he stress proteins in these disorders (selmaj et al., 1992; prabhaker et al., 1994; gao et al, 1995; van noort et al., 1995) . it has also been demonstrafed that 1fn-'f is capable of potentiating the induction of stress protein synthesis (morange et al., 1986; ferm et al., 1992) , such that this effect might facilitate the induction of the immune response to the heat-shock proteins in immune-mediated disorders. the characterlzation of the heat-shock response in these disorders should provide further insight into the role these proteins play in the pathogenesis of disease. a potential direct effect of ifn-? on oligodendrocytes in immune-mediated demyelinating disorders is consistent with recent studies that examined biopsy specimens from patients with acute ms (rodriguez and scheithauer, 1994) . they observed degeneration of inner myelin loops with preservation of outer loops and axons in early plaques, as well as areas in which well-myelinated and demyelinated axons were in close proximity. these observations suggest that the early pathological lesions that occur in ms are compatible with a primary disturbance in the myelinating function of oligodendrocytes, not a nonspecific inflammatory reaction that affects the myelin sheath, as might occur if microglia were activated subsequently to phagocytose the myelin sheath. astrocytes, which have been shown to express the receptor for ifn-7 (rubio and de felipe, 1991) , respond to the presence of this cytokine in a variety of ways. as mentioned previously, mhc class i expression on astrocytes increases substantially in the presence of ifn-7 (wong et al., 1984; mauerhoff et al., 1988; massa et al., 1993) . this expression appears functional in that astrocytes are susceptible to lysis by cytotoxic t-cells in an antigen-specific manner (skias et al., 1987) . ifnq,-induced mhc class ii expression by astrocytes has also been demonstrated in vitro and in vivo (fontana et al, 1984; fierz et al., 1985; pulver et al., 1987) , although fine in vivo levels are low relative to that detected on microglial cells (reviewed in benveniste, 1992) . the biological significance of the antigen-presenting potential of astrocytes remains to be resolved in vivo. there is also evidence that suggests that ifn-7 plays a role in the reactive astrogliotic response, particularly in immune-mediated disorders. reactive astrogliosis is the response in which astrocytes express increased levels of gfap, undergo hypertrophy, and proliferate (eng, 1988) . there are dramatically elevated levels of geap expression in the cns of the volume i4, 1997 mbp/ifn-7 transgenic animals, although it is unclear whether this represents a direct response of astrocytes to i~-7 or an indirect response elicited by the myelin abnormalities that occur in these mice (corbin et al., 1996) . moreover, as mentioned, yong et al. (1991) showed that if1n<7, as well as a number of other cytokines (balasingam et al., 1994) , increased trauma-induced gliosis in mice. these investigators also demonstrate that ifn-? promotes the proliferation of adult human astrocytes in vitro (yong et al., 1991) . nevertheless, in a follow-up study, yong et al. (1992) were unable to reproduce these results using mouse astrocytes, suggesting that there are species differences in the astrocytic response to ifn-7. ifn-7 has also been shown to increase the expression of intercellular adhesion molecules on astrocytes. human fetal (frohman et al., 1989) and adult (satoh et al., 1991a) astrocytes, as well as mouse astrocytes (satoh et al., 1991b) , have been shown to express icam-1 in the presence of ifn-7. such expression might facilitate the interaction of these cells with lymphocytes. ifn-~,-stimulated human and rat astrocytes have also been shown to express vcam-1, possibly suggesting a role in lymphocytic infiltration across the blood-brain barrier into the cns (rosenman et al., i995) . although ifn-7 readily induces the expression of mhc molecules in astrocytes, oligodendrocytes, and resident cns microglia, !fn-7-induced expression of mhc molecules in neurons is under tighter control. early evidence demonstrating the ability of brain cells, including neurons, to express mhc molecules includes experiments in which ifn-y was either added to mixed brain cultures or i~ected directly into the cns (wong et al., 1984 (wong et al., , 1985 . as detected by immunohistochemical analysis, the addition of ifn-7 to brain cells in culture results in 9% of neurons expressing mhc class i, whereas direct injection into the cns results in approx 15% of neurons expressing mhc class i. moreover, the response of the olb21 neuronal cell line to ifn-? has been investigated (joly and oldstone, 1992) . the olb21 cell line was developed by retroviral-mediated oncogene delivery to cells from the olfactory bulb (ryder et al., 1990) . in these cells, ifn-7 stimulates the expression of mhc class i both at the mrna level and on the cell surface. furthermore, in the presence of ifn-7, the peptide transporters ham 1 and ham 2, which are involved in the processing of class i antigens, are induced in these cells, whereas an increase in the expression of ~2-microglobulin also occurs (joly and oldstone, 1992) . in other studies, cell lines derived from two medulloblastomas, which are immunoreactive against neurofilament antibodies, but unreactive against gfap and s-100 antibodies, have also been shown to empress mhc class i and class ii antigens following exposure to ifn-y (tamura et al., 1989) . although these studies further demonstrated that cells of neuronal origin are intrinsically capable of expressing mhc molecules and suggest that this expression may serve a functional role, the physiological conditions under which functional neuronal mhc expression can occur have not yet been fully elucidated. toward this goal, neumann et al. (1995) have combined whole-cell patch-clamp recording techniques with single-cell rt-pcr analysis to demonstrate that in the presence of ifn-7, both mhc class i and 132-microglobulin are significantly more inducible on electrically silent neurons compared to neurons spontaneously firing action potentials. this result suggests that functional mhc class i expression may occur only in neurons that are no longer biologically active. this would then allow these cells to be recognized and killed by cytotoxic t-lymphocytes, thus permitting active virus to be cleared from inactive neurons. this result also suggests that active neurons may not possess the potential to express functional mhc, thus allowing neurons to survive at the cost of continued viral infection (joly et al., 1991) . future investigation in this area will likely yield additional insight into our understanding of the functional interactions between neurons and cells of the immune system. although the majority of the studies addressing the effect of ifn-~, on neurons have been directed at examining the expression of mhc glycoproteins, several studies have reported other ifn-~,-induced neuronal consequences. as previously discussed, brosnan et al. (1989) reported a significant reduction in neural conduction and modest axonal pathology in the visual system of the rabbit following injection of ifn-,f. recently, mcmillian et al. (1995) reported that the treatment of rat primary basal forebrain mixed cultures with ifn-7 decreased the number of choline acetyl transferase (chat) immunopositive neurons, whereas the number of chat immunonegative neurons was unaffected. this selective neuronal killing was shown to be mediated through ifn-~,-induced microglia activation. moreover, birdsall (1991) demonstrated that following exposure to ifn-~[, two human neuroblastoma cell lines, but not cultured human cortical neurons, expressed icam. ifn-y also enhanced tnf-el-induced binding of these cells to neutrophils. finally, collins (1995) demonstrated that susceptibility to viral infection of a human cerebral cortical neuronal cell line was dramatically increased following treatment with human ifn-~, and that this increased susceptibility was owing to membrane expression of hla class i molecules. ifn--f has also been shown to affect neuronal differentiation in vitro. chang et al. (1990) demonstrated that ifn-~, retarded the death of cultured rat sympathetic neurons following nerve growth factor (ngf) deprivation. moreover, ifn-y has been shown to potentiate ngfstimulated differentiation of pc12 cells (improta et al., 1988) . barish et al. (1991) also demonstrated that ifn-y increased the differentiation of cultured cortical and hippocampal neurons. nevertheless, it is unlikely that the developing nervous system is exposed to appreciable levels of ifn-7 under normal conditions, such that the in vivo relevance of these observations is unclear. in contrast, there is in vivo evidence that suggests that ifn-~, might have a detrimental effect on the developing nervous system. as mentioned earlier, transgenic mice in which ifn-y was targeted to the cns demonstrate a disruption in cerebellar granule cell migration (corbin et al., 1996) . during development, cerebellar granule cells migrate from the external granule cell layer (egl), through the molecular layer, to the internal granule cell layer (igl) along processes extended by radial gliai cells (chuong, 1990) . in transgenic mice expressing ifn-? within the cns, many granule cells are still observed in the egl and in the molecular layer. this observation is reminiscent of other mouse mutants (e.g., weaver, staggerer) in which the interaction between granule cells and radial glia is disrupted (chuong, 1990) . perhaps the ectopic expression of ifn-~, in the cerebellum of transgenic mice has a deleterious effect on lhe interaction between granule cells and radial glia, thus disrupting the migration of these cells from the egl to the igl. further investigation is warranted to characterize the potentially deleterious developmental effects of ifn-g on the cns in more detail. recently, meda et al. (1995) suggested a role for ifn-y in alzheimer's disease. the senile plaques that develop in alzheimer's patients are characterized by the extracellular deposits of [3-amyloid protein. meda et al. (1995) demonstrated that ifn-y acted synergistically with fj-amyloid in the activation of microglia, which produce tnf-o~ and nitric oxide. in coculture experiments, it was shown that microglia activated by ifnq, and ~-amyloid caused neuronal injury. clearly, these are exciting findings that lead to the speculation that ifn-~/might also be involved in other neurodegenerative disorders. ifn-? is a multifunctional cytokine involved in regulating a variety of immune responses. this cytokine also has a multitude of effects in the cns (table 2) . although normally excluded from the cns, this cytokine is present during many disorders in which the cns is affected, including the most common human demyelinating disease, ms. the presence of table 2 effects of ifn-? on the cns disruption of myelination exacerbation of reactive gliosis disruption of the blood-brain barrier recruitment of inflammatory cells from the periphery induction of mhc expression stimulation of macrophage/microglial cells disruption of cerebellar granule cell migration this cytokine in the cns leads to an abundance of pathological effects, including decreased myelination, increased gliosis, disruption of blood-brain barrier func~on, increased mhc expression, stimulation of macrophage/microglial activation, and disruption of cerebellar granule cell migration. taken as a whole, these observations demonstrate that ifn-? can either directly or indirectly mediate many of the cns pathological effects that are observed in disorders in which immune cell traffic in the cns increases. understanding the mechanism(s) by which ifn-~,r exerts its actions on cns function will undoubtedly prove useful toward generating rational therapeutic approaches for the treatment of cns disorders in which this cytokine plays a harmful role. we thank numerous colleagues for many helpful discussions that guided the formulation of the ideas presented here. we also thank kathy toews for assistance in preparing the manuscript. the ifn-? work in brian popko's laboratory has been supported by the national institutes of health (nih) research grant roi ns34939, and the national multiple sclerosis society research grant rg 2089. j. d. is supported by a training grant hd07201 from the nih. b. p. is a recipient of an nih research career development award (ns 01637) from ninds. reversible inhibitory effects of interferon-y and tumouar necrosis factor-o: on oligodendroglial lineage cell proliferation and differentiation in vitro molecular cloning and expression of the human interferon-? receptor experimental allergic encephalomyelitis: a usefid model for multiple sclerosis ligand-induced assembly and activation of the gamma interferon receptor in intact cells reactive astrogliosis in the neonatal mouse brain and its modulation by cytokines, f neurosci gamma-interferon promotes differentia* tion of cultured cortical and hippocampal neurons increased production of interferon gamma and tumor necrosis factor precedes clinical manifestation in multiple sclerosis: do cytokines trigger off exacerbations? inflammatory cytokines within the central nervous system: sou~, traction, and mechanism of action tnf-~ and ifn-7-mediated signal transduction pathways: effects on glial cell gene expression and function in the presence of dexamethasone, y interferon induces rat oligodendrocytes to express major histcycompatibiliw complex class ii molecules enhancement of experimental allergic encephalomyelitis in mice by antibodies against ifn-y induction of icam-i on human neural cells and mechanisms of neutrophilmediated injury localization of aldosterone and corticosterone in the central nervous system, assessed by quantitative autoradiography induction of nitric oxide synthase in demyelinating regions of multiple sclerosis brains immunohistochemical analysis of t-lymphocyte subsets in the central nervous system in chronic progressive multiple sclerosis preliminary studies of cytokine-induced functional effects on the visual pathways in the rabbit the differentiation of 0-2a progenitor cells into oligodendrocytes is associated with a loss of inducibility of ia antigens interferon suppresses sympathetic neuronal cell death caused by nerve growth factor deprivation. l neurochem differential roles of multiple adhesion molecules in cell migrafion: granule cell migration in cerebellum interferon g potentiates human coronavirus ~43 infection of neuronal cells by modulation of hla class i expression targeted cns expression of interferon-gamma in transgenic mice leads to hypomyelination, reactive gliosis, and abnormal cerebellar development the blood-brain barrier; a modified fight epithelium, in the blood-brain barrier in health and disease (suckling multiple defects of immune cell function in mice with disrupted interferon-y genes i994) jak-stat pathways and transcriptional activation in response to ifns and other extracellular signaling proteins transcription factors in interferon signaling inhibition of cellular responsiveness to interferon-y (ifnt) induced by overexpression of inactive forms of the ifn7 recepton tissue-specific targeting of cytokine unresponsiveness in transgenic mice effect of anti-interferon-y and anti-interleukin-w monoclonal antibody treatment on the development of actively and passively induced experimental allergic encephalomyelitis in the sjl/j mouse i994) effect of anti-interferon-gamma monoclonal antibody treatment on the development of experimental allergic encephalomyelitis in resistant mouse strains three-dimensional structure of recombinant human ifn-y regulation of g!ial intermediate filaments in astrogliosis the effect of cytokines on the expression and function of fc receptors for igg on human myeloid cells nervous tissue as an im_mune compartment: the dialect of the immune response in the cns the molecular cell biology of interferon-y and its receptor mice with a disrupted interferon-y gene are susceptible to the induction of experimental autoimmune encephalomyelitis (eae) induction of human hsp60 expression in monocytic cell lines astrocytes as antigen-presenting cells. i. induction of ia antigen expression on astrocytes by t cells via immune interferon and its effect on antigen presentation astrocytes present myelin basic protein to encephalitogenic t-cell lines stoichiometry of interaction between interferon y and its receptor the induction of intercellular adhesion molecule 1 (icam-1) expression on human fetal astrocytes by interferon-gamma, tumor necrosis factor alpha, lymphotoxin, and interleukin-l: relevance to intracerebral antigen presentation regulation of t-cell activation: differences among t cell subsets experimental autoimmune encephalomyelitis. qualitative and semiquanfitative differences in heat shock protein 60 expression in the central nervous system. 1~ lmmunol i982) structure of the human immune interferon gene cloning and expression of murine immune interferon cdna expression of human immune interferon cdna in e. coli and monkey ceils in terferon-y induces receptor dimerization in solution and on cells inflammatory mediators in demyelinating disorders of the cns and pns expression of the neu oncogene under the transcriptional control of the myelin basic protein gene in transgenic mice: generation of transformed glial cells i994) a novel member of the interferon receptor family complements functionality of the murine interferon "i receptor in human cells migration of hematogenous cells through the blood-brain barrier and the initiation of effects of interferon-gamma on primary cultures of human brain microvesset endothelial cells ifn-gamma facilitates ngf-induced neuronal differentiation in pct2 cells heat shock proteins: applications in health and disease neuronal cells are deficient in loading peptides onto mhc class i molecules !) viral persistence in neurons explained by lack of major histocompatibility class i expression effects of glycosidase treatment on the physicochemical properties and biological actiw ity of human interferon-) in vivo expression of inducible nitric oxide synthase in experimentally induced neurologic diseases differential regulation of oligodendrocyte markers by glucocorticoids: post-h-anscriptional regulation of both proteolipid protein and myelin basic protein and transcriptional regulation of glycerol phosphate dehydrogenase morphological and molecular response of the moch-1 oligodendrocyte cell line to serum and lnterferon-y: possible implications for demyelinating disorders immunological aspects of experimental allergic encephalomyelitis and multiple sclerosis immunological aspects of demyelinating diseases cell type-specific regulation of major histocompatibility complex (mhc) class i gene expression in astrocytes, oligodendrocytes, and neurons differential expression and regulation of major histocompatibility complex (mhc) products in neural and glial cells of the human fetal brain selective killing of cholinergic neurons by microglial activation in basal forebrain mixed neuronal/glial cultures cytokine-regulated adhesion between encephalitogenic t lymphocytes and cerebrovascular endothelial cells induction of active and adoptive relapsing experimental autoimmune encephalomyelitis (eae) using an encephalitogenir epitope of proteolipid protein activation of microglial cells by beta-amyloid protein and interferon-gamma microglial cell cytotoxicity of oligodendrocytes is mediated through nitric oxide evdution of the t-cell d~ng the course of experimental imanune-mediated demyelinating disea~s nitric oxide induces necrotic but not apoptotic cell death in oligodendrocytes differential expression of ia and ia-associated invariant chain in mouse tissue after in vivo treatment with ifn-7 interferon pretreatment lowers the threshold for maximal heat-shock response in mouse cells the protein tyrosine kinase jak1 complements defects in interferon-alpha/beta andgamma signal transduction induction of mhc class i genes in neurons cytokines in neuroinflammatory disease: role of myelin autoreactive t cell production of interferon-gamma autoreactive t lymphocytes in multiple sclerosis determined by antigen induced secretion of interferon-gamma interferons in multiple sclerosis treatment of multiple sclerosis with game, ha interferon: exacerbations associated with activation of the immune system lymphocyte responses and cytokines mochq cells: an oligodendrocyte cell line generated using a transgenic approach trauma and multiple sclerosis the pathogenesis of multiple sclerosis. additional considerations major histocompatibility complex class i expression in oligodendrocytes induces hypomyelination in transgenic mice heat shock protein immunoreactivity in csf: correlation with oligoclonal banding and demyelinating disease~ neurology cultured human fetal astrocytes can be induced by interferon-gamma to express hla-dr the dale e. mcfarlin memorial lecture: the immunology of the multiple sclerosis lesion presidential address: multiple sclerosis: immune system molecule expression in the central nervous system t helper cell differentiation in immune response cytokine production by cells in cerebrospinal fluid during experimental allergic encephalomyelitis in sjl/j mice ultrastructure of multiple sclerosis cytokineinduced expression of vascular cell adhesion molecule-1 (vcam-t) by astrocytes and astrocytoma cell lines demonstration of the presence of a specific interferon-~, receptor on murine astrocyte cell surface establishment and characterization of multipotent neural cell lines using retrovirus vectormediated oncogene transfer a common nuclear signal transduction pathway activated by growth factor and cytokine receptors the jak kinases differentially associate with the ot and [5 (accessory factor) chains of the interferon i receptor to form a functional receptor unit capable of activating stat transcription factors cytokine-induced expression of intercellular adhesion molecule-1 (icam-1) in cultured human oligodendrocytes and astrocytes expression and induction of intercellular adhesion molecules (icams) and major histocompatibility complex (mhc) antigens on cultured murine oligodendrocytes and astrocytes molecular characterization of interferon gamma as a macrophage activating factor acquisition of lymphokine-producing phenotype by cd4+ t cells cytokine cytotoxicity against oligodendrocytes. apoptosis induced by lymphotoxin expression of heat shock protein-65 by oligodendrocytes in vivo and in vitro: implications for multiple sclerosis immune modulation within the brain: recruitment of inflammatory cells and increased major histocompatibility antigen expression following intracerebral injection of interferon-gamma a conserved au sequence from the 3 ~ untranslated region of gm-csf ml-hna mediates selective mrna degradation i994) interferon-activated signal transduction to the nucleus a single phosphotyrosine residue of stat91 required for gene activation by interferongamma direct injection of cytokines into the spinal cord causes autoimmune encephalomyelitis-like inflammation susceptibility of astrocytes to class i mhc antigen-specific cytotoxicity interferongamma and b cell stimulatory factor-1 reciprocally regulate ig isotype production identification and sequence of an accessory factor required for activation of the human interferon %, receptor evidence for involvement of icam-1 and vcam-1 in lymphocyte interaction with endothelium in experimental autoimmune encephalomyelitis in the cen~al nervous system in the sjl/j mouse rat ependyma and microglia cells express class ii mhc antigens after intravenous infusion of recombinant gamma interferon the expression of mhc antigens on oligodendrocytes: induction of polymorphic h-2 expression by lymphokines helper t-cell subsets: phenotype, function and the role of lymphokines in regulating their development expression of major histocompatibility complex on human medulloblastoma cells with neuronal differentiation rg-2 glioma growth attenuation and severe brain edema caused by local production of interleuk~u-2 and interferon-gamma expression of interferon-~[ receptors on murine oligodendrocytes and its regulation by cytokines and mitogens deu, non~stration of ct, t3, and ~[ interferon in active chronic multiple sclerosis lesions multiple sclerosis: distribution of t-cell subsets within active chronic lesions~ immune interferon: a pleiotropic lymphokine with multiple effects myelin proteotipid protein-induced experimen--tal allergic encephalomyelitis a synthetic peptide from mye identification of an encephalitogenic determinant of myelin proteolipid protein for sjl mice i992) myelin proteolipid protein: minimum sequence requirements for active induction of autoimmune encephalomyelitis in swr/j and sjl/j mice dysmyelination in class i mhc transgenic mice regulation of mhc molecules on mbp positive oligodendrocytes in mice by ifn-7 and tnf-a dysmyelialation in transgenic mice resulting from expression of class i histocompatibility molecules in oligodendrocytes the role oft cells in multiple sclerosis: implications for therapies targeting the t cell receptor the small heat-shock protein alpha b-crystalline as candidate autoantigen in multiple sclerosis interferon-y-induced oligodendrocyte cell death: implications for the pathogenesis of multiple sclerosis interferon-1 potentiates anti~ body-mediated demyelination in vivo recent progress in the elucidation of interferon-gamma actions: molecular biology and biological functions interferon gamma: a 1}nnphokine for all seasons suppression of experimental allergic encephalomyelitis by intraventricular administration of interferon-gamma in lewis rats t helper 1 (tftl) functional phenotype of human myelin basic protein-specific t lyrnphocytes crystal structure of a complex between interferon-gamma and its soluble high-affinity receptor complementation by the protein tyrosine kinase jak2 of a mutant cell line defective in the interferon-gamma signal transduction pathway interferon-like virus-inhibitor induced in human leukocytes by phytohernagglutinin cytokines and murine autoimmune encephalomyelitis: inhibition or enhancement of disease with antibodies to select cytokines, or by delivery of exogenous cytokines using a recombinant vaccinia virus system inducible expression of h-2 and ia antigens on brain cells interferon-gamma induces the expression of h-2 and la antigens on brain ceils r (1991) y-interferon promotes proliferation of adult human astrocytes in vitro and reactive gliosis in the adult mouse brain in vivo differential proliferafive response of human and mouse astrocytes to gala-interferon ~1) try_ ~nsgewic mouse model for central nervo~is system demyelknation role of interferon-y in immune cell regulation the t lymphocyte in experimental allergic encephalomyelitis key: cord-012909-o6t2srim authors: chaudhari, jayeshbhai; liew, chia-sin; workman, aspen m.; riethoven, jean-jack m.; steffen, david; sillman, sarah; vu, hiep l. x. title: host transcriptional response to persistent infection with a live-attenuated porcine reproductive and respiratory syndrome virus strain date: 2020-07-28 journal: viruses doi: 10.3390/v12080817 sha: doc_id: 12909 cord_uid: o6t2srim both virulent and live-attenuated porcine reproductive and respiratory syndrome virus (prrsv) strains can establish persistent infection in lymphoid tissues of pigs. to investigate the mechanisms of prrsv persistence, we performed a transcriptional analysis of inguinal lymphoid tissue collected from pigs experimentally infected with an attenuated prrsv strain at 46 days post infection. a total of 6404 differentially expressed genes (degs) were detected of which 3960 degs were upregulated and 2444 degs were downregulated. specifically, genes involved in innate immune responses and chemokines and receptors associated with t-cell homing to lymphoid tissues were down regulated. as a result, homing of virus-specific t-cells to lymphoid tissues seems to be ineffective, evidenced by the lower frequencies of virus-specific t-cell in lymphoid tissue than in peripheral blood. genes associated with t-cell exhaustion were upregulated. likewise, genes involved in the anti-apoptotic pathway were upregulated. collectively, the data suggested that the live-attenuated prrsv strain establishes a pro-survival microenvironment in lymphoid tissue by suppressing innate immune responses, t-cell homing, and preventing cell apoptosis. porcine reproductive and respiratory syndrome virus (prrsv) is a positive sense, single stranded rna virus that belongs to the family arteriviridae, under the order nidovirales [1] . based on phylogenetic analysis, prrsv was originally classified into two types: type 1 or prrsv-1, which originated in europe, and type 2 or prrsv-2, which originated in north america. the international committee on taxonomy of viruses (ictv) recently updated the arterivirus taxonomic structure in which prrsv-1 and prrsv-2 are now respectively classified as two species: betaarterivirus suid 1 and betaarterivirus suid 2 [2] . prrsv infects pigs of all ages; however, clinical manifestations are more severe when the virus infects pregnant sows and young pigs, causing reproductive failure and respiratory distress, respectively (reviewed in [3] ). prrsv is endemic in most swine producing countries worldwide, causing significant economic losses to swine producers [4] . genes involved in innate immune response and genes encoding chemokines and receptors important for t-cell homing and trafficking were downregulated. on the other hand, genes involved in the anti-apoptotic pathway and t-cell exhaustion were upregulated. functional studies revealed that the frequencies of virus-specific ifn-γ-secreting cells are lower in lymphoid tissue than in peripheral blood mononucleated cells (pbmcs). collectively, the results shed important insight into the mechanisms of prrsv persistence in the host. the attenuated prrsv strain designated con90 used in this study was recovered from an infectious cdna clone constructed using viral rna extracted from the attenuated con-p90 [27] . marc-145 cells, a monkey kidney cell line [28] , were cultured in dulbecco's modified eagle's medium (dmem) supplemented with 10% fetal bovine serum (fbs) and 1 × penicillin-streptomycin (100 units/ml of penicillin, and 100 µg/ml of streptomycin (life technologies, grand island, ny, usa). pbmcs and lymph node-derived cells were cultured in complete rpmi-1640 medium (crpmi) supplemented with 10% fbs, 1 × of glutamax-1 (life technologies, grand island, ny, usa), 100 units/ml of penicillin, and 100 µg/ml of streptomycin (sigma-aldrich, st. louis, mo, usa). mouse monoclonal antibody specific to prrsv n protein sdow17 was purchased from the national veterinary services laboratories (ames, ia, usa). anti-porcine cd3ε (clone bb23-8e6-8c8; fitc-conjugated), anti-porcine cd4 (clone 74-12-4; pecy7 -conjugated), anti-porcine cd8 (clone 76-2-11; alexa flour 647 -conjugated), and anti-porcine ifn-γ (clone p2g10; pe-conjugated), were purchased from bd biosciences (san diego, ca, usa). alexa flour 488-conjugated goat anti-mouse igg was purchased from invitrogen (eugen, or, usa). the pig experiment conducted in this study was approved by the university of nebraska-lincoln (unl) institutional animal care and use committee under the protocol number 1360. ten 3-week-old prrsv, porcine circovirus type 2 (pcv2), and siv negative pigs were purchased from the midwest research swine (glencoe, mn, usa). the pigs were randomly assigned to two groups of five pigs, which were accommodated in two separate rooms in the animal biosecurity level 2 (absl-2) research facilities at unl. after 1 week of acclimation, pigs in group 1 were injected with dmem to serve as negative controls, whereas pigs in groups 2 were inoculated intramuscularly with 10 5.0 tcid50 of live-attenuated prrsv strain con90. whole blood samples with anticoagulant edta were collected to obtain plasma and pbmcs. plasma samples were stored at −70 • c for measurement of viremia and antibody response. a portion of freshly isolated pbmcs were used for measurement of t cell responses while the rest of pbmcs were cryopreserved for future analysis. at 46 days post-infection (dpi), sample of inguinal lymph node (iln) was aseptically collected from the pigs under anesthesia. one half of the inl was stored in crpmi for lymphocyte isolation while the other half of the inl was minced and stored in trizol reagent (life technologies, carlsbad, ca, usa) for rna extraction. viral loads in plasma and tissues were measured by a commercial rt-qpcr kit (tetracore inc., rockville, md, usa). viral loads in plasma was reported as log 10 copies per ml, whereas viral loads in tissues were reported as log 10 copy per µg of total rna used in the rt-qpcr reaction. for statistical purposes, samples that had no detectable levels of viral rna were assigned a value of 0 log 10 copies. pbmcs were isolated from edta-whole blood as previously described [29, 30] . single cell suspension was isolated from iln immediately after collection as follows. the tissue was processed to viruses 2020, 12, 817 4 of 22 remove connecting tissue and cut into small pieces which were placed in a 70-µm nylon cell strainer (corning, durham, nc, usa) in the presence of crpmi. the tissue pieces were pressed against the nylon mesh by using the plunger of a 3 ml syringe. the resulting cell suspension was collected into a 50 ml conical tube which was passed through a 70-µm cell strainer one more time to remove large tissue debris. cells were pelleted by centrifugation at 700× g for 10 min at room temperature and treated with a 5 ml red blood cell (rbc) lysis buffer (life technologies, carlsbad, ca, usa). rbc lysis reaction was ceased by adding ice cold pbs containing 4% fbs, followed by centrifugation at 700× g for 10 min at room temperature. cell pellet was resuspended in crpmi. to determine cell concentration and viability, samples of pbmc and iln-derived cells were stained with acridine orange and propidium iodide (viastaintm aopi staining solution, nexcelom, lawrence, ma, usa) and counted using an automatic cell counter (cellometer auto 2000, nexcelom, lawrence, ma, usa). freshly isolated cells were used for elispot and flow cytometric analysis. the remaining cells were cryopreserved in 10% dmso, 40% fbs, and 50% rpmi-1640 and stored in liquid nitrogen. prrsv antibody levels in plasma were measured at the veterinary diagnostic center of the university of nebraska by using the commercial elisa idexx prrs x3 ab test (idexx laboratories, westbrook, me, usa) following the manufacturer's instructions. the serum-virus neutralization (svn) assay was performed as previously described [31] using plasma rather than serum. results were expressed as the log 2 of the reciprocal of the highest dilution that showed a ≥90% reduction in the number of fluorescent foci presenting in the control wells. the frequencies of ifn-γ-secreting cells (ifn-γ scs) in pbmcs and iln-derived cells were measured by using an ifn-γ elispot assay as previously described [32, 33] . briefly two replicates of 250,000 pbmcs or iln cells freshly collected from each pig were plated into two wells of a 96-well plate with pvdf membrane that were coated with anti-porcine ifn-γ antibody. the cells were stimulated with 100 µl of crpmi containing 2.5 × 10 4 tcid50 of con90. for positive control, cells were plated at 5000 cells per well, followed by stimulation with a 100 µl crpmi containing 10 ng/ml of phorbol 12 myristate 13-acetate (pma) and 1 µg/ml of ionomycin. for negative control, cells were simply cultured in crpmi. at 18 h post stimulation, the plate was washed with pbs-containing 0.05% tween 20 (pbs-t) followed by incubation with biotin-labeled antibody against porcine ifn-γ (clone p2c11, bd biosciences pharmingen, san diego, ca, usa). spots were developed by using alkaline phosphatase-conjugated streptavidin (southern biotech, birmingham, al, usa) in conjunction with alkaline phosphatase substrate (vector laboratories, burlingame, ca, usa). spots were counted and analyzed using an aid elispot reader version 7.0 (aid gmbh, strassberg, germany). freshly isolated pbmcs and iln cells were ex vivo stimulated with 1 × 10 5 tcid50 of con90 as described above. a cocktail solution consisting of 10 ng/ml of pma and 1 µg/ml of ionomycin was included as a positive control while crpmi served as a negative control. at 12 hrs post-stimulation, 100 µl of crpmi containing 1 µg/ml of golgi-plug brefeldin a (bd bioscience, san jose, ca, usa) was added and further incubated for 6 hrs. samples were centrifuged at 500× g for 10 min at room temperature. cells were resuspended in flow cytometry staining buffer (facs buffer) (pbs with 4% fbs and 0.1% sodium azide), and stained with anti-porcine cd3ε, cd4, and cd8 monoclonal antibodies and incubated on ice for 30 min in the dark. cells were washed thrice using facs buffer. the cells were fixed and permeabilized with 4% paraformaldehyde and 0.1% triton x-100, respectively, followed by intracellular staining with an ifn-γ antibody. cells were analyzed by using a cytek dxp10 cytometer (cytek biosciences, fremont, ca, usa) and acquired data were analyzed using the flowjo software (bd biosciences, san jose, ca, usa) with gating based upon fluorescence minus one (fmo) control. for each sample, 100,000 events were acquired. relevant cell population was gated on cd3 + prior to analyzing the cd4 + and cd8 + cells ( figure s1a ). ifn-γ positive cells were counted from individual cd4 + , cd8 + , and cd4 + cd8 + double positive (dp) cells ( figure s1b ). total rna was isolated from iln collected from the infected and non-infected using the trizol reagent according to the manufacturer's protocol. rna was re-purified using rneasy mini kit (qiagen, hilden, nrw, germany) according to the manufacturer instructions, followed by a dnase treatment using turbo dna-free tm kit (life technologies, v.a. graiciuno 8, vilnius, lithuania) to remove dna contamination. purified rna was submitted to novogene bioinformatics technology co.ltd for rna sequencing. sequencing libraries were generated using nebnext ® ultratm rna library prep kit for illumina ® (neb, ipswich, ma, usa) following manufacturer's recommendations and index codes were added to attribute sequences to each sample. briefly, mrna was purified from 1 mg of total rna using poly-t oligo-attached magnetic beads. fragmentation was carried out using divalent cations under elevated temperature in nebnext first strand synthesis reaction buffer (5x). first strand cdna was synthesized using random hexamer primer and m-mulv reverse transcriptase (rnase h). second strand cdna synthesis was subsequently performed using dna polymerase i and rnase h. remaining overhangs were converted into blunt ends via exonuclease/polymerase activities. after adenylation of 3 ends of dna fragments, nebnext adaptor with hairpin loop structure were ligated to prepare for hybridization. in order to select cdna fragments of preferentially 150~200 bp in length, the library fragments were purified with ampure xp system (beckman coulter, beverly, ma, usa). then 3 µl user enzyme (neb, ipswich, ma, usa) was used with size-selected, adaptor-ligated cdna at 37 • c for 15 min followed by 5 min at 95 • c before pcr. then pcr was performed with phusion high-fidelity dna polymerase, universal pcr primers and index (x) primer. finally, pcr products were purified using ampure xp system and library quality was assessed on the agilent bioanalyzer 2100 system. the libraries were sequenced on an illumina platform hiseq 2000 and approximately 40 million raw 125 bp/150 bp paired-end) reads were generated. rna-seq reads were first trimmed with trim galore version 0.6.4 [34, 35] , which include filtering reads shorter than 30 bp and low-quality ends from reads (phred score: <20) and the option of removing reads with ns. the quality of reads before and after trimming was checked with fastqc version 0.11.7 [36] . the filtered reads were aligned to the sus scrofa genome (sscrofa11.1; gcf_000003025.6) using tophat version 2.0.14 [37] [38] [39] with a read mismatch of 0 and a maximum multi hits of 5 and further default parameters. alignment files generated by tophat were then used to generate gene counts using htseq version 0.9.1 (htseq-count), with a minimum alignment quality of 10 [40] . a data matrix of gene counts for all samples was created using a custom python script and the data matrix was used to run the differential gene expression analysis in deseq2 version 1.22.1 [41] . genes with an adjusted p value smaller than 0.05 and a log 2 fold change larger than 1 were considered as differentially expressed. the differential expression result table generated by deseq2 was annotated with gene information obtained from ensembl biomart for sus scrofa, supplemented by annotation from the gtf file. to visualize the level of prrsv rna genome, reads mapped to the prrsv con90 genome were counted base by base, normalized to counts per million and subsequently used to generate a coverage track using deeptools version 3.4.3 [42] . gene ontology (go) in the database (http://www.geneontology.org/) is an international standardized classification system for gene function, and it supplies a set of controlled vocabulary to comprehensively describe the properties of genes and gene products. go enrichment analysis of degs was implemented by the goseq r package [43] , in which gene length bias was corrected. go terms with viruses 2020, 12, 817 6 of 22 corrected p-value less than 0.05 were considered significantly enriched by differentially expressed genes. kyoto encyclopedia of genes and genomes (kegg) is a database resource for understanding high-level functions and utilities of the biological system, such as the cell, the organism, and the ecosystem, from molecular-level information, especially large-scale molecular datasets generated by genome sequencing and other high-throughput experimental technologies (http://www.genome.jp/kegg/). kobas version 3.0 [44] was used to test the statistical enrichment of differential expression genes in kegg pathway. virus-specific t-cell data were analyzed by unpaired t-test analysis using graphpad prism software version 8.3.1 (graphpad software, llc, san diego, ca, usa). a p < 0.05 was considered significant. sequencing data from rna-seq were deposited in the ncbi geo and are available under accession number geo: gse153174 all pigs inoculated with con90 became viremic starting from 2 dpi. the viremia level increased and peaked at 7 dpi ( figure 1a ). pigs inoculated with con90 did not displayed any clinical signs throughout the course of this study. at 46 dpi, iln was collected and rna was extracted. rt-pcr analysis revealed that viral rna genome was detected in all five con90-infected pigs but none of the sham-inoculated pigs ( figure 1b) . subsequently, the rna samples were subjected to rna-seq. an average of 32 million paired-end reads were obtained for each rna sample (table s1 ). to confirm the presence of viral rna during persistent infection in pigs, the reads were mapped to the con90 genome. viral rna reads were only detected from iln of con90-infected pigs which mapped throughout the viral genome ( figure 1c ). the results demonstrate that all five pigs in this study were persistently infected with the attenuated prrsv strain con90. to examine the host responses to con90 persistent infection, rna reads were mapped to the reference pig genome (sscrofa11.1; gcf_000003025.6). principal component analysis (pca) indicated that control and con90-infected pigs formed separated clusters indicating that they had distinct transcriptional profiles ( figure 2a ). the infected pigs showed more variability than the control pigs. in addition, one pig in the con90-infected group (con_429) showed a unique transcriptional profile, as it associated closer to the control pigs. this association is likely due to the different genetic makeup of this pig than the other four animals within this group. direct comparison of rna transcripts between control and con90-infected animals revealed a profound change in the transcriptional profiles in the iln tissue of con90-infected pigs. out of 17553 genes in the annotated porcine genome, there were 6404 degs (fdr < 5%, |log 2 | fold change ≥ 1), of which 3960 degs were upregulated and 2444 degs were downregulated ( figure 2b and table s2 ). to examine the host responses to con90 persistent infection, rna reads were mapped to the reference pig genome (sscrofa11.1; gcf_000003025.6). principal component analysis (pca) indicated that control and con90-infected pigs formed separated clusters indicating that they had distinct transcriptional profiles (figure 2a ). the infected pigs showed more variability than the control pigs. in addition, one pig in the con90-infected group (con_429) showed a unique transcriptional profile, as it associated closer to the control pigs. this association is likely due to the different genetic makeup of this pig than the other four animals within this group. direct comparison of rna transcripts between control and con90-infected animals revealed a profound change in the transcriptional profiles in the iln tissue of con90-infected pigs. out of 17553 genes in the annotated porcine genome, there were 6404 degs (fdr < 5%, |log2| fold change ≥ 1), of which 3960 degs were upregulated and 2444 degs were downregulated ( figure 2b and table s2 ). the control animals are indicated by red dots labeled "dmem_ animal number," while the con90-infected animals are indicated by cyan dots labeled "con_animal number"; (b) the volcano plot of differentially expressed genes between con90-infected and control pigs. red dots denote significantly down-regulated genes (p < 0.05, log2 fold change ≤−1), green dots indicated significantly up-regulated genes (p < 0.05, log2 fold change ≥1), and grey dots represent genes that were not differentially expressed. to understand their biological functions, degs were subjected to go enrichment analysis. go terms having a corrected value of p < 0.05 were considered statistically significant (table s3) . significantly enriched go terms were grouped under three categories: biological processes, molecular function, and cellular components. thirty highly enriched go terms in con90-infected pigs are shown (figure 3a) . a large number of degs are enriched into the category of biological processes (cytoskeleton organization, multicellular organism development, regulation of intracellular signal transduction, cell communication, cell surface receptor signaling, regulation of programmed cell death etc.), followed by molecular function (cellular macromolecular catabolic processes, kinase activity, phosphotransferase activity, dna binding, metal ion binding) and cellular components (ribonucleoprotein complexes, bounding membrane of organelles). kegg pathway analysis revealed multiple enriched pathways including apoptosis, chemokine signaling, cellular, senescence, mitophagy, lysosome, endocytosis, and mapk ( figure 3b and table s4 ). detailed analysis of selected enriched pathways is presented in the respective sections below. to understand their biological functions, degs were subjected to go enrichment analysis. go terms having a corrected value of p < 0.05 were considered statistically significant (table s3) . significantly enriched go terms were grouped under three categories: biological processes, molecular function, and cellular components. thirty highly enriched go terms in con90-infected pigs are shown (figure 3a) . a large number of degs are enriched into the category of biological processes (cytoskeleton organization, multicellular organism development, regulation of intracellular signal transduction, cell communication, cell surface receptor signaling, regulation of programmed cell death etc.), followed by molecular function (cellular macromolecular catabolic processes, kinase activity, phosphotransferase activity, dna binding, metal ion binding) and cellular components (ribonucleoprotein complexes, bounding membrane of organelles). kegg pathway analysis revealed multiple enriched pathways including apoptosis, chemokine signaling, cellular, senescence, mitophagy, lysosome, endocytosis, and mapk ( figure 3b and table s4 ). detailed analysis of selected enriched pathways is presented in the respective sections below. the innate immune system is the first line of defense against invading pathogens, with the major influence on the development of strong adaptive immune responses. con90 is capable of inducing type i ifns both in vitro and in vivo [27] . in the current study, we did not detect differential expression of type i ifn or interferon stimulated genes (isg) rna transcripts, suggesting that the type-i ifn was not induced in iln of con-infected pigs at 46 dpi. we observed an upregulation of rna-sensing molecules including tlr3 (dsrna), tlr7, and tlr8 (ssrna) (figure 4a ). on the other hand, no differential expression of signaling molecules downstream of tlr pathways including interferon regulatory factors (irf) irf3 and irf7, was observed (table s2 ). in addition, we observed an upregulation of tlr10 mrna in con90-infected pigs. while the ligands and signaling pathway involving tlr10 remain poorly understood, it has been demonstrated that tlr10 can act as an anti-inflammatory receptor which can also suppress tlr3-induced ifn-β production [45] . interestingly, we observed an increased expression of cd200 (ox2) and its receptor cd200r (figure 4a ). cd200r is an inhibitory immune receptor that is expressed on myeloid cells and b-and t-lymphocytes [46] , while cd200 is widely expressed on multiple cell types including endothelial cells, neurons, and lymphocytes [47] . cd200/cd200r interaction suppresses cytokine production, and inflammatory responses [48] . the innate immune system is the first line of defense against invading pathogens, with the major influence on the development of strong adaptive immune responses. con90 is capable of inducing type i ifns both in vitro and in vivo [27] . in the current study, we did not detect differential expression of type i ifn or interferon stimulated genes (isg) rna transcripts, suggesting that the type-i ifn was not induced in iln of con-infected pigs at 46 dpi. we observed an upregulation of rna-sensing molecules including tlr3 (dsrna), tlr7, and tlr8 (ssrna) (figure 4a ). on the other hand, no differential expression of signaling molecules downstream of tlr pathways including interferon regulatory factors (irf) irf3 and irf7, was observed (table s2 ). in addition, we observed an upregulation of tlr10 mrna in con90-infected pigs. while the ligands and signaling pathway involving tlr10 remain poorly understood, it has been demonstrated that tlr10 can act as an antiinflammatory receptor which can also suppress tlr3-induced ifn-β production [45] . interestingly, we observed an increased expression of cd200 (ox2) and its receptor cd200r (figure 4a ). cd200r is an inhibitory immune receptor that is expressed on myeloid cells and b-and t-lymphocytes [46] , while cd200 is widely expressed on multiple cell types including endothelial cells, neurons, and lymphocytes [47] . cd200/cd200r interaction suppresses cytokine production, and inflammatory responses [48] . the complement system is another crucial component of the innate immunity. it acts as an important link between innate and adaptive immune system. thus, viruses have developed a mechanism to modulate complement responses by down-regulating activation proteins and upregulation of regulatory proteins [49] . in the current study, increased expression of regulatory proteins (cfh, cfi, and cd55) was observed from con90-infected animals (figure 4b) . conversely, c1q, a classical complement component involved in increasing virus neutralizing ability of antibodies [50] was down-regulated ( figure 4b) . collectively, the data suggest that the con90 virus suppresses both complement activation and inflammatory responses at the site of persistent infection. apoptosis is a powerful innate immunity mechanism to curtail viral spread through eliminating virally infected cells. apoptosis can be triggered by both extrinsic and intrinsic stimuli [51] . it is well documented that prrsv induces apoptosis in tissues of infected pigs, especially during an acute phase of the infection [15, 16, 52] . in this study, expression of tnfrsf1a (tnfα receptor 1) gene that the complement system is another crucial component of the innate immunity. it acts as an important link between innate and adaptive immune system. thus, viruses have developed a mechanism to modulate complement responses by down-regulating activation proteins and up-regulation of regulatory proteins [49] . in the current study, increased expression of regulatory proteins (cfh, cfi, and cd55) was observed from con90-infected animals (figure 4b) . conversely, c1q, a classical complement component involved in increasing virus neutralizing ability of antibodies [50] was down-regulated ( figure 4b) . collectively, the data suggest that the con90 virus suppresses both complement activation and inflammatory responses at the site of persistent infection. apoptosis is a powerful innate immunity mechanism to curtail viral spread through eliminating virally infected cells. apoptosis can be triggered by both extrinsic and intrinsic stimuli [51] . it is well documented that prrsv induces apoptosis in tissues of infected pigs, especially during an acute phase of the infection [15, 16, 52] . in this study, expression of tnfrsf1a (tnfα receptor 1) gene that contains a death domain [53] , was downregulated in the iln of infected pigs ( figure 5) . similarly, expression of pro-apoptotic genes including aifm2, chac1, and osr1, was downregulated ( figure 5 ). on the other hand, expression of birc3 and bfl-1/a1 (bcl2a1), two apoptosis inhibitors that interfere with caspase activation [54] , was upregulated. furthermore, expression of several negative regulators of apoptotic genes including bcl-2-associated killer 1 (bak1), damage induced apoptosis suppressor (ddias), x-linked inhibitor of apoptosis protein (xiap), mcl1, api, bnip2, and faim was upregulated. collectively, these results suggest that the pro-apoptotic signaling pathway was suppressed in iln tissue of pigs persistently infected with con90. contains a death domain [53] , was downregulated in the iln of infected pigs ( figure 5) . similarly, expression of pro-apoptotic genes including aifm2, chac1, and osr1, was downregulated ( figure 5 ). on the other hand, expression of birc3 and bfl-1/a1 (bcl2a1), two apoptosis inhibitors that interfere with caspase activation [54] , was upregulated. furthermore, expression of several negative regulators of apoptotic genes including bcl-2-associated killer 1 (bak1), damage induced apoptosis suppressor (ddias), x-linked inhibitor of apoptosis protein (xiap), mcl1, api, bnip2, and faim was upregulated. collectively, these results suggest that the pro-apoptotic signaling pathway was suppressed in iln tissue of pigs persistently infected with con90. lymphocyte activation occurs in the secondary lymphoid organs. activated t-cells then migrate to the sites of infection where they exert their effector functions to eliminate infected cells [55] . celladhesion molecules, chemokines, and receptors play a central role in regulating t-cell migration [56] . prrsv mainly persists in lymphoid tissue of infected pigs [17, 18] . in this study, expression of several important chemokine ligands (ccl19, ccl21, ccl24, ccl22, cx3cl1, and ccl14) and chemokine receptors (ccr6 and ccr10) which play an essential role in migration and localization of lymphocytes and antigen-presenting cells (apcs) to the lymphoid tissues was down regulated in iln of con90-infected pigs (figure 6a ). on the other hand, expression of cd274 (pd-1), a marker of tcell exhaustion, was upregulated ( figure 6b) . likewise, expression of inhibitory receptors havcr2 (also known as tim3) and tgit, which transmit the inhibitory signals for t-cell differentiation and effector activities [57] , was upregulated. we also found increased expression of other co-inhibitory molecules (btla, faslg, fas, and ido1) that are associated with the regulation of t-cell exhaustion during chronic viral infection (figure 6b) . together, the results suggest that t-cell migration to iln, one of the sites of prrsv persistence, might be affected because of the down regulation of important chemokines and receptors, and that the t-cells in iln might be exhausted. interestingly, markers of lymphocyte activation occurs in the secondary lymphoid organs. activated t-cells then migrate to the sites of infection where they exert their effector functions to eliminate infected cells [55] . cell-adhesion molecules, chemokines, and receptors play a central role in regulating t-cell migration [56] . prrsv mainly persists in lymphoid tissue of infected pigs [17, 18] . in this study, expression of several important chemokine ligands (ccl19, ccl21, ccl24, ccl22, cx3cl1, and ccl14) and chemokine receptors (ccr6 and ccr10) which play an essential role in migration and localization of lymphocytes and antigen-presenting cells (apcs) to the lymphoid tissues was down regulated in iln of con90-infected pigs (figure 6a ). on the other hand, expression of cd274 (pd-1), a marker of t-cell exhaustion, was upregulated ( figure 6b) . likewise, expression of inhibitory receptors havcr2 (also known as tim3) and tgit, which transmit the inhibitory signals for t-cell differentiation and effector activities [57] , was upregulated. we also found increased expression of other co-inhibitory molecules (btla, faslg, fas, and ido1) that are associated with the regulation of t-cell exhaustion during chronic viral infection (figure 6b) . together, the results suggest that t-cell migration to iln, one of the sites of prrsv persistence, might be affected because of the down regulation of important chemokines and receptors, and that the t-cells in iln might be exhausted. interestingly, markers of regulatory t-cells (t reg ) were downregulated in iln of infected pigs (figure 6c) , suggesting that t reg might not be present in iln at 46 dpi. regulatory t-cells (treg) were downregulated in iln of infected pigs (figure 6c ), suggesting that treg might not be present in iln at 46 dpi. since the expression of chemokines and receptors important for t-cell migration was downregulated in iln, we sought to compare the frequencies of virus-specific t-cells in pbmcs and in iln using the ifn-γ sc elispot assay. the number of spots was similar when pbmcs and iln cells were stimulated with non-specific t-cell activator pha/ionomycin. however, the number of spots was significantly lower in iln than in pbmcs when the cells were stimulated with whole prrsv antigen (figure 7a ). the results clearly indicate that the frequencies of prrsv-specific ifn-γ scs were significantly lower in iln than in pbmcs. to further elucidate the phenotypes of prrsvspecific t-cells, we used a multi-color flow cytometric assay to identify subsets of t-cells that secrete ifn-γ in response to prrsv activation ex vivo. cd4 + cd8 + dp cells were the major t-cell population secreting ifn-γ, both in pbmcs and iln. swine have a significant population of extrathymic cd4 + cd8 + dp t cells that represent memory t-cells [58] . similar to the elispot assay, the flow cytometric assay also indicated that the percentage of t-cell secreting ifn-γ was comparatively lower in iln than in pbmcs (figure 7b ). cells collected from dmem-inoculated pigs did not show any significant t-cell reactivities after ex vivo stimulation with con90, both in elispot and in flow cytometry assays (data not shown). since the expression of chemokines and receptors important for t-cell migration was downregulated in iln, we sought to compare the frequencies of virus-specific t-cells in pbmcs and in iln using the ifn-γ sc elispot assay. the number of spots was similar when pbmcs and iln cells were stimulated with non-specific t-cell activator pha/ionomycin. however, the number of spots was significantly lower in iln than in pbmcs when the cells were stimulated with whole prrsv antigen (figure 7a ). the results clearly indicate that the frequencies of prrsv-specific ifn-γ scs were significantly lower in iln than in pbmcs. to further elucidate the phenotypes of prrsv-specific t-cells, we used a multi-color flow cytometric assay to identify subsets of t-cells that secrete ifn-γ in response to prrsv activation ex vivo. cd4 + cd8 + dp cells were the major t-cell population secreting ifn-γ, both in pbmcs and iln. swine have a significant population of extrathymic cd4 + cd8 + dp t cells that represent memory t-cells [58] . similar to the elispot assay, the flow cytometric assay also indicated that the percentage of t-cell secreting ifn-γ was comparatively lower in iln than in pbmcs (figure 7b ). cells collected from dmem-inoculated pigs did not show any significant t-cell reactivities after ex vivo stimulation with con90, both in elispot and in flow cytometry assays (data not shown). viruses 2020, 12, x for peer review 13 of 22 a number of genes associated with th2, or humoral response, were upregulated in the iln of con90-infected pigs (figure 8a ). il-21, rgs13, and nuggc are involved in the development of germinal center (gc) and activation of b-cell follicles [59] . tnfsf13b and tnfsf8 are potent activators of b-cell lineage and ig class switching, respectively [60] . b-cell surface antigens ms4a1 (cd20), activation-induced cytidine deaminase (aicda) [61] , and rafting family member 2 (rftn2) are associated with b-cell receptor signaling. the upregulated expression of these genes in iln of infected animals suggested that the humoral immune response to con90-infection was not affected. to corroborate transcriptome data, we measured both virus-specific antibody levels at various time points post-infection. high levels of non-neutralizing antibodies specific to viral n protein (measured by a commercial elisa) were detected in all pigs (figure 8b ). however, only minimal levels of virusneutralizing antibodies (titer 1:2) were detected in the serum of infected pigs at 46 dpi (figure 8c ). together, both transcriptomic and serological data indicate that b-cell development and antibody production are not affected by con90-infection. however, low levels of virus-neutralizing antibodies are likely due to the virus ability to escape antibody neutralization (see below). a number of genes associated with th2, or humoral response, were upregulated in the iln of con90-infected pigs (figure 8a ). il-21, rgs13, and nuggc are involved in the development of germinal center (gc) and activation of b-cell follicles [59] . tnfsf13b and tnfsf8 are potent activators of b-cell lineage and ig class switching, respectively [60] . b-cell surface antigens ms4a1 (cd20), activation-induced cytidine deaminase (aicda) [61] , and rafting family member 2 (rftn2) are associated with b-cell receptor signaling. the upregulated expression of these genes in iln of infected animals suggested that the humoral immune response to con90-infection was not affected. to corroborate transcriptome data, we measured both virus-specific antibody levels at various time points post-infection. high levels of non-neutralizing antibodies specific to viral n protein (measured by a commercial elisa) were detected in all pigs (figure 8b ). however, only minimal levels of virus-neutralizing antibodies (titer 1:2) were detected in the serum of infected pigs at 46 dpi (figure 8c ). together, both transcriptomic and serological data indicate that b-cell development and antibody production are not affected by con90-infection. however, low levels of virus-neutralizing antibodies are likely due to the virus ability to escape antibody neutralization (see below). viruses 2020, 12, x for peer review 14 of 22 prrsv persists in lymphoid tissue of infected pigs for several months [17, 18] . the mechanism of prrsv persistence is not fully understood. we performed genome-wide transcriptome analysis of lymphoid tissue collected from pigs persistently infected with an attenuated prrsv strain using rna-seq technology that detects both host and viral rna. viral rna reads were detected in iln of all five infected pigs. it was reported previously that prrsv genome mainly exists in dsrna forms in lymphoid tissues during persistent infection [26] . since only mrna (purified by using poly-t oligo-attached magnetic beads) was used for library construction and rna sequencing, the viral rna reads detected in this study must be derived from either viral genomic rna or sub-genomic mrna, not from dsrna. the viral rna reads map throughout the viral genome. however, we are not able to discern whether these reads are derived from genomic or sub-genomic mrna because we used a short-read rna sequencing platform. it would be interesting to use long-read rna sequencing to study the viral transcriptome at different states of infection in pigs [62] . it was reported previously that no significant degs were observed in lymphoid tissue of persistently infected animals [26] . in the current study, we identified a large number of degs in persistently infected animals (figure 2b ). this might be due to the difference in the experimental setup. in the previous study, pigs were infected with a wild-type prrsv-1 whereas in this current prrsv persists in lymphoid tissue of infected pigs for several months [17, 18] . the mechanism of prrsv persistence is not fully understood. we performed genome-wide transcriptome analysis of lymphoid tissue collected from pigs persistently infected with an attenuated prrsv strain using rna-seq technology that detects both host and viral rna. viral rna reads were detected in iln of all five infected pigs. it was reported previously that prrsv genome mainly exists in dsrna forms in lymphoid tissues during persistent infection [26] . since only mrna (purified by using poly-t oligo-attached magnetic beads) was used for library construction and rna sequencing, the viral rna reads detected in this study must be derived from either viral genomic rna or sub-genomic mrna, not from dsrna. the viral rna reads map throughout the viral genome. however, we are not able to discern whether these reads are derived from genomic or sub-genomic mrna because we used a short-read rna sequencing platform. it would be interesting to use long-read rna sequencing to study the viral transcriptome at different states of infection in pigs [62] . it was reported previously that no significant degs were observed in lymphoid tissue of persistently infected animals [26] . in the current study, we identified a large number of degs in persistently infected animals (figure 2b ). this might be due to the difference in the experimental setup. in the previous study, pigs were infected with a wild-type prrsv-1 whereas in this current study, pigs were infected with an attenuated synthetic prrsv strain [27, 63] . besides, the previous study looked at a small set of selected genes while in this study we look at the genome-wide rna transcriptome. go terms and kegg analysis revealed that genes involved in the innate immune (complement and tlr) pathways, apoptosis, cytokine-chemokine signaling, t-cell exhaustion, and humoral responses are highly differentially expressed in prrsv-infected animals compared to control. the complement system is a constituent of innate immunity that serves by neutralizing cell-free viruses, lysing virus-infected cells, and boosting virus specific responses [64] . it also links the innate and adaptive immune responses, enhances humoral immunity, regulates antibody effector mechanisms, and modulates t-cell function [65] . many viruses have developed a strategy to evade the complement pathway by recruiting or enhancing the production of host transcription regulatory components. during acute infection (7 dpi), prrsv significantly represses the expression of complement regulatory components (cd55 and c4bpb) in lung tissue [16] . early complement activation during acute infection facilitates the release of newly formed virions from infected cells, yet, chronic viral infection is reported to suppress the activity of complement activation proteins and increases the activity of regulatory proteins (reviewed [49] ). in the current study, we found overexpression of cfh and cfi along with another regulatory factor cd55 (decay-accelerating factor). overexpression of cfh, a major soluble regulator of the alternative pathway, results in inhibition of c3 and c5 convertase enzymes. cfi suppresses the complement active proteins c3b (opsonin) and c4b via mediating cleavage to their inactive form [66, 67] . regulatory factor cd55 is an inhibitor of c3 convertase which prevents c3b deposition on the cell surface [68] . apart from that, we also see the downregulation of c1q, which increases neutralizing and hemagglutination inhibition activity of anti-influenza antibodies [50] . available data from previous studies and the current study suggest that activation of complement pathway during acute infection helps to disseminate virus, while suppression of complement components like c1q, c1r, and c5, and upregulation in regulatory components during persistent infection facilitates the virus to escape from the complement system to maintain persistence in lymphoid tissues. most naturally occurring prrsv strains suppress type i ifn production by inhibiting the activation and nuclear translocation of irf3/irf7 and nf-kb [69, 70] . deficiency of irf3/7 results in severe mortality to infection with west nile virus (wnv), chikungunya virus (chikv), and ross river virus infection, and promotes viral persistence in the infected hosts [71] [72] [73] . it has been reported that prrsv nsp1β inhibits irf3 phosphorylation and nuclear translocation; thus, inhibiting ifn production [69, 74, 75] . interestingly, the synthetic prrsv-con and its attenuated form con90 were able to induce type i ifns [27, 76] . contrary to its nature to induce type i ifn, in the current study no upregulation of canonical type i ifn signaling pathway-associated genes was observed. however, expression of rna sensing molecules including tlr3, tlr7, and tlr8 was upregulated in con90-infected animals. on the basis of available data, we hypothesize that reduced levels of viral replication and sequestration of viral rna in infected cells during persistent infection might limit further activation of type i ifn signaling pathway by preventing interaction with cytoplasmic pattern recognition receptors (prrs). apoptosis or programmed cell death is a potential host immune mechanism against virally infected cells to curtail the spread of newly formed viral progenies. apoptosis is induced by two distinct, yet inter-connected signaling pathways, the extrinsic and intrinsic pathways [51] . in order to successfully establish persistent infection, viruses have developed mechanisms to inhibit apoptosis. for instance, adenovirus (e1b-19k), human cytomegalovirus (ul37), poxviruses (f1l), and myxoma virus (m11l) have an inhibitory effect on proapoptotic proteins bak/bax [77] . it appears that prrsv can also modulate apoptosis. studies of pulmonary alveolar macrophages (pam) infected with prrsv in vitro reveal that the virus stimulates anti-apoptotic pathways early in infection while it induces apoptosis late in infection [78] . on the other hand, the virus induces apoptosis in the tissues of infected animals during acute infection, but the frequencies of apoptotic cells reduced to normal levels observed in control, non-infected pigs from day 20 post-infection [52] . in this study, expression of multiple anti-apoptotic genes including xiap, bfl-1/a1 (bcl2a1), and birc3 was upregulated while expression of pro-apoptotic genes (aifm2, chac1, and osr1) was downregulated. xiap is an endogenous caspase 9 and 3 inhibitor [79] . bfl-1/a1 (bcl2a1) is a transcriptional target of nuclear factor-kb (nf-kb) that suppresses caspase activation and apoptosis in response to death-inducing stimuli like tnfα [54] . birc3 is an interaction partner to tnfrsf1b and inhibits apoptosis by interfering with the caspase activation [53] . the pro-apoptotic bcl-2 family member like bcl-2-associated killer 1 (bak1), which allows the release of cytochrome c via formation of homo-oligomers and stable insertion into outer mitochondrial membrane was significantly suppressed. thus, the data suggest that apoptosis was suppressed in the iln of con90-infected animals during persistent infection. chemokine molecules ccl19 and ccl21 play a crucial role in migration, activation, expansion, and survival of antiviral t-cells. suppression of the ccr7 and ccl19/ccl21 axis results in dysfunction of t-cells during viral infection [80, 81] . ccl19-ccr7 axis plays role in protection against multiple viruses, such as hiv [82] , herpes simplex virus (hsv-1) [83] , hsv-2, hepatitis c virus [84] , and pseudorabies [85] . acute prrsv infection increases the expression of chemokines, leading to the infiltration of immune cells toward the sites of infection [16, 86] . in this study, expression of both ccl19 and ccl21 was significantly downregulated in con90-infected lymph node (figure 6a ). additionally, expression of different chemoattractant molecules (fractalkine/cx3cl1, ccl14, ccl16, ccl17, ccl22, and ccl8) and receptors (ccr6 and ccr10) was also downregulated. it is possible that downregulation of these chemokines and receptors might impair t-cells trafficking to inguinal lymph node, the site of prrsv persistence. our transcriptional data are supported by the functional data which demonstrate that the frequencies of ifn-γ sc in iln were significantly lower than in pbmcs (figure 7) . chronic or persistent viral stimulation results in hierarchical loss of effector functions of t-lymphocytes, including proliferation, cytokine production (e.g., ifn-γ and il-2), and cytolytic responses [57, 87, 88] . although the molecular signatures involved in t-cell exhaustion are not completely understood, the overexpression of cell surface inhibitory receptors (e.g., pd-1, ctla-4, and others) primarily mediates cd8+ t-cell dysfunction [87] [88] [89] . t-cell exhaustion seems to be a common phenomenon caused by arteriviruses, as t-cell exhaustion was also reported in horses persistently infected with eav [24] . recently, it was shown that prrsv infection alone or co-infection with porcine circovirus type 2 (pcv2) can significantly upregulate surface expression of pd-l1 (cd274) on porcine monocytes-derived dendritic cells (modcs) [90] . increased expression of pd-l1 on the surface of antigen presenting cells (apcs) possibly contributes to the ineffective t-cell responses during the prrsv infection. in the present study, expression of several markers for t-cells exhaustion such as pd-1 (cd279) and its ligand, cd274 (pd-l1) was upregulated in con90-infected animals, suggesting that the t-cells in lymph nodes of persistently infected animals might be exhausted (figure 6b) . however, additional studies (e.g., t-cell cytotoxicity and cytokine production) are required to fully assess the functionality of t-cells during prrsv persistence. germinal centers (gcs) are the specialized locations in the secondary lymphoid tissues where b-cell maturation, differentiation, somatic hypermutation, and class switching of isotypes take place [91] . in the current study, the gc development-associated genes, which includes il-21 produced by follicular helper t-cells [61] , the regulator of g protein signaling (rgs13) [92] , and gc-associated nuclear gtpase (nuggc) [59] , were upregulated in the lymph node of con90-infected animals. thus, prrsv infection does not seem to impair gc formation and b-cell activation. this is supported by the fact that all five con90-infected pigs developed a robust antibody response, as measured by the idexx elisa ( figure 8b) . however, only minimal titers of virus-neutralizing antibodies were detected at 46 dpi. perhaps, prrsv does not suppress the humoral immune response. instead, the virus evades from antibody-mediated neutralization through different mechanisms such as glycan shielding and decoy epitopes [93] [94] [95] . in summary, we identified a robust host transcriptional response in the inguinal lymphoid tissue of pigs persistently infected with the attenuated prrsv strain con90. genes involved in the innate immune responses are downregulated. similarly, chemokines and receptors associated with t-cell homing to the lymphoid tissue are downregulated. this might lead to the lower frequencies of virus-specific t-cells in lymphoid tissue than in peripheral blood. additionally, the genes involved in the anti-apoptotic pathways are upregulated. collectively, the data suggest that prrsv can create a pro-survival microenvironment at the lymphoid tissue which allows the virus to persist for an extended period. supplementary materials: the following are available online at http://www.mdpi.com/1999-4915/12/8/817/s1. figure s1 . representative flow cytometry gating strategy used to identify ifn-γ secreting cd4α + , cd8α + , and cd4α + cd8α + (dp) t cells from pbmcs and iln. table s1 . summary of sequencing quality control and mapping data of samples. arterivirus molecular biology and pathogenesis ictv-international committee on taxonomy of viruses. virus taxonomy: 2019 release. ec 51 porcine reproductive and respiratory syndrome assessment of the economic impact of porcine reproductive and respiratory syndrome virus on united states pork producers effects of origin and state of differentiation and activation of monocytes/macrophages on their susceptibility to porcine reproductive and respiratory syndrome virus (prrsv) cd163 expression confers susceptibility to porcine reproductive and respiratory syndrome viruses in vivo detection of porcine reproductive and respiratory syndrome virus rna by in situ hybridization at different times postinfection apoptosis induced in vivo during acute infection by porcine reproductive and respiratory syndrome virus type 2 porcine reproductive and respiratory syndrome virus infection increases apoptosis at the maternal-fetal interface in late gestation pregnant gilts open reading frame 5 of porcine reproductive and respiratory syndrome virus as a cause of virus-induced apoptosis characterization of the porcine reproductive and respiratory syndrome virus glycoprotein 5 (gp5) in stably expressing cells induction of apoptosis by the nonstructural protein 4 and 10 of porcine reproductive and respiratory syndrome virus pig immune response to general stimulus and to porcine reproductive and respiratory syndrome virus infection: a meta-analysis approach expression of toll-like receptor mrna and cytokines in pigs infected with porcine reproductive and respiratory syndrome virus analysis of the swine tracheobronchial lymph node transcriptomic response to infection with a chinese highly pathogenic strain of porcine reproductive and respiratory syndrome virus understanding prrsv infection in porcine lung based on genome-wide transcriptome response identified by deep sequencing porcine reproductive and respiratory syndrome virus: description of persistence in individual pigs upon experimental infection duration of infection and proportion of pigs persistently infected with porcine reproductive and respiratory syndrome virus humoral immune responses and viral shedding following vaccination with modified live porcine reproductive and respiratory syndrome virus vaccines simian hemorrhagic fever virus: recent advances lactate dehydrogenase-elevating virus equine viral arteritis genome-wide association study among four horse breeds identifies a common haplotype associated with in vitro cd3+ t cell susceptibility/resistance to equine arteritis virus infection equine arteritis virus long-term persistence is orchestrated by cd8+ t lymphocyte transcription factors, inhibitory receptors, and the cxcl16/cxcr6 axis identification of factors associated with virus level in tonsils of pigs experimentally infected with porcine reproductive and respiratory syndrome virus double-stranded viral rna persists in vitro and in vivo during prolonged infection of porcine reproductive and respiratory syndrome virus development of a broadly protective modified-live virus vaccine candidate against porcine reproductive and respiratory syndrome virus enhanced replication of porcine reproductive and respiratory syndrome (prrs) virus in a homogeneous subpopulation of ma-104 cell line gradual development of the interferon-gamma response of swine to porcine reproductive and respiratory syndrome virus infection or vaccination location of t-cell epitopes in nonstructural proteins 9 and 10 of type-ii porcine reproductive and respiratory syndrome virus a 10-kda structural protein of porcine reproductive and respiratory syndrome virus encoded by orf2b use of interleukin 12 to enhance the cellular immune response of swine to an inactivated herpesvirus vaccine cross reactivity of immune responses to porcine reproductive and respiratory syndrome virus infection a wrapper script to automate quality and adapter trimming cutadapt removes adapter sequences from high-throughput sequencing reads a quality control tool for high throughput sequence data ultrafast and memory-efficient alignment of short dna sequences to the human genome differential gene and transcript expression analysis of rna-seq experiments with tophat and cufflinks accurate alignment of transcriptomes in the presence of insertions, deletions and gene fusions htseq-a python framework to work with high-throughput sequencing data moderated estimation of fold change and dispersion for rna-seq data with deseq2 manke, t. deeptools2: a next generation web server for deep-sequencing data analysis gene ontology analysis for rna-seq: accounting for selection bias kobas 2.0: a web server for annotation and identification of enriched pathways and diseases tlr10 is a negative regulator of both myd88-dependent and -independent tlr signaling the inhibitory cd200r is differentially expressed on human and mouse t and b lymphocytes cd200 and membrane protein interactions in the control of myeloid cells cd200/cd200r paired potent inhibitory molecules regulating immune and inflammatory responses; part ii: cd200/cd200r potential clinical applications complement evasion strategies of viruses: an overview. front influenza a virus m1 blocks the classical complement pathway through interacting with c1qa apoptosis: a review of programmed cell death apoptosis in the lungs of pigs infected with porcine reproductive and respiratory syndrome virus and associations with the production of apoptogenic cytokines nf-kappab antiapoptosis: induction of traf1 and traf2 and c-iap1 and c-iap2 to suppress caspase-8 activation bfl-1/a1 functions, similar to mcl-1, as a selective tbid and bak antagonist activation, differentiation, and migration of naive virus-specific cd8+ t cells during pulmonary influenza virus infection regulation of t cell migration during viral infection: role of adhesion molecules and chemokines molecular and cellular insights into t cell exhaustion functional and phenotypic analysis of porcine peripheral blood cd4/cd8 double-positive t cells speckled-like pattern in the germinal center (slip-gc), a nuclear gtpase expressed in activation-induced deaminase-expressing lymphomas and germinal center b cells member of the tumor necrosis factor family and b lymphocyte stimulator il-21 and il-4 collaborate to shape t-dependent antibody responses the architecture of sars-cov-2 transcriptome a synthetic porcine reproductive and respiratory syndrome virus strain confers unprecedented levels of heterologous protection viral mimicry of the complement system the complement system in regulation of adaptive immunity control of the amplification convertase of complement by the plasma protein beta1h human complement c3b inactivator: isolation, characterization, and demonstration of an absolute requirement for the serum protein beta1h for cleavage of c3b and c4b in solution decay accelerating factor (cd55): a target for cancer vaccines? porcine reproductive and respiratory syndrome virus nonstructural protein 1beta modulates host innate immune response by antagonizing irf3 activation the cysteine protease domain of porcine reproductive and respiratory syndrome virus nonstructural protein 2 possesses deubiquitinating and interferon antagonism functions regulation of interferon regulatory factor-3 by the hepatitis c virus serine protease infectious chikungunya virus in the saliva of mice, monkeys and humans induction of ifn-beta and the innate antiviral response in myeloid cells occurs through an ips-1-dependent signal that does not require irf-3 and irf-7 interplay between interferon-mediated innate immunity and porcine reproductive and respiratory syndrome virus the cysteine protease domain of porcine reproductive and respiratory syndrome virus non-structural protein 2 antagonizes interferon regulatory factor 3 activation identification of viral genes associated with the interferon-inducing phenotype of a synthetic porcine reproductive and respiratory syndrome virus strain bax insertion, oligomerization, and outer membrane permeabilization in brain mitochondria: role of permeability transition and sh-redox regulation porcine reproductive and respiratory syndrome virus modulates apoptosis during replication in alveolar macrophages structural biology. controlling the caspases ccr7 and its ligands: balancing immunity and tolerance ccr7 coordinates the primary immune response by establishing functional microenvironments in secondary lymphoid organs ccl28 induces mucosal homing of hiv-1-specific iga-secreting plasma cells in mice immunized with hiv-1 virus-like particles influence of ccr7 ligand dna preexposure on the magnitude and duration of immunity enhancement of immune responses by co-delivery of ccl19/mip-3beta chemokine plasmid with hcv core dna/protein immunization genetic co-transfer of ccr7 ligands enhances immunity and prolongs survival against virulent challenge of pseudorabies virus molecular characterization of transcriptome-wide interactions between highly pathogenic porcine reproductive and respiratory syndrome virus and porcine alveolar macrophages in vivo molecular signature of cd8+ t cell exhaustion during chronic viral infection t cell exhaustion t cells and viral persistence: lessons from diverse infections pd-l1 expression is increased in monocyte derived dendritic cells in response to porcine circovirus type 2 and porcine reproductive and respiratory syndrome virus infections role of nf-kappa b in cell survival and transcription of latent membrane protein 1-expressing or epstein-barr virus latency iii-infected cells influence of n-linked glycosylation of porcine reproductive and respiratory syndrome virus gp5 on virus infectivity, antigenicity, and ability to induce neutralizing antibodies immune evasion of porcine reproductive and respiratory syndrome virus through glycan shielding involves both glycoprotein 5 as well as glycoprotein 3 identification of neutralizing and nonneutralizing epitopes in the porcine reproductive and respiratory syndrome virus gp5 ectodomain this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license we would like to thank dirk anderson at flow cytometry core facility, nebraska center for biotechnology for assistance on flow cytometric analysis and the staff members of unl life sciences annex and veterinary diagnostic center for the care of animals. the use of product and company names is necessary to accurately report the methods and results; however, the united states department of agriculture (usda) neither guarantees nor warrants the standard of the products. the use of names by the usda implies no approval of the product to the exclusion of others that may also be suitable. the usda is an equal opportunity provider and employer. key: cord-005077-ejgzfzd2 authors: vogel, wolfgang title: treatment of chronic hepatitis c patients not responding to combination therapy with ribavirin and interferon α — hype or hope? date: 2004 journal: wien klin wochenschr doi: 10.1007/bf03217702 sha: doc_id: 5077 cord_uid: ejgzfzd2 nan infection with the hepatitis c virus (hcv) is still a major cause of chronic liver disease resulting in need for liver transplantation and of hepatocellular carcinoma in the western world. the prevalence of infection is believed to be 0.1% in central europe with figures as high as 5% reported in endemic areas such as egypt. historically, the major route of infection was poor medical practice. currently, intravenous drug abuse probably accounts for the majority of new infections. our observations suggest that about 4000 new infected patients will be diagnosed in austria annually. if all patients were treated with the best regimen available, based on a markov model, the number of decompensated cirrhotic patients could be reduced by almost two-thirds [1] . interferon-a (ifn) was introduced as therapy in the late eighties of the last century and since then has proved to be the mainstay of treatment. the sustained virological response rates (svr), defined as pcr negative 6 months after the end of therapy, which is considered as proof of final elimination of the virus, were initially dismal. therapy prolongation up to 12 months and administration of ribavirin, a nucleoside analogon with non-specific anti-hcv effects, to ifn improved svr from 6% to 36%. treatment with pegylated interferons in combination with ribavirin results in > 50% svr after 12 months of therapy in genotype 1 patients and> 80% after 6 months of therapy in genotype 2 and 3 patients. despite this dramatic improvement of therapy results, for 20 to 50% of the treated patients, we will have to look for new treatment options. a particularly difficult group of patients are nonresponders to ifn therapy. these patients typically will remain hcv-rna positive during the whole period of therapy. patients achieving hcv-rna negativity during treatment, but becoming positive again during the treatment phase or after the end of therapy are defined as break-through or relapse patients. on the basis of prospective studies, patients not becoming negative after 12 weeks of therapy or whose hcv-rna titre failed to drop by at least two log, have a likelihood of achieving svr of only 1.6%, whereas patients with a significant fall in hcv-rna have a 68% likelihood of svr [2] . re-treatment of these patients employing pegylated ifn in combination with ribavirin will result in svr only in approximately 10% of the patients. patients pretreated only with ifn monotherapy, however, have a 30% probability of svr when retreated with ifn-ribavirin combination therapy. relapsers or partial responders to treatment with conven-tional ifn plus ribavirin are believed to have a more favourable prognosis, but prospective controlled studies are lacking. factors associated with non-response are genotype 1 or 4, high serum hcv-rna concentration at baseline, cirrhosis, current alcohol abuse, race, dose reduction and non-compliance. only a few of these factors are correctable. a svr of 30% upon re-treatment in patients infected with genotype 2 or 3 and in patients with base-line hcv-rna concentration of < 1.5 million iv/ml can be achieved. the search for more efficacious treatment options has brought amantadine (ama) to the forefront. in 1997 a pilot study of patients with chronic hepatitis c who failed treatment with ifn monotherapy and subsequently treated with ama was carried out [3] . in this study a reduction of necro-inflammation and a decrease of transaminase activity in 64% of patients were observed. four out of 22 patients cleared the virus and achieved svr. however, this favourable effect of ama could be reproduced in subsequent trials neither in non-responders to ifn nor in naive patients [4, 5] . the observed reduction in transaminase levels is reminiscent of the effects of ribavirin, which improves liver biochemistry but has no effect on viral replication. amantadine, a tricyclic amine, has antiviral activity against toga, myxo, arena, flavi and corona viruses [6] . known mechanisms of action include an early step in viral replication and interaction with the influenza a viral matrix protein [6] . indirect assessment of the effect of ama on hcv protease, helicase, atpase, rna polymerase, and hcv internal ribosomal entry site (ires) translation has been performed by in vitro biochemical assays [7] . although no inhibition was observed, adenylyl cyclase associated protein (cap) and ires reporter genes were suppressed at higher levels probably by non-specific inhibition of translation. on the basis of these results it was concluded that ama has no direct specific inhibitory effect on hcv replication. in the hcv replicon system, ifn induces a dose-dependent inhibition of hcv rna levels, while ama and ribavirin had no effect. a competent immune response is mandatory for the efficient clearance of the virus. ama can more effectively suppress the hcv specific proliferative response of pbmcs than ifn [8] . in summary, ama has some weak anti-inflammatory properties without direct anti-viral effects. the lack of specific anti-viral drugs encouraged a number of large randomized clinical trials, which, however, had conflicting results. a comparative study in which wolfgang vogel 119 naive patients were investigated for assessing the effectiveness of combination therapy ifn and ama on the one hand, and of ifn plus placebo, on the other, demonstrated the former to be significantly superior. in this german study, 22% of patients had svr compared to 10% in the monotherapy arm after 48 weeks of treatment [9] . a similar study performed in italy on a cohort of 200 naive patients who were administered slightly higher doses of ifn, 6 mu t.i.w, reported svr in 29% and 17% of patients on the combined vs. monotherapy, respectively. the ama dose of 2 x 100 mg daily was identical in both studies [10] . two further studies, one from italy and another from the uk of a total of 359 naive patients confirmed svrs of 23% versus 17% similar to those reported in the german study. in all these studies ama had no effect on the safety profile of ifn treatment. another approach to increase the efficacy of ifn treatment would be to improve the initial virological response by induction therapy with high-dose ifn. the initial decline in viral load predicts the outcome of therapy as patients with svr are characterized by a greater than 90% drop in viral titre within 4 weeks of initiation of treatment. to test this concept a prospective randomized trial was performed by the austrian hepatitis study group [11] . in this pivotal study of 373 naive patients receiving 10 mu ifn induction followed by ifn-ribavirin combination therapy, genotype 1 patients had a significantly better svr of 44% versus 29% with induction therapy than without. however, no difference was observed in genotype 3 patients. the issue of three-drug combination therapy was first studied by brillanti [12] . in a randomised prospective trial 60 patients with chronic hepatitis c not responding to a previous course with inf were either treated with 5 mu ifn on alternate days in combination with ribavirin (800-1200 mg daily) or additionally with ama (200 mg daily) for 12 months. an encouraging 57% of the patients on triple therapy achieved svr but only 10% of the patients on dual therapy. however, a german study of 134 nonresponders found no difference between the two treatment regimens [13] . in a number of further smaller studies of non-responders to ifn monotherapy, no benefit of adding ama to ifn-ribavirin combination therapy could be demonstrated, either with or without induction therapy [14] . the difference in the studies is difficult to explain, aspects to consider are definition of non-response, patient selection with respect to genotype and stage of liver fibrosis. the interesting question of whether the addition of pegylated interferons to the combination of ribavirin and ama can improve svr in non-responders is still a matter of ongoing studies. preliminary results are promising. in this issue of the journal, stauber and the austrian hepatitis group present the findings of a prospective trial in the difficult-to-treat group of patients who have failed previous therapy with standard ifn and ribavirin [15] . the study included 67 non-responders, 19 relapsers after a standard treatment period and 16 patients, who had experienced a break-through of disease after an initial response while on therapy. eighty percent of the patients were infected with genotype 1 and 19% were cirrhotics. the novel approach was to combine an induction period of daily ifn therapy for 16 weeks with standard dose ama and ribavirin. interestingly, 34% of patients were negative at week 12 of therapy whereas only 15% had svr. relapsers and break-through patients had a higher svr than non-responders, but these differences did not reach statistical difference. the tolerability of the triple therapy was again not different from that reported for the dual therapy in previous trials. although the authors felt that the slightly higher svr of 15% compared to 11% in other trials was somewhat disappointing, the findings of the study offer some hope for this group of patients and raises interesting points. the surprisingly high response rate after 12 weeks of therapy suggests that modification of ifn dosing is efficacious in non-responders. approximately 50% of the responders were lost during treatment phase and another 50% during follow-up. because of the trial design it is difficult to separate clearly the effect of the induction phase and the effect of ama. but it is obvious that the gain in responders during the early phase of the trial was lost in the long run. this implies that ama is not capable of maintaining the early advantage by boosting the immune-response and increasing the clearance rate of infected hepatocytes. the conclusion therefore would be that this kind of patients could benefit from either longer therapy or higher ifn dose or both. however, the question of the extent to which addition of ama can increase the response rate still remains open. with the advent of more efficacious pegylated ifn, studies of longer treatment periods with and without ama are warranted. in summary, the question if ama offers additional benefit in the treatment of non-responders to combination therapy is still open. this inexpensive and well-tolerated drug holds some promises which need further evaluation. the current outlook for new treatment options is poor, and as the new designer drugs to specifically inhibit viral replication enter phase ii clinical trials, the likelihood that ifn with all its limitations and side effects will remain the mainstay of therapy for the foreseeable future is very high. so researchers are challenged to expand the existing treatment options for improved results, particularly in the difficult-to-treat group of patients. projecting future complications of chronic hepatitis c in the united states monitoring of viral levelsduringtherapy of hepatitis c treatment of chronic hepatitis c with amantadine treatment of chronic hepatitis c with amantadine treatment of chronichepatitis c with amantadine hydrochloride in patients who had not responded to previous treatment with interferon-alpha and! or ribavirin antiviral drugs: current state of the art amantadine and rimantadine haveno direct inhibitory effects against hepatitis c viral protease, heli-vogel, treatment of chronic hepatitis c patients case, atpase, polymerase, and internal ribosomal entry site-mediated translation in vitro effect of amantadine and interferon alpha-2a on hepatitis c virus markers in cultured peripheral blood mononuclear cells from hepatitis c virus-infected patients randomized, double-blind, placebo-controlled trial of interferon alfa2a with and without amantadine as initial treatment for chronic hepatitis c a randomized trial of amantadine and interferon versus interferon alone as initial treatment for chronic hepatitis c combination of interferon induction therapy and ribavirin in chronic hepatitis c triple antiviral therapy as a new option for patients with interferon non-responsive chronic hepatitis c randomized, placebo-controlled, double-blind trial with interferon-alpha with and without amantadine sulfate in primary ifn-alpha non-responders with chronic hepatitis c amantadine for chronic hepatitis c: a magic bullet or yet another dead duck? retreatment of patients with chronic hepatitis c not responding to interferonlribavirin combination therapy with daily interferon plus ribavirin plus amantadine key: cord-001542-f089bs8r authors: lai, kang yiu; ng, wing yiu george; cheng, fan fanny title: human ebola virus infection in west africa: a review of available therapeutic agents that target different steps of the life cycle of ebola virus date: 2014-11-28 journal: infect dis poverty doi: 10.1186/2049-9957-3-43 sha: doc_id: 1542 cord_uid: f089bs8r the recent outbreak of the human zaire ebolavirus (ebov) epidemic is spiraling out of control in west africa. human ebov hemorrhagic fever has a case fatality rate of up to 90%. the ebov is classified as a biosafety level 4 pathogen and is considered a category a agent of bioterrorism by centers for disease control and prevention, with no approved therapies and vaccines available for its treatment apart from supportive care. although several promising therapeutic agents and vaccines against ebov are undergoing the phase i human trial, the current epidemic might be outpacing the speed at which drugs and vaccines can be produced. like all viruses, the ebov largely relies on host cell factors and physiological processes for its entry, replication, and egress. we have reviewed currently available therapeutic agents that have been shown to be effective in suppressing the proliferation of the ebov in cell cultures or animal studies. most of the therapeutic agents in this review are directed against non-mutable targets of the host, which is independent of viral mutation. these medications are approved by the food and drug administration (fda) for the treatment of other diseases. they are available and stockpileable for immediate use. they may also have a complementary role to those therapeutic agents under development that are directed against the mutable targets of the ebov. electronic supplementary material: the online version of this article (doi:10.1186/2049-9957-3-43) contains supplementary material, which is available to authorized users. the recent outbreak of the human zaire ebolavirus (ebov) infection starting in west african countries has resulted in 15,351 infected patients, as of 18 th of november 2014. a total of 5,459 deaths have been reported in six affected countries (guinea, liberia, mali, sierra leone, spain, and the united states of america) and two previously affected countries (nigeria and senegal) [1] . apart from supportive care, neither a licensed vaccine nor a specific therapy is available for the treatment of the human ebov infection [2] . the world health organization (who) has considered that it is ethically acceptable to offer unproven interventions that have shown promising results in laboratory and animal models, but have not yet been evaluated for safety and efficacy in humans as potential sources of treatment or prevention [3] . several promising therapeutic agents have been identified for the treatment and immunization of the ebov. these may include monoclonal antibody (mabs)-based therapies (e.g. zmapp), anti-sense phosphorodiamidate morpholino oligomers (pmo avi-6002), lipid nanoparticle small interfering rna (lnp-sirna: tkm-ebola), and an ebov glycoprotein-based vaccine using live-attenuated recombinant vesicular stomatitis virus (rvsv-ebogp) or a chimpanzee adenovirus (rchad-ebogp)-based vector. human trial results of these agents would not be available until next year. moreover, existing supplies of all these experimental medications and vaccines for compassionate use are either extremely limited or exhausted [4] [5] [6] . to combat such an unprecedented global public-health crisis before these experimental agents are available, alternative available interventions that can target different steps in the replication cycle of the ebov should be explored in the management of the human ebov infection as contingency preparation for the international dissemination of the ebov outbreak in west africa. we have reviewed currently available therapeutic agents that have shown to be effective in suppressing the proliferation of the ebov in cell cultures or animal studies. we propose a therapeutic regimen to supplement the current supportive therapy aiming to reduce viral load, the most important factor in the determination of mortality. through viral load suppression, we may be able to prolong a patient's survival in order to provide a better chance for the patient to develop natural immune defense against the ebov. the ebov is an enveloped filamentous rna virus belonging to the family filoviridae. the 19-kb linear, non-segmented, negative-sense, single-stranded rna genome of the ebov encodes seven structural proteins and two non-structural proteins in the following order within the genome: 3′ non-coding region (leader), nucleoprotein (np), virion protein 35 (vp35), vp40, 3 glycoproteins (sgp/ssgp/gp1,2), vp30, vp24, rnadependent rna-polymerase protein (l-polymerase), and 5′ non-coding region [7] . the ebov genome encodes one transmembrane protein gp1,2 (gp1-gp2) and two secreted non-structural proteins: secretary glycoprotein (sgp) and small soluble glycoprotein (ssgp). a small soluble delta peptide (δ-peptide) is secreted from ebov-infected cells after the carboxylterminal cleavage of sgp [8] . gp1,2 is produced through transcriptional rna editing as a precursor for 676 amino acid polyprotein (gp0), which is post-translationally cleaved by furin into two disulfide-linked subunits; a surface subunit, gp1; and a membrane-spanning subunit, gp2. gp1 contains the receptor-binding domain (rbd) for host cell attachment and a mucin-like domain to protect the rbd from humoral and cell-mediated immunity. the rbd responsible for receptor binding, viral entry, and cellular tropism is covered by a heavily glycosylated "glycan cap". the transmembrane gp2 contains a helical heptad-repeat region, transmembrane anchor, and a 4-residue cytoplasmic tail. the gp2 drives fusion of the viral membrane with the endosomal membrane of the target cell. this gp1-gp2 heterodimer then assembles as a trimer on the viral surface. this homotrimeric gp1,2 complex forms the spike on the envelope membrane of the mature viral particles. during processing, gp1,2 are unstable, and an abundant amount of a soluble non-virion form of gp1 and a scanty amount of gp1,2 are released into the circulation [9] [10] [11] [12] . the virusassociated gp1,2 and not the other soluble glycoproteins released during the virus infection are responsible for primary target cell activation [13] . the highly glycosylated mucin-like region of gp1 is cytotoxic to the host cells [14] . the shedding of souble gp1,2-like protein due to cleavage of ebov glycoprotein on the surface of ebov-infected cells by tumor necrosis factor-alpha converting enzyme (tace) can activate non-infected dendritic cells and macrophages to induce cytokine dysregulation and endothelial cell dysfunction [15] . the gp2 of the ebov is able to counter the interferon (ifn)-inducible antiviral protein tetherin which restricts the vp40-dependent budding of the progeny viral particles from infected cells [16] [17] [18] . the sgp is produced from non-edited mrna species through furin cleavage from a precursor pre-sgp. the sgp shares the n-terminal 295 amino acids with gp1, but differs in the carboxyl terminus by 69 amino acids. the sgp is released into the circulation in the form of homodimers in antiparallel orientation [19] to evade an antibody-associated innate immune response [20, 21] . the sgp has an antiinflammatory function and impairs the transmigration and activation of neutrophils [22, 23] . while the gp1,2 in its particle-associated form mediates endothelial cell activation and a decrease in endothelial cell barrier function, sgp protects the endothelial cell against cytokine-induced barrier dysfunction. the sgp constitutes at greater than 80% of the total gp synthesized during infection. hence, the hypersecretion of the sgp may protect the ebov against host humoral immune defense and the host endothelial cell against cytokine-induced cytotoxicity during the early phase of the ebov infection [15, 24, 25] . δ-peptide released in ebov-infected cells joins cathepsins and integrins to inhibit further entry of the ebov in a dose-dependent manner to prevent superinfection of ebov-infected cells. δ-peptide inhibits entry of both marburgviruses and the ebov, indicating that they might interfere with a common pathway used by filoviruses to gain entry into target cells [26] . the ssgp of a yet undefined function is produced through transcriptional editing and secreted in the form of a disulfide-linked homodimer that is exclusively n-glycosylated. while ssgp appears to share similar structural properties with sgp, it does not appear to have the same anti-inflammatory function as sgp [22, 23, 27] . the ebov, being a rna virus with limited coding capacity, has utilized the host's unique metabolic pathway for its viral entry, replication, and egress. the entry of the ebov into cells is initiated by interaction of the viral gp1 with host cell surface t-cell immunoglobulin and mucin domain 1 (tim-1) receptors. upon receptor binding, the ebov is internalized into endosomes primarily via macropinocytosis [28] [29] [30] . within the acidified endosome compartment of the host cell, the heavily glycosylated gp1 is cleaved to a smaller 19-kda fusogenic form by the low ph-dependent cellular proteases cathepsin l (catl) and b (catb), exposing residues in the receptor binding site. this allows the binding of gp1 to cholesterol transporter niemann-pick c1 (npc1), a step in the late endosome phase essential for virus-host membrane fusion and viral entry [31] [32] [33] [34] . cells where the npc1 function has been biochemically disrupted or cells lacking npc1 showed resistance to the ebov infection. cells from subjects with npc1 disease were resistant to the ebov because of defects in the npc1 protein [35] [36] [37] [38] . after complete fusion of the viral and host endosomal membranes via conformational change in gp2, viral rna and its associated proteins are released into the host cell cytoplasm [39] . once inside the cytoplasm of the host cell, the ebov suppresses the innate immune response via vp35 and vp24 proteins [40] , and hijacks transcription and translation for robust genome replication and the production of new virions. the ribonucleoprotein (rnp) complex that mediates transcription and replication of the ebov genome comprises np, vp35, vp30, and l protein [41] [42] [43] [44] . vp30 is essential in the initiation of the ebov transcription, but is not required for viral replication. however, dynamic phosphorylation of vp30 is an important mechanism to regulate the balance between the transcription and replication processes in the ebov replication cycle [45] [46] [47] . this unique property of vp30 allows the development of a genetically stable vp30 deleted ebov vaccine with protective efficacy in the mice and guinea pig models [48] . the matrix proteins vp40 and vp24 associated with the viral lipid coat are important for virus structure and stability. both matrix proteins vp24 and vp40 contribute to the regulation of viral genome replication and transcription [49] and the budding of the virus [50] [51] [52] , an important step prior to viral egress [53, 54] . this distinct replication cycle of the ebov serves as an attractive target for the development of therapeutic agents against the ebov (see figure 1 and table 1 ). human ebov hemorrhagic fever, characterized by uncontrolled viral replication together with immune and vascular dysregulation, has a case fatality rate of up to 90% [7] . type i alpha/beta interferons (ifn-α/β), encoded by a single ifn-β and 13 homologous ifn-α genes in humans, represent an essential element of host defense against virus infections, including the ebov [55] . the human ebov infection is associated with robust ifn-α production-with plasma concentrations of ifn-α that greatly (60-to 100-fold) exceed those observed in other viral infections-but limited ifn-β production [56] . the upon receptor binding of ebov gp 1 with host tim-1 receptor, ebov is internalized into endosome via macropinocytosis. within the acidified endosome compartment of the host cell, under the action of the low ph-dependent cellular proteases cathepsins, the receptor binding site of gp 1 to cholesterol transporter niemann-pick c1 (npc1) is exposed. this results in conformational change in gp 2 , leading to complete fusion of the viral and host endosomal membranes in the late endosome and the release of viral rna and its associated proteins into the host cell cytoplasm. ebov then hijacks transcription and translation for robust genome replication and viral protein production under the action of ribonucleoprotein polymerase complex (rnp polymerase). the accumulation of gp 1,2 in the endoplasmic reticulum leads to endoplasmic reticulum overload response (er-overload) which, in turn, induces cytokine dysregulation via the activation of nuclear factor kappa b (nfκb) through the production of reactive oxygen species (ros). new virions are released through atp-dependent budding and egress from host cell membrane. currently available therapeutic agents that target the different steps of the ebov life cycle are described in table 1. ebov, protected from the host interferon response by its encoded vp35 and vp24 proteins [40, [57] [58] [59] , produced a heavy viral load [60] [61] [62] , cytopathic damages [14, 63, 64] , and cytokine dysregulation in humans [65] [66] [67] [68] . the efficient productive replication of the ebov inside monocyte and macrophages leads to a massive release of proinflammatory cytokines/chemokines and reactive oxygen species (ros) [13, 15, 65, 66, [69] [70] [71] , which in turn leads to diffuse endothelial cell dysfunction [72] [73] [74] [75] [76] , disseminated intravascular coagulation [77] [78] [79] , and vasomotor collapse [80] [81] [82] . the infection of the antigen presenting dendritic cells [83] [84] [85] [86] and profound bystander apoptosis of lymphocytes [63, [87] [88] [89] impairs the development of adaptive immunity [90, 91] and ebov-specific cd8+ t [92] [93] [94] , as well as cd4+ t cells [95] that are important for the clearance of, and protection from, the ebov infection. infected monocyte-derived dendritic cells were impaired in the secretion of pro-inflammatory cytokines, the up-regulation of co-stimulatory molecules, and the stimulation of t cells [96] . numbers of cd4+ and cd8+ t cells are substantially reduced in fatal human and nonhuman primate (nhp) infections before death [63, 88, 97] . immune evasion by the glycoproteins of the ebola virus: implications on passive immunization and vaccine development the ebov is able to counteract both humoral and cellmediated immunity through its gp1,2 and sgp [11, 98] . the overexpression of mature gp1,2 on the plasma membrane results in the masking of antigenic epitopes on gp1,2 itself and the shielding of mhc-i and integrin β, leading to evasion of antiviral immunity. steric shielding of surface epitopes by the heavily glycosylated gp impairs the recognition and killing of ebov-infected cells by the natural killer and cytotoxic cd8+ t cell during an acute viral infection. it may also contribute to the persistent infection in the natural reservoir host to perpetuate the spread of the ebov [99] [100] [101] . the sgp can evade host antibody-mediated response through "antigenic subversion" by eliciting non-neutralizing antibodies that cross-react with gp1,2. thus, the massive secretion of sgp by the ebov may prevent effective neutralization of the virus during an ebov infection and reduce the effectiveness of vaccines that rely upon neutralizing antibody responses against gp1,2 [20, 21] . some of the antibodies against gp1 may lead to enhancement of infectivity of the ebov via interaction with complement component c1q, a phenomenon known as the antibody-dependent enhancement. the ebov initiates infection by binding its gp1 to its specific human receptor sites on the surface of human cells. the interaction of c1q enhances binding between the virus-antibody complex and the c1q ligands on the cell surface, promoting interaction between the ebov and its receptor. these infectivity-enhancing antibodies were virus species specific and were primarily correlated with immunoglobulin igg2a and igm levels, but not with igg1 levels [102, 103] . the presence of infectivity-enhancing antibodies against gp1,2 in the ebov infection raises concerns about the effectiveness of gp-based ebov vaccines, and the use of passive prophylaxis or treatment with gp-based antibodies [104, 105] . antibodies against gp1 of the ebov can be neutralizing, enhancing, or non-neutralizing and non-enhancing. neutralizing antibodies are produced in infection by the ebov at a relatively low frequency [106] . some anti-ebov antibodies are known to be neutralizing in vitro but not protective in vivo, whereas other antibodies are known to be protective in animal models in vivo, but not neutralizing in vitro [107] . investigations of anti-gp antibodies against the ebov showed that non-neutralizing antibodies induce interferon-inducible transmembrane proteins (ifitmp) production to restrict entry of ebov. favipiravir suppress viral rna polymerase. inhibit na + /k + -atpase that are important in the budding and egress of encapsulated ebov. ouabain digoxin digitoxin anti-oxidants suppress ros-dependent nfκb activation and cytokine dysregulation induced by gp 1,2 -induced er-overload. high dose n-acetylcysteine infusion 1 chloroquine, amiodarone, dronedarone and toremifene administration is associated with an increased risk of qt prolongation and torsades de pointes. 2 verapamil should be avoided in patient with hypotension. recognized gp epitopes in the sgp or non-essential mucin-like domain of gp1, while neutralizing antibodies were specific to rbd in gp1 or conformation-dependent epitopes at the base of the gp1,2 spike where gp1 meets gp2. two neutralizing antibodies (kz52 and jp3k11) against ebov-that recognize conformation-dependent epitopes comprising residues in gp1 and gp2-were identified to have quite distinct mechanisms of neutralization. kz52 is a human recombinant igg1 neutralizing antibody derived from a human survivor of a natural ebov infection during the 1995 outbreak in kikwit, democratic republic of congo. kz52 has impaired recognition for the sgp and binding was dependent on the presence of gp2 residues which are not present in the sgp. kz52 is able to inhibit cathepsin cleavage of gp1,2. jp3k11, a monkey derived neutralizing monoclonal antibody against ebov, recognized the cleaved, fusion-active form of gp [108] . 16 f6 is a mice derived monoclonal igg1 antibody that neutralizes sudan ebov by preventing the conformational changes in gp1,2 required for membrane fusion. both 16 f6 and kz52 recognize gp1-gp2-bridging epitopes at the base of the gp1,2 trimer, indicating that this overlapping epitope may be one of the key sites for neutralization of the ebov, and is thus a target for immunotherapy and a key goal of vaccine design [109] . antibody subclass may be another important factor in protection against the ebov. igg2 isotype may offer more effective protection against ebov [110, 111] . although fully protecting guinea pigs from infection, kz52 fails to slow viral replication and protect nhps from the ebov infection [112] . in contrast, rvsv-ebogp [113] [114] [115] [116] and rchad-ebogp [117] [118] [119] [120] based vaccination have demonstrated both prophylactic and post-exposure protection in nhps [121] . this was previously attributed to the protective action of ebov-specific cd4+ and cd8+ t-cell response induced by these vaccines in limiting infection and the inability of kz52 to completely block all entries of the ebov into cells and its subsequent explosive replication [112] . rchad-ebogpbased vaccination is able to generate potent humoral and cell-mediated responses. significant antibody titers are detectable at 48 weeks post vaccination [122, 123] . cd8+ cellmediated immunity has been shown to play a critical role in protection against the ebov infection in nhps in rchad-ebogp-based vaccination [124] . on the other hand, humoral rather than the cell-mediated response contributes to protection against the ebov infection in nhps in rvsv-ebogp-based vaccination [125, 126] . candidate vaccines expressing the ebov gp or np protect rodents and nhps from the lethal ebov infection [127] [128] [129] . humoral and cell-mediated immune responses are working together to provide protection against the lethal ebov infection. either response alone may be able to limit virus replication but both arms of the immune response are required to clear the infection [97, 130] . vp proteins (vp24, vp30, vp35, and vp40) are poor inducers of cell-mediated immunity and are inaccessible to the protective effect of vp-induced neutralizing antibodies because they are not found on the surface of virions or infected cells [131] . however, the genetic sites of these internal proteins are susceptible to sirna and pmo interference. tkm-ebola (a sirna targeting l-polymerase, vp24, and vp35) can be administered intravenously or subcutaneously in a lyophilized lipid nanoparticle formulation. tkm-ebola offers post-exposure protection against the ebov infection in nhps. the fda has approved an "expanded access" program for the use of tkm-ebola in patients with confirmed or suspected infections [132, 133] . anti-sense phosphorodiamidate morpholino oligomers avi-6002 effectively reduce viral load, diminish virallyinduced pathology, and improve survival of nhps with the ebov infection by targeting vp24 and vp35 mrna. through judicious placement of positive charges on the drug backbone, the drug is able to bind to a negative charge on the virus even if binding at one or more drugvirus base pairs are lost through mutation. this integration of dual targeting and charge complementation significantly lowers the likelihood of drug resistance through viral mutagenesis [134, 135] . currently available therapeutic agents that are effective in targeting the ebov infection in cell or animal studies may include convalescent plasma, favipiravir, chloroquine, amiodarone, dronedarone, verapamil, clomiphene, toremifene, ifn-β, na + /k + exchangers, na + /k + -atpase pump inhibitors, and antioxidants. except for convalescent plasma and favipiravir, most of the therapeutic agents under review are acting against the non-mutable targets of the host cells which participate in the replication cycle of the ebov. they may also have a complementary role to conventional therapy in the management of the current ebov outbreak in west african countries (see table 1 ). the who issued a consensus statement that the use of whole blood therapies and convalescent blood serum needs to be considered as a matter of priority in the recent ebov outbreak in west african countries [2] . the development of neutralizing antibodies and t-cell responses are important for recovery from the ebov infection [97, 136] . patients who are able to mount an immune response to the ebov will begin to recover in seven to ten days and start a period of prolonged convalescence [137] . in survivors, early and increasing levels of igg, directed mainly against the np and the vp40, were followed by the clearance of circulating viral antigen and activation of cytotoxic t cells. in contrast, fatal infection was characterized by impaired humoral responses, with absent specific igg and barely detectable igm [63] . convalescent blood has been shown to improve survival of ebov-infected patients during the outbreak in kikwit in 1995 [138] . immunity against ebov gp is sufficient to protect individuals against infection, and several vaccines based on ebov gp are under development including recombinant adenovirus, parainfluenza virus, venezuelan equine encephalitis virus, vesicular stomatitis virus, and virus-like particles [139] . neutralizing human monoclonal antibodies is able to protect mouse and guinea pigs from lethal ebov. however, the protection was achieved only by treatment shortly before or after viral infection [140] [141] [142] . the ebov can rapidly mutate to produce antibody-escape mutants. hence, antibody therapy may require hyperimmune polyclonal serum or a panel of monoclonal antibodies of different epitope specificities to be successful [143, 144] . these studies have laid the foundation for subsequent clinical research on the development of monoclonal antibodies [145] [146] [147] [148] and utilization of a monoclonal antibody cocktail such as mb-003 [149] , zmab [150] , and zmapp [151] in the treatment of the ebov infection in nhps. it is interesting to note that all three monoclonal antibody cocktails include one antibody that binds to or close to the glycan cap and that two of the three monoclonal antibody cocktails include at least one antibody that binds the gp1/ gp2 interface, indicating that these two regions may be especially important in protection against ebov [148] . the treatment window of monoclonal antibody therapy can be extended by the co-administration of adenovirus-vectored interferon therapy. in a guinea pig model, monoclonal antibodies combined with adenovirus-vectored interferon given three days after infection resulted in 100% survival, a significant improvement over either treatment alone [152] . a subsequent study showed that such a combination therapy is capable of saving 100% of ebov-infected nhps when initiated after the presence of detectable viremia along with symptoms [153] . (2) favipiravir (t-705; 6-fluoro-3-hydroxy-2pyrazinecarboxamide) favipiravir is a broad-spectrum inhibitor of viral rna polymerase that is able to inhibit the replication of many rna viruses. it is registered in japan for the treatment of influenza virus infection [154, 155] . favipiravir is able to suppress the replication of the ebov in cell culture. favipiravir, initiated at day 6 after ebov infection, induced rapid virus clearance, reduced the biochemical parameters of disease severity, and prevented a lethal outcome in 100% of mice lacking the type i interferon receptor [156] . oral favipiravir taken twice daily for 14 days is able to give 100% protection against an aerosol ebov infection in an immune-deficient mice model [157, 158] . the survival benefit was suboptimal in nhps. only one of the six animals tested survived. studies using dosages that are two to five times higher and have duration longer than shown in influenza studies are being conducted for the human ebov infection [5] . bcx4430, a synthetic adenosine analogue with a viral rna polymerase inhibitor function, is active against the ebov and marburg virus in rodent and cell culture. bcx4430 completely protects nhps from the marburg virus infection when administered as late as 48 hours after infection [159, 160] . the antimalarial drug chloroquine is able to increase endosomal ph. an acidic endosomal environment is important for the ph-dependent activation of cysteine proteases catb and catl, the proteases responsible for the cleavage of ebov gp1,2 essential for endosomal virus-host membrane fusion [35, 39, [161] [162] [163] . however, proteolytic processing of the ebov glycoprotein has been demonstrated to be not critical for ebov replication in cell culture [164] or nhps [165] . a recent study using a catb and catl deficient mouse model for the study of the ebov infection demonstrates that catb and catl activity is not absolutely required for ebov replication. the ebov glycoprotein cleavage seems to be mediated through a broader spectrum of proteases making therapeutic approaches targeting limited proteases unlikely to be beneficial to combat ebov infections [166] . a broad-spectrum small molecule that targets the catl cleavage of the ebov and inhibits the entry of a wide variety of viruses has recently been identified. it has been examined for the potential to develop into a potent broad-spectrum antiviral medication [167] . multiple cationic amphiphiles including amiodarone, dronedarone, verapamil, clomiphene, and toremifene have been identified as potent inhibitors of the entry of the ebov in an npc1-dependent fashion [38, 168] . amiodarone used for the treatment of atrial fibrillation and ventricular cardiac arrhythmia can induce lipidosis with features similar to niemann-pick c disease [169] . amiodarone and dronedarone, having basic pka and high water solubility at acidic ph, accumulates within late endosomal compartments, blocking fluid-phase endocytosis, proteolysis and lipid trafficking, and inducing a niemann-pick c-like phenotype. in contrast to the niemann-pick type-c disease, they are not alleviated by cholesterol removal [170, 171] . amiodarone, at concentrations that are routinely reached in human serum during anti-arrhythmic therapy (1.5-2.5 μg/ml), is a potent inhibitor of filovirus cell entry through late endosomes (ic50 0.25 μg/ml for ebov), when induced as a niemann-pick c-like phenotype. significant inhibition is observed in most endothelial and epithelial cells (e.g. macrophage, monocyte, vascular endothelial cell), except for primary hepatocyte and fibroblast. the inhibitory effect of amiodarone on the entry of the ebov was dose-dependent and reversible upon removal of the drug. prolonged exposure to amiodarone will not lead to a compensatory change in the host cell. a similar inhibitory property is observed with the amiodarone-related agent dronedarone and the l-type calcium channel blocker verapamil [38, 168, 172, 173] . both clomiphene and toremifene have anti-ebov activity in both the vero e6 (interferon-deficient african green monkey kidney epithelial cells) and hepg2 (human hepatocellular carcinoma) cell lines. the anti-ebov activity of clomiphene and toremifene is dependent not on its estrogen receptor antagonistic action but upon the ability of both drugs to induce a niemann-pick c-like phenotype to inhibit viral entry at late endosome. clomiphene and toremifene do not disrupt the interaction between primed gp1 and npc1, but mediate the entry block indirectly through npc1 by targeting other endosomal/lysosomal proteins involved in the cholesterol uptake pathway whose functions may be regulated by npc1. clomiphene and toremifene at 60 mg/kg every other day have been shown to result in a 90% and 50% survival rate, respectively, in ebov-infected mice compared with 100% mortality in the control group in an in vivo murine ebov infection model. they are effective in both male and female mice [38, 174] . however, the therapeutic dose against ebov cannot be achieved with the oral clomiphene dose used for inducing ovulation in humans [175] [176] [177] . the therapeutic dose against ebov with tolerable side effects can be achieved with toremifene at an oral dose used in the human trial for the treatment of advanced carcinoma of the breast [178] [179] [180] [181] . toremifene is well absorbed and >99.5% bound to plasma protein. toremifene undergoes extensive liver metabolism and enterohepatic recirculation. the majority of the toremifene dose is excreted as metabolites in feces. the long terminal half-life of oral toremifene may be due to both plasma protein binding and enterohepatic recirculation [182, 183] . interferon-induced transmembrane proteins (ifitms) are expressed basally in the absence of ifn induction in both primary tissues and cell lines [184] . an ifitm is able to inhibit the entry of viruses to the host cell cytoplasm; permit endocytosis, but prevent subsequent viral fusion; and release viral contents into the cytosol. the human ifitm locus is located on chromosome 11 and composed of four functional genes: ifitm1, ifitm2, ifitm3, and ifitm5. ifitm4p is a pseudogene. viruses that are restricted by ifitm proteins tend to fuse with host cell membranes in a late endosome or lysosome that precedes the induction of type i ifn in infected cells. viral escape from restriction by ifitm proteins could be more challenging than for antagonizing inhibitory factors that function at later stages of the virus life cycle because the opportunity for de novo synthesis of viral inhibitors is not available. all four human ifitm proteins are induced robustly by both type i and type ii ifns. ifitm1 is active against multiple viruses, including the ebov and hepatitis c viruses [185] [186] [187] . ifnβ is able to induce interferon-inducible transmembrane protein production to restrict entry of the ebov [188] . early post-exposure treatment with ifn-β significantly increased survival time of rhesus macaques infected with a lethal dose of the ebov, although ifn-β alone failed to alter the mortality rate. ifn-β treatment was associated with a trend towards lower plasma and tissue viral burden and pro-inflammatory cytokines production [56] . amiloride and its derivatives are used as potassiumsparing diuretics to treat hypertension and congestive heart failure. apart from inhibiting epithelial na + channel and cellular na + /k + exchangers, these drugs could also affect the function of other less well-defined ionexchangers (na + /ca 2+ and na + /mg 2+ ), and disturb the equilibrium of other ions, such as mn 2+ [189] [190] [191] [192] . the entry of the ebov into host cells is the first step of infection and a crucial determinant of pathogenicity. upon receptor binding between gp1 and host tim-1 receptors, the ebov is internalized into endosomes primarily via the macropinocytic pathway. amiloride is able to inhibit the uptake of many viruses that utilize the macropinocytic pathway for host cell entry [193] [194] [195] [196] . amiloride at non-cytotoxic dosages leads to potent dosedependent inhibition of the entry and infection of the ebov [197, 198] . amiloride can lead to dose-dependent inhibition of rna synthesis. this may be due to a direct blockage of a nucleotide entry tunnel or catalytic site, or due to its effect on the equilibrium of mg 2+ and mn 2+ that are essential co-factors for polymerase activity and nucleotide insertion [199, 200] . these novel antiviral mechanisms of amiloride may uncover new targets for drug discovery against the ebov. adenosine triphosphate (atp) is essential in multiple steps in the replication cycle of many viruses. na + /k + -atpase pump is located in the plasma membrane of all animal cells to maintain the cell membrane potential. budding of enveloped viruses is a complex phenomenon that requires concerted actions of many viral and host components. atp may affect multiple steps in the budding process [201] . atp is required for the assembly and maturation of a number of enveloped viruses such as the influenza virus, vaccinia virus, retrovirus, and herpes simplex virus. the na + /k + -atpase pump inhibitors, ouabain, lanatoside c, strophanthidin, and digoxin are able to inhibit the replication of the influenza virus, newcastle disease virus, and vesicular stomatitis virus through an interferonindependent mechanism [202] . digoxin and lanatoside c have been shown to inhibit vaccinia virus replication at non-cytotoxic doses [203] . ouabain has shown antiviral activity against the influenza virus [204] , herpes simplex virus [205] , sendai virus [206] , murine leukemic virus [207] , cytomegalovirus porcine reproductive and respiratory syndrome virus [208] , and human cytomegalovirus virus [209] . one common feature shared by these viruses is that they all possess a lipid envelope. the ebov is an enveloped filamentous rna virus. the secondary matrix protein vp24-apart from its role in the evasion of host immune response, nucleocapsid formation, and regulation of replication-has an important role in viral budding and egress. na + /k + -atpase atp1a1 is detected to have a close interaction with vp24 of ebov during replication. ouabain, at a non-cytotoxic concentration of 20nm, is able to suppress the replication of the ebov in human mrc-5 cells [210, 211] . among the three cardiac glycosides that may include digoxin, digitoxin, and ouabain, only digoxin is commonly used in clinical practice. ouabain, because of its poor oral availability, is used primarily as a research tool. further research should be conducted to investigate whether digoxin and other na + /k + -atpase inhibitors might play a role in the management of the ebov or other enveloped virus infections. the virus-associated glycoprotein gp1,2 is responsible for the activation of human macrophages [13] . the highly glycosylated mucin-like region of the gp1 subunit of gp1,2 is cytotoxic to the host cells [14] . the mucin-like region in gp1 leads to an accumulation of gp1,2 at the endoplasmic reticulum, induces endoplasmic reticulum stress [212] , and activates nuclear factor kappa b (nf-κb) [213] . mutations of the ebov that lead to an enhanced accumulation of gp1,2 in the endoplasmic reticulum were significantly more cytotoxic than wild-type virus [214] . in human cells, the accumulation of protein in the endoplasmic reticulum will lead to endoplasmic reticulum overload response (eroverload) which activates nf-κb through the production of ros [215] . as a major transcription factor for antiviral and immune stimulatory activities, nf-κb is thought to play an important role in the induction of pro-inflammatory molecules, such as interleukin-1β (il-1β), and tumor necrosis factor α (tnf-α), upon cellular responses against a virus infection [216] . the cytokine dysregulation of the ebov involves massive ros, nf-κb, tnf-α, and il-1β activation [65, 66] . the effectiveness of antioxidant therapy for the ebov infection indicates the importance of ros in the pathogenesis of the ebov [217] . the activation of nf-κb by er-overload is ros-dependent [218] . nf-κb-induced cytokine dysregulation of novel h1n1 pneumonia has been shown to be suppressible by high-dose n-acetylcysteine (nac) antioxidant therapy at 100 mg/kg continuous infusion daily [219] . given the poor oral availability of nac in the range of 6% to 10% in humans [220] , a therapeutic dose of nac equivalent to the intravenous route can hardly be delivered by oral preparation. nac is a category b drug for pregnancy and is affordable, with a wide therapeutic window. nac has an established safety profile even in high doses and prolonged use in humans [221] [222] [223] . cytokine dysregulation is a common feature in the ebov infection and is associated with an enhanced mortality [65] [66] [67] [68] . antiviral medications directed against the mutable viral determinants of the ebov cannot directly prevent cytokine dysregulation. the early endothelial vascular damage characteristic of the ebov infection is not a direct effect of virus-induced cytolysis of endothelial cells, but is due to cytokine dysregulation resulting from massive release of proinflammatory cytokines/chemokines and ros by infected macrophage and monocytes [70] [71] [72] . lymphocytes are resistant to the ebov infection. cytokine dysregulation may also contribute to the diffuse bystander apoptosis of lymphocytes [63, [87] [88] [89] . with the safety profile of nac, if the therapeutic efficacy of a high-dose nac antioxidant therapy to manage ebov-induced cytokine dysregulation is confirmed, it may revamp the future management of the ebov infection. there is a desperate need for a viable treatment regimen in africa to engender hope and encourage people with symptoms and their close contacts to seek medical treatment, so as to limit the spread of the disease. this also helps to recruit and maintain adequate medical staff who are at high risk of contracting the disease. a proposed regimen against the human ebov infection based on available medications and information from in vivo animal testing and in vitro cell culture is attached (see tables 2 and 3 ). this regimen contains a cocktail of currently available medications that can target the different steps in the replication cycle of the ebov aiming to suppress viral proliferation. it has been shown that viral load is major contribution to survival in both human and animal studies [60] [61] [62] 136] . through viral load suppression, we may be able to prolong a patient's survival in order to allow the development of natural body immune defense against the ebov. the ebov has undergone a rapid mutation during its spread through humans [224] [225] [226] . the ebov is an rna virus the replication of which is highly error prone with nearly one viral mutation occurring during each cycle of replication. this extremely high mutation rate leads to significant genetic and antigenic diversity that allows the ebov population to evolve resistance to antiviral medications and vaccines [227, 228] . a combination therapy has been used in the treatment of rna virus infections, such as the human immunodeficiency virus (hiv) [229, 230] and hepatitis c [231, 232] to minimize the development of drug resistance. given the broad cell tropism and high replication rate of the ebov due to the potent suppression of both innate and adaptive immune responses of the host, patients with the ebov infection have an extremely high viral load. the selective pressure in the presence of the high mutation rate and viral load during the human ebov infection make the evolution of the ebov viral strains resistant to a single drug inevitable. the currently available medications in the proposed regimen-which is a treatment regimen containing a cocktail of antiviral medications targeting the different steps of the ebov replication in order to achieve maximal suppression of viral replication and to prevent the rapid development of resistance to favipiravir, the only drug in the regimen that is directed against a mutable target of the ebov-has been shown to reduce the replication of the ebov. [233] [234] [235] . the current ebov vaccine (rvsv-ebogp and rchad-ebogp) and therapeutic agents (zmapp, tkm-ebola, pmo avi-6002, and favipiravir) under development are directed against the mutable targets of the ebov, and their effectiveness is limited by viral mutation. the ebov, being a rna virus with limited coding capacity, has utilized the host's unique metabolic pathway for its viral entry, replication, and egress. most of the therapeutic agents in this current review are directed against nonmutable targets of the host which is independent of viral mutation. these medications are fda-approved for the treatment of other diseases. they are available and stockpileable for immediate use. they may also have a complementary role to those therapeutic agents under development that are directed against the mutable targets of the ebov. the primary target of the ebov is the mononuclear phagocytic system. the spectrum of target cells increases to include endothelial cells, fibroblasts, hepatocytes, and many other cells during the advanced stage of the disease [6, 236, 237] . the ebov may produce a viral load of up to 10 10 virions per ml serum in terminally ill patients [80] . oral amiodarone prophylaxis, by inducing a niemann-pick c-like phenotype in the cells of the mononuclear phagocytic system, may prevent viral entry into these cells during needle stick injury. through protection of the mononuclear system by our prophylaxis and cocktail therapy, we hope to offer a better chance of survival to these patients by allowing them to develop a natural body immune defense against the ebov infection. the liver, containing the largest number of fixed tissue macrophages (kupffer cells), as part of the reticuloendothelial immune defense system of the body, is a major target for the ebov infection [238, 239] . the ebov replicates to high titer in the liver [240] . hepatic apoptosis may play a role in the pathogenesis of the ebov infection [88] . toremifene is added to the treatment regimen for hepatic protection because amiodarone does not exert inhibitory action against the ebov in hepatocyte. however, both amiodarone and toremifene can increase qtc and the risk of torsades de pointes. therefore electrocardiogram should be carefully monitored if both drugs are to be used. amiodarone, favipiravir, and toremifene are available and stockpileable in oral preparations. these properties are advantageous in outbreak situations and contingency planning of a potential ebov epidemic or pandemic. the avoidance of intravenous administration will prevent needle stick injury in healthcare workers caring for the infected patients. ifn-β may have potential as an adjunctive postexposure therapy for high-risk exposure, such as needle stick injury, by inducing ifitm1 to limit entry of the ebov. post-exposure ifn-β treatment was associated with a trend towards lower plasma and tissue viral burden and pro-inflammatory cytokines production [56] . the reduction in viral load and cytokine dysregulation coupled with optimal supportive therapy may improve the chance of survival of the host to allow the development of natural immunity to control the underlying ebov infection. ifitm1 is active against multiple viruses, including the ebov [185, 188] and hepatitis c 1 ml of blood may contain 10 9 to 10 10 virions in terminally ill patient. prophylactic amiodarone therapy may protect macrophage, monocyte and endothelial cells immediately from ebov during needle stick injury and accidental exposure and allow time for the consideration of ifn-β, toremifene, favipiravir and convalescent blood serum therapy. 2 amiodarone is unable to protect hepatocyte from ebov infection. 3 both amiodarone and toremifene can increase the risk of qt prolongation and torsades de pointes. 4 the recommended dosage for treatment of human ebov infection may be 2 to 5 times higher than influenza studies. please confirm the recommended dose with the drug company. 5 n-acetylcysteine intravenous infusion at 100 mg/kg/day to control cytokine dysregulation (e.g. add 5 g of intravenous preparation of n-acetylcysteine into each liter of intravenous replacement fluid). [186, 187, 241, 242] . interferon induced ifitm1 plays an important role in the treatment of human hcv infection by inhibiting entry of hcv into the host cell [243] . six million international units (miu) of ifn-β intravenous administration is as effective as a three miu twice-daily regimen for treatment of the hcv infection [244] , but has lesser side effects that require discontinuation of the medication [245, 246] . as the aim of ifn-β therapy in our regimen for post needle stick prophylaxis against the ebov infection is to induce ifitm1 to limit viral entry, the dose of ifn-β for the post needle stick prophylaxis [247, 248] or induction therapy [249, 250] for hcv infection in humans is chosen. once infection is fully established, ifn-β are replaced by convalescent blood serum and high-dose nac infusion for providing passive humoral immunity and for the control of ros-dependent nf-κb-induced cytokine dysregulation respectively. the ebov is classified as biosafety level 4 pathogen and is classified by centers for disease control and prevention as a category a agent of bioterrorism with no approved therapies and vaccines for its treatment but carrying a high potential for large-scale dissemination. recent political, economic, military, and religious turbulence around the world raises concerns that the ebov might be used as an agent of bioterrorism [251] [252] [253] . the recent ebov epidemic is spiraling out of control in west africa. the containment measures that worked in the past, such as isolating those who are infected and tracing their contacts, have failed due to an exponential rise in infected patients. although the short-term (threeand six-week) probability of international spread outside the african region is small, the risk of the extension of the outbreak to other african countries followed by international dissemination on a longer time scale is not negligible, indicating that this public health emergency has the potential to grow to extraordinarily destructive dimensions [254, 255] . although several promising therapeutic agents and vaccines against the ebov are undergoing the phase i human trial, the current epidemic might be outpacing the speed at which drugs and vaccines can be produced [5] . to combat such an unprecedented global public-health crisis before these experimental agents are available, alternative available interventions capable of managing the enhanced viral replication and cytokine dysregulation of the human ebov infection should be explored and stockpiled as contingency preparation for the worst-case scenario of an impending human ebov pandemic [256] . like all viruses, the ebov largely relies on host cell factors and physiological processes for its entry, replication, and egress which, in turn, lead to cytopathic damage, cytokine dysregulation, and death of the host. these non-mutable key steps inside the host may be novel targets for future therapeutic strategies against these rapidly mutating viruses. if the efficacy of amiloride, digoxin, amiodarone, and high-dose nac antioxidant therapy against the human ebov infection is confirmed, the availability and affordability of these stockpileable oral regimen are for those workers who are already on amiodarone prophylaxis with a loading dose of amiodarone 600 mg p.o. twice daily for 8 days followed by maintenance amiodarone 600 mg p.o. daily. electrocardiogram and thyroid function should be monitored. 2 monitor for side effect of thrombocytopenia and proteinuria. 3 intravenous dosage of ifn-β that are used for human hepatitis c virus infection to induce ifitm1 to limit viral entry. 4 intravenous regimen is for those workers who have not been on amiodarone prophylaxis and agreed for the insertion of a central venous line for drug administration. intravenous amiodarone should be administered via central venous line to avoid phlebitis. the dosage for treatment of frequently recurring ventricular fibrillation and hemodynamically unstable ventricular tachycardia is recommended because it can achieve therapeutic drug level immediately after the first dose of amiodarone. agents make them ideal medications in pandemic situation and in countries with limited resources. they may have a complementary role to other antiviral medications to prevent the emergence of resistant strains. this may also signify a major breakthrough in future management of the ebov infection. additional file 1: multilingual abstracts in the six official working languages of the united nations. the authors declare that they have no competing interests. authors' contributions kyl and gwyn contributed to the conception, drafting, and writing of the paper. kyl, gwyn and fc revised the draft paper. all authors read and approved the revised paper. world health organization ebola response roadmap situation reports world health organization statement on the who consultation on potential ebola therapies and vaccines world health organization: ethical considerations for use of unregistered interventions for ebola viral disease emerging targets and novel approaches to ebola virus prophylaxis and treatment world health organization: potential ebola therapies and vaccines clinical features and pathobiology of ebolavirus infection ebola virus: unravelling pathogenesis to combat a deadly disease delta-peptide is the carboxyterminal cleavage fragment of the nonstructural small glycoprotein sgp of ebola virus the glycoproteins of marburg and ebola virus and their potential roles in pathogenesis release of viral glycoproteins during ebola virus infection the secret life of viral entry glycoproteins: moonlighting in immune evasion the pathogenesis of ebola hemorrhagic fever role of ebola virus secreted glycoproteins and virus-like particles in activation of human macrophages identification of the ebola virus glycoprotein as the main viral determinant of vascular cell cytotoxicity and injury shed gp of ebola virus triggers immune activation and increased vascular permeability ebola virus glycoprotein counteracts bst-2/tetherin restriction in a sequence-independent manner that does not require tetherin surface removal anti-tetherin activities of hiv-1 vpu and ebola virus glycoprotein do not involve removal of tetherin from lipid rafts ebolavirus replication and tetherin/bst-2 the nonstructural small glycoprotein sgp of ebola virus is secreted as an antiparallel-orientated homodimer antigenic subversion: a novel mechanism of host immune evasion by ebola virus a novel mechanism of immune evasion mediated by ebola virus soluble glycoprotein distinct cellular interactions of secreted and transmembrane ebola virus glycoproteins ebola virus secretory glycoprotein (sgp) diminishes fc gamma riiib-to-cr3 proximity on neutrophils effects of ebola virus glycoproteins on endothelial cell activation and barrier function ebola virus glycoprotein gp is not cytotoxic when expressed constitutively at a moderate level ebolavirus delta-peptide immunoadhesins inhibit marburgvirus and ebolavirus cell entry a new ebola virus nonstructural glycoprotein expressed through rna editing ebola virus enters host cells by macropinocytosis and clathrin-mediated endocytosis the ebola virus glycoprotein mediates entry via a non-classical dynamindependent macropinocytic pathway ebolavirus is internalized into host cells via macropinocytosis in a viral glycoprotein-dependent manner ebola virus entry requires the cholesterol transporter niemann-pick c1 small molecule inhibitors reveal niemann-pick c1 is essential for ebola virus infection ebola virus entry requires the host-programmed recognition of an intracellular receptor niemann-pick c1 (npc1)/npc1-like1 chimeras define sequences critical for npc1's function as a flovirus entry receptor host cell factors in filovirus entry: novel players, new insights. viruses a new player in the puzzle of filovirus entry a mutation in the ebola virus envelope glycoprotein restricts viral entry in a host species-and cell-type-specific manner multiple cationic amphiphiles induce a niemann-pick c phenotype and inhibit ebola virus entry and infection filovirus entry: a novelty in the viral fusion world how ebola virus counters the interferon system the importance of the np: vp35 ratio in ebola virus nucleocapsid formation knockdown of ebola virus vp24 impairs viral nucleocapsid assembly and prevents virus replication the assembly of ebola virus nucleocapsid requires virion-associated proteins 35 and 24 and posttranslational modification of nucleoprotein the ebola virus ribonucleoprotein complex: a novel vp30-l interaction identified role of ebola virus vp30 in transcription reinitiation role of vp30 phosphorylation in the ebola virus replication cycle phosphorylation of ebola virus vp30 influences the composition of the viral nucleocapsid complex: impact on viral transcription and replication replication-deficient ebolavirus as a vaccine candidate both matrix proteins of ebola virus contribute to the regulation of viral genome replication and transcription contribution of ebola virus glycoprotein, nucleoprotein, and vp24 to budding of vp40 virus-like particles biochemical and functional characterization of the ebola virus vp24 protein: implications for a role in virus assembly and budding could the ebola virus matrix protein vp40 be a drug target? the ebola virus matrix protein deeply penetrates the plasma membrane: an important step in viral egress transcriptional activation of alpha/beta interferon genes: interference by nonsegmented negative-strand rna viruses interferon-β therapy prolongs survival in rhesus macaque models of ebola and marburg hemorrhagic fever evasion of interferon responses by ebola and marburg viruses filoviral immune evasion mechanisms ebola virus vp24 targets a unique nls binding site on karyopherin alpha 5 to selectively compete with nuclear import of phosphorylated stat1 ebola virus haemorrhagic fever analysis of human peripheral blood samples from fatal and nonfatal cases of ebola (sudan) hemorrhagic fever: cellular responses, virus load, and nitric oxide levels rapid diagnosis of ebola hemorrhagic fever by reverse transcription-pcr in an outbreak setting and assessment of patient viral load as a predictor of outcome defective humoral responses and extensive intravascular apoptosis are associated with fatal outcome in ebola virus-infected patients ebola virus pathogenesis: implications for vaccines and therapies proinflammatory response during ebola virus infection of primate models: possible involvement of the tumor necrosis factor receptor superfamily ebola virus selectively inhibits responses to interferons, but not to interleukin-1beta, in endothelial cells inflammatory responses in ebola virus-infected patients human asymptomatic ebola infection and strong inflammatory response infection and activation of monocytes by marburg and ebola viruses monocyte-derived human macrophages and peripheral blood mononuclear cells infected with ebola virus secrete mip-1alpha and tnf-alpha and inhibit poly-icinduced ifn-alpha in vitro markedly elevated levels of interferon (ifn)-gamma, ifn-alpha, interleukin (il)-2, il-10, and tumor necrosis factor-alpha associated with fatal ebola virus infection pathogenesis of ebola hemorrhagic fever in primate models: evidence that hemorrhage is not a direct effect of virus-induced cytolysis of endothelial cells ebola virus glycoprotein toxicity is mediated by a dynamin-dependent protein-trafficking pathway filovirus-induced endothelial leakage triggered by infected monocytes/ macrophages ebola virus glycoproteins induce global surface protein down-modulation and loss of cell adherence requirements for cell rounding and surface protein down-regulation by ebola virus glycoprotein mechanisms underlying coagulation abnormalities in ebola hemorrhagic fever: overexpression of tissue factor in primate monocytes/ macrophages is a key event treatment of ebola virus infection with a recombinant inhibitor of factor viia/tissue factor: a study in rhesus monkeys recombinant nematode anticoagulant protein c2 and other inhibitors targeting blood coagulation factor viia/tissue factor immunopathology of highly virulent pathogens: insights from ebola virus ebola haemorrhagic fever pathogenesis of viral hemorrhagic fever pathogenesis of ebola hemorrhagic fever in cynomolgus macaques: evidence that dendritic cells are early and sustained targets of infection molecular basis for ebola virus vp35 suppression of human dendritic cell maturation ebola virus: the role of macrophages and dendritic cells in the pathogenesis of ebola hemorrhagic fever the role of antigen-presenting cells in filoviral hemorrhagic fever: gaps in current knowledge human fatal zaire ebola virus infection is associated with an aberrant innate immunity and with massive lymphocyte apoptosis mechanisms and consequences of ebolavirus-induced lymphocyte apoptosis ebola virus does not block apoptotic signaling pathways characterization of host immune responses in ebola virus infections filoviruses and the balance of innate, adaptive, and inflammatory responses functional cd8+ t cell responses in lethal ebola virus infection cd8-mediated protection against ebola virus infection is perforin dependent induction of humoral and cd8+ t cell responses are required for protection against lethal ebola virus infection induction of immune responses in mice and monkeys to ebola virus after immunization with liposome-encapsulated irradiated ebola virus: protection in mice requires cd4(+) t cells cutting edge: impairment of dendritic cells and adaptive immunity by ebola and lassa viruses correlates of immunity to filovirus infection the multiple roles of sgp in ebola pathogenesis ebolavirus glycoprotein gp masks both its own epitopes and the presence of cellular surface proteins steric shielding of surface epitopes and impaired immune recognition induced by the ebola virus glycoprotein influences of glycosylation on antigenicity, immunogenicity, and protective efficacy of ebola virus gp dna vaccines antibody-dependent enhancement of viral infection: molecular mechanisms and in vivo implications infectivity-enhancing antibodies to ebola virus glycoprotein antibody-dependent enhancement of ebola virus infection epitopes required for antibody-dependent enhancement of ebola virus infection ebola virus can be effectively neutralized by antibody produced in natural human infection structural basis for differential neutralization of ebolaviruses. viruses antibody-mediated neutralization of ebola virus can occur by two distinct mechanisms saphire eo: a shared structural solution for neutralizing ebolaviruses characterization of zaire ebolavirus glycoprotein-specific monoclonal antibodies epitopes involved in antibody-mediated protection from ebola virus neutralizing antibody fails to impact the course of ebola virus infection in monkeys recombinant vesicular stomatitis virus-based vaccines against ebola and marburg virus infections effective post-exposure treatment of ebola infection vesicular stomatitis virus-based vaccines protect nonhuman primates against aerosol challenge with ebola and marburg viruses vesicular stomatitis virus-based ebola vaccine is well-tolerated and protects immunocompromised nonhuman primates chimpanzee adenovirus vaccine generates acute and durable protective immunity against ebolavirus challenge recombinant adenovirus serotype 26 (ad26) and ad35 vaccine vectors bypass immunity to ad5 and protect nonhuman primates against ebolavirus challenge demonstration of cross-protective vaccine immunity against an emerging pathogenic ebolavirus species airway delivery of an adenovirus-based ebola virus vaccine bypasses existing immunity to homologous adenovirus in nonhuman primates progress in filovirus vaccine development: evaluating the potential for clinical use immune protection of nonhuman primates against ebola virus with single low-dose adenovirus vectors encoding modified gps vrc 205 study team: a replication defective recombinant ad5 vaccine expressing ebola virus gp is safe and immunogenic in healthy adults nabel gj: cd8+ cellular immunity mediates rad5 vaccine protection against ebola virus infection of nonhuman primates live attenuated recombinant vaccine protects nonhuman primates against ebola and marburg viruses antibodies are necessary for rvsv/zebov-gp-mediated protection against lethal ebola virus challenge in nonhuman primates protection from ebola virus mediated by cytotoxic t lymphocytes specific for the viral nucleoprotein ebola virus-like particles stimulate type i interferons and proinflammatory cytokine expression through the toll-like receptor and interferon signaling pathways ebola virus-like particle-based vaccine protects nonhuman primates against lethal ebola virus challenge protective cytotoxic t-cell responses induced by venezuelan equine encephalitis virus replicons expressing ebola virus proteins vaccine potential of ebola virus vp24, vp30, vp35, and vp40 proteins postexposure protection of non-human primates against a lethal ebola virus challenge with rna interference: a proof-of-concept study ebolavirus in west africa, and the use of experimental therapies or vaccines gene-specific countermeasures against ebola virus based on antisense phosphorodiamidate morpholino oligomers discovery and early development of avi-7537 and avi-7288 for the treatment of ebola virus and marburg virus infections clinical virology of ebola hemorrhagic fever (ehf): virus, virus antigen, and igg and igm antibody findings among ehf patients in kikwit, democratic republic of the congo a current review of ebola virus: pathogenesis, clinical presentation, and diagnostic assessment treatment of ebola hemorrhagic fever with blood transfusions from convalescent patients. international scientific and technical committee ebola virus glycoprotein fc fusion protein confers protection against lethal challenge in vaccinated mice passive transfer of antibodies protects immunocompetent and imunodeficient mice against lethal ebola virus infection without complete inhibition of viral replication pre-and postexposure prophylaxis of ebola virus infection in an animal model by passive transfer of a neutralizing human antibody protective efficacy of neutralizing antibodies against ebola virus infection identification of protective epitopes on ebola virus glycoprotein at the single amino acid level by using recombinant vesicular stomatitis viruses production and characterization of monoclonal antibodies against different epitopes of ebola virus antigens protective efficacy of neutralizing monoclonal antibodies in a nonhuman primate model of ebola hemorrhagic fever an update on the use of antibodies against the filoviruses do therapeutic antibodies hold the key to an effective treatment for ebola hemorrhagic fever? immunotherapy molecular characterization of the monoclonal antibodies composing zmab: a protective cocktail against ebola virus therapeutic intervention of ebola virus infection in rhesus macaques with the mb-003 monoclonal antibody cocktail successful treatment of ebola virus-infected cynomolgus macaques with monoclonal antibodies reversion of advanced ebola virus disease in nonhuman primates with zmapp monoclonal antibodies combined with adenovirus-vectored interferon significantly extend the treatment window in ebola virus-infected guinea pigs mabs and ad-vectored ifn-α therapy rescue ebola-infected nonhuman primates when administered after the detection of viremia and symptoms favipiravir (t-705), a novel viral rna polymerase inhibitor t-705 (favipiravir) and related compounds: novel broad-spectrum inhibitors of rna viral infections successful treatment of advanced ebola virus infection with t-705 (favipiravir) in a small animal model post-exposure efficacy of oral t-705 (favipiravir) against inhalational ebola virus infection in a mouse model lethality and pathogenesis of airborne infection with filoviruses in a129 α/β −/− interferon receptor-deficient mice protection against filovirus diseases by a novel broad-spectrum nucleoside analogue bcx4430 meeting report: 27th international conference on antiviral research a systematic screen of fda-approved drugs for inhibitors of biological threat agents cathepsins b and l activate ebola but not marburg virus glycoproteins for efficient entry into cell lines and macrophages independent of tmprss2 expression filoviruses require endosomal cysteine proteases for entry but exhibit distinct protease preferences reverse genetics demonstrates that proteolytic processing of the ebola virus glycoprotein is not essential for replication in cell culture proteolytic processing of the ebola virus glycoprotein is not critical for ebola virus replication in nonhuman primates cathepsin b & l are not required for ebola virus replication identification of a broad-spectrum antiviral small molecule against severe acute respiratory syndrome coronavirus and ebola, hendra, and nipah viruses by using a novel high-throughput screening assay the clinically approved drugs amiodarone, dronedarone and verapamil inhibit filovirus cell entry amiodarone induced lipidosis similar to niemann-pick c disease. biochemical and morphological study amiodarone impairs trafficking through late endosomes inducing a niemann-pick c-like phenotype the niemann-pick c proteins and trafficking of cholesterol through the late endosomal/lysosomal system abnormal cholesterol metabolism in imipramine-treated fibroblast cultures. similarities with niemann-pick type c disease niemann-pick c1 functions in regulating lysosomal amine content fda-approved selective estrogen receptor modulators inhibit ebola virus infection josh farkas 8/6/ 2014 serum concentrations of enclomiphene and zuclomiphene across consecutive cycles of clomiphene citrate therapy in anovulatory infertile women rostami-hodjegan a: evaluation of the relationship between plasma concentrations of en-and zuclomiphene and induction of ovulation in anovulatory women being treated with clomiphene citrate pharmacokinetics of toremifene and its metabolites in patients with advanced breast cancer phase i clinical and pharmacokinetics study of high-dose toremifene in postmenopausal patients with advanced breast cancer phase i study of the tolerance and pharmacokinetics of toremifene in patients with cancer a pharmacological review of selective oestrogen receptor modulators clinical pharmacokinetics of toremifene pharmacokinetic evaluation of toremifene and its clinical implications for the treatment of osteoporosis ifitm/mil/fragilis family proteins ifitm1 and ifitm3 play distinct roles in mouse primordial germ cell homing and repulsion the broad-spectrum antiviral functions of ifit and ifitm proteins isg56 and ifitm1 proteins inhibit hepatitis c virus replication hepatitis c virus infection modulates expression of interferon stimulatory gene ifitm1 by upregulating mir-130a distinct patterns of ifitm-mediated restriction of filoviruses, sars coronavirus, and influenza a virus amiloride and its analogs as tools in the study of ion transport mechanisms of intracellular mg2+ regulation affected by amiloride and ouabain in the guinea-pig taenia caeci ca2 + −dependent modulation of intracellular mg2+ concentration with amiloride and kb-r7943 in pig carotid artery species-specific mn2+/mg2+ antiport from mg2(+)-loaded erythrocytes defining macropinocytosis virus entry by macropinocytosis kaposi' s sarcoma-associated herpesvirus utilizes an actin polymerization-dependent macropinocytic pathway to enter human dermal microvascular endothelial and human umbilical vein endothelial cells amiloride derivatives inhibit coxsackievirus b3 rna replication cellular entry of ebola virus involves uptake by a macropinocytosis-like mechanism and subsequent trafficking through early and late endosomes the tyro3 receptor kinase axl enhances macropinocytosis of zaire ebolavirus fidelity variants of rna dependent rna polymerases uncover an indirect, mutagenic activity of amiloride compounds amiloride inhibits the initiation of coxsackievirus and poliovirus rna replication by inhibiting vpg uridylylation role of atp in influenza virus budding modulation of influenza virus replication by alteration of sodium ion transport and protein kinase c activity identification of novel antipoxviral agents: mitoxantrone inhibits vaccinia virus replication by blocking virion assembly the cardioactive glycoside ouabain inhibits influenza a viral replication inhibitors of the sodium potassium atpase that impair herpes simplex virus replication identified via a chemical screening approach inhibition of virus growth by ouabain: effect of ouabain on the growth of hvj in chick embryo cells suppression of murine leukaemia virus production by ouabain and interferon in mouse cells natural compounds inhibiting the replication of porcine reproductive and respiratory syndrome virus human cytomegalovirus infection increases the number of ouabain-binding sites in human fibroblasts elucidation of the ebola virus vp24 cellular interactome and disruption of virus biology through targeted inhibition of host cell protein function mechanisms of ouabain toxicity full-length ebola glycoprotein accumulates in the endoplasmic reticulum ebola virus-like particle-induced activation of nf-kappab and erk signaling in human dendritic cells requires the glycoprotein mucin domain recovery of infectious ebola virus from complementary dna: rna editing of the gp gene and viral cytotoxicity the er-overload response: activation of nf-kappa b intrinsic cellular defenses against virus infection by antiviral type i interferon identification of an antioxidant small-molecule with broad-spectrum antiviral activity activation of nf-kappa b by er stress requires both ca2+ and reactive oxygen intermediates as messengers high-dose n-acetylcysteine therapy for novel h1n1 influenza pneumonia pharmacokinetics of n-acetylcysteine in man mechanisms of n-acetylcysteine in the prevention of dna damage and cancer, with special reference to smoking-related end-points paracetamol overdose in pregnancy analysis of the outcomes of 300 cases referred to the teratology information service management of paracetamol overdose: current controversies genomic surveillance elucidates ebola virus origin and transmission during the 2014 outbreak emergence of zaire ebola virus disease in guinea -preliminary report check hayden e: ebola virus mutating rapidly as it spreads. nature news quasispecies theory and the behavior of rna viruses viral quasispecies evolution optimal antiviral switching to minimize resistance risk in hiv therapy hiv quasispecies dynamics during pro-active treatment switching: impact on multi-drug resistance and resistance archiving in latent reservoirs mixing the right hepatitis c inhibitor cocktail exploratory study of oral combination antiviral therapy for hepatitis c quasispecies and the development of new antiviral strategies error thresholds and the constraints to rna virus evolution molecular approaches for the treatment of hemorrhagic fever virus infections ultrastructural pathology of experimental ebola haemorrhagic fever virus infection ebola virus: from discovery to vaccine ebola virus: a comparison, at ultrastructural level, of the behaviour of the sudan and zaire strains in monkeys the pathology of experimental ebola virus infection in monkeys ultrastructure of ebola virus particles in human liver impaired hcv clearance in hiv/ hcv coinfected subjects treated with pegifn and rbv due to interference of ifn signaling by ifnαr2a gene expression profile associated with superimposed non-alcoholic fatty liver disease and hepatic fibrosis in patients with chronic hepatitis c twice-daily administration of interferon-beta for chronic hepatitis c is not superior to a once-daily regimen is β-interferon a promising therapeutic option for the management of hepatitis c? is interferon-beta an alternative treatment for chronic hepatitis c? sustained viral response of a case of acute hepatitis c virus infection via needlestick injury complete response to twice-a-day interferon-beta with standard interferon-alpha therapy two week induction of interferon-beta followed by pegylated interferon alpha-2b and ribavirin for chronic infection with hepatitis c twenty four-week peginterferon plus ribavirin after interferon-β induction for genotype 1b chronic hepatitis c centers for disease control and prevention: emergency preparedness and response for bioterrorism agents/diseases ebola hemorrhagic fever in the era of bioterrorism medical aspects of bio-terrorism assessing the international spreading risk associated with the 2014 west african ebola outbreak. plos currents outbreaks early epidemic dynamics of the west african 2014 ebola outbreak: estimates derived with a simple two-parameter model filoviruses": a real pandemic threat? submit your next manuscript to biomed central and take full advantage of: • convenient online submission • thorough peer review • no space constraints or color figure charges • immediate publication on acceptance • inclusion in pubmed, cas, scopus and google scholar • research which is freely available for redistribution key: cord-001109-xs7df6a7 authors: tapia, karla; kim, won-keun; sun, yan; mercado-lópez, xiomara; dunay, emily; wise, megan; adu, michael; lópez, carolina b. title: defective viral genomes arising in vivo provide critical danger signals for the triggering of lung antiviral immunity date: 2013-10-31 journal: plos pathog doi: 10.1371/journal.ppat.1003703 sha: doc_id: 1109 cord_uid: xs7df6a7 the innate immune response to viruses is initiated when specialized cellular sensors recognize viral danger signals. here we show that truncated forms of viral genomes that accumulate in infected cells potently trigger the sustained activation of the transcription factors irf3 and nf-κb and the production type i ifns through a mechanism independent of ifn signaling. we demonstrate that these defective viral genomes (dvgs) are generated naturally during respiratory infections in vivo even in mice lacking the type i ifn receptor, and their appearance coincides with the production of cytokines during infections with sendai virus (sev) or influenza virus. remarkably, the hallmark antiviral cytokine ifnβ is only expressed in lung epithelial cells containing dvgs, while cells within the lung that contain standard viral genomes alone do not express this cytokine. together, our data indicate that dvgs generated during viral replication are a primary source of danger signals for the initiation of the host immune response to infection. the recognition of virus-specific pattern associated molecular patterns (pamps) is a pivotal event in the initiation of the host innate response to infection. in recent years, it has been established that most viral danger signals are derived from oligonucleotide structures exposed during the replication of the viral genomes [1, 2, 3, 4, 5, 6] . however, most viruses produce proteins that antagonize and effectively delay signaling by the primary viral oligonucleotide sensor molecules retinoic acid inducible gene i (rig-i) and melanoma differentiation-associated gene 5 (mda5), allowing the virus to replicate to high titers and produce large amounts of danger signals prior to host intervention [7, 8] . it is currently unclear how the host immune response overcomes viral evasion to initiate a protective antiviral response. defective viral genomes (dvgs) arise when the viral polymerase loses processivity during virus replication at high titers, thereby generating truncated versions of the viral genome that contain deletions and/or complementary ends (the later known as copyback or snap-back genomes) [9, 10] . dvgs with the ability to interfere with standard virus replication were first described by von magnus in the early 1950s as the genomes of incomplete forms of influenza virus called defective interfering (di) viral particles [11] . dvgs have been identified in multiple distinct viral families when the viruses are grown in the laboratory at high multiplicity of infection and span a broad range of hosts, from plants to mammals [12] . importantly, dvgs are found in patients infected with hepatitis a [13] , hepatitis b [14, 15] , hepatitis c [16] , hiv [17] , dengue virus [18] , and influenza virus [19] . however, the biological role of dvgs in the context of natural infections is not well understood. we and others have shown that stocks of sendai virus (sev) with a high content of copy-back dvgs with interfering activity trigger enhanced production of cytokines in vitro and more potently induce antigen presentation by mouse and human dendritic cells than do virus stocks lacking this kind of dvgs [20, 21, 22, 23, 24, 25] . our group has also demonstrated that in contrast to standard viral genomes, sev copy-back dvgs induce the expression of mda5 and of a number of other interferonstimulated genes in the absence of type i ifn positive feedback [23, 26, 27] . remarkably, sev copy-back dvgs show this potent in vitro stimulatory activity even in the presence of functional viral encoded antagonists of the host response [23, 24] . here, we demonstrate that dvgs that trigger a robust activation of the transcription factors irf3 and nf-kb accumulate at a high rate in infected cells becoming the main source of viral pamps. these dvgs arise naturally during acute respiratory viral infections in mice and provide essential stimuli for the initiation of the antiviral innate immune response in the lung. these data demonstrate the generation of dvgs in vivo during acute respiratory viral infections and suggest a critical role of these kinds of viral genomes in determining the quality of the host response to infection. sev copy-back dvgs trigger a robust and sustained activation of irf3 and nf-kb independent of type i ifn feedback to further investigate the cellular mechanisms responsible for the efficient activation of the antiviral response by sev dvgs, we evaluated the phosphorylation of transcription factors that are critical for the expression of type i ifns in cells infected with equivalent amounts of infectious particles of a sev strain cantell stock containing high levels of copy-back dvgs (sev cantell hd) or with sev cantell depleted of dvgs (sev cantell ld). virus stocks were prepared from the same parental virus and their content of dvgs was determined by calculating the ratio of infectious particles to total particles (ratios are specified in the material and methods section). in addition, copy-back dvgs of these stocks were identified by pcr. one predominant copy-back genome was present in cells infected with sev cantell hd (amplicon of 278 bp), while no copy-back defective genome was detected in cells infected with sev cantell ld up to six hours after infection (figs. 1a and s1). cloning and sequencing of the 278 nt long amplicon confirmed that it corresponded to a previously described sev cantell copy-back dvg of 546 nt in length (dvg-546) [28] . phosphorylation of irf3 and of the nf-kb repressor ikba in response to sev cantell hd occurred rapidly and was sustained even in type i ifn receptor ko cells (ifnar1 2/2 ) ( fig. 1b and c), while no phosphorylation of irf3 or ikba was observed for up to ten hours post-infection with sev cantell ld despite equivalent or higher expression of the viral protein np (fig. 1d) . corresponding with the strong activation of transcription factors, ifnb mrna was expressed in ifnar1 2/2 cells infected with sev cantell hd (fig. 1e ). in contrast, type i ifn signaling was required for the cellular response to newcastle disease virus (ndv), an avian virus that only partially inhibits the type i ifn pathway, triggering the expression of type i ifn and other cytokines in the absence of dvgs. to further validate the role of sev copy-back dvgs as triggers of type i ifn-independent antiviral responses, we cloned dvg-546 under the control of the t7 polymerase promoter and used this construct to prepare a sev stock containing a single recombinant dvg (rdvg). for this purpose we used sev strain 52 that normally does not produce highly immunostimulatory copy-back dvgs [24] . equivalent infectious units of sev 52 and sev 52 plus rdvgs had similar levels of total rna (fig. 1f ) but infection with virus containing rdvgs strongly induced the antiviral response while virus that lacked dvgs did not (fig. 1g) confirming the dvg immunostimulatory activity. in addition, presence of rdvgs significantly reduced the expression of sev np mrna, demonstrating their strong interfering capacity (fig. 1g) . notably, mouse embryo fibroblasts lacking the type i ifn receptor expressed ifnb mrna in response to sev 52 containing rdvg (fig. 1h ) and virus containing rdvgs triggered irf3 phosphorylation independently of type i ifn feedback (fig. 1i) , mirroring the response to sev cantell hd. altogether, this evidence conclusively shows that sev copy-back dvgs confer potent immunostimulatory ability to sev stocks, independent of type i ifn feedback. notably, potent ifnb mrna expression in response to sev dvgs was independent of irf1, irf5, and irf8 while only partially dependent on irf7 (fig. s2 ). this response was maintained in a variety of cell types (fig. s3) . dvgs accumulate at a high rate in infected cells and are a primary source of pathogen associated molecular patterns that trigger rlr signaling to determine whether standard viral genomes and dvg rnas have distinct intrinsic properties that explain their differential immunostimulatory activities, we compared naked rna purified from a stock of sev cantell ld with in vitro transcribed dvg-546. rnas were transfected into cells before or after treatment with phosphatase or with rnase a that cleaves 39 of single stranded c and u residues, and/or rnase v1 that cleaves base paired nucleotides. both genomic rna and dvg rna were susceptible to treatment with phosphatase, as well as to treatment with rnases ( fig. 2a) , corresponding with the literature that demonstrates a crucial role for 59-triphosphate-rna in the induction of type i ifns. while transfected dvgs induced stronger expression of ifnb than an equivalent concentration of gsev ( fig. 2a) , transfection of equivalent molar amounts of genomic and dvg rna resulted in higher immunostimulatory activity of genomic rna compared to dvg rna (fig. 2b) , demonstrating that sev ld rna can strongly trigger the host response to infection when delivered naked into the cells. paradoxically, cells infected with sev cantell ld alone failed to induce strong type i ifn production even when used at a 10 times higher infectious dose than sev cantell hd (fig. 2c) . although the amount of gsev rna was significantly higher in cells infected with an moi of 15 of sev cantell ld compared with ten times less sev cantell hd at 6 h post-infection (fig. 2c) , dvgs were only detected in cells infected with sev cantell hd, confirming a strong correlation between the presence of dvgs in the infected cells and the induction of the host response to infection. to determine whether the amount of total input viral rna affected the immunostimulatory activity of sev cantell ld and hd, we measured the rna content in equivalent infectious doses of these stocks. sev cantell hd had less than two fold higher the amount of total rna than sev cantell ld and total rna levels were equivalent between sev cantell ld and hd when ld was at twice the infectious dose (fig. 2d) . thus, differences in the net input amount of viral rna cannot explain the more than .1000 fold difference in the expression of ifnb mrna between cells infected with equivalent infectious doses of sev cantell ld and hd. dvgs have an increased rate of replication compared to standard viral genomes due to their shorter size and promoter properties [29] . to determine whether dvgs replicate faster than gsev, we calculated the rate of replication of gsev and dvgs in cells infected with sev cantell hd. although at an early time point more copies of gsev than dvgs were detected in the cells, dvgs dominated by 12 h post-infection (fig. 2e ) accumulating at a 4 times faster rate than gsev (fig. 2f) . these data demonstrate that dvgs rapidly surpass the number of gsev in infected cells, providing large quantities of pathogen associated molecular patterns. in infections with viruses well adapted to the host virusencoded proteins that delay the cellular response allow the virus to replicate to high titers prior to host intervention. the mechanisms overcoming viral evasion of the immune system and leading to the production of the primary antiviral cytokine ifnb are not well established. here, we demonstrate that truncated forms of viral genomes that are generated in situ during virus replication are a primary source of danger signals for the initiation of the host immune response to respiratory viral infections in vivo. defective viral genomes (dvgs) are able to function as triggers of the immune response even in the absence of type i ifn signaling and are strong triggers of the host response to infection while overcoming viral antagonism. supporting previous observations that dvgs from sev stimulate the cellular antiviral response through signaling by rig-i like receptors (rlrs) [1, 23, 24] , the essential rlr adaptor protein mitochondrial antiviral signaling protein (mavs) was required for the activation of the transcription factors irf3 and nf-kb and for expression of numerous antiviral and pro-inflammatory molecules upon infection with sev cantell hd. in contrast, mavs was not required for the response to herpes simplex virus, which can trigger the host response independently of rlrs (fig. s4 ). in addition, only dvg rna, but not standard viral genomes, could be amplified from endogenous rig-i and mda5 complexes immunoprecipitated from infected cells (fig. s4c ), supporting published evidence that dvgs bind to rig-i preferentially over the standard viral genomes in infected cells [1] . as predicted, association of dvgs with rlrs correlated with type i ifn induction, but not with the level of virus replication (fig. s4d ). importantly, in addition to the primary role of rig-i in the response to sev dvgs, mda5 participates in the induction of type i ifn in primary mouse lung fibroblasts infected with sev hd (fig. s4e) , similar to what we have observed in dcs [23, 27] . overall, these data demonstrate that dvgs are produced in the infected cells at a higher rate than genomic rna and that dvgs are the predominant ligands for both rig-i and mda5 during sev infection. based on the potent ability of sev stocks containing a high content of copy-back dvgs to induce the host response to infection in vitro [23, 24, 25, 28] (fig. 1 ) and on our prior reports of strong host responses to dvgs regardless of the presence of functional virus-encoded antagonists [23, 24] , we hypothesized that dvgs that arise in situ during viral infections provide essential stimuli to initiate an antiviral immune response. to test this hypothesis, we first determined if sev strains that accumulate copy-back dvgs early in infection induced faster ifnb mrna expression in vitro than viruses with delayed dvg accumulation. for these experiments we used sev preparations that did not show immunostimulatory activity or evidence of copy-back dvg accumulation by 2 h post-infection and all the viruses were used at a multiplicity of infection of 1.5 tcid 50 /cell. while standard viral genomes of all the different sev strains used were detected at all tested time points, copy-back dvgs of different sizes were detected starting at 6 h post-infection in cells infected with sev z and at later time points in cells infected with sev 52, enders, or cantell ld in both murine lung epithelial cells (tc-1) and bone marrow-derived dendritic cells (bmdcs) (fig. 3a and b and data not shown). sequences of the starred pcr products confirming the amplification of copy-back dvgs are shown in fig. s5 . remarkably, accumulation of dvgs was directly associated with phosphorylation of irf3 (fig. 3c ) and with the expression of ifnb mrna (fig. 3d ), demonstrating that standard viral genomes alone are not sufficient to initiate this response during infection in vitro and strongly supporting a unique ability of naturally arising dvgs to initiate the cellular antiviral response. to evaluate the impact of dvgs during sev infection in vivo, we infected mice with sev cantell hd or ld. mice infected with sev cantell hd showed diminished morbidity than mice infected [30, 31, 32, 33, 34, 35] . reduced virulence of sev cantell hd was associated with a stronger stimulation of the host antiviral response as shown by the expression of ifnb mrna (fig. 4c ). to conclusively demonstrate the role of dvgs in diminishing virulence in vivo, we co-infected mice with sev cantell ld and purified viral particles containing dvgs (defective particles; dps). confirming their critical role, dvgs reduced the pathogenicity of sev cantell ld in mice, while uv-inactivated dp particles did not provide significant protection (figs. 4d-f). interestingly, infection in the presence of dps resulted in reduced expression of sev np protein in the lung at day 7 post-infection, suggesting that in this system, dps reduce virulence by interfering with virus replication. to determine whether immunostimulatory dvgs were generated in situ in the lung during infection, we infected mice with sev cantell ld, and we followed the appearance of copy-back dvgs in the lung by pcr. sev copy-back dvgs were detected in whole lung homogenates at the time of high viral replication (fig. 5a) . notably, upon infection with sev cantell ld, a copy-back dvg of high molecular weight was detected at day 3 post-infection in the lung, while a dvg of low molecular weight (amplicon of 278 bp) that predominates in the parent stock of sev cantell hd (fig. 1a) was only detectable at day 5 post-infection. copy-back dvgs also appeared in the lung of mice infected with sev 52 (fig. s6) , showing that dvgs naturally arise during infection in vivo independent on the virus strain. interestingly, accumulation of copy-back dvgs during infection with sev cantell ld was associated with the expression of ifnb and il-6 mrna in the lung (fig. 5b) . to determine whether dvgs were necessary for the expression of antiviral cytokines in vivo, we took advantage of ifnb-yfp reporter mice. to demonstrate that yfp expression serves as readout for dvg activity, we first infected bmdcs prepared from ifnb-yfp reporter mice with sev cantell ld alone, or together with increasing doses of purified dps. as shown in fig. 5c , at 6 h post-infection, yfp was expressed only in the presence of dps and in a dose-dependent manner, and the yfp expression was lost when uv-treated dps were used. dps alone were also able to induce yfp in a dose-dependent manner, albeit at much lower levels than during co-infection with sev. these data agree with our previous reports that demonstrate that the immunostimulatory activity of dps is greatly amplified during dvg replication by the cognate polymerase provided by co-infecting sev [23] and validate the ifnb-yfp reporter system as a readout for dvg activity. we then infected ifnb-yfp reporter mice with sev cantell ld and analyzed viral genomes in yfp + cells. we focused our analysis on the cd45 2 (non-hematopoietic) cellular fraction of the lung as sev replicates predominantly in the lung epithelium [36] . although full-length viral genomes were detected in both yfp + and yfp 2 cd45 2 populations sorted three days after infection, dvgs were only found in yfp + cells (fig. 5d) , suggesting that the presence of dvgs promotes ifnb production in response to virus infection in vivo. together, these findings show that dvgs are normally generated in situ in the lung during respiratory infection with sev, and that their accumulation is associated with the expression of ifnb in the lung. sev copy-back dvgs are generated in the lung independently of type i ifn signaling to determine whether type i ifns produced early upon infection promoted the generation of dvgs in the lung, we infected wild type or type i ifn receptor deficient mice (ifnar1 2/2 ) with sev cantell ld and analyzed the lungs at different times post-infection. as shown in fig. 5e , dvgs accumulated in the lung at a higher rate in mice unable to respond to type i ifns compared with wild type mice, corresponding with the predicted enhanced rate of virus replication in the lack of type i ifn signaling and demonstrating that type i ifns are not required for the generation of sev copy-back dvgs in vivo. to investigate whether the content of dvgs in iav stocks affects virulence similar to sev, we obtained iav strain pr8 stocks with a high content of dvgs (hd) or lacking dvgs (ld). the stock of iav pr8 hd produced two predominant dvgs derived from the pa and pb1 genomic segments in infected cells, while no dvgs were detected in cells infected with iav pr8 ld (fig. 6a ) (strategy for iav detection and sequences for the iav dvgs present in infected cells can be found in fig. s7 ). mice infected with iav pr8 hd showed reduced morbidity compared to mice infected with iav pr8 ld (fig. 6b ) despite similar levels of virus replication (fig. 6c ). similar to sev cantell hd, reduced morbidity was associated with enhanced ifnb mrna expression in the lung (fig. 6d) . to determine whether iav dvgs were generated in situ in the infected lung, we tracked their appearance in mice infected with iav pr8 ld. accumulation of dvgs was clearly observed at day 3 post-infection (fig. 6e) . representative sequences of starred iav dvgs products are shown in fig. s8 . similar to sev infection, accumulation of dvgs corresponded with enhanced expression of mrna for ifnb and il-6 ( fig. 6f ) despite evidence of reduced genomic viruses at that time point (figs. 6e and f) . these data demonstrate that dvgs are generated de novo in the lung during infections with iav, and suggest an important role of these types of genomes in promoting the host response to iav in vivo. we have shown that dvgs are naturally generated in the lung during infection with sev and iav and provide primary danger signals for the triggering of the host response to infection. the generation of dvgs during virus growth in tissue culture is a highly conserved phenomenon among viruses of different species and is tempting to speculate that dvgs provide an evolutionary advantage to the virus by contributing to the preservation of both the virus and the host. interestingly, immunostimulatory dvgs result from drastic truncations in the genome of the virus that render it a dead end product unable to persist in the absence of helper virus. it will be relevant to determine how dvgs relate to viral ''quasispecies'' that result from mutations as a consequence of having a viral polymerase with a lower fidelity and processivity [37] . viral quasispecies have been shown to be essential for viral fitness and virulence [37] . whether dvgs a tradeoff of this viral polymerase characteristic that enables more rapid virus evolution but makes the virus more vulnerable to innate immune detection remains to be established. our data demonstrate that dvg recognition is not necessary for the response to ndv, while is required for the response to sev. we speculate that the differential dvg requirement may be explained by the poor adaptation of the avian ndv to grow in mice while the murine sev is fully adapted to grow in this species. a critical factor of this adaptation is the activity of the virally encoded v and c proteins that effectively block the induction of type i ifns, as well as the type i ifn-mediated amplification of the type i ifn pathway [38, 39, 40, 41] . as ndv is adapted to grow in birds, its antagonistic proteins are not fully functional in mammalian cells [42] allowing unrestricted production and amplification of type i ifns. in contrast, the sev c and v proteins very effectively block the cellular response to sev [40, 42, 43, 44] and no cellular response is observed unless dvgs are present. interestingly, in the absence of type i ifn feedback (or of the ifn-inducible transcription factor irf7) sev dvgs induce a more potent cellular response compared to ndv, suggesting that dvgs have a unique ability to bypass both virus antagonism and the requirement for irf7 for strong type i ifn production. we have reported that sev cantell hd, but not ndv, has the ability to induce the expression of the viral sensor mda5 independently of type i ifn feedback [27] , and that mda5 is involved in the recognition of sev dvgs ( [23] and data in fig. s4 ). although it is unclear why this newly synthetized mda5 is less susceptible to inhibition by the sev v protein, preferential binding of dvgs to both rig-i and mda5 compared to standard sev genomes, together with the availability of high levels of mda5, may explain the strong activation of transcription factors and type i ifn expression in response to dvgs, regardless of type i ifn feedback. remarkably, dvgs arise in vivo independently of type i ifn feedback, demonstrating that dvgs do not appear in response to host pressure via type i ifns. notably, viruses containing dvgs have significantly reduced virulence. in a previous study, we reported that a sev strain with a lower propensity to produce dvgs (sev 52) persisted longer in the lung than a sev strain able to produce high levels of dvgs (cantell) [26] . consistently, we observed higher levels of viral np protein at day 7 post-infection in the lung of mice infected with sev cantell ld, compared to mice infected with sev cantell ld plus dps (fig. 4g) . in additional studies, we have not observed significant differences in the rate of sev-specific t cells in the lung of mice infected with sev ld alone or in the presence of dps (data not shown and [26] ). although interpretation of this observation is complicated by the reduced amount of sev antigen (np protein) present in infection with sev ld plus dps, we favor the hypothesis that dps diminish virulence by competing for the viral polymerase, thus interfering with the replication of the standard virus, a well-defined characteristic of dvgs. additionally, enhanced production of type i ifns in response to dvgs likely contributes to dampened viral replication. highly immunostimulatory sev dvgs are of the copy-back type. intriguingly, during sev infections in vivo, accumulation of dvgs of high molecular weight preceded the appearance of low molecular weight dvgs, suggesting that the smaller ones may be secondary to longer defective genomic products. copy-back dvgs are not transcribed due to their promoter properties [10] , thus their stimulatory activity likely derives solely from their genomic composition. iav dvgs are truncated versions of one of the genomic segments that have natural complementarity among their 39 and 59 ends providing the theoretical capacity to form structures similar to copy-back dvgs. notably, it is apparent that both sev genomic and dvg rnas have the potential to induce a host response when delivered naked into the cells. based on our data, we predict that in the context of infection, dvg rna is more available for detection due to their enhanced rate of replication compared to standard viral genomes (fig. 2) . interestingly, we have shown that dvgs have the ability to bypass viral-encoded antagonists of the immune response even upon overexpression of viral antagonistic proteins [24] . it remains to be investigated what is the molecular mechanism behind this dvg property. notably, dvgs of different forms and compositions have been described in the sera of patients chronically infected with a number of different viruses [13, 14, 15, 16, 17] and dvgs of various viruses have been shown to promote persistent infections in tissue cultures [45, 46, 47, 48, 49, 50, 51, 52, 53] supporting a role for dvgs in the maintenance of chronic viruses. the role of naturally arising dvgs in promoting virus persistence in vivo remains to be investigated. in summary, we have demonstrated that dvgs arise naturally during an acute respiratory virus infection and that they play a critical role in regulating the virus-host cycle in vivo. importantly, the recognition of dvgs as stimuli for the onset of immunity has multiple practical implications, most directly: (i) dvgs represent novel determinants of virus pathogenesis that could be targeted for therapy, and (ii) dvgs are novel candidate biomarkers to predict the outcome of infections and the rate of virus spread in the population. this study was carried out in strict accordance with the recommendations in the guide for the care and use of laboratory animals of the national institutes of health. the protocol (803176) was approved by the institutional animal care and use committee, university of pennsylvania animal welfare assurance number a3079-01. sev strains cantell, 52, enders, and z, and influenza a/pr8/ 34 virus were grown in 10 days hen embryonated eggs (spafas; charles river laboratories). sev cantell was passaged to retain its original high di particle content (hd) or to deplete it of di particles (ld) as we previously described [24] . in brief, sev 52, enders, z, and cantell hd were grown in embryonated hen eggs inoculated with 30,000 medium tissue culture infectious dose (tcid 50 ) for 40 h. sev cantell hd tcid 50 was calculated by end point dilution in llcmk2 cells in the presence of trypsin, as described below [24] . sev cantell hd total particles were calculated by end point dilution of hemagglutination of chicken red blood cells. sev cantell hd stocks had consistently an infectious:total particle ratio of 5,000-15,000. sev 52 had an infectious:total particle ratio of 24,472. sev enders had an infectious:total particle ratio of 38,746. sev z had an infectious:total particle ratio of 391,553. sev cantell ld was prepared by inoculating embryonated hen eggs with 3 tcid 50 for 40 h. under these conditions only 33% of the eggs grew virus. allantoic fluid from those eggs was pooled and diluted 1/10 6 for subsequent inoculation into embryonated hen eggs in a total volume of 100 ml. allantoic fluid containing virus (80% of the inoculated eggs) was pooled and tittered as described below. sev cantell ld stocks had consistently an infectious:total particle ratio of 100,000-200,000. iav strain pr8 (ld) was grown by inoculating hen embryonated eggs with 30,000 tcid 50 obtained directly from infected lung homogenates. allantoic fluid containing the virus was collected 40 h later. egg's allantoic fluid was snap frozen in an ethanol/dryice bath and stored at 280uc. iav pr8 containing high dose of di particles (hd) was kindly provided by dr. laurence c. eisenlohr, v.m.d., ph.d (thomas jefferson university). iav hd was grown by inoculating hen embryonated eggs with 10,000 pfu of egg-passed virus. eggs were incubated at 35uc and allantoic fluid containing the virus was collected 48 h later. iav pr8 hd and ld stocks were originated from the same parent stock but were extensively passaged in the different conditions described. permissive cells were infected with serial 1:10 dilutions of lung homogenates or virus stocks in the presence of 2 mg/ml of trypsin to determine the medium tissue culture infectious dose (tcid 50 ). llcmk2 cells were used for sev titration, while mdck cells were used for iav titration. after 72 h of incubation at 37uc, 50 ml of supernatant from each well was tested by hemagglutination of chicken red blood cells (rbcs) for the presence of virus particles at the end point dilution. to do this, 1:4 dilutions of the cell supernatant were incubated in 0.5% chicken rbcs at 4uc for 30 min. hemagglutination of rbcs indicated the presence of sev or influenza virus particles. bmdcs were generated as previously described [24] . detailed procedure can be found in the supplemental information material and methods. bmdcs were infected after 4 days in culture with viruses at an multiplicity of 1.5 as we have previously described [24] . for sev infections, mice were anesthetized with tribromoethanol (avertinh; acros organics) and inoculated in the nostrils with 30 ml of pbs containing 10 4 or 10 5 tcid 50 of sev. for iav infections animals were infected intranasally with 100 tcid 50 / mouse in a 30 ml volume. lungs were extracted at different times post-infection, homogenized in 0.1% w/v gelatin-pbs and snap frozen in dry-ice/ethanol for preservation. total rna was extracted from cell lines or lungs with trizol (invitrogen) according to the manufacturer's specifications and total rna was reversed transcribed using the high capacity rna to cdna kit from applied biosystem. for sorted cells, 500 ng of rna were reversed transcribed, for all other experiments 1-2 mg of rna were reversed transcribed. cdna was diluted to a concentration of 10 mg/ml and amplified with specific primers in the presence of sybr green (applied biosystem). for the detection of dvgs, isolated total rna was reverse transcribed using superscript iii without rnase h activity, to avoid selfpriming by the dvgs complementary ends and recombinant rnase h (invitrogen) was added later to the samples. for the detection of the standard virus genome, the negative strand of the full-length genome was reverse transcribed with transcriptor first strand cdna synthesis kit (roche). pcr detection for iav was performed using as it has been previously described [54] . primers and detailed pcr conditions can be found in the supplemental information material and methods. detailed primers and pcr conditions can be seen in the supplemental information material and methods. whole cellular extracts were prepared by lysing 3610 6 of cells in a np-40-based lysis buffer containing phosphatase inhibitors, proteinase inhibitors (roche and thermo scientific), and 0.5 m edta. the concentration of protein was measured by bradford assay (themo scientific). samples (25 mg) were boiled for 5 min and resolved on 10% bis-tris pre-cast gels (bio-rad). resolved proteins were transferred to a polyvinylidene fluoride (pvdf) membrane (millipore). the membrane was blocked with 5% nonfat milk and immunoblotted with the indicated antibodies. antirabbit irf3, anti-rabbit phospho-irf3 (ser396), anti-mouse ikba, anti-mouse phospho-ikba (ser32/36), and anti-rabbit igg (hrp-conjugated) were purchased from cell signaling. antimouse gapdh was purchased from sigma. anti-mouse igg and anti-mouse igg 1 (hrp-conjugated) were purchased from jackson immunologicals. lumi-light western blotting substrate was used for hrp detection (roche). dp purification was performed as previously described [24] . in short, allantoic fluid from 100 infected hen eggs was pooled and concentrated by high-speed centrifugation. pellets were suspended in 0.5 ml of pbs/2 mm edta and incubated overnight at 4uc in a 5-45% sucrose (fisher) gradient that was prepared using a gradient maker (biocomp). gradients were centrifuged at 4uc for 1.5 h at 28,000 rpm and fractions containing low-density viral particles were collected, pelleted, suspended and re-purified using the same procedure. collected low-density fractions were concentrated by centrifugation at 4uc for 2 h at 21,000 rpm. pellets were suspended in pbs, snap frozen, and stored at 280uc. the content of di particles was determined by calculating the ratio of infectious over non-infectious particles as described above. a 591 nt long product containing the sequence of the t7 promoter followed by the 546-nucleotide long copy back dvg from sev cantell, and flanked by the restriction enzymes spei and sapi at the 39 an 59 ends was synthetically synthetized (dna 2.0) and clone into the psl1180 vector (amersham pharmacia biotech) containing the sequences for the hepatitis delta virus ribozyme and the t7 polymerase terminator. in order to optimize the transcription of the dvg, 3 g residues were introduced downstream of the t7 promoter by site-directed mutagenesis (stratagene, ca) using the oligonucleotides 59ccactagttaa-tacgactcactatagggaccagacaagagtttaagag-39 and 59ctcttaaactcttgtctggtccctatagtgag tcgtattaactagtgg-39. bsr-t7 cells were infected with a moi of approximately 66 of partially inactivated sev strain 52. virus inactivation was performed by exposing diluted virus to uv light (254 nm model mrl-58, uvp upland, ca) for 53 sec at a distance of 9 inches from the light source. virus inactivation diminished the virus replication rate, while allowing the expression of viral proteins necessary for the replication of dvgs. cells were incubated at 37uc for 1 h before transfection of 3 mg of vector encoding dvg. transfection was performed with xtremegene transfection reagent (roche) according to manufacturer instructions. cells were cultured in dulbecco's modified eagle medium (dmem) supplemented with 1% bovine serum albumin, 2% naco 3 , 0.5 mg/ml trypsin (worthington) and 0.1% penicillinstreptomycin (invitrogen) and incubated in 7% co 2 at 37uc. cells and supernatant containing sev 52 and rdps were harvested after 48 h and 200 ml of the suspension were inoculated in the allantoic cavity of 10-day embryonated hen eggs (b & e eggs, silver springs, pa). after 40 h allantoic fluid was harvest and 200 ml of undiluted fluid were inoculate in 10-day embryonated eggs for virus growth and egg inoculation was repeated for three consecutive passages. allantoic fluid from the third passage was quick-frozen in dried ice/ethanol and used for infections. presence of recombinant dvg was confirmed by pcr. no other dvgs were detected. dvg rna was in vitro transcribed (ambion) from the t7-dvg 546 plasmid. standard genomic rna was extracted from sev cantell ld stocks. to remove 59-triphosphates, 1 mg of rna was incubated with 10 u of calf intestinal phosphatase (new england biolabs) for 60 min at 37uc. to cleave single stranded rna, 1 mg of rna was incubated with 1 ng of rnase a (ambion) for 15 min at room temperature. to cleave double stranded rna, rna was incubated with 0.1 u of rnase v1 (ambion) for 15 min at room temperature. after treatments, rna was purified using trizol or precipitation/inactivation buffer according to the manufacturer's specifications. llc-mk2 cells were transfected with 250 ng or indicated doses of dvg and genomic rna using lipofectamin 2000 (invitrogen). at 4 hours post transfection, the cells were harvested and total rna was isolated using trizol according to the manufacturer's specifications. infected ifnb-yfp cells were collected at 6 h post-infection. reporter mice were sacrificed 3 days post-infection. lungs were collected and dissociated with collagenase (roche), followed by suspension on 0.5 m edta and rbc lysis buffer. single cell suspensions were then incubated with cd16/cd32 fcblock for 20 min at 4c, followed by incubation with biotinylated mouse anti-cd45.2. washed cells were incubated with anti-biotin microbeads (miltenyi) for 20 min and passed through a magnetic column for negative selection. cd45 2 cells were sorted based on yfp expression using a facs vantage se sorter. statistical analyses were performed as indicated in each figure. graphpad prism version 5.00 for windows, graphpad software, san diego california usa, www.graphpad.com, was used for analysis. genes ncbi id numbers. tuba1b: 22143; rps11:27207; ifnb: 15977; ifnl2: 330496; illb: 16176; il12b: 16160; tnf: 2926; il-6, 16193. text s1 supporting materials and methods. the text includes in detail descriptions of the procedures, as well as specific material and methods and references for figures included as supporting information. (docx) preference of rig-i for short viral rna molecules in infected cells revealed by next-generation sequencing 59-triphosphate rna is the ligand for rig-i lengthdependent recognition of double-stranded ribonucleic acids by retinoic acidinducible gene-i and melanoma differentiation-associated gene 5 rig-imediated antiviral responses to single-stranded rna bearing 59-phosphates rig-i detects viral genomic rna during negative-strand rna virus infection recognition of 59 triphosphate by rig-i helicase requires short blunt doublestranded rna as contained in panhandle of negative-strand virus cutting edge: stealth influenza virus replication precedes the initiation of adaptive immunity antagonism of the interferon-induced oas-rnase l pathway by murine coronavirus ns2 protein is required for virus replication and liver pathology defective viral particles and viral disease processes the origins of defective interfering particles of the negative-strand rna viruses incomplete forms of influenza virus defective interfering rnas: foes of viruses and friends of virologists detection of defective genomes in hepatitis a virus particles present in clinical specimens hepatitis b defective virus with rearrangements in the pres gene during chronic hbv infection in vivo and in vitro expression of defective hepatitis b virus particles generated by spliced hepatitis b virus rna characterization of hepatitis c virus deletion mutants circulating in chronically infected patients mechanisms associated with the generation of biologically active human immunodeficiency virus type 1 particles from defective proviruses defective interfering viral particles in acute dengue infections sequence analysis of in vivo defective interfering-like rna of influenza a h1n1 pandemic virus the characteristics required for a sendai virus preparation to induce high levels of interferon in human lymphoblastoid cells interferon induction by viruses. xix. vesicular stomatitis virus-new jersey: high multiplicity passages generate interferoninducing, defective-interfering particles activation of the beta interferon promoter by unnatural sendai virus infection requires rig-i and is inhibited by viral c proteins mda5 participates in the detection of paramyxovirus infection and is essential for the early activation of dendritic cells in response to sendai virus defective interfering particles a novel role for viral-defective interfering particles in enhancing dendritic cell maturation differential type i ifn-inducing abilities of wild-type versus vaccine strains of measles virus sendai virus infection induces efficient adaptive immunity independently of type i interferons cytokine-independent upregulation of mda5 in viral infection sendai virus defective-interfering genomes and the activation of interferon-beta nucleotide sequences that affect replicative and transcriptional efficiencies of sendai virus deletion mutants prevention of death in semliki forest virusinfected mice by administration of defective-interfering semliki forest virus defective interfering viruses: modulators of infection protection of mice from lethal influenza: evidence that defective interfering virus modulates the immune response and not virus multiplication protection of three strains of mice against lethal influenza in vivo by defective interfering virus defective interfering influenza a virus protects in vivo against disease caused by a heterologous influenza b virus defective interfering virus protects elderly mice from influenza melanoma differentiation-associated gene 5 (mda5) is involved in the innate immune response to paramyxoviridae infection in vivo quasispecies diversity determines pathogenesis through cooperative interactions in a viral population the v proteins of paramyxoviruses bind the ifn-inducible rna helicase, mda-5, and inhibit its activation of the ifn-beta promoter ) mda-5, but not rig-i, is a common target for paramyxovirus v proteins the paramyxovirus, sendai virus, v protein encodes a luxury function required for viral pathogenesis c and v proteins of sendai virus target signaling pathways leading to irf-3 activation for the negative regulation of interferon-beta production newcastle disease virus v protein is a determinant of host range restriction the sendai paramyxovirus accessory c proteins inhibit viral genome amplification in a promoter-specific fashion longer and shorter forms of sendai virus c proteins play different roles in modulating the cellular antiviral response persistent infection. i interferon-inducing defective-interfering particles as mediators of cell sparing: possible role in persistent infection by vesicular stomatitis virus role of defective interfering particles of sendai virus in persistent infections comparative study of rabies virus persistence in human and hamster cell lines characterization of west nile virus persistent infections in genetically resistant and susceptible mouse cells. i. generation of defective nonplaquing virus particles characterization of a cell culture persistently infected with the da strain of theiler's murine encephalomyelitis virus persistent infection of some standard cell lines by lymphocytic choriomeningitis virus: transmission of infection by an intracellular agent human cytomegalovirus persistent infection in a human central nervous system cell line: production of a variant virus with different growth characteristics defective interfering particles of human parainfluenza virus type 3 are associated with persistent infection in cell culture establishment of respiratory syncytial virus persistence in cell lines: association with defective interfering particles singlereaction genomic amplification accelerates sequencing and vaccine production for classical and swine origin human influenza a viruses the authors wish to thank dr. laurence eisenlohr for hd influenza virus, luis muñ oz for technical support, and the flow cytometry and cell sorting resource laboratory and the dna sequencing facility at the university of pennsylvania. we also thank drs. thomas moran, christopher basler, and benjamin tenoever (icahn school of medicine at mount sinai), and marco colonna (washington university) for providing invaluable reagents. conceived and designed the experiments: kt wk cbl. performed the experiments: kt wk xml ys ed ma mw. analyzed the data: kt wk cbl. wrote the paper: kt wk cbl. key: cord-002021-67ao8chx authors: kim, seong bum; choi, jin young; uyangaa, erdenebileg; patil, ajit mahadev; hossain, ferdaus mohd altaf; hur, jin; park, sang-youel; lee, john-hwa; kim, koanhoi; eo, seong kug title: blockage of indoleamine 2,3-dioxygenase regulates japanese encephalitis via enhancement of type i/ii ifn innate and adaptive t-cell responses date: 2016-04-18 journal: j neuroinflammation doi: 10.1186/s12974-016-0551-5 sha: doc_id: 2021 cord_uid: 67ao8chx background: japanese encephalitis (je), a leading cause of viral encephalitis, is characterized by extensive neuroinflammation following infection with neurotropic je virus (jev). indoleamine 2,3-dioxygenase (ido) has been identified as an enzyme associated with immunoregulatory function. although the regulatory role of ido in viral replication has been postulated, the in vivo role of ido activity has not been fully addressed in neurotropic virus-caused encephalitis. methods: mice in which ido activity was inhibited by genetic ablation or using a specific inhibitor were examined for mortality and clinical signs after infection. neuroinflammation was evaluated by central nervous system (cns) infiltration of leukocytes and cytokine expression. ido expression, viral burden, jev-specific t-cell, and type i/ii interferon (ifn-i/ii) innate responses were also analyzed. results: elevated expression of ido activity in myeloid and neuron cells of the lymphoid and cns tissues was closely associated with clinical signs of je. furthermore, inhibition of ido activity enhanced resistance to je, reduced the viral burden in lymphoid and cns tissues, and resulted in early and increased cns infiltration by ly-6c(hi) monocytes, nk, cd4(+), and cd8(+) t-cells. je amelioration in ido-ablated mice was also associated with enhanced nk and jev-specific t-cell responses. more interestingly, ido ablation induced rapid enhancement of type i ifn (ifn-i) innate responses in cd11c(+) dendritic cells (dcs), including conventional and plasmacytoid dcs, following jev infection. this enhanced ifn-i innate response in ido-ablated cd11c(+) dcs was coupled with strong induction of prrs (rig-i, mda5), transcription factors (irf7, stat1), and antiviral isg genes (mx1, mx2, isg49, isg54, isg56). ido ablation also enhanced the ifn-i innate response in neuron cells, which may delay the spread of virus in the cns. finally, we identified that ido ablation in myeloid cells derived from hematopoietic stem cells (hscs) dominantly contributed to je amelioration and that hsc-derived leukocytes played a key role in the enhanced ifn-i innate responses in the ido-ablated environment. conclusions: inhibition of ido activity ameliorated je via enhancement of antiviral ifn-i/ii innate and adaptive t-cell responses and increased cns infiltration of peripheral leukocytes. therefore, our data provide valuable insight into the use of ido inhibition by specific inhibitors as a promising tool for therapeutic and prophylactic strategies against viral encephalitis caused by neurotropic viruses. japanese encephalitis (je) is an acute zoonotic, mosquitoborne disease caused by je virus (jev), a single-stranded, positive-sense rna (~11 kb, monopartite, linear) virus belonging to the family flaviviridae and the genus flavivirus [1] . infection with neurotropic flaviviruses of the je serotype, which include je, murray valley encephalitis, st. louis encephalitis, and west nile virus (wnv), results in debilitating neurological disorders in a significant proportion of clinical cases [2, 3] . je is a leading cause of viral encephalitis manifested by extensive neuroinflammation in the central nervous system (cns) and disruption of the blood-brain barrier (bbb). in humans, the clinical presentation of jev infection ranges from mild febrile illness to severe meningoencephalitis [4] . due to rapid changes in climate and demography, vector-transmitted je poses an increasing threat to global health and welfare with nearly 70,000 cases reported annually [5] [6] [7] . the incubation period of je ranges from 5 to 15 days, and most jev infections in endemic regions manifest as mild febrile, subclinical disease which leads to protective adaptive immune responses [4] . however, approximately 25-30 % of je cases, mostly in infants, are lethal and 50 % of cases result in permanent neuropsychiatric sequelae [4] . thus, je is considered more fatal than encephalitis caused by wnv infection, which has a fatality rate of 3-5 % (1100 deaths/ 29,000 symptomatic infections) [7, 8] . currently, more than 60 % of the world's population inhabits je endemic areas, such as eastern and southern asia, and the virus is spreading to previously unaffected regions, including indonesia, pakistan, and northern australia [5, 6] . however, despite the importance of je, little is known regarding potential therapeutic strategies for regulating je progression. indoleamine 2,3-dioxygenase (ido) has been identified as an enzyme associated with powerful immunoregulatory function, likely derived from its enzymatic activity, which leads to catabolism of the essential amino acid l-tryptophan (l-trp) [9] [10] [11] [12] [13] [14] . therefore, ido-mediated depletion of l -trp and the resulting metabolites (l-kynurenine, l-kyn) induces an immunosuppressive environment through provoking tolerogenicity of antigenpresenting cells (apcs), t-cell anergy, and immune cell death [9, 10] . ido can be induced in a variety of cell types, including dendritic cells (dcs) [15] , macrophages [16] , and epithelial cells [17] . these cell types play an important role in controlling viral replication and facilitating antigenspecific adaptive immune responses [9, 10] . in various tissues, ido activity has been induced by several cytokines after viral infection, and its enzymatic activity can be blocked using the pharmacological competitive inhibitor, 1-methyl-d,l-tryptophan (1-mt) [18] . thus, inhibition of ido with the competitive inhibitor 1-mt may be a promising strategy for enhancing immune responses in various viral infection models, including human immunodeficiency virus (hiv) and influenza virus [19, 20] . also, ido ablation was shown to suppress viral replication through the upregulation of type i interferon (ifn-i) production in a retrovirus-infected murine model [21] . although the regulatory role of ido in viral replication has been reported in a few studies using an in vivo viral infection model, the in vivo role of ido activity is not fully understood in viral encephalitis caused by infection with neurotropic viruses such as jev and wnv. the molecular pathogenesis of viral encephalitis, including je, remains unclear. however, je is considered an immunopathological disease because cns invasion by jev drives the stimulation of microglia/glia and infiltrating leukocytes, leading to indirect death of neuron cells via the uncontrolled secretion of pro-inflammatory cytokines (such as il-6 and tnf-α) and soluble mediators [22, 23] . furthermore, jev infects and kills neuron cells directly in the cns. this complicated progression of je after viral infection in the host prompted us to explore the role of ido in je progression. here, we found that ido expression in myeloid cells and in neurons of the lymphoid and cns tissues was closely associated with clinical signs of je. furthermore, the inhibition of ido activity using genetic ablation or 1-mt provided enhanced resistance to je, along with a reduced viral burden and the early and increased cns infiltration of myeloid and lymphoid leukocytes. je amelioration was also associated with enhanced nk and antigen-specific t-cell responses. more interestingly, the rapid enhancement of ifn-i innate immune responses in cd11c + dcs (conventional and plasmacytoid dcs) and neuron cells likely contributed to the protective role of ido ablation in je. therefore, our data suggest that the inhibition of ido with pharmacological inhibitors could be a promising therapeutic strategy for regulating je progression. all animal experiments described in the present study were conducted at chonbuk national university according to the guidelines set by the institutional animal care and use committees (iacuc) of chonbuk national university and were pre-approved by the ethical committee for animal experiments of chonbuk national university (permission code 2013-0028). the animal research protocol used in this study followed the guidelines set up by the nationally recognized korea association for laboratory animal sciences (kalas). all experimental protocols requiring biosafety were approved by institutional biosafety committees (ibc) of chonbuk national university. c57bl/6 (h-2 b ) mice (4-5 weeks old) were purchased from samtako (o-san, korea). ido (h-2 b ) knockout (ko) mice were obtained from the jackson laboratory (bar harbor, me, usa). all mice were genotyped and bred in the animal facilities of chonbuk national university. jev beijing-1 strain was obtained from green cross research institute (suwon, korea) and propagated in the mosquito cell line, c6/36, using dmem supplemented with 2 % fbs, penicillin (100 u/ml), and streptomycin (100 u/ml) [24] . the c6/36 cultures were infected with jev beijing-1 at a multiplicity of infection (moi) of 0.1 and were incubated in a humidified co 2 incubator for 1 h at 28°c. after absorption, the inoculum was removed, and 7 ml of a maintenance medium containing 2 % fbs was added. approximately 6-7 days post-infection (dpi), cultures of host cells showing an 80-90 % cytopathic effect were harvested. the virus stocks were titrated using either a conventional plaque assay or a focus-forming assay and were stored in aliquots at −80°c until use. the mabs used for flow cytometric analyses and other experiments were obtained from ebioscience (san diego, ca, usa) or r&d systems (minneapolis, mn, usa): fluorescein isothiocyanate (fitc)-conjugated anti-cd4 (rm4-5), cd8 (53-6.7), ly-6g (1a8), b220 (ra3-6b2); phycoerythrin (pe)-conjugated anti-mouse-cd11b (m1/70), foxp3 (fjk-16s), cd154 (mr1); peridinin chlorophyll protein complex (percp)-conjugated anti-ly-6c (hk1.4); pe-cyanine dye (cy5.5)-conjugated anti-mouse ifn-γ (xmg1.2); pecyanine dye (cy7)-conjugated anti-mouse nk1.1 (pk136), il-5 (trfk4); and allophycocyanin (apc)-conjugated antimouse-cd3ε (145-2c11), cd25 (pc62.5), cd49b (dx5), ly-6g (gr-1), tnf-α (mp6-xt22), il-4 (bvd6-24g2), and il-17a (ebio17b7). the peptides of i-a b -restricted epitopes (jev ns1 132-145 [tfvvdgpetkecpd] and ns3 563-574 [wcfdgprtnail]) and h-2d b -restricted epitopes (jev ns4b 215-223 [savwnstta]) were chemically synthesized at peptron inc. (daejeon, korea). jev-specific primers for the detection of viral rna (jev10,564-10,583 forward, 5′-ccc tca gaa ccg tct cgg aa-3′ and jev10,862-10,886 reverse, 5′-cta ttc cca ggt gtc aat atg ctg t-3′) and primers specific for cytokines, ifn-i (ifnα/β), and rlrs, irfs, isgs (table 1) were synthesized at bioneer corp. (daejeon, korea) and used for pcr amplification of target genes. quantitative real-time rt-pcr for viral burden and cytokine expression viral burden and the expression of cytokines (il-6, tnf-α, and ifn-α/β), cc chemokines, and ido in inflammatory and lymphoid tissues were determined by conducting quantitative sybr green-based real-time rt-pcr (real-time qrt-pcr). mice were infected intraperitoneally (i.p.) with jev (3.0 × 10 7 plaque-forming unit (pfu)) and tissues, including brain, spinal cord, and spleen, were harvested at 2 and 4 dpi following extensive cardiac perfusion with hank's balanced salt solution (hbss). total rna was extracted from tissues using easy-blue (intron, inc., daejeon, korea) and subjected to real-time qrt-pcr using a cfx96 real-time pcr detection system (bio-rad laboratories, hercules, ca, usa). following reverse transcription of total rna with the high-capacity cdna reverse transcription kits (applied biosystems, foster, ca, usa), the reaction mixture contained 2 μl of template cdna, 10 μl of 2× sybr premix ex taq, and 200 nm primers, yielding a final volume of 20 μl. the reactions were denatured at 95°c for 30 s and then subjected to 45 cycles of 95°c for 5 s and 60°c for 20 s. after the reaction cycle was complete, the temperature was increased from 65 to 95°c at a rate of 0.2°c/15 s, and the fluorescence was measured every 5 s to construct a melting curve. a control sample that contained no template dna was run with each assay, and all determinations were performed at least in duplicates to ensure reproducibility. the authenticity of the amplified product was determined by melting curve analysis. all data were analyzed using the bio-rad cfx manager, version 2.1 analysis software (bio-rad laboratories). viral burden was expressed by the copy number of viral rna per microgram of total rna, after calculating the absolute copy number of viral rna in comparison with the standard cdna template of viral rna. the levels of l-kynurenine in the sera and brain homogenates following jev infection were measured via hplc after deproteination using a c18 reverse phase column [25] . the levels of l-kynurenine in the sera and brain homogenates were expressed as micromolars and picomoles per milligram tissue, respectively. levels of l-kynurenine were used for an in vivo index of ido enzyme activity. mice infected with jev were perfused with 30 ml of hbss at 3 or 5 dpi via cardiac puncture of the left ventricle. brains were then harvested and homogenized by gently pressing them through a 100-mesh tissue sieve, after which they were digested with 25 μg/ml collagenase type iv (worthington biochem, freehold, nj, usa), 0.1 μg/ml trypsin inhibitor nα-p-tosyl-l-lysine chloromethyl ketone, 10 μg/ml dnase i (amresco, solon, oh, usa), and 10 mm hepe in hbss for 1 h at 37°c, under shaking conditions. cells were separated using an optiprep density gradient (18/10/5 %) with centrifugation at 800×g for 30 min (axis-shield, oslo, norway), after which cells were collected from the 18 to the 10 % interface and washed twice with pbs. cells were counted and stained for cd11b, ly6g, ly6c, cd3ε, cd4, cd8α, dx5, and nk1.1 with directly conjugated antibodies (ebioscience) for 30 min at 4°c. finally, the cells were fixed with 10 % formaldehyde. data collection and analysis were performed with a facscalibur flow cytometer (becton dickson medical systems sharon, ma, usa) and flowjo (tree star, san carlos, ca, usa) software. jev-specific cd4 + and cd8 + t-cell responses were determined by intracellular cd154 [26, 27] , ifn-γ, and tnf-α staining in response to stimulation with jev epitope peptides. surviving mice infected with 3.0 × 10 7 pfu jev were sacrificed at 7 dpi, and splenocytes were prepared. the erythrocytes were depleted by treating single-cell suspensions with ammonium chloride-containing tris buffer (nh 4 cl-tris) for 5 min at 37°c. the splenocytes were cultured in 96-well culture plates (5 × 10 5 cells/well) in the presence of synthetic peptide epitopes (ns1 132-145 , ns3 563-574 , and ns4b 215-225 ) for 12 and 6 h, in order to observe cd4 + and cd8 + t-cell responses, respectively. monensin at a concentration of 2 μm was added to antigenstimulated cells 6 h before harvest. cells were washed twice with pbs and surface-stained with fitc-anti-cd4 or cd8 antibodies for 30 min at 4°c, and then washed twice with pbs containing monensin. after fixation, the cells were washed twice with permeabilization buffer (ebioscience) and stained with pe cy5.5-anti-ifn-γ or apc-anti-tnf-α in permeabilization buffer for 30 min at room temperature. intracellular cd154 was detected by the addition of cd154 mab to culture media during peptide stimulation. finally, the cells were washed twice with pbs and fixed using fixation buffer. sample analysis was performed with a facscalibur flow cytometer (becton dickson medical systems) and flowjo (tree star) software. intracellular staining for analysis of cd4 + th1, th17, and treg cells to monitor cd4 + th subsets, mice were infected i.p. with 3.0 × 10 7 pfu of jev, sacrificed at 5 dpi, and splenocytes were prepared. splenocytes were then cultured in 96-well culture plates (10 6 cells/well) with pma/ionomycin (th1 and th17) in the presence of monensin (2 μm) for 5 h at 37°c. the stimulated cells were washed twice with pbs and surface stained with fitc-anti-cd4 for 30 min at 4°c and then washed twice with pbs containing monensin. after fixation, the cells were washed twice with permeabilization buffer (ebioscience) and stained with percp-anti-ifn-γ and apc-anti-il-17α in permeabilization buffer for 30 min at room temperature. finally, the cells were washed twice with pbs and fixed using fixation buffer. to monitor treg cells, splenocytes were stained by surface staining for fitc-anti-cd4 markers for 30 min on ice and then fixed with fixation/permeabilization concentrate buffer (ebioscience) for 6 h at 4°c. after fixation, the cells were washed twice with permeabilization buffer (ebioscience) and stained with pe-anti-foxp3 in permeabilization buffer for 30 min at room temperature. the sample analysis was performed using a facscalibur flow cytometer. bone marrow-derived conventional dcs (bmdcs), plasmacytoid dcs (pdcs), and macrophages (bmdms) were prepared from bm cells from bl/6 and ido ko mice, as described earlier with some modifications [24] . briefly, for bmdcs and pdcs, bm cells (3 × 10 5 cells/ml) from femurs and tibiae were cultured in rpmi 1640 supplemented with gm-csf (2 ng/ml) plus il-4 (10 ng/ml) and flt3-l (10 ng/ ml), respectively. on day 3, another 6 ml of fresh complete medium containing gm-csf plus il-4 and flt3-l was added, and half of the medium was changed on day 6. on day 8, non-adherent and loosely adherent dcs were harvested by vigorous pipetting. cells were then characterized by flow cytometric analysis, which revealed that the culture generally consisted of >85 % cd11c + cells (25 % cd11c + cd11b + and 65 % cd11c + cd8α + ), and >90 % cd11c + pdca-1 + b220 + . bmdms were prepared by culturing bone marrow cells in dmem supplemented with 30 % l929 cell-conditioned medium (lccm) as a source of macrophage colony-stimulating factor (m-csf). on day 3, another 6 ml of fresh complete medium containing 30 % lccm was added, and half of the medium was changed on day 6. the cultured cells were harvested following an 8-day incubation and were analyzed by flow cytometry. the prepared bmdms were composed of >85 % f4/80 + cells that consisted of 99.2 % f4/80 + cd11b + and~1 % f4/80 + cd11c + cells. prepared bmdcs, pdcs, and bmdms were infected with jev at mois of 0.1, 1.0, and 10 for analysis of viral replication and 10 moi for evaluating cytokine expression. primary microglia cells were recovered from the brains of fetal bl/6 and ido ko mice (1-3 days old), as described previously [28] . primary microglia cells recovered from the brains via trypsin + edta digestion were initially seeded on poly-d-lysine/laminin-coated plates in dmem containing 10 % fbs and mechanically isolated from glia cells by a brief duration of shaking (100 rpm, 1 h). primary microglia cells were infected with jev at mois of 1.0 and 10 for analysis of viral replication and 10 moi for the evaluation of cytokine, ifn-i, rlr, and isg gene expression. primary cortical neurons were prepared from 15-day-old embryos, as described previously [24] . cortical neurons were seeded in 12-well poly-d-lysine/laminin-coated plates in dmem containing 5 % fbs and 5 % horse serum for 24 h, and then cultured for 4 days with neurobasal medium containing b27 supplement and l-glutamine (invitrogen, carlsbad, ca, usa). primary cortical neurons were infected with jev at mois of 0.001 and 0.01 for analysis of viral replication and 0.1 moi for the evaluation of cytokine expression. generation of bm chimeric mice and determination of serum ifn-β bl/6 mice (5 weeks old) and ido ko mice were irradiated with one dose of 900 rads. within 12 h, recipient mice were reconstituted with 10 7 donor bm cells derived from bl/6 and ido ko mice. the recipient mice were given sulfamethoxazole and trimethoprim in their drinking water for 10 days after irradiation. mice were infected with jev 4-6 weeks after irradiation. a commercial elisa kit (pbl biomedical laboratories, piscataway, nj, usa) was used to measure levels of secreted ifn-β protein in sera according to the manufacturer's protocol. all data were expressed as the average ± standard deviation, and statistically significant differences between groups were analyzed by unpaired two-tailed student's t tests for ex vivo experiments and immune cell analyses or anova and post hoc test for multiple comparisons of the mean. the statistical significance of viral burden was evaluated using the mann-whitney test or unpaired two-tailed student's t test. kaplan-meier survival curves were analyzed with the log-rank test. a p value of ≤0.05 was considered significant. all data were analyzed using prism software (graphpad prism4, san diego, ca, usa). to determine whether ido expression changes during je progression, several tissues obtained from jevinfected bl/6 mice were used to evaluate ido mrna expression at the early stage of infection (from 0 to 3 dpi) prior to the presentation of neurological disorders. as shown in fig. 1a , jev infection induced no apparent changes in the expression of ido in the examined tissues, including lymphoid (spleen, mesenteric ln, bone marrow), extraneural (liver), and cns tissues (brain, spinal cord) prior to the onset of neurological disorders. in general, infected mice showed clinical signs starting with generalized piloerection, paresis, and rigidity, which then progressed to severe neurological signs, such as postural imbalance, ataxia, and generalized tonic-clonic seizure, by 4 to 5 dpi. thus, we were interested in testing whether ido expression varies between mice showing clinical signs, such as paralysis, and mice displaying no clinical signs. to this end, mice were divided into two populations showing either paralysis or no paralysis 5 dpi, the point at which approximately 30-40 % of infected mice showed neurological disorders, and the expression of ido mrna was evaluated in several tissues. interestingly, enhanced induction of ido expression was observed only in lymphoid tissues (spleen and bone marrow) and the cns (brain and spinal cord) of mice showing paralysis compared to mice showing no paralysis (fig. 1b) . however, other extraneural tissues, including mesenteric ln and liver, showed no apparent increases in expression of ido in paralyzed mice. ido expression at the protein level was also confirmed by western blotting using total lysates derived from several tissues. in agreement with the enhanced induction of ido mrna expression in paralyzed mice, mice showing paralysis displayed an apparent increase in the expression of ido protein in lymphoid tissues (spleen, bone marrow) and neural tissues (brain and spinal cord) (fig. 1c) . dcs and macrophages in peripheral tissues are the primary target cells of jev, and neuron cells support jev replication after the virus gains access into the cns. also, microglia, tissue-resident macrophages in the cns are believed to regulate neuroinflammation caused by various insults. therefore, we evaluated ido expression in primary myeloid-derived dcs, pdcs, macrophages, microglia, and primary cortical neuron cells after jev infection. conventional dcs (bmdcs), pdcs, macrophages (bmdms), microglia, and neuron cells displayed different dynamic patterns of ido expression, depending on the time after jev infection (fig. 1d) . pdcs showed the most rapid expression of ido, with levels peaking at 24 h pi, whereas bmdcs displayed a delayed expression pattern with the levels peaking at 48 h pi. ido was also expressed in macrophages with gradual and moderate increases in levels up to 72 h pi, and microglia showed basal levels of ido expression up to 24 h pi and increased levels thereafter. neuron cells showed a gradual increase in ido expression with approximately fivefold higher levels at 72 h pi compared to levels in the mockinfected group. this result indicates that the primary target cells in the periphery and cns tissues can actively express ido with different intrinsic patterns in response to jev infection. collectively, these results indicate that ido expression in myeloid cells and neurons of the lymphoid and cns tissues is closely associated with the clinical signs of je. notably, ido expression in the neural tissues (brain and spinal cord) was likely to be coupled with neurological disorders such as paralysis. blockage of ido provides enhanced resistance to je because ido expression in lymphoid and neural tissues was closely associated with je progression, we were interested in investigating the role of ido during je progression. bl/6 mice infected with jev showed gradually elevated release of the tryptophan catabolite l-kynurenine by cells expressing functional ido enzyme activity in the sera and brain homogenates relative to basal levels in ido ko mice (fig. 2a) , which suggests that ido expression increases during je progression. also, we examined and compared the susceptibility of ido-deficient (ko) mice to je caused by neurotropic jev infection with that of wild-type bl/6 mice (fig. 2b) . the ablation of ido resulted in a markedly increased survival rate of 55 % in ido ko mice vs. 18 % in bl/6 mice after jev infection (3.0 × 10 7 pfu) (left curve in fig. 2b) , thereby signifying significantly enhanced resistance to je. likewise, ido ko mice showed delayed signs of neurological disorder starting 5-6 dpi compared to bl/6 mice, which displayed signs of neurological disorder sooner and in a higher proportion of animals (middle graph in fig. 2b) , and ido ablation resulted in less change in body weight (right graph in fig. 2b) . these results indicate that , and liver (liv), 1, 2, and 3 days following jev infection. b comparison of ido expression between aparalytic and paralytic hosts. ido expression was determined by real-time qrt-pcr using total rna extracted from several tissues in mice showing a neurological disorder such as paralysis (paralytic) and mice showing no paralytic symptoms (aparalytic) 5 dpi. c detection of ido protein during je progression. ido protein levels in several tissues of jev-infected mice were evaluated by western blot using a monoclonal antibody specific for ido. differences in ido protein levels between aparalytic and paralytic hosts were evaluated after mice were divided into groups showing either aparalytic (ap) or paralytic symptoms (p) 5 dpi. d ido expression in primary myeloid cells, microglia, and cortical neurons after jev infection. bone marrow-derived dcs (bmdcs), plasmacytoid dcs (pdcs), macrophages (bmdms), microglia, and primary cortical neurons were infected with jev (10 moi for bmdcs, pdcs, bmdms, and microglia; 0.01 moi for neurons) and ido expression was determined by real-time qrt-pcr at the indicated time points. the levels of ido expression were expressed as fold relative to mock-infected cells (0 h). *p < 0.05; **p < 0.01; ***p < 0.001 compared to levels in the indicated groups ido ablation ameliorates je progression. furthermore, we tested the effect of the ido-specific inhibitor 1-methyl-[d]tryptophan (1-mt) on je progression. as suggested by our other results, oral treatment with 1-mt resulted in reduced mortality, delayed neurological disorder, and less change in body weight compared to untreated bl/6 mice (fig. 2c) . therefore, this result strengthens our finding that ido ablation provides enhanced resistance to je. to better understand je progression in ido-ablated mice, we examined viral burden in peripheral lymphoid and cns tissues after jev infection. ido ko mice were observed to contain less amount of virus, with around a tenfold decrease in the spleen and brain compared to levels in bl/6 mice (fig. 2d) . ultimately, these results suggest that ido ablation ameliorates je progression by regulating the viral burden in peripheral lymphoid and cns tissues. cns infiltration by cd11b + ly-6c hi monocytes is a hallmark of cns inflammation caused by neurotropic viral infection [29] . these cells migrate into the infected brain, where they differentiate into dc, macrophage, and microglia populations [30] [31] [32] [33] . although the potential contribution of cd11b + ly-6c hi monocytes to neuroinflammation remains controversial, cns infiltration by cd11b + ly-6c hi monocytes is believed to support their protective role during lethal neuroinflammation [34] [35] [36] [37] . therefore, in order to better understand the amelioration of je in ido ko mice, we examined cns infiltration by leukocytes, including cd11b + ly-6c hi monocytes, during je progression. wild-type bl/6 and ido ko mice contained comparable levels of cd11b + ly-6c hi monocytes and cd11b + ly-6g hi granulocytes in the brain before jev infection. however, the frequency of cd11b + ly-6c hi monocytes gradually increased in the cns of ido ko mice during je progression (fig. 3a) . also, cns infiltration by cd11b + ly-6g hi granulocytes was transiently increased in ido ko mice 3 dpi, after which the ly-6g hi granulocyte frequency was comparable in both bl/6 and ido ko mice 5 dpi. similarly, the accumulated total number of cns-infiltrated ly-6c hi monocytes and ly-6g hi granulocytes was increased in ido ko mice, compared to those of bl/6 mice (fig. 3b) . furthermore, because cd4 + th1, cd8 + , and nk cells may play beneficial roles in controlling je progression [38] [39] [40] [41] , we examined cns infiltration of nk, cd4 + , and cd8 + tcells. as with cns infiltration by ly-6c hi monocytes, cns infiltration by nk, cd4 + , and cd8 + t-cells was observed to gradually increase in ido ko mice compared to levels observed in bl/6 mice (fig. 3c, d) . notably, cd8 + t-cells showed marked infiltration with threefold increased levels in the cns of ido ko mice. with respect to cns inflammation, the expression of cytokines and chemokines within the cns may be required to fully explain encephalitis, because encephalitis caused by neurotropic viruses is indirectly derived from cns degeneration caused by robust immunological responses, such as the uncontrolled secretion of cytokines, including tnf-α, and the resultant activation of microglia and astrocytes [22, 23] . therefore, we examined the expression of tnf-α and cc chemokines in the cns. our results revealed that the expression of cc chemokines was comparable in both bl/6 and ido ko mice, except that the expression levels of tnf-α and ccl2 were modestly decreased in ido ko mice (fig. 3e) . this result implies that cc chemokine expression may not play a role in increased cns infiltration by ly-6c hi monocytes, nk, cd4 + , and cd8 + t-cells. collectively, these results suggest that ido ablation allows for early and increased cns infiltration by myeloid and lymphoid leukocytes, which may mediate the early control of viral replication in the cns. antiviral innate nk cell activation is believed to play an important role in regulating je progression through the control and clearance of jev in extraneural and neural tissues [38] . therefore, in order to characterize the immunological parameters associated with control of jev replication in ido ko mice, we examined and compared nk cell responses in both bl/6 and ido ko mice. because jev was administered intraperitoneally, analysis of the spleen provides insight into how ido modulates the innate immune and inflammatory responses during the early phase of infection. analysis of splenic nk cells revealed that bl/6 mice exhibited a reduction in cd3 − nk1.1 + dx5 + nk cells following jev infection (fig. 4a) , as (see figure on previous page.) fig. 2 blockage of ido enhances resistance to je and reduces viral burden. a levels of l-kynurenine in the sera and brain of bl/6 and ido ko mice. following jev infection, levels of l-kynurenine in the sera and brain homogenates were estimated by hplc at different time points. b susceptibility of idoablated mice to je. bl/6 and ido ko mice (4 to 5 weeks old, n = 25-30) were inoculated i.p. with jev (3.0 × 10 7 pfu) and examined over 15 days for their survival. c enhanced resistance to je by ido inhibition. bl/6 mice were infected i.p. with jev and administered an ido inhibitor (1-mt, 1 and 2 mg per mouse) every day. left graph, curve showing survival rates; middle graph, proportion of mice showing neurological disorders during je progression every 6 h from 4 to 11 dpi; right graph, changes in body weight. changes in body weight were expressed as the mean percentage ± sd of body weight relative to the time of challenge. d viral burden in lymphoid and inflammatory tissues during je. viral burden in the spleen and brain of infected mice was assessed by real-time qrt-pcr at the indicated time points post-infection. the viral rna load was expressed as viral rna copy number per microgram of total rna (n = 5-7). *p < 0.05; **p < 0.01; ***p < 0.001 compared with levels in the indicated groups or in bl/6 control mice if wild-type mice previously showed decreased number of nk cells [24] . however, ido ko mice showed less of a reduction in the number of cd3 − nk1.1 + dx5 + nk cells. consequently, an increase in the total number of splenic nk cells was detected in ido ko mice compared to bl/6 mice. moreover, when the activation of nk cells was evaluated by the production of ifn-γ and granzyme b from nk cells, the frequency and the total number of cd3 − nk1.1 + dx5 + nk cells producing ifn-γ and granyzme b were apparently increased in ido ko mice (fig. 4b, c) . therefore, this result indicates that increased activation of nk cells plays a role in the early control of jev replication, thereby resulting in the amelioration of je progression. in addition to nk cells, adaptive immune responses specific for jev antigen are required for the regulation of je progression through peripheral control of jev replication [38] [39] [40] [41] . furthermore, ido may in some cases affect antigen-specific antibody responses [42, 43] , which contribute to the control of jev dissemination and replication in the brain. our data revealed that ido ablation induced no significant changes in serum igm and igg specific for jev antigen (fig. 5a) . because ido is known to suppress t-cellmediated adaptive immune responses in a range of clinically relevant syndromes, including autoimmune, allergic, and infectious diseases [9, 10] , we also evaluated cd4 + and cd8 + t-cell responses specific for jev antigen in surviving bl/6 and ido-ablated mice 7 dpi. ido ablation resulted in moderately increased jev-specific cd4 + t-cell responses when cd4 + t-cell responses were evaluated by intracellular cd154, ifn-γ, and tnf-α staining in response to stimulation with two epitope-peptides (ns1 132-145 and ns3 563-574 ) derived from jev (fig. 5b) . consistent with this finding, the total number of cd4 + t-cells producing ifn-γ or tnf-α in response to jev epitope stimulation was higher in idoablated mice (fig. 5c) . furthermore, somewhat interestingly, ido-ablated mice displayed markedly increased cd8 + tcell responses with three-to fivefold higher levels, compared to bl/6 mice (fig. 5d) , and consistently contained a higher total number of jev-specific cd8 + t-cells producing the frequency and number of ly-6c hi monocytes and ly-6g hi granulocytes in the cns. the frequency (a) and total number (b) of ly-6c hi monocytes and ly-6g hi granulocytes in the cns were determined by flow cytometric analyses 3 and 5 dpi using vigorous heart perfusion. values presented in the representative dot plots denote the average percentage of the indicated population after gating on cd11b + cells (n = 4-5). c, d accumulated number of nk cells, cd4 + , and cd8 + t-cells in the cns. total accumulated number of nk cells (cd3 − nk1.1 + dx5 + ), cd4 + (cd3 + cd4 + ), and cd8 + (cd3 + cd8 + ) t-cells in the cns were enumerated by flow cytometric analysis 3 and 5 dpi. e the expression of tnf-α and cc chemokines in the cns. the expression levels of tnf-α and cc chemokines were determined by real-time qrt-pcr using total rna extracted from brain tissue 2 dpi. data show the average ± sd of the indicated cell populations derived from at least five mice per group. *p < 0.05; **p < 0.01; ***p < 0.001 compared with levels in the indicated groups ifn-γ or tnf-α in response to a jev cd8 + t-cell epitope (ns4b 215-223 ) (fig. 5e) . these results indicate that the increased cd4 + and cd8 + t-cell responses generated in ido-ablated mice could contribute to the control of je progression during the late stage of infection. cd4 + cd25 + foxp3 + treg cells may contribute to controlling lethal neuroinflammation caused by neurotropic viruses [44] . in contrast, il-17 + cd4 + th17 cells play a critical role in autoimmune and virus-caused immunopathologic diseases by facilitating pathologic consequences through neutrophil recruitment [45, 46] . the generation of cd4 + cd25 + foxp3 + tregs is reciprocally linked with il-17 + cd4 + th17 and ifn-γ + cd4 + th1 cells [47, 48] . because ido may regulate the equilibrium of cd4 + foxp3 + tregs and il-17 + cd4 + th17 cells in inflammatory diseases [49, 50] , we examined the generation of each cd4 + th subset, including cd4 + foxp3 + tregs, il-17 + cd4 + th17, and ifn-γ + cd4 + th1 cells, early during je progression. our results revealed that ido-ablated mice showed no significant alterations in the frequency or number of cd4 + cd25 + foxp3 + tregs, compared to wild-type bl/6 mice (fig. 6a, b) . in contrast, ifn-γ + cd4 + th1 cells were detected at higher levels in ido-ablated mice, and the frequency and number of il-17 + cd4 + th17 cells were not changed by ido ablation (fig. 6c, d) . therefore, this result suggests that the increase in ifn-γ + cd4 + th1 cells in ido-ablated mice contributes in part to the control of je progression during the early stage of infection. myeloid cells, including dcs and macrophages, are the primary target cells of jev infection in the peripheral tissues and function to regulate the spread of virus to distant tissues such as the cns [23] . furthermore, myeloid cells can produce ifn-i via prr recognition upon jev infection, which plays a crucial role in controlling viral replication [51, 52] . additionally, ido ablation appears to regulate viral replication via ifn-i production [21] . because viral loads at the periphery in ido-ablated mice were lower than in wild-type bl/6 mice, we evaluated the contribution of dc subsets (conventional and plasmacytoid) and macrophages to the ifn-i innate immune responses caused by jev infection in the idoablated environment. in order to assess the role of ido in inducing the ifn-i innate response of jev-infected dc subsets and macrophages, bmdcs, pdcs, and macrophages (bmdms) prepared from ido-ablated mice were infected with jev and used to evaluate viral replication and the induction of ifn-i and pro-inflammatory cytokines. interestingly, ido-ablated bmdcs and pdcs, but not bmdms, showed significantly lower jev replication (fig. 7a-c) . notably, bmdcs derived from ido-ablated mice exhibited impaired jev replication throughout the examination period compared to replication in cells from wild-type bl/6 mice. furthermore, the inhibition of jev replication in bmdcs and pdcs derived from idoablated mice was closely associated with enhanced expression of ifn-i (ifn-α/β) following jev infection (fig. 7d) . ido-ablated bmdcs and pdcs, but not bmdms, showed rapid induction of ifn-α/β in response to jev infection compared to levels measured in cells from wild-type bl/6 mice. however, it was curious that pdcs showed less apparent regulation of viral replication than bmdcs, despite pdcs inducing stronger ifn-i innate responses. presumably, this discrepancy is related to cell-intrinsic properties or other mechanisms involved in regulating viral replication. in addition, rapid and increased induction of il-6 and tnf-α mrnas was observed upon jev infection in ido-ablated bmdcs (fig. 7e) . collectively, these results imply that rapid and increased ifn-i innate responses in cd11c + dc subsets (conventional dcs and pdcs) may contribute to the early control of viral replication in the absence of ido. to further characterize ifn-i innate responses in jev-infected dc subsets derived from ido-ablated mice, we also measured the induction levels of isg genes. we specifically focused on pattern recogni). our results revealed that bmdcs and pdcs derived from ido-ablated mice showed rapid and enhanced expression of the stat1, oas1, mx1, mx2, and isg genes with slightly different patterns at 24 h pi, whereas bmdms derived from ido-ablated mice showed either no alteration or a slight decrease in the expression of prrs, ifr3 and irf7, and isg genes (fig. 8) . also, it was interesting that bmdcs and pdcs derived from ido-ablated mice displayed early enhanced induction of prrs (rig-i and mda5) and their transcription factor (irf7) at 24 h pi. this indicates that rapid and increased expression of isg genes in jev-infected bmdcs and pdcs follows the inhibition of jev replication via enhanced expression of ifn-α/β. collectively, ido ablation appears to provide rapid and increased responses of ifn-i innate immunity in myeloid-derived dcs and pdcs upon jev infection, thereby contributing to the amelioration of je through early control of viral replication. microglia cells are cns-resident macrophages that play an important role in regulating the neuroinflammation caused by sterile and non-sterile insults [53] . bystander damage caused by pro-inflammatory mediators released from microglia likely contributes to the exacerbation of je progression. furthermore, because ido-ablated microglia are considered to show stronger inflammatory responses after the virus gains access to the cns, we examined the innate immune responses of primary microglia cells derived from the brain of fetal bl/6 and ido ko mice in response to jev infection. our results revealed that primary microglia recovered from the brain of ido-ablated mice showed similar innate immune responses to jev infection as shown in macrophages derived from bm cells of ido ko mice. ido-ablated microglia were observed to allow jev replication with moderately but not significantly lower levels compared to those of bl/6 mice (fig. 9a) . consistent with this, ido-ablated microglia displayed no apparent enhancement of ifn-i innate responses when the expression of ifn-α/β and isg genes (isg49, isg54, isg56) was examined (fig. 9b, c) . also, no significant differences in the expression of rlrs and transcription factors irf3 and irf7 were observed between microglia derived from ido ko and bl/6 mice (fig. 9d, e) , and both idoablated and wild-type microglia showed comparable expression of pro-inflammatory cytokines tnf-α and il-6 in response to jev infection (fig. 9f ) . therefore, these results indicate that microglia could not provide dominant contribution to the regulation of jev (see figure on previous page.) fig. 5 ido ablation enhances cd4 + and cd8 + t-cell responses specific for jev antigen. a jev e-specific igm and igg response. sera were collected from surviving mice 7 dpi and used in elisa to detect igm and igg levels specific for the jev e protein. the data show the average ± sd of the jev e-specific igm and igg levels derived from surviving mice (n = 6-8). b, c cd4 + t-cell response specific for jev antigen. the splenocytes prepared from surviving mice (n = 4-5) were stimulated with the jev epitope peptides of cd4 + t-cells (ns1 132-145 and ns3 563-574 ) for 12 h. the frequency (b) and absolute number (c) of cd4 + t-cells specific for the jev epitope peptides were determined by intracellular cd154, ifn-γ, or tnf-α staining, combined with surface cd4 staining. d, e cd8 + t-cell response specific for jev antigen. the frequency (d) and absolute number (e) of cd8 + t-cells specific for the jev epitope peptide (ns4b 215-223 ) were determined by intracellular ifn-γ or tnf-α staining after an 8-h stimulation with peptide. values in the representative dot plots denote the average percentage of the indicated cell population, and the bar charts show the average ± sd of the values derived from at least four mice per group. *p < 0.05; **p < 0.01; ***p < 0.001 compared with levels in the indicated groups dissemination and je progression in the cns under an ido-ablated environment. neurons are the main target cell of jev replication within the cns, and their death is a key factor in the pathogenesis and neurological sequelae of jev [23] . furthermore, neuron cells have also been shown to produce antiviral ifn-i in response to viral infection and are consequently involved in controlling viral replication in the cns [54, 55] . therefore, we examined viral replication and ifn-i innate immune responses in primary cortical neuron cells generated from wild-type bl/6 and ido-ablated mice after jev infection. consistent with the results obtained from bmdcs and pdcs, primary cortical neurons derived from ido-ablated mice showed transiently reduced jev replication (fig. 10a) . this transient reduction of jev replication in ido-ablated neurons was associated with significantly increased induction of ifn-i (ifn-α/β), relative to the levels measured in cells from wild-type bl/6 mice (fig. 10b) . also, it seemed that the expression of antiviral isg genes (isg49, isg54, isg59) in ido-ablated neurons followed ifn-i innate responses and the transient reduction in viral replication (fig. 10c) , whereas the expression levels of transcription factors (irf3, irf7), but not prrs (rig-i, mda5), were decreased by jev infection (fig. 10d, e) . therefore, these results suggest that the ablation of ido could provide enhanced ifn-i innate immune responses in neuron cells to regulate the spread of jev in the cns. the frequency and number of cd4 + foxp3 + tregs in the spleen of ido-ablated mice. the frequency (a) and absolute number (b) of cd4 + foxp3 + treg cells in the spleen of wild-type and ido ko mice were determined by flow cytometric analysis 5 dpi. the values in the representative dot plots show the average percentage of cd25 + foxp3 + cells in cd4 + t-cells; the bar charts denote the average number ± sd of cd4 + foxp3 + tregs in the spleen derived from at least four mice per group. c, d the frequency and number of ifnγ + cd4 + th1 and il-17 + cd4 + th17 cells in the spleen of ido-ablated mice. the frequency and number of ifn-γ + cd4 + th1 and il-17 + cd4 + th17 cells were determined by intracellular cytokine staining of the splenocytes prepared from wild-type and ido ko mice 5 dpi in response to pma + ionomycin stimulation. the values in the representative dot plots show the average percentage of the indicated cell populations after gating on cd4 + t-cells; the bar charts denote the average number ± sd of ifn-γ + cd4 + th1 and il-17 + cd4 + th17 cells in the spleen derived from at least four mice per group. **p < 0.01 compared with levels in the indicated groups ido ablation in hsc-derived leukocytes alleviates je via enhancement of the ifn-i innate response our results support the idea that ido ablation ameliorates je progression by provoking potent antiviral ifn-i innate responses in myeloid-derived dcs and pdcs at the periphery. therefore, we were interested in confirming whether myeloid cells derived from hematopoietic stem cells (hscs) play a dominant role in regulating je progression in ido-ablated mice via the induction of enhanced ifn-i innate responses. to this end, we used a bm chimeric model of wild-type bl/6 and ido-ablated mice. in support of our hypothesis, our results revealed that myeloid cells derived from hscs played a dominant role in conferring amelioration of je in the ido-ablated environment. specifically, wild-type bl/6 recipients of ido ko bm donor cells (ko-wt) showed enhanced resistance to je, compared to the ido ko recipients of wild-type bl/6 bm donor cells (wt-ko) and wild-type bl/6 recipients of wild-type bl/6 bm donor cells (wt-wt) (fig. 11a) . furthermore, ko-wt and ko-ko bm chimeric models showed less change in body weight after jev infection compared to the other bm chimeric models (fig. 11b) . also, potent and rapid ifn-i innate immune responses were observed in ko-wt and ko-ko bm chimeric models compared to those observed in the wt-wt bm chimeric model, as evaluated by the determination of serum ifn-β (fig. 11c) . this result indicates that myeloid cells derived from hscs of ido-ablated mice play a dominant role in the ifn-i innate response in ido ko hosts. collectively, it appears that the ablation of ido in myeloid cells derived from hscs has an important role in ameliorating je by inducing a potent and rapid ifn-i innate immune response. in this study, ido expression from myeloid and neuron cells located in extraneural and neural tissues during je progression appears to be closely associated with clinical signs such as neurological disorders. furthermore, we demonstrated that inhibition of ido activity through genetic ablation or use of an ido inhibitor (1-mt) enhanced the resistance to je progression induced by jev infection. in our mechanistic studies on the role of ido in regulating je fig. 8 the expression of rlr, irf, and isg genes in bmdcs, bmdms, and pdcs following jev infection. bmdcs, bmdms, and pdcs recovered from wt and ido ko mice were infected with jev at a moi of 10 and used to analyze the induction of irf, isg, and rlr genes at 24 and 48 h pi. the expression of each irf, isg, and rlr gene was normalized to that of β-actin after determining the mrna levels by real-time qrt-pcr and displayed as the average of at least four independent samples, according to the indicated color on a log 2 scale (see figure on previous page.) fig. 7 ifn-i innate immune responses of ido-ablated myeloid-derived cells after jev infection. primary bone marrow-derived conventional dcs (bmdcs), plasmacytoid dcs (pdcs), and macrophages (bmdms) recovered from bl/6 and ido ko mice were infected with jev at mois of 0.1, 1.0, and 10 for viral replication and 10 for cytokine expression. jev replication and the expression of cytokines and ifn-α/β were evaluated by real-time qrt-pcr using extracted total rna. a-c jev replication in bmdcs, bmdms, and pdcs. d, e ifn-α/β, il-6, and tnf-α expression in bmdcs, pdcs, and bmdms. the bar charts show the average ± sd of the values derived from bmdcs, bmdms, and pdcs assayed in quadruplicates. *p < 0.05; **p < 0.01; p < 0.001 compared with levels in the indicated groups progression, ido-ablated mice showed early and increased cns infiltration by myeloid cells (ly-6c hi monocytes and ly-6g hi granulocytes) and lymphoid cells (cd3 − nk1.1 + dx5 + nk, cd4 + , and cd8 + t-cells), which were associated with a reduced viral burden in the cns. ido ablation increased the activity of cytolytic effector cells following jev infection, due to the enhancement of ifn-γ or granzyme b-producing nk cells and jev-specific cd8 + t-cell responses in ido-ablated mice. in addition, the enhanced production of ifn-γ from cd4 + th1 cells in response to jev antigen might contribute to early viral clearance from extraneural and neural tissues of ido-ablated mice. also, somewhat intriguingly, our data revealed that potent and rapid ifn-i innate immune responses in cd11c + dcs and neuron cells following jev infection could effectively regulate the spread of jev in ido-ablated mice. this finding was supported by the results that ido ablation in myeloid cells derived from hscs played a dominant role in ameliorating je and that ido-ablated hsc-derived leukocytes were major contributors to ifn-i innate responses in the host. collectively, our data suggest that the ablation of ido activity with specific inhibitors, such as 1-mt, could be a valuable therapeutic tool for the treatment of je. ido is considered a negative regulator of the immune system because ido activity in apcs, including dcs and macrophages, is a potent mechanism for inducing immunosuppression and tolerance. therefore, it is probable that the attenuation of immunosuppressive ido activity leads to enhanced innate and adaptive immune responses for viral clearance. indeed, the inhibition of ido activity with a specific inhibitor resulted in enhanced cd4 + th1type and cd8 + t-cell responses specific to the influenza virus [20] . furthermore, the inhibition of ido activity reportedly modifies the immunodominance hierarchy by increasing the number of cd8 + t-cells specific to a subdominant epitope derived from the influenza virus. the ablation of ido also suppresses viral replication in retrovirus-infected mice through ifn-i production [21] . these findings strongly support our data that ido ablation ameliorates je progression through enhanced type i/ ii ifn innate and adaptive immune responses. although the in vivo role of ido after viral infection has been investigated in a few studies using murine infection models, the regulatory role of ido in neuroinflammation caused by neurotropic viruses, such as jev, has not been addressed to date. to the best of our knowledge, our findings provide the first evidence that ido inhibition could regulate the in vivo progression of viral encephalitis caused by neurotropic viruses such as jev and wnv. the molecular pathogenesis of je remains unclear, but je is considered an immunopathological disease since uncontrolled over-activation of microglia/glia and infiltrated leukocytes after cns invasion of jev drives neurological disorders [22, 23] . although cns infiltration by peripheral innate and adaptive immune cells appears to play a critical role in host defense against infections with neurotropic viruses such as jev, control of cns infiltration is also required because excessive or inappropriate cns infiltration can cause profound damage. therefore, the outcome of je pathogenesis appears to depend on the complicated interplay between virus-induced tissue damage and host immune response against jev antigens. cns infiltration by cd11b + ly-6c hi monocytes is a hallmark of neuroinflammation caused by neurotropic viruses. these cd11b + ly6c hi monocytes migrate into the inflamed cns, where they differentiate into dcs, macrophages, and microglia to regulate neuroinflammation [30] [31] [32] [33] [34] [35] [36] [37] . although conflicting roles have been postulated for ly-6c hi monocytes in cns inflammation, cns infiltration by cd11b + ly-6c hi monocytes is essential in the control of neuroinflammation caused by neurotropic viruses, which supports their protective role during cns inflammation [34] [35] [36] [37] . notably, the differentiation state of ly-6c hi monocytes that infiltrate into the cns appears to affect the progression of neuroinflammation caused by various insults [56] [57] [58] . supportively, ido ablation was observed to lead to enhanced cns infiltration of cd11b + ly-6c hi monocytes, which may contribute to a beneficial outcome in je. furthermore, early and increased infiltration of nk, cd4 + , and cd8 + t-cells in the cns of ido-ablated mice appears to play a role in reducing the viral burden, because infiltration of nk, cd4 + th1, and cd8 + t-cells producing ifn-γ is required for early clearance of the virus from the cns [38] [39] [40] [41] . after peripheral introduction of jev via mosquito bites, jev initially replicates in its primary target cells, including dcs and macrophages, at the periphery and subsequently gains entry into the cns through the bbb. presumably, jev-specific cd4 + th1 and cd8 + t-cell responses generated in the peripheral lymphoid tissues of ido-ablated mice would likely provide more effective control of viral replication, thereby reducing the amount of virus supplied from the periphery. in support of this notion, our data revealed that the viral burden in the extraneural and neural tissues of ido-ablated mice were lower than those in wild-type bl/6 mice. the depletion or adoptive transfer of specific cell populations of nk and cd8 + t-cells was previously reported to provide no significant contribution to host survival and viral clearance [38] . furthermore, co-transfer of immune cd4 + and cd8 + tcells, but not individual transfer of either t-cell subpopulation, significantly contributed to a favorable je outcome and promoted host survival [40] . these facts suggest the possibility that enhancement of broad immunity, including nk, cd4 + th1, and cytotoxic cd8 + t-cells, in idoablated mice accounts for the effective regulation of early viral clearance in extraneural and neural tissues, thereby providing protection against je progression without tissue injury. however, ido ablation did not result in any significant change in the humoral immune responses specific for jev antigen. the effects of ido on the humoral immune response appear to be variable, depending on the experimental model used [42, 43] . because b-cell-deficient (μmt) mice uniformly succumbed to viral encephalitis caused by flaviviruses in a previous study, one obvious conclusion is that an early and sustained humoral immune response is absolutely essential to surviving infection with jev and other related flaviviruses [59] . this notion suggests a limitation that ido inhibition may not provide the perfect protection against je progression due to the lack of substantial enhancement in the humoral response against jev antigen. thus, further studies on how to enhance the humoral immune response are needed in the context of ido inhibition. the requirement of ifn-γ in the recovery from infections with flaviviruses has been shown to be variable. ifn-γ provides an early protective immune response against a virulent north american isolate of wnv [60] and mouseadapted strains of dengue virus [61, 62] , whereas ifn-γ is dispensable in the control of infection with less virulent strains of wnv [63] or of yellow fever virus [64, 65] . additionally, ifn-γ shows only a modest protective role against murray valley encephalitis [66] . similarly, in the je model, fig. 11 ido ablation in hsc-derived leukocytes alleviates je via enhancement of the ifn-i innate response. bm cells from wt or ido ko mice were grafted to lethally irradiated wt or ido ko recipient mice, which were then infected with jev (3.0 × 10 7 pfu). a susceptibility of ido ko bm chimeric models to je. infected recipient mice (n = 12) were examined over 18 days to determine the survival rate. b changes in body weight. data are expressed as the average percentage of body weight relative to the time of challenge. c systemic ifn-β levels in an ido ko bm chimera. the amount of serum ifn-β was determined by elisa at the indicated time points. *p < 0.05; **p < 0.01 compared with levels in wt-wt bm chimera 48 h pi. #p < 0.05; ##p < 0.01 compared with levels in wt-wt bm chimera 72 h pi il-12-mediated induction of ifn-γ has been reported to result in suppressed protective immunity [67] , whereas ifn-γ was associated with a beneficial effect on the outcome of je [38] . our data favor the latter result, showing a beneficial role of ifn-γ in je progression, because ido ablation markedly increased ifn-γ-producing nk, cd4 + th1, and cd8 + t-cells. ifn-γ is believed to play diverse roles in infectious diseases, including the activation and polarization of cd4 + th cells, upregulation of fas in infected target cells, upregulation of mhc i and ii-restricted ag-presentation pathways, macrophage activation, and direct antiviral activity that overlaps with activities triggered by ifn-i [68] . also, ifn-γ produced from cd4 + th1 and cd8 + t-cells in ido-ablated mice is believed to induce the maturation of ly-6c hi monocytes, which may contribute to better je outcomes [69, 70] . however, ifn-γ-producing nk cells do not appear to significantly contribute to host survival, because nk-cell-depleted mice showed no change in viral burden or survival [38] . furthermore, flaviviruses including wnv exhibit immune escape from nk cell attack via the upregulation of mhc-i in infected cells [71] . in contrast, antigen-specific cd8 + t-cell cytolytic activity on infected target cells is thought to play a crucial role in disease recovery, given that depletion of cd8 + t-cells resulted in an increased viral burden in the cns [40] . supportively, our data revealed that ido ablation provided marked enhancement of cd8 + t-cells producing ifn-γ and tnf-α upon jev ag stimulation. in addition, ido ablation induced an increase in cd4 + th1 cells producing ifn-γ at the early stage, but il-17 + cd4 + th17 and cd4 + foxp3 + tregs were not altered by the ablation of ido. collectively, these findings suggest that ifn-γ produced from cd4 + th1 and cd8 + t-cells play an important role in regulating je progression in ido-ablated mice. the role of ido in antiviral activity has been postulated in different in vitro experiments. for example, ido activity in astrocytes is induced by the tlr3 ligand polyinosinicpolycytidylic acid, which is involved in antiviral function via the exhaustion of the essential amino acid l-trp in the environment [72] . this finding is inconsistent with our data, but it is notable that this finding was derived from an in vitro model. the intriguing result in the present study was that ido-ablated cd11c + dcs, including conventional and plasmacytoid dcs, showed potent and rapid ifn-i innate immune responses to jev infection. also, ko-ko and ko-wt bm chimeric models showed systemic ifn-β production at levels higher than in wt-wt and wt-ko bm chimeric models. this supports the notion that myeloid cells derived from hscs, including cd11c + dcs, may dominantly contribute to the rapid ifn-i innate immune response. this result is strengthened by the finding that ido ko and 1-mt-treated mice showed induction of a markedly increased ifn-i innate response in the spleen following retrovirus infection [21] . conventional and plasmacytoid dcs exhibited potent and rapid ifn-i innate responses with different dynamic patterns in the expression of prrs, transcription factors, and isg genes following jev infection. these different ifn-i innate responses in both types of cd11c + dcs are thought to be derived from the intrinsic properties of the specific cell type. although we did not investigate the detailed mechanism by which idoablated cd11c + dcs displayed a potent ifn-i response to jev infection, our data suggest the involvement of prrs (rig-i, mda5) and transcription factor irf7 because the expression levels of rig-i, mda5, and irf-7 were rapidly enhanced in ido-ablated cd11c + dcs. also, considering that only a small fraction (10-20 %) of myeloid-derived cells are infected by jev [73] , uninfected myeloid-derived cells are thought to contribute substantially to the induction of antiviral isgs through early stimulation of the stat1 transcription factor which induces isg49, isg54, and isg56 [24] . also, it is worth noting that primary cortical neuron cells derived from ido-ablated mice showed potent ifn-i innate responses resulting in delayed replication of jev. although the induction patterns of prrs and their transcription factors in neuron cells differed from those of cd11c + dcs following jev infection, the induction of isgs in ido-ablated neurons definitely followed potent ifn-i innate immune responses, such as ifn-αβ production. however, because cortical neurons are relatively insensitive to the antiviral effects of ifn-i [74] , the regulation of viral replication in primary cortical neurons by ifn-i innate responses may ultimately show some limitations. indeed, our data indicating that primary cortical neuron cells derived from ido ko mice showed transient inhibition of viral replication support this speculation. therefore, further studies are needed to define the mechanistic and functional intermediates that link ido to the regulation of ifn-i innate immune responses in different cell types. je pathogenesis in the murine model may be affected by the route of administration, dosage, and strain of the virus, and the virus propagation conditions used [23] . ido was expressed with different dynamic patterns in conventional and plasmacytoid dcs, macrophages, and neuron cells upon jev infection. thus, it is thought that je pathogenesis may also be affected by how the innate responses of specific cell types are exhibited after jev infection. rapid ifn-i/ii innate immune responses are considered more crucial in the control of je progression than adaptive tcell responses, which take time to develop. considering that ido ablation provided early and enhanced ifn-i/ii innate responses and subsequent adaptive t-cell response in the host, the inhibition of ido may be a valuable tool for regulating je progression. the inhibition of ido ameliorates je progression via rapid enhancements in ifn-i/ii innate immune responses and molecular epidemiological studies of veterinary arboviral encephalitides immunopathology of flavivirus infections zoonotic encephalitides caused by arboviruses: transmission and epidemiology of alphaviruses and flaviviruses overview: japanese encephalitis review of climate, landscape, and viral genetics as drivers of the japanese encephalitis virus ecology flaviviruses, an expanding threat in public health: focus on dengue, west nile, and japanese encephalitis virus japanese encephalitis: status of surveillance and immunization in asia and the western pacific west nile virus activity-human disease cases reported the role of indoleamine 2, 3-dioxygenase in immune suppression and autoimmunity. vaccines(basel) tryptophancatabolizing enzymes-party of three the kynurenine pathway is involved in bacterial meningitis activation of the kynurenine pathway and increased production of the excitotoxin quinolinic acid following traumatic brain injury in humans allogeneic mesenchymal stem cells inhibited t follicular helper cell generation in rheumatoid arthritis performance-enhanced mesenchymal stem cells via intracellular delivery of steroids functional expression of indoleamine 2,3-dioxygenase by murine cd8 alpha(+) dendritic cells flavivirus infection induces indoleamine 2,3-dioxygenase in human monocyte-derived macrophages via tumor necrosis factor and nf-kappab ifn-gamma amplifies il-6 and il-8 responses by airway epithelial-like cells via indoleamine 2,3-dioxygenase toxicology and pharmacokinetics of 1-methyl-d-tryptophan: absence of toxicity due to saturating absorption inhibition of indoleamine 2,3-dioxygenase (ido) enhances elimination of virus-infected macrophages in an animal model of hiv-1 encephalitis indoleamine 2,3-dioxygenase (ido) activity during the primary immune response to influenza infection modifies the memory t cell response to influenza challenge the absence of ido upregulates type i ifn production, resulting in suppression of viral replication in the retrovirus-infected mouse an insufficient anti-inflammatory cytokine response in mouse brain is associated with increased tissue pathology and viral load during japanese encephalitis virus infection flavivirus encephalitis: pathological aspects of mouse and other animal models distinct dictation of japanese encephalitis virus-induced neuroinflammation and lethality via triggering tlr3 and tlr4 signal pathways more rapid method for simultaneous measurement of tryptophan and kynurenine by hplc direct access to cd4+ t cells specific for defined antigens according to cd154 expression live-cell assay to detect antigenspecific cd4+ t-cell responses by cd154 expression isolation, purification, and culture of primary murine microglia cells inflammatory monocytes and the pathogenesis of viral encephalitis the plasticity of inflammatory monocyte responses to the inflamed central nervous system direct induction of ramified microglia-like cells from human monocytes: dynamic microglial dysfunction in nasu-hakola disease molecular and cellular neuroinflammatory status of mouse brain after systemic lipopolysaccharide challenge: importance of ccr2/ccl2 signaling ly6c + "inflammatory monocytes" are microglial precursors recruited in a pathogenic manner in west nile virus encephalitis chemokine receptor ccr2 is critical for monocyte accumulation and survival in west nile virus encephalitis west nile virus neuroinvasion and encephalitis induced by macrophage depletion in mice recruited inflammatory monocytes stimulate antiviral th1 immunity in infected tissue lack of ccr2 results in increased mortality and impaired leukocyte activation and trafficking following infection of the central nervous system with a neurotropic coronavirus cytolytic effector pathways and ifn-gamma help protect against japanese encephalitis th1 immune response takeover among patients with severe japanese encephalitis infection pivotal role of antibody and subsidiary contribution of cd8+ t cells to recovery from infection in a murine model of japanese encephalitis impaired t helper 1 function of nonstructural protein 3-specific t cells in japanese patients with encephalitis with neurological sequelae suppression of humoral immune response to hepatitis b surface antigen vaccine in balb/c mice by 1-methyl-tryptophan co-administration deficiency of indoleamine 2,3-dioxygenase enhances commensal-induced antibody responses and protects against citrobacter rodentium-induced colitis tregs control the development of symptomatic west nile virus infection in humans and mice stat4 controls gm-csf production by both th1 and th17 cells during eae t-cell-mediated regulation of neuroinflammation involved in neurodegenerative diseases il-1beta promotes th17 differentiation by inducing alternative splicing of foxp3 th17 and th17/treg ratio at early hiv infection associate with protective hiv-specific cd8(+) t-cell responses and disease progression the regulation of the treg/th17 balance by mesenchymal stem cells in human systemic lupus erythematosus ido upregulates regulatory t cells via tryptophan catabolite and suppresses encephalitogenic t cell responses in experimental autoimmune encephalomyelitis leukocytederived ifn-alpha/beta and epithelial ifn-lambda constitute a compartmentalized mucosal defense system that restricts enteric virus infections the yin and yang of viruses and interferons systemic inflammation and microglial activation: systematic review of animal experiments neurons produce type i interferon during viral encephalitis soluble amyloid triggers a myeloid differentiation factor 88 and interferon regulatory factor 7 dependent neuronal type-1 interferon response in vitro activation of invariant nkt cells in early phase of experimental autoimmune encephalomyelitis results in differentiation of ly6chi inflammatory monocyte to m2 macrophages and improved outcome induction of inhibitory central nervous system-derived and stimulatory blood-derived dendritic cells suggests a dual role for granulocytemacrophage colony-stimulating factor in central nervous system inflammation the blood-brain barrier induces differentiation of migrating monocytes into th17-polarizing dendritic cells b cells and antibody play critical roles in the immediate defense of disseminated infection by west nile encephalitis virus gamma interferon plays a crucial early antiviral role in protection against west nile virus infection ifngamma production depends on il-12 and il-18 combined action and mediates host resistance to dengue virus infection in a nitric oxide-dependent manner interferondependent immunity is essential for resistance to primary dengue virus infection in mice, whereas t-and b-cell-dependent immunity are less critical cd8(+) t cell-mediated immune responses in west nile virus (sarafend strain) encephalitis are independent of gamma interferon yellow fever virus encephalitis: properties of the brainassociated t-cell response during virus clearance in normal and gamma interferon-deficient mice and requirement for cd4+ lymphocytes a mouse model for studying viscerotropic disease caused by yellow fever virus infection role of type i and type ii interferon responses in recovery from infection with an encephalitic flavivirus suppression of immune response and protective immunity to a japanese encephalitis virus dna vaccine by coadministration of an il-12-expressing plasmid interferons: success in anti-viral immunotherapy ifn-gamma, as secreted during an alloresponse, induces differentiation of monocytes into tolerogenic dendritic cells, resulting in foxp3+ regulatory t cell promotion timing and intensity of exposure to interferon-gamma critically determines the function of monocyte-derived dendritic cells mhc class i up-regulation by flaviviruses: immune interaction with unknown advantage to host or pathogen astrocyte indoleamine 2,3-dioxygenase is induced by the tlr3 ligand poly(i:c): mechanism of induction and role in antiviral response impaired cross-presentation of cd8alpha + cd11c + dendritic cells by japanese encephalitis virus in a tlr2/ myd88 signal pathway-dependent manner pkr and rnase l contribute to protection against lethal west nile virus infection by controlling early viral spread in the periphery and replication in neurons challenges in the discovery of indoleamine 2,3-dioxygenase 1 (ido1) inhibitors research progress of indoleamine 2,3-dioxygenase inhibitors this study was supported by a national research foundation of korea (nrf) grant funded by the korean government (misp) (2013r1a4a1069486). the funder had no role in study design, data collection and analysis, the decision to publish, or preparation of the manuscript. cns infiltration by ly-6c hi monocytes. also, ido ablation may accelerate viral clearance from the host at a later stage via the enhancement of jev-specific ifn-γ-producing tcell responses. therefore, these results suggest that inhibition of ido activity by specific inhibitors [75, 76] may be a promising tool for therapeutic and prophylactic strategies against je. the authors declare that they have no competing interests.authors' contributions sbk and jyc conceived the study and discussed the data with ske. eu, amp, and fmah conceived the study and contributed to the experimental design. jh, syp, jhl, and kk contributed to the reagent/materials/analysis and tools and provided critical conceptual guidance. sbk and ske wrote the paper with contributions from all authors. all of the authors reviewed and approved the final version of the manuscript.• we accept pre-submission inquiries • our selector tool helps you to find the most relevant journal submit your next manuscript to biomed central and we will help you at every step: key: cord-007796-zggk0x2q authors: lindemans, caroline a.; kimpen, jan l. l. title: the immune response to viral lower respiratory tract infection date: 2005 journal: hot topics in infection and immunity in children ii doi: 10.1007/0-387-25342-4_4 sha: doc_id: 7796 cord_uid: zggk0x2q viruses are responsible for the majority of respiratory infections in childhood,causing considerable morbidity and mortality. it is estimated that in the united states approximately $ 652 million per year is spent on medical costs for respiratory syncytial virus (rsv) related disease alone (paramore et al., 2004). viruses cause a variety of respiratory diseases in children from the common cold to life-threatening pneumonia and bronchiolitis. the host reacts to a viral infection with a combination of innate and adaptive immune mechanisms, usually resulting in the clearance of the virus and clinical recovery. however, there is an accumulating evidence for a number of viral infections that the host immune response actually enhances disease in the course of clearing virus from the infected organs. interestingly, the effectiveness of the immune response seems to be dependent on the age and probably genetic background of the child. this has important implications for treatment as well as vaccine development. viruses are responsible for the majority of respiratory infections in childhood, causing considerable morbidity and mortality. it is estimated that in the united states approximately $ 652 million per year is spent on medical costs for respiratory syncytial virus (rsv) related disease alone (paramore et al., 2004) . viruses cause a variety of respiratory diseases in children from the common cold to life-threatening pneumonia and bronchiolitis. the host reacts to a viral infection with a combination of innate and adaptive immune mechanisms, usually resulting in the clearance of the virus and clinical recovery. however, there is an accumulating evidence for a number of viral infections that the host immune response actually enhances disease in the course of clearing virus from the infected organs. interestingly, the effectiveness of the immune response seems to be dependent on the age and probably genetic background of the child. this has important implications for treatment as well as vaccine development. viral infections play an important role in both childhood and adult asthma. they might be instrumental in the inception of asthma and are associated with the majority of exacerbations in asthmatic individuals (johnston et al., 1995; bont et al., 2000) . in respect to the role of viruses in the pathogenesis of acute and chronic airway disease in children, it is of utmost importance that we gain a proper understanding of the underlying mechanisms involved in order to design effective therapeutic and preventive strategies. although viral respiratory tract infections are considered to be mainly pediatric diseases, there is an increasing acknowledgement of their pathogenic potential in the immunocompromised host of all age groups and in the elderly. causative agent is not identified. when an upper respiratory tract infection in an infant progresses to lower respiratory tract disease, bronchiolitis and pneumonia are most common. both disease entities are hard to differentiate and no clinically relevant differences with regard to outcome have been identified (van woensel et al., 2003) . rsv belongs to the paramyxoviridae family and the genus of pneumovirus. it is an enveloped unsegmented single-stranded rna-virus of which two subtypes are known (a and b) . a clear relationship between subtype and disease severity has not been established (kneyber et al., 1996) . since rsv infection does not lead to complete immunity, reinfection is common. immaturity of the immune system during initial infection seems to be the main cause of incomplete memory-response although an as yet undefined mechanism of partial immune evasion by rsv cannot be ruled out . recently, it was suggested that rsv could cause persistent infection or latency (dakhama et al., 1997; schwarze et al., 2004) , although the significance of this is not clear. rsv affects 70% of infants in the first year of life, and by the age of two nearly all children have been infected. (figure 4 .1) it is likely that a specific balance and timeframe of changes in air temperature and humidity are responsible for the well-defined yearly winter outbreaks of rsv (stensballe et al., 2003) . in most infants as well as in older children and adults, rsv is the cause of upper respiratory tract infection with mild symptoms. however, in the very young, the infection spreads to the lower respiratory tract in approximately 40% of cases. one to three percent of infants develop bronchiolitis or pneumonia requiring hospitalization, with a considerable number requiring mechanical ventilatory support. apart from the obvious respiratory symptoms, very young children frequently present with atypical symptoms such as impaired feeding, vomiting, lethargy, and apnea (kneyber et al., 1998) . several risk factors for more severe disease have been identified, including age less than 6 weeks, prematurity, pre-existent cardiorespiratory disease and immunological impairment (bont and kimpen, 2002) . respiratory symptoms are directly related to airway pathology. necrosis of the airway epithelium is a key phenomenon resulting in sloughing of the epithelial cells. together with a dramatic influx of inflammatory cells into the airways and increased mucus production, this leads to the formation of copious secretions that block the small airways. mucosal edema and bronchospasm through irritation of subepithelial nerve endings further compromise airway diameter. although antibiotics are prescribed for up to 50% of children with lower respiratory tract infections, proof of bacterial superinfection is only found in a minority of patients and the role of this event in the pathogenesis of severe disease remains controversial (purcell and fergie, 2002; bloomfield et al., 2004) . on the other hand, it has been demonstrated that rsv infection of airway epithelial cells in vitro enhances adherence of s. pneumoniae (hament et al., 2004) . the influenza virus belongs to the orthomyxoviridae family and is an enveloped segmented single-stranded rna (ssrna) virus (table 4 .1). the structural proteins of the virion are encoded by separate gene segments and include three viral rna polymerases, nucleoprotein, matrix, and the hemagglutinin (ha) and neuraminidase (na) surface glycoproteins. on the basis of their nucleocapsid and matrix protein antigens, the influenza viruses are divided into three distinct immunological types (a, b, and c). although all three influenza viruses cause respiratory disease in humans, only a and b are known to cause epidemics. induction of a memory-response results in long-lasting immunity and it is the antigenic variation that is responsible for frequent reinfection with the virus. the most antigenic variation is seen in the virus that infects both animals and humans, influenza a. fourteen subtypes of hemagglutinin (h1-h14) and nine types of neuraminidase (n1-n9) are circulating in nature. the segmentation of the genome makes exchange of genetic material between subtypes possible, resulting in the structural changes observed in influenza, which has caused pandemics in the past. when two different subtypes of influenza a virus infect the same cell, major changes (antigenic shifts) can occur through rearrangement of genetic segments from both infecting viruses. minor changes (antigenic drifts) in na and/or ha proteins occur through accumulation of point mutations, and provide a mechanism for the virus to escape protective antibodies and cause respiratory symptoms every year. influenza virus epidemics are difficult to separate in time from rsv epidemics, and the diseases caused by both viruses can also be difficult to differentiate (zambon et al., 2001) . (figure 4 .1) compared to other viruses, morbidity caused by influenza is high in all age groups. children with influenza infection of the respiratory tract are more likely to present with fever. infants aged less than 6 months and older children with an impaired immune system, or other serious health problems, have a higher risk of hospitalization and mortality. subclinical infections with influenza in children are common, suggesting children can be an important reservoir and source of transmission. yearly updated vaccines are available and effective, and recently it has been proposed to extend the current recommendations to children less than 2 years of age, children with recurrent acute otitis media or respiratory tract infections, and healthy children attending day-care centers or elementary schools (principi and esposito, 2004 ). adenoviruses, belonging to the adenoviridae family and the genus mastadenovirus, are a group of dna-viruses of which at least 47 serotypes are known. for lower respiratory disease, subtypes 1, 2, 3, 4, 5, 7, and 21 are most important. it is an icosahedral capsid virus with extruding fiber proteins, which are required for viral entry to epithelial cells (howitt et al., 2003) . although human adenoviruses are ubiquitous, and cause primary infection in the first year of life, there is geographical variation in the distribution of serotypes and in the association of serotypes with different age groups. in europe, adenovirus is the cause of infection in approximately 5% of hospitalized patients with viral lower respiratory tract disease. however, in some south american and asian countries, adenovirus is the second most prevalent pathogen for acute lower respiratory tract infection in children after rsv (carballal et al., 2002) . although adenovirus infections in general occur the whole year round, respiratory adenovirus infections are most common during late winter, spring, and early summer. adenovirus type 7, acquired by inhalation, has been associated with more severe lower respiratory tract disease (larranaga et al., 2000) . subtype-specific immunity occurs. however, some types are capable of establishing persistent asymptomatic infections in tonsils, adenoids, and intestines of infected hosts, and shedding can occur for months or years. children under 6-years old. though it is the causative agent of similar disease entities, hospitalizations occur four times less frequently than for rsv infections (hall, 2001) . two subtypes are clinically important respiratory pathogens in children, piv-1 and piv-3. piv-1 is the main cause of croup in 2-6-year olds, while piv-3 is responsible for parainfluenza bronchiolitis in children under 6-months old. piv-4 only causes mild upper respiratory tract infections. because of acute narrowing of the subglottic region of the larynx, moderate to severe croup may require emergency management with systemic or inhaled corticosteroids, which are effective in improving stridor in a few hours (cetinkaya et al., 2004) . the hallmark cytopathic effect of acute infection with piv-3 is comparable to that of rsv with extensive cell fusion resulting in syncytium formation. for fusion to occur, two piv glycoproteins are required, including the hemagglutinin-neuraminidase (hn) glycoprotein interacting with host cell sialic acid receptor and the viral fusion (f) glycoprotein. rhinovirus infections account for the largest number of respiratory tract infections in children. however, rhinovirus infections produce mild symptoms compared to rsv, piv, and influenzavirus. most of the symptoms caused by rhinovirus are confined to the upper respiratory tract. although present in the community the whole year round, rhinovirus infections peak at the onset of fall, which is probably related to schools starting after summer break. rhinoviruses can cause severe lower respiratory tract infection (guittet et al., 2003; papadopoulos, 2004) and in immunocompromised patients, life-threatening pneumonia. rhinovirus is a positive-stranded rna-virus belonging to the picornavirus family and over 100 serotypes exist, making it difficult to develop an effective vaccine. rhinovirus subtypes have been divided into a major and minor group with respect to the receptor used for cell entry (table 4 .1). major group rhinoviruses use epithelial intracellular adhesion molecule l (icam-1) for cell entry, while minor group viruses bind to the low-density lipoprotein (ldl) receptor. during rhinovirus infection, a predominant granulocyte and monocyte recruitments are observed. while specific antibody production occurs, it is probably not required for viral clearance, although neutralizing antibodies can provide some temporary protection against rhinovirus reinfection (van kempen et al., 1999) . icam-1 blocking antibodies have also been utilized and have been shown to decrease inflammation in vitro. there are, however, indications that rhinovirus can adapt to this with changes in receptor usage (reischl et al., 2001 ). in 2001, a new respiratory virus was identified in the netherlands causing infections similar to rsv in children (van den hoogen et al., 2001) . the reported incidence rate of human metapneumovirus (hmpv) infection in children with acute respiratory symptoms varies between 4% and 16%, of which three-quarters occur in children less than 1-year old (williams et al., 2004) . by the age of 5 years, approximately 70% of children have developed antibodies to hmpv. hmpv very much resembles rsv in its clinical spectrum, varying from coryza to bronchiolitis and pneumonia. however, hmpv is less likely to cause pneumonia than rsv and influenza virus. children with hmpv infection present less frequently with atypical symptoms such as vomiting, and on physical examination, rales and wheezing are found less often. co-infection with rsv and hmpv does occur and has been suggested to result in more severe disease (greensill et al., 2003) . there is some evidence that secondary hmpv infection occurs frequently in childhood, probably accompanied only by mild symptoms (ebihara et al., 2004) . several investigators have found chemokine profiles during acute infection to be different in children with hmpv infections compared to those with rsv infections, with higher interleukin-8 (il-8) and lower rantes concentrations in hmpv patients. however in another study, inflammatory cytokine (il-8, tnf-␣, il-1␤) levels in respiratory secretions were 6-fold lower than in children infected with rsv (jartti et al., 2002; laham et al., 2004) . tthe physiological relevance of these observations remains unclear. the outbreak of severe acute respiratory distress syndrome (sars), which started in late 2002 in east asia, and spread throughout the world during that winter, was found to affect mainly health-care workers and close contacts of diseased individuals. sars, which was proved to be caused by a new coronavirus, induces an atypical pneumonia with fever, dry cough, and shortness of breath. many adults also suffer from myalgia, dizziness, chills, and rigors. it was concluded from postmortem examinations that sars-pathology is primarily caused by immunological damage to the lungs. the interstitial space of the lungs was mainly filled with mononuclear infiltrates and there was diffuse hemorrhage on the lung surface. sars coronavirus (sars-cov) spreads mainly via the respiratory route, the epithelial cell being its primary target cell. as for other coronaviruses, the spike proteins, s1 for cell entry and s2 for fusion, also seem to be important for sars entry of host cells, despite the fact that sars is only 20-30% homologous to other coronaviruses. very recently, angiotensine converting enzyme (ace2) was identified as the host cell receptor for sars-cov (li et al., 2003) and surface expression of ace2 on alveolar epithelial cells was demonstrated (hamming et al., 2004) . transmission of sars-cov occurs by droplets and most cases have occurred through close contact exposure. however, recently evidence of airborne transmission has emerged (yu et al., 2004) . although many individuals were infected in the initial 2 weeks of the epidemic and the disease spread rapidly over several countries, the numbers of infected children stayed relatively low in all regions (ͻ5%). furthermore, children tend to develop less severe disease. after an incubation period of 5-10 days, similar to adults, infected children developed symptoms of a mild upper respiratory tract infection, clinically indistinguishable from other common colds. none of the 100 pediatric sars cases in hong kong turned out to be fatal and only one adolescent required mechanical ventilation (leung et al., 2003) . adolescents are more likely to develop severe disease, as observed in adult sars patients (leung et al., 2004) . a sore throat and a high initial and peak peripheral blood neutrophil count were found to be independent risk factors for severe disease in children with a laboratory-confirmed sars infection. furthermore, children seemed to spread the disease less easily to others and there have appeared no reports in the literature demonstrating transmission from children to other individuals. many children with laboratory-confirmed sars do not meet the who criteria for diagnosis of sars. as children seem to have a much milder clinical course, the term "sars" may not represent the disease in children very well. the role of the innate immune system in viral lower respiratory tract infection has not been studied intensively until recently. studies have focused on the adaptive immunity with the goal of developing a vaccine, for example, for rsv. understanding the mechanisms underlying primary and recurrent viral infection has attracted increased attention. the immunological response against viral invasion of the lower respiratory tract comprises both adaptive and innate immune response mechanisms with both beneficial as well as detrimental characteristics. the innate response occurs in the early phase of the infection and increasing evidence suggests that these early events determine disease course and possibly even long-term outcome (garofalo and haeberle, 2000; tasker et al., 2000) . for the rest of the discussion on immunological phenomena, focus will be on rsv as a prototype. epithelial cells are key regulators of the innate immune response against viral infections (garofalo and haeberle, 2000) , producing a number of inflammatory mediators in response to rsv infection. these include cytokines (interleukin-6, -1, tumor necrosis factor (tnf)-␣), several chemokines (il-8, macrophage inflammatory protein (mip)-1␣, monocyte chemotactic protein (mcp-1), rantes), type-l interferon (ifn-␣/␤), and growth factors (gm-csf, g-csf). epithelial-derived levels of chemokines correlate with disease severity (bont et al., 1999; smyth et al., 2002) . surfactant proteins produced by epithelial cells (sp-a and sp-d) may also play a role as opsonins for viruses and bacteria. thus, epithelial cells provide a potential mechanism for serum-independent phagocytosis. many of these mediators are induced both at the level of secretion and transcription. interestingly, some mediators (e.g., il-8 and rantes) are also upregulated by inactive forms of the virus (harrison et al., 1999) . rsv uptake by immune and non-immune cells is a receptor-mediated process. experiments with blocking antibodies against g-protein revealed inhibition of binding of rsv to epithelial cells. the fractalkine receptor, also known as the cx3cr1 chemokine receptor, is involved in g-protein-mediated uptake by epithelial cells . other receptors may very well be involved in uptake by dendritic cells, macrophages, and other cells of the innate immune system (harris and werling, 2003) , and toll-like receptors (tlrs), especially tlr-4, are being investigated as possible candidates for mediating viral uptake (haeberle et al., 2002a, b; monick et al., 2003) . several groups have demonstrated activation of the transcription factor nf-kb in rsv-infected epithelial cells (tian et al., 2002) . many of the exhibited effects observed in epithelial cells can be explained by activation of nf-kb. several cytokines associated with rsv infection have nf-kb binding sites in their promoter or enhancer regions (bitko et al., 1997) . epithelial nf-kb activation has also been observed in other viral infections, including parainfluenza, influenza a, and rhinovirus (pahl and baeuerle, 1995; kim et al., 2000; bose et al., 2003) . nf-kb could be an exciting target for therapy development and experiments in which balb/c mice were treated with perflubron have confirmed this concept. perflubron has already been shown to be effective in clinical trials of patients with respiratory distress syndrome because of its physical characteristics. besides the beneficial physical effect in improvement of gas exchange and of lung compliance, this agent was found to have anti-inflammatory effects. rsv-infected balb/c mice treated with perflubron intranasally showed a reduction in cellular inflammatory infiltrates and decreased chemokine expression in the lung tissue. both the anti-inflammatory effects were directly linked to interference of perflubron with nf-kb-mediated transcription (haeberle et al., 2002a, b) . the chemokines produced by epithelial cells attract t-cells, neutrophils, monocytes, and possibly eosinophils to the respiratory tract. besides induction of secreted products, epithelial cells upregulate expression of adhesion molecules for neutrophils on their surface, allowing neutrophils to adhere firmly to infected cells (wang and forsyth, 2000) . furthermore, neutrophils are the dominant cell type found in bronchoalveolar lavage (bal) fluid of rsv patients (everard et al., 1994 . however, their role in fighting viral infection is not as well established as in bacterial infections. pathological studies of lungs of rsv-infected calves have shown a major influx of neutrophils in the infected airway mucosa, observed earlier than any other cell type involved. furthermore, neutrophils are the dominant cell type found in bronchoalveolar lavage (bal) fluid of rsv patients (everard et al., 1994 " several chemokines and cytokines involved in neutrophil activation have been associated with rsv lower respiratory tract infections. recently, local neutrophil il-9 production has been linked to rsv bronchiolitis (mcnamara et al., 2004) . as shown by wang et al., a major increase in epithelial damage occurs, when rsvinfected epithelial cells are co-cultured with neutrophils (wang and forsyth, 2000) . this is suggestive of a detrimental role for neutrophil-induced immunopathology in lower respiratory tract infections. rantes and mip-1␣ are produced by the epithelium in response to rsv infection and these chemoattractants recruit eosinophils to the inflammatory site. the analogy between clinical features of virus-induced wheezing illnesses and asthma has made eosinophils an attractive subject for studies aimed at improving understanding of rsv pathogenesis. however, mainly because of their absence in bal of rsv patients, their involvement remains controversial. however, eosinophil-derived cationic protein (ecp) has been linked to bronchiolitis and postbronchiolitic wheezing pifferi et al., 2001; dimova-yaneva et al., 2004) . in vitro, eosinophils have also been shown to be susceptible to rsv. eosinophil priming, superoxide production, and degranulation were induced by incubation with rsv kimpen et al., 1992; olszewska-pazdrak et al., 1998; tachibana et al., 2002) . rosenberg and domachowske (2001) have suggested a beneficial role for eosinophils in rsv bronchiolitis. they identified antiviral properties for the eosinophil based on ribonuclease activity of eosinophil-derived neurotoxin (edn) and ecp. this enzymatic activity leads to destruction of extracellular ssrna virions and delayed replication both in vitro and in vivo. recently, there has also been great interest in the involvement of macrophages and dendritic cells in rsv pathogenesis. these cells were already appreciated for their role in antigen presentation, at which dendritic cells are by far superior. macrophages express phagocyte activity, which may be of importance in clearance of infected epithelial cellular debris. fascinating new players in host defense against viruses are pattern recognition receptors. toll-like receptor-4 (tlr-4) and cd14, both present in a complex on these cells, have been found to interact with rsv and receptor-binding results in triggering of the innate immune system. tlr-4 has been shown to activate nf-kb in macrophages of rsv-infected mice (haeberle et al., 2002a, b) and tlr-4-deficient mice have impaired nk-cell and cd14ϩ cell trafficking and delayed viral clearance . furthermore, intracellular pattern recognition receptors tlr-3 and -7, may be involved in recognizing doubleand single-stranded rna (dsrna/ssrna), respectively (akira and hemmi, 2003; lund et al., 2004) . dsrna is produced during replication of rna-viruses and is a potent inducer of ifn-␣/␤. all human cells can produce ifn-␣/␤ in response to viral infection, while only t-cells and nk-cells produce ifn-␥. dsrna also activates dsrna-dependent protein kinase r (pkr) and nf-kb via distinct pathways. transcription of pkr is under control of ifn-␣/␤. pkr controls enzymes directly involved in protein synthesis, thereby inhibiting cellular and viral protein translation. ifn-␣/␤-deficient mice as well as pkrϫ/ϫ mice are extremely sensitive to influenza infection (balachandran et al., 2000) . several viruses, including rsv, have evolved mechanisms to escape the interferon system, which will be discussed below. respiratory epithelial cells are the principal host cells for viral pathogens in lower respiratory tract disease. the degree of replication and the mechanism of spread along the epithelial layer depend on the virus family characteristics. through the fusion (f) protein, rsv is capable of syncytium formation, which allows it to replicate and spread relatively undetected by the immune system for a relatively long period. the virus itself is directly responsible for cytopathology and viral envelope proteins are expressed on the surface of infected epithelial cells. dendritic cells, lining the basal membrane of the respiratory epithelium encounter rsv, pick up viral antigens and migrate to mediastinal lymph nodes where viral antigen is presented to naïve cd4ϩ t-cells. antigen presentation and co-stimulatory molecule expression lead to maturation to the t-helper phenotype. this then induces b-cell proliferation with the production of specific antibodies as well as proliferation of virus-specific cytotoxic cd8-cells. cellular responses are responsible for controlling and terminating acute infection with rsv. in primary infections, the adaptive cellular immune response develops within 10 days. these cd8ϩ cells can recognize and eliminate virus-infected epithelial cells resulting in perforin-mediated cytotoxity. epithelial cells are nonprofessional antigen presenting cells (apc) expressing mhc class l on the surface (garofalo et al., 1996) . when infected, epithelial cells present viral antigen in association with mhc class l molecules. mhc class l restricted antigen presentation to cd8ϩ cells, among other factors, may determine the strength of the cytotoxic response. in cd8-deficient mice, there is delayed viral clearance; however, these mice also exhibit decreased disease severity (graham et al., 1991) . therefore, it is conceivable that cd8ϩ t-cells are crucial in viral clearance while a surplus of cytotoxicity may result in pulmonary injury. in humans, a cytotoxic t-cell response is elicited against all viral proteins, except the g-(attachment)-protein, which is required for cell entry (bangham et al., 1986; hacking and hull, 2002) . it is suggested that a defective response against g-protein is directly associated with enhanced disease. however, g-protein can induce a cd4ϩ response in mice, which is associated with th2-cytokine production and eosinophilia both during primary and secondary infection (openshaw, 1995) . the immune response to the f-protein is dominated by ifn-␥ production and subsequent polarization toward a th1-type cellular response, and therefore it has been postulated that responses to the other viral proteins can modulate the strong th2response to g-protein (graham et al., 2000) . a stronger th1-response seems to induce a more rapid viral clearance and milder disease (bont et al., 1999; legg et al., 2003) . besides activated t-cells, nkcells also produce considerable amounts of ifn-␥ (hussell and openshaw, 1998) . ifn-␥ has important antiviral effects and provides a link between adaptive and innate immune system. it can induce expression of tnf-related apoptosis inducing ligand (trail) on immune cells, which has the potential to trigger apoptosis of virus-infected cells (sedger et al., 1999) . in vitro findings suggest that rsv-infected cells in vivo are susceptible to killing by immune cells through the trail pathway (kotelkin et al., 2003) . nk-cells are also thought to play a role in activating cd8ϩ cells, further modulating the degree of cytotoxicity (hussell and openshaw, 1998) . in summary, in rsv lower respiratory tract infections, cytotoxic cd8ϩ t-cells are involved in viral clearance while the humoral response is required for the protection against reinfection. however, as has been discussed before, memory is incomplete and repeated infections with rsv are common. both igm and igg as well as secretory iga against rsv are formed in infants, and a more vigorous antibody response seems to be protective against rsv infections (meurman et al., 1984; welliver et al., 1989 ). rsv infections are most severe in the youngest age group, which is the least mature in terms of immunity to infections. relative deficiencies in both innate and antigen-specific immunity in infancy have been characterized. these include delayed trafficking of immune cells, less-efficient antigen presentation by dendritic cells, and impaired production of ifn-␥ by t-cells in response to antigen presentation (bont and kimpen, 2002) . the fetus derives maternal igg-antibodies via the placenta fairly late in gestation. this partly explains why prematurity is an important risk factor for severe disease caused by rsv, as well as the physiological characteristics of the small airways. antibody titers produced by infants are relatively low compared to older children. trials with humanized monoclonal antibodies against rsv-f-protein have shown a 50% reduction in rsv lower respiratory tract-related hospitalizations in this highrisk group for severe disease (impact study group, 1998). the cytokine milieu at the time of infection is another factor possibly contributing to the occurrence of severe rsv bronchiolitis especially in the youngest age group. at birth, there is skewing toward a th2-phenotype and rsv bronchiolitis was long thought to be a th2-type disease. this role of th2-skewing is an attractive concept, because it provides some explanation for the association between rsv bronchiolitis and the development of asthma. asthma and allergy have long been acknowledged to beth2-mediated conditions. however, convincing evidence that primary rsv infections are mediated by th2 cytokines is lacking. dendritic cells are thought to have an important function in skewing the th1/th2-ratio. viruses may be important in maturation of dendritic cells, which can then drive differentiation of naive t-cells into either a th2-or a th1-phenotype. the role of regulatory t-cells that suppress both th1 and th2 differentiation has not been studied in rsv bronchiolitis so far. gene polymorphism studies have been undertaken to identify a genetic background to explain the individual susceptibility to rsv lower respiratory tract infection. several polymorphisms situated in genes relevant for the adaptive and innate immunity have been found to correlate with occurrence of rsv infection. polymorphisms of interleukin-4, il-4r, and its receptor, have been associated with rsv bronchiolitis, which is consistent with the th2-hypothesis (choi et al., 2002; hoebee et al., 2003) . very recently, a polymorphism of the gene coding for interleukin-10 (il-10) was found as well (hoebee et al., 2004) . this is particularly interesting since il-10 is a cytokine produced by t-regulatory cells and monocytes, thought to be primarily involved in development of allergy. gene polymorphisms involved in innate immunity include surfactant proteins spa and d (lahti et al., 2002) , the chemokine il-8 (hull et al., 2001) , tlr-4 (tal et al., 2004) , and the chemokine receptor for rantes and mip-1␣, ccr5 (hull et al., 2003) . immunocompromised patients have a higher risk of developing severe disease from viral respiratory tract infections. in particular, the presence of defects in cellular immunity result in an increased duration of viral shedding and enhanced risk of developing severe disease. most cellular immunodeficiencies are iatrogenic in nature. an important cause is intensive immunosuppressive treatment. the number of pediatric patients undergoing organ or stem-cell transplantation is increasing and high doses of chemotherapeutic and immunosuppressive agents are often used in the pre-and posttransplant regimens. immunosuppressive drugs are used in cancer treatment regimens and for a number of inflammatory conditions. community acquired respiratory viruses such as rsv, rhinovirus, adenovirus, influenza a, influenza b, and the parainfluenza group are frequent causes of respiratory disease in these patients (soldatou and davies, 2003) . adenovirus infections have a particularly high risk of adverse outcome, mortality rates are high, and no effective treatment exists. the presence of lower respiratory tract infection and infection in the pre-engraftment phase of hsct is believed to have a particularly poor prognosis (khushalani et al., 2001) . the risk of severe disease is higher during allogenic hsct than autologous hsct. besides causing increased morbidity and mortality, respiratory tract infections are associated with a greater risk of delayed engraftment (abdallah et al., 2003) . in solid organ transplant patients, respiratory virus infections are also associated with a higher incidence of rejection (wendt, 1997) . prolonged shedding of respiratory viruses for weeks or months has been documented in hiv-infected adults and children. this has important implications for infection control in medical facilities. in addition, respiratory viral infection may result in increased hiv replication and, theoretically, hiv disease progression (king, 1997) . in hiv-infected children, rsv infections are less limited by season (madhi et al., 2000) . however, generally, the course of rsv infections in hiv patients is not more severe, unless there is profound lymphopenia or pre-existing lung disease (soldatou and davies, 2003) . other viruses may also cause respiratory complications in the immunocompromised patients. in particular herpesviruses, such as cytomegalovirus (cmv) and varicella zoster virus, can cause severe pneumonia. with a cmv-negative donor and a cmv-positive recipient, there is an especially high risk of reactivation which may lead to severe disease. this reactivation also occurs with epstein barr virus (ebv), human herpes virus (hhv)-6, -7, and -8, although these are much less frequent causative agents of pneumonia. the innate immune defense to viral respiratory tract infections consists of the mucosal layer, type 1 interferons, activated phagocytes, and nk-cells. the impact of primary defects in the innate immune defense has not been well documented. phagocyte defects are primarily related to a higher incidence of bacterial infections. one indication that impaired phagocyte function also leads to increased severity of respiratory viral infection can be derived from a report of severe abnormalities on lung-ct-scans of rsv patients with phagocyte defects (uzel et al., 2000) . interferon-gamma receptor deficiency, which may have implications for both the adaptive and innate immune system, has also been associated with increased susceptibility to viral respiratory pathogens (dorman et al., 1999) . chronic lung disease also increases susceptibility to respiratory viruses (meert et al., 1989; griffin et al., 2002) . premature patients with bronchopulmonary dysplasia are candidates for rsv-immunoprophylaxis because of their increased risk of developing severe lower respiratory tract infections. in children with cystic fibrosis (cf), 39% are already hospitalized with respiratory virus infection in their first year of life. furthermore, there is a correlation between viral infections in infancy and disease progression. infants with cf suffering from a respiratory virus infection are at significant risk for lower respiratory tract disease, hospitalization, and deterioration in lung function that persists months after the acute illness (hiatt et al., 1999) . cf infants were found to be four times more likely to develop an lrti compared with controls. it has been shown that cfderived airway epithelial cells allow a higher degree of piv replication and have an increased production of pro-inflammatory cytokines (zheng et al., 2003) . cfderived epithelial cells are also unable to express no-synthase 2, which results in a decrease production of nitric oxide (no), which has antiviral capacity, reducing effects on replication. furthermore, in cf-cells there is no viral induction of 2ј5јoligoadenylate synthetase (oas), an enzyme that is normally induced by dsrna and ifn-␥. oas is involved in inhibition of cellular protein synthesis, thereby inhibiting viral replication. respiratory viruses can be isolated from the secretions of approximately 75% of children and of more than half of adults during asthma exacerbations (johnston et al., 1995; lemanske, 2003) . recently, copd exacerbations have also been attributed to viral infection by rhinovirus, rsv, and piv (seemungal and wedzicha, 2003) . the underlying mechanisms for this are, however, still a matter of debate. from experimental rhinovirus (rv) infections in humans, it has been shown that rv infection causes increased bronchoconstriction in atopic non-asthmatic and asthmatic individuals, while symptoms in normal individuals are relatively mild. this implies that induction of a wheezing episode requires both rv infection and a preexisting tendency to develop allergic or asthmatic disease (message and johnston, 2001) . rv-specific t-cell responses can be activated by either serotype-specific or shared viral epitopes. cross-reactivity between rv-subtypes could result in vigorous t-cell responses and may amplify allergic inflammation. other proposed mechanisms linking viral infections to asthma exacerbation are epithelial dysregulation, airway remodeling, the immune response to virus, and alterations of neural responses (message and johnston, 2001; gern, 2002) . upregulation of icam-1-expression, which is the entry receptor for major group rhinoviruses, has been found in susceptible individuals. this may be one mechanism predisposing atopic individuals to rv-induced exacerbations. rhinovirus can induce a number of inflammatory mediators (kinins, arachidonic acid) and cytokines (e.g., il-1, il-6, ifn-␣/␤, gm-csf, tnf-␣) that can further enhance inflammation. th1 cytokines seem to have a general antiviral effect while a predominant th2-cytokine response leads to enhanced disease, failure to clear the virus, and amplification of allergic inflammation (message and johnston, 2001) . eosinophil numbers were found to be increased in bronchial biopsies from both healthy and asthmatic human volunteers after experimental rhinovirus infection. this cell type is associated with allergic inflammation in the lung. in allergic rhinitis patients, the increased level of eosinophils in bal even persisted for 6 weeks. these data suggest a potential role for eosinophils in virus-induced asthma, which can be either pathogenic or protective. virus-induced exacerbations of asthma tend to be resistant to treatment with corticosteroids and may require a different therapeutic approach. in vitro, blocking icam-1 has been tried with positive results, which may be of particular relevance to rhinovirus infections. the possibility of other immunomodulating drugs is being investigated and may be of significant benefit to future asthma treatment. a causal relationship between viral respiratory tract infections and asthma exacerbations is generally acknowledged. however, the suggestion that respiratory virus infection is a causal determinant in the development of asthma is highly controversial. according to the hygiene hypothesis, viral infection would be expected to have an inhibitory effect on the development of asthma (an allergy), and this is supported by a study from matricardi et al. (1997) , showing an inverse relation between hepatitis a seropositivity and atopy among soldiers. the hygiene hypothesis is based on the theory that the immune system is directed toward a more th1-skewed immune response with each viral infection. however, this hypothesis is not supported by the observation that rsv infections, severe enough to cause bronchiolitis, are significantly associated with a higher incidence of asthma up to the age of 7-11 years (stein et al., 1999; sigurs et al., 2000) . these data convincingly show a link between rsv bronchiolitis and recurrent wheezing in childhood. in a recent study, wheezing following rsv lower respiratory tract infection was found to develop independent of atopy . it may, however, be true that the transmission route, the organs involved, and exposure to microbial products may be important in determining the final effect of a virus infection on the development of asthma and allergy (gern and busse, 2002) . the link between rsv infection and atopy is even less clear than the one with recurrent wheezing and asthma, at least in humans. animal studies have yielded conflicting results. one group found that rsv infection in mice enhances subsequent allergic inflammation (schwarze et al., 1997) , while others reported a decrease in allergic sensitization and bhr after rsv infection (peebles et al., 2001) . no proof exists that severe rsv infections are associated with atopy that persists into adulthood (peebles, 2004) . a key question is whether the association with the development of asthma is merely an expression of increased susceptibility to both asthma and rsv-induced lower respiratory tract infections or whether true causality is involved. the prevailing theory on this subject involves maturation effects of the th1/th2-balance. the system shifts from a th2-polarization in fetal life, which is an optimal environment for the placenta, to a more balanced th1/th2-phenotype in adulthood. most viruses are known to induce a th1 cytokine response (ifn-␥). this theory states that when infections occur early in infancy, there is a reduced ability to react with an appropriate antiviral th1-response. low ifn-␥ production may result in spread of the virus to the lower respiratory tract. this is in agreement with findings that in children with severe rsv lower respiratory tract infections, lower amounts of ifn-␥ are produced . the dynamics of this shift toward a more balanced th1/th2 immune response may differ between individuals. both environmental factors and genetic make-up may contribute to a slower maturation of th1 competence in some individuals. respiratory virus infections in infancy and an atopic sensitization to aero-allergens, both of which are related to th2-skewed responses and intermittent wheeze, may than synergistically result in persistent wheeze (holt and sly, 2002) . it is likely that more links between atopic sensitization and respiratory infections exist, while preventive rsv-ivig treatment of children results in a decreased sensitization to aeroallergens as well. rsv-prophylaxis may therefore have a long-term benefit in the development of persistent wheeze (piedimonte and simoes, 2002; wenzel et al., 2002) . another theory linking viral infections in childhood to the development of asthma involves the pathologic effects of viral lower respiratory tract infections on airway physiology. wall thickening with consequent increased resistance may predispose the airway to more infections and thus influence bronchial hyperreactivity (bardin et al., 1992) . however, it may also be true that small airways predispose to both asthma and airway symptoms from viral infections. remodeling of the submucosal neural networks by rsv, as observed by piedimonte et al., is also proposed to result in increased responsiveness to airway irritants (piedimonte, 2002) . walter et al. (2002) have proposed that paramyxoviral infection has the ability not only to induce acute hyperresponsiveness, but also to result in long-lasting changes in airway behavior. from mouse-studies with a piv (sev), it has been concluded that viruses cause long-term effects in epithelial cells, associated with airway reactivity and goblet cell hyperplasia. long-term effects are induced by the virus in the acute phase, and later on, the presence of virus is no longer required for the persistence of symptoms. it is speculated that primary paramyxoviral infection within the proper genetic background may result in chronic dysfunction of epithelial cell behavior. their results have indicated that different mechanisms are responsible for the induction of the acute and the chronic response (walter et al., 2002) . several theories on the induction of asthma have thus been proposed. the current view is that virus infection modulates the development of an asthmatic phenotype in a susceptible host. the relationship is, therefore, not purely causal but certainly requires an intrinsic vulnerability. the effectiveness of respiratory virus infections in the host depends partly on the ability to evade the immune system (figure 4 .2). while several viral evasion mechanisms have evolved, not all have been intensively studied in respiratory viruses. viral entry into host cells is one of the first obstacles viruses have to overcome. since the cell membrane is in principle impermeable to macromolecules, viruses 56 caroline a. lindemans and jan l. l. kimpen must first have an effective method to attach to the cell membrane. some viruses bind putative cell surface receptors that do not simply play a role in viral attachment, but also allow viral entry by inducing endocytosis. for some viral pathogens, such as rhinovirus, these receptors have been identified (icam-1 and ldl-r), while for others, such as rsv, the receptor that is used for cellular entry has not been unequivocally defined. the cx3cr1-chemokine receptor, also known as fractalkine-receptor, may be involved and tlrs have also been proposed to play a role. many enveloped viruses have glycosylated proteins, which not only bind to cellular receptors, but also have additional functions as membrane fusion factors, or receptor-destroying enzymatic activity. membrane fusion factors such as the rsv fprotein also allow cell-to-cell transmission of virus, which keeps it relatively hidden from the cellular immune system (smith and helenius, 2004) . escaping the "interferon signaling system" is one of the common mechanisms most viruses have acquired. as mentioned earlier in this chapter, both ifn-␣/␤ and ifn-␥ have potent antiviral properties. both types of interferon regulate transcription of a variety of target genes through activation of interferon inducible transcription factors. ifn-stimulated genes encode a variety of cellular enzymes, including pkr and 2ј5 ј-oligoadenylate synthetase, both involved in inhibition of viral protein synthesis. furthermore, interferons induce cellular apoptosis and upregulate mhc1 expression, targeting cells for cd8ϩ t-cell-mediated cytotoxicity. additionally, ifn-␥ activates the adaptive cellular immune system. rsv infection leads to an increase in ifn-␣/␤, but does not induce ifn-␥ from mononuclear and nk-cells as efficiently. despite the fact that interferons are known for their antiviral properties, intranasal administration of either lfn-␣/␤ or ifn-␥ in the airway does not lead to reduction of symptoms of viral respiratory infections (ramaswamy et al., 2004) . this is suggestive of a viral mechanism to evade the host's interferon response. the paramyxoviridae family (figure 4 .3), responsible for a large part of children's respiratory infections, consists of two subfamilies based on the structural differences in the gene encoding for the polymerase complex (p-protein). the paramyxovirinae subfamily members are able to block interferon-mediated promoter activity. these paramyxovirinae, to which parainfluenza-, measles-, and mumps virus belong to, have a p-gene that encodes for additional proteins besides the p-protein, the v-proteins. it is these v-proteins that have been found to be responsible for evading the interferon signaling pathways in this group of viruses. ifnmediated transcription is predominantly mediated by signal transducers and activators of transcription (stat). v-proteins have the ability to block interferon-mediated signaling by targeting stats for proteosomal degradation (horvath, 2004) . in contrast, rsv, belonging to the pneumovirinae subfamily, fails to inhibit ifn-induced promoter activity. the pneumovirinae consist of only one genus, the pneumovirus, which also includes hmpv. in this subfamily, p-genes only encode for the p-protein and therefore rsv cannot block interferon-mediated signaling (young et al., 2000) . however, recently it was demonstrated that, although rsv does not inhibit interferon-induced promoter activity, rsv replication is still resistant to ifn treatment of infected cells. apparently, an alternative mechanism to circumvent the interferon antiviral response exists. this has been attributed to additional proteins, characteristic of these pneumoviruses (spann et al., 2004) . these are nonstructural proteins (ns1 and ns2) that have no homologs in the paramyxovirinae. however, the underlying molecular pathway has not yet been elucidated. rsv infection of epithelial has been shown to lead to an upregulation of trail-receptor expression on these cells (kotelkin et al., 2003) . apoptosis of infected cells is an effective way to eliminate intracellular pathogens without damage to the surrounding tissue (figure 4.4) . however, several respiratory viruses have developed mechanisms to inhibit apoptosis. it has been demonstrated that rsv is able to effectively inhibit apoptosis of epithelial cells in vitro, in accordance with the limited pathology induced by rsv in epithelial cells during the first few days of the infection (thomas et al., 2002) . eventually, necrosis is observed when mature viral particles are released from the cells, after 2-3 days. furthermore, experiments with both adult and cord blood monocytes have shown a prolonged longevity of cells, when cultured in the presence of rsv (krilov et al., 2000) . of all respiratory viruses, viral evasion techniques of adenoviruses have been studied most intensively. it appears that approximately a third of the adenovirus genome is devoted to counteracting innate and adaptive immune defenses (burgert et al., 2002) . adenoviruses encode the protein e1a that blocks interferon-induced gene transcription. through the va-rna protein that blocks activation of pkr, they also interfere with the antiviral enzymes that are synthesized under interferon control. additionally, adenoviruses have developed several mechanisms to inhibit both constitutive and death receptor induced apoptosis (figure 4.4) . the e1b/55k protein inhibits p53-mediated apoptosis, e3/19k interacts with pro-apoptotic proteins bax and bak, whereas several e3 proteins are involved in removing fas and trail (death) receptors from the cell surface by promoting their degradation in lysosomes (wold et al., 1999) . finally, adenoviruses interfere with recognition of infected cells by cytotoxic lymphocytes. the adenovirus e3/19k protein inhibits transport of mhc1 molecules to the cell surface, resulting in decreased viral antigen presentation to cd8ϩ cells. future studies may unravel further mechanisms of immune evasion that may also be important in viruses involved in lower respiratory tract disease. this knowledge extrinsic and intrinsic signaling pathways of apoptosis. some viruses such as adenovirus interfere with cellular apoptosis. cellular apoptosis can normally occur via activation of the extrinsic (death receptor) pathway or the intrinsic pathway. both lead to activation of effector caspases 3 and 7, which will inevitably result in apoptosis. via production of external factors, such as tnf-␣, fas-ligand, and trail, death-receptors are cross-linked, which leads to apoptosis. adenovirus e3 proteins can remove these death receptors from the surface, thereby inhibiting extrinsic apoptosis. the intrinsic pathway is regulated by pro-and anti-apoptotic proteins of the bcl2-family. the transcription factor p53 has anticarcinogenic activity, induces the intrinsic pathway and promotes the transcription of pro-apoptotic proteins such as bax and bak. these proteins induce mitochondrial leakage of cytochrome c, which activates caspase 9, finally leading to effector caspase activation and apoptosis. adenovirus proteins are involved in both inhibition of p53 and the functioning of bax and bak. an outbreak of respiratory syncytial virus infection in a bone marrow transplant unit: effect on engraftment and outcome of pneumonia without specific antiviral treatment recognition of pathogen-associated molecular patterns by tlr family essential role for the dsrna-dependent protein kinase pkr in innate immunity to viral infection human and murine cytotoxic t cells specific to respiratory syncytial virus recognize the viral nucleoprotein (n), but not the major glycoprotein (g), expressed by vaccinia virus recombinants viruses as precipitants of asthma symptoms. ii. physiology and mechanisms transcriptional induction of multiple cytokines by human respiratory syncytial virus requires activation of nf-kappa b and is inhibited by sodium salicylate and aspirin bacteraemia and antibiotic use in respiratory syncytial virus infections long-term consequences of respiratory syncytial virus (rsv) bronchiolitis peripheral blood cytokine responses and disease severity in respiratory syncytial virus bronchiolitis immunological mechanisms of severe respiratory syncytial virus bronchiolitis seasonality of long term wheezing following respiratory syncytial virus lower respiratory tract infection natural reinfection with respiratory syncytial virus does not boost virus-specific t-cell immunity temporal activation of nf-kappab regulates an interferon-independent innate antiviral response against cytoplasmic rna viruses subversion of host defense mechanisms by adenoviruses adenovirus type 7 associated with severe and fatal acute lower respiratory infections in argentine children a comparison of nebulized budesonide, and intramuscular, and oral dexamethasone for treatment of croup a common haplotype of interleukin-4 gene il4 is associated with severe respiratory syncytial virus disease in korean children persistence of respiratory syncytial virus (rsv) infection and development of rsv-specific igg1 response in a guinea-pig model of acute bronchiolitis eosinophil activation and cysteinyl leukotriene production in infants with respiratory syncytial virus bronchiolitis viral infections in interferon-gamma receptor deficiency human metapneumovirus infection in japanese children analysis of cells obtained by bronchial lavage of infants with respiratory syncytial virus infection eosinophil degranulation in the respiratory tract during naturally acquired respiratory syncytial virus infection respiratory syncytial virus infection of human respiratory epithelial cells up-regulates class i mhc expression through the induction of ifn-beta and il-1 alpha epithelial regulation of innate immunity to respiratory syncytial virus rhinovirus respiratory infections and asthma relationship of viral infections to wheezing illnesses and asthma role of t lymphocyte subsets in the pathogenesis of primary infection and rechallenge with respiratory syncytial virus in mice immune-mediated disease pathogenesis in respiratory syncytial virus infection human metapneumovirus in severe respiratory syncytial virus bronchiolitis winter viruses: influenza-and respiratory syncytial virus-related morbidity in chronic lung disease rhinovirus and acute respiratory infections in hospitalized children. retrospective study respiratory syncytial virus--viral biology and the host response perflubron reduces lung inflammation in respiratory syncytial virus infection by inhibiting chemokine expression and nuclear factor-kappa b activation respiratory syncytial virus-induced activation of nuclear factor-kappab in the lung involves alveolar macrophages and toll-like receptor 4-dependent pathways respiratory syncytial virus and parainfluenza virus enhanced adherence of streptococcus pneumoniae to human epithelial cells infected with respiratory syncytial virus tissue distribution of ace2 protein, the functional receptor for sars coronavirus. a first step in understanding sars pathogenesis binding and entry of respiratory syncytial virus into host cells and initiation of the innate immune response respiratory syncytical virus-induced chemokine expression in the lower airways: eosinophil recruitment and degranulation involvement of toll-like receptor 4 in innate immunity to respiratory syncytial virus effects of viral lower respiratory tract infection on lung function in infants with cystic fibrosis influence of promoter variants of interleukin-10, interleukin-9, and tumor necrosis factor-alpha genes on respiratory syncytial virus bronchiolitis association of severe respiratory syncytial virus bronchiolitis with interleukin-4 and interleukin-4 receptor alpha polymorphisms interactions between rsv infection, asthma, and atopy: unraveling the complexities silencing stats: lessons from paramyxovirus interferon evasion adenovirus interaction with its cellular receptor car unusual haplotypic structure of il8, a susceptibility locus for a common respiratory virus variants of the chemokine receptor ccr5 are associated with severe bronchiolitis caused by respiratory syncytial virus intracellular ifn-gamma expression in natural killer cells precedes lung cd8ϩ t cell recruitment during respiratory syncytial virus infection metapneumovirus and acute wheezing in children community study of role of viral infections in exacerbations of asthma in 9-11 year old children respiratory syncytial virus infection in the late bone marrow transplant period: report of three cases and review role of nf-kappa b in cytokine production induced from human airway epithelial cells by rhinovirus infection activation of human eosinophils in vitro by respiratory syncytial virus community respiratory viruses in individuals with human immunodeficiency virus infection risk factors for respiratory syncytial virus associated apnoea relationship between clinical severity of respiratory syncytial virus infection and subtype respiratory syncytial virus infection sensitizes cells to apoptosis mediated by tumor necrosis factor-related apoptosisinducing ligand alterations in apoptosis of cord and adult peripheral blood mononuclear cells induced by in vitro infection with respiratory syncytial virus differential production of inflammatory cytokines in primary infection with human metapneumovirus and with other common respiratory viruses of infancy surfactant protein d gene polymorphism associated with severe respiratory syncytial virus infection adenovirus surveillance on children hospitalized for acute lower respiratory infections in chile (1988-1996) type 1 and type 2 cytokine imbalance in acute respiratory syncytial virus bronchiolitis viruses and asthma: inception, exacerbation, and possible prevention severe acute respiratory syndrome among children severe acute respiratory syndrome (sars) in children: epidemiology, presentation and management angiotensin-converting enzyme 2 is a functional receptor for the sars coronavirus recognition of single-stranded rna viruses by toll-like receptor 7 increased burden of respiratory viral associated severe lower respiratory tract infections in children infected with human immunodeficiency virus type-1 cross sectional retrospective study of prevalence of atopy among italian military students with antibodies against hepatitis a virus human metapneumovirus-an important new respiratory virus interleukin 9 production in the lungs of infants with severe respiratory syncytial virus bronchiolitis bronchoalveolar lavage cellularity in infants with severe respiratory syncytial virus bronchiolitis clinical characteristics of respiratory syncytial virus infections in healthy versus previously compromised host the immunology of virus infection in asthma immunoglobulin class-specific antibody response in respiratory syncytial virus infection measured by enzyme immunoassay respiratory syncytial virus up-regulates tlr4 and sensitizes airway epithelial cells to endotoxin respiratory syncytial virusinfected pulmonary epithelial cells induce eosinophil degranulation by a cd18-mediated mechanism immunity and immunopathology to respiratory syncytial virus. the mouse model expression of influenza virus hemagglutinin activates transcription factor nf-kappa b do rhinoviruses cause pneumonia in children? economic impact of respiratory syncytial virus-related illness in the us: an analysis of national databases viral infections, atopy, and asthma: is there a causal relationship? immune interaction between respiratory syncytial virus infection and allergen sensitization critically depends on timing of challenges pathophysiological mechanisms for the respiratory syncytial virus-reactive airway disease link respiratory syncytial virus and subsequent asthma: one step closer to unravelling the gordian knot? eosinophil cationic protein in infants with respiratory syncytial virus bronchiolitis: predictive value for subsequent development of persistent wheezing are we ready for universal influenza vaccination in paediatrics? concurrent serious bacterial infections in 2396 infants and children hospitalized with respiratory syncytial virus lower respiratory tract infections specific inhibition of type i interferon signal transduction by respiratory syncytial virus viral evolution toward change in receptor usage: adaptation of a major group human rhinovirus to grow in icam-1-negative cells eosinophils, eosinophil ribonucleases, and their role in host defense against respiratory virus pathogens respiratory syncytial virus infection results in airway hyperresponsiveness and enhanced airway sensitization to allergen latency and persistence of respiratory syncytial virus despite t cell immunity ifngamma mediates a novel antiviral activity through dynamic modulation of trail and trail receptor expression viral infections in obstructive airway diseases respiratory syncytial virus bronchiolitis in infancy is an important risk factor for asthma and allergy at age 7 how viruses enter animal cells respiratory syncytial virus bronchiolitis: disease severity, interleukin-8, and virus genotype respiratory virus infections in the immunocompromised host suppression of the induction of alpha, beta, and gamma interferons by the ns1 and ns2 proteins of human respiratory syncytial virus in human epithelial cells and macrophages respiratory syncytial virus in early life and risk of wheeze and allergy by age 13 years respiratory syncytial virus epidemics: the ups and downs of a seasonal virus respiratory syncytial virus enhances the expression of cd11b molecules and the generation of superoxide anion by human eosinophils primed with platelet-activating factor association between common toll-like receptor 4 mutations and severe respiratory syncytial virus disease time course of severe respiratory syncytial virus infection in mechanically ventilated infants palivizumab, a humanized respiratory syncytial virus monoclonal antibody, reduces hospitalization from respiratory syncytial virus infection in high-risk infants respiratory syncytial virus inhibits apoptosis and induces nf-kappa b activity through a phosphatidylinositol 3-kinase-dependent pathway identification of nf-kappab-dependent gene networks in respiratory syncytial virus-infected cells cx3c chemokine mimicry by respiratory syncytial virus g glycoprotein respiratory syncytial virus infection in patients with phagocyte defects a newly discovered human pneumovirus isolated from young children with respiratory tract disease an update on the pathophysiology of rhinovirus upper respiratory tract infections viral lower respiratory tract infection in infants and young children replication and clearance of respiratory syncytial virus: apoptosis is an important pathway of virus clearance after experimental infection with bovine respiratory syncytial virus viral induction of a chronic asthma phenotype and genetic segregation from the acute response the interaction of neutrophils with respiratory epithelial cells in viral infection respiratory syncytial virus-specific antibody responses in immunoglobulin a and e isotypes to the f and g proteins and to intact virus after natural infection community respiratory viruses: organ transplant recipients respiratory outcomes in high-risk children 7 to 10 years after prophylaxis with respiratory syncytial virus immune globulin human metapneumovirus and lower respiratory tract disease in otherwise healthy infants and children immune responses to adenoviruses: viral evasion mechanisms and their implications for the clinic paramyxoviridae use distinct virusspecific mechanisms to circumvent the interferon response evidence of airborne transmission of the severe acute respiratory syndrome virus contribution of influenza and respiratory syncytial virus to community cases of influenza-like illness: an observational study impaired innate host defense causes susceptibility to respiratory virus infections in cystic fibrosis is likely to be crucial to the improvement of immunotherapies for prevention and treatment of viral respiratory tract infections. key: cord-004400-li1sc47z authors: ma, jingjiao; wu, rujuan; xu, guanlong; cheng, yuqiang; wang, zhaofei; wang, heng’an; yan, yaxian; li, jinxiang; sun, jianhe title: acetylation at k108 of the ns1 protein is important for the replication and virulence of influenza virus date: 2020-02-24 journal: vet res doi: 10.1186/s13567-020-00747-3 sha: doc_id: 4400 cord_uid: li1sc47z non-structural protein 1 (ns1) of influenza virus is a multifunctional protein that plays an important role in virus replication and virulence. in this study, an acetylation modification was identified at the k108 residue of the ns1 protein of h1n1 influenza virus. to further explore the function of the k108 acetylation modification of the ns1 protein, a deacetylation-mimic mutation (k108r) and a constant acetylation-mimic mutation (k108q) were introduced into the ns1 protein in the background of a/wsn/1933 h1n1 (wsn), resulting in two mutant viruses (wsn-ns1-108r and wsn-ns1-108q). in vitro and mouse studies showed that the deacetylation-mimic mutation k108r in the ns1 protein attenuated the replication and virulence of wsn-ns1-108r, while the constant acetylation-mimic mutant virus wsn-ns1-108q showed similar replication and pathogenicity as the wild-type wsn virus (wsn-wt). the results indicated that acetylation at k108 of the ns1 protein has an important role in the replication and virulence of influenza virus. to further explore the potential mechanism, the type i interferon (ifn-i) antagonistic activity of the three ns1 proteins (ns1-108q, ns1-108r, and ns1-wt) was compared in cells, which showed that the k108r mutation significantly attenuated the ifn-β antagonistic activity of the ns1 protein compared with ns1-wt and ns1-108q. both ns1-wt and ns1-108q inhibited the ifn-β response activated by rig-i card domain, mavs, tbk1, and irf3 more efficiently than the ns1-108r protein in cells. taken together, the results indicated that acetylation at ns1 k108 is important for the ifn antagonistic activity of the ns1 protein and virulence of the influenza virus. influenza virus non-structural protein 1 (ns1) is a multifunctional protein that is responsible for interacting with cellular factors to antagonize the host antiviral response during viral infection [1] . the major role of the ns1 protein is inhibition of both interferon (ifn) and ifn-stimulated proteins by different mechanisms. ns1 inhibits the transcription of type i ifn by binding the 5′ triphosphate viral double-stranded rnas generated during viral replication to prevent the recognition of viral genomic material by host pattern recognition receptors (prrs), including rig-i, dsrna-dependent protein kinase r (pkr), and 2′5′-oligoadenylate synthetase (oas)/rnase l [2] [3] [4] . ns1 can also interact directly with rig-i in the absence of rna binding to inhibit the conformational change of rig-i required for mavs activation [5] . moreover, ns1 is able to interrupt mrna maturation by inhibiting the nuclear export of host mrnas by binding to host poly(a)-binding protein ii (pabpii) and cleavage and polyadenylation specific factor 30 (cpsf30), which are required for host mrna processing, resulting in the accumulation of ifn pre-mrnas in the nucleus of infected cells [6] . in addition, ns1 also antagonizes the open access *correspondence: lijinxiang@caas.cn; sunjhe@sjtu.edu.cn 1 shanghai key laboratory of veterinary biotechnology, key laboratory of urban agriculture (south), ministry of agriculture, school of agriculture and biology, shanghai jiao tong university, shanghai 200240, china 2 chengdu national agricultural science and technology center, sichuan, china full list of author information is available at the end of the article ifn signalling response by regulating other host factors, such as phosphoinositide 3-kinase (pi3k) activity, crklike protein (crkl), and the jak-stat signalling pathway [7] [8] [9] [10] [11] . the ns1 protein, typically 202-237 aa in length depending on the strain, contains four functional regions: an rna binding domain (rbd, 1-73 aa), linker region (lr, 74-88 aa), effector domain (ed, 89-202 aa), and c-terminal "tail" (ctt, 207-237 aa) [12] . multiple basic amino acids (e.g., 35r, 38r, 41k, and 46r) in the rbd are important for rna binding activity and suppressing the activation of pkr [12, 13] . the ed plays an important role by targeting multiple host factors, such as pkr, cpsf30, and p85β (pi3k), to inhibit antiviral responses and enhance viral replication [1] . the residue 187w in the ed domain is important for the dimerization of the ns1 protein, and the w187r substitution impaired ns1 dimerization and attenuated the virus in vivo [14] . in addition, residues 186e, 189d, and 194v play important roles in the binding of ns1 to cleavage and polyadenylation specificity factor 30 (cpsf30), and mutations in those residues weaken the binding of ns1 to cpsf30 and impair the ability of the ns1 protein to shut off host gene expression [15, 16] . post-translational modifications, such as phosphorylation, sumoylation, and acetylation, are important for protein function. phosphorylation at 49t, 80t, and 215t of the ns1 protein are important for interferon antagonistic activity and replication of human influenza virus [17, 18] . sumoylation at positions 219 and 221 of ns1 are crucial for host protein expression shutoff and replication of h5n1 influenza virus [19] . acetylation is an important post-translational modification that occurs in two forms [20] . one is the co/post-translational acetylation at the n α -termini of the nascent polypeptide chains [21] . the other form is acetylation of the ε-amino group of lysine, which was first recognized in histones regulating gene translation [22] and later was found in non-histone proteins [23] . the acetylation status is reversible and well balanced by lysine (k) acetyltransferases (kats) and lysine deacetylases (kdacs), which are tightly regulated to perform many cellular functions [24] . dysfunction of the acetylation machinery can inhibit protein functions and consequently lead to severe diseases [25, 26] . acetylation has been found in multiple proteins of influenza viruses. acetylation was identified in the np protein of influenza virus, and deacetylation of the np protein prevented the virus from assembling functional virus particles [27] . a histone-like sequence (histone mimic) was identified in the ns1 protein of influenza a h3n2, which contributes to suppression of the antiviral response [28] . the n-terminal acetylation of pa-x is required for the host shutoff activity of pa-x and for viral polymerase activity [29, 30] . acetylation of pa has been reported to be crucial for polymerase activity, and deacetylated pa protein restricts iav rna transcription and replication. the influenza virus haemagglutinin (ha) has three conserved cysteine residues (551, 559, and 562) at its c terminus serving as acylation sites that are essential for the formation of fusion pores and infectivity [31] . in the present study, an acetylation modification at k108 of the ns1 protein was identified and characterized. the results showed that the deacetylation-mimic mutation k108r in the ns1 protein attenuated the replication and virulence of the virus in vitro and in vivo. ifn-β antagonist assays indicated that the k108r mutation attenuated ifn antagonistic ability compared with the ns1-wt or ns1-108q (constant acetylation-mimic) proteins. overall, this study indicated that acetylation at k108 of the ns1 protein plays an important role in the replication and virulence of influenza virus. madin-darby canine kidney (mdck) cells were maintained in eagle's minimal essential medium (emem, hyclone, grand island, usa) with 5% foetal bovine serum (fbs, gibco, grand island, usa), l-glutamine (gibco), and 1% antibiotic (gibco). human embryonic kidney (hek) 293t cells and adenocarcinomic human alveolar basal epithelial cells (a549 cells) were maintained in dulbecco's modified eagle's medium (dmem, hyclone) supplemented with 10% fbs (gibco), l-glutamine (gibco), and 1% antibiotic (gibco). the virus strain a/wsn/1933 h1n1 (wsn), a mouse-adapted human influenza virus, was propagated and titrated in mdck cells. to identify the putative acetylation sites in influenza viral proteins, mass spectrometry was conducted with concentrated influenza virus. briefly, the h1n1 virus was propagated in mdck cells, and then a total of 100 ml of virus stock was prepared. to concentrate the virus, the collected virus was pelleted by centrifugation at 2000 g for 10 min to remove the cell debris. clarified virus supernatants were layered on a 30% (w/v) sucrose cushion and centrifuged at 200 000 g for 3 h. the virus pellet was suspended in water and subjected to mass spectrometry analysis performed by ptm biolabs llc (hangzhou, china). to generate mutant viruses, the site mutations k108r (deacetylation-mimic mutation) and k108q (constant acetylation-mimic) were introduced into the reverse genetic plasmid phw2000-wsn-ns by a commercial site-directed mutagenesis kit (invitrogen, grand island, usa). the mutant viruses were rescued in the background of wsn-h1n1 virus as described previously [32] , resulting in wsn-ns1-108r and wsn-ns1-108q viruses, and all the mutant viruses were verified by sequencing. then, the ns1-wt, ns1-108r, and ns1-108q genes were amplified and cloned into the pcdna3.0-flag expression vector (flag-ns1-wt, flag-ns1-108r, and flag-ns1-108q). the three viruses were inoculated on monolayer mdck and a549 cells cultured in 12-well plates with multiplicities of infection (mois) of 0.001 and 0.01 for each virus, respectively. each time point was set up in triplicate, and then the samples were collected at 12, 24, 36, and 48 hours post-inoculation (hpi). the supernatants were titrated on mdck cells cultured in 96-well plates following the reed and muench method to calculate tcid 50 /ml. the cells were collected and subjected to western blotting. briefly, the cell lysates were separated on a sodium dodecyl sulfate (sds)-polyacrylamide gel and transferred to a pvdf membrane. the membrane was blocked in pbs containing 5% skim milk and then incubated with rabbit anti-ns1 polyclonal antibody (genscript, piscataway, usa) and then with horseradish peroxidase-conjugated secondary anti-mouse antibody (thermo fisher, grand island, usa). the proteins were visualized by using an ecl kit (yeasen, shanghai, china). additionally, to determine the protein expression levels of the three ns1 expression plasmids, the flag-ns1-wt, flag-ns1-108q, and flag-ns1-108r plasmids were transfected into 293t cells. forty-eight hours post-transfection, the cells were collected and subjected to western blotting. forty-eight 4-week-old female balb/c mice were randomly allocated into four groups, and each group contained 12 mice. three groups were challenged with the indicated viruses, and one group was challenged with pbs as a control. the mice were inoculated with virus intranasally with 10 5.5 tcid 50 of virus in 50 µl solution under slight anaesthesia with co 2 . the mice were monitored for body weight, clinical signs, and survival rate each day until 14 days post-infection (dpi), and they were euthanized if they lost more than 25% of their original body weight. three mice from each group were euthanized at 3 and 5 dpi. the mouse lungs were collected for viral titration and cytokine analysis. to quantify the cytokine levels of il-1β, ifn-β, and tnf-α in mouse lungs, total rna was extracted from lung tissues, reverse-transcribed and subjected to quantitative realtime polymerase chain reaction as described previously [33] . to detect the ifn-β antagonistic ability of ns1 proteins, 293t cells were transfected with the indicated ns1 expression plasmids (0.2 μg/well) together with a plasmid expressing firefly luciferase under the control of the ifn-β promoter (pgl-ifn-β-luc, 0.2 μg/well), the renilla luciferase expressing plasmid prl-tk (0.07 μg/ well), and the ifn-β stimulator poly(i:c) (0.2 μg/well) or a plasmid expressing the active caspase recruitment domain (card) of rig-i (pcdna-rig-i 0.2 μg/well), pcdna-mavs (0.2 μg/well), pcdna-tbk1 (0.2 μg/well) or pcdna-irf3 (0.2 μg/well) as described previously [34] . twenty-four hours post-transfection, the cells were lysed and subjected to a dual-luciferase reporter assay kit (promega, madison, usa). mdck cells cultured on glass slides were infected with wsn-ns1-k108r, wsn-ns1-k108q, or wsn-wt at an moi of 1. all cells were fixed with 4% paraformaldehyde (pfa) and permeabilized with 0.1% triton x-100 in pbs at 12, 24, and 36 hpi. to detect the ns1 proteins, the fixed cells were incubated with rabbit anti-ns1 polyclonal antibody (genscript), followed by fitc-conjugated antirabbit igg antibody (yeasen), and then the cells were stained with dapi. all images were obtained on a leica tcs sp2 confocal microscope (leica microsystems inc., buffalo grove, usa). the animal study was conducted in accordance with the guidelines of the animal care and use committee of shanghai jiao tong university, and the animal study protocols were approved by shanghai jiao tong university (approval no. 20181012). all data were analysed using analysis of variance (two-way anova) in graph-pad prism version 5.0 (graphpad software inc., la jolla, usa); a p-value of 0.05 or less was considered significant. the mass spectrometry results showed that one acetylation modification was identified at position k108 of the ns1 protein of the wsn-wt virus (figure 1 ). to further explore whether k108 is subtype-specific, we compared the ns1 amino acid sequences of 1000 randomly selected influenza virus strains of each endemic subtype in birds and humans from genbank. most avian h9n2 (100%), h5n1 (99.9%), h7n9 (100%), human h3n2 (99.4%), and human h1n1 (99.9%, isolated before 2009) viruses contained k108 in the ns1 genes, whereas 99.9% of 2009 pandemic h1n1 viruses contained 108r in the ns1 protein. these data demonstrated that the k108 residue is relatively conserved in influenza viruses except the 2009 pandemic h1n1. since the ns1 protein is an important virulence marker and antagonist of host innate immunity, we chose to further explore the influence of acetylated k108 on virus replication and virulence in this study. to mimic deacetylated lysine, a k108r substitution was introduced into the ns1 protein, since an r substitution prevents acetylation but preserves the positive charge, and a mutant virus containing the ns1-k108r substitution was generated in the background of wsn virus (wsn-ns1-108r). moreover, to mimic constantly acetylated lysine at k108 of the ns1 protein, a k108q substitution that is a known acetylation mimic was introduced into ns1, resulting in a mutant virus containing ns1-k108q (wsn-ns1-108q). to determine the effect of acetylated k108 on virus replication, mdck and a549 cells were infected with wsn-wt or the two mutant virus wsn-ns1-108r (deacetylation mimic) or wsn-ns1-108q (constant acetylation mimic) at the indicated mois to obtain multicycle growth curves. all three viruses replicated efficiently in mdck and a549 cells, and wsn-ns1-108q and wsn-wt replicated to similar levels at each time point. however, the growth of the deacetylated mutant virus wsn-ns1-108r was significantly impaired compared with that of the other two viruses in mdck and a549 cells at 36 and 48 hpi, which indicated that the acetylated k108 of ns1 is important for virus replication in vitro at the late stage of infection (figures 2a and b) . similarly, ns1 protein levels were detected by western blotting. the results showed that the ns1 expression levels of wsn-ns1-108r were lower than those of wsn-wt and wsn-ns1-108q at different time points in mdck and a549 cells (figures 2c and d) . all the mice infected with viruses showed clinical signs such as ruffled fur, depression, and inappetence. the mice infected with constantly acetylated wsn-ns1-108q displayed more severe clinical signs and started to show mortality earlier than wsn-wt-infected mice. both wsn-ns1-108q and wsn-wt caused 100% mortality in infected mice. however, the deacetylated wsn-ns1-108r virus infection resulted in 80% mortality, and the mortality was delayed by 2 days compared with other viruses, which indicated that the deacetylation-mimic k108r substitution attenuated the wsn-ns1-108r virus in mice ( figures 3a and b) . virus titers were slightly lower in the lungs of mice infected with wsn-ns1-108r than in the other two groups ( figure 3c) . notably, significantly higher levels of ifn-β, il-1β, and tnf-α mrna were detected in wsn-ns1-108r-infected mice than in the other two groups at 3 dpi but not at 5 dpi ( figures 3d-f) , which indicated that wsn-ns1-108r was less efficient at inhibiting the innate immune response than wsn-ns1-108q and wsn-wt at 3 dpi in mice. to determine whether the k108r and k108q mutations affect the expression of the ns1 protein, the expression levels of flag-ns1-wt, flag-ns1-108q, and flag-ns1-108r in 293t cells were compared. the three proteins were expressed at similar levels in transfected 293t cells, which indicated that the k108r and k108q mutations did not influence protein expression ( figure 4e) . the major function of the ns1 protein is inhibition of type i ifn induction, and the acetylated k108 residue is located in the effector domain of ns1, which is important for its ifn antagonistic ability. to determine the effect of the acetylated k108 residue on ifn suppression by the ns1 protein, the inhibition of ifn-β promoter activity by flag-ns1-wt, flag-ns1-108q, and flag-ns1-108r was evaluated. the results showed that flag-ns1-wt and acetylation-mimic flag-ns1-108q suppressed the ifn-β promoter activity stimulated by poly(i:c) ( figure 4a) ; however, the deacetylation-mimic flag-ns1-108r protein was significantly less capable of inhibiting the activation of the ifn-β promoter compared with the acetylated ns1 proteins ( figure 4a ). this result indicated that the impaired ifn-β antagonistic ability might be responsible for the attenuation of the wsn-ns1-108q virus in vitro and in vivo. influenza virus infection stimulates type i ifn production by signal transduction from rig-i to tbk1 to irf3. to further explore how the k108r mutation attenuated the ifn-β antagonistic ability of the ns1 protein, we co-transfected ns1 expression plasmids, an ifn-β reporter plasmid and different type i interferon pathway components, including rig-i card, tbk1, and the active form of irf3, into 293t cells. the results showed that ns1-108q and ns1-wt inhibited the ifn-β response stimulated by each component more efficiently than ns1-108r, which suggested that the acetylated k108 residue is important for inhibiting the ifn-β response of ns1 that targets factors downstream of irf3 or other proteins (figures 4b-d) . two nuclear localization signals (35-41 and 216-221) have been identified in the ns1 protein, which drive ns1 to the nucleus during the early stage of infection. to determine whether k108r changes the subcellular localization of ns1 during infection, mdck cells were infected with the three viruses. the ns1 protein of wsnwt was located in the nucleus and cytoplasm of infected cells at 12 and 24 hpi ( figures 5a and b) . at the late stage of infection (36 hpi), the ns1 protein was mainly located in the nucleus and perinuclear area of infected cells (figure 5c ). the ns1-108q protein was located in both the nucleus and cytoplasm at 12 hpi and 24 hpi, while it was mainly located in the perinuclear area of infected cells at 36 hpi. in contrast, the ns1-108r protein accumulated mostly in the cytoplasm during the whole infection course. this result indicated that the deacetylation-mimic k108r substitution retained ns1 protein in the cytoplasm of infected cells, suggesting that the acetylated k108 residue is important for the nuclear localization of the ns1 protein ( figures 5a-c ). post-translational modification is important for protein function, stability, cellular localization, and protein-protein interactions. recent studies have shown that posttranslational modifications of viral proteins modulate the virus life cycle, e.g., phosphorylation of influenza viral proteins (ns1, m1, and np) plays important roles in virus replication [35] [36] [37] [38] . the ubiquitination of np and m2 proteins is crucial for viral rna replication and the production of infectious virus particles [37] . acetylation is an important post-translational modification in eukaryotes, but the occurrence and function of acetylation in influenza viral proteins remain largely unclear. giese et al. reported that acetylation of 77 k, 113 k, and 229 k of np proteins is important for virus polymerase activity and replication [27] . in the present study, we identified and characterized the acetylation of k108 in the ns1 protein, and the deacetylation of k108q affected viral replication in cells at 36 and 48 hpi. the expression levels of ns1-k108q and ns1-k108r in transfected 293t cells were similar ( figure 4e ), while the ns1 levels in infected mdck and a549 cells were different ( figures 2c and d) . this result could be attributed to the deacetylation affecting virus replication, which resulted in low expression of ns1 in infected cells. moreover, the acetylation of 108q contributes to the ifn antagonistic ability of the ns1 protein. the mrna levels of ifn-β, il-1β, and tnf-α in mouse lungs of the wsn-ns1-k108r-infected group were significantly higher than those in the other two groups at 3 dpi, which indicated that ns1-108r was less efficient at inhibiting the production of innate antiviral cytokines at 3 dpi in mice. the ns1 protein of influenza virus is a virulence factor that inhibits the antiviral immunity of the infected host, and c-terminal truncation has been widely used as a strategy to generate attenuated virus vaccine candidates [39] . one mechanism used by ns1 to inhibit the ifn response is through direct binding and sequestration of rna as well as direct interaction with trim25 and complex formation with the rna sensor rig-i, resulting in inhibition of the activation of the rig-i card and hence inhibition of irf3 activation [12] . the rna binding, rig-i and trim25 interacting domains are located in the n-terminus (1-73 aa) of the ns1 protein. however, the acetylated k108 is located in the ed domain, and acetylation of k108 may not affect the rna binding capability of the ns1 protein. the ns1-k108r substitution impaired the suppression of ifn promoter activation by poly(i:c), rig-1 card, tbk-1, and irf3, which suggested that the ns1-k108r substitution affected the ifn antagonism of ns1 either through targeting downstream of irf3 or a general mechanism that ns1 uses to inhibit ifn, such as interaction with cpsf30, resulting in inhibition of the processing of mrna, including ifn mrna [6] . notably, the cpsf30 protein is mainly located in the nucleus and is required for the 3′ end processing of all host pre-mrnas. interestingly, the deacetylation-mimic k108r substitution retained ns1 protein in the cytoplasm of infected cells, resulting in a possible impaired interaction between cpsf30 and the ns1 protein, subsequently leading to attenuated ifn antagonism. benjamin hale and colleagues found that the a/california/04/2009 (h1n1) virus has 108r in ns1, and ns1 was unable to suppress general host gene expression. nevertheless, the rk108 substitution in the ns1/2009 protein restored its ability to block general gene expression and bind cpsf30 [40] . this could explain why attenuation of the ifn antagonistic ability of ns1-k108r is independent of rig-i card, tbk-1, and irf3 activation. in addition, anastasina et al. [41] reported that the ns1 protein binds to cellular dna to block the cellular transcription of ifns and isgs; thus, ns1 proteins retained in the cytoplasm lose their cellular dna binding function, resulting in impaired ifn-β antagonistic ability. two known nuclear localization sequences (nlss) of ns1 proteins are located at the 35-41 and 219-232 positions. the 35-41 nls of the ns1 protein is highly conserved among influenza a virus strains [42] . the second nls (219-232) is virus strain specific, and the 2009 pandemic h1n1 lacks the second nls in the ns1 protein [43] . single point mutations, either r35a, r38a, or k41a, completely eliminated importin protein binding, which transports target proteins to the nucleus [42] . notably, in this study, acetylation of k108 located outside of the nls affected the cellular localization of ns1 protein, and the deacetylation-mimic k108r substitution blocked the nuclear localization of the ns1 protein in infected cells; however, the underlying mechanism remains unknown. interestingly, the ns1-k108 residue is relatively conserved in most influenza viruses, except for the 2009 pandemic h1n1. the 2009 pandemic h1n1 has 108r in ns1, which causes inefficient general host gene expression shutoff, while r108k restores its ability to block general host genes and bind cpsf30 [40] . potentially, the 2009 pandemic h1n1 virus might use different strategies to overcome the ifn response compared with the other influenza viruses. overall, we identified an acetylation of k108 of the ns1 protein of influenza virus, and the acetylation of k108 plays an important role in the cellular localization, ifn antagonistic ability, replication, and virulence of influenza virus. conformational plasticity of the influenza a virus ns1 protein inhibition of retinoic acid-inducible gene i-mediated induction of beta interferon by the ns1 protein of influenza a virus rig-i-mediated antiviral responses to single-stranded rna bearing 5′-phosphates immunogenicity and protection efficacy of replication-deficient influenza a viruses with altered ns1 genes structural basis for a novel interaction between the ns1 protein derived from the 1918 influenza virus and rig-i structural basis for suppression of a host antiviral response by influenza a virus influenza virus non-structural protein 1 (ns1) disrupts interferon signaling a site on the influenza a virus ns1 protein mediates both inhibition of pkr activation and temporal regulation of viral rna synthesis the primary function of rna binding by the influenza a virus ns1 protein in infected cells: inhibiting the 2′-5′ oligo (a) synthetase/rnase l pathway influenza a virus inhibits type i ifn signaling via nf-kappabdependent induction of socs-3 expression influenza a virus abrogates ifn-gamma response in respiratory epithelial cells by disruption of the jak/stat pathway the multifunctional ns1 protein of influenza a viruses influenza a virus virulence depends on two amino acids in the n-terminal domain of its ns1 protein to facilitate inhibition of the rnadependent protein kinase pkr contribution of ns1 effector domain dimerization to influenza a virus replication and virulence ns1 protein amino acid changes d189n and v194i affect interferon responses, thermosensitivity, and virulence of circulating h3n2 human influenza a viruses the k186e amino acid substitution in the canine influenza virus h3n8 ns1 protein restores its ability to inhibit host gene expression roles of the phosphorylation of specific serines and threonines in the ns1 protein of human influenza a viruses threonine 80 phosphorylation of non-structural protein 1 regulates the replication of influenza a virus by reducing the binding affinity with rig-i modification of nonstructural protein 1 of influenza a virus by sumo1 50 years of protein acetylation: from gene regulation to epigenetics, metabolism and beyond proteomics analyses reveal the evolutionary conservation and divergence of n-terminal acetyltransferases from yeast and humans acetylation and methylation of histones and their possible role in the regulation of rna synthesis acetylation and deacetylation of non-histone proteins the world of protein acetylation genetic dissection of histone deacetylase requirement in tumor cells the many roles of histone deacetylases in development and physiology: implications for disease and therapy role of influenza a virus np acetylation on viral growth and replication suppression of the antiviral response by an influenza histone mimic n-terminal acetylation by natb is required for the shutoff activity of influenza a virus pa-x hdac6 restricts influenza a virus by deacetylation of the rna polymerase pa subunit acylation-mediated membrane anchoring of avian influenza virus hemagglutinin is essential for fusion pore formation and virus infectivity analysis of recombinant h7n9 wild-type and mutant viruses in pigs shows that the q226l mutation in ha is important for transmission quantification of murine cytokine mrnas using real time quantitative reverse transcriptase pcr herpes simplex virus 1 ubiquitinspecific protease ul36 inhibits beta interferon production by deubiquitinating traf3 mapping the phosphoproteome of influenza a and b viruses by mass spectrometry effects of the s42 residue of the h1n1 swine influenza virus ns1 protein on interferon responses and virus replication ubiquitination of the cytoplasmic domain of influenza a virus m2 protein is crucial for production of infectious virus particles phosphorylation and dephosphorylation of threonine 188 in nucleoprotein is crucial for the replication of influenza a virus attenuation of the virulence of a recombinant influenza virus expressing the naturally truncated ns gene from an h3n8 equine influenza virus in mice inefficient control of host gene expression by the 2009 pandemic h1n1 influenza a virus ns1 protein influenza virus ns1 protein binds cellular dna to block transcription of antiviral genes nuclear and nucleolar targeting of influenza a virus ns1 protein: striking differences between different virus subtypes influenza a h3n2 subtype virus ns1 protein targets into the nucleus and binds primarily via its c-terminal nls2/nols to nucleolin and fibrillarin this study was supported by the national natural science authors' contributions jm, jl, and js designed the study; jm was involved in the acquisition of data, analysis, and figure preparation; rw, gx, yc, and zw contributed to some of the laboratory experiments and data analysis; hw and yy helped revise the manuscript; jl and js supervised the study; jm drafted the original paper. all authors read and approved the final manuscript. the authors declare that they have no competing interests. key: cord-005664-n4xv247l authors: plötz, frans b.; vreugdenhil, harriet a.; slutsky, arthur s.; zijlstra, jitske; heijnen, cobi j.; van vught, hans title: mechanical ventilation alters the immune response in children without lung pathology date: 2002-01-15 journal: intensive care med doi: 10.1007/s00134-002-1216-7 sha: doc_id: 5664 cord_uid: n4xv247l objective: this study was undertaken to examine the hypothesis that mechanical ventilation in association with anesthesia would alter the cytokine profile in infants without preexisting lung pathology. design and setting: prospective observational study in pediatric intensive care unit in a university hospital. patients: twelve infants who were subjected to an uncomplicated diagnostic cardiac catheterization procedure were studied. all subjects were ventilated with a volume control mode, 0.3 fio(2), 4 cmh(2)o peep, and 10 ml/kg tidal volume. volatile (servoflurane) anesthetics were given. measurements and results: tracheal aspirates and blood samples were obtained before and after 2 h of mechanical ventilation. in tracheal aspirates and in supernatants of stimulated whole-blood cultures cytokine concentrations were measured. in the tracheal aspirates the immune balance was characterized by a proinflammatory response pattern, with a significant increase in tnf-α and il-6 concentrations; concentrations of anti-inflammatory mediators remained very low. the functional capacity of peripheral blood leukocytes to produce inf-γ, tnf-α, and il-6 in vitro was significantly decreased. this was accompanied by a significant decrease in the killing activity of natural killer cells. conclusions: two hours of servoflurane and mechanical ventilation using a tidal volume of 10 ml/kg is associated with remarkable changes in the immune response in infants without preexisting lung pathology undergoing cardiac catheterization. in the lungs the immune balance favors a proinflammatory response pattern without detectable concentrations of anti-inflammatory mediators. the th1 immune response by peripheral blood leukocytes was decreased. the observed change in th1/th2 balance in favor of th2 cytokine activity may be a systemic adaptation to the proinflammatory milieu in the lung. blood leukocytes to produce inf-γ, tnf-α, and il-6 in vitro was significantly decreased. this was accompanied by a significant decrease in the killing activity of natural killer cells. conclusions: two hours of servoflurane and mechanical ventilation using a tidal volume of 10 ml/kg is associated with remarkable changes in the immune response in infants without preexisting lung pathology undergoing cardiac catheterization. in the lungs the immune balance favors a proinflammatory response pattern without detectable concentrations of antiinflammatory mediators. the th1 immune response by peripheral blood leukocytes was decreased. the observed change in th1/th2 balance in favor of th2 cytokine activity may be a systemic adaptation to the proinflammatory milieu in the lung. it has become clear that alterations in the immune balance may prevent an appropriate and effective response to various stimuli [1, 2] . cd4 + t-cells can be divided functionally into th1 and th2 cells based on their cytokine profiles [3] . th1 cells secrete interferon (ifn) γ while th2 cells secrete interleukin (il) 4, il-5, il-10, and il-13. macrophages secrete proinflammatory and anti-inflammatory cytokines such as il-1β, tumor necro-sis factor (tnf) α, il-12, and il-10. for example, an alteration in the th1/th2 balance, resulting in a th2 dominance, is thought to contribute to enhanced pulmonary disease in respiratory syncytial virus bronchiolitis [4] . on the other hand, new evidence indicates that a disturbance of the balance between proinflammatory mediators and anti-inflammatory mediators may initiate or amplify the inflammatory response in patients with the acute respiratory distress syndrome (ards) [1, 5] . for example, the ratio of il-1β to il-1 receptor antagonist is markedly elevated in patients with ards, favoring the unopposed proinflammatory activity of il-1β. the observation that low intrapulmonary concentrations of il-10 and il-1 receptor antagonist at the onset of ards are associated with a poor outcome suggests that a lack of inhibitory cytokines is correlated with a poor prognosis. it has also been suggested that mechanical ventilation produces alterations in the immune balance [6] . experimental studies have demonstrated that mechanical ventilation results in an inflammatory reaction in the lungs and that the degree of inflammation depends on the ventilatory strategy and mode [7, 8, 9, 10, 11] . this inflammatory reaction may not be limited to the lungs but may initiate or propagate multiple system organ failure [12, 13, 14] . a possible explanation for the spillover of inflammatory mediators as a result of mechanical ventilation is loss of compartmentalization [15] . the important concept of compartmentalization refers to the fact that the inflammatory response remains compartmentalized in the area of the body were it is produced [16, 17] . haitsma et al. [18] have shown in rats that injurious ventilatory strategies, although not conclusive, disturb the compartmentalization of the early cytokine response in both the lung and the systemic circulation [15] . infants who undergo cardiac catheterization may have multiple risk factors that may affect the inflammatory milieu in their lungs, including mechanical ventilation, exposure to anesthetic agents, and the stress of the procedure. the present study was designed to examine the hypothesis that mechanical ventilation in association with anesthesia would alter the cytokine profile in the lungs, and/or systemic circulation, of patients without preexisting lung pathology. the study included 12 children (median age 3.5 years, range 1-11) who were undergoing a diagnostic cardiac catheterization procedure. the children had a history of a congenital heart disease, some of whom had been (partially) corrected: atrial-ventricular septal defect, transposition of the great arteries [2] , aortic valve insufficiency, ventricular septal defect [2] , tetralogy of fallot, coarctation of aorta, tricuspid atresia, pulmonary atresia [2] , double outlet right ventricle. patients with a history of allergic or respiratory diseases, known chromosomal or immunological disorders, and patients recently hospitalized or mechanically ventilated were excluded. all subjects were intubated and ventilated with a volume control mode and a fractional inspiratory oxygen of 0.3, a maximum peak inspiratory pressure of 19.1±2.0 cm h 2 o, a mean positive end-expiratory pressure (peep) of 3.8±1.0 cm h 2 o, and a mean tidal volume of 9.95±0.95 ml/kg (measured body weight). the end-tidal co 2 was maintained between 35-45 mmhg. if peep, inspiratory oxygen concentration, or tidal volume needed to be adjusted to maintain an adequate oxygenation or to maintain normocapnia, the patient was excluded from the study. heart rate and blood pressure of the individual patients remained constant during the procedure. all patients received servoflurane (3.75%) anesthetic during the procedure. the study was approved by the medical ethics committee, and parents gave informed consent. tracheal aspirates and blood samples were obtained immediately after intubation, before the start of mechanical ventilation, and after 2 h of mechanical ventilation. tracheal aspirates were obtained as previously described [19] . the suction catheter was rinsed with 0.5 ml sterile normal saline and added to the suction trap. the aspirate was placed immediately on ice. thereafter 10% dithiothreitol (10%; 100 µl per 1 ml aspirate) was added, and the samples were centrifuged at 1500 rpm for 5 min. supernatants were stored at -80°c until analysis. blood samples were drawn from a venous catheter. heparinized blood was diluted 1:10 in rpmi-1640 medium (roswell park memorial institute life technologies, grand island, n.y., usa), and whole-blood cultures were set up. the whole-blood culture stimulated with lipopolysaccharide (lps) is a suitable ex vivo method to study monocyte cytokine production under conditions in which many of the physiologically relevant cellular interactions remain intact [20, 21] . to induce lymphocyte cytokine production (il-4, ifn-γ) anti-cd2,1 and anti-cd2,2 (1:12000) plus anti-cd28 (1:3000) monoclonal antibodies (clb, amsterdam, the netherlands) were added, and cultures were incubated for 72 h at 37°c in 5% co 2 in air. all cultures were performed in quadruplicate. to induce the production of monocyte il-6, il-8, tnf-α, lps (difco laboratories, detroit, mich., usa) (1 ng/ml) was added to the diluted blood samples and cultures were incubated for 24 h at 37°c in 5% co 2 in air. to induce monocyte il-10 production lps (1 ng/ml) was added, and cultures were incubated for 48 h at 37°c in 5% co 2 in air. to induce monocyte il-12 production lps (100 ng/ml) and ifn-γ (20 ng/ml) were added, and cultures were incubated for 24 h at 37°c in 5% co 2 . addition of ifn-γ results in a more optimal il-12 response in the presence of lps. cytokine assays tnf-α, il-4, il-6, il-8, il-10, il-12, and ifn-γ were measured via enzyme-linked immunosorbent assay (clb). the detection limit was 4-6 pg/ml for tnf-α, 0.6 pg/ml for il-4, 1 pg/ml for il-6, 4-8 pg/ml for il-8, 3-5 pg/ml for il-10, 3 pg/ml for il-12, and 4-6 pg/ml for ifn-γ. when cytokines were not detectable, the minimum detectable level was used in the calculations. the composition of peripheral leukocytes was determined by analyzing the forward-sideward scatter. for lymphocyte subset analysis, whole blood was incubated with conjugated monoclonal antibodies under saturating conditions specific for cd3, cd4, cd8, and cd16/56 (simultest, becton and dickinson, mountain view, calif., usa). subsequently, red blood cells were lysed and samples were analyzed with a flow cytometer (facs-star+, becton and dickinson). the difference between negative and positive fluorescence was determined by measuring unstained cells and cells stained with an irrelevant isotype control body. natural killer cell activity natural killer cell (nk) cell activity was analyzed by determining the capacity of peripheral blood cells to kill 51 cr-labeled k562 target cells as described previously [22] . cortisol was measured by a chemiluminescence immunoassay performed on the fully automated advia centaur immunoanalyzer (bayer, leverkusen, germany). all values were expressed as mean ±sd and were analyzed by the nonparametric wilcoxon signed-rank test. differences were considered significant at the level of p<0.05. the concentrations of tnf-α in the supernatant of the tracheal aspirates increased significantly 2 h after me-chanical ventilation (p=0.01; fig. 1 ). there was a trend towards an increase in il-6 levels (p=0.05; fig. 1 ). il-8 concentrations showed high interindividual variation both before and after mechanical ventilation. the concentrations of the anti-inflammatory cytokines il-10 and ifn-γ remained unchanged just above the detection level (fig. 1 ). the capacity of lymphocytes to produce cytokines was determined in whole-blood cultures stimulated with anti-cd2/cd28 [20, 21] . after 2 h of mechanical ventilation a significant decrease in ifn-γ production was observed in the cultured supernatants (p=0.01), but no significant changes in il-4 concentrations were observed (fig. 2) . the capacity of monocytes to produce cytokines was determined in whole-blood cultures stimulated with lps. after 2 h of mechanical ventilation there was a decrease in the production of proinflammatory cytokines il-6 (p<0.05) and tnf-α by peripheral blood monocytes (p<0.05; fig. 2 ). il-8 concentrations showed high interindividual variation before and after mechanical ventilation. the amount of il-10 and il-12 produced by monocytes was unaltered in all patients as a result of 2 h of mechanical ventilation (fig. 2) . we observed significant changes in the cellular composition of the whole-blood samples. there was a increase in the percentage of granulocytes (p<0.05) and a decrease in the percentage of lymphocytes (p<0.05; table 1 ). the percentage of cd3 and cd4 increased slightly but significantly (p<0.05). the percentage of cd16/56 tended to decrease (table 1) . as a result of 2 h of mechanical ventilation the killing capacity of nk cells to lyse k562 target cells decreased significantly (p<0.01). the mean percentage of activity decreased from 35.1±5.1 to 22.3±4.3. this remarkable decrease in killing capacity of nk cells cannot be explained by a decrease in the total numbers of nk cells (table 1) . the major finding of the present study is that exposing infants with normal lung function to 2 h of volatile anesthetics, mechanical ventilation, and cardiac catheterization is associated with remarkable changes in immune responses. we observed a proinflammatory response in the lungs with a significant increase in tnf-α, while antiinflammatory cytokine concentrations in tracheal aspirates remained virtually unchanged, just above the detection level. in addition, the functional capacity of peripheral blood leukocytes to produce proinflammatory cytokines in vitro was significantly decreased, in particular ifn-γ, tnf-α, and il-6. this was accompanied by a significant decrease in the activity of nk cells. this indicates that this procedure is associated with a change in the th1/th2 balance with a decreased th1 immune response. a major question from our study is which aspect of the total procedure consisting of exposure to volatile anesthetics, ventilation, and catheterization is responsible for the observed changes in the inflammatory response of our patients. a recent review article summarized the effect of anesthetic agents on the immune response and concluded that there is little evidence to support the concept of clinically relevant immune modulation by anesthetics during major surgery [23] . no clinical study has examined the effect of servoflurane on the immune response in infants and young children. experimental studies have shown that during mechanical ventilation of uninjured lungs several volatile anesthetics may augment gene expression of proinflammatory cytokines in rat alveolar macrophages [24] . however, servoflurane was not associated with a significant increase in gene expression of proinflammatory cytokines or with concentrations of tnf-α in the lavage fluid of these rats over that with mechanical ventilation alone [24] . kotani et al. [25] demonstrated in mechanically ventilated adult patients that intravenous propofol or volatile isoflurane produced a similar increase in gene expression of all proinflammatory cytokines on alveolar macrophages. this is remarkable since the route of administration of these anesthetics are completely different. one would have expected that by directly acting on alveolar macrophages the volatile anesthetic -isoflurane -would induce faster and probably more pronounced gene expression. it therefore remains questionable whether these clinical observed effects are all attributable to general anesthesia. any effect of anesthesia is likely to be overwhelmed by the neuroendocrine stress response during major sur-gery [23] . however, in our study the response of the hypothalamo-pituitary-adrenal axis to the catheterization procedure is probably negligible. serum cortisol levels measured before and after mechanical ventilation were similar. other factors such as hemorrhage, hypotension, ischemia/reperfusion, and blood transfusion, which may affect immune competence during major surgery, were negligible in our study. thus the catheterization procedure can therefore not considered to be major surgery. we are therefore left with the possibility that the changes in the immune response in our study were the result of mechanical ventilation, although we are aware that definitive conclusions cannot be made. several experimental studies have reported that injurious ventilatory strategies increase tnf-α mrna expression and lung lavage levels of tnf-α protein [7, 9, 11] . in these studies tidal volumes were very high (40 ml/kg), and/or there was preexisting lung injury. pretreatment with intratracheal instillation of anti-tnf-α antibodies improved oxygenation, reduced infiltration of leukocytes, and ameliorated pathological findings [26] . the results of the experimental studies clearly demonstrated that tnf-α plays a pivotal role in initiating an inflammatory cascade induced by mechanical ventilation. the lung macrophage may be the critical mechanosensor cell capable of producing tnf-α in response to stretching mechanical forces [27], although there is evidence that the pulmonary epithelium may also be a key player in this regard [28] . it is remarkable, however, that such a significant proinflammatory response was observed with the ventilatory strategy we adopted. our patients had normal lungs, and a tidal volume of 10 ml/kg should not cause overdistention, since the patients would not have the marked heterogeneities in pulmonary compliance that exist in patients with ards [29, 30] . this is supported by the observation that peak inspiratory pressures remained low (19.1±2.0 cmh 2 o) throughout the 2-h period. to our knowledge, only one other study has examined the effect of mechanical ventilation on release of cytokines in patients with normal lung function [31] . wrigge et al. [31] observed that after 1 h of mechanical ventilation plasma levels of pro-and anti-inflammatory mediators remained low and did not differ from baseline. unfortunately, the local production of cytokines in the lung was not measured. the observed effects in our study may be explained by a two-hit hypothesis in which any one factor by itself does not induce an effect, but a combination of factors act synergistically to cause the changes in immune response, i.e., mechanical ventilation and volatile anesthetics. it remains speculative what causes the onset of the peripheral immune response. one of the mechanisms could be that tnf-α produced locally in the lung causes leukocyte redistribution from the systemic circulation into the alveolar space [9, 11] . mechanical ventilation may recruit t cell subsets with distinctive properties with respect to homing and trafficking into inflamed sites [32] . we observed that the functional capacity of peripheral blood leukocytes to produce proinflammatory cytokines in vitro was significantly decreased, in particular inf-γ, tnf-α, and il-6. ifn-γ is associated with a th1 response, which is considered to be beneficial in terms of an appropriate and effective response to various stimuli, including trauma, infection, and perhaps mechanical ventilation [2] . ifn-γ is also important in stimulating the cytolytic activity of nk cells and cd8 + cytotoxic t lymphocytes. the decrease in ifn-γ production was also accompanied by a significant decrease in the killing activity of nk cells. the altered th1/th2 balance in favor of th2 cytokine activity may be a systemic adaptation to the proinflammatory milieu in the lung. in conclusion, 2 h of servoflurane and mechanical ventilation with a tidal volume of 10 ml/kg is associated with remarkable changes in the immune response in infants without preexisting lung pathology undergoing cardiac catheterization. in the lungs a proinflammatory response pattern dominates without detectable concentrations of anti-inflammatory mediators. we observed a decrease in the th1 immune response by peripheral blood leukocytes. the altered th1/th2 balance in favor of th2 cytokine activity may be a systemic adaptation to the proinflammatory milieu in the lung. further studies possibly using different anesthetic agents, different operative procedures, and different ventilatory strategies are needed to establish the mechanisms and clinical relevance of our findings. cytokines and the acute respiratory distress syndrome (ards): a question of balance dominance of t-helper 2-type cytokines after severe injury human th1 and th2 subsets: doubt no more anti-il-4 treatment at immunization modulates cytokine expression, reduces illness, and increases cytotoxic t lymphocyte activity in mice challenged with respiratory syncytial virus the acute respiratory distress syndrome ventilator-induced lung inflammation: is it always harmful? injurious ventilatory strategies increase cytokines and c-fos m-rna expression in an isolated rat lung model role of high-frequency ventilation in surfactant-depleted lung injury as measured by granulocytes inflammatory chemical mediators during conventional ventilation and during high frequency oscillatory ventilation ventilator pattern influences neutrophil influx and activation in atelectasis-prone rabbit lung intraalveolar expression of tumor necrosis factor-alpha gene during conventional and high-frequency ventilation multiple system organ failure: is mechanical ventilation a contributing factor? effect of mechanical ventilation on inflammatory mediators in patients with acute respiratory distress syndrome: a randomized clinical trial mechanical ventilation as a mediator of multisystem organ failure in acute respiratory distress syndrome compartmentalized lung cytokine release in response to intravascular and alveolar endotoxin challenge loss of compartmentalization of alveolar tumor necrosis factor after lung injury ventilator-induced lung injury measurement of interleukin 10 in bronchoalveolar lavage from preterm ventilated infants a convenient whole blood culture system for studying the regulation of tumor necrosis factor release by bacterial lipopolysaccharide monocyte il-10 production during respiratory syncytial virus bronchiolitis is associated with recurrent wheezing in a one year follow-up study the authors thank the pediatric cardiologists and cardioanesthetists for their technical assistance. key: cord-003514-yyzbv7ys authors: arslan, mehboob; yang, xin; santhakumar, diwakar; liu, xingjian; hu, xiaoyuan; munir, muhammad; li, yinü; zhang, zhifang title: dynamic expression of interferon lambda regulated genes in primary fibroblasts and immune organs of the chicken date: 2019-02-14 journal: genes (basel) doi: 10.3390/genes10020145 sha: doc_id: 3514 cord_uid: yyzbv7ys interferons (ifns) are pleiotropic cytokines that establish a first line of defense against viral infections in vertebrates. several types of ifn have been identified; however, limited information is available in poultry, especially using live animal experimental models. ifn-lambda (ifn-λ) has recently been shown to exert a significant antiviral impact against viral pathogens in mammals. in order to investigate the in vivo potential of chicken ifn-λ (chifn-λ) as a regulator of innate immunity, and potential antiviral therapeutics, we profiled the transcriptome of chifn-λ-stimulated chicken immune organs (in vivo) and compared it with primary chicken embryo fibroblasts (in vitro). employing the baculovirus expression vector system (bevs), recombinant chifn-λ3 (rchifn-λ3) was produced and its biological activities were demonstrated. the rchifnλ3 induced a great array of ifn-regulated genes in primary chicken fibroblast cells. the transcriptional profiling using rna-seq and subsequent bioinformatics analysis (gene ontology, differential expressed genes, and keggs analysis) of the bursa of fabricious and the thymus demonstrated an upregulation of crucial immune genes (viperin, ikkb, ccl5, il1β, and ap1) as well as the antiviral signaling pathways. interestingly, this experimental approach revealed contrasting evidence of the antiviral potential of chifn-λ in both in vivo and in vitro models. taken together, our data signifies the potential of chifn-λ as a potent antiviral cytokine and highlights its future possible use as an antiviral therapeutic in poultry. viral pathogens pose significant threats to the poultry industry around the globe. this necessitates the development of novel and alternative antiviral therapies to contain the impacts of pathogens. avian influenza viruses (aivs) are a particular threat, which cause severe damage to the poultry industry, especially in developing countries where huge monetary losses are incurred [1, 2] . public health is also threatened by aivs, owing to their zoonotic importance. active preventive strategies would minimize the risk of viral transmission to humans and also benefit the poultry industry. interferons (ifns) are pleiotropic functional cytokines with antiviral, antitumor, and natural immune-boosting effects. ifns play a significant role in eliciting an antiviral state in vertebrates [3] . ifns are broadly categorized into three distinct types based on their molecular structure, receptor specificity, and induction pathway [4] . type i ifns include ifn-α, ifn-β, ifn-ε, ifn-κ, and ifnω, and all signal via common cell surface receptors (ifnαr-1) and (ifnαr-2), which are situated on a broad range of cells [3] . type ii ifns consist of ifn-γ, which is activated through highly specific ligand interactions with distinct ifn-γ receptors (ifn-γr1) and (ifn-γr2). the third family of ifns consists of ifn lambda, which interacts with a heterodimeric receptor complex (il-28rα and il-10β). ifn-λ was first discovered in mammals and subdivided into ifn-λ1 (also known as il-29), ifn-λ2 (il-28a), ifn-λ3 (il-28b), and ifn-λ4 [5] . ifns are crucial in an innate immune response, as their expression and antiviral potential is dependent on their cognate receptor interaction in a particular system [6] . in chickens, type i ifns primarily interact in fibroblasts, whereas epithelial cells (gastrointestinal and respiratory tract) are the primary site for the actions of type iii ifns [7] . despite morphological diversity, ifns share integrated, interconnected, and a precisely coordinated cascade in immunity pathways [3] . ligand recognition and interaction by ifn receptors results in rapid activation of janus kinase/signal transducers and activators of transcription (jak-stat pathway). this leads to phosphorylation of stat1 and stat2, activation of interferon stimulated gene factor 3 (isgf3), binding of ifn-stimulated response elements (isres), and expression of ifn stimulated genes (isgs) [8] . once expressed, these isgs demonstrate an essential role in the antiviral response. it is evident from published data that ifns upregulate identical sets of isgs, which in turn express antiviral proteins. ifn-induced transmembrane protein (ifitms), viperin and myxovirus resistance protein (mx) are some of the potent antiviral proteins expressed in response to viral infections [9] . once expressed, these isgs control viral replication, which provides an antiviral atmosphere to limit viral propagation in infected cells. compared to the mammalian ifn-λ repertoire (ifn-λ1, ifn-λ2, ifn-λ3, and ifn-λ4), chicken ifn-λ is the sole member in birds and demonstrates structural identity with human ifn-λ3. ifn-λ is chiefly involved in protection against viral infection of the respiratory and gastrointestinal tract epithelia (aiv, ndv, ibv), and due to the distribution of il-28rα in epithelium-rich organs, ifn-λ demonstrates significant potential to limit viral propagation [10] . while most of the current studies in chickens are mainly focused on type i and type ii ifns, we investigated the potential of type iii ifns in innate and adaptive immunity. previously, it was established that chifn-α presented a significant delay in the propagation of rous sarcoma virus and confirmed in vivo [11] . it was also revealed that chifn-α treatment ameliorates infection progression in experimental chickens with highly pathogenic influenza a virus (hpaiv) subtype h5n1 [12] . compared to type i ifns, chifn-λ has also been shown to elicit moderate antiviral response in both the chicken macrophage cell line hd11 and primary chicken embryo fibroblasts (cef) [13] . another published study demonstrated that cefs treated with recombinant chifn-λ induced isgs in a temporal fashion [14] . however, the antiviral potential of chifn-λ in live animals (e.g., chickens) has not yet been investigated, which could provide evidence for the potential of chifn-λ in animals per se. to investigate the impact of exogenous chifn-λ on the innate immune system in chickens, we first expressed chifn-λ in a silkworm bioreactor platform utilizing a baculovirus expression vector system (bevs) [15] . compared to the autographa californica nucleopolyhedrovirus (acmnpv)-sf9 cell expression system, the bombyx mori nucleopolyhedrovirus (bmnpv)-silkworm system possesses greater post-translational modifications and enhanced expression efficiency [16, 17] . comparative transcriptomic profiling revealed the key mechanisms, signaling pathways, and expression patterns of genes involved in interferon-induced immunity. our results highlight the dynamics of chifn-λ roles in chicken innate immunity. bm5 cells (bombyx mori-derived cell line) were cultured and maintained at 27 • c with 10% fetal bovine serum (fbs, gibco, usa) in tc100 (insect cell culture medium) (applichem, darmstadt, germany) as per the published literature [18] . for co-transfection, bm5 cells were cultured at a constant density of 1 × 10 6 cells per well in six well plates for 12 hours with tc100 media containing fbs. tc100 media without fbs was used to wash the cells twice and a mixture of transfection and co-transfection was introduced to cells. between 4-6 h post-transfection, fbs was introduced to the cell culture media. for viral amplification and expression, cells were infected with a multiplicity of infection (moi) of 0.1 for 1-2 h. the ensembl chicken genome database (ftp://ftp.ensembl.org/pub/release-93/fasta/gallus_ gallus/dna/) was extensively screened for homologues of chifn-λ by employing the blast algorithm (http://www.ncbi.nlm.nih.gov/blast/). a stretch of sequences demonstrating high sequence identity was identified and characterized. 1] were acquired from the national center for biotechnology information (ncbi) and aligned using the clustalw program, and phylogenetic analysis was performed using the neighbor-joining method with bootstrap n = 1000 in mega software (version 7). amino acid sequences of ifn-λ from multiple species were aligned using the clustalw algorithm. the espript 3.0 (http://espript.ibcp.fr/espript/cgi-bin/espript.cgi) was utilized to analyze the sequences. in our previous study, we developed a novel defective-rescue recombinant bombyx mori bacmid (rebmbac) expression system [15] . we used this in-house built and developed system to express chifn-λ. the rebmbac-silkworm expression system was employed to construct chifn-λ (interferon lambda-3 [gallus gallus]; sequence id: xp_015144667.1; length: 186). briefly, in order to enhance expression efficiency by codon optimization, chifn-λ genes were optimized for expression in the silkworm (bombyx mori) and synthesized by genscript company (china). plasmid-containing orf1629+ with gene of interest (chifn-λ) and pph as a promoter was co-transfected with rebmbac in the bm5 cell line. recombinant virus containing the chifn-λ gene was harvested 4-5 days post co-transfection. expression product was acquired after 4-5 days of silkworm/pupae infection. the plaque assay was performed to evaluate the recombination efficiency [18] . luciferase assay kit (promega, usa) was employed to analyze expression quantity of luciferase in 50 µg of protein lysate. the bradford method was used to measure the amount of protein [19] . antiviral activity of chifn-λ was assayed in the gfp-reduction assay using recombinant vesicular stomatitis virus (vsv-gfp) [20] . cefs were prepared from 9-11 days old specific pathogen free (spf) chicken eggs and maintained in cell culture flasks [21] . after 24 hours, cefs were stimulated with chifn-λ and cells were harvested after 12 hours post treatment, snap frozen, and stored at −80 • c for further processing. all experiments were performed in triplicate. the present study was conducted in accordance with animal ethics guidelines and approval was given by the beijing administration office of laboratory animals, china. a total of 60 newly hatched spf chicks were obtained from beijing arbor acre company ltd., p.r. china. chicks were reared in cages (n = 10 birds/cage) and placed in six cages in a temperature-controlled environment at the biotechnology research institute, chinese academy of agricultural sciences (caas), p.r. china. birds were offered standard commercial feed obtained from cp group ltd., p.r. china. unrestricted access to water was provided via nipple drinker lines and ad libitum feed was offered. a treatment group of 14-day old chicks were injected daily with chifn-λ (10,000 iu/kg body weight) (105 iu/ml). phosphate buffer saline (pbs) was injected intramuscularly to the control group. the bursa of fabricious and thymus were obtained by euthanizing the chickens at five days post-treatment. tissue samples were rapidly collected, snap-frozen in liquid nitrogen, and stored at −80 • c for further processing. total rna was extracted from virus-infected or mock-treated cefs (in triplicates), as per manufacturer's guidelines [22] . similarly, a total of five immune organs (bursa of fabricious and thymus) were pooled (in duplicates) from randomly selected chicken from each virus-or mock-infected group. total rna extraction was performed as per manufacturer's instructions [22] . extracted rna quality was analyzed by employing 1% agarose gel and rna integrity was assured using rna nano 6000 assay kit from bioanalyzer 2100 system (agilent technologies, ca, usa). extracted samples were sent to novogene beijing for sequencing. samples were sequenced using hiseq x ten (ilumina) and pe150 platforms. rna-seq generated from cef, bursa of fabricious and thymus samples of chicken (both chifnλ-treated and control groups) are presented in supplementary table s1 . reads were mapped to the reference genome database (ftp://ftp.ensembl.org/pub/release-89/fasta/gallus_gallus/dna/). individually mapped reads for each sample were assembled by stringtie (v1.3.3b) using a reference-based approach. featurecountsv1.5.0-p3 was utilized to estimate read numbers mapped to each gene. fragments per kilo base of transcript sequence per million base pairs sequenced (fpkm) of each gene was analyzed on the basis of length of gene and read count mapped to this gene. differential expression analysis was accomplished by employing deseq2 r package (1.16.1). using benjamini and hochberg's approach, p-values were adjusted for controlling false discovery rate (fdr). genes with (p < 0.05, |log2fold change|>1) observed by deseq2 were designated as differentially expressed. for differentially expressed genes, both gene ontology (go) enrichment analysis and kyoto encyclopedia of genes and genomes (kegg) pathway enrichment was conducted using the clusterprofiler r package. go terms with adjusted p-values < 0.05 were considered as significantly enriched (http://www.genome.jp/kegg/). using the chicken ifn gene as a query, we constructed the phylogenetic tree by employing the neighbor joining method (bootstrap n = 1000). this demonstrates the relationship of chifnλ with its mammalian orthologues by illustrating that chifn-λ is distinct in its evolution. furthermore, this revealed the contrasting consensus sequence from databases including ensembl and genbank. chifn-λ encodes a putative protein of 186 amino acids and further demonstrates typical characteristics of type iii ifns. a pairwise blast analysis demonstrated that chifn-λ shares 36%, 34%, 39%, 34% and 33% sequence similarity with recently characterized pig, mouse, human, cattle, and frog ifn-λ, respectively. based on amino acid homology, conserved amino acids among distinct avian and mammalian ifn-λ are identified. taken together, this comparative characterization further shows that chifn-λ shares characteristic features of type iii ifns (supplementary figure s1a ,b). in order to construct chifn-λ, we employed a bevs study. in order to determine the expression efficiency, we used a luciferase reporter gene for quality control as we described previously [15] . the luciferase gene was acquired from pgl3-basic vector by employing bglii/xbai digestion and insertion into the bamhi/xbai-digested pvl1393 vector to construct pvl1393-luc vector. a combination of pvl1393-luc and rebmbac dna was co-transfected in bm5 cells (figure 1) . a viral plaque assay was used to determine a suitable virus strain with which to express luciferase. supernatant from bm5 cells containing recombinant bmnpv (rebm-luc) was harvested five days post-transfection before inoculation into silkworms. after four to five days, protein was harvested from silkworms and 50 µg protein from lysed larval haemolymph was subjected to luciferase assays. luminescence detected from silkworm larval haemolymph was approximately 3.42 ± 0.52 × 10 8 relative light units (rlu), compared to 150-300 rlu from luc-negative virus-infected samples. pcr amplification (qpcr) further verified and validated the chifn-λ gene expression in bevs (supplementary figure s1c ). in order to construct chifn-λ, we employed a bevs study. in order to determine the expression efficiency, we used a luciferase reporter gene for quality control as we described previously [15] . the luciferase gene was acquired from pgl3-basic vector by employing bglii/xbai digestion and insertion into the bamhi/xbai-digested pvl1393 vector to construct pvl1393-luc vector. a combination of pvl1393-luc and rebmbac dna was co-transfected in bm5 cells (figure 1) . a viral plaque assay was used to determine a suitable virus strain with which to express luciferase. supernatant from bm5 cells containing recombinant bmnpv (rebm-luc) was harvested five days post-transfection before inoculation into silkworms. after four to five days, protein was harvested from silkworms and 50 μg protein from lysed larval haemolymph was subjected to luciferase assays. luminescence detected from silkworm larval haemolymph was approximately 3.42 ± 0.52 × 10 8 relative light units (rlu), compared to 150-300 rlu from luc-negative virus-infected samples. pcr amplification (qpcr) further verified and validated the chifn-λ gene expression in bevs (supplementary figure s1c) . in order to investigate the possible biological, cellular, and molecular mechanisms involved in the cascade of interferon-induced immunity, we performed transcriptomic analysis on chicken embryo fibroblasts and organs of live chickens. transcriptomes from the bursa of fabricious and thymus (most important immune organs in chicken) were compared with the control group to identify differentially expressed genes (degs) among all groups. experimentation started at day 14 post-hatch as this is a phase of rapid growth and development, and we hoped to achieve biologically active transcriptional changes. the differences in degs observed in the present study control cellular architecture, immune function, metabolic pathway, and muscular function. it has previously been established that huifn-λ signals via il-10 and il-28r exhibit typei-like antiviral potential [23] . protection from simian foamy virus (sfv) and avian influenza (ai) augments the antiviral functioning and further postulates its diverse antiviral potential against avian pathogens. in this context, we stimulated chickens with silkworm-expressed chifn-λ and profiled the gene expression in immune organs (thymus and bursa) and compared it with that in primary chicken fibroblasts using rna-seq. an overall low isg expression was noticed in chifn-λstimulated cef; out of 26,616 genes, 161 were degs (84 upregulated and 77 downregulated) (p< 0.05, │ log2fold change │ >1) (figure 2a) . although cef do not possess receptors for ifn-λ, slight temporal expression of degs in response to chifn-λ treatment signifies its antiviral potential in primary cells. next, we monitored the gene expression in the thymus and bursa. between the chifn-λ-treated and non-treated thymus, a total of 23,801 genes were expressed. among them, 331 genes were degs, in which 177 genes were upregulated and 154 genes were downregulated (figure 2b) . in the bursa of fabricious, 289 out of 23,951 genes were differentially expressed (130 upregulated and 159 in order to investigate the possible biological, cellular, and molecular mechanisms involved in the cascade of interferon-induced immunity, we performed transcriptomic analysis on chicken embryo fibroblasts and organs of live chickens. transcriptomes from the bursa of fabricious and thymus (most important immune organs in chicken) were compared with the control group to identify differentially expressed genes (degs) among all groups. experimentation started at day 14 post-hatch as this is a phase of rapid growth and development, and we hoped to achieve biologically active transcriptional changes. the differences in degs observed in the present study control cellular architecture, immune function, metabolic pathway, and muscular function. it has previously been established that huifn-λ signals via il-10 and il-28r exhibit typei-like antiviral potential [23] . protection from simian foamy virus (sfv) and avian influenza (ai) augments the antiviral functioning and further postulates its diverse antiviral potential against avian pathogens. in this context, we stimulated chickens with silkworm-expressed chifn-λ and profiled the gene expression in immune organs (thymus and bursa) and compared it with that in primary chicken fibroblasts using rna-seq. an overall low isg expression was noticed in chifn-λ-stimulated cef; out of 26,616 genes, 161 were degs (84 upregulated and 77 downregulated) (p < 0.05, |log2fold change|>1) (figure 2a ). although cef do not possess receptors for ifn-λ, slight temporal expression of degs in response to chifn-λ treatment signifies its antiviral potential in primary cells. pathways [24] (figure 3) . due to the induction of a distinct subset of genes, a lower level of antiviral activity is observed as compared to type-i ifns. it is speculated that the activation of chifn-λ is similar to type-i ifns but they are diverse in functional capability. the chifn-λ have particular significance in viral infections of epithelial origin, where they are optimally active by eliciting a broad antiviral state. using conventional approaches, we have confirmed the expression of selected genes as shown in supplementary figure s2a and s2b. next, we monitored the gene expression in the thymus and bursa. between the chifn-λ-treated and non-treated thymus, a total of 23,801 genes were expressed. among them, 331 genes were degs, in which 177 genes were upregulated and 154 genes were downregulated ( figure 2b ). in the bursa of fabricious, 289 out of 23,951 genes were differentially expressed (130 upregulated and 159 downregulated) ( figure 2c ). interestingly, a relatively low number of genes overlapped among these three groups ( figure 2d ). in order to confirm the expression of degs, we used a conventional approach (qpcr) and show (supplementary figure s2a,b) a scenario corresponding to the rna-seq data. on the basis of abundance and fold change, degs were further characterized (supplementary table s1 ). cumulatively, a significant upregulation of crucial cytokine and chemokine genes (il1-β, ccl4, ccl5, and cx3cl1) was observed. these are broadly involved in antiviral response, apoptosis, cellular proliferation and differentiation, cytokine-cytokine receptor interaction and inflammation pathways [24] (figure 3 ). due to the induction of a distinct subset of genes, a lower level of antiviral activity is observed as compared to type-i ifns. it is speculated that the activation of chifn-λ is similar to type-i ifns but they are diverse in functional capability. the chifn-λ have particular significance in viral infections of epithelial origin, where they are optimally active by eliciting a broad antiviral state. using conventional approaches, we have confirmed the expression of selected genes as shown in supplementary figure s2a degs were further analyzed for go terms and the kegg pathway by utilizing deseq2 [25] . of 956 go terms associated with chifn-λ-treated cef, 112 go terms were significant (p < 0.05) ( figure 4a ). in the bursa, among biological processes, we observed the wnt signaling pathway (wif1/camk2a), cytokine-cytokine receptor interactions (tnfsf11), the apelin signaling pathway (ryr2/myl4), and the significant antiviral pathway (novel gene) in cellular components (figure 4b) . in the thymus, out of 1712 go terms, we observed 309 significant, and in the bursa, out of 2298 go terms, 637 were significant (p < 0.05). in order to understand the biological functions associated with degs, we further analyzed the data in three distinct categories, including biological processes (bp), cellular components (cc), and molecular functions (mf) (figure 4c) . degs were further analyzed for go terms and the kegg pathway by utilizing deseq2 [25] . of 956 go terms associated with chifn-λ-treated cef, 112 go terms were significant (p < 0.05) ( figure 4a ). in the bursa, among biological processes, we observed the wnt signaling pathway (wif1/camk2a), cytokine-cytokine receptor interactions (tnfsf11), the apelin signaling pathway (ryr2/myl4), and the significant antiviral pathway (novel gene) in cellular components ( figure 4b ). in the thymus, out of 1712 go terms, we observed 309 significant, and in the bursa, out of 2298 go terms, 637 were significant (p < 0.05). in order to understand the biological functions associated with degs, we further analyzed the data in three distinct categories, including biological processes (bp), cellular components (cc), and molecular functions (mf) ( figure 4c ). further to gene ontology and differential expression, we investigated kegg pathway enrichment. in cefs, significant enrichment was seen in pathways including the mapk signaling pathway ( further to gene ontology and differential expression, we investigated kegg pathway enrichment. in cefs, significant enrichment was seen in pathways including the mapk signaling pathway (fos/il1b/fosb), the toll-like receptor signaling pathway (fosb, il8l1, il1b, fos, ccl5), influenza a (il8l1/il1b/ccl5), cytokine-cytokine receptor interactions (ccl20/il8l1/il1b/cx3cr1/ccl5), salmonella infection (fosb/il8l1/il1b/fos), the nod-like receptor signaling pathway (il8l1/il1b/ccl5), and herpes simplex infection (fosb/il1b/fos/ccl5) ( figure 5a ). in bursa, wnt signaling (wif1), the apelin signaling pathway (ryr2), and the calcium signaling pathway (ryr2) were significantly observed ( figure 5b ). for the thymus, the nod-like receptor signaling pathway (plcb1/mapk11), the mapk signaling pathway (srf/mapk11), influenza a (rsad2/mapk11), and mapk11 (salmonella, toll-like, herpes simplex infection) were observed ( figure 5c ). collectively, apoptosis (jun/birc5/ctsc/actg1), rna degradation (eno1/btg2/c1d), the tca cycle (mdh1/idh3a), the p53 signaling pathway (perp1/ccnb2), biosynthesis of amino acid (eno1/idh3a), influenza a (rsad2/jun/actg1), and the toll-like receptor signaling pathway (jun) were among the most significant. here, we present the first comprehensive report on cloning and expression of chifn-λ by employing bevs and demonstrate that it is biologically active in both cef (in vitro) and live chickens here, we present the first comprehensive report on cloning and expression of chifn-λ by employing bevs and demonstrate that it is biologically active in both cef (in vitro) and live chickens (in vivo). the identification of this potentially significant ifn among the ifn family advances fundamental aspects and functionality of chifn-λ in avian type-iii ifns. it is evident from the data that this ifn, like human interferon lambda (huifn-λ) , demonstrates similar type-i ifn-like properties. however, a distinct pattern of expression of isgs in chifn-λ contrasts it from other type-i ifns. knowledge regarding ifns is fundamental as rapid outbreaks of viral pathogens cause huge economic losses to the poultry industry every year. the present study investigates the isgs and signaling pathways associated with avian immunity and will bring new horizons to target problematic viral pathogens, e.g., aivs, circulating within the poultry industry. interferon lambda is a biologically active type-iii interferon which primarily acts on epithelial tissues [3] . studies have demonstrated the antiviral potential of ifn-λ against highly pathogenic avian influenza by eliciting a broad antiviral state [10] . ifn-λ is structurally peculiar as it possesses five exonic regions located on chromosome 7, contrary to type-i ifns, which are intronless and situated on the z sex chromosome in chicken [5, 26] . this is in agreement with human ifn-λ subfamily which are anatomically identical by possessing five exonic regions on chromosome 1 of the human genome [5] . furthermore, 36% of amino acids are identical between huifn-λii and chifn-λ, which signifies the similarity of these two ifns. however, unlike mammals, only one member exists in chicken (chifn-λ). this is in agreement with the other types of chicken ifns, which have fewer members compared to mammalian ifns [27] . reduced expression of isgs in response to chifn-λ in our experiment demonstrates the fact that cefs are optimally less receptive to ifn-λ, which is in agreement with published reports [10] . one study revealed that chifn-λ can actively inhibit the viral replication of ai in primary embryonic tracheal organ cultures and clec-213 (chicken lung cell line). it is further postulated that with treatment of chifn-λ, isgs are expressed significantly, especially mx gene, which is primarily expressed in epithelial rich organs (i.e., trachea, lungs, and intestine) was also observed in the present study [10] . furthermore, studies have also revealed that a high degree of cell type specificity in receptor-ligand interactions make avian ifns distinct from mammalian ifns. recently, it has been established that chicken ifn-λ inhibits low pathogenic influenza virus replication in cefs; however, as compared to chifn-γ and chifn-β, higher doses are required to induce isgs and maintain the strong antiviral state in the cells [14] . go and kegg analysis of each experimental group demonstrated overlapping biological functions. an important gene involved in the host response of infected samples is rsad2, also termed viperin, which is one of the potent interferon stimulated genes (isgs) responsible for eliciting a broad antiviral state against a variety of viral and bacterial pathogens [28] . in mammals, it is highly expressed in response to invading viral infections [29] . elevated expression of viperin in chifn-λ-treated organs further augments the expression of isgs in response to injected ifn in vivo. viperin was upregulated in response to chifn-λ treatment, which is symbolic for all isgs. ifn-inducible transmembrane protein-1 (ifitm-1) is one of the potent isgs expressed in response to either type of ifn and plays an antiviral role by blocking cytoplasmic entry [30] . it is further demonstrated that ifitm alters membrane fluidity, hence producing curvature in the outer leaflets of the membrane or by interfering with intracellular cholesterol homeostasis [31, 32] . significant upregulation of ifitm3 in the chifn-λ-treated thymus augments the temporal expression of isgs in response to ifn treatment. further studies are needed to investigate the possible future role of chifn-λ as a potent and novel therapeutic in the poultry industry. although the immune response elicited by type iii ifns is still not very clear, in the present study we also found some novel genes involved in the cascade of the avian immune response. furthermore, in vitro exposure of cef to chifn-λ demonstrated a rapid surge of pro inflammatory cytokines. considering their vital role in immune pathways, cytokine gene expression is widely employed as an indicator for the immune response. we did observe some genes that were previously illustrated in publications; one such example is chemokine (c-c motif) ligand 1 (ccl1, ensgalt00000003670) [33] . chemokines are secreted chemotactic cytokines that play a fundamental role in the recruitment and migration of lymphoid and myeloid cells in target tissues, and hence govern the avian immune response [34] . ccl1 is a chemokine secreted by monocytes that is capable of activating macrophages and t lymphocytes [35] . ccl20, like its mammalian orthologue, is responsible for recruiting lymphoid cells and is involved in the early immune response in chickens [36] . likewise, ccl1, ccl4, and ccl5 were also upregulated in cef and are chiefly involved in the innate avian immune response. the present study describes the transcriptomic analysis of differential gene expression following exposure to chifn-λ and the resultant pro-inflammatory response in both cef and chicken tissues. this response ostensibly is due to rapid and sustained signaling via cell surface receptors and a surge of chemokines and cytokines, which in turn create an antiviral environment. a contrasting feature of the present study is the upregulation of the toll-like receptor (tlr) signaling pathway in all three treatment groups, where it is evident that numerous genes are upregulated in tlr mediated cytotoxicity. tlr15, a unique chicken receptor expressed on the surface of fibroblasts, heterophils, and macrophages, shares 30% sequence identity with tlr2 [37] . it is evident from experimentation that tlr15 is a broad spectrum tlr that has the capability to recognize heat stable components of both gram-positive and gram-negative bacteria, cpg oligonucleotides, lipopolysaccharide (lps), and tripalmitoylated lipopeptide [38] . tlr15, an avian-specific tlr, plays a significant role in avian immune responses against bacterial and viral pathogens. recently, it has been demonstrated that diacylated lipopeptide from mycoplasma synoviae activated tlr15 and regulated innate immune responses [39] . similarly, significant upregulation of tlr15, observed in the present study, highlights a possible role of chifn-λ against mycoplasma infections in chicken. however, it warrants future studies to delineate the molecular processes. it has also been established by repeated experimentation that chifn-λ has been seen to cause delay in viral excretion and the spread of highly pathogenic avian influenza (hpai) h5n1 [10] . it is evident that in mammals, ifn-λ elicits a protective antiviral response toward ai, whereas ifn-λ plays a minor role in lung epithelia [40] . similarly, in the respiratory tract of chickens, not all mucosal cells are responsive to chifn-λ. therefore, treatments can only delay, but do not significantly support the complete removal of viral loads of h5n1 or halt the virus crossing the epithelial barrier [41] . however, for low-pathogenic avian influenza (lpai), it is evident that chifn-λ has demonstrated significant antiviral activities [42] . recent reports revealed another contrasting feature of ifn-λ, where it significantly elicited strong antiviral potential on intestinal epithelial cells to control murine rotaviruses [43, 44] . it will be fascinating to investigate in the future whether the same antiviral phenomena occurs, and chifn-λ might also demonstrate epitheliotropism like rotaviruses and halt viral pathogens of the gastrointestinal tract in chickens. nuclear factor kappa-b (nf-kb) is the most significant, evolutionarily conserved, pleiotropic, inducible transcription factor responsible for regulating genetic expression in a variety of fundamental processes, including apoptosis, growth, immune response, inflammation, stress response, etc. [45] . notably, the upregulation of nf-kb in response to chifn-λ treatment on cef signifies their potent role in the immune response. activator protein 1 (ap-1) is a transcription factor complex highly responsive for cytokine signaling and growth promotion [46] . formed through noncovalent dimerization between the fos and jun family of nuclear oncogenes, this complex activates ap-1-dependent genes, hence controlling cell proliferation, differentiation, and apoptosis [47] . consistent with these observations, our study demonstrated that many genes associated with this pathway were upregulated. this finding suggests a link between ap1 and the transcriptional cascade associated with recombinant interferon treatment. overall, transcriptomic analysis revealed significant upregulation of fos and jun in cef and bursa, and thymus of chicken. the innate immune response is a highly complex, precise, interconnected, and integrated response that relies on many factors. the genes investigated in our study control direct protein interactions and are significantly involved in the avian innate immunity cascade. however, further validation of a broad set of immunity-related genes will also be required to elucidate the mechanism of interferon-induced immunity. a more comprehensive study including a larger set of immune genes and multiple recombinant ifns, which will correlate their integrated role, will enable researchers to provide comprehensive insight into the avian innate response. other future studies involving backyard poultry to assess whether similar patterns of innate immunity prevail in indigenous breeds in response to chifnλ are also important and will further develop our understanding of avian immunity. in the current study, we employed rna-seq to illustrate vital transcriptomes involved in the cascade of avian biology and observed divergent results in recombinant interferon-treated chickens compared to a control group chickens. our data suggest that significant antiviral, cell cycle regulators, and biologically active genes are expressed in response to administered chicken ifn. functional characterization of these vital genes warrants further investigation to determine the future possible role for recombinant chicken ifn in the poultry industry. the authors declare no conflict of interest. phylogeography and evolutionary history of reassortant h9n2 viruses with potential human health implications h9n2 avian influenza virus in korea: evolution and vaccination avian interferons and their antiviral effectors characterization and transcriptional analysis of the mouse chromosome 16 cytokine receptor gene cluster ifn-λs mediate antiviral protection through a distinct class ii cytokine receptor complex lambda interferon (ifn-λ), a type iii ifn, is induced by viruses and ifns and displays potent antiviral activity against select virus infections in vivo advances in avian immunology-prospects for disease control: a review interferon lambda genetics and biology in regulation of viral control partial antiviral activities detection of chicken mx jointing with neuraminidase gene (na) against newcastle disease virus antiviral activity of lambda interferon in chickens protective effects of type i and type ii interferons toward rous sarcoma virus-induced tumors in chickens highly pathogenic avian influenza viruses do not inhibit interferon synthesis in infected chickens but can override the interferon-induced antiviral state molecular cloning, expression, and characterization of chicken ifn-λ biological effects of chicken type iii interferon on expression of interferon-stimulated genes in chickens: comparison with type i and type ii interferons a highly efficient and simple construction strategy for producing recombinant baculovirus bombyx mori nucleopolyhedrovirus comparison of the n-linked glycosylation of human beta1,3-n-acetylglucosaminyltransferase 2 expressed in insect cells and silkworm larvae comparison of recombinant protein expression in a baculovirus system in insect cells (sf9) and silkworm a manual of methods for baculovirus vectors and insect cell culture procedures the bradford method for protein quantitation generating vesicular stomatitis virus pseudotype bearing the severe acute respiratory syndrome coronavirus spike envelope glycoprotein for rapid and safe neutralization test or cell-entry assay laboratory manual for the isolation and identification of avian pathogens purification of rna using trizol (tri reagent) interferons α and λ inhibit hepatitis c virus replication with distinct signal transduction and gene regulation kinetics transcriptional innate immune response of the developing chicken embryo to newcastle disease virus infection moderated estimation of fold change and dispersion for rna-seq data with deseq2 type i interferons and the innate immune response-more than just antiviral cytokines a genomic analysis of chicken cytokines and chemokines characterisation of chicken zap the antiviral response. microbes infect palmitoylome profiling reveals s-palmitoylation-dependent antiviral activity of ifitm3 ifitm proteins restrict viral membrane hemifusion the antiviral effector ifitm3 disrupts intracellular cholesterol homeostasis to block viral entry marek's disease virus-induced immunosuppression: array analysis of chicken immune response gene expression profiling cloning, expression and functional characterization of chicken ccr6 and its ligand ccl20 unique chemotactic response profile and specific expression of chemokine receptors ccr4 and ccr8 by cd4+ cd25+ regulatory t cells identification, mapping, and phylogenetic analysis of three novel chicken cc chemokines unique features of chicken toll-like receptors expression of the avian-specific toll-like receptor 15 in chicken heterophils is mediated by gram-negative and gram-positive bacteria, but not tlr agonists diacylated lipopeptide from mycoplasma synoviae mediates tlr15 induced innate immune responses ifn-λ: a new spotlight in innate immunity against influenza virus infection differential responses of innate immunity triggered by different subtypes of influenza a viruses in human and avian hosts type iii interferon gene expression in response to influenza virus infection in chicken and duck embryonic fibroblasts distinct roles of type i and type iii interferons in intestinal immunity to homologous and heterologous rotavirus infections interferon-λ and interleukin 22 act synergistically for the induction of interferon-stimulated genes and control of rotavirus infection interferons and viral infections innate immune sensing of dna viruses neuronal activity-dependent local activation of dendritic unfolded protein response promotes expression of brain-derived neurotrophic factor in cell soma key: cord-005119-vsvc437g authors: georgiades, jerzy a.; fleischmann, w. robert title: oral application of cytokines date: 1996 journal: biotherapy doi: 10.1007/bf01877206 sha: doc_id: 5119 cord_uid: vsvc437g a number of different laboratories reported on studies with orally administered interferons and cytokines. their observations extend previous observations which showed that orally administered interferons and cytokines can exert both local and systemic effects. as difficult as it may be to understand how orally administered interferons and cytokines may exert both effects, the increasing number of laboratories that demonstrate biological effects with orally administered cytokines suggests that serious consideration be given to the possibility that orally administered interferons and cytokines can indeed exert effects. they also raise the possibility that these effects may have biological relevance for the treatment of human disease. moreover, they may indicate that the nasal/oral region is a window on the environment. it is most important, however, to assure that these experiments are performed with special care to avoid presenting preliminary data that is not properly controlled. it is essential to carry out these studies with sufficient animals or patients to ascertain their significance; and to plan the studies as double-blind evaluations to avoid misinterpretations when subjective tests are used. nevertheless, the overall data presented give one the impression of an area that should be pursued. oral or nasal administration of interferons has been shown in a number of studies to have a protective effect against viral infections. the majority of the studies suggested that orally or nasally administered interferons exerted their antiviral activity locally [cummins and hutcheson, 1983; cummins and rosenquist, 1980; greenberg et al., 1978; hayden et al., 1986; higgins etal., 1983; merigan etal., 1973; schaferetal., 1972; smith et al., 1987; turner et al., 1986] . more recently, a number of studies have suggested that the orally administered interferons might be exerting their antiviral activity through a systemic effect [cummins et al., 1988; hutchinson and cummins, 1987; koech et al., 1990] . further studies have extended the original observations with the antiviral activity of orally administered interferons to include demonstrations of immunoregulatory and antiparasitic activities of orally administered interferons [cummins and hutcheson, 1986; fleischmann et al., 1991; young et al., 1990] . additional studies have further extended the observations with oral administration of interferons to include immunoregulatory and antibacterial activities mediated by oral administration of other cytokines [baqar et al., 1993; koren and fleischmann, 1994] . the concept that oral administration of interferons could have a systemic effect has been a difficult one to evaluate. it is difficult to understand how orally administered interferons, particularly ph sensitive ifn-7 could survive passage through the acidic and/or peptidase rich environment of the stomach and intestinal tract to trigger a systemic effect. one concern involves the lack of parallel controls in many of the studies. a further concern involves questions about the biological relevance of the often relatively modest effects obtained with orally administered interferons. other concerns relate to the lack of universality in the observation of biological effects of orally administered interferons [sperber et al., 1993; witt et al., 1992] . the efficacy of the interferon therapy may depend upon the form of disease (acute or persistent), the daily dosage of interferon , and the type of interferon preparation (whether it is made from natural or recombinant sources) . furthermore, the type of disease and the duration of therapy may influence the final response to treatment . these concerns are real and appropriate. it will be attendant upon the researchers investigating the oral administration of cytokines to address all of them effectively. the oral administration of cytokines was the topic of a number of presentations at a recent workshop presented in conjunction with the 1994 annual meeting of the international society for interferon and cytokine research in budapest, hungary. the mechanism by which orally administered ifn-a might exert an antiviral effect was probed in vitro in studies with cultured primary human buccal epithelial cells [smith etal., 1994] . david chi and his colleagues used florescence studies to show that ifn-a treatment significantly increased the number of hla-dr positive cells. a trend toward greater expression of hla-dr by hla-dr positive cells was also observed. the indication that ifn-a causes an upregulation of hla-dr expression by human buccal epithelial cells and peripheral white blood cells suggests that orally administered ifn-a may exert an antiviral activity against local viral infections. this anfiviral activity may be medicated at least in part by a greater degree of recognition of viral infected cells in the context of hla-dr by the host immune system. the mechanism by which orally administered ifn-a might exert antimicrobial action was examined in studies reported by kunihiro ohashi and his colleagues . it was found that the saliva of healthy volunteers contained il-la and il-8. established epithelial cell lines were also found to produce il-la and il-8 spontaneously. the possible immunological roles of ifn-a in combination with these lymphokines were evaluated in vitro. il-1 a, used at a level equivalent to that in the saliva, and ifn-c~ were examined for cooperative effects on activation of neutrophil mediated anticandidal action. they were found to have an additive effect. the production of il-8 was found to be enhanced by treatment of established epithelial cell lines by ifn-c~ treatment. the ifn-a was also found to partially restore il-8 production in'a monocyte cell line that had been latently infected with human immunodeficiency virus-1 (hiv-1) . finally, the ifn-a treatment of detroit 562 (a pharyngeal cancer cell line) and kb cells (oral cancer cell line) caused enhanced binding to a human t cell line, suggesting that ifn-a caused an enhanced recognition of the tumors by t cells. in this regard, the investigators showed an enhanced expression of icam-1, cd29, and cd49wb on some oral-mucosal epithelial cells treated with ifn-a in vitro. taken together, the work provides some in vitro evidence to support the concepts that in vivo oral administration of ifn-a may (a) locally activate neutrophils in the buccal cavity to exhibit a greater antimicrobial action, (b) affect local lymphokine production by epithelial cells in contact with the ifn-a, and (c) increase cell surface antigens in mucosal epithelial cells making them more sensitive to tumor surveillance mechanisms. mary tompkins reported on the effects of intranasally administered ifn-a on lymphoid cell phenotype and function in lymph nodes and the spleen [tonkonogy et al., 1994] . she and her colleagues showed that lymphocytes isolated from periglandular lymph nodes responded to in vitro stimulation with immobilized anti-cd3 activated t cells to produce 4-fold more ifn-7 than control mice. lymphocytes from superficial cervical lymph nodes of oral ifn-a-treated mice produced 2-fold more ifn-7 than those from control mice. no differences in ifn-7 production were detected for lymphocytes from more distal lymphoid organs such as axillary lymph nodes, mesenteric lymph nodes, peyer's patches, or spleens from ifn-a and control mice. effects of orally administered ifn-a on the expression of cell surface markers of lymphocytes in the lymphoid tissue were also measured. no differences in proportion of expressing cells or degree of expression of cell surface cd4, cd8, mhc class ii or immunoglobulin were seen in the lymphocytes from any of the lymphoid tissues from ifn-a treated and control mice. the results indicate that intranasal administration of ifn-a establishes a greater responsiveness of lymphocytes in periglandular and superficial lymph nodes to ifn-7 induction by exposure to anti-cd3. this greater responsiveness occurs in the absence of a change in phenotype of the lymphocytes. further, the results suggested that the effect of intranasal administration of ifn-a and ifn-7 production may be primarily a local effect, since it diminishes with increasing distance from the site of ifn-a administration. a paper presented by robert fleischmann summarized his published work on the myelosuppressive effects of orally administered interferons in mice [fleischmann et al., 1991 [fleischmann et al., , 1992 koren and fleischmann, 1993] . the presentation reviewed work showing that oral administration of each of three interferons (ifn-a, ifn-/3 and ifn-7) exerted a dose dependent suppressive effect on peripheral white blood cell counts [fleischmann et al., 1991] . moreover, the peripheral white blood cell suppression was shown to be reflective of a suppression of bone marrow function by the orally administered interferons [koren and fleischmann, 1993] . of importance in addressing the concerns about the biological relevance of orally administered interferons, the magnitudes of the peripheral white blood cell and bone marrow suppressions induced by oral interferons were equivalent to those induced by subcutaneous or intraperitoneal administration of the interferons. studies on the kinetics of establishment of peripheral white blood cell and bone marrow suppression indicated that the suppressive effects developed more slowly for orally administered interferons (2 days for peripheral white blood cell suppression and 3 days for bone marrow suppression) than for injected interferons (1 day and 2 days, respectively). finally, the mechanisms of establishment of peripheral white blood cell and bone marrow suppression by orally administered interferons and injected interferons were examined [fleischmann et al., 1992] . in contrast to observations with injected interferons, the systemic activities of orally administered interferons were not blocked by the presence of circulating antibody. further, the peripheral white blood cell suppressive effects of orally administered interferons were shown to be able to be adoptively transferred by inoculation of white blood cells to otherwise untreated mice. these studies show in a mouse model that orally administered interferons are as potent as injected interferons in suppressing both the peripheral white blood cell counts and the bone marrow. moreover, they indicate that the mechanism by which orally administered interferons exert these systemic effect, is different from that of injected interferons. in a study reported earlier [georgiades et al., 1988] , georgiades and colleagues found that the antiviral state could be transferred from the oral cavity of mice treated with molfn-a or rat rifn-7 to peripheral blood lymphocytes and spleen cells. they demonstrated that white blood cells of mice treated with 5-10 iu and 0.005-1 iu ifn-~, could transfer antiviral resistance in vitro to fetal fibroblasts. such a transfer apparently happened by direct cell to cell contact when the spleen cells from mice orally treated with interferon were cocultured with fetal fibroblasts in the absence of interferon as previously described [blalock and baron, 1977] . the phenomenon appeared within 5 hrs after initiation of oral interferon treatment and, with continued oral interferon treatment, persisted through at least 15 days. neither thymus cells nor plasma of orally interferon-207 treated mice had the ability to transfer the antiviral effect. taken together, these and other controlled experiments suggest that interferon may be absorbed quickly in the oral cavity by cells of mucosa, somehow activating leukocytes present in mucosal tissue resulting in the activation of leukocytes in the lymphatic system and in the peripheral circulation. a paper presented by srecko koren reported parallel observations for il-2 [koren and fleischmann, 1994] . oral administration of il-2 was shown to exert systemic effects by suppressing both the peripheral white blood cell counts and the bone marrow in a dose dependent manner. moreover, the potency of orally administered il-2 was equivalent to that of injected il-2. taken together with the results of fleischmann, these observations suggest that the induction of systemic effects by orally administered cytokines may be a general phenomenon. taking together all of the above information, it appears that not only ifn-a but also several other interferons and cytokines introduced to the mucosal membrane of the oral cavity can transfer signals through the mucous membrane and induce systemic responses that can be measured by a variety of techniques. this evidence about systemic effects was confirmed by clinical studies carried out on patients with chronic active hepatitis b, and c and on patients with aids as discussed below. a paper presented by shahida baqar reported on the effects of orally administered il-2, il-5 and il-6 on gut mucosal immunity to campylobacter jejuni [baqar et al., 1993; b aqar et al., 1994] . she and her colleagues showed that oral administration of il-6 resulted in a 3 log reduction (relative to control mice) in the amount of c. jejuni excreted in the feces within 48 hours after administration of the c. jejuni. oral administration of il-5 showed a similar reduction though the time course for the reduction was delayed relative to il-6. no reduction in c. jejuni was observed with oral administration of il-2. these results indicate that oral administration of il-5 and il-6 can reduce the initial colonization by c. jejuni. the duration of the effect of the three lymphokines was probed by rechallenging mice that had been treated with oral administration of il-2, il-5 or il-6 to establish a recolonization with c. jejuni. the results indicate that, for all three lymphokines, the initial level of recolonization was 2 logs lower than in control animals; however, the recolonization ultimately progresses to the level of the control for il-5 and il-6 treated animals. only il-2 treated animals showed a durable suppression of c. jejuni colonization. specific iga antibody response to c. jejuni was also measured for mice treated orally with the three lymphokines. il-6 treated mice, but not il-2 or il-5 treated mice, showed enhanced levels of c. jejuni-specific iga antibodies in the intestine and in the blood. in further experiments on the effects of orally administered lymphokines on iga production, il-2 was evaluated for its potential as an oral mucosal adjuvant for vaccination with formalin-killed c. jejuni. a three-fold enhancement in siga was observed relative to control mice. this enhancement in slga activity was biologically relevant since it led to an enhanced rate of clearance of c. jejuni from the intestine and a reduced number of c. jejuni that were present in the feces. oral administration of ifn-a has previously been reported to be efficacious in the treatment of feline leukemia [cummins et al., 1988] . thomas toth reported on a double-blind study evaluating the effects of orally administered il-2 or ifn-a on various hematological variables in feline leukemia virus-positive cats [toth et al., 1994] . the hematological parameters measured included total red blood cell counts, total white blood cell counts, differential white blood cell counts, total hemoglobin, mean corpuscular hemoglobin, mean corpuscular-hemoglobin concentration, mean corpuscular volume and hematocrit. oral treatment with il-2 was given 3 times (days 1, 3 and 5) at 2 dosage levels (40 and 400 units/treatment). the hematological variables were monitored on days 7 and 14. orally administered il-2 affected only one hematological variable as it reduced the mean corpuscular hemoglobin concentration to a significant level by day 7 in cats treated with 400 units il-2. by day 14 the mean corpuscular hemoglobin concentration returned to the control level. oral treatment with ifn-a was given 8 times (days 0, 3, 7, 10, 14, 17, 21 and 28) at 3 dosage levels (0.05, 0.5 and 5.0 iu/treatment). the hematological variables were monitored on days 0, 7, 14, 21 and 28. oral administration of ifn-a caused significant and dose dependent elevations in total red blood cell count, total hemoglobin, and hematocrit on days 14, 21 and 28. interestingly, with 0.5 iu/treatment, monocyte counts were also significantly elevated on these days. the other hematological variables were at the control level. these results indicate that oral administration of il-2 and ifn-a can affect several hematological variables, suggesting that these biological response modifiers cause systemic effects. the biological importance of these systemic effects and their relevance for the control of the feline leukemia virus infection are as yet unknown. finally, miklos degre [1994] reported on his studies on the effect of oral administration of two biological response modifiers on the course of an enteric bacterial infection in mice [degre, 1994] . he found that orally administered tumor necrosis factor did not have an effect on the course of infection. however, orally administered ifn-a reduced the mortality of infected mice relative to controls. the degree of mortality with orally administered ifn-a was the same as that observed for intraperitoneally administered ifn-c~. the results suggest that oral administration of ifn-a may exert a protective effect against enteric bacterial pathogens. race horses suffer from inflammatory airway disease that impairs their ability to perform on the race track. bonnie moore presented the results of a double-blind, placebo controlled study on the efficacy of oral ifna in the treatment of inflammatory airway disease . horses were randomly assigned (8 horses/group) into a placebo group and three ifn-a dosage groups (50, 150 and 450 iu/day for 5 days). fifteen days after initiation of therapy, horses were evaluated for the degree of inflammatory airway disease by semi-quantitative endoscopic examination score and by cytologic evaluation of bronchoalveolar lavage fluid. oral administration of 50 and 150 iu of ifn-a resulted in a reduction in the endoscopic examination score as well as a reduction in the number of white blood cells in the bronchoalveolar lavage fluid. administration of 450 iu of ifn-a was not as efficacious. the relative percentages of the various lymphocyte subpopulations was unaffected by the ifn-a treatments. the results suggest that orally administered ifn-a may have a beneficial effect on inflammatory airway disease in horses. previously published work suggested that oral administration of ifn-a may be useful as a treatment of patients with aids [hutchinson and cummins, 1987; koech et al., 1990] . two presentations discussed work evaluating the efficacy of orally administered ifna against hiv, while one presentation evaluated the efficacy of orally administered ifn-a against feline leukemia virus. musabbir mian reported on a study in poland of 48 patients with aids or arc (aids related complex) who had been given low dose oral treatment with ifn-a [babiuch and mian, 1994] . this report represented the results of studies initiated four years earlier. part of these studies were already published [babiuch et al., 1993; georgiades and babiuch, 1994] and found confirmation by others [jordan, 1994] . it was reported that most patients with initial cd4+ cell counts above 200 became asymptomatic, gained weight, had improved karnofsky performance scores and had either increased or stable cd4+ cell counts and total lymphocyte counts. patients with initial cd4+ cell counts below 200 did not respond to oral ifn-o~. martin cummins reported on a double-blind, placebo controlled study in zambia involving 147 patients 209 divided randomly and equally into three treatment groups: group 1, placebo; group 2, 150 iu/day ifn-a administered on an alternate weekly basis with placebo for 24 weeks; and, group 3, 150 iu/day ifn-a administered continuously for 24 weeks [mukunyandela et al., 1994] . the study showed that oral ifna treatment had a significant effect in reducing hivrelated signs and infections. both groups 2 and 3 had a significantly greater resolution of pre-existing hivrelated skin infections than the placebo treated group 1. group 3 had a greatly reduced incidence of new, mucocutaneous hsv infections (2%) compared to the placebo treated group 1 (25%), with group 2 having an intermediate value (13%). interestingly, patients in the alternate ifn-a/placebo group 2 (but not the continuous ifn-a group 3) showed a reduction in global symptom score and small, but significant, increases in absolute and mean cd4+ counts compared with placebo treated group 1. taken together, these studies provide significant support for the concept that orally administered ifn-a may have beneficial effects for aids patients, provided they are treated in the early stage of disease. as a caution, however, several negative studies have been reported showing a lack of efficacy of orally administered ifn-a in the treatment of hiv infected patients [hulton et al., 1992; sperber et al., 1993] . these negative observations may relate to the limited period of oral ifn-a treatment employed in these studies or to the use of recombinant as opposed to natural ifn-a. while it is not now possible to be certain of the anti hiv effects of oral ifn-a in humans, several studies provide additional support. the observation that aids patients with baseline cd4+ cell counts below 200 cells/mm 3 responded poorly to oral ifn-a therapy was in agreement with reports of kaiser et al. [ 1992] in germany, hutton et al. [ 1992] in canada, and sperber et al. [ 1993] in the usa. all of these studies reported similar findings: patients having < 200 cd4+ cells/mm 3 did not respond very well to ifn-a therapy. however, patients with baseline cd4+ cell counts > 200 cells/mm 3 survived longer than control patients and required much less medical attention than patients in the control group [babiuch and mian, 1994; georgiades and babiuch, 1994] . these observations were confirmed by a report from california where physicians had similar experiences with hiv-l-infected patients [jordan, 1994] . interestingly, the beneficial effects of ifn-a therapy may not be constant through the course of ifna therapy. babiuch reported that, from time to time, patients may apparently lose their sensitivity to oral ifn-a therapy [babiuch and mian, 1994; georgiades and babiuch, 1994] . this apparent loss of sensitivity to ifn-a therapy (or deterioration of clinical status) can be reversed by slightly increasing the dose of ifn-c~ [babiuch and mian, 1994] . these observations may represent clinical manifestations of a phenomenon described in vitro by yamamoto and colleagues [yamamoto et al., 1993] . these investigators examined the effects of ifn-a using long-term culture of hiv-1-infected white blood cells. they observed a transient loss of sensitivity of the white blood cells to ifn-a. the transient loss was observed twice during a 60 day culture period. further studies will be needed to determine if hiv-1 resistance observed in vivo and in vitro occur by the same mechanism. small scale trials with 14 hepatitis b patients with chronic active hepatitis have previously suggested that oral administration of ifn-a might be of therapeutic benefit . similar beneficial results were seen in a small scale trial with six hepatitis c patients with chronic active hepatitis who were treated with oral ifn-a . zielinska reported on the long-term follow-up of 30 hepatitis b patients with chronic active hepatitis who were treated with oral ifn-a [zielinska et al., 1994] . the diagnosis of chronic active hepatitis was based on clinical and biochemical signs of active disease. oral ifn-a was given daily at a dose of 25-150 iu/day, depending on the body weight of the patients, for an average of 7 months. seroconversion from hbeag to antihbe was observed in 18 of 30 patients. the median time to seroconversion was 36.2 weeks. in addition, two patients seroconverted from hbsag to antihbs. in all 18 patients who seroconverted to antihbe, hbv dna was not found to be present in serum. also, biochemical markers associated with liver function returned to the normal range. further, 7 of the 12 patients who did not seroconvert to antihbe were negative for the presence of hbv dna in the serum. the remaining 5 patients had serum hbv dna levels that were significantly lower than at the initiation of oral ifn-a treatment. taken together, these observations suggest that oral ifn-a may be an effective therapy for control of chronic active hepatitis. the annual meeting of the international society for interferon and cytokine research (isicr-94) held in budapest provided, for the first time, compelling evidence that oral doses of cytokines modulate immune functions and increase resistance to infectious disease. studies were presented by investigators from a number of different laboratories. clinical data, especially in humans, indicates that ifn, given though the oral mucosa, is capable of inducing systemic reactions and may have potential as a new therapeutic method. a number of different laboratories reported on studies with orally administered interferons and cytokines. their observations extend previous observations which showed that orally administered interferons and cytokines can exert both local and systemic effects. as difficult as it may be to understand how orally administered interferons and cytokines may exert both effects, the increasing number of laboratories that demonstrate biological effects with orally administered cytokines suggests that serious consideration be given to the possibility that orally administered interferons and cytokines can indeed exert effects. they also raise the possibility that these effects may have biological relevance for the treatment of human disease. moreover, they may indicate that the nasal/oral region is a window on the environment. it is most important, however, to assure that these experiments are performed with special care to avoid presenting preliminary data that is not properly controlled. it is essential to carry out these studies with sufficient animals or patients to ascertain their significance; and to plan the studies as double-blind evaluations to avoid misinterpretations when subjective tests are used. nevertheless, the overall data presented give one the impression of an area that should be pursued. an interim report on the effect of natural human interferon alpha (ifna) lozenges in patients seropositive for the human immunodeficiency virus type 1 (hiv-1) oral mucosal administration of natural human alpha-interferon in hiv-1 seropositive patients modulation of mucosal immunity against campylobacter jejuni by orally administered cytokines modulation of mucosal immunity against campylobacter jejuni by orally administered cytokines. workshop presentation interferon-induced transfer of viral resistance between animal cells the effect of low dose oral human interferon alpha therapy on diarrhea in veal calves oral therapy with human interferon alpha in calves experimentally injected with infectious bovine rhinotrachetis virus effect of interferon on feedlot cattle low dosage of interferon to enhance vaccine efficiency in feedlot calves protection of calves against rhinovirus infection by nasal secretion interferon induced by infectious bovine rhinotracheitis virus oral use of human alpha interferon in cats interferons and other eytokines in infectious diseases: molecular mechanisms and clinical value. review speech modulation of peripheral leukocyte counts in mice by oral administration of interferons orally administered interferons exert their wbc suppressive effects via a novel mechanism results of the prolonged use of natural human interferon alpha (nhuifna) lozenges in the treatment of human immune deficiency virus infection natural human interferon alpha (nhulfna) given orally has different effects on patients with distinct forms of chronic viral hepatitis b (chbv) transfer of antiviral resistance by spleen and blood cells of mice receiving low oral doses of ifn alpha or gamma antiviral activity of intranasally applied human leukocyte interferon prevention of natural colds by contact prophylaxis with intranasal alpha2-interferon intranasal interferon as protection against experimental respiratory coronavirus infection in volunteers randomized, placebocontrolled double-blind study of low-dose oral interferon-a in hiv-1 antibody positive patients low-dose oral interferon in patient with aids three open-label studies of oral interferon alpha in the treatment of hiv disease low-dose oral natural human interferon-a in 29 patients with hiv-1 infections: a double-blind randomized, placebo-controlled trial low dose oral alpha-interferon therapy for patients seropositive for the human immunodeficiency virus type-1 (h/v-1) modulation of peripheral leukocyte counts and bone marrow function in mice by oral administration ofinterleukin-2 administered interferons suppress bone marrow function inhibition of respiratory virus infection by locally applied interferon oral administration of interferon-alpha for treatment of inflammatory airway disease in standard-bred racehorses treatment of symptomatic hiv-1 infected patients with low dose oral natural human interferon alpha effects of interferon-a on a reduced release of interleukin-8 from latenfly hiv-1 infected monocytic cell line u937 cells interferon alpha (ifna) in antimicrobial mechanisms in the oral cavity: possibility of oral-mucosal use of ifna to treat fungal infections in the oral cavity interferon administered orally: protection of neonatal mice from lethal virus challenge intranasally administered alpha/beta interferon prevents extension of mouse hepatitis virus strain jhm into the brains of balb/cbyj mice effect of ifn a on hla-dr expression in human buccal epithelial ceils. workshop presentation low-dose oral recombinant interferon-alpha a in patients with hiv-1 infection: a blinded pilot study effect of intranasal administration of interferon-alpha on lymphoid cell phenotype and function. workshop presentation effects of very-low-dose oral cytokine treatment on hematological values in feline leukemia positive cens prevention of experimental coronavirus colds with intranasal alpha-2b interferon absence of biological effect of orally administered interferon-bser inhibition of human immunodeficiency virus type 1 replication by human interferons alpha, beta, and gamma low-dose oral administration of human interferon alpha can control the development of theileria parva infection in cattle treatment of fourteen chronic active hbsag+, l-ibeag+ hepatitis patients with low dose natural human interferon alpha administered orally treatment of six patients with chronic active hcv hepatitis with low dose natural human interferon alpha administered orally long-term follow-up of 30 chronic active hepatitis b patients treated with low dose natural human interferon alpha administered orally key: cord-020717-niiib47f authors: kristensson, krister title: neuronal targeting and functional effects of infectious agents transmitted from animals to man date: 2003 journal: nan doi: 10.1007/bf02904487 sha: doc_id: 20717 cord_uid: niiib47f the nervous system is an «immune-privileged» site and can provide a reservoir to harbor as persistent or latent infections certain microbes that find their way to the brain. from an evolutionary standpoint, such infections are characterized at most times by low levels of the infectious agent in the systemic domain, except when multiplication has just taken place. hence the ability for transmission of the pathogens from animals to man will be determined by the availability of microbes to be transferred by a vector (e.g. in trypanosomiasis), or the amount of infective forms of the microbes shed into an environment (e.g. in toxoplasmosis). using african trypanosomes, toxoplasma,listeria and influenza a virus as examples, mechanisms by which microbes can spread and be targeted to and within the brain to cause various types of nervous system dysfunctions is reviewed. newly revealed potentials of certain cytokines to stimulate neurons to control the growth, and even kill, microbes in their cell bodies is also described. za, west nile encephalitis, borna disease, rabies, japanese encephalitis, sars), and prions (e.g. bovine spongiform encephalopathy, variant creutzfeldt-jakob disease). while the detection of the negri bodies in rabies marks the beginning of the 20 th century, the threat of a prion epidemic spreading from cows to humans has created a wave of anxiety at the end of the century. prion infections are covered in the lecture by a. aguzzi in this symposium, and this presentation will focus on transmissible diseases caused by the other categories of pathogens. these pathogens will be exemplified by african trypanosomes, listeria monocytogenes and influenza a virus, which have been the subject of experimental studies in our laboratory, and by toxoplasma gondii, which in spite of its high prevalence in the human population (10-25% of the world's population), seems to have been largely neglected in neuroscience research. the choose of these infections is also given by the fact that they illustrate how microbes can use different pathways for spread and targeting to and within the brain as well as how they can use different strategies to interact with neurons to cause degeneration or functional disturbances of the host brain. by excluding most macromolecules from passing into the brain, the blood-brain barrier (bbb) also prevents most microbes from entering the nerve parenchyma. in spite of this, a number of pathogens have adopted mechanisms for a hematogenous spread to the brain. for instance, toxoplasma gondii are carried by macrophages in the blood. from these macrophages they can be released to infect and multiply in brain endothelial cells for further passage into the nerve parenchyma. other pathogens, e.g. rabies virus, spread instead to the central nervous system along peripheral nerves and some, e.g. influenza a virus and listeria monocytogenes, may spread both via the blood and along peripheral nerves as will be discussed in a following section. we have focused our interest on mechanisms by which subspecies of the extracellular parasite trypanosoma brucei (tb) cross the bbb. tb causes african trypanosomiasis (sleeping sickness), which is an example of a re-emerging zoonosis. although the disease in humans, human african trypanosomiasis, had almost disappeared in the early 1960 ths , the incidence of the disease has now almost returned to the same levels as in the 1920 ths . the african trypanosomes spread by the tsetse fly in sub-saharan africa and use both wild game and domestic animals, e.g. cattle and pigs, as reservoirs. the subspecies tb rhodesiense in east and southern africa causes a relatively rapid disease, while tb gambiense in west and central africa causes a more protracted disease hallmarked by disturbances in sleep patterns. infections with both subspecies lead invariable to death when left untreated. for therapeutic purposes, the question of how and when the parasites pass the bbb during the course of disease has important consequences. at an early stage of the disease relatively non-toxic drugs, which do not pass the bbb, are available, but for the late stage with involvement of the nervous system arsenic preparations are the drug of choice. these arsenic compounds are trypanocidal and pass the bbb, but they are also neurotoxic and cause lethality in a high proportion, up to 10 %, of the patients. the diagnosis of the late stage relies on the finding of parasites and/or white blood cells in the cerebrospinal fluid. the mechanisms of trypanosome entry into the brain are still not known, and the proper staging of the disease, i.e. determination of the stage when the parasites has passed beyond the bbb, remains to be clarified. with the aim of determining how and when trypanosomes pass the bbb, we are undertaking a series of studies on invasion of the rodent-pathogenic tb brucei strain into the brains of mice and rats. in both animals, the trypanosomes localize early during infection to regions in the brain that lack a bbb, i.e. the choroid plexus and the circumventricular organs. at later stages, however, we have recently found that the parasites can penetrate into the brain parenchyma through the cerebral vessels ( fig. 1 ), although the tight junction proteins of the endothelial cells are preserved (mulenga et al., 2001) . this indicates that the penetration of trypanosomes into the brain parenchyma is an active process and not a consequence of a damaged bbb. we have obtained preliminary evidence that the pro-inflammatory cytokine, interferon (ifn)-g may be involved in regulating the neuroinvasion. in ifn-g receptor knock out mice, the parasites stay in or around the cerebral vessels and do not spread into the neuropil (kristensson et al., unpublished observation) . the mechanisms by which ifn-g may facilitate invasion of the trypanosomes may involve effects on the brain tissue or on the trypanomes. for instance, this cytokine is a most potent inducer of expression of chemokines in astrocytes and microglial cells in the brain and of proteases, and may regulate cell junctions as well. such factors are involved in attraction and penetration of different populations of white blood cells across the bbb, and may -invasion of african trypanosomes (tb brucei) through the blood-brain barrier. brains from rats sampled at 12 (left) and 55 (right) after intraperitoneal injection of the parasites. double-immunolabelling of the variable surface glycoprotein of the parasites (red) and glucose transporter-1 of the cerebral endothelial cells (green). at 12 days after infection, the parasites are confined to the vessel lumens, while at 55 days after infection they have invaded the brain parenchyma. hypothetically therefore facilitate the invasion of trypanosomes. the trypanosomes may have an advantage to pass through an intact bbb into the brain, because this would protect them from neutralization by circulating antibodies. in fact, treatment of mice with drugs that do not pass the bbb can clear the infection from the organism except from the brain, where the parasites have been suggested to persist for extended periods to cause late relapses (jennings et al., 1979) . the most characteristic clinical feature of nervous system involvement by the parasites is the sleep disturbances manifested as a disruption of the sleep pattern and circadian rhythms. in collaboration with prof. m. bentivoglio we have described marked dysfunctions of the suprachiasmatic nucleus, which is the main pacemaker for circadian rhythms, in an experimental model of the disease. these findings have been reviewed elsewhere (bentivoglio et al., 1994; kristensson et al., 2002) . trypanosome infections evoke a marked inflammatory response in the brain, which in humans is most intense in the white matter; a leukoencephalitis. during this process, inflammatory cytokines are induced in astrocytes and microglial cells (hunter et al., 1992; quan et al., 2000) . in spite of the long-standing induction of proinflammatory cytokines, which potentially may be neurotoxic, there are only minor signs of neurodegeneration in trypanosome-infected brains, both in human clinical materials and in experimental rodents (kristensson and bentivoglio, 1999) . this is interesting in view of the fact that over-expression of inflammatory cytokines have been implicated in the pathogenesis hiv dementia and in neurodegenerative diseases, such as alzheimer's disease. many of these cytokines are activated by nuclear factor-kb (nf-kb). aspirin and its metabolite sodium salicylate inhibit nf-kb-initiated transcription of immune response genes and treatment regimes with this drug have been considered in neurodegenerative diseases. since a chronic rodent infection with trypanosomes would provide an in vivo model of long-term inflammatory neurodegeneration, we have examined the effects of chronic treatment with sodium salicylate on neurodegeneration and cytokine rna expression in trypanosome-infected rats. in brains from untreated trypanosome-infected rats, degeneration, as detected by a modified cupric silver stain, was limited to certain nerve fibers in the vagus, lateral olfactory tract and some other white matter tracts, while sodium salicylate treatment resulted in extensive terminal and neuronal cell body degeneration in the cortex, hippocampus, striatum, thalamus and anterior olfactory nucleus. this exaggerated neurodegeneration was temporally and spatially associated with enhanced mrna expression of interleukin-1b, interleukin-1b converting enzyme, tumor necrosis factor-a, and inhibitory factor kba in the brain. restricted areas showed elevations in mrna expression of interleukin-1 receptor antagonist, interleukin-6, inducible nitric oxide synthase, ifn-g, and inducible cyclooxygenase (quan et al., 2000) . our results reveal an unexpected, serious complication in using aspirin drugs for treatment of chronic inflammatory brain conditions. in contrast to the effect of ifn-g in facilitating invasion of the extracellular african trypanosomes into the brain parenchyma, we have found that the same cytokine plays a crucial role in preventing spread of the intracellular bacterium, listeria monocytogenes, to the brainstem along the trigeminal nerves. listeria is widespread in nature and can infect several animal species. the bacteria pose a particular problem in sheep farming. they cause the so-called «circling disease» in sheep, which is a lethal disease with signs of brainstem encephalitis. by electron microscope, listeria has been found within axons of the trigeminal nerve in infected sheep (otter and blakemore, 1989 ). an encephalitis similarly targeted to the brainstem has, since its first description by eck (1957) , been reported in hundreds of human patients and the disease has commonly followed the eating of listeria-contaminated, unpasteurized cheese (armstrong and fung, 1993) . for study of factors that control spread of listeria monocytogenes along the trigeminal nerve to the brainstem, we have developed a mouse model in which the bacteria are injected into the snout of different immuno-deficient mice and the appearance of the bacteria in neurons of the trigeminal ganglia and brainstem recorded. in recombination activating gene 1 (rag-1)-deficient mice a neural route of infection was suggested after snout injection of the bacteria (jin et al., 2001) . this suggestion was based on immunostaining of listeria monocytogenes in the trigeminal ganglia and brainstem, but not in other areas of the brain; the kinetics of bacterial loads in snout, trigeminal ganglia and brain; and the increased resistance of mice infected with the plcb bacterial mutant (unable to spread from cell to cell). by using mice genomically lacking the mononuclear phagocytic growth factor colony-stimulating factor 1, and thereby deficient in macrophage and dendritic cell populations, we found that these cells play a dual role in listeria infections (jin et al., 2002) . on the one hand, they constitute a major defense against systemic infections, but on the other hand, they facilitate the invasion of the bacteria along the trigeminal nerve after snout injection. since dendritic cells of the cd11c 1 phenotype are in direct contact with peripheral nerve fibers, it may be suggested that these cells serve as an amplifier of listeria replication and thereby increasing the chances of bacterial spread to nerve fibers, which is dependent on direct cell contacts ( fig. 2) . ifn-g plays a protective role against listeria neuroinvasion along the trigeminal nerve and inducible nitric oxide synthase accounts partially for this protection. this was shown by comparison of the susceptibility to infection by listeria monocytogenes in the snout of mice deleted for the genes of both the ifn-g receptor and the inducible nitric oxide synthase with wild-type mice. the source of ifn-g appeared to be natural killer cells as shown by the use of combined rag-1-deficient, g-chain receptor gene-deficient mice. in vitro experiments showed that ifn-g can directly inhibit growth of listeria in dorsal root ganglia cells (jin et al., 2001) , and recent experiments ha. taken together these observations show that listeria monocytogenes after initial replication in macrophages/dendritic cells have the propensity to infect peripheral sensory neurons and spread to the central nervous system. at the same time, however, sensory neurons can control the replication of listeria and ifn-g may induce neurons to kill bacteria. this observed new function of a neuron, i.e. a bacterocidal activity, may be of relevance for an understanding of how spread and replication of infectious agents can be controlled in the immuno-priveledged nervous system. during infections with the parasite toxoplasma gondii, direct effects of ifn-g on replication of a pathogen in somatic cells have also been observed. this parasite replicates rapidly in macrophages, and ifn-g activation of these cells was previously considered as the major effectors for resistance to this intracellular pathogen. however, recent studies employing experiments on chimeric mice have shown that also nonhemopoietic cells can directly mediate ifn-g-dependent resistance of the host during toxoplasma gondii infections. in fact, it was speculated that control of intracellular parasitic replication is a reason why the ifn-g receptor is retained in nucleated somatic cells (yap and sher, 1999) . after infection of neurons (and skeletal muscle cells), the growth of toxoplasma gondii in a host is much retarded and the parasites persist in the brain as slowly replicating bradyzoites (fagard et al., 1999; lüder et al., 1999) . also in the nervous system, growth of toxoplasma may be under the control of ifn-g (fagard et al., 1999) , although the precise role of the cytokine remains to be determined (schlüter et al., 2001) . in spite of the long-standing infections with release of cytokines that control the intracellular growth of the parasites there are, like in african trypanosome-infected brains, no or only minor signs of neurodegeneration. the question how potentially neurotoxic cytokines can control growth of the parasite in neurons without causing their damage has been addressed in cell culture systems. both astrocytes and neurons are infected by the toxoplasma parasites, but supernatant from infected astrocytic cultures could prevent infected neuronal cultures from degeneration (rozenfeld et al., 2003) . the suggestion of a neuroprotective effect of astrocytes during infections is accordance with our studies showing such an effect in mumps virus-infected hippocampal neurons (owe-larsson et al., 1997) . these studies emphasis that infections in the brain are controlled not only by molecules that arrest intraneuronal microbe growth, but also by molecules that may protect neurons from damage. since microbes, like toxoplasma, may persist in the brain for the life span of the host, the questions arise whether the balance in release of microbe growth control and neuroprotective molecules can be altered to cause neurodegeneration later in life, and whether the infected neurons or the release of the molecules have any effect on neuronal function and behavior of the host. the latter question, i.e. potential behavior consequences of latent toxoplasma infections in the brain, was recently addressed by berdoy et al. (2000) . the definite host animal of toxoplasma gondii is the cat, while rodents, birds and by accident humans are intermediate hosts ( fig. 3) . although the brain provides a protected site for hiding of the parasite, it may also be a cul-de-sac: if the intermediate host dies, the parasite will die, unless the predator eats the intermediate host. according to the hypothesis of parasite manipulation of host behavior, a parasite may alter the behavior of a host to favor its transmission to another animal (poulin, 1994) . rats fear the smell of urine of their predator, the cat, but not that of a rabbit. however, this fear was lost in toxoplasma-infected rats; the infected rats were even attracted to the cat urine, and this «fatal attraction» will increase the chances of predation (berdoy et al., 2000) . in a review of human latent toxoplasma infections, it was noted that infected children may have a somewhat reduced iq, and that both men and women may have certain traits in their personality profile (webster, 2001) . infected men had a higher tendency to disregard rules of the society, they were more suspecting, jealous and dogmatic, while infected women were more warm-hearted, out-and easy-going, but also more conscientious, persistent, moralistic and staid (flegr and hrdá, 1994; flegr et al., 2000) . whether these findings are reproducible in other studies and societies remains to be evaluated as well as whether they represent causes or consequences of the infection. however that may be, these very common infections of the brain deserve studies in more detail. another common infectious agent transmissible from animals to man, which has been associated with human behavior disturbances is influenza a virus. there are well-documented cases of transmission from pigs to humans, and a few years ago direct spread of influenza a virus from hens to humans caused an epidemic in hong kong. pigs, ducks and humans are considered as a melting pot for this virus, and since the virus is segmented, reassorted variants can appear. thus, there is always the threat that new variants with neurotropic properties may araise. even in current epidemics, encephalitis due to influenza is not uncommon; in a recent study of 3,231 patients in finland (population 4 million) with acute central nervous system disease of suspected viral origin collected during 1995-1996, varizella-zoster virus was the main cause of disease followed by herpes simplex virus, enteroviruses and influenza a virus (koskiniemi et al., 2001) . there are anecdotal reports that psychiatric disturbances followed influenza in past epidemics; for instance, it was said that the world was never the same again, mentally, after the 1890 epidemic, and a reversible schizophrenia-like syndrome was associated with the 1918/19 epidemic (menninger, 1926) . there are also a number of studies, although the data are equivocal, suggesting an association between influenza a virus infections during pregnancy (the second trimester) and schizophrenia. although, taken together the epidemiological studies seem to have shown such an association, these type of studies may not contribute more than to indicate a relationship and the «the initiative now rests on cellular anatomy and neurophysiology» (munk-jørgensen and ewald, 2001) to forward our knowledge. in order to study the potentials of influenza a virus to cause developmental and/or functional disturbances of the nervous system, we have used a mouse-neuroadapted strain, wsn/33, of influenza a virus, which is derived from the first influenza a virus isolate 1933, in a series of studies in mice. this virus causes a lethal disease in mice with virus targeting to neurons in the hippocampus and substantia nigra following intracerebral or systemic injections (takahashi et al., 1995) . following injection into the olfactory bulbs, the virus spread to the anterior olfactory, the midline thalamic and medial habenular nuclei as well as to the ventral tegmentum areas. following this way of injection, the mice survive and the virus is cleared from the brain (mori et al., 1999) . our preliminary studies have shown that these surviving mice show certain disturbances consisting of learning disabilities and altered behavior in an elevated plus maze test (aronsson et al., unpublished data) . in immunodefective transporter associated with antigen processing 1 (tap1) mutant mice similarly infected with the virus, all segments of the genome of the virus could persist in the brain for more than 17 months after infection. viral rna encoding the nonstructural ns1 protein was detected in sections from the brain at midbrain levels by rt-pcr in almost all animals. both negative-strand genomic rna (vrna) and positive-strand rna, including mrna, were found (aronsson et al., 2001) . this observation shows that certain regions of the brain in immunodefective mice may harbor the genome of influenza a virus, including the ns1 gene the products of which may play a regulatory role in a host cell metabolism. in order to investigate if maternal influenza a virus has the potential to cause brain dysfunctions in the offspring, the wsn/33 strain was instilled intranasally into mice at day 14 of pregnancy (aronsson et al., 2002) . when the fetuses were examined 3 days later, viral nucleoprotein and rna were detected in their brains. after showing that influenza a virus could pass the placenta and be targeted to the fetal brains we studied in another series of experiments the effect of the maternal infection on the offspring. at an infectious dose of 7,500 plaque forming units (pfu) intranasally instilled during pregnancy, mice were born but all died within 2-8 days after birth. when the viral dose was reduced to 750 pfu, all offspring survived. about 25 % of brains sampled from these offspring at 10, 20, 35, 60 and 90 days after birth showed presence of viral rna encoding the matrix and nucleocapsid proteins by rt-pcr ( fig. 4) . these studies show that influenza a virus can persist in the brain even of non-immunocompromised individuals after an infection at early life. in our preliminary studies, we have observed that there are certain changes in the gene expression profile, verified by real time pcr, in brains from the offspring. interestingly some of these changes did not appear until 90 days of age, which may indicate that a maternal influenza a virus infection can cause gene expression changes that progress over time to become manifested later in life. new observations on the neuropathogenesis of infectious agents that can be transmitted from animals to man have been highlighted and these include: i) the role of cytokines for spread and targeting to the brain. while the host derived inflammatory molecule ifn-g seems to inhibit the invasion of the intracellular listeria monocytogenes along trigeminal nerves, it had the paradoxical effect of facilitating entry of the extracellular african trypanosomes into the brain through the bbb. ii) populations of neurons that are attacked by pathogens have the capacity to control growth, and even to kill, the microbes. this occurs by mechanisms that at the same time protect neurons from being damaged. pharmacological treatment may tilt a balance between potentially neurotoxic and neuroprotective molecules in an unpredictable direction to cause severe neurodegeneration in the brain. iii) microbes may induce imbalances in neuronal circuits and gene expression changes that may progress in later life. questions may therefore be addressed whether microbes transmitted from animals could be included brainstem encephalitis (rhombencephalitis) due to listeria monocytogenes: case report and review persistence of the influenza a/wsn/33 virus rna at midbrain levels of immunodefective mice persistence of viral rna in the brain of offspring to mice infected with influenza a/wsn/33 virus during pregnancy trypanosoma brucei and the nervous system fatal attraction in rats infected with toxoplasma gondii encephalomyelitis listeria apostematosa differential development of toxoplasma gondii in neural cells influence of chronic toxoplasmosis on some human personality factors. folia parasit correlation of duration of latent toxoplasma gondii infection with personality changes in women astrocyte activation correlates with cytokine production in central nervous system of trypanosoma brucei brucei-infected mice the brain as a source of relapsing trypanosoma brucei infection in mice after chemotherapy a neural route of cerebral listeria monocytogenes murine infection -role of immune mechanisms in the control of bacterial neuroinvasion colony-stimulating factor-1 dependent cells protect against systemic infection with listeria monocytogenes, but facilitate neuroinvasion infections of the central nervous system of suspected viral origin: a collaborative study from finland pathology of african trypanosomiasis parasites and the brain: neuroinvasion, immunopathogenesis and neuronal dysfunctions toxoplasma gondii in primary rat cns cells: differential contribution of neurons, astrocytes, and microglial cells for the intracerebral development and stage differentiation influenza and schizophrenia. an analysis of post-natal 'dementia precox', as of 1918, and five years later selective targeting of habenular, thalamic midline and monoaminergic brainstem neurons by neurotropic influenza a virus in mice trypanosoma brucei brucei crosses the blood-brain barrier while tight junction proteins are preserved in a rat chronic disease model epidemiology in neurobiogical research: exemplified by the influenza-schizophrenia theory observation on the presence of listeria monocytogenes in axons reduction of voltage-dependent calcium currents in mumps virus-infected cultures of rat hippocampal neurons the evolution of parasite manipulation of host behaviour: a theoretical analysis chronic sodium salicylate treatment exacerbates brain neurodegeneration in rats infected with trypanosoma brucei soluble factors released by toxoplasma gondii-infected astrocytes down-modulate nitric oxide production by gamma interferon-activated microglia and prevent neuronal degeneration toxoplasma gondii infection of neurons induces neuronal cytokine and chemokine production, but gamma interferon-and tumor necrosis factor-stimulated neurons fail to inhibit the invasion and growth of t. gondii the substantia nigra is a major target for neurovirulent influenza a virus rats, cats, people and parasites: the impact of latent toxoplasmosis on behaviour effector cells of both nonhemopoietic and hemopoietic origin are required for interferon (ifn)-g and tumor necrosis factor (tnf)-a-dependent host resistance to the intracellular pathogen, toxoplasma gondii in the list of potential causes of behavior disturbances or even neuropsychiatric diseases in man. key: cord-003315-r1wkx0ml authors: jacobs, sophie; wavreil, fanny; schepens, bert; gad, hans henrik; hartmann, rune; rocha-pereira, joana; neyts, johan; saelens, xavier; michiels, thomas title: species specificity of type iii interferon activity and development of a sensitive luciferase-based bioassay for quantitation of mouse interferon-λ date: 2018-11-01 journal: j interferon cytokine res doi: 10.1089/jir.2018.0066 sha: doc_id: 3315 cord_uid: r1wkx0ml the type iii interferon (ifn-λ) family includes 4 ifn-λ subtypes in man. in the mouse, only the genes coding for ifn-λ2 and -λ3 are present. unlike mouse and human type i ifns (ifn-α/β), which exhibit strong species specificity, type iii ifns were reported to act in a cross-specific manner. we reexamined the cross-specificity and observed that mouse and human ifn-λ exhibit some species specificity, although much less than type i ifns. mouse ifn-λ3 displayed clear species specificity, being 25-fold less active in human cells than the closely related mouse ifn-λ2. this specificity likely depends on amino acids in α helices a and f that diverged from other ifn-λ sequences. human ifn-λ4, in contrast, retained high activity in mouse cells. we next developed a firefly luciferase-based reporter cell line, named fawa-λ-luc, to detect ifn-λ in biological fluids with high specificity and sensitivity. fawa-λ-luc cells, derived from mouse epithelial cells that are responsive to ifn-λ, were made nonresponsive to type i ifns by inactivation of the ifnar2 gene and strongly responsive to ifn-λ by overexpression of the mouse ifnlr1. this bioassay was as sensitive as a commercially available enzyme-linked immunosorbent assay in detecting mouse ifn-λ in cell culture supernatant, as well as in serum and bronchoalveolar lavage samples of virus-infected mice. the assay also enabled the sensitive detection of human ifn-λ activity, including that of the divergent ifn-λ4 with a bias, however, due to variable activity of ifn-λ subtypes. t ype i and iii interferons (ifns) are typically produced in response to viral infection, and these cytokines induce an antiviral state in target cells (isaacs and lindenmann 1957; kotenko and others 2003; sheppard and others 2003) . type i ifns consist of 13 ifn-a subtypes and ifn-b, -o (human) , and -z (mouse) -e and -k. the type iii family of ifns in humans comprises 3 functional genes that express ifn-l1 (il-29), ifn-l2 (il-28a), and ifn-l3 (il-28b) (kotenko and others 2003; sheppard and others 2003) . a fourth ifn-l subtype (ifn-l4) is present in a part of the human population, depending on a dinucleotide frameshift polymorphism upstream of the ifnl3 gene, which creates or disrupts the open reading frame (orf) encoding ifn-l4 (prokunina-olsson and others 2013) . in the mouse, ifnl1 is a pseudogene, and only ifn-l2 and ifn-l3 are expressed. unlike mouse and human type i ifns that exhibit strongly species-specific activity (veomett and veomett 1979) , type iii ifns were reported to act on cell types from both origins (lasfar and others 2006; hermant and others 2014) . the type i ifn family members signal through a unique heterodimeric receptor (ifnar), composed of the ifnar1 and ifnar2 subunits. type iii ifns engage a distinct receptor (ifnlr), composed of 2 chains: the ifn-l-specific ifnlr1, and il10rb which is shared by other il10-related cytokines (kotenko and others 2003; sheppard and others 2003) . while ifnar is ubiquitously expressed, ifnlr is expressed by a restricted range of cell types and mostly acts at mucosal surfaces. epithelial cells are well-established targets of ifn-l in vivo (sommereyns and others 2008) , although some immune cells such as neutrophils and dendritic cells have been characterized as ifn-l responders (koltsida and others 2011; blazek and others 2015; broggi and others 2017; espinosa and others 2017) . although type i and type iii ifns act on distinct receptors, they activate a similar jak-stat transduction pathway, leading to the phosphorylation of stat1 and stat2 that associate with irf9 to form the isgf3 complex. type i and type iii ifn signaling leads to the transcription of an overlapping set of ifn-stimulated genes (isgs) (dumoutier and others 2004; ank and others 2006) . type i ifn in biological samples can be measured by a variety of bioassays, but efficient techniques for type iii ifn quantification are largely lacking. enzyme-linked immunosorbent assay (elisa) for ifn-l detection is time and cost intensive and fails to detect ifn-l4. conventional cytopathic effect reduction bioassays to quantify antiviral activity as a proxy for the presence of ifn require the manipulation of infectious virus. luciferase reporter cells that have previously been used for recombinant human ifn-l detection were still responsive to type i ifn and would not allow specific ifn-l detection from biological samples (uze and monneron 2007) . in this work, we reexamined the species specificity of ifn-l activity, and we developed a very sensitive luciferase-based bioassay specific for type iii ifn detection. the lkr10 cell line (kind gift from guido bommer, de duve institute, brussels, belgium) is derived from lung adenocarcinoma tissues from a k-rasla1 mouse ( johnson and others 2001) . a549 cells (atcc) were kindly provided by pierre coulie, balb/3t3 fibroblasts (aaronson and todaro 1968) those cells, and derivatives, were maintained in dulbecco's modified eagle's medium (dmem) (lonza, vervier, belgium) containing 4.5 g/l glucose, supplemented with 10% fetal calf serum (fcs) (sigma-aldrich, overijse, belgium). african green monkey kidney (vero) cells (atcc) were cultured in dmem supplemented with 10% fcs, nonessential amino-acid, l-glutamine, and sodium pyruvate. bhk-21 cells (atcc) were cultured in glasgow's minimum essential medium (gibco; thermo fisher scientific, asse, belgium) supplemented with 10% newborn calf serum and 2.95 g/l tryptose phosphate broth. all media were supplemented with 50 u/ml penicillin and 50 mg/ml streptomycin (lonza). expression vectors used in this study are listed in table 1 . plasmids psj1 and psj7 are pcdna3 derivatives (invitrogen; thermo fisher scientific) used for mouse ifn-l2 and ifn-l3 expression in 293t cells. these plasmids were constructed by cloning the orf encoding ifn-l2 (psj1) and between the bamhi and xbai sites of the vector. coding sequences were polymerase chain reaction (pcr)-amplified from liver cdna samples of a c57bl/6 mouse infected with influenza turh (h7/n1) strain (hermant and others 2014) , using the following primers: forward (fw): bamhi-agei-kozak-mifn-l2/3, 5¢-aaa agg atc cac cgg tgc cac cat gct cct cct gct gtt gcc tct g-3¢; reverse (rev): mifn-l2/3-stop-xbai, 5¢-aaa atc tag att atc aga cac act ggt ctc cay tgg c-3¢. the sequence of psj7 matches the genomic ifnl3 sequence but diverges from a few nucleotides from that of the previously constructed pcs59 plasmid (sommereyns and others 2008) . both plasmids encode functional ifn-l3. pph23 was obtained by pcr-cloning the ifna2 orf from hela-m cell cdna, between the ecori and noti sites of pcdna3 (fw: ecori-kozak-ifna2, 5¢-aaa aga att cac cat ggc ctt gac ctt tgc ttt-3¢; rev: ifna2noti, 5¢-aaa agc ggc cgc tca ttc ctt act tct taa act tt-3¢). pmk03 is a lentiviral vector, derived from ptm897, that carries the mouse mx1 gene promoter driving the expression of the firefly luciferase gene. it is derived from pcclsin. cppt.hpgk.gfp.pre (follenzi and others 2000) in which a cloning polysite was first inserted to replace the coding sequences, yielding ptm897. the firefly luciferase gene from pgl4.16 (promega) was inserted between the xhoi and xbai sites of the vector, and the mx1 promoter was then cloned from pbsk-mx1, a gift from peter staeheli (freiburg university, freiburg, germany), as a bamhi/bsabi fragment. the lentiviral vector psj12, used for expression of the mouse ifn-l receptor, was obtained by cloning the ifnlr1 orf between the bamhi and xbai sites of ptm945, a lentiviral vector allowing the coexpression of the cloned gene and of mcherry (hermant and others 2013) . the ifnlr1 orf sequence was subcloned in this plasmid from pcr2.1-licr2, kindly provided by laure dumoutier (de duve institute, brussels, belgium). gene inactivation was done with px461 plasmid (pspcas9n-2a-gfp) coding for the cas9 nickase and green fluorescent protein (gfp) (ran and others 2013a). the 2 single guide rnas (sgrnas) were designed using the mit clustered regularly interspaced short palindromic repeats (crispr) design tool website (http://crispr.mit.edu). the selected sgrna pair (sgrna1-fw: 5¢-cac cgt caa att ctg gcg gct caa g-3¢; rev: 5¢-aaa cct tga gcc gcc aga att tga c-3¢; sgrna2-fw: 5¢-cac cga gac cac ata aac gtg acg a-3¢; rev: 5¢-aaa ctc gtc acg ttt atg tgg tct c-3¢) was targeting exon 6 of the mouse ifnar2 gene (genbank: y09865.1) and exhibited no expected off-target cleavage site. the sgrnas were cloned into px461 to form the plasmids psj37 and psj38. these constructs were cotransfected in lkr10 cells, using transit ò -lt1 transfection reagent (mirus bio llc, madison), according to the manufacturer's instructions. after 48 h, gfp-positive cells were sorted by fluorescence activated cell sorting (facs aria iii; bd biosciences) and cloned in 96-well plates. clones were screened for loss of type i ifn response with an antiviral assay. genome editing of the targeted exon was further confirmed by sequencing (genewiz). isg expression was measured by reverse transcriptionquantitative pcr. ifnar2-knockout (ko) and wild-type (wt) lkr10 cells were treated with 100 u/ml mouse ifn-aa, 700 pg/ml mouse ifn-l3, or control supernatant (mock) for 8 h before rna extraction. total rna extraction, reverse transcription, and sybr green quantitative pcr for mrna encoding mouse b-actin, oasl2, and usp18 were performed as previously described (paul and michiels 2006) . for infection analysis, cells were dissociated with trypsin-edta and suspended in phosphate-buffered saline (pbs) containing 5% of filtered fcs and 0.5% of paraformalde-hyde. data acquisition was done with an lsrfortessa flow cytometer (bd biosciences) using facsdiva software. data were analyzed using flowjo 9.6.4. the rate of infection was defined as the percentage of mcherry-positive cells 24 h postinfection with 0.5 pfu/cell tm967. for cell sorting, transduced cells were suspended in pbs containing 1% fcs and 1 mm edta. mcherry-or gfppositive cells were cloned at 1 cell per well in 96-well plates using the facs aria iii (bd biosciences). immunoblotting stat1, p-stat1, and b-actin were detected by western blot using sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels containing 10% acrylamide and run in a tris-glycine buffer. blots were probed with anti-stat1 polyclonal (sc-346; santa cruz biotechnology, heidelberg, germany), p-stat1 monoclonal (#9167; cell signaling technology, leiden, the netherlands), and anti-b-actin monoclonal (a5441; sigma-aldrich) antibodies. mouse ifn-aa, ifn-b, ifn-l2, ifn-l3, and human ifn-a2 were produced as described previously from 293t cells transfected with pcdna3-ifn-aa, pcdna3-ifn-b (van pesch and others 2004), pcdna3-ifn-l2 (psj1), and pcdna3-ifn-l3 (psj7). human ifn-a2 was produced similarly, using pcdna3-ifn-a2 (pph23). supernatant collected from 293t cells transfected in parallel with the empty pcdna3 vector was used for control treatment (mock) of cells and diluted as for ifns. recombinant mouse ifn-l3 (1789-ml-025) and human ifn-l1 (1598-il-025), ifn-l2 (1587-il-025), and ifn-l3 (5259-il-025) were purchased from r&d (r&d systems, minneapolis). recombinant human ifn-l4 was produced as described (hamming and others 2013) . mouse (ref 315-05) and human (ref 300-02) ifn-g were purchased from pe-protech (london, united kingdom). genbank accession numbers for mouse and human ifn-l are listed in table 2 . ifns were diluted in culture medium for cell treatment or in reagent diluent for elisa. mouse ifn-l2 and ifn-l3 were quantified by elisa. mouse ifn-aa and human ifn-a2 antiviral activities were quantified, as described previously, by a cytopathic effect reduction assay in mouse balb-3t3 and human hela-m cells, respectively, using mengovirus (van pesch and michiels 2003) . similar assays were conducted in human a549 cells and mouse lkr10 cells for quantification of ifn-l cross-species activity. ifnl4 was quantified by a cytopathic effect reduction assay in a549 cells, using recombinant human ifn-l3 as a standard, which was reported to have a similar specific activity (hamming and others 2013). mouse ifn-l2/3 measurement by elisa was performed using the mouse ifn-l2/3 duoset (r&d systems), according to the manufacturer's protocol. mouse sera were diluted 5-to 10-fold, and bronchoalveolar lavage fluids (balfs) were diluted 5-fold. when required (in case of samples derived from infected cells or mice), infectious virus present in biological samples was ultraviolet light (uv)-inactivated. for uv inactivation, 2.5 mm thick fluid samples were exposed at fixed uv dose (0.25-0.5-1-2 j/cm 2 ) with the uv irradiation system biolink 254 (vilber lourmat, eberhardzell, germany), either in 24-(470 ml) or in 96-well (80 ml) plate. uv-exposed samples were titrated on bhk-21 cells by a standard plaque assay to confirm virus inactivation. to confirm that the uv irradiation procedure that was established for picornaviruses effectively inactivates respiratory syncytial virus (rsv), 3 samples of 470 ml containing 1.5 · 10 6 pfu of rsv were exposed to 2 j/cm 2 with the uv irradiation system bio-link 254 at room temperature in a 24-well plate (uv irradiated samples). as controls 3 similar samples of 470 ml containing 1.5 · 10 6 pfu of rsv were incubated at room temperature in a 24-well plate (untreated samples). the titers of replicating rsv in the uv irradiated and untreated samples were determined by plaque assay using vero cells. cells were seeded at 160,000 cells per well in 24-well plates for cell supernatant and recombinant ifn analysis or at 32,000 cells per well in 96-well plates for mouse serum and bal analysis. forty-eight hours after seeding, cells were treated with ifn-l2/3 or mock-treated or incubated with uv-treated mouse serum (50-and 100-fold dilutions) or bal (5-fold dilutions). samples were diluted in culture medium. recombinant mouse ifn-l3 provided in the elisa kit was used as a standard. firefly luciferase activity was measured 6 h after treatment using the luciferase assay system (promega). twenty microliters and 100-ml lysis buffer were used in 96-and 24-well plates, respectively; 10 ml of the lysate was mixed with 25 ml of substrate for detection. luminescence was measured with a glomax 20/20 luminometer (promega). the limit of detection (lod) for each test was established based on the mean of mock-treated samples, plus 3 standard deviations, in all assays. tm967 is a theiler's murine encephalomyelitis virus (tmev) (strain da) derivative that carries a capsid adapted to infect l929 cells and the orf encoding the mcherry fluorescent protein cloned as a xbai/bsiwi fragment to replace codons 5-67 of the leader protein coding region, in the pkj6 vector ( jnaoui and michiels 1998) . mengovirus (a strain of encephalomyocarditis virus) used for ifn bio-assay is an attenuated variant that has been described previously (hermant and others 2013) . those viruses were quantified by plaque assay on bhk21 cells. rsv propagation and enrichment were performed as described in schepens and others (schepens and others 2014) . mouse experiments were conducted according to the national (belgian law 14/08/1986 and 22/12/2003, belgian royal decree 06/04/2010) and european (eu directives 2010/ 63/eu, 86/609/eeg) animal regulations. animal protocols were approved by the ethics committee of ghent university (permit no. la1400091, approval id 2013-030). all efforts were made to avoid or ameliorate suffering of animals. specific pathogen-free female balb/c mice at the age of 7-8 weeks were purchased from charles river. the mice were housed in a specific-pathogen-free temperature controlled environment with 12-h light/12-h dark cycles and given water and food ad libitum. mice were used at 9 weeks of age after adaptation in the animal room for 1 week. for challenge, the mice were sedated with isoflurane and infected intranasally with 4 · 10 6 pfu of human rsv. mock infection of the control group was performed with hank's balanced salt solution. five days postinfection, bal was isolated under anesthesia with an intraperitoneal injection of avertin (2.5% in pbs). a 23gauge cannula was inserted into the trachea, and cells were collected by washing the airway lumen twice with 0.5 ml pbs containing 0.05 mm edta. after removing the cells by centrifugation (5 min at 400g), the balf was stored at -80°c. serum samples from norovirus-infected and plasmidelectroinjected mice were reused from a previously published experiment (rocha-pereira and others 2018) to minimize animal experimentation. statistical analysis was performed using prism 7 software (graphpad software, san diego, ca). [***p < 0.001, **p < 0.01, *p < 0.05, and ns no statistically significant difference (p ‡ 0.05)]. unlike mouse and human type i ifns (ifn-a/b) that exhibit strong species specificity, type iii ifns were reported to act in a cross-species manner. to confirm whether a mouse cellbased bioassay would permit the detection of ifn-l subtypes from 2 different mammalian species, the species specificity of mouse and human type iii ifns was reexamined. crossreactive antiviral activity was systematically compared in a cytopathic effect reduction assay, using epithelial cell lines from mouse (lkr10) and human (a549) origin. as expected, type i human (ifn-a2) and mouse (ifn-aa) ifns were extremely species-specific in the antiviral assay, with an activity difference higher than 10,000-fold when applied to mouse and human cells. ifn-l exhibited some level of species specificity although much less than type i ifns (fig. 1a) . mouse ifn-l3 displayed the most pronounced species specificity. for a quantity that yielded the same antiviral activity in mouse cells, ifn-l3 was 25 times less active in human cells than the closely related mouse ifn-l2. human ifn-l1, ifnl-2 and, to a lesser extent, ifnl-3 exhibited significant antiviral effect on mouse epithelial cells. as ifn-l4 has been shown to have a specific activity similar to that of human ifn-l3 in human cells, it was quantified accordingly using the antiviral assay (hamming and others 2013) . remarkably, human ifn-l4 displayed much more antiviral activity on mouse cells than equivalent amounts of human ifn-l3. accordingly, treatment of lkr10 cells with concentrations of human ifn-l3 and ifn-l4 that yielded equivalent antiviral activities in human cells resulted in clear stat1 phosphorylation after ifn-l4 but not after ifn-l3 treatment (fig. 1b) . in line with their epithelial origin, mouse lkr10 cells respond to ifn-l. to ensure a selective detection of type iii ifns, the gene coding for the ifnar2 subunit of the type i ifn receptor was inactivated using the double nickase crispr/crispr-associated (cas) 9 technology (ran and others 2013b). an lkr10-ifnar2-ko clone was selected for the absence of response to type i ifn and intact response to type iii ifn. sequencing of the targeted exon revealed frameshift mutations in both ifnar2 alleles, resulting in premature stop codon appearance. in contrast to ifn-l treatment, ifn-a treatment of this clone failed to induce the expression of the ifn-stimulated genes, oasl2 ( fig. 2a) and usp18 (fig. 2b) , and to protect against infection with tm967, a tmev derivative expressing mcherry (fig. 2c) . to develop ifn-l reporter cells, the lkr10-ifnar2-ko clone was transduced with a lentiviral vector encoding the firefly luciferase gene under the control of the mx1 pro-moter. among the clones tested for luciferase activity induction after ifn-l treatment, the best one displayed not more than 10-fold luciferase activity induction. to try to boost the reporter gene responsiveness to ifn-l, this clone was transduced with sj12, a lentiviral vector that coexpresses the mouse ifnlr1 and mcherry. transduced cells were sorted, based on mcherry expression, and cloned. a selected clone, named ''fawa-l-luc,'' responded vigorously to ifn-l3. this clone displayed increased constitutive stat1 expression and strong stat1 phosphorylation after ifn-l3 but not after ifn-a treatment (fig. 2d) . a significant luciferase activity increase was measured in fawa-lluc cells as soon as 1 h after treatment with 700 pg/ml (quantified by elisa) of mouse ifn-l3. maximal induction was reached within 6 h and lasted up to 16 h posttreatment (fig. 3a) . the sensitivity of the fawa-l-luc assay was compared to that of an available elisa test for mouse ifn-l. mouse ifn-l2 and -3 produced by transfected 293t cells and recombinant mouse ifn-l3 were quantified in parallel by elisa and the fawa-l-luc assay. in the elisa, responses were linear between 1,000 and 16.25 pg/ml, the experimental lod of this test (fig. 3b ). in the fawa-l-luc assay, linearity was reached in a narrower concentration range (1.95-62.5 pg/ml for mouse ifn-l2; 3.9-125 pg/ml for mouse ifn-l3), but a higher sensitivity was observed with the detection limit being below 1.95 pg/ml and 3.9 pg/ml, for ifn-l2 and -3, respectively (fig. 3c, d) . based on elisa quantification, mouse ifn-l2 bioactivity in this assay was 2-fold higher than that of mouse ifn-l3. importantly, after up to 29 passages, the intensity of the luciferase signal in the fawa-l-luc cells tended to decrease, but the sensitivity of the bioassay was preserved because table showing the relative antiviral activity of mouse and human ifn-l on mouse lkr10 and human a549 cells. species specificity index was calculated as the ratio between the relative antiviral activity on cells of homologous to nonhomologous species (eg, for mouse ifn: relative activity in mouse cells/relative activity in human cells). relative activities in a given cell line were calculated as the ifn dilutions (starting from 1 ng/ml) that yielded similar antiviral activities. *due to the low amount of ifn-l4 available, the initial concentration of this ifn was estimated by comparison with human ifn-l3 antiviral activity that was reported to have a similar specific activity. (b) western blot showing stat1 phosphorylation in mouse lkr10 cells 30 min after treatment with control medium (mock) or ifn-l. concentrations of human (hu) ifn-l3 (615 pg/ml) and ifn-l4 that yielded the same antiviral activity on human a549 cells were used. as a control, mouse ifn-l3 was used at a concentration (2.5 ng/ml) showing equivalent antiviral activity as human ifn-l4 on mouse cells. results are representative of 3 experiments. the background activity also decreased with high passage numbers. to confirm the specificity of fawa-l-luc cells for ifn-l, we also evaluated any possible luciferase induction after treatment with type i and type ii (ie, ifn-g) ifns. as expected, no signal was observed after treatment with up to 2,500 iu/ml of either mouse ifn-aa or ifn-b. human ifn-g also failed to induce a detectable luminescent signal in the fawa-l-luc cells in the tested concentration range (0-25 ng/ml). the lod for mouse ifn-g was 12.55 ng/ml, a concentration at least 10-fold higher than what is observed in mouse fluids in infectious contexts (pomeroy and others 1998; claser and others 2017) . ifn-g is thus not expected to interfere with ifn-l detection in biological samples. we tested the sensitivity of our bioassay for human ifn-l detection. responses were analyzed individually for all 4 human ifn-l subtypes (fig. 4a-d) . as their cross-species activity differed, different detection sensitivities were reached with our mouse cell-based reporter assay: 15.125 pg/ml for ifn-l1, 500 pg/ml for ifn-l2, 31.25 pg/ml for ifn-l3, and 1.95 pg/ml for ifn-l4. the bioassay turned out to be extremely sensitive for human ifn-l4 detection. it is, however, worth noting that the recombinant ifn-l4 stock concentration was determined by comparison of its activity with that of a human ifn-l3 standard. when testing biological samples, potential occurrence of infectious virus may interfere with the viability of reporter cells. it is therefore important to inactivate infectious viruses in such samples. as ifn-l had previously been shown to be acid labile (kugel and others 2011) , uv inactivation of the virus was preferred. we thus tested whether virucidal uv doses would affect ifn-l activity. to this end, tm967 virus, mouse ifn-l2, and mouse ifn-l3 were irradiated with increasing uv doses. mock-and uv-exposed samples were then analyzed with the bioassay for mouse ifn-l2 and ifn-l3 detection and by plaque assay to determine viral titers. a 2 j/cm 2 uv exposure was sufficient to ensure a virus titer reduction of more than 100,000-fold (5.5 · 10 5 pfu to <1 pfu). at this uv dose, mouse ifn-l2 activity was not significantly reduced and mouse ifn-l3 showed a bioactivity reduction of less than 25% (fig. 5a) . similarly, ifn-l1 and ifn-l4 bioactivities were reduced by 25%, while human ifn-l2 and ifnl-3 were slightly more sensitive to irradiation, with about 2-fold activity decrease (fig. 5b) . we then compared the sensitivities of the fawa-l-luc and of the elisa assays to detect ifn-l in mouse serum and bal samples. ifn-l was first quantified in the serum of mice that were either injected intramuscularly with an ifn-l3 expressing plasmid (pcs59), thus expected to produce circulating ifn-l, or infected with mouse norovirus (rocha-pereira and others 2018). a preliminary test revealed that the minimal serum dilution that did not interfere with spiked-in ifn-l detection was 16fold for the bioassay and 5-fold for the elisa (fig. 5c, d) with both the fawa-l-luc and elisa assays. ifn-l was clearly detected at day 2 and 4 in the serum of mice that were electroinjected with the ifn-l3 expressing plasmid, as well as at day 3 postnorovirus infection (fig. 6a, b) . at day 2 after a single ifn-l3 injection, the cytokine was detectable in 1 out of 4 mice by elisa and in 3 out of 4 mice by the bioassay. the detection limit was higher for the elisa (194 pg/ml) than for the bioassay (9.29 pg/ml), showing the higher sensitivity of the luciferase-based bioassay (fig. 6a, b) . next, we compared ifn-l detection by the fawa-l-luc assay and by elisa in balf of balb/c mice that were mock infected or infected with rsv. balf and lungs were collected 5 days postinfection. plaque assay revealed that balf of all infected mice contained between 1.8 · 10 2 and 7.33 · 10 2 pfu/ ml of live replicating virus. the used uv irradiation procedure proved to readily inactivate rsv (rsv titer reduction of more than 46,000-fold; 4.6 · 10 4 pfu to <1 pfu). five-fold diluted control balf did not modify ifn-l2/3 detection in any of the 2 assays. using 5-fold dilutions of the balf as in the bioassay, we failed to detect any ifn-l by elisa (fig. 6c ). in contrast (fig. 6d) , ifn-l was detected by fawal-luc assay at a very low concentration in the balf of the rsv infected mice (5/6) and not detected in the control group. percentage of ifn-l activity (mean and sd) remaining after uv treatment (n = 3) at 2 j/cm 2 . ifn activity was measured in fawa-l-luc cells for ifn concentrations that yielded equivalent luciferase activities (20,000 rlu) before uv treatment (250 pg/ml mifn-l2 and mifn-l3, 125 pg/ml human ifn-l1, 10 ng/ml huifn-l2, 500 pg/ml huifn-l3, and 62.5 pg/ml huifn-l4). (c, d) influence of serum dilution on ifn-l detection by fawa-l-luc cells (c) and elisa (d). (c) fawa-l-luc cells were treated in triplicate with fixed doses of 15 and 250 pg/ml mifn-l2 supernatant and with 2-fold serial dilutions (2-128-fold) of control mouse serum. (d) 125 pg/ml recombinant mifn-l3 was mixed with 2-fold serial dilutions of control mouse serum (2.5-10-fold) before detection by elisa. (a-c) reporter cells were exposed to ifn for 6 h before luciferase assay. student's t-test: */**/***denotes a significant difference in signal compared to no uv exposure (a) or the absence of serum (c). uv, ultraviolet light. we conclude that the fawa-l-luc-based assay described in this study allows to quantify ifn-l from biological samples in a highly sensitive way. we reexamined the species specificity of ifn-l and highlighted a previously overlooked difference in bioactivity between mouse and human type iii ifns. mouse ifn-l3 displayed a surprising species specificity, with a 50-fold difference in relative antiviral activity when applied to lkr10 (mouse) and a549 (human) cells. this contrasted strongly with the closely related mouse ifn-l2 (93% amino acid sequence identity), which displayed little species specificity and was 25 times more active in human cells than mouse ifn-l3. in contrast, human ifn-l4 displayed a strikingly strong activity on mouse cells compared to human ifn-l3. although ifn-l4 is not expressed in mouse and rat, it is highly conserved in many mammalian species (key and others 2014) where it was shown to be cross-reactive. the nonhuman ifn-l4 orthologs were reported to activate ifnlr1 in human cells, sometimes with a higher efficacy than human ifn-l4 (eg, dog ifn-l4) (paquin and others 2016). the mouse cell response to human ifn-l4 is thus in line with the ability of ifn-l4 to be cross-reactive among mammalian species, although we do not have any physiological explanation for this phenomenon. ifn-l proteins follow the typical class ii cytokine structure, consisting of 6 secondary structure elements (a-f) (pestka and others 2004) . helix a is involved in both ifnlr1 and il10rb binding, while helix d binds il10rb and helix f binds ifnlr1 (gad and others 2010). among amino acid residues that have been characterized as crucial for ifnlr1 binding and activation (gad and others 2010), a few divergences are observed between sequences from mouse and human ifn-l (fig. 7) . notably, the more species-specific mouse ifn-l3 has 4 amino acid residues in a helices a and f that diverge from the 5 other ifn-l sequences, including the related mouse ifn-l2. in helix a, gly37 uniquely replaces the asp residue present in the other sequences. in helix f, 3 residues are unique to mouse ifn-l3: asp 150 (ala / asp), gln158 and bioassay (b) in the serum of ag129 mice 2 or 4 days after electroinjection of mouse ifn-l3 (mifn-l3) expressing (pcs59) or empty (pcdna3) plasmids, 2 days after injection of pcs59, or 3 days after infection with mouse norovirus. ifnl detection in the serum by elisa was performed without uv exposure to keep maximal sensitivity. (c, d) ifn-l2/3 detection by elisa (c) and bioassay (d) in the bronchoalveolar lavage of balb/c mice, 5 days postinfection with rsv, compared to control mice (mock). balf were uv-exposed before testing. (a-d) the horizontal dotted line represents the lod. mann-whitney: */**indicates a significant difference compared to pcdna3 group at days 2 or 4 (a, b) or compared to mock (d). balf, bronchoalveolar lavage fluid; rsv, respiratory syncytial virus. (arg / gln), and leu161 (thr / leu). future site-directed mutagenesis studies could help defining the key residues involved in the species specificity. we designed a highly sensitive bioassay named ''fawa-lluc'' based on luciferase reporter cells, specific for ifn-l detection and quantification. the bioassay is based on a cell line that is naturally responsive to ifn-l in which the type i ifn receptor gene was inactivated. overexpression of the mouse ifnlr1 receptor in those cells led to increased induction of the reporter gene after ifn-l treatment. unlike previously described bioassays, such as hl-116 fibroblasts stably transfected with the human ifnlr1 (uze and monneron 2007), fawa-l-luc cells permit the selective detection of type iii ifn because they lack the type i ifn receptor. they also fail to respond to ifn-g concentrations that are reached in infected mouse serum. this novel bioassay is efficient in measuring ifn-l from mouse bal and serum. ifn-l activity was hardly affected by uv exposure at virucidal doses, thus allowing detection in infected biological fluids. the high sensitivity of the assay permits sparing biological material as 10-fold more diluted serum samples could be used for bioassay detection (50-100-fold dilution) compared to the elisa (5-10-fold). when cell toxicity of the tested samples is suspected, cell viability may independently be confirmed with nonlytic viability assays such as resazurin-based tests, which are compatible with luciferase detection. importantly, our bioassay offers a physiologic analysis, attesting of the functionality of the ifn-l present in the samples, irrespective of specific activity. the bioassay also allows to detect human ifn-l. given the divergent cross-species activity and the higher uv lability of the human type iii ifns, the bioassay might be used for individual subtype analysis, but would not fit for their detection in complex biological samples. finally, it offers a sensitive detection of the divergent ifn-l4, for which no efficient commercial test is available. development of 3t3-like lines from balb-c mouse embryo cultures: transformation susceptibility to sv40 lambda interferon (ifn-lambda), a type iii ifn, is induced by viruses and ifns and displays potent antiviral activity against select virus infections in vivo ifn-lambda resolves inflammation via suppression of neutrophil infiltration and il-1beta production ifn-lambda suppresses intestinal inflammation by non-translational regulation of neutrophil function host resistance to plasmodiuminduced acute immune pathology is regulated by interleukin-10 receptor signaling basis for regulated rna cleavage by functional analysis of rnase l and ire1p role of the interleukin (il)-28 receptor tyrosine residues for antiviral and antiproliferative activity of il-29/interferon-lambda 1: similarities with type i interferon signaling type iii interferon is a critical regulator of innate antifungal immunity gene transfer by lentiviral vectors is limited by nuclear translocation and rescued by hiv-1 pol sequences the structure of human interferon lambda and what it has taught us interferon lambda 4 signals via the ifnlambda receptor to regulate antiviral activity against hcv and coronaviruses ifn-l sequence alignments. sequences of human and mouse ifn-l regions implicated in receptor binding were aligned. key amino acid residues involved in receptor activation that differs between mouse and human ifn-l2 and ifn-l3 sequences are indicated in bold in mouse sequences. residues unique to mouse ifn-l3 in helices a and f are indicated in bold red. *indicates identical amino acids between human ifn-l3 and ifn-l4 human but not mouse hepatocytes respond to interferon-lambda in vivo ifnepsilon is constitutively expressed by cells of the reproductive tract and is inefficiently secreted by fibroblasts and cell lines virus interference. i. the interferon adaptation of theiler's virus to l929 cells: mutations in the putative receptor binding site on the capsid map to neutralization sites and modulate viral persistence somatic activation of the k-ras oncogene causes early onset lung cancer in mice selection on a variant associated with improved viral clearance drives local, adaptive pseudogenization of interferon lambda 4 (ifnl4) il-28a (ifn-lambda2) modulates lung dc function to promote th1 immune skewing and suppress allergic airway disease ifn-lambdas mediate antiviral protection through a distinct class ii cytokine receptor complex novel nonviral bioassays for mouse type i and type iii interferon characterization of the mouse ifn-lambda ligand-receptor system: ifn-lambdas exhibit antitumor activity against b16 melanoma comparative functional analysis of 12 mammalian ifn-lambda4 orthologs cardiovirus leader proteins are functionally interchangeable and have evolved to adapt to virus replication fitness interferons, interferonlike cytokines, and their receptors role of interferon-gamma in murine cytomegalovirus infection a variant upstream of ifnl3 (il28b) creating a new interferon gene ifnl4 is associated with impaired clearance of hepatitis c virus double nicking by rna-guided crispr cas9 for enhanced genome editing specificity genome engineering using the crispr-cas9 system interferon lambda (ifn-lambda) efficiently blocks norovirus transmission in a mouse model protection and mechanism of action of a novel human respiratory syncytial virus vaccine candidate based on the extracellular domain of small hydrophobic protein il-28, il-29 and their class ii cytokine receptor il-28r ifnlambda (ifn-lambda) is expressed in a tissue-dependent fashion and primarily acts on epithelial cells in vivo il-28 and il-29: newcomers to the interferon family characterization of the murine alpha interferon gene family characterization of interferonalpha 13, a novel constitutive murine interferon-alpha subtype species specificity of interferon action: maintenance and establishment of the antiviral state in the presence of a heterospecific nucleus the authors are grateful to nicolas dauguet for his help in flow cytometry (ludwig institute for cancer research). work was supported by the eos joint programme of fonds de la recherche scientifique-fnrs and fonds wetenschapellijk onderzoek-vlaanderen-fwo (eos id: 30981113), by interuniversitary attraction poles program initiated by the belgian science policy office (iap-p7/45 belvir), by actions de recherche concertées (arc), and by a pdr grant (t.0185.14) of fnrs to t.m., and b.s. is a doctoral assistant at the department of biomedical molecular biology of ghent university. no competing financial interests exist. key: cord-000554-p4ufea6x authors: gao, wei; sun, wenkui; qu, bingqian; cardona, carol j.; powell, kira; wegner, marta; shi, yi; xing, zheng title: distinct regulation of host responses by erk and jnk map kinases in swine macrophages infected with pandemic (h1n1) 2009 influenza virus date: 2012-01-18 journal: plos one doi: 10.1371/journal.pone.0030328 sha: doc_id: 554 cord_uid: p4ufea6x swine influenza is an acute respiratory disease in pigs caused by swine influenza virus (siv). highly virulent siv strains cause mortality of up to 10%. importantly, pigs have long been considered “mixing vessels” that generate novel influenza viruses with pandemic potential, a constant threat to public health. since its emergence in 2009 and subsequent pandemic spread, the pandemic (h1n1) 2009 (h1n1pdm) has been detected in pig farms, creating the risk of generating new reassortants and their possible infection of humans. pathogenesis in siv or h1n1pdm-infected pigs remains poorly characterized. proinflammatory and antiviral cytokine responses are considered correlated with the intensity of clinical signs, and swine macrophages are found to be indispensible in effective clearance of siv from pig lungs. in this study, we report a unique pattern of cytokine responses in swine macrophages infected with h1n1pdm. the roles of mitogen-activated protein (map) kinases in the regulation of the host responses were examined. we found that proinflammatory cytokines il-6, il-8, il-10, and tnf-α were significantly induced and their induction was erk1/2-dependent. ifn-β and ifn-inducible antiviral mx and 2′5′-oas were sharply induced, but the inductions were effectively abolished when erk1/2 was inhibited. induction of ccl5 (rantes) was completely inhibited by inhibitors of erk1/2 and jnk1/2, which appeared also to regulate fasl and tnf-α, critical for apoptosis in pig macrophages. we found that nfκb was activated in h1n1pdm-infected cells, but the activation was suppressed when erk1/2 was inhibited, indicating there is cross-talk between map kinase and nfκb responses in pig macrophages. our data suggest that map kinase may activate nfκb through the induction of rig-1, which leads to the induction of ifn-β in swine macrophages. understanding host responses and their underlying mechanisms may help identify venues for effective control of siv and assist in prevention of future influenza pandemics. swine influenza is an acute respiratory disease caused by swine influenza viruses (siv). the symptoms and signs generally include fever, sneezing, nasal rattles, and respiratory distress in pigs. pigs recover within a few days, but severe signs can develop and mortality can reach up to 10% when highly virulent strains are involved [1] or pigs are infected at young ages [2, 3] . pigs have long been considered to be the intermediate host of various subtype viruses and ''mixing vessels'' for the evolution and genesis of influenza viruses with pandemic potential because of their susceptibility to swine, avian, and human influenza viruses [4, 5, 6] . this broad susceptibility is due to the presence of both sialic acid (sa)2,3 gal-and sa2,6-gal receptors present in the respiratory epithelium. three major siv subtypes are prevalent: h1n1 (classical swine h1n1 and avian-like h1n1), h3n2 (triple reassortant h3n2 and human-like h3n2), and h1n2 [2, 7, 8, 9, 10, 11] . pigs are also susceptible to and show clinical signs when infected with pandemic (h1n1) 2009 virus (referred to hereafter as h1n1pdm) [12] , which emerged in april 2009 in north america [13] , arising at least in part from contemporaneous siv. to date h1n1pdm has been found in a few swine farms [12, 14, 15] , which further demonstrates a two-way process of both gene and virus trafficking between humans and pigs. though h1n1pdm has remained antigenically and genetically stable in humans since its emergence, a novel reassortant siv containing a h1n1pdm-like na and seven other genes from triple-reassortant h1n2 and european ''avian-like'' h1n1 viruses was identified in early 2010 [16] , and that same year h1n1pdm was shown to be evolving genetically at a faster pace in pigs than it was in humans [12, 15, 17] . effective control of circulating influenza viruses in swine populations is key to reducing consequent genesis of novel pandemic strains that threaten the health of both humans and animals. studies have been conducted to identify proinflammatory cytokines including tnf-a, il-6, il-12, and ifn-a or ifn-c, which are upregulated in lung or bronchoalveolar secretions in siv-infected pigs [18, 19, 20, 21] and may be correlated with clinical manifestations. in an alveoli macrophage-depleted pig model, macrophages appeared to be indispensible to effective clearance of siv from lungs. a higher frequency of cytotoxic t, cd t, and treg cells were also detected in infected pig lungs [18] , which together with the induction of cytokines, contribute to pathogenesis of influenza infection in pigs. exploring the mechanism of regulation of host responses is crucial for understanding the pathogenesis of siv and for controlling swine influenza in pigs. macrophages residing beneath the respiratory epithelium and surrounding alveoli are part of the first line defenses against influenza viruses. during influenza viral replication in bronchial epithelial cells, macrophages are one of the earliest targets to be infected. together with dendritic cells, macrophages coordinate innate immune responses, which subsequently lead to adaptive immunity by initiating antigen presentation and lymphocyte activation. macrophages are indispensable in alveolar host defense and controlling influenza virus in pulmonary organs in pigs [22] . while protective in launching host antiviral responses and restricting virus spread, induced proinflammatory cytokines and chemokines are also the cause of pathogenicity for the host and may lead to acute respiratory failure (arf), a major cause of death in highly pathogenic h5n1 or h1n1pdm-infected humans [23] . needless to say, the roles of macrophages are critical to pathogenicity as well as host protection in siv-infected pigs. however, little is known about the mechanisms of how host responses are regulated in pigs or their macrophages. considering the critical role macrophages play in siv infections, and the threat that h1n1pdm could further evolve higher virulence in pigs and subsequently infect humans, we were interested in profiling host responses of swine macrophages to h1n1pdm, and more importantly, in exploring the underlying mechanism of host response regulation including antiviral, proinflammatory responses, and apoptosis in pigs. in this report, we will demonstrate that swine macrophages are susceptible to infection by h1n1pdm. we will show a unique pattern of proinflammatory cytokine responses to the infection, which are distinctly regulated by swine mitogen-activated protein (map) kinases. we have also observed cross-talk between map kinase and nfkb pathways, and our data indicate that map kinase erk1/2 and jnk1/2 may impact the activation of nfkb through the induction of rig-1, leading to ifn-b induction in h1n1pdm-infected swine macrophages. the 3d/4 cells used in our study are a spontaneouslytransformed line of swine macrophages purchased from atcc (manassas, va) and grown in rpmi 1640 medium (invitrogen) containing 10% fetal bovine serum (fbs). mouse anti-erk and anti-jnk antibodies as well as rabbit anti-phospho erk and antiphospho jnk antibodies (cell signaling), anti-cytochrome c, anti-influenza ns1, and alkaline phosphatase (ap)-conjugated anti-rabbit and anti-mouse igg antibodies (santa cruz biotechnology) were obtained from their respective providers. anticleaved caspase antibody was obtained from cell signaling technology, and anti-bak antibody was obtained from emd chemicals. the chemicals purchased from emd chemicals also included inhibitors for map kinases, u0126 (erk1/2), sb203580 (p38), and insolution jnk inhibitor ii (jnk1/2), and the inhibitors for nfkb and ikk (6-amino-4-(4-phenoxyphenylethylamino) quinazoline (cat. 481406) and wedelolactone (cat. 401474), respectively). a/nanjing/108/2009 (h1n1), a pandemic (h1n1) 2009 virus, was isolated from a swab sample of an outpatient febrile child at the nanjing children's hospital during the pandemic in 2009, nanjing, china. the sampling procedure was performed in accordance with the rules set by the institutional review board at the hospital. the eight genomic segments of this virus have been fully sequenced and the raw data are deposited at genbank under accession numbers jq173100 through jq173107. the virus was grown in 9-day-old embryonating chicken eggs; virus allantoic fluid (vaf) was harvested 48 hrs after inoculation, then titrated with standard haemagglutination tests (ha) and plaque assays in mdck cells for ha and infectious viral titers, respectively [24] . for viral infection, the 3d/4 cells were trypsinized, resuspended in rpmi 1640 medium containing 10% fbs, and plated on 6-cm tissue culture plates at 5610 6 cells per plate 12 hrs before infection. the cells were infected with h1n1pdm inocula in vaf at a multiplicity of infection (moi) of 1. after 1 hr of adsorption, the virus inocula were discarded and 3 ml of serum-free rpmi 1640 medium containing tpck-trypsin (1 mg/ml, sigma) was added. the cells were incubated at 37uc and 5% co 2 for various time points before cell lysates or total rna extraction were prepared. mrna transcript levels of ifn-b, il-1b, il-6, il-8, ccr5, ip-10, tnf-a, fasl, trail, mx, 2959-oas, retinoic acid-inducible gene i (rig-1), melanoma differentiation-associated antigen 5 (mda-5), and glyceraldehyde-3-phosphate dehydrogenase (gapdh) genes were analyzed by a two-step real-time rt-pcr assay as described previously [25] . 1 mg of total rna, prepared from the cells using the rneasy kit (qiagen), was reverse transcribed with the quantitect reverse transcription kit (qiagen) following the manufacturer's instructions. the sequences of primers used in the study are listed in table 1 . the rt reaction was carried out with the rna after treatment with dnase i at 42uc for 2 min. real-time pcr was conducted with 1 ml of cdna in a total volume of 25 ml with the iq sybr green supermix (bio-rad) following the manufacturer's instructions. relative expression values were normalized using an internal gapdh control. the fold change of relative gene expression levels was calculated following the formula: 2 (dct of gene2dct of gapdh) [25, 26] . for each reaction, melting curves were analyzed to determine the specificity of each amplicon. to determine the viral rna level, the total rna from infected cells was reverse transcribed and cdna used for taqman-based real-time pcr (applied biosystems) to measure viral m gene transcripts in the infected cells [27] . cell lysates were prepared by lysing uninfected and infected 3d/4 cells in 1% np-40 lysis buffer containing 1 mm pmsf, 1% aprotinin, 20 mg leupeptin ul 21 and 1 mm sodium vanadate (sigma) as described previously [10] . cell lystes were clarified by low speed centrifugation (1000 g, 5 min at 4uc) and subjected to sds-page (10 to 12%). proteins were transferred to the immuno-blot pvdf membrane (bio-rad), and western blot analysis was performed following standard protocols [28] using rabbit or mouse anti-map kinase or phosphor-map kinase antibodies (1:500) in tbst containing 5% fat-free milk powder for 90 mins incubation at rt. after washes, incubation with apconjugated anti-rabbit or anti-mouse igg antibody for another 90 mins followed. after incubation and thorough washes, bcip/ nbt reagents (sigma) were used for the development of colorimetric signals on the membrane. the membrane was also blotted with a monoclonal anti-actin antibody (santa cruz biotechnology) for input control. for statistical analysis, a two-tailed student's t-test was used to evaluate realtime rt-pcr data. an x 2 analysis was used to evaluate significant differences of the data in two and more groups. the 0.05 level of probability (p,0.05) was considered statistically significant. to examine the susceptibility of pig macrophages to h1n1pdm originating from a human host, we infected 3d/4 cells with the a/ nanjing/108/2009 (h1n1). typical cytopathic effect (cpe) appeared 16 hrs post infection and the cell monolayer was destroyed 32 hrs post infection (fig. 1a) . this result demonstrated that h1n1pdm retains the ability to infect and replicate in swine macrophages, and can reach 1.8610 4 pfu/ml as shown in a replicative curve (fig. 1b) . apoptosis occurred and proceeded through the course of the infection, as we observed cleaved/ activated caspase-9 as well as the emergence of downstream executioner caspases-6, -7, and -3, which eventually destroyed the infected swine macrophages (fig. 1c) . clearly, cytochrome c was released into the cytosol (fig. 1e) , which activated mitochondriamediated intrinsic apoptosis as early as 3 hrs post infection. bak, a pro-apoptotic bcl-2 family member, was upregulated as detected in the infected cells (fig. 1d) , and may be involved in the release of cytochrome c from mitochondria in swine macrophages. to elucidate the pathogenesis of h1n1pdm in pigs, we examined the pattern of cytokine responses in ph1n1-infected swine macrophages. total rna from infected and uninfected 3d/ 4 cells collected at different time points post infection (p.i.) were prepared and used for realtime rt-pcr analyses with specific primers to swine cytokines. we found that the levels of proinflammatory cytokines il-6 and il-8 were upregulated up to 51-and 38-fold at 16 hrs, respectively, and the level of il-8 continued to rise up to 142-fold at 36 hrs p.i.. however, the level of il-1b remained unchanged throughout the infection ( fig. 2a) , indicating that il-6 and il-8, as well as tnf-a (fig. 2b ) as described later, were the main proinflammatory cytokines upregulated. we observed a robust induction of antiviral ifn-b, which rose up to 620-and 5,100-fold at 16 and 36 hrs p.i., respectively (fig. 2c ). ifn-inducible antiviral proteins mx and 295.-oas were induced accordingly up to 910-and 12,510-fold, respectively, at 36 hrs p.i. (fig. 2c) . tnf family members were also induced in response to h1n1pdm infection, which may be attributable to cell death. we found that in pig macrophages the levels of fasl and tnf-a remained undetectable, while tnf-related apoptosis-inducing ligand (trail) seemed to be most abundant before infection, based on ct values from realtime rt-pcr (data not shown). fasl and tnf-a were induced most robustly, but trail was only mildly induced in response to infection (fig. 2b) . among the induced, the level of tnf-a, critical in both cell death and inflammation, was sharply upregulated up to 14-and 162-fold, and fasl up to 43-and 22-fold at 16 and 36 hrs p.i., respectively. fasl and tnf-a may play a major role in h1n1pdm-triggered extrinsic apoptosis. to understand the mechanism of proinflammatory cytokine and tnf family ligand induction in h1n1pdm-infected swine macrophages, we investigated how map kinases were activated and whether their signaling pathways were involved in the regulation of various cytokines and tnf family ligands in pig immune cells. 3d/4 cells were infected with h1n1pdm, and cell lysates were prepared at various time points for sds-page and western blot analyses with specific anti-erk1/2 and anti-jnk1/2 antibodies. activated forms of erk and jnk (phospho-erk1/2 and phosphor-jnk1/2) were detected by anti-phospho-erk1/2 and anti-phospho-jnk antibodies. as shown in figure 3a , erk1/2 was basally phosphorylated at a low level before infection, but further phosphorylated between 9 and 18 hrs and thereafter p.i.. phosphorylation and activation of jnk1/2 appeared at 9 hrs and increased to the peak around 18 hrs p.i. (fig. 3b) . although both erk1/2 and jnk1/2 were activated in response to h1n1pdm infection in swine macrophages, erk1/2 remained active at basal level even before infection, so did jnk1/2 as shown in some of our experiments (fig. 4b) . however, our data showed that basal level phosphorylation of both erk1/2 and jnk1/2 remained unchanged in uninfected 3d/4 cells through the period of our infection. in addition to erk1/2 and jnk1/2, we have also observed the phosphorylation and activation of p38 map kinase in h1n1pdm-infected cells (data not shown). to evaluate the role of map kinases in the regulation of proinflammatory cytokine responses in h1n1pdm-infected swine macrophages, we pre-treated 3d/4 cells with specific inhibitors for erk1/2, p38, and jnk1/2 1 hr prior to infection. we then infected the cells with the virus and observed how infectioninduced activation of map kinases was affected by inhibition of the respective map kinases. as shown in figure 4a , 3d/4 cells were pre-treated with inhibitors of erk1/2 (u0126), p38 (sb230058), and jnk1/2 (jnk insolution), at concentrations of 10 mm, 5 mm, and 50 mm, respectively. while the phosphorylation of erk1/2 was unaffected by treatment with the p38 and jnk inhibitors, it was completely abolished at both 18 and 30 hrs p.i. (lines 4-5) by the erk1/2 inhibitor u0126 (fig. 4a) . we noted that the basal level phosphorylation of erk1/2 diminished in the presence of u0126. on the other hand, in light of the p38 and jnk inhibition with their specific inhibitors, the phosphorylation of erk1/2 appeared to be enhanced (fig. 4a, lines 6-9 ), indicating that a compensatory mechanism may exist among map kinases. we observed a similar response in which a complete suppression of jnk1/2 phosphorylation was observed (lines 8-9) when the cells were pre-treated with the jnk1/2 inhibitor (fig. 4b) . however, the phosphorylation of jnk1/2 was not suppressed at all by the inhibitors of erk1/2 and p38. we noted that there were double bands for jnk1, and a lower band of jnk1 usually appeared at a later stage of infection (30 hrs p.i.). this band was detected mainly by anti-jnk1/2, but not by anti-phospho-jnk1/ 2, indicating that jnk activation was transient and dephosphorylation of jnk occurred at later stages of infection, probably by an uncharacterized map kinase phosphatase (mkp) present in pigs. a basal level phosphorylation of nfkb was also observed in 3d/4 pig macrophages, and was further enhanced upon h1n1pdm infection, indicating that the nfkb pathway was activated as well in infected pig macrophages (fig. 4c) . when the cells were pre-treated with specific inhibitors of nfkb (10 nm) or ikk (10 mm), the phosphorylation/activation of nfkb was effectively decreased or diminished. map kinases and nfkb pathways were activated in h1n1pdm infected pig macrophages, which could be reversed or inhibited by their specific inhibitors. we used these inhibitors to study the regulation of host responses, which may be controlled by these pathways. to determine how cytokine responses are regulated by individual map kinases, we pre-treated the cells with erk1/2 and jnk1/2 inhibitors, respectively, and measured the induction of the cytokines after infection with realtime rt-pcr. we observed that il-1b was barely detected and not induced during h1n1pdm infection. interestingly, we noticed that il-1b was upregulated in the presence of the jnk inhibitor, although no change was observed after the treatment by the erk inhibitor, indicating that il-1b could have been induced in swine macrophages infected with h1n1pdm, but was virtually suppressed by jnk1/2 (fig. 5a) . we observed that the induction of il-6, il-8, and il-10 was completely suppressed in the presence of the erk1/2 inhibitor, which indicates that il-6, il-8, and il-10 inductions are all dependent on the erk signaling pathway (fig. 5b-d) . it is interesting to note that jnk1/2 may play different roles in the induction of il-6, il-8, and il-10 based on their responses in the presence of the jnk inhibitor. jnk1/2 may have moderate effects in the induction of il-6 ( fig. 5b ), but may be not relevant at all to the induction of either il-8 or il-10 ( fig. 5c-d) . we also noted that ccl5 (rantes) was strongly regulated by erk1/2 and jnk1/2 in swine immune cells. as shown in figure 5e , induction of ccl5 was efficiently blocked in the presence of either erk1/2 or jnk1/2 inhibitors, indicating that ccl5 is induced by h1n1pdm infection through erk and jnk signaling pathways. as for antiviral ifn-b, which was robustly induced with h1n1pdm infection in swine macrophages, erk1/2 appeared to be essential since the induction of its mrna transcripts was virtually abolished in the presence of the erk inhibitor (fig. 6a ). jnk1/2 may also play a role in ifn-b induction because of its significant decrease at the earlier stage of infection (16 hrs p.i.) when 3d/4 cells were pre-treated with the jnk inhibitor. however, erk1/2 seemed to be the primary pathway in the ifn-b induction in swine macrophages. the distinct contributions to the induction of ifn-b by erk1/2 and jnk1/2 were also reflected in the decreased mrna transcript levels of ifn-inducible antiviral proteins, mx and 2959-oas, in the presence of erk and jnk inhibitors, respectively (fig. 6b-c) , which is in accordance with the suppression of the ifn-b induction by these same compounds in the infected cells. both mx and 2959-oas were suppressed significantly by the erk inhibitor, but only the in contrast to the abundance of trail transcripts, mrna levels of fasl and tnf-a were barely detectable by realtime rt-pcr in swine macrophages (data not shown). however, both fasl and tnf-a were induced profoundly in response to ph1n1 infection ( fig. 2b and 7a-c) , while the change of trail was mild. by using inhibitors, we concluded that the induction of fasl and tnf-a are mainly controlled by the erk1/2 and jnk1/2 pathways in pig macrophages. the nfkb pathway could also be critical in host responses, as has been shown in humans and mice infected with influenza a virus. nfkb can be phosphorylated and activated in swine macrophages in response to h1n1pdm infection ( fig. 8a and 4c ), albeit at a later stage. interestingly, when the cells were pre-treated with erk1/2 or jnk1/2 inhibitors, the phosphorylation of nfkb was also suppressed. however, when the cells were pre-treated with the p38 inhibitor, nfkb phosphorylation decreased much less than with erk1/2 or jnk1/2 inhibitors (fig. 8a) . this result suggests that a cross-talk may exist between map kinase and nfkb pathways, and that among the map kinases, erk1/2 and jnk1/2 are mainly involved. figure 5 . regulation of swine proinflammatory cytokine gene transcripts by map kinases. 3d/4 cells were pretreated with u0126 and insolution jnk inhibitor, which are inhibitors of erk1/2 and jnk1/2, respectively, 1 hr before h1n1pdm infection. total rna was prepared at 24 and 36 hrs post infection for reverse transcription. cdna was used for realtime pcr with specific primers to measure fold changes of cytokine transcripts at different time points. each assay was repeated at least twice. a-e. regulation of il-1b, il-6, il-8, il-10, and ccl5, respectively, by erk1/2 and jnk1/ 2 inhibitors. data show mean fold changes plus standard deviation of two or three independent assays. *p,0.05, student's t-test. doi:10.1371/journal.pone.0030328.g005 we next examined the expression levels of rig-1 and mda-5, the rlr family members and cytosolic sensors for rna viruses. we found that rig-1 in particular was significantly induced up to 1280-fold, while mda-5 was also upregulated up to 42-fold in infected pig macrophages (fig. 8b) . we further examined the induction of rig-1 and mda-5 and their relevance to map kinases. to do this, we pre-treated the cells with inhibitors of map kinases. as shown in figure 8c , the induction of rig-1 was completely abolished by the inhibition of erk1/2 or jnk1/2 inhibitors, and to a much lesser extent, by the p38 inhibitor, suggesting that the induction of rig-1 was dependent on erk1/ 2 and jnk1/2, but not as much on p38. this differentially regulated pattern of rig-1 induction by erk1/2, p38, and jnk1/2 was similar to the suppression of nfkb phosphorylation/activation by map kinases (fig. 8a) , suggesting that the induction of rig-1 was associated with erk1/2 or jnk1/2 activation, but to a much lesser extent with p38. since nfkb could be downstream activated by rig-1/ips-1 [29, 30] , we postulate that erk1/2 or jnk1/2 may activate nfkb through the activation of rig-1/ips-1 during h1n1pdm infection in pig macrophages. a similar, albeit less dramatic, induction and suppression of mda-5 expression was also observed (fig. 8d) , which indicated that mda-5 might also be an intermediate adaptor bridging the map kinases erk1/2 and jnk1/2 to the nfkb pathway activation. the cells were pretreated with u0126, sb230058, and insolution jnk inhibitor, 1 hr before h1n1pdm infection. total rna was prepared from infected cells for reverse transcription. cdna was used for realtime pcr with rig-1 and mda-5 primers to measure fold changes of rig-1 and mda-5 transcripts in treated swine macrophages. each assay was repeated at least twice. data show mean fold change plus standard deviation of two or three independent assays. *p,0.05, student's t-test. doi:10.1371/journal.pone.0030328.g008 in the present study, we have demonstrated a pattern of host responses in swine macrophages to h1n1pdm infection. strong proinflammtory and antiviral cytokine responses including il-6, il-8, tnf-a, as well as ifn-b, were observed. in contrast, il-1b was not induced, and was barely detectable in pig macrophages. this pattern differs from that in bronchoalveolar secretions of siv-infected pigs in which il-1b was induced but il-8 was not [19, 20, 21, 22, 25] . the different cell types involved (macrophages and epithelial cells) may account for the difference. it has previously been reported that in human immune cells and patients a weak innate immune response, evidenced by a poor induction of proinflammatory and antiviral cytokines including ifn-b and tnf-a, has been observed in human monocyte-derived dcs and macrophages infected with h1n1pdm, compared to seasonal h1n1 infection [31] . highly pathogenic h5n1 viral infection in human macrophages induced higher expression of il-6 and ccl5 (rantes) than ph1n1 [32] , which may explain generally mild clinical disease among h1n1pdm-infected patients. in human macrophages, similar to our findings, il-1b was not detected. map kinase signaling pathways and their roles in the regulation of cytokines and viral replications have not been characterized in influenza-infected pig immune cells. in this study, we found that erk1/2 and jnk1/2 could both be activated in swine macrophages. we noted that erk1/2 was phosphorylated and active at a low level constitutively, which may be important for the rapid physiological responses required upon infection. to elucidate the mechanism that regulates swine host responses, we used specific inhibitors of map kinases to pre-treat macrophages before infection. we determined that the induction of ifn-b, il-6, il-8, and il-10 were regulated by erk1/2, while jnk1/2 may only play a minor or no role in the regulation of these cytokines. as described earlier, il-1b was not induced in response to the ph1n1 infection, which could be explained by our data indicating that its induction was in fact efficiently suppressed by jnk1/2 in swine macrophages. this may be the first time that jnk1/2 inhibitory effects on the induction of proinflammatory cytokines have been demonstrated. previous studies found that ifn induction was dependent on the jnk1/2 signaling pathway in epithelial cells infected with influenza virus infection [33] . however, our data clearly demonstrate that erk1/2 plays a major role in the regulation of ifn-b in pig macrophages, which may indicate that the regulation of ifn differs in different cell types. we noted that basal level activities of both erk1/2 and jnk1/2 were constitutively present in non-infected 3d/4 cells, which may be important in the induction of proinflammatory and antiviral cytokines at the early stages of infection. our data indicate that the induction of il-6, il-8, il-10, ccl-5, as well as ifn-b, were apparent at the earliest stages of viral infection even before erk1/2 was further activated. we realized that a transformed monocytic cell line, instead of primary cells, was used in the study, which may compromise the significance of our data. basal level phosphorylation of both erk1/ 2 and jnk1/2, which may affect certain cytokine production, would be minimal in primary monocytes. however, specific inhibitors used in the study completely wiped out phosphorylation of both erk1/2 and jnk1/2 (fig. 4) . the effect of map kinase phosphorylation and activation on the regulation of affected cytokines as observed in our study with the inhibitors is, therefore, valid, even though the cells were not primary cultures. macrophages appear to die inevitably of apoptosis when infected with influenza virus [26] . the fas-mediated extrinsic apoptotic pathway is apparently triggered by tnf family ligands. while both fasl and tnf-a were induced vigorously upon the viral infection, induction of trail was rather mild in h1n1pdminfected swine macrophages. we knew previously that fasl and tnf-a were barely detectable, while the level of trail remained high prior to the infection based on our realtime rt-pcr data (ct) (xing et al., unpublished data). we can therefore presume that h1n1pdm-induced apoptosis may be mainly attributed to fasl and tnf-a, while pig macrophages could be resistant to trail, since the cells remained intact despite the presence of a high level of trail before infection. furthermore, we were also able to determine that both erk1/2 and jnk1/2 were involved in the induction of fasl, tnf-a, and trail. fasl is also regulated by erk1 in chicken macrophages infected with an h9n2 avian influenza virus [34] . both toll-like receptors (tlr) and rna helicases, such as rig-1 and mda-5, are critical to antiviral innate immunity [35, 36] . as a cytosolic sensor, rig-1 binds to dsrna and viral ssrna that contain a 59-triphosphate not present in host rna, and then is recruited to mitochondrial protein ips via the card domain, leading to activation of nfkb, irf-3/-7, and induction of ifn [37, 38, 39] . rig-1 can be induced by viral infection [40] . in this study, we observed a robust induction of rig-1 and mda-5 in h1n1pdm-infected swine macrophages, which appeared to be suppressed completely by inhibitors of erk1/2 or jnk1/2, but to be a much lesser extent, by the inhibitor of p38. this indicates that the induction of rig-1 or mda-5 depends on the activation of erk1/2 and jnk1/2 in pig macrophages. we postulate a mechanism, therefore, that the cross-talk between map kinase and nfkb pathways is through the regulation of rig-1 and maybe mda-5, and that erk1/2 controls the activation of nfkb, leading to the induction of ifn in swine macrophages. characterization of an influenza a virus isolated from pigs during an outbreak of respiratory disease in swine and people during a county fair in the united states pathogenic and antigenic properties of phylogenetically distinct reassortant h3n2 swine influenza viruses cocirculating in the united states protection against a european h1n2 swine influenza virus in pigs previously infected with h1n1 and/or h3n2 subtypes evidence for the natural transmission of influenza a virus from wild ducts to swine and its potential importance for man genetic reassortment between avian and human influenza a viruses in italian pigs influenza-a model of an emerging virus disease the epidemiology and evolution of influenza viruses in pigs antigenic and genetic diversity among swine influenza a h1n1 and h1n2 viruses in europe evolution of swine h3n2 influenza viruses in the united states genetic characterization of novel reassortant h1n2 influenza a viruses isolated from pigs in southeastern china genetic characterization of h1n1 swine influenza a viruses isolated in eastern china genetic and pathobiologic characterization of pandemic h1n1 2009 influenza viruses from a naturally infected swine herd antigenic and genetic characteristics of swine-origin 2009 a(h1n1) influenza viruses circulating in humans an investigation into human pandemic influenza virus (h1n1) 2009 on an alberta swine farm evidence of human-to-swine transmission of the pandemic (h1n1) 2009 influenza virus in south korea reassortment of pandemic h1n1/2009 influenza a virus in swine pandemic (h1n1) 2009: update 89. world health organization swine influenza h1n1 virus induces acute inflammatory immune responses in pig lungs: a potential animal model for human h1n1 influenza virus cytokines and acute phase proteins associated with acute swine influenza infection in pigs porcine innate and adaptative immune responses to influenza and coronavirus infections cytokines in the pathogenesis of influenza alveolar macrophages are indispensable for controlling influenza viruses in lungs of pigs pathology of the swine-origin influenza a (h1n1) flu replication and plaque assay of influenza virus in an established line of canine kidney cells immune-related gene expression in response to h11n9 low pathogenic avian influenza virus infection in chicken and pekin duck peripheral blood mononuclear cells modulation of the immune responses in chickens by low-pathogenicity avian influenza virus h9n2 genetic and phenotypic characterization of a low-pathogenicity avian influenza h11n9 virus host immune and apoptotic responses to avian influenza virus h9n2 in human tracheobronchial epithelial cells rna recognition and signal transduction by rig-i-like receptors rig-i-like receptors: sensing and responding to rna virus infection pandemic h1n1 2009 influenza a virus induces weak cytokine responses in human macrophages and dendritic cells and is highly sensitive to the antiviral actions of interferons cytokine profiles induced by the novel swine-origin influenza a/h1n1 virus: implications for treatment strategies influenza virus-induced ap-1-dependent gene expression requires activation of the jnk signaling pathway roles of the erk mapk in the regulation of proinflammatory and apoptotic responses in chicken macrophages infected with h9n2 avian influenza virus toll-like receptors and rna helicases: two parallel ways to trigger antiviral responses intracellular pattern recognition receptors in the host response 59-triphosphate rna is the ligand for rig-i recognition of rna virus by rig-i results in activation of card9 and inflammasome signaling for interleukin 1 beta production rig-imediated antiviral responses to single-stranded rna bearing 59-phosphates an rna helicase, rhiv -1, induced by porcine reproductive and respiratory syndrome virus (prrsv) is mapped on porcine chromosome 10q13 we thank members of xing laboratory for their technical assistance in the study and helpful discussions for manuscript preparation. we appreciate excellent editing work performed by sandy shanks. key: cord-023373-6wh1kb3p authors: melchjorsen, j.; bowie, a. g.; matikainen, s.; paludan, s. r. title: differential requirements for toll‐like receptor signalling for induction of chemokine expression by herpes simplex virus and sendai virus date: 2008-06-28 journal: scand j immunol doi: 10.1111/j.0300-9475.2004.01423r.x sha: doc_id: 23373 cord_uid: 6wh1kb3p toll‐like receptors (tlrs) are pattern recognition receptors of the innate immune system, which recognize molecular structures on pathogens or cellular stress‐associated molecules. tlr–ligand interactions trigger activation of inflammatory signal transduction and expression of genes involved in host defense. in this study, we have examined the requirement for different tlr adaptor molecules in virus‐induced chemokine expression and are currently trying to identify the tlr involved. we have found that both a herpesvirus [herpes simplex virus (hsv)] and a paramyxovirus (sendai virus) require a functional genome to induce expression or proinflammatory chemokines in human and murine monocytic cell lines. for both viruses, this is independent of the tlr adaptor molecules trif and mal. however, overexpression of the vaccinia virus‐encoded inhibitor of tlr‐signalling a52r or dominant‐negative myd88 totally inhibited hsv‐induced rantes expression but only partially prevented sendai virus from inducing this chemokine. this suggests that hsv‐induced rantes expression occurs via a tlr pathways, whereas sendai virus utilizes both tlr‐dependent and ‐independent pathways to stimulate expression of rantes. we are currently trying to identify the tlrs involved. data from these studies will also be presented at the meeting. the nuclear receptor heterodimers of liver x receptors (lxrs) are recently identified as key transcriptional regulators of genes involved in lipid homeostasis and inflammation. lxrs and their ligands are negative regulators of macrophage inflammatory gene expression. multiple sclerosis (ms), a demyelinating disease of the central nervous system of unknown cause, is characterized by recurrent inflammation involving macrophages and their inflammatory mediators. sweden belongs to the countries with a high ms incidence. in italy, incidence is lower, with an exception for sardinia where the incidence is even higher than that in sweden. subjects from sardinia are ethnically more homogeneous and differ from swedes, also regarding genetic background and environment. we studied lxrs and their related molecules of blood mononuclear cells (mncs) from female patients with untreated relapsing-remitting ms from sassari, sardinia and stockholm, sweden. sex-and age-matched healthy controls (hcs) were from both areas. mrna expression was evaluated by real-time pcr. lxr-a was lower (p < 0.05) in ms (mean ae sem: 3.1 ae 0.2; n ¼ 37) compared to hc (3.6 ae 0.1; n ¼ 37). lxr-a was lower in ms from stockholm (2.6 ae 0.2; n ¼ 22) compared to corresponding hc (3.4 ae 0.1; n ¼ 22; p < 0.01) and compared to ms (3.8 ae 0.2; n ¼ 15; p < 0.001) and hc (4 ae 0.2; n ¼ 15; p < 0.001) from sardinia. ms patients from stockholm, but not from sassari, also expressed lower (p < 0.05) lxr-b (à4.1 ae 0.4) compared to corresponding hc (à2.9 ae 0.3). ms from stockholm was associated with higher abca-1 (6.1 ae 0.4 versus 5.0 ae 0.3; p < 0.05) and higher estrogen receptor-b-cx (2.4 ae 0.4 versus 0.8 ae 0.4; p < 0.01) compared to corresponding hc. the hc from sassari had higher androgen receptor (2.9 ae 0.2) compared to ms from sassari (1.4 ae 0.3; p < 0.01), ms (1.3 ae 0.4; p < 0.01) and hc from stockholm (1.2 ae 0.3; p < 0.01). ms from sassari had lower cyclooxygenase-1 compared to corresponding hc (5.1 ae 0.4 versus 6.6 ae 0.3; p < 0.01) and lower prostaglandin-e (à0.03 ae 0.5) compared to the hc (1.4 ae 0.5; p < 0.05) and ms (2.7 ae 0.4; p < 0.05) and hc from stockholm (1.9 ae 0.4, p < 0.001). our findings identify lxrs and their related molecules as being involved in ms from stockholm but not from sassari, while sex hormone receptors seem to be involved in ms in sassari. multiple sclerosis: ifn-b induces cd123 + bdca2 -dendritic cells that produce il-6 and il-10 and have no enhanced type i interferon production y. m. huang, 1 s. adikari, 1 u. båve, 2 a. sanna 1,3 & g. alm 4 dc antigens (bdca) and investigate their ability to produce type i ifn in response to virus stimulation. we show that ifn-b induces development of cd123 þ dc from human blood monocytes, which coexpress bdca4 þ but are negative for bdca2 -, a specific marker for plasmacytoid dc. such ifn-b-modulated dc produce large amounts of il-6 and il-10, but no il-12p40 and have no enhanced ifn-b and ifn-b production. the findings indicate that ifn-bmodulated dc represent a myeloid dc subset with diminished cd11c, bdca-1 and cd1a expression, having potent th2-promoting function but lacking antiviral capacity. the association of psoriasis with throat infections by streptococcus pyogenes suggests a potential antigenic target for the t cells that are known to infiltrate dermis and epidermis of psoriatic skin. streptococcal m protein shares an extensive sequence homology with human epidermal keratins. keratins 16 (k16) and 17 (k17) are mostly absent from uninvolved skin but are upregulated in psoriatic lesions. there is increasing evidence that cd8 þ t cells play an important effector role in psoriasis and m proteinprimed t cells may recognize these shared epitopes in skin via molecular mimicry. to identify candidate epitopes, peptides with sequences from k17 were selected on the basis of predicted binding to hla-cw6 and sequence similarities with m6 protein. matched peptides from the sequence of m6 protein and a set of peptides with poor predicted binding were also selected. cw6 þ individuals with psoriasis and cw6 þ healthy controls, having a family history of psoriasis, were recruited. pbmcs were incubated with the peptide antigens. t-cell activation in the cd4 þ , cd8 þ and later the skin-homing cutaneous lymphocyteassociated antigen (cla)-expressing subset of cd8 þ t cells was evaluated by cd69 expression and intracellular ifn-g accumulation using flow cytometry. we demonstrate that cw6 þ psoriasis patients had significant cd8 þ t-cell ifn-g responses to peptides from k17 and m6 protein selected on the basis of sequence homology and predicted hla-cw*0602 binding. these responses were about 10 times more frequent in the skin-homing cutaneous lymphocyte-associated antigen-expressing (cla þ ) subset of cd8 þ t cells. cd4 þ t cells showed only borderline responses. cd8 þ t cells from cw6 þ nonpsoriatic individuals responded to some m6 peptides but very rarely to k17 peptides, and this also applied to the cla þ cd8 þ subset. these findings indicate that psoriatic individuals have cd8 þ t cells that recognize keratin self-antigens and that epitopes shared by streptococcal m protein and human keratin may be targets for the cd8 þ t cells that infiltrate psoriatic skin lesions. autoantibodies directed against citrulline-containing proteins have an impressive specificity of nearly 100% in ra patients and a suggestive involvement in the pathogenesis. the targeted epitopes are generated by a post-translational modification catalysed by the calcium-dependent enzyme peptidyl arginine deaminase that converts the positively charged arginine to polar but uncharged citrullin. the aim of this study was to analyse the presence of citrulline in the joints at different time points of collagen-induced arthritis in da rats by immunohistochemistry and to investigate how immunogenicity and arthritogenicity was affected by citrullination of rat serum albumin (rsa) and collagen type ii (cii). our results indicate that citrulline could be detected in joints of arthritic animals, first appearance at the onset of disease and increasing as disease progressed into a chronic state. unimmunized animals or time points before clinical signs of arthritis were negative. by morphology, we state that some infiltrating macrophages as well as the cartilage surface stain positive for citrulline, while the major source of citrullinated proteins appears to be fibrin depositions. a specific cit-rsa t-cell response was observed in animals challenged by citrullinated rsa, no response was recorded when rsa was used as a stimulus. the igg analysis reveals not only a response towards the modified protein but also cross-reactivity to native rsa. no t-cell or b-cell response was noted in animals injected with unmodified rsa. cit-cii induced a disease with higher incidence and earlier onset than did the native counterpart. we conclude that, in contrast to the human disease, citrulline does not seem to appear before clinical signs. as inflammation proceeds, citrulline is detected specifically in the joints. all other organs investigated were negative. we also conclude that citrullination of a protein can break tolerance and increase its arthritogenic properties. ectopic germinal centers (gcs) can be detected in the salivary glands of approximately 1/5 of patients with sjögren's syndrome (ss) and appear in both primary and secondary ss. previously, ectopic gc have been associated with increased local autoantibody production. the aim of this study was to determine whether gc in primary sjögren's syndrome (pss) defines a distinct seroimmunological phenotype. retrospectively, a material of 130 haematoxylin and eosin-stained paraffin-embedded tissue sections of minor salivary gland tissue from patients with pss was morphologically screened for the presence of ectopic gc. gc-like lesions were detected in 33/130 (25%) of the pss patients. seventy-two pss patients lacking these structures (gc-) were randomly selected for comparison. focus score was significantly increased in the gc þ patients compared to the gcpatients (p ¼ 0.035). in the gc þ group, 54.5% of the patients presented with anti-ro/ssa compared to 43.7% in the gcgroup. anti-la/ssb was detected in 31.3% of the gc þ patients compared to 25.7% of the gcpatients. sixty-one percentage of gc þ patients presented with increased levels of igg, a nonsignificant difference when compared to 39.4% in the gcpatients (p ¼ 0.089). levels of rf, ana, ena, igm and iga were similar in both patient groups, as were esr and crp. in conclusion, patients with ectopic gc have a higher focus score and more often present with autoantibodies and increased levels of igg compared to pss patients with regular focal infiltration (gc -). our findings may indicate a certain seroimmunological phenotype and warrant for further prospective studies. association between mannose-binding lectin and vascular complications in type 1 diabetes complement activation and inflammation have been suggested in the pathogenesis of diabetic vascular lesions. we investigated serum mannose-binding lectin (mbl) levels and polymorphisms in the mbl gene in type 1 diabetic (t1dm) patients with and without diabetic nephropathy and associated macrovascular complications. polymorphisms in the mbl gene and serum mbl levels were determined in 199 t1dm patients with overt nephropathy and 192 t1dm patients with persistent normoalbuminuria matched for age, sex and duration of diabetes as well as in 100 healthy control subjects. the frequencies of high and low expression mbl genotypes were similar in patients with t1dm and healthy controls. high mbl genotypes were significantly more frequent in diabetic patients with nephropathy than in the normoalbuminuric group, and the risk of having nephropathy, given a high mbl genotype, assessed by odds ratio was 1.52 (1.02-2.27), p ¼ 0.04. median serum mbl concentrations were significantly higher in patients with nephropathy than in patients with normoalbuminuria [2306 mg/l (iqr 753-4867 mg/l) versus 1491 mg/l (iqr 577-2944), p ¼ 0.0003], and even when comparing patients with identical genotypes, serum mbl levels were higher in the nephropathy group than in the normoalbuminuric group. patients with a history of cardiovascular disease had significantly elevated mbl levels independently of nephropathy status [3178 mg/l (iqr 636-5231 mg/l) versus 1741 mg/l (iqr 656-3149 mg/l), p ¼ 0.02]. the differences in mbl levels between patients with and without vascular complications were driven primarily by pronounced differences among carriers of high mbl genotypes (p < 0.0001). our findings suggest that mbl may be involved in the pathogenesis of microvascular and macrovascular complications in type 1 diabetes and that determination of mbl status might be used to identify patients at increased risk of developing these complications. neuroimmunology unit, center for molecular medicine, karolinska institutet, stockholm, sweden. e-mail: judit.wefer@cmm.ki.se dna vaccine coding for the encephalitogenic peptide mog 91-108 protects lew.1av1 from subsequent development of experimental autoimmune encephalomyelitis (eae). protection is associated with a type 1 immune response and is dependent on the presence of cpg dna motifs. the mechanisms underlying the observed reduction of eae development in protected rats have not been fully clarified. we investigated immunological characteristics of lymphocytes after dna vaccinaton and subsequent eae induction. we confirm that protection was not associated with suppression of t1 cells, as transcription of the novel molecule rat t-cell immunoglobulin-and mucindomain-containing molecule (tim-3), reported to be exclusively expressed on differentiated t1 cells, was not altered by dna vaccination. we did not note any clonal deletion upon tolerization, but detected an antigen-specific lymphocyte population upregulating ifng upon recall stimulation 3 weeks after protective dna vaccination. in protected rats, we observed (1) no alterations in antigenspecific th2 or th3 responses, (2) reduced mhc ii expression on splenocytes early after eae induction, (3) antigen-specific upregulation of ifnb upon recall stimulation and (4) reduced il-12rb2 on lymphocytes. we thus demonstrate an association of the protective effect of dna vaccination with expression of ifnb. we are currently investigating the cellular mechanisms behind this ifnb-mediated protection. multiple sclerosis (ms) is an autoimmune condition characterized by degeneration of nerve fibre myelin sheets. a candidate autoantigen, myelin basic protein (mbp), has especially attracted attention. the presence of anti-mbp antibodies is a predictor of definite ms, but their role in the pathogenesis remains obscure. t cells have long been known to play a pivotal role in the pathogenesis of ms. recently, an important role for b cells as autoantigen-presenting cells has been demonstrated in other autoimmune diseases, including rheumatoid arthritis and diabetes. the uptake of mbp by b cells and the presentation of mbp-derive peptides to t helper (th) cells by b cells may be promoted by the formation of complement (c) activating immune complexes (ics) between mbp and natural autoantibodies in healthy individuals and disease-associated anti-mbp antibodies in ms patients, respectively. we have investigated the formation of mbp-containing ic, the binding of mbp to b cells, the mbp-elicited induction of th-cell and b-cell proliferation and the cytokine production in peripheral blood mononuclear cells (pbmcs) from healthy donors grown in the presence of intact or c-inactivated serum from healthy donors or patients with ms. while mbp did not induce measurable proliferation of b cells nor cd4 þ t cells, we observed the production of tnf-a, ifn-g and il-10 by pbmc in response to incubation with mbp in the presence of sera from healthy controls as well as sera from ms patients. by contrast, no production of il-2, il-4 and il-5 was detected. we are currently investigating the capability of ms sera to promote the formation of mbp-containing ic and thereby enhance the cytokine responses, by virtue of elevated anti-mbp contents. the phagolysosomally localized acid sphingomyelinase (asmase) activated by proinflammatory cytokines such as tnf and ifn-g generates the signalling molecule ceramide which in turn results in the activation of proteases like cathepsin d. these characteristics of asmase suggest a possible role of this molecule in the phagocytotic uptake and phagosomal degradation processes of antigens or in antigen presentation. we show here that asmase -/mice fail to eliminate the noncytopathic lymphocytic choriomeningitis (lcm) virus as rapidly as littermate wildtype mice. investigation of the immune response revealed a reduced expansion of cd8 þ t cells. the secretion of ifn-g in response to contact with target cells as well as the cytolytic activity of virus-specific cd8 þ t cells was severely impaired. additionally, both phases of the lcm virus-specific dth response, mediated by cd8 þ and cd4 þ t cells consecutively, were diminished in asmase -/mice. however, the secondary memory response of virus-specific ctl was not altered, and the 614 abstracts ................................................................................................................................................................................................. virus was effectively controlled for at least 3 months by asmase -/mice. in conclusion, the results of this study suggest an involvement of the asmase in the activation, expansion or maturation of virus-specific cd8 þ t cells during the acute infection of mice with the lcm virus. novel markers for alternative activation of macrophages: macrophage galactose-type c-type lectins 1 and 2 in parallel with the th1/th2 dichotomy, macrophages are capable of developing into functionally and molecularly distinct subpopulations, due to differences in, for example cytokine environment and pathological conditions. while the best-studied, classically activated macrophage is induced by type i stimuli such as ifn-g, a type ii cytokine environment antagonizes the classical activation of macrophages and is capable of alternatively activating macrophages. however, molecular markers associated with these type ii cytokine-dependent, alternatively activated macrophages remain scarce. besides the earlier documented markers macrophage mannose receptor and arginase 1, we recently demonstrated that murine alternatively activated macrophages are characterized by increased expression of fizz1 and ym. we now report that expression of the two members of the mouse macrophage galactose-type c-type lectin gene family, termed mmgl1 and mmgl2, is induced in diverse populations of alternatively activated macrophages, including peritoneal macrophages elicited during infection with the protozoan trypanosoma brucei or the helminth taenia crassiceps, and alveolar macrophages elicited in a mouse model of allergic asthma. we also demonstrate that, in vitro, interleukin-4 and interleukin-13 upregulate mmgl1 and mmgl2 expression and that, in vivo, induction of mmgl1 and mmgl2 is dependent on interleukin-4 receptor signalling. moreover, we show that regulation of mgl expression is similar in human monocytes and monocyte-derived macrophages. hence, macrophage galactose-type c-type lectins represent novel markers for both murine and human alternatively activated macrophages; thus, paving the way for further characterization of the phenotype of macrophages occurring in th2 conditions. background: human parvovirus b19 (b19) is a ubiquitous pathogen, normally causing a mild self-limiting disease, but also capable of causing both significant pathology and long-term persistence. the small size and stability of the virus makes it suitable for mapping of the full breath and the kinetics of the cellular immune responses following acute viral infection. methods: five patients with acute primary b19 infection were included in the study and followed consecutively for up to 200 weeks. cellular immune responses were mapped by ifng enzyme-linked immunospot to overlapping peptides spanning the whole b19 genome. results: in all five acutely infected patients, we were able to monitor the kinetics of a strong specific cellular immune reaction. responses peaked at levels of 850-1850 sfc/ million pbmcs, roughly corresponding to 0.3-0.6% b19specific cd8 þ cells circulating in peripheral blood at 10-80 weeks post-infection. the responses in individual patients were directed to three or four different peptide pools, and the specificity was confined to the same cd8 epitopes present in the pools throughout the follow-up period. the majority of responses were directed to the virus nonstructural protein, only two patients showed any response to the capsid proteins, elicited by the same epitope in both cases. conclusion: the cellular immune responses to acute b19 infection are surprisingly narrow in distribution and remain at high levels for up to 80 weeks post-infection. the initial epitope specificity is maintained, and the majority of responses target the virus nonstructural protein, which is not included in vaccine preparations, evaluated against the infection. the relationship between malnutrition and malaria is controversial. on one hand, malaria may cause malnutrition, while on the other, malnutrition itself may modulate susceptibility to the disease. we investigated the association between plasmodium falciparum malaria and malnutrition in a cohort of children living on the coast of kenya. the study involved longitudinal follow-up for clinical malaria episodes and anthropometric measurements at four cross-sectional surveys. we used poisson regression analysis to investigate the association between malaria and nutritional status. compared to baseline (children with a waz or haz score of !à2), the crude incidence rate ratios (irrs) for malaria in children with low haz or waz scores (<à2) during the period prior to assessment were 1.17 (95% ci 0.91-1.50; 0 ¼ 0.21) and 0.94 (0.71-1.25; 0.67), respectively, suggesting no association between malaria and the subsequent development of pem. however, we found that age was acting as an effect modifier in the association between malaria and malnutrition. the irr for malaria in children 0-2 years old who were subsequently characterized as wasted was 1.65 (1.10-2.20; p ¼ 0.01), and a significant overall relationship between malaria and low-haz was found on regression analysis when adjusting for the interaction with age (irr 1.89; 1.01-3.53; p < 0.05). although children living on the coast of kenya continue to suffer clinical episodes of uncomplicated malaria throughout their first decade, the association between malaria and malnutrition appears to be limited to the first 2 years of life. a. astrinidou-vakaloudi, 1 s. xytsas, 1 i. diamanti, 1 h. ioannidis 2 & p. pangidis 2 1 microbiology department of general hospital of thessaloniki 'agios pavlos', thessaloniki, greece, and 2 nefrology, 2 nd ika hospital of thessaloniki, thessaloniki, greece. e-mail: stasa@hol.gr aim: renal dysfunction may influence the colonization of gastric mucosa by urea-splitting bacteria such as helicobacter pylori, by increasing urea concentrations in the gastric juice. our aim was to investigate the prevalence of h. pylori in patients with end-stage renal disease (esrd), receiving long-term haemodialysis treatment. methods: this study included 40 sera from patients with esrd (29 male and 11 female) undergoing periodic haemodialysis; mean time of treatment was 42.6 months. using elisa technique, we investigated the presence of igg and iga antibodies against h. pylori as well as igg caga (antibodies specific for caga(þ) strains of h. pylori). sera from 40 healthy blood donors were used as a control group. results: h. pylori igg antibodies were detected in 32 out of 40 (80%) patients in the dialysis group, while 31/40 (77.5%) tested positive for iga. igg caga antibodies were present in 13 out of 40 (32.5%). prevalence of h. pylori igg, iga and caga igg antibodies in the control group was 33, 7 and 15%, respectively. conclusions: although international data suggest that prevalence of h. pylori infection is the same in esrd patients as in healthy individuals, in our study that seems not to be the case. the higher blood and gastric juice urea levels may be a risk factor (among many others), but more studies are required in order to understand the relation of h. pylori infection in this group of patients. flanders interuniversity institute for biotechnology, department of molecular and cellular interactions, free university of brussels, brussels, and 2 pasteur institute of brussels, mycobacterial immunology, brussels, belgium. e-mail: tgartner@vub.ac.be immunity against tuberculosis (tb), caused by mycobacterium tuberculosis, depends largely on activation and maintenance of strong cell-mediated immune responses involving both cd4 þ and cd8 þ t cells and the ability to respond with th1-type cytokines, particularly ifn-g. recent studies suggested that bcg, the only licensed vaccine against m. tuberculosis, may fail to induce t-cell responses in the lung mucosa and may therefore not protect against pulmonary tb. a decrease in tb mortality may be achieved by enhancing immunity in the lung. the present study evaluated the induction of antigen-specific immunity in the lung by intranasal (i.n.) delivery of the lipoprotein i (opri) from pseudomonas aeruginosa. opri has shown to be a toll-like receptor 2/4 agonist that, when given subcutaneously, induces type-1 immune responses against heterologous antigens. here, a fusion of opri to ag85a of mtb (opri-ag85a) was used as a subunit vaccine in homologous prime-boost immunizations. in addition, opri-ag85a was combined with an ag85a-encoding dna vaccine (ag85a dna) or with bcg in heterologous prime-boost vaccinations. intranasal and parenteral delivery with opri-ag85a elicited comparable t-cell responses in the spleen; in addition, i.n. delivery elicited specific t-cell responses in the lung lymph nodes (llns). intramuscular delivery of ag85a dna induced significant systemic th1 immune responses. intranasal boosting with opri-ag85a enhanced this response and in addition induced an antigen-specific ifn-g response in lln. opri may therefore be an efficient adjuvant for mucosal boosting. we continue to evaluate the protection induced by opri-based prime-boost vaccinations against pulmonary tb. results on the immunogenicity and protection against intravenous mtb h37rv infection will be presented. toll-like receptors (tlrs) are pattern recognition receptors of the innate immune system, which recognize molecular structures on pathogens or cellular stress-associated molecules. tlr-ligand interactions trigger activation of inflammatory signal transduction and expression of genes involved in host defense. in this study, we have examined the requirement for different tlr adaptor molecules in virus-induced chemokine expression and are currently trying to identify the tlr involved. we have found that both a herpesvirus [herpes simplex virus (hsv)] and a paramyxovirus (sendai virus) require a functional genome to induce expression or proinflammatory chemokines in human and murine monocytic cell lines. for both viruses, this is independent of the tlr adaptor molecules trif and mal. however, overexpression of the vaccinia virus-encoded inhibitor of tlr-signalling a52r or dominant-negative myd88 totally inhibited hsv-induced rantes expression but only partially prevented sendai virus from inducing this chemokine. this suggests that hsv-induced rantes expression occurs via a tlr pathways, whereas sendai virus utilizes both tlr-dependent and -independent pathways to stimulate expression of rantes. we are currently trying to identify the tlrs involved. data from these studies will also be presented at the meeting. 2 0 -5 0 -oligoadenylate synthetases are interferon-induced, double-stranded rna-activated antiviral enzymes which are the only proteins known to catalyse 2 0 -specific nucleotidyl transfer. this first crystal structure of a 2 0 -5 0oligoadenylate synthetase reveals a structural conservation with the 3 0 -specific poly(a) polymerase that, coupled with structure-guided mutagenesis, supports a conserved catalytic mechanism for the 2 0 -and 3 0 -specific nucleotidyl transferases. comparison with structures of other superfamily members indicates that the donor substrates are bound by conserved active site features while the acceptor substrates are oriented by nonconserved regions. the 2 0 -5 0oligoadenylate synthetases are activated by viral doublestranded rna in infected cells and initiate a cellular response by synthesizing 2 0 -5 0 -oligoadenylates, that in turn activate rnase l. this crystal structure suggests that activation involves a domain-domain shift and identifies a putative dsrna activation site that is probed by mutagenesis. we demonstrated that this site is required both for the binding of dsrna and for the subsequent activation of oas. this rna-binding site is different from known rna-binding site; rather than forming a defined three-dimensional domain, it is located at the interface of the two major domains in oas. this novel architecture ensures that the dsrna helix can make simultaneously contact with both domains of oas and ensure the subsequent structural rearrangement leading to the activation of oas. our work provides structural insight into cellular recognition of double-stranded rna of viral origin and identifies a novel rna-binding motif. bacteria-specific iga antibodies are efficient opsonins for neutrophils and mononuclear phagocytes, provided that the phagocytes express the fca receptor (cd89). expression of cd89 can be stimulated by inflammatory cytokines, activated complement factors and certain microbial components. in one study, unstimulated phagocytes were able to ingest iga antibody-treated pneumococci, but only in the presence of complement, which was found to be activated by the iga antibodies along the alternative pathway. pneumococci produce iga1 protease that cleaves human iga1, but not iga2, molecules in the hinge region. this leaves iga1 as faba (monovalent) deprived of fca which contains the docking site for cd89. iga1 is the vastly predominant subclass of iga in the upper airways and circulation of humans. aims: to examine the effects of iga1 protease activity and complement on phagocytosis of iga antibody-coated pneumococci by an unstimulated human phagocytic cell line (hl60). materials and methods: iga1 and iga2 monoclonal antibodies to serotype 4 pneumococcal capsular polysaccharide (ps) were generated by heterohybridoma technique involving b cells from human vaccinees. isogenic serotype 4 pneumococci with and without iga1 protease activity, respectively, were obtained after inactivation of the iga gene of the tigr4 strain. opsonophagocytosis was quantitated using the assay described by romero-steiner et al. based on enumeration of surviving bacteria by culture. the integrity of iga molecules was examined by western blotting. results: both iga1 and iga2 antibody to type-4 polysaccharide-induced phagocytosis of iga1 protease-deficient type-4 pneumococci equally well in the absence as in the presence of complement. iga1 antibody to type-4 polysaccharide displayed a fourfold higher opsonophagocytosis titer against iga1 protease deficient compared to homologous wildtype target bacteria. a similar effect of iga1 protease activity of the target bacteria was not observed in a parallel experiment where iga2 antibody to type-4 polysaccharide served as opsonin. iga1 antibody extracted from iga1 protease-producing target bacteria was almost exclusively in the form of faba. conversely, iga1 from protease-deficient bacteria and iga2 from both types of bacteria were intact. conclusions: these results indicate that the iga1 protease activity of s. neumoniae may help the bacteria escape iga1 antibody-mediated opsonophagocytosis. besides, in these experiments, iga-mediated opsonophagocytosis was independent of complement. vitamins e and c have been found to increase the cellular and humeral immunity of pigs. vitamin e deficiency has also been found to predispose pigs to different diseases, e. coli infection is one among them. after weaning, the vitamin e status of pigs often decreases to a critical low level. in this experiment, we studied whether vitamin c supplementation would be a possible feeding strategy to optimize the immune status of weaners. the interaction between vitamin e and c is interesting due to the reported sparing action on vitamin e or synergism between these to vitamins. piglets were weaned at day 28 of age from sows fed increasing dietary vitamin e during lactation, and piglets were during the following 3 weeks fed either a control diet or this diet supplemented with 500 mg stay-c per kg. blood sampling was obtained weekly from day 28 and until day 49 of age. on the same days, one piglet per dietary treatment was killed and alveolar macrophages (am) were harvested. vitamin c supplementation increased the concentration of igm in serum of piglets throughout the weaning period. although the vitamin e concentration in am decreased with increasing age of the piglets, the concentration was numerically higher in piglets of sows fed the high dietary level of vitamin e. however, vitamin c supplementation tended to increase the total am concentration of vitamin e after weaning and increased the proportion of the biologically most active isomer of vitamin e [rrr-(a-tocopherol)] in the am. the eicosanoid synthesis by am was not influenced by the vitamin c supplementation, but the synthesis of leukotriene b4 was decreased 2 weeks after weaning compared to other days of am harvesting. in conclusion, dietary vitamin c supplementation improved the immune responses of piglets after weaning. a whole blood stimulation assay with escherichia coli (o111:b4) endotoxin was established to measure the capacity of dairy cows to produce the proinflammatory cytokine tumour necrosis factor-a (tnf-a) ex vivo. initially, a time-and dose-dependent study was carried out to find the optimal stimulation conditions for the tnf-a response. the tnf-a response peaked between 3 and 4 h at 38.5 c. a dose in the range of 5-10 g of e. coli lipopolysaccharide (lps)/ml whole blood was found to give the maximum tnf-a response. thirty-eight danish-holstein dairy cows were investigated for their tnf-a responsiveness ex vivo in the periparturient period. heparin-stabilized blood samples were collected seven times over a period of 4 months (weeks à3, à1, 2, 3, 5, 9 and 13 around calving) and stimulated with 5 g/ml of e. coli lps. indeed, fluctuations in the tnf-a responsiveness occurred over time. moreover, the mean tnf-a responsiveness of 38 cows was found to be significantly increased (p < 0.001) in the weeks close to calving. however, in the more stabile physiological periods, some cows had a consistently low tnf-a response, whereas others had high a tnf-a response. we are currently investigating whether high and low tnf-a responders to e. coli lps also exist in dairy cows in vivo. moreover, the importance of tnf-a responsiveness ex vivo to dairy cows' susceptibility and clinical response to experimental e. coli infections in the udder is being investigated. coelomic cytolytic factor (ccf) is a 42 kda invertebrate pattern recognition molecule isolated from the coelomic fluid of the earthworm eisenia foetida (oligochaeta, annelida). ccf displays a number of similarities with the mammalian cytokine tumour necrosis factor-a (tnfa) as a result of a shared n,n 0 -diacetylchitobiose lectin-like domain. however, these similarities are solely functional and are not based on any (dna or amino acid) sequence homology, thus suggesting a form of convergent evolution. in particular, the lectin-like domain of tnf-a has been shown to induce membrane depolarization in various mammalian cell types, through interactions with endogenous amiloride-sensitive ion channels. this nonreceptor-mediated activity of tnf-a has been reported to be involved in the resorption of oedema. likewise, the lectin-like domain of ccf also induces membrane depolarization in mammalian cells. here, we show that ccf appears to be able to induce oedema resorption in an alveolar epithelial cell line through its lectin-like domain. this lectin-like domain of ccf interacts (directly or indirectly) with endogenous sodium and/or chloride channels, and not potassium channels, on mammalian cells. additionally, we suggest that the jnk/sapk and erk1/2 pathways are involved in ccf-induced macrophage activation. these results further establish the functional analogy between an invertebrate pattern recognition molecule and a mammalian cytokine and, from a more applied point of view, suggest the possibility of utilizing ccf in the treatment of oedema. release of svegf and svegfr1 from white blood cells and platelets during surgery and stimulation with bacterial antigens introduction: the influence of surgery on release of soluble vascular endothelial growth factor (svegf) and the soluble vascular endothelial growth factor inhibitory receptor 1 (svegfr1) is unknown. we studied the effect of major and minor surgery on potential variations in svegf and svegfr1 concentrations in vivo and on bacterial antigen-induced release of svegf and svegfr1 from whole blood in vitro. methods: sixty-one patients with abdominal diseases undergoing five different surgical procedures were included. blood samples were drawn from anaesthetized patients before and after the operation. white blood cells and platelets were counted, and plasma svegf and svegfr1 was determined by an elisa method. whole blood from each blood sample was stimulated in vitro with bacteria-derived antigens (lps or protein-a) and svegf and svegfr1 levels were subsequently determined in the supernatants. stimulation with isotonic saline served as control assay. neither svegf or svegfr1 in plasma changed during surgery. in vitro stimulation of blood samples with bacteria-derived antigens resulted in a significant increase in svegf (p < 0.0001) and a less pronounced but still significant increase in svegfr1. release of svegf due to stimulation was significantly higher after the operation (nonsignificant), whereas svegfr1 release remained largely unchanged after surgery. correlation between bacterial antigen-induced release of svegf and neutrophile cell count was highly significant (p < 0.0001). there was no correlation between svegf and platelet cell count, and bacterial antigen-induced svegfr1 release did not correlate with counts of neutrophils and platelets. conclusions: plasma svegf and svegfr1 concentrations did not change during surgery. in vitro bacterial stimulation led to increased release of svegf and svegfr1, which was not significantly amplified during surgery and which may be related to number of circulating neutrophils. natural killer cell functions and subsets after in vitro stimulation with il-2 and il-12, with special emphasis on intracellular ifn-g and nk-cell cytotoxicity r. nyboe, 1,2 t. rix, 1,2 j. krog, 1,2 e. tønnesen 1 & m. hokland 2 1 department of anaesthesiology and intensive care, aarhus university hospital, and 2 institute of medical microbiology, and immunology, university of aarhus, aarhus, denmark. e-mail: rnsr@studmed.au.dk materials and methods: isolated cryopreserved human peripheral blood mononuclear cells (pbmcs) were stimulated with il-2 and il-12. this stimulation has previously been shown to activate nk cells. cell cytotoxicity was measured by flow cytometry after incubation with k562 cells. this method was compared to the current standard 51cr release assay. cells were treated with bfa to accumulate ifn-g, stained for surface markers, permeabilized and stained for intracellular ifn-g. flow cytometry was then performed to measure intracellular ifn-g production in pbmc, especially in nk cells. results: we have demonstrated that stimulation with il-2 and il-12 is effective in increasing the number of ifn-gpositive cells. there is a distinct difference between the cd3-cd56dim and the cd3-cd56bright subsets, with a much greater proportion of ifn-g-positive cells in the cd3-cd56bright subset. the effects of stimulation with il-2 and il-12 on cytotoxicity will be presented, as will the relation between ifn-g production and cytotoxicity. in addition, we will present results of these assays applied to porcine cells. discussion: in combination, these tests will address nk cell function by combining cytotoxicity with ifn-g production in nk cell subsets. the results will demonstrate whether this could serve as a useful tool in describing nkcell function, which could be of value in clinical and experimental settings. culture of regulatory t-cell lines from bronchial mucosa t lymphocytes play a major role in many immune responses. in the last decade, special focus has been on the function of th1 and th2 effector cells. now the importance of regulatory cd4 þ cd25 þ t cells in maintenance of the immunological homeostasis emerges. sarcoidosis is a multisystem granulomatous disorder often affecting the lungs. the typical sarcoid granulomas consists of epitheloid cells, macrophages and lymphocytes, mainly cd4 þ t cells of th1 phenotype. we have cultured t cells from bronchial biopsies of patients with sarcoidosis as well as from controls in high levels of interleukin 2 (il-2) and il-4 and demonstrate spontaneously arising cd4 þ cd25 þ populations and high concentrations of il-10 in these cultures. the main difference between cultures of sarcoid origin compared to controls is a very much higher concentration of the inflammatory cytokines il-6 and tnf-a in cultures of sarcoid origin. the effects of hyperbaric exposure on human peripheral blood mononuclear cells, with special emphasis on natural killer cell cytotoxicity and subsets materials and methods: as an experimental physiological stress model, we examined the effects of hyperbaric exposure on peripheral blood mononuclear cells (pbmcs) obtained from venous blood drawn from eight divers during a simulated heliox saturation dive. eight persons working in normobar atmosphere outside the pressurized chamber served as control donors. the spontaneous cytotoxicity of the pbmcs was estimated in a 4 h 51cr-release assay using k562 as nk-sensitive target cells. the pbmcs were characterized, using 4-colour flow cytometry, with special emphasis on the nk-cell subsets. the data were statistically analysed using a multivariate regression model (stata 8.2). p values <0.05 was considered statistically significant. results: the estimated cytotoxicity increased significantly in both the group of divers and control donors during the dive (pdivers < 0.01 and pcontrols < 0.01). although the cytotoxicity increased relatively more (p < 0.01) in the group of divers compared to the group of control donors between day 1 and 2. discussion: the increased cytotoxicity of pbmc estimated in the group of divers indicate that parts of the cellular immune system are affected during the extreme physiological conditions induced during the initial phase of the presented experimental hyperbaric setup. the increase in cytotoxicity observed in the group of control donors could hypothetically reflect the stress level in persons working outside the pressurized chamber during the dive. the interaction with adhesion molecules expressed by vascular endothelium is the first step in lymphocyte infiltration into tissues. at both cutaneous and mucosal sites interleukin-10 (il-10), il-12 and transforming growth factor (tgf)-b are important regulators of chronic inflammatory disease, where cutaneous lymphocyteassociated antigen (cla) and ae integrin (cd103) may be expressed. unlike cla, cd103 is not believed to play a role in tissue-specific homing but may help to retain t cells within epithelial layers. we have previously shown that il-12 alone can together with an unknown cofactor increase the expression of cla. stimulation with streptococcal pyrogenic exotoxin c (spec) increased the expression of cd103 by cd8 þ but not cd4 þ t cells. while il-12 increased superantigen-stimulated expression of cla, this cytokine strongly inhibited the cd103 expres-sion, and a combination of il-12 and tgf-b completely abrogated the induced cd103 expression. conversely, il-10 suppressed cla but increased cd103 expression. these findings indicate that, in addition to suppressing the development of th1-mediated inflammatory responses, il-10 may also inhibit the migration of cd8 þ t cells into the skin while il-12 promotes such migration. thus, the expression of cla and cd103 may be antagonistically regulated by il-10 and il-12, and the balance between these cytokines could influence the t-cell migration of inflammatory cells into epithelial tissues. during contact sensitivity reaction, immune cells proliferate. in order to study the histological picture of these proliferation phases, we used a mouse model of contact sensitivity in the oral mucosa and on skin. we also used bromodeoxyuridin (brdu, an analogue to thymidin) that is incorporated into the nucleus during cell replication. the hapten oxazolone (oxa) was used to sensitize and elicit the oral mucosa and/or the ear skin. mice were killed at various times after elicitation, and unsensitized animals were also exposed to the hapten as controls. brdu (25 mg/ kg animal) was injected i.p. 2 h before the kill. specimens from the oral mucosa, ear skin and submandibular and auricular lymph nodes were cut and fixed in 4% paraformaldehyde. they were then treated with acid and biotinylated anti-brdu antibody and developed using abc-kit and dab. the analyses were performed using a leica light microscope and the computer program analysis. in the oral mucosa, the frequency of proliferating cells were increasing during the observation period, 4-24 h after elicitation, regardless of site of sensitization. the proliferating cells were found mainly in the basal cell layer of the epithelium. similar patterns were found in ear skin. the regional lymph nodes demonstrated a few scattered proliferating cells 4 h after elicitation. after 24 h, these cells were found frequently in the whole lymph node. control animals exhibited considerable less proliferating cells at all times. we conclude that most proliferating cells were found 24 h after elicitation locally at the hapten-exposed sites (the oral mucosa or the ear skin) as well as in the regional lymph nodes. the endogenous nucleoside adenosine is released in excess during inflammation or other metabolic stress and is generally known to deliver tissue protective anti-inflammatory effects. adenosine acts via four adenosine receptors of which the a2a receptor is the predominant form in t cells. adenosine levels are elevated in asthmatic lung, and adenosine can directly induce mast cell degranulation and bronchoconstriction in these patients. instead, the role of anti-inflammatory mechanisms of adenosine on t cells in asthma is unclear. aim: to study the a2a receptor expression in peripheral blood cd4 þ t cells in asthmatic and healthy individuals using flow cytometric and quantitative real-time pcr methods. results: unstimulated cd4 þ cells of asthmatic patients expressed significantly lower levels (p < 0.001) of a2a receptor in protein level (mean percentage of cells positive ae sem: 76.8 ae 1.2, n ¼ 6) compared to healthy individuals (90.4% ae 1.9, n ¼ 4). double staining for cd69 expression showed that stimulation of cd4 þ cells decreased a2a expression in both groups but indicated that the detected lower levels of a2a in unstimulated cells of asthmatics was not due to preactivation in these patients. surprisingly, a2a mrna expression in unstimulated cd4 þ cells was significantly higher (p < 0.05) in asthmatics (n ¼ 28) compared to healthy controls (n ¼ 7). the expression did not correlate with serum total ige levels. conclusions: asthmatic individuals express less a2a adenosine receptor on their peripheral cd4 þ t cells. the higher mrna levels instead may point to a negative feedback regulation in the receptor expression. the role of possibly decreased adenosine-mediated anti-inflammatory effects in asthma pathogenesis require further studies on this t-cell mediated disease. the chronic inflammatory skin disease atopic eczema (ae) affects almost 15% of the population in many countries today. the pathogenesis of ae is not fully understood. a combination of genetic predisposition and environmental factors like microorganisms seems to contribute to the symptoms. the yeast malassezia sympodialis is part of our normal skin micro flora but can act as an allergen and elicit specific ige and t-cell reactivity in patients with ae. recently, we identified a novel major m. sympodialis allergen, designated mala s 11 (22.4 kda), with sequence similarity to the mitochondrial enzyme manganese superoxide dismutase (mnsod). interestingly, mala s 11 has a high degree of homology to human mnsod. the aim of this study was to examine the effects of recombinant mala s 11 on antigen-presenting dendritic cells. monocytederived dendritic cells (mddcs) from healthy blood donors were cultured with or without mala s 11 for different time periods. it was found that the maturation marker cd83 and the costimulatory molecules cd80 and cd86 were upregulated on the mddcs exposed to mala s 11 for 24 h, as demonstrated by flow cytometry. furthermore, coculture of mddcs with mala s 11 for 9 h induced an increased production of the inflammatory cytokines il-6 (200-fold), tnf-a (100-fold) and il-8 (sixfold), as detected by the cytometric bead array (cba) analysis. our results suggest that mala s 11 affects the immune response through dc maturation and production of inflammatory cytokines. the potential cross-reactivity with human mnsod needs to be explored and the exact role of mala s 11 in the pathogenesis of ae assessed in clinical studies involving skin prick and atopy patch tests. allergen-specific immunotherapy (sit) is commonly conducted with allergen extracts adsorbed to aluminium hydroxide (alum). drawbacks linked to the use of alum, such as the formation of granuloma at the site of injection, have led to suggestions of novel allergen carriers. an alternative carrier is 2 mm carbohydrate-based particles (cbps). in mouse, allergen-coupled cbps have been demonstrated to skew the allergen-specific immune response towards a th1-like activity (grönlund et al. immunology, 2002) . we here coupled the recombinant major cat allergen fel d 1 to cbps (cbp-fel d 1) by cyanogen-bromide activation, resulting in covalent binding. the effect of cbp-fel d 1 on monocyte-derived dendritic cells (mddcs) from healthy human blood donors was studied. we found that the majority of the cd1a þ mddcs were capable of taking up fitc-labelled cbp-fel d 1, as demonstrated by flow cytometry and confocal laser scanning microscopy. furthermore, incubation with cbp-fel d 1 resulted in an upregulation of the costimulatory molecule cd86 on the mddcs, which was not observed with fel d 1 or cbps alone. finally, cbp-fel d 1 induced a fivefold increase in the release of the pro-inflammatory cytokine tumour necrosis factor (tnf)-a and a fourfold increase in the release of the chemokine interleukin-8 from mddcs. taken together, the effects cbps possess make them interesting as novel allergen carriers for sit. the cysteine protease der p1 from dust mite of the genus dermatophagoides pteronyssinus is a major type i allergen. about 80% of house dust mite (hdm) allergic individuals are reactive to this protease in standard assays for detection of ige. a curative treatment for atopic allergy is immunotherapy (it) with hdm extracts which are complex mixtures occasionally resulting in anaphylactic reactions. novozymes focuses on developing a recombinant variant of der p1 which exhibit lowered risk of ige-mediated allergic reactions, while maintaining its ability to trigger proper th-cell responses. this may provide a safer alternative for specific it of hdm allergy. a secreted recombinant form of pro-der p 1 expressed by saccharamyces cerevisiae was obtained by fusion of the pro-enzyme to a fungal signal peptide. the n-glycosylation site of der p1 was mutated resulting in a deglycosylated pro-enzyme with a molecular mass of 35 kda. protein purification procedure was developed to obtain nearly pure der p1 protein followed by determination of concentration by active-site-titration with the cysteine protease inhibitor e64. the deglycosylated recombinant pro-der p 1 revealed immunologic similarity to the native der p 1 molecule when compared in basophile histamine release, ige-binding assays and t-cell proliferation assays. by in silico epitope mapping of a modelled 3-dimensional structure of der p1, five putative igg and ige epitopes were predicted. by protein engineering, the predicted epitopes were removed one by one in der p1 and screening for hypoallergenic variants was performed. combining inhaled long-acting b-2 agonist (laba) and inhaled corticosteroid (ics) seems to offer asthma control at a lower dose of ics than achieved by ics alone. fine mapping of t-cell surface markers by flow cytometry offers a detailed status of the individual's inflammatory response. the frequency of mt2 (cd4 þ cd45ra -cd62l þ cd11adim) and mt1 (cd4 þ cd45ra -cd62l -cd11abright) cells in peripheral blood, and their ratio, has been shown to differ predictably in atopics and patients with leprosy, where mt2 correlates with a th2 phenotype and mt1 with a th1 phenotype. stable asthmatics, requiring fluticasone propionate (fp) 750-1000 mg daily or equivalent, were randomized to receive, double-blinded, either seretide 1 [salmeterol and fluticasone propionate (sfc, n ¼ 16)] 50 mg/500 mg bd or fp 500 mg bd (n ¼ 17). if asthma was controlled based on lung function and symptoms at clinic visits every 6 weeks, ics dose was tapered until asthma exacerbated or 0 mg was reached. the frequency and ratio of mt2 and mt1 t cells of the patients was monitored at 6 week intervals. as treatment tapered, the frequency of mt2 cells decreased (p ¼ 0038 from first to final visit), whereas that of mt1 cells increased. the ratio of mt2/mt1 decreased (p ¼ 0049 from first to final visit). in patients receiving laba þ ics, the fall in mt2/mt1 ratio appeared to be more pronounced than in patients receiving ics alone. thus, the mt2 phenotype may be associated with stable asthma, whereas an imminent exacerbation may associate with an increase in the mt1 phenotype. laba may allow for a greater effect of fp on the mt ratio. activation of complement pathways, leading to production of c3a and c5a anaphylatoxins, has been postulated in the pathogenesis of asthma and allergic airway inflammation. the present study was undertaken to investigate the role of mannan-binding lectin (mbl), an initiator of the lectin pathway of complement, in asthma and allergic rhinitis. mbl levels and mbl-induced complement activity were determined in 19 patients of bronchial asthma with allergic rhinitis and 20 unrelated, age-matched controls of indian origin. mbl levels and activity were correlated with percent eosinophilia and percent predicted fev1 values of the patients. association of single nucleotide polymorphisms (snps) in exon 1 and intron 1 of the mbl with the disease, clinical markers, mbl levels and mbl-induced complement activity was analysed using standard statistical tools. significantly higher mbl levels and activity were observed in patients of bronchial asthma with allergic rhinitis as compared to the controls. we identified five snps, of which two, a816g in exon 1 and g1011a in intron 1 of the mbl, were novel. snp g1011a was significantly associated with the disease (p ¼ 0.0024, or ¼ 5.8696, 95% ci: 1.7316 < or < 19.8963). individuals with 'a' allele at position 1011 showed increased mbl levels, activity and disease severity. our results suggest that 'a' allele at position 1011 leading to high mbl levels and complement activity may be contributing to the severity of bronchial asthma and allergic airway inflammation. serum resistance of borrelia burgdorferi strains belonging to the b. afzelii and b. burgdorferi sensu stricto genospecies is dependent on binding of complement inhibitor factor h. we recently reported that factor h binding by b. burgdorferi is due to inducible expression of several approximately 20 kda plasmid-encoded, surface-exposed lipoproteins related to ospe (e.g. erpa, erpp and p21). in addition, a second class of factor h-binding proteins of approximately 27-35 kda has been described. the ospe-related lipoproteins are dramatically induced by b. burgdorferi during transmission from its tick vector into the mammalian host. the induction of ospe-related lipoproteins during mammalian infection may play a key a role in the borrelial evasion of the host's immune system. the goal of the present study was to define the factor h-binding regions of ospe-related proteins using mutagenesis, peptide mapping and surface plasmon resonance analysis (biacore). the combined studies revealed that the c-terminal regions of both human and mouse factor h (scrs 18-20) specifically bind to ospe-related lipoproteins. we also found fhr-1, whose c-terminal scrs 3-5 are homologous to scrs 18-20 of factor h, to bind to ospe. peptide mapping revealed five putative regions (designated i-v) in ospe that could directly interact with factor h. deleting the c-terminal 15 amino acid residues from region v of p21 abolished its ability to bind factor h. at the same time, however, synthetic peptides corresponding to the c-termini of ospe, p21 and erpp did not inhibit factor h binding to ospe. thus, the c-terminal-binding region v appears to be necessary but not sufficient for factor h binding. when a more specific mutation strategy was employed, where single amino acid residues in peptides spanning over the factor h-binding regions were mutated to alanines, we observed that lysines in the factor h-binding regions of ospe were required for factor h binding. the combined data have revealed that key lysine residues in ospe-related lipoproteins and ionic interactions are crucial for factor h interactions. furthermore, binding of ospe to the c-termini of both mouse and human factor h suggests that borrelia spirochetes utilize analogous complement resistance mechanisms in both rodents and man. in borrelia garinii strains, which in in vitro analyses have been found to be sensitive to complement killing, differences in the ospe sequences as well as in the expression of factor h-binding proteins may account for their susceptibility to serum lysis. role of yada, ail and lipopolysaccharide in serum resistance of yersinia enterocolitica serotype o:3 mannan-binding lectin (mbl), l-ficolin and h-ficolin are pattern recognition molecules of the innate immune system. we investigated the ability of these molecules to bind to different serotypes and noncapsulated variants of streptococcus pneumonia and staphylococcus aureus. we found that mbl binds to noncapsulated s. aureus strain (wood) but not any of the examined s. pneumoniae serotypes. l-ficolin binds to some capsulated s. pneumoniae serotypes (11a, 11d and 11f) as well as some capsulated s. aureus serotypes (type-1, -8, -9, -11 and -12). h-ficolin does not bind to any of the examined s. pneumoniae and s. aureus serotypes included in this study but did bind to a strain of aerococcus viridans. when bound to bacteria, mbl and h-ficolin initiated activation of complement factor c4, whereas l-ficolin did not. during this study, quantitative assays for the three proteins were developed and the concentration in 97 plasma samples were determined and the median values were estimated at 0.8 mg of mbl/ml, 3.3 mg of l-ficolin/ml and 18.4 mg of h-ficolin/ ml, respectively. the absence of early complement components (c1, c4 and c2 but not c3) is a predisposing factor for systemic lupus erythematosus (sle). recently, we demonstrated that, in c4-deficient (c4 def.) mice, igm-containing immune complexes (igm-ic) are filtered by the splenic barrier of marginal zone macrophages (mzm), resulting in an increased immune response against antigens within these igm-ic, but this could not be observed in wildtype or c3 def. mice. we hypothesized that splenic cd11b þ mzm play an important role in the induction of autoimmunity, and we therefore analysed their cytokine profile after isolation with the help of magnetic antibody cell sorting. mrna was isolated, and real-time pcr was performed with specific primers for murine ifn-g (ifn-g), interleukin-12 (il-12) and ifn-a (ifn-a). we observe a moderate increase of il-12 and ifn-g mrna in cd11b þ cells of c4 def. mice compared to wildtype cells. surprisingly, the concentration of ifn-a mrna is six times higher in c4 def. mice. preliminary results suggest that mrna in cd11b þ cells of c3 def. mice is even lower than that in wt. six hours following i.v. application of 20 mg of a abstracts 625 .................................................................................................................................................................................................. murine monoclonal igm anti-dsdna antibody, production of il-12, ifn-g and ifn-a mrna is increased in cd11b þ cells of both c4 def. and wt mice. several references described increased levels of inf-a in patients with sle. dendritic cells are discussed as a major source of ifn-a. our observation that c4-deficient, sle-susceptible mice demonstrate an increased spontaneous ifn-a production by splenic cd11b þ marginal zone macrophages could be an early sign and a trigger for the development of sle. this is supported by the fact that the absence of c3 is not a predisposing factor for sle and our observation that c3 def. animals display low levels of ifn-a mrna. 200-400 million people worldwide and represents one of the leading causes for liver cirrhosis and hepatocellular carcinoma. control over the hbv infection is achieved mainly by vaccination with hepatitis b surface antigen (hbsag). hbsag contains n-linked glycosylation side and is recognized by both mbl-a and mbl-c in a cadependent manner. hbsag-mbl complexes activate complement and may thus affect humoural immunity. to investigate the role of mbl in humoural responses to hbsag, we immununized mice that lack both mbl-a and mbl-c proteins with soluble hbsag. it has been shown that deficiencies in other complement components like c1q, c4 and c3 result in decreased antibody responses. however, mbl double ko animals mounted dramatically increased humoural responses. after priming, mbl double kos mounted hbsag-specific igm responses, which were threefold higher than wt controls. after boosting the hbsag, total igg was 10-fold higher in mbl ko than in wt control animals. similar to the response to hbsag, other glycosylated soluble antigens (e.g. invertase) induced better humoural responses in mbl double ko animals, suggesting that mbl plays an important role in a negative feedback regulation of adaptive immunity. reconstitution experiments with rmbl partially rescued the ko phenotype. we propose that the clearance of glycoprotein antigens in mbl ko is handled differently from the wt, resulting in better stimulation of humoural responses. alternatively, glycoprotein-ag-mbl-rich complexes inhibit b-cell responsiveness via putative mbl receptors. the complement system is an important part of the innate immune system. the activation of complement proceeds through three different pathways that converge in the generation of c3-activating enzyme complexes. complement activation via the lectin pathway is initiated when recognition molecules, mannan-binding lectin (mbl) or ficolin, bind to carbohydrate structures characteristic for microbial surfaces. in the circulation, mbl and ficolins are found in association with three structurally related mblassociated serine proteases (masp)-1, -2 and -3 and a small, nonenzymatic component, map19. masp-2 has been shown to elicit complement activation through the sequential proteolytic cleavage of c4 and c2 upon binding of mbl/masp-2 complexes to microbial surfaces. we have recently uncovered a polymorphism in the masp-2/map19 gene in a patient shown to be deficient in the lectin pathway of complement activation. the polymorphism results in a single amino acid substitution in the n-terminal part of the masp-2 protein. recombinant wildtype masp-2 and masp-2 containing the amino acid substitution in question was produced, and the ability to activate complement was studied. the mutation had a profound impact on masp-2 function, resulting in the lack of complement activation through the lectin pathway. elisa-based experiments showed that the mutation leads to the impairment of complement activation through influencing the binding of masp-2 to mbl or ficolins. deficiencies in the lectin pathway of complement activation have so far been accounted for only by lack of functional mbl. the mutation described above is the first defect described affecting both activation through mbl and the ficolins. .................................................................................................................................................................................................. th1, th2 and treg cell balance. dcs are present in the gut mucosa and may thus be target for modulation by gut microbes, including ingested probiotics. here, we tested the hypothesis that species of lactic acid bacteria, important members of the gut flora, differentially activate dc. a large panel of human gut-derived lactobacillus and bifidobacterium spp. was screened for dc-polarizing capacity by exposing bone marrow-derived murine dc to lethally irradiated bacteria. cytokines in culture supernatants and dc-surface maturation markers were analysed. substantial differences were found among strains in the capacity to induce interleukin-12 (il-12) and tumour necrosis factor (tnf)-a, while the differences for il-10 and il-6 were less pronounced. bifidobacteria tended to be weak il-12 and tnf-a inducers, while both strong and weak il-12 inducers were found among the strains of lactobacillus. remarkably, strains weak in il-12 induction inhibited il-12 and tnf-a production induced by an otherwise strong cytokine-inducing strain of lactobacillus casei, while il-10 production remained unaltered. selected strains were tested for induction of dc maturation markers. those lactobacilli with greatest capacity to induce il-12 were most effective in upregulating surface mhc class ii and cd86. moreover, l. casei-induced upregulation of cd86 was reduced in the presence of a weak il-12inducing l. reuteri. in conclusion, human lactobacillus and bifidobacterium spp. polarize differentially dc maturation. thus, the potential exists for th1/th2/treg-driving capacities of the gut dc to be modulated according to composition of gut flora including ingested probiotics. the intestinal micro flora is indispensable in developing and maintaining homeostasis of the gut-associated immune system. evidence indicates that lactic acid bacteria (lab), e.g. lactobacilli and bifidobacteria, have beneficial effects on the host. established health effects include increased gut maturation, antagonisms towards pathogens and immune modulation. the objective of this study is to evaluate the immunomodulating properties of a range of lab of human origin. as dendritic cells (dcs) play a pivotal role in the balance between tolerance and immunity to commensal microorganisms, in vitro-generated immature dcs serve as a suitable model for studying the immunomodulating effects of lab. human immature dcs were generated in vitro from monocytes and exposed to lethally uv-irradiated lab. the effect of various species of lab on dcs in direct contact was evaluated. furthermore, the maturation pattern of dcs separated from the bacteria by an epithelial cell layer (caco-2 cells), which should mimic the intestinal environment, was studied. cytokine secretion (il-12, il-10 and tnf-a) and upregulation of maturation surface markers on dcs (cd83 and cd86) was measured. different lab induced diverse cytokine responses. some strains were strong il-12 and tnf-a inducers and others weak. all strains induced il-10. different lab also differentially modulated expression of cd83 and cd86 on dcs. although some variation in the response to lab of dcs generated from different blood donors was observed, general differences in the effect of the various lab was revealed. experiments with the dc caco-2 coculture system are ongoing. different species of lab differentially affect dc maturation; this suggets that the gut flora plays a pivotal role in polarization of the immune response. natural killer (nk) cells are cells of the nonspecific immune system lysing altered self-cells. a noncytolytic subset of nk cells may serve a regulatory role by secreting cytokines. bacteria translocating across the gastrointestinal mucosa are presumed to gain access to nk cells, as consumption of certain lactic acid bacteria has been shown to increase in vivo nk cytotoxicity. here, we investigated how human gut flora-derived lactobacilli affect nk cells in vitro, by measuring proliferation and ifn-g production of human nk cells upon bacterial stimulation. cd3 -cd56 þ nk cells were isolated from buffy coats by negative isolation using non-nk lineage-specific antibodies and magnetic beads. nk cells were incubated with 10mg/ml uv-inactivated bacteria or 10mg/ml phytohemagglutinin (pha) for 4 days. proliferation was assessed by incorporation of radioactive thymidine into nk-cell dna. the ifn-g concentration was measured by elisa. incubation of nk cells with a lactobacillus acidophilus strain increased the proliferation of the nk cells and induced ifn-g production, both to levels comparable to pha stimulation. the proliferative response was further enhanced with autologous monocytes present, probably because cytokines, secreted by monocytes having engulfed bacteria, stimulated the nk cells. in contrast, a lactobacillus paracasei strain caused the nk cells to proliferate only in the presence of monocytes. these results demonstrate that various strains of lactobacilli have the capacity to activate nk cells in vitro, in a monocyte-dependent or -independent way. hence, the encounter of nk cells with lactic acid bacteria will affect nk-cell activation. such activation of nk cells may potentially skew an on-going or subsequent immune response towards a th1 response. lactobacilli are nonpathogenic gram-positive inhabitants of the normal human intestine known for their healthpromoting effects. in our earlier work, it is shown that human monoclonal antibody isolated from sera of a patient with waldenstrom macroglobulinaemia possess innate antibody characteristics and binds to lactic acid bacteria. according to the immune network model, immunization with this bacteria could induce the perturbations in immune system that might result in production of anti-lactobacillus antibodies, human monoclonal antibody like (ab1) and anti-idiotypic antibody (ab2). in this study, balb/c mice were immunized with two doses of bacteria lactobacillus acidophilus in complete and incomplete freund's adjuvant and phosphate-buffered saline (pbs), respectively. seven days after the last immunization, sera from immunized mice were collected and the presence of lactobacillus-specific ab1 and ab2 were determined by elisas. in the sera of immunized mice, antibodies specific to bacteria lactobacillus acidophilus were shown. the concentration of lactobacillus-specific antibodies was higher in the sera of hyperimmunized mice (mice immunized with 1 mg of igm dj) than in sera of mice immunized with 100 times lower doses of immunogen (0.01 mg per doses). moreover, ab1 and ab2 antibodies were detected in the sera of lactobacillus-hyperimmunized mice. in this study, we have shown the idiotypic network interactions in mice immunized with bacteria lactobacillus acidophilus. the normal gastrointestinal flora is crucial for the maturation of the acquired immunity via effects on antigenpresenting cells (apcs). here, we have investigated how two types of apcs, monocytes and dendritic cells (dcs), react to different bacterial strains typical of the commensal intestinal flora. purified monocytes and monocyte-derived dcs were stimulated with uv-inactivated gram-positive (lactobacillus plantarum and bifidobacterium adolescentis) and gram-negative (escherichia coli and veillonella parvula) bacterial strains. monocytes produced higher levels of il-12p70 and tnf, as detected by elisa, in response to l. plantarum than to e. coli and v. parvula. in contrast, dcs secreted high amounts of il-12p70, tnf, il-6 and il-10 in response to e. coli and v. parvula but were practically unresponsive to l. plantarum and b. adolescentis. the lack of response to the gram-positive strains correlated with a lower surface expression of toll-like reseptor 2 (tlr2) on dcs compared to monocytes. the surface expression of tlr4 on dcs was undetectable when analysed by flow cytometry, but blocking this receptor decreased the tnf production in response to v. parvula, indicating that low tlr4 expression on dcs is sufficient to mount an inflammatory response to gram-negative bacteria. ifn-g increased the expression of tlr4 on dcs and also potentiated the cytokine response to gram-negative bacteria. our results indicate that, when monocytes differentiate into dcs, their ability to respond to different commensal bacteria dramatically changes, thereby becoming unresponsive to probiotic gram-positive bacteria. these results may have important implications for the capacity of different groups of commensal bacteria to regulate mucosal and systemic immunity. probiotic bacteria, e.g. lactobacillus spp., may improve diseases such as chronic inflammatory bowel disease. we examined cytokine production and phenotypic change after in vitro stimulation of t cells from healthy volunteers using different probiotic strains. methods: t cells were cultured from colonic biopsies from eight healthy volunteers (agnholt and kaltoft, exp clin immunogenet 2001; 18:213-25) , and dendritic cells were matured from their peripheral blood mononuclear cells. t-cell cultures were stimulated with autologous bacterial sonicate or strains of lactobacillus spp., with and without the addition of dendritic cells. cytokine levels (tnf-a, ifn-g, il-10 and gm-csf) and phenotype (cd3, cd4, cd25 and cd69) were measured on day 4. results: lactobacillus spp. induced higher productions of tnf-a and il-10 than did autologous bacteria. in presence of dendritic cells, the production of all cytokines increased. however, the increases of ifn-g and tnf-a were more pronounced in wells with autologous bacteria than in wells with lactobacillus spp. the addition of dendritic cells upregulated cd25 expression without simultaneous upregulation of cd69. the upregulation was pronounced after stimulation with lactobacillus rhamnosus gg compared with autologous bacteria and other lactobacilli. discussion: in presence of dendritic cells, autologous bacteria induced inflammatory cytokines, while probiotics mainly induced regulatory cytokines. lactobacillus rhamnosus gg induced a regulatory phenotype (cd25 þ ), in part mediated by dendritic cells. future studies will address whether this shift to a cd25 þ phenotype represents a differentiation into competent regulatory t cells. in a clinical context, such cells might be used for treatment of inflammatory diseases. protein microarrays will play a key role in the postgenomic era and offer a unique possibility to perform highthroughput global proteome analysis. a chip can be printed with thousands of protein probes (e.g. antibodies), the biological sample added (e.g. a proteome) and any binding detected. we aim to develop protein microarrays based on human recombinant scfv antibody fragments for global proteome analysis. the concept of comparing proteomic maps of healthy versus diseased samples will allow diseasespecific proteins to be detected. in fact, antibody microarrays will allow us to perform comparative proteome analysis on any sample format in a species-independent manner, as long as a proteome can be isolated. however, the complexity of proteomes, containing several thousands of different proteins, is a problem. here, we have designed antibody microarrays targeting the water-soluble fraction of a proteome. to this end, an anticytokine antibody array was developed and human dendritic cells (aeactivation) was used as model system. the results showed that our antibody microarrays could be used to examine the cytokine profile in complex samples. furthermore, we have taken the first steps towards comparing our results with those of other technologies on both the protein and gene level. due to the complexity of the model proteome, we also examined the possibility to prefractionate the proteome in a simple one-step procedure (based on size) prior to the labelling step. in more detail, the sample proteome was fractionated into two fractions using membrane devices with different molecular weight cut-offs. the results showed that the fractionation considerably enhanced the assay sensitivity allowing cytokines in the pg/ml range to be readily detectable. the immunomodulatory effect of heat shock protein 70: immunization with a dna construct based on the malarial antigen fused with a fragment of hsp 70 primes for a th-1 type of response finding an appropriate adjuvant for human vaccination is crucial. heat shock proteins (hsps) act as adjuvants when coadministered with peptide antigens or given as fusion proteins. however, there is a potential risk of autoimmunity when using the complete molecules, because hsps are evolutionary conserved. to overcome this, we first evaluated the adjuvant effect against two different antigens of a less-conserved fraction of plasmodium falciparum hsp70 (pf70c) and compared it to the whole hsp70 molecule from trypanosoma cruzi (tchsp70). we found that pf70c exhibited similar adjuvant properties as the whole molecule. we later evaluated the adjuvant potential of pf70c against the malarial antigen eb200 in a chimeric dna construct. no appreciable levels of eb200-specific abstracts 629 .................................................................................................................................................................................................. antibodies were detected in mice immunized only with the dna constructs. however, dna primed the immune system, because subsequent challenge with the corresponding recombinant fusion proteins elicited a strong th-1 antibody response. in contrast, no priming effect was observed for ex vivo ifn-g production but stimulation with the hsp-chimeric fusion protein induced a stronger secretion of ifn-g in vitro than other proteins used. these results indicate that the use of hsps is promising in the design of new vaccines. high-throughput proteomics on antibody-based microarrays: the importance of probe and surface design in analogy to dna microarrays, protein microarrays offer a new distinct possibility to perform sensitive highthroughput global proteome analysis. however, the development of the protein microarray technology will place high demands upon the design of both probes and solid supports. the analysis of thousands of heterogeneous proteins on a single microarray requires the use of uniform probes, such as antibodies, directly designed for protein microarray applications. we have recently generated a human recombinant single-chain fv antibody library, genetically constructed around one framework, the ncoder-library, containing 2 â 1010 clones. single framework antibody fragments (sinfabs) selected from this library were successfully applied as probes for microarrays providing sensitive detection in the 600 attomol (mass spectrometry) and the 300 zeptomole range (fluorescence). however, the choice of framework is critical. we have shown that the selected ncoder framework displayed excellent functional on-chip stability and arrayed dehydrated probes retained their activity for several months. furthermore, we have addressed the issues of biocompatibility of the solid support and immobilization strategies for our microarray setup. an in-house-designed substrate, macroporous silicon coated with nitrocellulose (map3-nc7), displayed properties equal to, or better than, those of five commercially available supports used as reference surfaces. we have also evaluated different coupling strategies, such as adsorption, covalent coupling, diffusion and affinity coupling. using a novel affinity tag, the double-(his)6-tag, we increased the binding efficiency of sinfab-molecules to ni2 þ -coated solid supports, thereby allowing nonpurified probes to be directly applied. the mannan-binding lectin (mbl) pathway is part of the innate immune system providing a first line of defence against infections. mbl and ficolins circulate in complexes with mbl-associated serine proteases (masp-1, -2 and -3). after recognition of a microorganism by mbl, activation of the complement system occurs. masp-1 and masp-3 share five domains (making up the so-called a-chain), whereas they have unique protease domains (b-chains). before the identification of masp-3, an assay for masp was presented, based on antibodies against the a-chain of masp-1. with the new knowledge of the three masps, and the sharing of domains by masp-1 and masp-3, assays specific for the protease domains have to be constructed, if one wishes to measure the proteins individually. we present an assay for quantifying total masp-3 in plasma and serum samples. the assay is a sandwich-type assay using as catching antibody a monoclonal antibody against the common a-chain of masp-1/3 and a developing secondary antibody against the c-terminal part of the protease domain of masp-3. we have used this assay for estimating the normal concentration of the protein as well as the concentration in patients and also for characterizing by gel permeation chromatography the masp-3 protein in serum. inducible costimulator ligand (icosl) is a costimulatory molecule related to b7.1 (cd80) and b7.2 (cd86). b cells, monocytes, dendritic cells and endothelial cells express icosl. inducible costimulator (icos) interacts with icosl, and this interaction leads to signals involved in isotype switching and the development of immunological memory. hitherto, no polymorphisms of this gene have been described. the aim of this study was to reveal variation of the icosl gene in normal individuals. all eight exons, except exon 1, were sequenced with flanking introns in 10 healthy blood donors. eight single nucleotide polymorphisms (snps) and two length polymorphisms were found. one of the snps was found in the coding regions of the gene. the base involved was located in exon 3 and caused a conservative amino acid change from valine (gtt) to isoleucine (att). three individuals were heterozygous g/a for the exon polymorphism, while the remaining seven individuals were homozygous for the wildtype g/g. exon 3 encodes the immunoglobulin variable (igv)-like domain of the molecule which is situated outside the cell. this means that the amino acid could be critical for the stability of the molecule or could constitute part of the binding site for icos. the results form the basis for further experiments to find possible associations of the alleles to diseases caused by immune dysregulation. especially, the exon 3 variant is interesting and could play a role for the development of immunological diseases. besides, it would be interesting to see whether both exon 3 alleles are expressed or only the wildtype allele is functional. interferon-a/b (ifn-a/b) is increasingly used as antiviral and immunomodulatory therapies. unfortunately, bioavailability varies with ifn species and mode of administration, and all ifn species are potentially immunogenic. assays for antiviral activity (ifn) and antiviral neutralization (antibodies, nab) have been used for some time to monitor patients on ifn biologicals. these assays require laborious titrations making them unsuitable for large-scale clinical use. myxovirus a (mxa) is a resistance gtpbinding protein that is specifically induced by treatment with type 1 ifns. for example, ifn-b-induced mxa in blood leucocytes has been used as a biomarker in ifn-btreated patients with multiple sclerosis. however, the degree of specificity of mxa in this regard is unclear, and measurements of mxa protein and/or mrna are not yet suitable for routine clinical use. in an attempt to find new and better reporter genes (and, hopefully, genes and gene products with proven specificity for ifn-a and -b), microarray screenings with u133a genechips (affymetrix) were carried using human blood leucocytes and the human lung carcinoma cell line a549. we studied the simultaneous expression of 22,000 transcripts before and after exposure to human recombinant ifn-a and ifn-b and other antiviral and immunomodulatory cytokines. the results will be presented at the conference. interferon-a/b (ifn-a/b) is increasingly used as antiviral and immunomodulatory therapies. unfortunately, bioavailability varies with ifn species and mode of administration, and all ifn species are potentially immunogenic. assays for antiviral activity (ifn) and antiviral neutralization (antibodies, nab) have been used for some time to monitor patients on ifn biologicals. these assays require laborious titrations making them unsuitable for large-scale clinical use. our laboratories have therefore modified the antiviral assays for ifn bioactivity and nab, so that they are suitable for large-scale screening in specialized laboratories. the read-out is survival of a subcloned a549 cell line in the presence of an otherwise lethal amount of virus. thus, survival increases in the presence of type 1 ifn and decreases in the presence of nab against the ifn added to the cells. mxa is induced by type 1 ifn and can be used for measuring the nab activity. in another assay, the mxa level in the a549 cell line is measured. in an attempt to find a new and better reporter gene for type 1 ifn than mxa and genes specific for either ifn-a or -b, a micro array screen was carried using the u133a chip from affymetrix. the expression of 22,000 genes can be studied simultaneous with this technology. the results will be presented at the conference. in our laboratory, we have developed a database system, which we believe is of immediate interest to the general scientific community. the database represents a computerbased replacement for the laboratory notebooks used in the majority of research laboratories worldwide. in addition, the database provides an effective tool for organizing and managing laboratory information at all levels, spanning from managing and revising standard operating procedures and producing documentation of research activities to keeping track of data and conclusions. using the commercially available database toolkit software filemaker pro, we have developed a relational database solution for management of laboratory information. the system consists of a hierarchy of five interrelated databases, each pertaining to a separate type of information, namely, overall project information, information relating to individual experiment setups, documentation of daily research activity, generated data and descriptions of standard operating procedures. like other databases, each individual database consists of a number of records, each comprised of a set of fields in which information is entered. in each record, a certain field is reserved to specify the relation of the record to a record in another database at a higher level. thus, the database is essentially five databases linked by a hierarchy of one-to-many relations, organizing information in a folder-like structure. importantly, the database system allows multiple users to access and edit records simultaneously, and the data entered in one database immediately becomes accessible through the other databases. the limitations of laboratory notebooks are apparent when looking for information, which is dispersed throughout one or more notebooks, or possibly on loose sheets of paper or printouts 'somewhere'. the often complicated process of gathering laboratory data or results when writing grant applications or research papers is made considerably easier with the database system. thus, the database solution presented should be broadly attractive to researchers, irrespective of their scientific discipline. an effective sars vaccine is likely to include components that can induce specific cytotoxic t-cell (ctl) responses. the specificities of such responses are governed by hlarestricted presentation of sars-derived peptide epitopes. exact knowledge of how the immune system handles protein antigens would allow for the identification of such linear sequences directly from genomic/proteomic sequence information. the latter was recently established when a causative coronavirus (sars cov) was isolated and full-length sequenced. here, we have combined advanced bioinformatics and high-throughput immunology to perform an hla supertype, genome-wide scan for sars-specific cytotoxic t cell epitopes. the scan includes all nine human hla supertypes in total covering >99% of all major human populations. for each hla supertype, we have selected the 15 top candidates for test in biochemical-binding assays. at this time (approximately 6 months after the genome was established), we have tested the majority of the hla supertypes and identified almost 100 potential vaccine candidates. these should be further validated in sars survivors and used for vaccine formulation. we suggest that immunobioinformatics may become a fast and valuable tool in rational vaccine design. rationale: major histocompatibility complex class i (mhc i) molecules monitor the protein content of the cell by binding small derived peptides and presenting them to cytotoxic cd8 þ t cells. the goal of the human mhc project is to predict the binding strength of any given peptide/mhc complex. this prediction allows the design of peptide-based vaccines. the prediction requires representative binding data from mhc alleles from all the nine hla supertypes. here, we describe the genetic construction, protein production and purification as well as the establishment-binding assays for two recombinant mhc supertype alleles, hla-b*1501 and hla-b*5801. methods: using the quikchange multisite directed mutagenesis kit (stratagene), codon-optimized genes encoding hla-b*1501 and hla-b*5801 are created. the two mhc i molecules are fermented and purified by ion exchange chromatography, hydrophobic interaction chromatography and size exclusion chromatography. the binding (kd) of natural t-cell epitopes, as well as predicted peptide ligands, is described by radioactive immunoassays (rias) and enzyme-linked immunosorbent assays (elisas). the mhc molecules are biotinylated during expression. results: the expression of mhc i resulted in multiple disulfide bond isomers, which are separated by hydrophobic interaction chromatography and used in subsequent binding studies resulting in the determination of kd for various peptide ligands ranging from strong binders we have previously demonstrated that bioinformatics tools such as artificial neural networks (anns) are capable of performing pathogen-, genome-and hlawide predictions of peptide-hla interactions. these tools may therefore enable a fast and rational approach to epitope identification and thereby assist in the development of vaccines and immunotherapy. a crucial step in the generation of such bioinformatics tools is the selection of data representing the event in question (in casu peptide-hla interaction). this is particularly important when it is difficult and expensive to obtain data. herein, we demonstrate the importance in selecting information-rich data and we develop a computational method, query-bycommittee, which can perform a global identification of such information-rich data in an unbiased and automated manner. furthermore, we demonstrate how this method can be applied to an efficient iterative development strategy for these bioinformatics tools. methods: a large panel of binding affinities of peptides binding to hla a*0204 was measured by a radioimmunoassay (ria). this data was used to develop multiple first generation anns, which formed a virtual committee. this committee was used to screen (or 'queried') for peptides, where the anns agreed ('low-qbc'), or disagreed ('high-qbc'), on their hla-binding potential. seventeen low-qbc peptides and 17 high-qbc peptides were synthesized and tested. the high-or low-qbc data were added to the original data, and new high-or low-qbc second generation anns were developed, respectively. this procedure was repeated 40 times. the high-qbc-enriched ann performed significantly better than the low-qbc-enriched ann in 37 of the 40 tests. conclusion: these results demonstrate that high-qbcenriched networks perform better than low-qbc-enriched networks in selecting informative data for developing peptide-mhc-binding predictors. this improvement in selecting data is not due to differences in network training performance but due to the difference in information content in the high-qbc experiment and in the low-qbc experiment. finally, it should be noted that this strategy could be used in many contexts where generation of data is difficult and costly. interleukin-18 (il-18), a pro-inflammatory cytokine that is produced by both lymphoid and nonlymphoid cells, has a critical role in modulation of innate and adaptive immunity. its primary function in stimulation of ifn-g production and stimulation of nk-cell-cytotoxic activities makes this cytokine a candidate for cancer immunotherapy. in oral cavity, this cytokine is produced by oral epithelia and carcinoma cells and is related to tumour regression in nude mice bearing salivary adenocarcinoma. however, direct effects of this cytokine on oral cancer cells have not been elucidated. in this project, we investigated il-18 effect on an oral carcinoma (kb) cell line. with rt-pcr technique, kb-cell line was found to express il-18 receptors (il-18ra and il-18rb), indicating that this oral carcinoma line is a target for il-18 study. we showed that recombinant human il-18 inhibited kb-cell proliferation by 17% at concentration of 100 ng/ml (p < 0.05), whereas ldh release by these cells in treatment group and control groups was comparable, indicating that il-18 suppression of cell proliferation was not mediated by the induction of cell death. to further address this hypothesis, we found that il-18 treatment did not induce apoptotic cell death, as studied by dna laddering and tunel assays. in addition, expression pattern of cell death-controlling genes (bcl-2 and bax) was not altered by this cytokine. findings in these studies indicated that suppression of kb-cell proliferation may be attributed to control of cell cycle, growth arrest or induction of cell differentiation. the data presented in this project could provide an insight of how cancer cell directly responds to il-18, as this cytokine is an important regulator of anticancer mechanisms. aloe emodin (ae) is a naturally occurring compound with wide spectrum of biological properties, including antimicrobial, vasorelaxant, immunosuppressive and anticancer actions. this anthraquinone induces apoptosis in several tumour cell lines with special affinity to tumours of neuroectodermal origin. high amounts of nitric oxide (no) released by activated macrophages induce tumour cell death. therefore, we explored the capacity of ae to modulate no-mediated antitumour response in vitro. interestingly, while ae markedly suppressed no release from macrophages alone, it significantly potentiated no production in cocultures of macrophages and c6 cells, after 48 h of cultivation. accordingly, the viability of c6 cells cocultivated with macrophages was reduced in the presence of ae. moreover, the observed ae-imposed potentiation of no production in macrophages was closely related to macrophage culture cell density. according to these data, we proposed that no modulator capacity of ae strongly depended on intercellular contact, indicating that macrophage antitumour response was not compromised but even potentiated by ae. immunotherapy represents an attractive fourth-modality therapeutic approach, especially in the light of the shortcomings of conventional surgery, radiation and chemotherapies in the management of metastatic cancer. to this end, a large number of peptide antigens derived from taa have been applied in immunotherapeutic trials for the treatment of various malignancies, e.g. cancers of the breast, prostate and kidney, in addition to haematological cancers. in some cases the response rates have been impressive and no adverse autoimmunity have been observed. a major strategic difficulty associated with these trials relates to the choice of best-suited peptide antigens. the vast majority of the antigens described thus far is not vital for survival and growth of the tumour cells, and immunoselection of antigen-loss variants may therefore prove to be an additional obstacle for the clinical applicability of most of the known peptide epitopes. in this respect, the development of acquired antigen loss during immunotherapy has been demonstrated in several cases. obviously, the development of loss-variant tumour cells implies that these cells acquire a pronounced growth advantage and are left unaffected by further treatment. ideally, target antigens should be derived from proteins required for survival and growth of tumour cells, as antigens with these characteristics would not be inflicted by the development of loss-variant tumour cells. in this respect, several inhibitors of apoptosis proteins (iaps) are universally expressed among tumours and play an important role in tumour cell escape from apoptosis. we have characterized spontaneous t-cell reactivity against iapderived peptides in cancer patients. from the iap survivin, we have characterized peptides restricted to the class i molecules hla-a1, a2, a3, a11, b7 and b35. furthermore, we have demonstrated that survivin-specific t cells infiltrate metastatic lesions and that isolated survivinspecific ctls are capable of killing hla-matched tumour cells. survivin-derived peptides are now in clinical trial, and continued work in our lab has demonstrated that other iaps are targets for spontaneous t-cell reactivity in cancer patients. we previously reported that in mice with large progressing t-cell lymphoma tumours, dysfunctions in the antitumour ctl activity occur, associated with an accumulation of splenic arginase-producing myeloid suppressor cells (mscs). in this study, we first demonstrate that both the presence and the activation state of these msc depends on tumour evolution. while in tumour regressors hardly any arginase-producing msc can be found, both the amount and the arginase activity of this population expands from early over late progressors. this gradual induction of mscs is paralleled by an increasing suppression of ctl activity and th1, but not th2, cytokine production. upon analysing the molecular repertoire of msc in vitro, we found, besides arginase1, a well-established marker for alternatively activated myeloid cells or m2, a strong upregulation of fizz1 and ym, two additional recently identified markers for m2. further evaluation of molecular markers by microarray analysis in msc yielded genes involved in wound healing (e.g. coagulation factor xiiia), anti-inflammation (e.g. selenoprotein p), immunomodulation (e.g. pd-l2) and fat and sugar metabolism (e.g. leptin receptor). of note, many of these genes are regulated by type 2 cytokines (il-4, il-13 and il-10) and are therefore rather m2 associated. overall, our data provide new markers for msc in cancer and further establish their m2 activation state. study. only sp-a showed a significant expression in normal mucosa which was downregulated in crc. as the absolute signal level was below the noise threshold, these results have to be interpreted with caution and require confirmation by direct measurenment of the proteins. our results suggest that there is no major role for the human collectins in colorectal cancer. tetramerization is visualized by sds-page. conclusion: an effective method for the production of highly pure mhc i molecules has been applied to hla-b*1501 and hla-b*5801, and ria and elisa binding assays for those alleles have been established background: proliferation, differentiation and apoptosis are essential processes in the normal functions of the mammary epithelium. the hypothesis examined in this study is that the transcription factor bcl-6 is critically important not only for regulating b-cell growth and development but also for mammary epithelial apoptosis. methodology: twenty breast cancer cases and 31 healthy controls were used to investigate whether bcl-6 protein in involved in breast cancer (grade iii). full length bcl-6 cdna was retrovirally transduced into eph-4 cell line. we then used flow cytometry of brdurd-stained cells to investigate the cell-cycle duration of the control and transduced cell lines. tunel was used as a marker of apoptosis to find out differences in the frequencies of apoptotic cells in the control and transduced cell lines. finally, immunohistochemistry staining was performed to detect bcl-6 in breast cancer (iii). results: restoration of bcl-6 into eph-4 cells not only inhibits apoptosis but also prolongs the cell cycle and results in increased cell size and protein content. the results also indicated that the cell-cycle time of bcl-6-transduced eph-4 cells is prolonged by about 3 h, presumably as a result of the action of bcl-6 at the bcl-6 at the g1/s transition. we found differences in the frequencies of viable and apoptotic cells in cultures of the parent eph-4 cells, control-transduced eph-4 cells and bcl-6-transduced eph-4 cells. consistently, we demonstrated that bcl-6 is expressed in 90% of high grade of breast carcinoma, which is considered as the most aggressive of tumours. conclusion: together, these results suggest that bcl-6 is likely to be involved in mammary gland development and carcinogenesis. inflammatory cytokines have a critical role in modulation of both innate and adaptive immunity in response to foreign antigen. they also play an important role in anticancer immunity. for example, they can promote cell-mediated immunity against cancer cells. with their immunostimulatory effects, these cytokines are being tested for cancer treatment in the form of dna vaccine or adjuvant or therapeutic cytokines. direct effect of these cytokines on cancer cell, however, is still unclear. in this project, we investigated whether il-1( and il-18 can modulate cancer cell proliferation. we employed a simple nonradioactive proliferation (mtt) assay and detection of lactate dehydrogenase (ldh) to test the effect of these recombinant human cytokines on various cancer cell lines, including breast cancer cell line (mcf-7), oral carcinoma cell line (kb), colon cancer cell line (caco-2) and choriocarcinoma cell line (jar). cytokines used in this study had both inhibitory and stimulatory effect on cell proliferation. findings in this project could provide an insight of cancer cell response to these cytokines and this could lead to a consideration on using cytokine as immunotherapy for cancer treatment.capacity of ae to modulate nitric oxide production depended on intercellular contact donor t cells are involved in the antitumour effects observed after bmt. thus, patients receiving t-celldepleted bmt have a higher risk of leukaemia relapse compared to patients receiving nonmanipulated bmt, and patients experiencing graft-versus-host disease (gvhd) have a lower risk of disease relapse than patients who do not experience gvhd. although the importance of donor t cells for the curative action of bmt has been established, the exact mechanisms and molecules involved in this graft-versus-tumour effect remain largely unknown. in a recently initiated project, we have conducted a longitudinal study of t-cell clonotypes in patients who received peripheral blood stem cell grafts after nonmyeloablative conditioning. peripheral blood samples were obtained sequentially after transplant, and the mononuclear cells (mncs) were isolated and cryopreserved. cd8 þ t cells were isolated from the mncs by use of immunomagnetic beads or facs and analysed for the presence of clonally expanded cells by t-cell receptor clonotype mapping based on rt-pcr and denaturing gradient gel electrophoresis (dgge). using this gel-based methodology, clonally expanded t cells were monitored after transplant and compared to the clinical data of the patients. the preliminary results demonstrates the presence of clonally expanded cd8 þ t cells at all time points analysed. furthermore, a number of clonotypes persisted for more than 6 months, and other clonotypes emerged during this period. the appearance of newly emerged clonotypes which coincided with clinical gvhd could indicate a role for these t cells in the pathogenesis of gvhd. background: deficiency of the mannan-binding lectin (mbl) pathway of innate immunity leads to increased susceptibility to infections. in patients with colorectal cancer, postoperative infection is associated with poor prognosis. the aim of the present study was to evaluate (1) the relation between the mbl pathway and postoperative infectious complications and survival of patients resected for colorectal cancer and (2) the role of mbl as acute phase reactant compared to crp. methods: preoperative mbl concentration, mbl/mblassociated serine protease (masp) activity and crp were determined in serum from 611 patients and 150 healthy controls. the patients were observed for 8 years. postoperative infections, recurrence and survival were recorded. results: the mbl pathway components were increased in the patients (p < 0.0001) compared to healthy controls. low mbl levels were predictive of pneumonia (p ¼ 0.01), and pneumonia (n ¼ 87) was associated with poor survival (p ¼ 0.003, hr ¼ 1.5, 95% ci 1.1-1.9). mbl and mbl/ masp activity could not predict postoperative overall infections. mbl showed no correlation (spearman's r ¼ 0.02, 95% ci à0.06-0.10) with crp. conclusions: low preoperative mbl levels are predictive of pneumonia, which is associated with poorer survival. mbl concentration and mbl/masp activity was not predictive of other postoperative infections or long-term prognosis. mbl apparently is not a surrogate measure of crp. department of surgery, university hospital of erlangen, erlangen, germany. e-mail: michael.siassi@rzmail.uni-erlangen.de introduction: the human collectins, mannan-binding lectin (mbl), surfactant protein-a (sp-a) and surfactantprotein-d (sp-d) play a central role in the innate immune system. immunological responses to malignant transformation of epithelial cells gained increasing interest recently. a former study could demonstrate binding of mbl to certain colorectal carcinoma (crc) cell lines in vitro. we therefore examined the expression of human collectins in normal colon mucosa and in colorectal carcinomas. materials and methods: colon samples from 20 crc patients and 10 normal mucosa samples were collected immediately after surgery. the tissue was microdissected and rna isolated (qiagen, rneasy-kit). gene expression profiles were analysed using gene-chips (affymetrix, hg-u133). we analysed the data for the expression of mbl, its associated serine proteases mannan-binding lectinassociated serine protease 1/2 (masp 1/2), sp-a and sp-d. the signal intensity of the genes of interest was compared using the mann-whitney u-test. results: the expression of human collectins in normal human colon mucosa was generally low. only the expression of sp-a and masp-2 reached the noise threshold of 250 signals. these genes were significantly downregulated in crc specimens. the expression of the other proteins showed no difference in normal mucosa and crc. conclusion: as demonstrated before, the expression of human collectins in normal colon was low in this being the first lymph node to receive drainage from the tumour area, the sentinel node offers a unique possibility to obtain tumour-reactive lymphocytes. we investigated antitumour immune responses in sentinel nodes from patients with bladder cancer, by assaying tumour-specific proliferation and tcr vb repertoires. during tumour surgery, sentinel lymph nodes were identified by peritumoural injection of blue dye. fresh specimens of tumour, sentinel and nonsentinel lymph nodes were obtained, and single-cell suspensions were prepared. cells were assayed for reactivity against autologous tumour extract in [ 3 h]-thymidine incorporation assays and characterized by flow cytometry. parallel analyses of the expression of vb gene families were performed with padlock probes, linear oligonucleotides which upon target recognition can be converted to circular molecules by a ligase. probes were reacted with cdna prepared from magnetically separated cd4 þ cells, and the tcr repertoire was determined by hybridizing the products to oligonucleotide microarrays. dose-dependent proliferation in response to tumour extract could be detected in sentinel lymph nodes. common clonal expansions were detected among tumourinfiltrating lymphocytes and in sentinel lymph nodes. nonsentinel lymph nodes displayed a divergent tcr vb repertoire. these results indicate an ongoing immune response against tumour antigens in sentinel nodes, draining urinary bladder cancer. identification of sentinel lymph nodes makes it possible to obtain tumour-reactive lymphocytes for use in adoptive immunotherapy. key: cord-023393-8nye3nc8 authors: krarup, a.; sørensen, u.; matsushita, m.; jensenius, j. c.; thiel, s. title: mannan‐binding lectin, l‐ficolin and h‐ficolin selectively binds to different bacteria date: 2008-06-28 journal: scand j immunol doi: 10.1111/j.0300-9475.2004.01423al.x sha: doc_id: 23393 cord_uid: 8nye3nc8 mannan‐binding lectin (mbl), l‐ficolin and h‐ficolin are pattern recognition molecules of the innate immune system. we investigated the ability of these molecules to bind to different serotypes and noncapsulated variants of streptococcus pneumonia and staphylococcus aureus. we found that mbl binds to noncapsulated s. aureus strain (wood) but not any of the examined s. pneumoniae serotypes. l‐ficolin binds to some capsulated s. pneumoniae serotypes (11a, 11d and 11f) as well as some capsulated s. aureus serotypes (type‐1, ‐8, ‐9, ‐11 and ‐12). h‐ficolin does not bind to any of the examined s. pneumoniae and s. aureus serotypes included in this study but did bind to a strain of aerococcus viridans. when bound to bacteria, mbl and h‐ficolin initiated activation of complement factor c4, whereas l‐ficolin did not. during this study, quantitative assays for the three proteins were developed and the concentration in 97 plasma samples were determined and the median values were estimated at 0.8 μg of mbl/ml, 3.3 μg of l‐ficolin/ml and 18.4 μg of h‐ficolin/ml, respectively. the nuclear receptor heterodimers of liver x receptors (lxrs) are recently identified as key transcriptional regulators of genes involved in lipid homeostasis and inflammation. lxrs and their ligands are negative regulators of macrophage inflammatory gene expression. multiple sclerosis (ms), a demyelinating disease of the central nervous system of unknown cause, is characterized by recurrent inflammation involving macrophages and their inflammatory mediators. sweden belongs to the countries with a high ms incidence. in italy, incidence is lower, with an exception for sardinia where the incidence is even higher than that in sweden. subjects from sardinia are ethnically more homogeneous and differ from swedes, also regarding genetic background and environment. we studied lxrs and their related molecules of blood mononuclear cells (mncs) from female patients with untreated relapsing-remitting ms from sassari, sardinia and stockholm, sweden. sex-and age-matched healthy controls (hcs) were from both areas. mrna expression was evaluated by real-time pcr. lxr-a was lower (p < 0.05) in ms (mean ae sem: 3.1 ae 0.2; n ¼ 37) compared to hc (3.6 ae 0.1; n ¼ 37). lxr-a was lower in ms from stockholm (2.6 ae 0.2; n ¼ 22) compared to corresponding hc (3.4 ae 0.1; n ¼ 22; p < 0.01) and compared to ms (3.8 ae 0.2; n ¼ 15; p < 0.001) and hc (4 ae 0.2; n ¼ 15; p < 0.001) from sardinia. ms patients from stockholm, but not from sassari, also expressed lower (p < 0.05) lxr-b (à4.1 ae 0.4) compared to corresponding hc (à2.9 ae 0.3). ms from stockholm was associated with higher abca-1 (6.1 ae 0.4 versus 5.0 ae 0.3; p < 0.05) and higher estrogen receptor-b-cx (2.4 ae 0.4 versus 0.8 ae 0.4; p < 0.01) compared to corresponding hc. the hc from sassari had higher androgen receptor (2.9 ae 0.2) compared to ms from sassari (1.4 ae 0.3; p < 0.01), ms (1.3 ae 0.4; p < 0.01) and hc from stockholm (1.2 ae 0.3; p < 0.01). ms from sassari had lower cyclooxygenase-1 compared to corresponding hc (5.1 ae 0.4 versus 6.6 ae 0.3; p < 0.01) and lower prostaglandin-e (à0.03 ae 0.5) compared to the hc (1.4 ae 0.5; p < 0.05) and ms (2.7 ae 0.4; p < 0.05) and hc from stockholm (1.9 ae 0.4, p < 0.001). our findings identify lxrs and their related molecules as being involved in ms from stockholm but not from sassari, while sex hormone receptors seem to be involved in ms in sassari. multiple sclerosis: ifn-b induces cd123 + bdca2 -dendritic cells that produce il-6 and il-10 and have no enhanced type i interferon production y. m. huang, 1 s. adikari, 1 u. båve, 2 a. sanna 1,3 & g. alm 4 dc antigens (bdca) and investigate their ability to produce type i ifn in response to virus stimulation. we show that ifn-b induces development of cd123 þ dc from human blood monocytes, which coexpress bdca4 þ but are negative for bdca2 -, a specific marker for plasmacytoid dc. such ifn-b-modulated dc produce large amounts of il-6 and il-10, but no il-12p40 and have no enhanced ifn-b and ifn-b production. the findings indicate that ifn-bmodulated dc represent a myeloid dc subset with diminished cd11c, bdca-1 and cd1a expression, having potent th2-promoting function but lacking antiviral capacity. the association of psoriasis with throat infections by streptococcus pyogenes suggests a potential antigenic target for the t cells that are known to infiltrate dermis and epidermis of psoriatic skin. streptococcal m protein shares an extensive sequence homology with human epidermal keratins. keratins 16 (k16) and 17 (k17) are mostly absent from uninvolved skin but are upregulated in psoriatic lesions. there is increasing evidence that cd8 þ t cells play an important effector role in psoriasis and m proteinprimed t cells may recognize these shared epitopes in skin via molecular mimicry. to identify candidate epitopes, peptides with sequences from k17 were selected on the basis of predicted binding to hla-cw6 and sequence similarities with m6 protein. matched peptides from the sequence of m6 protein and a set of peptides with poor predicted binding were also selected. cw6 þ individuals with psoriasis and cw6 þ healthy controls, having a family history of psoriasis, were recruited. pbmcs were incubated with the peptide antigens. t-cell activation in the cd4 þ , cd8 þ and later the skin-homing cutaneous lymphocyteassociated antigen (cla)-expressing subset of cd8 þ t cells was evaluated by cd69 expression and intracellular ifn-g accumulation using flow cytometry. we demonstrate that cw6 þ psoriasis patients had significant cd8 þ t-cell ifn-g responses to peptides from k17 and m6 protein selected on the basis of sequence homology and predicted hla-cw*0602 binding. these responses were about 10 times more frequent in the skin-homing cutaneous lymphocyte-associated antigen-expressing (cla þ ) subset of cd8 þ t cells. cd4 þ t cells showed only borderline responses. cd8 þ t cells from cw6 þ nonpsoriatic individuals responded to some m6 peptides but very rarely to k17 peptides, and this also applied to the cla þ cd8 þ subset. these findings indicate that psoriatic individuals have cd8 þ t cells that recognize keratin self-antigens and that epitopes shared by streptococcal m protein and human keratin may be targets for the cd8 þ t cells that infiltrate psoriatic skin lesions. autoantibodies directed against citrulline-containing proteins have an impressive specificity of nearly 100% in ra patients and a suggestive involvement in the pathogenesis. the targeted epitopes are generated by a post-translational modification catalysed by the calcium-dependent enzyme peptidyl arginine deaminase that converts the positively charged arginine to polar but uncharged citrullin. the aim of this study was to analyse the presence of citrulline in the joints at different time points of collagen-induced arthritis in da rats by immunohistochemistry and to investigate how immunogenicity and arthritogenicity was affected by citrullination of rat serum albumin (rsa) and collagen type ii (cii). our results indicate that citrulline could be detected in joints of arthritic animals, first appearance at the onset of disease and increasing as disease progressed into a chronic state. unimmunized animals or time points before clinical signs of arthritis were negative. by morphology, we state that some infiltrating macrophages as well as the cartilage surface stain positive for citrulline, while the major source of citrullinated proteins appears to be fibrin depositions. a specific cit-rsa t-cell response was observed in animals challenged by citrullinated rsa, no response was recorded when rsa was used as a stimulus. the igg analysis reveals not only a response towards the modified protein but also cross-reactivity to native rsa. no t-cell or b-cell response was noted in animals injected with unmodified rsa. cit-cii induced a disease with higher incidence and earlier onset than did the native counterpart. we conclude that, in contrast to the human disease, citrulline does not seem to appear before clinical signs. as inflammation proceeds, citrulline is detected specifically in the joints. all other organs investigated were negative. we also conclude that citrullination of a protein can break tolerance and increase its arthritogenic properties. ectopic germinal centers (gcs) can be detected in the salivary glands of approximately 1/5 of patients with sjögren's syndrome (ss) and appear in both primary and secondary ss. previously, ectopic gc have been associated with increased local autoantibody production. the aim of this study was to determine whether gc in primary sjögren's syndrome (pss) defines a distinct seroimmunological phenotype. retrospectively, a material of 130 haematoxylin and eosin-stained paraffin-embedded tissue sections of minor salivary gland tissue from patients with pss was morphologically screened for the presence of ectopic gc. gc-like lesions were detected in 33/130 (25%) of the pss patients. seventy-two pss patients lacking these structures (gc-) were randomly selected for comparison. focus score was significantly increased in the gc þ patients compared to the gcpatients (p ¼ 0.035). in the gc þ group, 54.5% of the patients presented with anti-ro/ssa compared to 43.7% in the gcgroup. anti-la/ssb was detected in 31.3% of the gc þ patients compared to 25.7% of the gcpatients. sixty-one percentage of gc þ patients presented with increased levels of igg, a nonsignificant difference when compared to 39.4% in the gcpatients (p ¼ 0.089). levels of rf, ana, ena, igm and iga were similar in both patient groups, as were esr and crp. in conclusion, patients with ectopic gc have a higher focus score and more often present with autoantibodies and increased levels of igg compared to pss patients with regular focal infiltration (gc -). our findings may indicate a certain seroimmunological phenotype and warrant for further prospective studies. association between mannose-binding lectin and vascular complications in type 1 diabetes complement activation and inflammation have been suggested in the pathogenesis of diabetic vascular lesions. we investigated serum mannose-binding lectin (mbl) levels and polymorphisms in the mbl gene in type 1 diabetic (t1dm) patients with and without diabetic nephropathy and associated macrovascular complications. polymorphisms in the mbl gene and serum mbl levels were determined in 199 t1dm patients with overt nephropathy and 192 t1dm patients with persistent normoalbuminuria matched for age, sex and duration of diabetes as well as in 100 healthy control subjects. the frequencies of high and low expression mbl genotypes were similar in patients with t1dm and healthy controls. high mbl genotypes were significantly more frequent in diabetic patients with nephropathy than in the normoalbuminuric group, and the risk of having nephropathy, given a high mbl genotype, assessed by odds ratio was 1.52 (1.02-2.27), p ¼ 0.04. median serum mbl concentrations were significantly higher in patients with nephropathy than in patients with normoalbuminuria [2306 mg/l (iqr 753-4867 mg/l) versus 1491 mg/l (iqr 577-2944), p ¼ 0.0003], and even when comparing patients with identical genotypes, serum mbl levels were higher in the nephropathy group than in the normoalbuminuric group. patients with a history of cardiovascular disease had significantly elevated mbl levels independently of nephropathy status [3178 mg/l (iqr 636-5231 mg/l) versus 1741 mg/l (iqr 656-3149 mg/l), p ¼ 0.02]. the differences in mbl levels between patients with and without vascular complications were driven primarily by pronounced differences among carriers of high mbl genotypes (p < 0.0001). our findings suggest that mbl may be involved in the pathogenesis of microvascular and macrovascular complications in type 1 diabetes and that determination of mbl status might be used to identify patients at increased risk of developing these complications. neuroimmunology unit, center for molecular medicine, karolinska institutet, stockholm, sweden. e-mail: judit.wefer@cmm.ki.se dna vaccine coding for the encephalitogenic peptide mog 91-108 protects lew.1av1 from subsequent development of experimental autoimmune encephalomyelitis (eae). protection is associated with a type 1 immune response and is dependent on the presence of cpg dna motifs. the mechanisms underlying the observed reduction of eae development in protected rats have not been fully clarified. we investigated immunological characteristics of lymphocytes after dna vaccinaton and subsequent eae induction. we confirm that protection was not associated with suppression of t1 cells, as transcription of the novel molecule rat t-cell immunoglobulin-and mucindomain-containing molecule (tim-3), reported to be exclusively expressed on differentiated t1 cells, was not altered by dna vaccination. we did not note any clonal deletion upon tolerization, but detected an antigen-specific lymphocyte population upregulating ifng upon recall stimulation 3 weeks after protective dna vaccination. in protected rats, we observed (1) no alterations in antigenspecific th2 or th3 responses, (2) reduced mhc ii expression on splenocytes early after eae induction, (3) antigen-specific upregulation of ifnb upon recall stimulation and (4) reduced il-12rb2 on lymphocytes. we thus demonstrate an association of the protective effect of dna vaccination with expression of ifnb. we are currently investigating the cellular mechanisms behind this ifnb-mediated protection. multiple sclerosis (ms) is an autoimmune condition characterized by degeneration of nerve fibre myelin sheets. a candidate autoantigen, myelin basic protein (mbp), has especially attracted attention. the presence of anti-mbp antibodies is a predictor of definite ms, but their role in the pathogenesis remains obscure. t cells have long been known to play a pivotal role in the pathogenesis of ms. recently, an important role for b cells as autoantigen-presenting cells has been demonstrated in other autoimmune diseases, including rheumatoid arthritis and diabetes. the uptake of mbp by b cells and the presentation of mbp-derive peptides to t helper (th) cells by b cells may be promoted by the formation of complement (c) activating immune complexes (ics) between mbp and natural autoantibodies in healthy individuals and disease-associated anti-mbp antibodies in ms patients, respectively. we have investigated the formation of mbp-containing ic, the binding of mbp to b cells, the mbp-elicited induction of th-cell and b-cell proliferation and the cytokine production in peripheral blood mononuclear cells (pbmcs) from healthy donors grown in the presence of intact or c-inactivated serum from healthy donors or patients with ms. while mbp did not induce measurable proliferation of b cells nor cd4 þ t cells, we observed the production of tnf-a, ifn-g and il-10 by pbmc in response to incubation with mbp in the presence of sera from healthy controls as well as sera from ms patients. by contrast, no production of il-2, il-4 and il-5 was detected. we are currently investigating the capability of ms sera to promote the formation of mbp-containing ic and thereby enhance the cytokine responses, by virtue of elevated anti-mbp contents. the phagolysosomally localized acid sphingomyelinase (asmase) activated by proinflammatory cytokines such as tnf and ifn-g generates the signalling molecule ceramide which in turn results in the activation of proteases like cathepsin d. these characteristics of asmase suggest a possible role of this molecule in the phagocytotic uptake and phagosomal degradation processes of antigens or in antigen presentation. we show here that asmase -/mice fail to eliminate the noncytopathic lymphocytic choriomeningitis (lcm) virus as rapidly as littermate wildtype mice. investigation of the immune response revealed a reduced expansion of cd8 þ t cells. the secretion of ifn-g in response to contact with target cells as well as the cytolytic activity of virus-specific cd8 þ t cells was severely impaired. additionally, both phases of the lcm virus-specific dth response, mediated by cd8 þ and cd4 þ t cells consecutively, were diminished in asmase -/mice. however, the secondary memory response of virus-specific ctl was not altered, and the 614 abstracts ................................................................................................................................................................................................. virus was effectively controlled for at least 3 months by asmase -/mice. in conclusion, the results of this study suggest an involvement of the asmase in the activation, expansion or maturation of virus-specific cd8 þ t cells during the acute infection of mice with the lcm virus. novel markers for alternative activation of macrophages: macrophage galactose-type c-type lectins 1 and 2 in parallel with the th1/th2 dichotomy, macrophages are capable of developing into functionally and molecularly distinct subpopulations, due to differences in, for example cytokine environment and pathological conditions. while the best-studied, classically activated macrophage is induced by type i stimuli such as ifn-g, a type ii cytokine environment antagonizes the classical activation of macrophages and is capable of alternatively activating macrophages. however, molecular markers associated with these type ii cytokine-dependent, alternatively activated macrophages remain scarce. besides the earlier documented markers macrophage mannose receptor and arginase 1, we recently demonstrated that murine alternatively activated macrophages are characterized by increased expression of fizz1 and ym. we now report that expression of the two members of the mouse macrophage galactose-type c-type lectin gene family, termed mmgl1 and mmgl2, is induced in diverse populations of alternatively activated macrophages, including peritoneal macrophages elicited during infection with the protozoan trypanosoma brucei or the helminth taenia crassiceps, and alveolar macrophages elicited in a mouse model of allergic asthma. we also demonstrate that, in vitro, interleukin-4 and interleukin-13 upregulate mmgl1 and mmgl2 expression and that, in vivo, induction of mmgl1 and mmgl2 is dependent on interleukin-4 receptor signalling. moreover, we show that regulation of mgl expression is similar in human monocytes and monocyte-derived macrophages. hence, macrophage galactose-type c-type lectins represent novel markers for both murine and human alternatively activated macrophages; thus, paving the way for further characterization of the phenotype of macrophages occurring in th2 conditions. background: human parvovirus b19 (b19) is a ubiquitous pathogen, normally causing a mild self-limiting disease, but also capable of causing both significant pathology and long-term persistence. the small size and stability of the virus makes it suitable for mapping of the full breath and the kinetics of the cellular immune responses following acute viral infection. methods: five patients with acute primary b19 infection were included in the study and followed consecutively for up to 200 weeks. cellular immune responses were mapped by ifng enzyme-linked immunospot to overlapping peptides spanning the whole b19 genome. results: in all five acutely infected patients, we were able to monitor the kinetics of a strong specific cellular immune reaction. responses peaked at levels of 850-1850 sfc/ million pbmcs, roughly corresponding to 0.3-0.6% b19specific cd8 þ cells circulating in peripheral blood at 10-80 weeks post-infection. the responses in individual patients were directed to three or four different peptide pools, and the specificity was confined to the same cd8 epitopes present in the pools throughout the follow-up period. the majority of responses were directed to the virus nonstructural protein, only two patients showed any response to the capsid proteins, elicited by the same epitope in both cases. conclusion: the cellular immune responses to acute b19 infection are surprisingly narrow in distribution and remain at high levels for up to 80 weeks post-infection. the initial epitope specificity is maintained, and the majority of responses target the virus nonstructural protein, which is not included in vaccine preparations, evaluated against the infection. the relationship between malnutrition and malaria is controversial. on one hand, malaria may cause malnutrition, while on the other, malnutrition itself may modulate susceptibility to the disease. we investigated the association between plasmodium falciparum malaria and malnutrition in a cohort of children living on the coast of kenya. the study involved longitudinal follow-up for clinical malaria episodes and anthropometric measurements at four cross-sectional surveys. we used poisson regression analysis to investigate the association between malaria and nutritional status. compared to baseline (children with a waz or haz score of !à2), the crude incidence rate ratios (irrs) for malaria in children with low haz or waz scores (<à2) during the period prior to assessment were 1.17 (95% ci 0.91-1.50; 0 ¼ 0.21) and 0.94 (0.71-1.25; 0.67), respectively, suggesting no association between malaria and the subsequent development of pem. however, we found that age was acting as an effect modifier in the association between malaria and malnutrition. the irr for malaria in children 0-2 years old who were subsequently characterized as wasted was 1.65 (1.10-2.20; p ¼ 0.01), and a significant overall relationship between malaria and low-haz was found on regression analysis when adjusting for the interaction with age (irr 1.89; 1.01-3.53; p < 0.05). although children living on the coast of kenya continue to suffer clinical episodes of uncomplicated malaria throughout their first decade, the association between malaria and malnutrition appears to be limited to the first 2 years of life. a. astrinidou-vakaloudi, 1 s. xytsas, 1 i. diamanti, 1 h. ioannidis 2 & p. pangidis 2 1 microbiology department of general hospital of thessaloniki 'agios pavlos', thessaloniki, greece, and 2 nefrology, 2 nd ika hospital of thessaloniki, thessaloniki, greece. e-mail: stasa@hol.gr aim: renal dysfunction may influence the colonization of gastric mucosa by urea-splitting bacteria such as helicobacter pylori, by increasing urea concentrations in the gastric juice. our aim was to investigate the prevalence of h. pylori in patients with end-stage renal disease (esrd), receiving long-term haemodialysis treatment. methods: this study included 40 sera from patients with esrd (29 male and 11 female) undergoing periodic haemodialysis; mean time of treatment was 42.6 months. using elisa technique, we investigated the presence of igg and iga antibodies against h. pylori as well as igg caga (antibodies specific for caga(þ) strains of h. pylori). sera from 40 healthy blood donors were used as a control group. results: h. pylori igg antibodies were detected in 32 out of 40 (80%) patients in the dialysis group, while 31/40 (77.5%) tested positive for iga. igg caga antibodies were present in 13 out of 40 (32.5%). prevalence of h. pylori igg, iga and caga igg antibodies in the control group was 33, 7 and 15%, respectively. conclusions: although international data suggest that prevalence of h. pylori infection is the same in esrd patients as in healthy individuals, in our study that seems not to be the case. the higher blood and gastric juice urea levels may be a risk factor (among many others), but more studies are required in order to understand the relation of h. pylori infection in this group of patients. flanders interuniversity institute for biotechnology, department of molecular and cellular interactions, free university of brussels, brussels, and 2 pasteur institute of brussels, mycobacterial immunology, brussels, belgium. e-mail: tgartner@vub.ac.be immunity against tuberculosis (tb), caused by mycobacterium tuberculosis, depends largely on activation and maintenance of strong cell-mediated immune responses involving both cd4 þ and cd8 þ t cells and the ability to respond with th1-type cytokines, particularly ifn-g. recent studies suggested that bcg, the only licensed vaccine against m. tuberculosis, may fail to induce t-cell responses in the lung mucosa and may therefore not protect against pulmonary tb. a decrease in tb mortality may be achieved by enhancing immunity in the lung. the present study evaluated the induction of antigen-specific immunity in the lung by intranasal (i.n.) delivery of the lipoprotein i (opri) from pseudomonas aeruginosa. opri has shown to be a toll-like receptor 2/4 agonist that, when given subcutaneously, induces type-1 immune responses against heterologous antigens. here, a fusion of opri to ag85a of mtb (opri-ag85a) was used as a subunit vaccine in homologous prime-boost immunizations. in addition, opri-ag85a was combined with an ag85a-encoding dna vaccine (ag85a dna) or with bcg in heterologous prime-boost vaccinations. intranasal and parenteral delivery with opri-ag85a elicited comparable t-cell responses in the spleen; in addition, i.n. delivery elicited specific t-cell responses in the lung lymph nodes (llns). intramuscular delivery of ag85a dna induced significant systemic th1 immune responses. intranasal boosting with opri-ag85a enhanced this response and in addition induced an antigen-specific ifn-g response in lln. opri may therefore be an efficient adjuvant for mucosal boosting. we continue to evaluate the protection induced by opri-based prime-boost vaccinations against pulmonary tb. results on the immunogenicity and protection against intravenous mtb h37rv infection will be presented. toll-like receptors (tlrs) are pattern recognition receptors of the innate immune system, which recognize molecular structures on pathogens or cellular stress-associated molecules. tlr-ligand interactions trigger activation of inflammatory signal transduction and expression of genes involved in host defense. in this study, we have examined the requirement for different tlr adaptor molecules in virus-induced chemokine expression and are currently trying to identify the tlr involved. we have found that both a herpesvirus [herpes simplex virus (hsv)] and a paramyxovirus (sendai virus) require a functional genome to induce expression or proinflammatory chemokines in human and murine monocytic cell lines. for both viruses, this is independent of the tlr adaptor molecules trif and mal. however, overexpression of the vaccinia virus-encoded inhibitor of tlr-signalling a52r or dominant-negative myd88 totally inhibited hsv-induced rantes expression but only partially prevented sendai virus from inducing this chemokine. this suggests that hsv-induced rantes expression occurs via a tlr pathways, whereas sendai virus utilizes both tlr-dependent and -independent pathways to stimulate expression of rantes. we are currently trying to identify the tlrs involved. data from these studies will also be presented at the meeting. 2 0 -5 0 -oligoadenylate synthetases are interferon-induced, double-stranded rna-activated antiviral enzymes which are the only proteins known to catalyse 2 0 -specific nucleotidyl transfer. this first crystal structure of a 2 0 -5 0oligoadenylate synthetase reveals a structural conservation with the 3 0 -specific poly(a) polymerase that, coupled with structure-guided mutagenesis, supports a conserved catalytic mechanism for the 2 0 -and 3 0 -specific nucleotidyl transferases. comparison with structures of other superfamily members indicates that the donor substrates are bound by conserved active site features while the acceptor substrates are oriented by nonconserved regions. the 2 0 -5 0oligoadenylate synthetases are activated by viral doublestranded rna in infected cells and initiate a cellular response by synthesizing 2 0 -5 0 -oligoadenylates, that in turn activate rnase l. this crystal structure suggests that activation involves a domain-domain shift and identifies a putative dsrna activation site that is probed by mutagenesis. we demonstrated that this site is required both for the binding of dsrna and for the subsequent activation of oas. this rna-binding site is different from known rna-binding site; rather than forming a defined three-dimensional domain, it is located at the interface of the two major domains in oas. this novel architecture ensures that the dsrna helix can make simultaneously contact with both domains of oas and ensure the subsequent structural rearrangement leading to the activation of oas. our work provides structural insight into cellular recognition of double-stranded rna of viral origin and identifies a novel rna-binding motif. bacteria-specific iga antibodies are efficient opsonins for neutrophils and mononuclear phagocytes, provided that the phagocytes express the fca receptor (cd89). expression of cd89 can be stimulated by inflammatory cytokines, activated complement factors and certain microbial components. in one study, unstimulated phagocytes were able to ingest iga antibody-treated pneumococci, but only in the presence of complement, which was found to be activated by the iga antibodies along the alternative pathway. pneumococci produce iga1 protease that cleaves human iga1, but not iga2, molecules in the hinge region. this leaves iga1 as faba (monovalent) deprived of fca which contains the docking site for cd89. iga1 is the vastly predominant subclass of iga in the upper airways and circulation of humans. aims: to examine the effects of iga1 protease activity and complement on phagocytosis of iga antibody-coated pneumococci by an unstimulated human phagocytic cell line (hl60). materials and methods: iga1 and iga2 monoclonal antibodies to serotype 4 pneumococcal capsular polysaccharide (ps) were generated by heterohybridoma technique involving b cells from human vaccinees. isogenic serotype 4 pneumococci with and without iga1 protease activity, respectively, were obtained after inactivation of the iga gene of the tigr4 strain. opsonophagocytosis was quantitated using the assay described by romero-steiner et al. based on enumeration of surviving bacteria by culture. the integrity of iga molecules was examined by western blotting. results: both iga1 and iga2 antibody to type-4 polysaccharide-induced phagocytosis of iga1 protease-deficient type-4 pneumococci equally well in the absence as in the presence of complement. iga1 antibody to type-4 polysaccharide displayed a fourfold higher opsonophagocytosis titer against iga1 protease deficient compared to homologous wildtype target bacteria. a similar effect of iga1 protease activity of the target bacteria was not observed in a parallel experiment where iga2 antibody to type-4 polysaccharide served as opsonin. iga1 antibody extracted from iga1 protease-producing target bacteria was almost exclusively in the form of faba. conversely, iga1 from protease-deficient bacteria and iga2 from both types of bacteria were intact. conclusions: these results indicate that the iga1 protease activity of s. neumoniae may help the bacteria escape iga1 antibody-mediated opsonophagocytosis. besides, in these experiments, iga-mediated opsonophagocytosis was independent of complement. vitamins e and c have been found to increase the cellular and humeral immunity of pigs. vitamin e deficiency has also been found to predispose pigs to different diseases, e. coli infection is one among them. after weaning, the vitamin e status of pigs often decreases to a critical low level. in this experiment, we studied whether vitamin c supplementation would be a possible feeding strategy to optimize the immune status of weaners. the interaction between vitamin e and c is interesting due to the reported sparing action on vitamin e or synergism between these to vitamins. piglets were weaned at day 28 of age from sows fed increasing dietary vitamin e during lactation, and piglets were during the following 3 weeks fed either a control diet or this diet supplemented with 500 mg stay-c per kg. blood sampling was obtained weekly from day 28 and until day 49 of age. on the same days, one piglet per dietary treatment was killed and alveolar macrophages (am) were harvested. vitamin c supplementation increased the concentration of igm in serum of piglets throughout the weaning period. although the vitamin e concentration in am decreased with increasing age of the piglets, the concentration was numerically higher in piglets of sows fed the high dietary level of vitamin e. however, vitamin c supplementation tended to increase the total am concentration of vitamin e after weaning and increased the proportion of the biologically most active isomer of vitamin e [rrr-(a-tocopherol)] in the am. the eicosanoid synthesis by am was not influenced by the vitamin c supplementation, but the synthesis of leukotriene b4 was decreased 2 weeks after weaning compared to other days of am harvesting. in conclusion, dietary vitamin c supplementation improved the immune responses of piglets after weaning. a whole blood stimulation assay with escherichia coli (o111:b4) endotoxin was established to measure the capacity of dairy cows to produce the proinflammatory cytokine tumour necrosis factor-a (tnf-a) ex vivo. initially, a time-and dose-dependent study was carried out to find the optimal stimulation conditions for the tnf-a response. the tnf-a response peaked between 3 and 4 h at 38.5 c. a dose in the range of 5-10 g of e. coli lipopolysaccharide (lps)/ml whole blood was found to give the maximum tnf-a response. thirty-eight danish-holstein dairy cows were investigated for their tnf-a responsiveness ex vivo in the periparturient period. heparin-stabilized blood samples were collected seven times over a period of 4 months (weeks à3, à1, 2, 3, 5, 9 and 13 around calving) and stimulated with 5 g/ml of e. coli lps. indeed, fluctuations in the tnf-a responsiveness occurred over time. moreover, the mean tnf-a responsiveness of 38 cows was found to be significantly increased (p < 0.001) in the weeks close to calving. however, in the more stabile physiological periods, some cows had a consistently low tnf-a response, whereas others had high a tnf-a response. we are currently investigating whether high and low tnf-a responders to e. coli lps also exist in dairy cows in vivo. moreover, the importance of tnf-a responsiveness ex vivo to dairy cows' susceptibility and clinical response to experimental e. coli infections in the udder is being investigated. coelomic cytolytic factor (ccf) is a 42 kda invertebrate pattern recognition molecule isolated from the coelomic fluid of the earthworm eisenia foetida (oligochaeta, annelida). ccf displays a number of similarities with the mammalian cytokine tumour necrosis factor-a (tnfa) as a result of a shared n,n 0 -diacetylchitobiose lectin-like domain. however, these similarities are solely functional and are not based on any (dna or amino acid) sequence homology, thus suggesting a form of convergent evolution. in particular, the lectin-like domain of tnf-a has been shown to induce membrane depolarization in various mammalian cell types, through interactions with endogenous amiloride-sensitive ion channels. this nonreceptor-mediated activity of tnf-a has been reported to be involved in the resorption of oedema. likewise, the lectin-like domain of ccf also induces membrane depolarization in mammalian cells. here, we show that ccf appears to be able to induce oedema resorption in an alveolar epithelial cell line through its lectin-like domain. this lectin-like domain of ccf interacts (directly or indirectly) with endogenous sodium and/or chloride channels, and not potassium channels, on mammalian cells. additionally, we suggest that the jnk/sapk and erk1/2 pathways are involved in ccf-induced macrophage activation. these results further establish the functional analogy between an invertebrate pattern recognition molecule and a mammalian cytokine and, from a more applied point of view, suggest the possibility of utilizing ccf in the treatment of oedema. release of svegf and svegfr1 from white blood cells and platelets during surgery and stimulation with bacterial antigens introduction: the influence of surgery on release of soluble vascular endothelial growth factor (svegf) and the soluble vascular endothelial growth factor inhibitory receptor 1 (svegfr1) is unknown. we studied the effect of major and minor surgery on potential variations in svegf and svegfr1 concentrations in vivo and on bacterial antigen-induced release of svegf and svegfr1 from whole blood in vitro. methods: sixty-one patients with abdominal diseases undergoing five different surgical procedures were included. blood samples were drawn from anaesthetized patients before and after the operation. white blood cells and platelets were counted, and plasma svegf and svegfr1 was determined by an elisa method. whole blood from each blood sample was stimulated in vitro with bacteria-derived antigens (lps or protein-a) and svegf and svegfr1 levels were subsequently determined in the supernatants. stimulation with isotonic saline served as control assay. neither svegf or svegfr1 in plasma changed during surgery. in vitro stimulation of blood samples with bacteria-derived antigens resulted in a significant increase in svegf (p < 0.0001) and a less pronounced but still significant increase in svegfr1. release of svegf due to stimulation was significantly higher after the operation (nonsignificant), whereas svegfr1 release remained largely unchanged after surgery. correlation between bacterial antigen-induced release of svegf and neutrophile cell count was highly significant (p < 0.0001). there was no correlation between svegf and platelet cell count, and bacterial antigen-induced svegfr1 release did not correlate with counts of neutrophils and platelets. conclusions: plasma svegf and svegfr1 concentrations did not change during surgery. in vitro bacterial stimulation led to increased release of svegf and svegfr1, which was not significantly amplified during surgery and which may be related to number of circulating neutrophils. natural killer cell functions and subsets after in vitro stimulation with il-2 and il-12, with special emphasis on intracellular ifn-g and nk-cell cytotoxicity r. nyboe, 1,2 t. rix, 1,2 j. krog, 1,2 e. tønnesen 1 & m. hokland 2 1 department of anaesthesiology and intensive care, aarhus university hospital, and 2 institute of medical microbiology, and immunology, university of aarhus, aarhus, denmark. e-mail: rnsr@studmed.au.dk materials and methods: isolated cryopreserved human peripheral blood mononuclear cells (pbmcs) were stimulated with il-2 and il-12. this stimulation has previously been shown to activate nk cells. cell cytotoxicity was measured by flow cytometry after incubation with k562 cells. this method was compared to the current standard 51cr release assay. cells were treated with bfa to accumulate ifn-g, stained for surface markers, permeabilized and stained for intracellular ifn-g. flow cytometry was then performed to measure intracellular ifn-g production in pbmc, especially in nk cells. results: we have demonstrated that stimulation with il-2 and il-12 is effective in increasing the number of ifn-gpositive cells. there is a distinct difference between the cd3-cd56dim and the cd3-cd56bright subsets, with a much greater proportion of ifn-g-positive cells in the cd3-cd56bright subset. the effects of stimulation with il-2 and il-12 on cytotoxicity will be presented, as will the relation between ifn-g production and cytotoxicity. in addition, we will present results of these assays applied to porcine cells. discussion: in combination, these tests will address nk cell function by combining cytotoxicity with ifn-g production in nk cell subsets. the results will demonstrate whether this could serve as a useful tool in describing nkcell function, which could be of value in clinical and experimental settings. culture of regulatory t-cell lines from bronchial mucosa t lymphocytes play a major role in many immune responses. in the last decade, special focus has been on the function of th1 and th2 effector cells. now the importance of regulatory cd4 þ cd25 þ t cells in maintenance of the immunological homeostasis emerges. sarcoidosis is a multisystem granulomatous disorder often affecting the lungs. the typical sarcoid granulomas consists of epitheloid cells, macrophages and lymphocytes, mainly cd4 þ t cells of th1 phenotype. we have cultured t cells from bronchial biopsies of patients with sarcoidosis as well as from controls in high levels of interleukin 2 (il-2) and il-4 and demonstrate spontaneously arising cd4 þ cd25 þ populations and high concentrations of il-10 in these cultures. the main difference between cultures of sarcoid origin compared to controls is a very much higher concentration of the inflammatory cytokines il-6 and tnf-a in cultures of sarcoid origin. the effects of hyperbaric exposure on human peripheral blood mononuclear cells, with special emphasis on natural killer cell cytotoxicity and subsets materials and methods: as an experimental physiological stress model, we examined the effects of hyperbaric exposure on peripheral blood mononuclear cells (pbmcs) obtained from venous blood drawn from eight divers during a simulated heliox saturation dive. eight persons working in normobar atmosphere outside the pressurized chamber served as control donors. the spontaneous cytotoxicity of the pbmcs was estimated in a 4 h 51cr-release assay using k562 as nk-sensitive target cells. the pbmcs were characterized, using 4-colour flow cytometry, with special emphasis on the nk-cell subsets. the data were statistically analysed using a multivariate regression model (stata 8.2). p values <0.05 was considered statistically significant. results: the estimated cytotoxicity increased significantly in both the group of divers and control donors during the dive (pdivers < 0.01 and pcontrols < 0.01). although the cytotoxicity increased relatively more (p < 0.01) in the group of divers compared to the group of control donors between day 1 and 2. discussion: the increased cytotoxicity of pbmc estimated in the group of divers indicate that parts of the cellular immune system are affected during the extreme physiological conditions induced during the initial phase of the presented experimental hyperbaric setup. the increase in cytotoxicity observed in the group of control donors could hypothetically reflect the stress level in persons working outside the pressurized chamber during the dive. the interaction with adhesion molecules expressed by vascular endothelium is the first step in lymphocyte infiltration into tissues. at both cutaneous and mucosal sites interleukin-10 (il-10), il-12 and transforming growth factor (tgf)-b are important regulators of chronic inflammatory disease, where cutaneous lymphocyteassociated antigen (cla) and ae integrin (cd103) may be expressed. unlike cla, cd103 is not believed to play a role in tissue-specific homing but may help to retain t cells within epithelial layers. we have previously shown that il-12 alone can together with an unknown cofactor increase the expression of cla. stimulation with streptococcal pyrogenic exotoxin c (spec) increased the expression of cd103 by cd8 þ but not cd4 þ t cells. while il-12 increased superantigen-stimulated expression of cla, this cytokine strongly inhibited the cd103 expres-sion, and a combination of il-12 and tgf-b completely abrogated the induced cd103 expression. conversely, il-10 suppressed cla but increased cd103 expression. these findings indicate that, in addition to suppressing the development of th1-mediated inflammatory responses, il-10 may also inhibit the migration of cd8 þ t cells into the skin while il-12 promotes such migration. thus, the expression of cla and cd103 may be antagonistically regulated by il-10 and il-12, and the balance between these cytokines could influence the t-cell migration of inflammatory cells into epithelial tissues. during contact sensitivity reaction, immune cells proliferate. in order to study the histological picture of these proliferation phases, we used a mouse model of contact sensitivity in the oral mucosa and on skin. we also used bromodeoxyuridin (brdu, an analogue to thymidin) that is incorporated into the nucleus during cell replication. the hapten oxazolone (oxa) was used to sensitize and elicit the oral mucosa and/or the ear skin. mice were killed at various times after elicitation, and unsensitized animals were also exposed to the hapten as controls. brdu (25 mg/ kg animal) was injected i.p. 2 h before the kill. specimens from the oral mucosa, ear skin and submandibular and auricular lymph nodes were cut and fixed in 4% paraformaldehyde. they were then treated with acid and biotinylated anti-brdu antibody and developed using abc-kit and dab. the analyses were performed using a leica light microscope and the computer program analysis. in the oral mucosa, the frequency of proliferating cells were increasing during the observation period, 4-24 h after elicitation, regardless of site of sensitization. the proliferating cells were found mainly in the basal cell layer of the epithelium. similar patterns were found in ear skin. the regional lymph nodes demonstrated a few scattered proliferating cells 4 h after elicitation. after 24 h, these cells were found frequently in the whole lymph node. control animals exhibited considerable less proliferating cells at all times. we conclude that most proliferating cells were found 24 h after elicitation locally at the hapten-exposed sites (the oral mucosa or the ear skin) as well as in the regional lymph nodes. the endogenous nucleoside adenosine is released in excess during inflammation or other metabolic stress and is generally known to deliver tissue protective anti-inflammatory effects. adenosine acts via four adenosine receptors of which the a2a receptor is the predominant form in t cells. adenosine levels are elevated in asthmatic lung, and adenosine can directly induce mast cell degranulation and bronchoconstriction in these patients. instead, the role of anti-inflammatory mechanisms of adenosine on t cells in asthma is unclear. aim: to study the a2a receptor expression in peripheral blood cd4 þ t cells in asthmatic and healthy individuals using flow cytometric and quantitative real-time pcr methods. results: unstimulated cd4 þ cells of asthmatic patients expressed significantly lower levels (p < 0.001) of a2a receptor in protein level (mean percentage of cells positive ae sem: 76.8 ae 1.2, n ¼ 6) compared to healthy individuals (90.4% ae 1.9, n ¼ 4). double staining for cd69 expression showed that stimulation of cd4 þ cells decreased a2a expression in both groups but indicated that the detected lower levels of a2a in unstimulated cells of asthmatics was not due to preactivation in these patients. surprisingly, a2a mrna expression in unstimulated cd4 þ cells was significantly higher (p < 0.05) in asthmatics (n ¼ 28) compared to healthy controls (n ¼ 7). the expression did not correlate with serum total ige levels. conclusions: asthmatic individuals express less a2a adenosine receptor on their peripheral cd4 þ t cells. the higher mrna levels instead may point to a negative feedback regulation in the receptor expression. the role of possibly decreased adenosine-mediated anti-inflammatory effects in asthma pathogenesis require further studies on this t-cell mediated disease. the chronic inflammatory skin disease atopic eczema (ae) affects almost 15% of the population in many countries today. the pathogenesis of ae is not fully understood. a combination of genetic predisposition and environmental factors like microorganisms seems to contribute to the symptoms. the yeast malassezia sympodialis is part of our normal skin micro flora but can act as an allergen and elicit specific ige and t-cell reactivity in patients with ae. recently, we identified a novel major m. sympodialis allergen, designated mala s 11 (22.4 kda), with sequence similarity to the mitochondrial enzyme manganese superoxide dismutase (mnsod). interestingly, mala s 11 has a high degree of homology to human mnsod. the aim of this study was to examine the effects of recombinant mala s 11 on antigen-presenting dendritic cells. monocytederived dendritic cells (mddcs) from healthy blood donors were cultured with or without mala s 11 for different time periods. it was found that the maturation marker cd83 and the costimulatory molecules cd80 and cd86 were upregulated on the mddcs exposed to mala s 11 for 24 h, as demonstrated by flow cytometry. furthermore, coculture of mddcs with mala s 11 for 9 h induced an increased production of the inflammatory cytokines il-6 (200-fold), tnf-a (100-fold) and il-8 (sixfold), as detected by the cytometric bead array (cba) analysis. our results suggest that mala s 11 affects the immune response through dc maturation and production of inflammatory cytokines. the potential cross-reactivity with human mnsod needs to be explored and the exact role of mala s 11 in the pathogenesis of ae assessed in clinical studies involving skin prick and atopy patch tests. allergen-specific immunotherapy (sit) is commonly conducted with allergen extracts adsorbed to aluminium hydroxide (alum). drawbacks linked to the use of alum, such as the formation of granuloma at the site of injection, have led to suggestions of novel allergen carriers. an alternative carrier is 2 mm carbohydrate-based particles (cbps). in mouse, allergen-coupled cbps have been demonstrated to skew the allergen-specific immune response towards a th1-like activity (grönlund et al. immunology, 2002) . we here coupled the recombinant major cat allergen fel d 1 to cbps (cbp-fel d 1) by cyanogen-bromide activation, resulting in covalent binding. the effect of cbp-fel d 1 on monocyte-derived dendritic cells (mddcs) from healthy human blood donors was studied. we found that the majority of the cd1a þ mddcs were capable of taking up fitc-labelled cbp-fel d 1, as demonstrated by flow cytometry and confocal laser scanning microscopy. furthermore, incubation with cbp-fel d 1 resulted in an upregulation of the costimulatory molecule cd86 on the mddcs, which was not observed with fel d 1 or cbps alone. finally, cbp-fel d 1 induced a fivefold increase in the release of the pro-inflammatory cytokine tumour necrosis factor (tnf)-a and a fourfold increase in the release of the chemokine interleukin-8 from mddcs. taken together, the effects cbps possess make them interesting as novel allergen carriers for sit. the cysteine protease der p1 from dust mite of the genus dermatophagoides pteronyssinus is a major type i allergen. about 80% of house dust mite (hdm) allergic individuals are reactive to this protease in standard assays for detection of ige. a curative treatment for atopic allergy is immunotherapy (it) with hdm extracts which are complex mixtures occasionally resulting in anaphylactic reactions. novozymes focuses on developing a recombinant variant of der p1 which exhibit lowered risk of ige-mediated allergic reactions, while maintaining its ability to trigger proper th-cell responses. this may provide a safer alternative for specific it of hdm allergy. a secreted recombinant form of pro-der p 1 expressed by saccharamyces cerevisiae was obtained by fusion of the pro-enzyme to a fungal signal peptide. the n-glycosylation site of der p1 was mutated resulting in a deglycosylated pro-enzyme with a molecular mass of 35 kda. protein purification procedure was developed to obtain nearly pure der p1 protein followed by determination of concentration by active-site-titration with the cysteine protease inhibitor e64. the deglycosylated recombinant pro-der p 1 revealed immunologic similarity to the native der p 1 molecule when compared in basophile histamine release, ige-binding assays and t-cell proliferation assays. by in silico epitope mapping of a modelled 3-dimensional structure of der p1, five putative igg and ige epitopes were predicted. by protein engineering, the predicted epitopes were removed one by one in der p1 and screening for hypoallergenic variants was performed. combining inhaled long-acting b-2 agonist (laba) and inhaled corticosteroid (ics) seems to offer asthma control at a lower dose of ics than achieved by ics alone. fine mapping of t-cell surface markers by flow cytometry offers a detailed status of the individual's inflammatory response. the frequency of mt2 (cd4 þ cd45ra -cd62l þ cd11adim) and mt1 (cd4 þ cd45ra -cd62l -cd11abright) cells in peripheral blood, and their ratio, has been shown to differ predictably in atopics and patients with leprosy, where mt2 correlates with a th2 phenotype and mt1 with a th1 phenotype. stable asthmatics, requiring fluticasone propionate (fp) 750-1000 mg daily or equivalent, were randomized to receive, double-blinded, either seretide 1 [salmeterol and fluticasone propionate (sfc, n ¼ 16)] 50 mg/500 mg bd or fp 500 mg bd (n ¼ 17). if asthma was controlled based on lung function and symptoms at clinic visits every 6 weeks, ics dose was tapered until asthma exacerbated or 0 mg was reached. the frequency and ratio of mt2 and mt1 t cells of the patients was monitored at 6 week intervals. as treatment tapered, the frequency of mt2 cells decreased (p ¼ 0038 from first to final visit), whereas that of mt1 cells increased. the ratio of mt2/mt1 decreased (p ¼ 0049 from first to final visit). in patients receiving laba þ ics, the fall in mt2/mt1 ratio appeared to be more pronounced than in patients receiving ics alone. thus, the mt2 phenotype may be associated with stable asthma, whereas an imminent exacerbation may associate with an increase in the mt1 phenotype. laba may allow for a greater effect of fp on the mt ratio. activation of complement pathways, leading to production of c3a and c5a anaphylatoxins, has been postulated in the pathogenesis of asthma and allergic airway inflammation. the present study was undertaken to investigate the role of mannan-binding lectin (mbl), an initiator of the lectin pathway of complement, in asthma and allergic rhinitis. mbl levels and mbl-induced complement activity were determined in 19 patients of bronchial asthma with allergic rhinitis and 20 unrelated, age-matched controls of indian origin. mbl levels and activity were correlated with percent eosinophilia and percent predicted fev1 values of the patients. association of single nucleotide polymorphisms (snps) in exon 1 and intron 1 of the mbl with the disease, clinical markers, mbl levels and mbl-induced complement activity was analysed using standard statistical tools. significantly higher mbl levels and activity were observed in patients of bronchial asthma with allergic rhinitis as compared to the controls. we identified five snps, of which two, a816g in exon 1 and g1011a in intron 1 of the mbl, were novel. snp g1011a was significantly associated with the disease (p ¼ 0.0024, or ¼ 5.8696, 95% ci: 1.7316 < or < 19.8963). individuals with 'a' allele at position 1011 showed increased mbl levels, activity and disease severity. our results suggest that 'a' allele at position 1011 leading to high mbl levels and complement activity may be contributing to the severity of bronchial asthma and allergic airway inflammation. serum resistance of borrelia burgdorferi strains belonging to the b. afzelii and b. burgdorferi sensu stricto genospecies is dependent on binding of complement inhibitor factor h. we recently reported that factor h binding by b. burgdorferi is due to inducible expression of several approximately 20 kda plasmid-encoded, surface-exposed lipoproteins related to ospe (e.g. erpa, erpp and p21). in addition, a second class of factor h-binding proteins of approximately 27-35 kda has been described. the ospe-related lipoproteins are dramatically induced by b. burgdorferi during transmission from its tick vector into the mammalian host. the induction of ospe-related lipoproteins during mammalian infection may play a key a role in the borrelial evasion of the host's immune system. the goal of the present study was to define the factor h-binding regions of ospe-related proteins using mutagenesis, peptide mapping and surface plasmon resonance analysis (biacore). the combined studies revealed that the c-terminal regions of both human and mouse factor h (scrs 18-20) specifically bind to ospe-related lipoproteins. we also found fhr-1, whose c-terminal scrs 3-5 are homologous to scrs 18-20 of factor h, to bind to ospe. peptide mapping revealed five putative regions (designated i-v) in ospe that could directly interact with factor h. deleting the c-terminal 15 amino acid residues from region v of p21 abolished its ability to bind factor h. at the same time, however, synthetic peptides corresponding to the c-termini of ospe, p21 and erpp did not inhibit factor h binding to ospe. thus, the c-terminal-binding region v appears to be necessary but not sufficient for factor h binding. when a more specific mutation strategy was employed, where single amino acid residues in peptides spanning over the factor h-binding regions were mutated to alanines, we observed that lysines in the factor h-binding regions of ospe were required for factor h binding. the combined data have revealed that key lysine residues in ospe-related lipoproteins and ionic interactions are crucial for factor h interactions. furthermore, binding of ospe to the c-termini of both mouse and human factor h suggests that borrelia spirochetes utilize analogous complement resistance mechanisms in both rodents and man. in borrelia garinii strains, which in in vitro analyses have been found to be sensitive to complement killing, differences in the ospe sequences as well as in the expression of factor h-binding proteins may account for their susceptibility to serum lysis. role of yada, ail and lipopolysaccharide in serum resistance of yersinia enterocolitica serotype o:3 mannan-binding lectin (mbl), l-ficolin and h-ficolin are pattern recognition molecules of the innate immune system. we investigated the ability of these molecules to bind to different serotypes and noncapsulated variants of streptococcus pneumonia and staphylococcus aureus. we found that mbl binds to noncapsulated s. aureus strain (wood) but not any of the examined s. pneumoniae serotypes. l-ficolin binds to some capsulated s. pneumoniae serotypes (11a, 11d and 11f) as well as some capsulated s. aureus serotypes (type-1, -8, -9, -11 and -12). h-ficolin does not bind to any of the examined s. pneumoniae and s. aureus serotypes included in this study but did bind to a strain of aerococcus viridans. when bound to bacteria, mbl and h-ficolin initiated activation of complement factor c4, whereas l-ficolin did not. during this study, quantitative assays for the three proteins were developed and the concentration in 97 plasma samples were determined and the median values were estimated at 0.8 mg of mbl/ml, 3.3 mg of l-ficolin/ml and 18.4 mg of h-ficolin/ ml, respectively. the absence of early complement components (c1, c4 and c2 but not c3) is a predisposing factor for systemic lupus erythematosus (sle). recently, we demonstrated that, in c4-deficient (c4 def.) mice, igm-containing immune complexes (igm-ic) are filtered by the splenic barrier of marginal zone macrophages (mzm), resulting in an increased immune response against antigens within these igm-ic, but this could not be observed in wildtype or c3 def. mice. we hypothesized that splenic cd11b þ mzm play an important role in the induction of autoimmunity, and we therefore analysed their cytokine profile after isolation with the help of magnetic antibody cell sorting. mrna was isolated, and real-time pcr was performed with specific primers for murine ifn-g (ifn-g), interleukin-12 (il-12) and ifn-a (ifn-a). we observe a moderate increase of il-12 and ifn-g mrna in cd11b þ cells of c4 def. mice compared to wildtype cells. surprisingly, the concentration of ifn-a mrna is six times higher in c4 def. mice. preliminary results suggest that mrna in cd11b þ cells of c3 def. mice is even lower than that in wt. six hours following i.v. application of 20 mg of a abstracts 625 .................................................................................................................................................................................................. murine monoclonal igm anti-dsdna antibody, production of il-12, ifn-g and ifn-a mrna is increased in cd11b þ cells of both c4 def. and wt mice. several references described increased levels of inf-a in patients with sle. dendritic cells are discussed as a major source of ifn-a. our observation that c4-deficient, sle-susceptible mice demonstrate an increased spontaneous ifn-a production by splenic cd11b þ marginal zone macrophages could be an early sign and a trigger for the development of sle. this is supported by the fact that the absence of c3 is not a predisposing factor for sle and our observation that c3 def. animals display low levels of ifn-a mrna. 200-400 million people worldwide and represents one of the leading causes for liver cirrhosis and hepatocellular carcinoma. control over the hbv infection is achieved mainly by vaccination with hepatitis b surface antigen (hbsag). hbsag contains n-linked glycosylation side and is recognized by both mbl-a and mbl-c in a cadependent manner. hbsag-mbl complexes activate complement and may thus affect humoural immunity. to investigate the role of mbl in humoural responses to hbsag, we immununized mice that lack both mbl-a and mbl-c proteins with soluble hbsag. it has been shown that deficiencies in other complement components like c1q, c4 and c3 result in decreased antibody responses. however, mbl double ko animals mounted dramatically increased humoural responses. after priming, mbl double kos mounted hbsag-specific igm responses, which were threefold higher than wt controls. after boosting the hbsag, total igg was 10-fold higher in mbl ko than in wt control animals. similar to the response to hbsag, other glycosylated soluble antigens (e.g. invertase) induced better humoural responses in mbl double ko animals, suggesting that mbl plays an important role in a negative feedback regulation of adaptive immunity. reconstitution experiments with rmbl partially rescued the ko phenotype. we propose that the clearance of glycoprotein antigens in mbl ko is handled differently from the wt, resulting in better stimulation of humoural responses. alternatively, glycoprotein-ag-mbl-rich complexes inhibit b-cell responsiveness via putative mbl receptors. the complement system is an important part of the innate immune system. the activation of complement proceeds through three different pathways that converge in the generation of c3-activating enzyme complexes. complement activation via the lectin pathway is initiated when recognition molecules, mannan-binding lectin (mbl) or ficolin, bind to carbohydrate structures characteristic for microbial surfaces. in the circulation, mbl and ficolins are found in association with three structurally related mblassociated serine proteases (masp)-1, -2 and -3 and a small, nonenzymatic component, map19. masp-2 has been shown to elicit complement activation through the sequential proteolytic cleavage of c4 and c2 upon binding of mbl/masp-2 complexes to microbial surfaces. we have recently uncovered a polymorphism in the masp-2/map19 gene in a patient shown to be deficient in the lectin pathway of complement activation. the polymorphism results in a single amino acid substitution in the n-terminal part of the masp-2 protein. recombinant wildtype masp-2 and masp-2 containing the amino acid substitution in question was produced, and the ability to activate complement was studied. the mutation had a profound impact on masp-2 function, resulting in the lack of complement activation through the lectin pathway. elisa-based experiments showed that the mutation leads to the impairment of complement activation through influencing the binding of masp-2 to mbl or ficolins. deficiencies in the lectin pathway of complement activation have so far been accounted for only by lack of functional mbl. the mutation described above is the first defect described affecting both activation through mbl and the ficolins. .................................................................................................................................................................................................. th1, th2 and treg cell balance. dcs are present in the gut mucosa and may thus be target for modulation by gut microbes, including ingested probiotics. here, we tested the hypothesis that species of lactic acid bacteria, important members of the gut flora, differentially activate dc. a large panel of human gut-derived lactobacillus and bifidobacterium spp. was screened for dc-polarizing capacity by exposing bone marrow-derived murine dc to lethally irradiated bacteria. cytokines in culture supernatants and dc-surface maturation markers were analysed. substantial differences were found among strains in the capacity to induce interleukin-12 (il-12) and tumour necrosis factor (tnf)-a, while the differences for il-10 and il-6 were less pronounced. bifidobacteria tended to be weak il-12 and tnf-a inducers, while both strong and weak il-12 inducers were found among the strains of lactobacillus. remarkably, strains weak in il-12 induction inhibited il-12 and tnf-a production induced by an otherwise strong cytokine-inducing strain of lactobacillus casei, while il-10 production remained unaltered. selected strains were tested for induction of dc maturation markers. those lactobacilli with greatest capacity to induce il-12 were most effective in upregulating surface mhc class ii and cd86. moreover, l. casei-induced upregulation of cd86 was reduced in the presence of a weak il-12inducing l. reuteri. in conclusion, human lactobacillus and bifidobacterium spp. polarize differentially dc maturation. thus, the potential exists for th1/th2/treg-driving capacities of the gut dc to be modulated according to composition of gut flora including ingested probiotics. the intestinal micro flora is indispensable in developing and maintaining homeostasis of the gut-associated immune system. evidence indicates that lactic acid bacteria (lab), e.g. lactobacilli and bifidobacteria, have beneficial effects on the host. established health effects include increased gut maturation, antagonisms towards pathogens and immune modulation. the objective of this study is to evaluate the immunomodulating properties of a range of lab of human origin. as dendritic cells (dcs) play a pivotal role in the balance between tolerance and immunity to commensal microorganisms, in vitro-generated immature dcs serve as a suitable model for studying the immunomodulating effects of lab. human immature dcs were generated in vitro from monocytes and exposed to lethally uv-irradiated lab. the effect of various species of lab on dcs in direct contact was evaluated. furthermore, the maturation pattern of dcs separated from the bacteria by an epithelial cell layer (caco-2 cells), which should mimic the intestinal environment, was studied. cytokine secretion (il-12, il-10 and tnf-a) and upregulation of maturation surface markers on dcs (cd83 and cd86) was measured. different lab induced diverse cytokine responses. some strains were strong il-12 and tnf-a inducers and others weak. all strains induced il-10. different lab also differentially modulated expression of cd83 and cd86 on dcs. although some variation in the response to lab of dcs generated from different blood donors was observed, general differences in the effect of the various lab was revealed. experiments with the dc caco-2 coculture system are ongoing. different species of lab differentially affect dc maturation; this suggets that the gut flora plays a pivotal role in polarization of the immune response. natural killer (nk) cells are cells of the nonspecific immune system lysing altered self-cells. a noncytolytic subset of nk cells may serve a regulatory role by secreting cytokines. bacteria translocating across the gastrointestinal mucosa are presumed to gain access to nk cells, as consumption of certain lactic acid bacteria has been shown to increase in vivo nk cytotoxicity. here, we investigated how human gut flora-derived lactobacilli affect nk cells in vitro, by measuring proliferation and ifn-g production of human nk cells upon bacterial stimulation. cd3 -cd56 þ nk cells were isolated from buffy coats by negative isolation using non-nk lineage-specific antibodies and magnetic beads. nk cells were incubated with 10mg/ml uv-inactivated bacteria or 10mg/ml phytohemagglutinin (pha) for 4 days. proliferation was assessed by incorporation of radioactive thymidine into nk-cell dna. the ifn-g concentration was measured by elisa. incubation of nk cells with a lactobacillus acidophilus strain increased the proliferation of the nk cells and induced ifn-g production, both to levels comparable to pha stimulation. the proliferative response was further enhanced with autologous monocytes present, probably because cytokines, secreted by monocytes having engulfed bacteria, stimulated the nk cells. in contrast, a lactobacillus paracasei strain caused the nk cells to proliferate only in the presence of monocytes. these results demonstrate that various strains of lactobacilli have the capacity to activate nk cells in vitro, in a monocyte-dependent or -independent way. hence, the encounter of nk cells with lactic acid bacteria will affect nk-cell activation. such activation of nk cells may potentially skew an on-going or subsequent immune response towards a th1 response. lactobacilli are nonpathogenic gram-positive inhabitants of the normal human intestine known for their healthpromoting effects. in our earlier work, it is shown that human monoclonal antibody isolated from sera of a patient with waldenstrom macroglobulinaemia possess innate antibody characteristics and binds to lactic acid bacteria. according to the immune network model, immunization with this bacteria could induce the perturbations in immune system that might result in production of anti-lactobacillus antibodies, human monoclonal antibody like (ab1) and anti-idiotypic antibody (ab2). in this study, balb/c mice were immunized with two doses of bacteria lactobacillus acidophilus in complete and incomplete freund's adjuvant and phosphate-buffered saline (pbs), respectively. seven days after the last immunization, sera from immunized mice were collected and the presence of lactobacillus-specific ab1 and ab2 were determined by elisas. in the sera of immunized mice, antibodies specific to bacteria lactobacillus acidophilus were shown. the concentration of lactobacillus-specific antibodies was higher in the sera of hyperimmunized mice (mice immunized with 1 mg of igm dj) than in sera of mice immunized with 100 times lower doses of immunogen (0.01 mg per doses). moreover, ab1 and ab2 antibodies were detected in the sera of lactobacillus-hyperimmunized mice. in this study, we have shown the idiotypic network interactions in mice immunized with bacteria lactobacillus acidophilus. the normal gastrointestinal flora is crucial for the maturation of the acquired immunity via effects on antigenpresenting cells (apcs). here, we have investigated how two types of apcs, monocytes and dendritic cells (dcs), react to different bacterial strains typical of the commensal intestinal flora. purified monocytes and monocyte-derived dcs were stimulated with uv-inactivated gram-positive (lactobacillus plantarum and bifidobacterium adolescentis) and gram-negative (escherichia coli and veillonella parvula) bacterial strains. monocytes produced higher levels of il-12p70 and tnf, as detected by elisa, in response to l. plantarum than to e. coli and v. parvula. in contrast, dcs secreted high amounts of il-12p70, tnf, il-6 and il-10 in response to e. coli and v. parvula but were practically unresponsive to l. plantarum and b. adolescentis. the lack of response to the gram-positive strains correlated with a lower surface expression of toll-like reseptor 2 (tlr2) on dcs compared to monocytes. the surface expression of tlr4 on dcs was undetectable when analysed by flow cytometry, but blocking this receptor decreased the tnf production in response to v. parvula, indicating that low tlr4 expression on dcs is sufficient to mount an inflammatory response to gram-negative bacteria. ifn-g increased the expression of tlr4 on dcs and also potentiated the cytokine response to gram-negative bacteria. our results indicate that, when monocytes differentiate into dcs, their ability to respond to different commensal bacteria dramatically changes, thereby becoming unresponsive to probiotic gram-positive bacteria. these results may have important implications for the capacity of different groups of commensal bacteria to regulate mucosal and systemic immunity. probiotic bacteria, e.g. lactobacillus spp., may improve diseases such as chronic inflammatory bowel disease. we examined cytokine production and phenotypic change after in vitro stimulation of t cells from healthy volunteers using different probiotic strains. methods: t cells were cultured from colonic biopsies from eight healthy volunteers (agnholt and kaltoft, exp clin immunogenet 2001; 18:213-25) , and dendritic cells were matured from their peripheral blood mononuclear cells. t-cell cultures were stimulated with autologous bacterial sonicate or strains of lactobacillus spp., with and without the addition of dendritic cells. cytokine levels (tnf-a, ifn-g, il-10 and gm-csf) and phenotype (cd3, cd4, cd25 and cd69) were measured on day 4. results: lactobacillus spp. induced higher productions of tnf-a and il-10 than did autologous bacteria. in presence of dendritic cells, the production of all cytokines increased. however, the increases of ifn-g and tnf-a were more pronounced in wells with autologous bacteria than in wells with lactobacillus spp. the addition of dendritic cells upregulated cd25 expression without simultaneous upregulation of cd69. the upregulation was pronounced after stimulation with lactobacillus rhamnosus gg compared with autologous bacteria and other lactobacilli. discussion: in presence of dendritic cells, autologous bacteria induced inflammatory cytokines, while probiotics mainly induced regulatory cytokines. lactobacillus rhamnosus gg induced a regulatory phenotype (cd25 þ ), in part mediated by dendritic cells. future studies will address whether this shift to a cd25 þ phenotype represents a differentiation into competent regulatory t cells. in a clinical context, such cells might be used for treatment of inflammatory diseases. protein microarrays will play a key role in the postgenomic era and offer a unique possibility to perform highthroughput global proteome analysis. a chip can be printed with thousands of protein probes (e.g. antibodies), the biological sample added (e.g. a proteome) and any binding detected. we aim to develop protein microarrays based on human recombinant scfv antibody fragments for global proteome analysis. the concept of comparing proteomic maps of healthy versus diseased samples will allow diseasespecific proteins to be detected. in fact, antibody microarrays will allow us to perform comparative proteome analysis on any sample format in a species-independent manner, as long as a proteome can be isolated. however, the complexity of proteomes, containing several thousands of different proteins, is a problem. here, we have designed antibody microarrays targeting the water-soluble fraction of a proteome. to this end, an anticytokine antibody array was developed and human dendritic cells (aeactivation) was used as model system. the results showed that our antibody microarrays could be used to examine the cytokine profile in complex samples. furthermore, we have taken the first steps towards comparing our results with those of other technologies on both the protein and gene level. due to the complexity of the model proteome, we also examined the possibility to prefractionate the proteome in a simple one-step procedure (based on size) prior to the labelling step. in more detail, the sample proteome was fractionated into two fractions using membrane devices with different molecular weight cut-offs. the results showed that the fractionation considerably enhanced the assay sensitivity allowing cytokines in the pg/ml range to be readily detectable. the immunomodulatory effect of heat shock protein 70: immunization with a dna construct based on the malarial antigen fused with a fragment of hsp 70 primes for a th-1 type of response finding an appropriate adjuvant for human vaccination is crucial. heat shock proteins (hsps) act as adjuvants when coadministered with peptide antigens or given as fusion proteins. however, there is a potential risk of autoimmunity when using the complete molecules, because hsps are evolutionary conserved. to overcome this, we first evaluated the adjuvant effect against two different antigens of a less-conserved fraction of plasmodium falciparum hsp70 (pf70c) and compared it to the whole hsp70 molecule from trypanosoma cruzi (tchsp70). we found that pf70c exhibited similar adjuvant properties as the whole molecule. we later evaluated the adjuvant potential of pf70c against the malarial antigen eb200 in a chimeric dna construct. no appreciable levels of eb200-specific abstracts 629 .................................................................................................................................................................................................. antibodies were detected in mice immunized only with the dna constructs. however, dna primed the immune system, because subsequent challenge with the corresponding recombinant fusion proteins elicited a strong th-1 antibody response. in contrast, no priming effect was observed for ex vivo ifn-g production but stimulation with the hsp-chimeric fusion protein induced a stronger secretion of ifn-g in vitro than other proteins used. these results indicate that the use of hsps is promising in the design of new vaccines. high-throughput proteomics on antibody-based microarrays: the importance of probe and surface design in analogy to dna microarrays, protein microarrays offer a new distinct possibility to perform sensitive highthroughput global proteome analysis. however, the development of the protein microarray technology will place high demands upon the design of both probes and solid supports. the analysis of thousands of heterogeneous proteins on a single microarray requires the use of uniform probes, such as antibodies, directly designed for protein microarray applications. we have recently generated a human recombinant single-chain fv antibody library, genetically constructed around one framework, the ncoder-library, containing 2 â 1010 clones. single framework antibody fragments (sinfabs) selected from this library were successfully applied as probes for microarrays providing sensitive detection in the 600 attomol (mass spectrometry) and the 300 zeptomole range (fluorescence). however, the choice of framework is critical. we have shown that the selected ncoder framework displayed excellent functional on-chip stability and arrayed dehydrated probes retained their activity for several months. furthermore, we have addressed the issues of biocompatibility of the solid support and immobilization strategies for our microarray setup. an in-house-designed substrate, macroporous silicon coated with nitrocellulose (map3-nc7), displayed properties equal to, or better than, those of five commercially available supports used as reference surfaces. we have also evaluated different coupling strategies, such as adsorption, covalent coupling, diffusion and affinity coupling. using a novel affinity tag, the double-(his)6-tag, we increased the binding efficiency of sinfab-molecules to ni2 þ -coated solid supports, thereby allowing nonpurified probes to be directly applied. the mannan-binding lectin (mbl) pathway is part of the innate immune system providing a first line of defence against infections. mbl and ficolins circulate in complexes with mbl-associated serine proteases (masp-1, -2 and -3). after recognition of a microorganism by mbl, activation of the complement system occurs. masp-1 and masp-3 share five domains (making up the so-called a-chain), whereas they have unique protease domains (b-chains). before the identification of masp-3, an assay for masp was presented, based on antibodies against the a-chain of masp-1. with the new knowledge of the three masps, and the sharing of domains by masp-1 and masp-3, assays specific for the protease domains have to be constructed, if one wishes to measure the proteins individually. we present an assay for quantifying total masp-3 in plasma and serum samples. the assay is a sandwich-type assay using as catching antibody a monoclonal antibody against the common a-chain of masp-1/3 and a developing secondary antibody against the c-terminal part of the protease domain of masp-3. we have used this assay for estimating the normal concentration of the protein as well as the concentration in patients and also for characterizing by gel permeation chromatography the masp-3 protein in serum. inducible costimulator ligand (icosl) is a costimulatory molecule related to b7.1 (cd80) and b7.2 (cd86). b cells, monocytes, dendritic cells and endothelial cells express icosl. inducible costimulator (icos) interacts with icosl, and this interaction leads to signals involved in isotype switching and the development of immunological memory. hitherto, no polymorphisms of this gene have been described. the aim of this study was to reveal variation of the icosl gene in normal individuals. all eight exons, except exon 1, were sequenced with flanking introns in 10 healthy blood donors. eight single nucleotide polymorphisms (snps) and two length polymorphisms were found. one of the snps was found in the coding regions of the gene. the base involved was located in exon 3 and caused a conservative amino acid change from valine (gtt) to isoleucine (att). three individuals were heterozygous g/a for the exon polymorphism, while the remaining seven individuals were homozygous for the wildtype g/g. exon 3 encodes the immunoglobulin variable (igv)-like domain of the molecule which is situated outside the cell. this means that the amino acid could be critical for the stability of the molecule or could constitute part of the binding site for icos. the results form the basis for further experiments to find possible associations of the alleles to diseases caused by immune dysregulation. especially, the exon 3 variant is interesting and could play a role for the development of immunological diseases. besides, it would be interesting to see whether both exon 3 alleles are expressed or only the wildtype allele is functional. interferon-a/b (ifn-a/b) is increasingly used as antiviral and immunomodulatory therapies. unfortunately, bioavailability varies with ifn species and mode of administration, and all ifn species are potentially immunogenic. assays for antiviral activity (ifn) and antiviral neutralization (antibodies, nab) have been used for some time to monitor patients on ifn biologicals. these assays require laborious titrations making them unsuitable for large-scale clinical use. myxovirus a (mxa) is a resistance gtpbinding protein that is specifically induced by treatment with type 1 ifns. for example, ifn-b-induced mxa in blood leucocytes has been used as a biomarker in ifn-btreated patients with multiple sclerosis. however, the degree of specificity of mxa in this regard is unclear, and measurements of mxa protein and/or mrna are not yet suitable for routine clinical use. in an attempt to find new and better reporter genes (and, hopefully, genes and gene products with proven specificity for ifn-a and -b), microarray screenings with u133a genechips (affymetrix) were carried using human blood leucocytes and the human lung carcinoma cell line a549. we studied the simultaneous expression of 22,000 transcripts before and after exposure to human recombinant ifn-a and ifn-b and other antiviral and immunomodulatory cytokines. the results will be presented at the conference. interferon-a/b (ifn-a/b) is increasingly used as antiviral and immunomodulatory therapies. unfortunately, bioavailability varies with ifn species and mode of administration, and all ifn species are potentially immunogenic. assays for antiviral activity (ifn) and antiviral neutralization (antibodies, nab) have been used for some time to monitor patients on ifn biologicals. these assays require laborious titrations making them unsuitable for large-scale clinical use. our laboratories have therefore modified the antiviral assays for ifn bioactivity and nab, so that they are suitable for large-scale screening in specialized laboratories. the read-out is survival of a subcloned a549 cell line in the presence of an otherwise lethal amount of virus. thus, survival increases in the presence of type 1 ifn and decreases in the presence of nab against the ifn added to the cells. mxa is induced by type 1 ifn and can be used for measuring the nab activity. in another assay, the mxa level in the a549 cell line is measured. in an attempt to find a new and better reporter gene for type 1 ifn than mxa and genes specific for either ifn-a or -b, a micro array screen was carried using the u133a chip from affymetrix. the expression of 22,000 genes can be studied simultaneous with this technology. the results will be presented at the conference. in our laboratory, we have developed a database system, which we believe is of immediate interest to the general scientific community. the database represents a computerbased replacement for the laboratory notebooks used in the majority of research laboratories worldwide. in addition, the database provides an effective tool for organizing and managing laboratory information at all levels, spanning from managing and revising standard operating procedures and producing documentation of research activities to keeping track of data and conclusions. using the commercially available database toolkit software filemaker pro, we have developed a relational database solution for management of laboratory information. the system consists of a hierarchy of five interrelated databases, each pertaining to a separate type of information, namely, overall project information, information relating to individual experiment setups, documentation of daily research activity, generated data and descriptions of standard operating procedures. like other databases, each individual database consists of a number of records, each comprised of a set of fields in which information is entered. in each record, a certain field is reserved to specify the relation of the record to a record in another database at a higher level. thus, the database is essentially five databases linked by a hierarchy of one-to-many relations, organizing information in a folder-like structure. importantly, the database system allows multiple users to access and edit records simultaneously, and the data entered in one database immediately becomes accessible through the other databases. the limitations of laboratory notebooks are apparent when looking for information, which is dispersed throughout one or more notebooks, or possibly on loose sheets of paper or printouts 'somewhere'. the often complicated process of gathering laboratory data or results when writing grant applications or research papers is made considerably easier with the database system. thus, the database solution presented should be broadly attractive to researchers, irrespective of their scientific discipline. an effective sars vaccine is likely to include components that can induce specific cytotoxic t-cell (ctl) responses. the specificities of such responses are governed by hlarestricted presentation of sars-derived peptide epitopes. exact knowledge of how the immune system handles protein antigens would allow for the identification of such linear sequences directly from genomic/proteomic sequence information. the latter was recently established when a causative coronavirus (sars cov) was isolated and full-length sequenced. here, we have combined advanced bioinformatics and high-throughput immunology to perform an hla supertype, genome-wide scan for sars-specific cytotoxic t cell epitopes. the scan includes all nine human hla supertypes in total covering >99% of all major human populations. for each hla supertype, we have selected the 15 top candidates for test in biochemical-binding assays. at this time (approximately 6 months after the genome was established), we have tested the majority of the hla supertypes and identified almost 100 potential vaccine candidates. these should be further validated in sars survivors and used for vaccine formulation. we suggest that immunobioinformatics may become a fast and valuable tool in rational vaccine design. rationale: major histocompatibility complex class i (mhc i) molecules monitor the protein content of the cell by binding small derived peptides and presenting them to cytotoxic cd8 þ t cells. the goal of the human mhc project is to predict the binding strength of any given peptide/mhc complex. this prediction allows the design of peptide-based vaccines. the prediction requires representative binding data from mhc alleles from all the nine hla supertypes. here, we describe the genetic construction, protein production and purification as well as the establishment-binding assays for two recombinant mhc supertype alleles, hla-b*1501 and hla-b*5801. methods: using the quikchange multisite directed mutagenesis kit (stratagene), codon-optimized genes encoding hla-b*1501 and hla-b*5801 are created. the two mhc i molecules are fermented and purified by ion exchange chromatography, hydrophobic interaction chromatography and size exclusion chromatography. the binding (kd) of natural t-cell epitopes, as well as predicted peptide ligands, is described by radioactive immunoassays (rias) and enzyme-linked immunosorbent assays (elisas). the mhc molecules are biotinylated during expression. results: the expression of mhc i resulted in multiple disulfide bond isomers, which are separated by hydrophobic interaction chromatography and used in subsequent binding studies resulting in the determination of kd for various peptide ligands ranging from strong binders we have previously demonstrated that bioinformatics tools such as artificial neural networks (anns) are capable of performing pathogen-, genome-and hlawide predictions of peptide-hla interactions. these tools may therefore enable a fast and rational approach to epitope identification and thereby assist in the development of vaccines and immunotherapy. a crucial step in the generation of such bioinformatics tools is the selection of data representing the event in question (in casu peptide-hla interaction). this is particularly important when it is difficult and expensive to obtain data. herein, we demonstrate the importance in selecting information-rich data and we develop a computational method, query-bycommittee, which can perform a global identification of such information-rich data in an unbiased and automated manner. furthermore, we demonstrate how this method can be applied to an efficient iterative development strategy for these bioinformatics tools. methods: a large panel of binding affinities of peptides binding to hla a*0204 was measured by a radioimmunoassay (ria). this data was used to develop multiple first generation anns, which formed a virtual committee. this committee was used to screen (or 'queried') for peptides, where the anns agreed ('low-qbc'), or disagreed ('high-qbc'), on their hla-binding potential. seventeen low-qbc peptides and 17 high-qbc peptides were synthesized and tested. the high-or low-qbc data were added to the original data, and new high-or low-qbc second generation anns were developed, respectively. this procedure was repeated 40 times. the high-qbc-enriched ann performed significantly better than the low-qbc-enriched ann in 37 of the 40 tests. conclusion: these results demonstrate that high-qbcenriched networks perform better than low-qbc-enriched networks in selecting informative data for developing peptide-mhc-binding predictors. this improvement in selecting data is not due to differences in network training performance but due to the difference in information content in the high-qbc experiment and in the low-qbc experiment. finally, it should be noted that this strategy could be used in many contexts where generation of data is difficult and costly. interleukin-18 (il-18), a pro-inflammatory cytokine that is produced by both lymphoid and nonlymphoid cells, has a critical role in modulation of innate and adaptive immunity. its primary function in stimulation of ifn-g production and stimulation of nk-cell-cytotoxic activities makes this cytokine a candidate for cancer immunotherapy. in oral cavity, this cytokine is produced by oral epithelia and carcinoma cells and is related to tumour regression in nude mice bearing salivary adenocarcinoma. however, direct effects of this cytokine on oral cancer cells have not been elucidated. in this project, we investigated il-18 effect on an oral carcinoma (kb) cell line. with rt-pcr technique, kb-cell line was found to express il-18 receptors (il-18ra and il-18rb), indicating that this oral carcinoma line is a target for il-18 study. we showed that recombinant human il-18 inhibited kb-cell proliferation by 17% at concentration of 100 ng/ml (p < 0.05), whereas ldh release by these cells in treatment group and control groups was comparable, indicating that il-18 suppression of cell proliferation was not mediated by the induction of cell death. to further address this hypothesis, we found that il-18 treatment did not induce apoptotic cell death, as studied by dna laddering and tunel assays. in addition, expression pattern of cell death-controlling genes (bcl-2 and bax) was not altered by this cytokine. findings in these studies indicated that suppression of kb-cell proliferation may be attributed to control of cell cycle, growth arrest or induction of cell differentiation. the data presented in this project could provide an insight of how cancer cell directly responds to il-18, as this cytokine is an important regulator of anticancer mechanisms. aloe emodin (ae) is a naturally occurring compound with wide spectrum of biological properties, including antimicrobial, vasorelaxant, immunosuppressive and anticancer actions. this anthraquinone induces apoptosis in several tumour cell lines with special affinity to tumours of neuroectodermal origin. high amounts of nitric oxide (no) released by activated macrophages induce tumour cell death. therefore, we explored the capacity of ae to modulate no-mediated antitumour response in vitro. interestingly, while ae markedly suppressed no release from macrophages alone, it significantly potentiated no production in cocultures of macrophages and c6 cells, after 48 h of cultivation. accordingly, the viability of c6 cells cocultivated with macrophages was reduced in the presence of ae. moreover, the observed ae-imposed potentiation of no production in macrophages was closely related to macrophage culture cell density. according to these data, we proposed that no modulator capacity of ae strongly depended on intercellular contact, indicating that macrophage antitumour response was not compromised but even potentiated by ae. immunotherapy represents an attractive fourth-modality therapeutic approach, especially in the light of the shortcomings of conventional surgery, radiation and chemotherapies in the management of metastatic cancer. to this end, a large number of peptide antigens derived from taa have been applied in immunotherapeutic trials for the treatment of various malignancies, e.g. cancers of the breast, prostate and kidney, in addition to haematological cancers. in some cases the response rates have been impressive and no adverse autoimmunity have been observed. a major strategic difficulty associated with these trials relates to the choice of best-suited peptide antigens. the vast majority of the antigens described thus far is not vital for survival and growth of the tumour cells, and immunoselection of antigen-loss variants may therefore prove to be an additional obstacle for the clinical applicability of most of the known peptide epitopes. in this respect, the development of acquired antigen loss during immunotherapy has been demonstrated in several cases. obviously, the development of loss-variant tumour cells implies that these cells acquire a pronounced growth advantage and are left unaffected by further treatment. ideally, target antigens should be derived from proteins required for survival and growth of tumour cells, as antigens with these characteristics would not be inflicted by the development of loss-variant tumour cells. in this respect, several inhibitors of apoptosis proteins (iaps) are universally expressed among tumours and play an important role in tumour cell escape from apoptosis. we have characterized spontaneous t-cell reactivity against iapderived peptides in cancer patients. from the iap survivin, we have characterized peptides restricted to the class i molecules hla-a1, a2, a3, a11, b7 and b35. furthermore, we have demonstrated that survivin-specific t cells infiltrate metastatic lesions and that isolated survivinspecific ctls are capable of killing hla-matched tumour cells. survivin-derived peptides are now in clinical trial, and continued work in our lab has demonstrated that other iaps are targets for spontaneous t-cell reactivity in cancer patients. we previously reported that in mice with large progressing t-cell lymphoma tumours, dysfunctions in the antitumour ctl activity occur, associated with an accumulation of splenic arginase-producing myeloid suppressor cells (mscs). in this study, we first demonstrate that both the presence and the activation state of these msc depends on tumour evolution. while in tumour regressors hardly any arginase-producing msc can be found, both the amount and the arginase activity of this population expands from early over late progressors. this gradual induction of mscs is paralleled by an increasing suppression of ctl activity and th1, but not th2, cytokine production. upon analysing the molecular repertoire of msc in vitro, we found, besides arginase1, a well-established marker for alternatively activated myeloid cells or m2, a strong upregulation of fizz1 and ym, two additional recently identified markers for m2. further evaluation of molecular markers by microarray analysis in msc yielded genes involved in wound healing (e.g. coagulation factor xiiia), anti-inflammation (e.g. selenoprotein p), immunomodulation (e.g. pd-l2) and fat and sugar metabolism (e.g. leptin receptor). of note, many of these genes are regulated by type 2 cytokines (il-4, il-13 and il-10) and are therefore rather m2 associated. overall, our data provide new markers for msc in cancer and further establish their m2 activation state. study. only sp-a showed a significant expression in normal mucosa which was downregulated in crc. as the absolute signal level was below the noise threshold, these results have to be interpreted with caution and require confirmation by direct measurenment of the proteins. our results suggest that there is no major role for the human collectins in colorectal cancer. tetramerization is visualized by sds-page. conclusion: an effective method for the production of highly pure mhc i molecules has been applied to hla-b*1501 and hla-b*5801, and ria and elisa binding assays for those alleles have been established background: proliferation, differentiation and apoptosis are essential processes in the normal functions of the mammary epithelium. the hypothesis examined in this study is that the transcription factor bcl-6 is critically important not only for regulating b-cell growth and development but also for mammary epithelial apoptosis. methodology: twenty breast cancer cases and 31 healthy controls were used to investigate whether bcl-6 protein in involved in breast cancer (grade iii). full length bcl-6 cdna was retrovirally transduced into eph-4 cell line. we then used flow cytometry of brdurd-stained cells to investigate the cell-cycle duration of the control and transduced cell lines. tunel was used as a marker of apoptosis to find out differences in the frequencies of apoptotic cells in the control and transduced cell lines. finally, immunohistochemistry staining was performed to detect bcl-6 in breast cancer (iii). results: restoration of bcl-6 into eph-4 cells not only inhibits apoptosis but also prolongs the cell cycle and results in increased cell size and protein content. the results also indicated that the cell-cycle time of bcl-6-transduced eph-4 cells is prolonged by about 3 h, presumably as a result of the action of bcl-6 at the bcl-6 at the g1/s transition. we found differences in the frequencies of viable and apoptotic cells in cultures of the parent eph-4 cells, control-transduced eph-4 cells and bcl-6-transduced eph-4 cells. consistently, we demonstrated that bcl-6 is expressed in 90% of high grade of breast carcinoma, which is considered as the most aggressive of tumours. conclusion: together, these results suggest that bcl-6 is likely to be involved in mammary gland development and carcinogenesis. inflammatory cytokines have a critical role in modulation of both innate and adaptive immunity in response to foreign antigen. they also play an important role in anticancer immunity. for example, they can promote cell-mediated immunity against cancer cells. with their immunostimulatory effects, these cytokines are being tested for cancer treatment in the form of dna vaccine or adjuvant or therapeutic cytokines. direct effect of these cytokines on cancer cell, however, is still unclear. in this project, we investigated whether il-1( and il-18 can modulate cancer cell proliferation. we employed a simple nonradioactive proliferation (mtt) assay and detection of lactate dehydrogenase (ldh) to test the effect of these recombinant human cytokines on various cancer cell lines, including breast cancer cell line (mcf-7), oral carcinoma cell line (kb), colon cancer cell line (caco-2) and choriocarcinoma cell line (jar). cytokines used in this study had both inhibitory and stimulatory effect on cell proliferation. findings in this project could provide an insight of cancer cell response to these cytokines and this could lead to a consideration on using cytokine as immunotherapy for cancer treatment.capacity of ae to modulate nitric oxide production depended on intercellular contact donor t cells are involved in the antitumour effects observed after bmt. thus, patients receiving t-celldepleted bmt have a higher risk of leukaemia relapse compared to patients receiving nonmanipulated bmt, and patients experiencing graft-versus-host disease (gvhd) have a lower risk of disease relapse than patients who do not experience gvhd. although the importance of donor t cells for the curative action of bmt has been established, the exact mechanisms and molecules involved in this graft-versus-tumour effect remain largely unknown. in a recently initiated project, we have conducted a longitudinal study of t-cell clonotypes in patients who received peripheral blood stem cell grafts after nonmyeloablative conditioning. peripheral blood samples were obtained sequentially after transplant, and the mononuclear cells (mncs) were isolated and cryopreserved. cd8 þ t cells were isolated from the mncs by use of immunomagnetic beads or facs and analysed for the presence of clonally expanded cells by t-cell receptor clonotype mapping based on rt-pcr and denaturing gradient gel electrophoresis (dgge). using this gel-based methodology, clonally expanded t cells were monitored after transplant and compared to the clinical data of the patients. the preliminary results demonstrates the presence of clonally expanded cd8 þ t cells at all time points analysed. furthermore, a number of clonotypes persisted for more than 6 months, and other clonotypes emerged during this period. the appearance of newly emerged clonotypes which coincided with clinical gvhd could indicate a role for these t cells in the pathogenesis of gvhd. background: deficiency of the mannan-binding lectin (mbl) pathway of innate immunity leads to increased susceptibility to infections. in patients with colorectal cancer, postoperative infection is associated with poor prognosis. the aim of the present study was to evaluate (1) the relation between the mbl pathway and postoperative infectious complications and survival of patients resected for colorectal cancer and (2) the role of mbl as acute phase reactant compared to crp. methods: preoperative mbl concentration, mbl/mblassociated serine protease (masp) activity and crp were determined in serum from 611 patients and 150 healthy controls. the patients were observed for 8 years. postoperative infections, recurrence and survival were recorded. results: the mbl pathway components were increased in the patients (p < 0.0001) compared to healthy controls. low mbl levels were predictive of pneumonia (p ¼ 0.01), and pneumonia (n ¼ 87) was associated with poor survival (p ¼ 0.003, hr ¼ 1.5, 95% ci 1.1-1.9). mbl and mbl/ masp activity could not predict postoperative overall infections. mbl showed no correlation (spearman's r ¼ 0.02, 95% ci à0.06-0.10) with crp. conclusions: low preoperative mbl levels are predictive of pneumonia, which is associated with poorer survival. mbl concentration and mbl/masp activity was not predictive of other postoperative infections or long-term prognosis. mbl apparently is not a surrogate measure of crp. department of surgery, university hospital of erlangen, erlangen, germany. e-mail: michael.siassi@rzmail.uni-erlangen.de introduction: the human collectins, mannan-binding lectin (mbl), surfactant protein-a (sp-a) and surfactantprotein-d (sp-d) play a central role in the innate immune system. immunological responses to malignant transformation of epithelial cells gained increasing interest recently. a former study could demonstrate binding of mbl to certain colorectal carcinoma (crc) cell lines in vitro. we therefore examined the expression of human collectins in normal colon mucosa and in colorectal carcinomas. materials and methods: colon samples from 20 crc patients and 10 normal mucosa samples were collected immediately after surgery. the tissue was microdissected and rna isolated (qiagen, rneasy-kit). gene expression profiles were analysed using gene-chips (affymetrix, hg-u133). we analysed the data for the expression of mbl, its associated serine proteases mannan-binding lectinassociated serine protease 1/2 (masp 1/2), sp-a and sp-d. the signal intensity of the genes of interest was compared using the mann-whitney u-test. results: the expression of human collectins in normal human colon mucosa was generally low. only the expression of sp-a and masp-2 reached the noise threshold of 250 signals. these genes were significantly downregulated in crc specimens. the expression of the other proteins showed no difference in normal mucosa and crc. conclusion: as demonstrated before, the expression of human collectins in normal colon was low in this being the first lymph node to receive drainage from the tumour area, the sentinel node offers a unique possibility to obtain tumour-reactive lymphocytes. we investigated antitumour immune responses in sentinel nodes from patients with bladder cancer, by assaying tumour-specific proliferation and tcr vb repertoires. during tumour surgery, sentinel lymph nodes were identified by peritumoural injection of blue dye. fresh specimens of tumour, sentinel and nonsentinel lymph nodes were obtained, and single-cell suspensions were prepared. cells were assayed for reactivity against autologous tumour extract in [ 3 h]-thymidine incorporation assays and characterized by flow cytometry. parallel analyses of the expression of vb gene families were performed with padlock probes, linear oligonucleotides which upon target recognition can be converted to circular molecules by a ligase. probes were reacted with cdna prepared from magnetically separated cd4 þ cells, and the tcr repertoire was determined by hybridizing the products to oligonucleotide microarrays. dose-dependent proliferation in response to tumour extract could be detected in sentinel lymph nodes. common clonal expansions were detected among tumourinfiltrating lymphocytes and in sentinel lymph nodes. nonsentinel lymph nodes displayed a divergent tcr vb repertoire. these results indicate an ongoing immune response against tumour antigens in sentinel nodes, draining urinary bladder cancer. identification of sentinel lymph nodes makes it possible to obtain tumour-reactive lymphocytes for use in adoptive immunotherapy. key: cord-001397-nrq4ncdf authors: mlera, luwanika; melik, wessam; bloom, marshall e. title: the role of viral persistence in flavivirus biology date: 2014-05-12 journal: pathogens and disease doi: 10.1111/2049-632x.12178 sha: doc_id: 1397 cord_uid: nrq4ncdf in nature, vector-borne flaviviruses are persistently cycled between either the tick or mosquito vector and small mammals such as rodents, skunks, and swine. these viruses account for considerable human morbidity and mortality worldwide. increasing and substantial evidence of viral persistence in humans, which includes the isolation of rna by rt-pcr and infectious virus by culture, continues to be reported. viral persistence can also be established in vitro in various human, animal, arachnid and insect cell lines in culture. although some research has focused on the potential roles of defective virus particles, evasion of the immune response through the manipulation of autophagy and/or apoptosis, the precise mechanism of flavivirus persistence is still not well understood. we propose additional research for further understanding of how viral persistence is established in different systems. avenues for additional studies include determining if the multifunctional flavivirus protein ns5 has a role in viral persistence, the development of relevant animal models of viral persistence as well as investigating the host responses that allow vector borne flavivirus replication without detrimental effects on infected cells. such studies might shed more light on the viral-host relationships, and could be used to unravel the mechanisms for establishment of persistence. defining mechanisms of viral persistence will be critical for understanding vector-borne flavivirus infections. these viral infections account for considerable human morbidity and mortality worldwide. furthermore, incidence is increasing and infections are being appreciated in previously nonendemic geographic locations. prominent vector-borne flaviviruses (vbfvs) associated with significant human infections include both tick-borne and mosquito-borne agents. the tick-borne flaviviruses (tbfvs) are exemplified by the tick-borne encephalitis virus (tbev) sero-complex group, omsk hemorrhagic fever virus, kyasanur forest disease virus, alkhurma virus, powassan virus (powv), and deer tick virus (dtv), the latter two occurring in the united states (holbrook et al., 2005; brackney et al., 2008b; ebel, 2010) . the mosquito-borne flaviviruses (mbfvs) are perhaps better known and include yellow fever virus (yfv), west nile virus (wnv), japanese encephalitis virus (jev), and dengue virus (denv) serotypes 1-4. infections with all of these viruses can lead to severe disease, prolonged debilitating neurological sequelae, hemorrhagic fever, and/ or death in some cases (solomon et al., 1998; glass et al., 2002; haglund & gunther, 2003; madden, 2003; van gerpen, 2003; carson et al., 2006) . viral persistence is a hallmark of the ecology of vbfvs. both tbfvs and mbfvs are cycled between arthropod and vertebrate hosts (figs 1 and 2) , and in many cases, they are maintained without deleterious effects on the hosts. in nature, tbfvs, such as powv and tbev, alternately infect small vertebrates such as rodents, hares, some carnivores, and a range of hard-bodied (ixodid) ticks, although recent evidence suggests that the soft-bodied ornithodoros (argasid) ticks can also support tbfv (rajagopalan et al., 1969; charrel et al., 2007) . similarly, mbfvs, such as wnv and jev, primarily alternate in nature between small mammals, birds, and mosquitoes ( fig. 1 ). in addition, there is evidence that mbfv and tbfv persistence also occurs in humans, and persistence in cell culture is well documented (poidinger et al., 1991; lancaster et al., 1998; bugrysheva et al., 2001; farfan-ale et al., 2009; murray et al., 2010) . although the hosts for the vbfvs are highly varied, the genomic and structural organization of the viruses themselves is remarkably similar. all flaviviruses are spherical, enveloped particles that contain a genome of (+) ssrna measuring approximately 11 kb. the genome also functions as mrna and encodes a single polyprotein that is cleaved into 10 proteins (table 1) . reasonable progress has been made toward understanding how these viruses replicate (chambers et al., 1990; mandl, 2005; miorin et al., 2008; tuplin et al., 2011; pierson & diamond, 2012; brinton, 2013; pierson & kielian, 2013) . we present a simplified overview of the general aspects of a vbfv replication cycle in fig. 3 . the individual proteins are three structural proteins: capsid (c), precursor membrane/memfig. 1 flavivirus maintenance and transmission cycle in ticks and vertebrate hosts. ticks are crucial for viral persistence as they remain infected once they acquire viral infection. infected ticks are capable of transmitting tbfvs to other ticks when they feed in close proximity on the same animal, as well as the different stages of the tick life cycle. tbfvs persist also in a cycle between small mammals (e.g. rodents) and the ticks that feed on them. large mammals and humans tend to be incidental, dead-end hosts. fig. 2 a representation of a mosquito-borne flavivirus amplification and transmission cycle. wnv is cycled between the mosquito and avian hosts that play an amplification and maintenance role. swine are important amplifying hosts for jev, and mosquitoes that acquire blood meals on infected pigs can become infected and transmit the virus. similar to tbfvs, mbfv infection in humans and large animals, such as horses, is accidental. the flavivirus proteins, untranslated regions (utrs), and their known functions the 5 0 utr contains conserved rna stem loops (sl), cis-elements located upstream and downstream of the aug region (uar and dar, respectively), and complementary sequence (cs) sponsoring cyclization of the genome by interacting with the 3 0 utr to form a 'panhandle' (alvarez et al., 2005; villordo & gamarnik, 2009; friebe & harris, 2010; friebe et al., 2011) . the 'panhandle' structure is essential for certain steps in the flavivirus replication cycle such as replication/translation and viral assembly (gritsun & gould, 2007b ). sl1-2 or (sl-a and sl-b) is located within the 5 0 utr, whereas the sl3-4 is extended and situated in the n-terminal of the open reading frame orf (alvarez et al., 2005; lodeiro et al., 2009; villordo & gamarnik, 2009; friebe & harris, 2010; friebe et al., 2011) . the sl1 structure is essential for viral replication and acts as a promoter which is targeted initially by ns5 and then delivered to the 3 0 end via cyclization (filomatori et al., 2006; zhang et al., 2008; lodeiro et al., 2009) although there is low nucleotide conservation between flaviviruses and different cs homology (hahn et al., 1987) , the 5 0 utrs of tbfvs share the same genomic organization as the mbfvs (kofler et al., 2006) structural proteins capsid (c) 11 kda, 114 aa cytosol/nucleus the capsid protein is a dimeric alpha-helical (jones et al., 2003) protein and assembles into an icosahedral structure, measuring 30 nm in diameter, which initiates encapsidation of the associated genomic rna in virus-induced membrane invaginations of the er (welsch et al., 2009 ). the c protein is also a pro-apoptotic factor in various cell lines (yang et al., 2002 (yang et al., , 2008 oh et al., 2006; netsawang et al., 2010; morchang et al., 2011) . c of jev was reported to limit viral neurovirulence (mori et al., 2005) the c-encoding nucleotide sequence of tbev contains conserved rna structures that function as replication enhancer elements (tuplin et al., 2011) † precursor membrane (prm) 469-972, 26 kda, 165 aa er lumen during virion assembly, the prm forms a heterodimer with the e protein and acts as a chaperone for correct e protein folding (kuhn et al., 2002; lorenz et al., 2002; zhang et al., 2003) . in the final step of virion maturation in the trans-golgi network prior, to viral release, the precursor is cleaved by furin and only the c-terminal region (m) is retained in the viral membrane (heinz et al., 1994; elshuber & mandl, 2005; lindenbach et al., 2007) . prm protein of jev also limits viral neurovirulence (kim et al., 2008 ) † (bressanelli et al., 2004) . the glycoprotein forms 'head to tail' homodimers that completely cover the surface of the mature icosahedral virions. it is also responsible for receptor binding, which leads to virus internalization by clathrin-mediated endocytosis (lindenbach et al., 2007) . the e protein contains three b-sheets domains (di-diii): dii includes the membrane fusion peptide (beasley & barrett, 2002; bressanelli et al., 2004; sukupolvi-petty et al., 2007) ; diii is associated with receptor binding and is also a target for several neutralizing monoclonal antibodies. crystal structures of the e protein from several flaviviruses all show similar secondary structure and domain organization (modis et al., 2003 (modis et al., , 2005 zhang et al., 2004; kanai et al., 2006; nybakken et al., 2006) . the e protein is important in determining neuroinvasiveness and neurovirulence for several mbfvs (cecilia & gould, 1991; hiramatsu et al., 1996; ni & barrett, 1996; sanchez & ruiz, 1996; chambers et al., 1998; beasley et al., 2004; lee et al., 2004 lee et al., , 2006 bordignon et al., 2007) . for wnv, jev, and denv, mutations in the e protein (located in the lateral surface of diii and the base of dii) affect fusion and receptor binding with target cells (lee et al., 2004) the e protein was first described in 1995 and later has been extensively defined and crystalized for many other flaviviruses (rey et al., 1995) . tbfv e protein is important for determining neurovirulence and neuroinvasiveness (holzmann et al., 1990; mcminn, 1997; beasley et al., 2005; mandl, 2005; engel et al., 2010) nonstructural proteins ns1 2461-3516/46-55 kda/325 aa er lumen/cytosol/ secreted low-resolution structural studies found the ns1 structure to be an open barrel configuration with d3 symmetry measuring 10 nm by 7.5 nm and with a central cavity approximately 4.5 nm in diameter (gutsche et al., 2011; edeling et al., 2014) . all flavivirus ns1 genes share a conserved degree of homology. ns1 is found at different cellular locations such as vesicular compartments associated with the cell membrane or on the cell surface and is also secreted as an extracellular † westaway et al., 1997; lindenbach et al., 2007) . each subunit forms a homodimer located in the er lumen, and it has been shown to colocalize with viral dsrna. secreted and cell surface associated ns1 is immunogenic and induces an antibody response that can serve as an important diagnosis marker (falgout et al., 1990; avirutnan et al., 2006; sun et al., 2007) . ns1 is also implicated in immune evasion functions as it activates human complement and induces host vascular leakage by specific inhibition of the classical and lectin pathways of complement activation. this occurs through a direct interaction with complement components c4 and c1s (avirutnan et al., 2010) . denv, wnv, and yfv ns1 are shown to limit c4b deposition and c3 convertase activity by enhancing cleavage of c4 through the recruitment of the complement-specific protease c1s (avirutnan et al., 2010) . ns2a (blitvich et al., 1995 (blitvich et al., , 1999 (assenberg et al., 2009 ). ns3 is a highly conserved and multifunctional protein: the protein has protease activity in one domain, rna-stimulated ntpase activity and rna triphosphate activity (rtpase) in the other domain (chambers et al., 1991; valle & falgout, 1998; lescar et al., 2008) . ns3 protein is an essential member of the rc and is activated in association with ns5 to bind the genomic rna 3 0 sl prior to replication (chen et al., 1997; mackenzie et al., 1998; lindenbach et al., 2007) . beside its essential role in the viral replication cycle, ns3 can also induce apoptosis (shafee & abubakar, 2003; ramanathan et al., 2006; yang et al., 2009; yiang et al., 2013) . ns3 is involved in the inhibition of the transcriptional factor ap1 signaling pathway, which also directly affects apoptosis (lin et al., 2006) . studies on neurovirulence associated with denv-1 ns3 have shown that mutations in ns3 (leu480ser) enhance the ability of replication in cns causing leptomeningitis and encephalitis in mice (bordignon et al., 2007) . this mutation was also shown to induce cell death in denv-1-infected cells (duarte dos santos et al., 2000) . the ns3 protein can be targeted by antiviral drugs such as ivermectin, ribavirin as inhibitors of the atpase activity of helicases (mastrangelo et al., 2012) the (lindenbach et al., 2007) . its interaction with ns1 is essential for viral rna replication as ns4a acts as a docking site (bartenschlager et al., 1995; miller et al., 2007) . ns4a interacts with the polypyrimidine tract-binding protein (ptb), with the latter being implicated in translation, transcription, and viral processes for various viruses (bieleski et al., 2004; florez et al., 2005; shi & lai, 2005) including the role in regulation of hcv replication (tischendorf et al., 2004; domitrovich et al., 2005; aizaki et al., 2006; chang & luo, 2006) . ns4a-ptb plays an indirect role in stabilization of the viral genome dsrna intermediates by binding to host cell ptb protein in a similar manner as stabilizing wnv rna synthesis by the elongation factor 1 alpha (davis et al., 2007; jiang et al., 2009) . ns4a together with ns4b and ns3 is also responsible for inducing the rearrangement of the host er membrane leading to the formation of virus-induced membranous spherules and vesicles encasing the dsrna and rc; this is thought to minimize exposure of the replicating rna to innate immune sensors such as retinoic acid-inducible gene-i, rig-i roosendaal et al., 2006; miller et al., 2007) . the mature ns4a can also induce the pi3k-dependent autophagy signaling, which in turn lead to protection from camptothecin-and staurosporine-induced cell death (mclean et al., 2011) . the role of ns4a in the up-regulation of the autophagy and er rearrangement makes the protein a potential target for antiviral drug development † 2k 6841-6909, 2 kda, 23 aa complete cleavage of the vbfv polyprotein generates an er-spanning 2k peptide located between ns4a and ns4b (lin et al., 1993; roosendaal et al., 2006; miller et al., 2007) . the 2k peptide functions as a signal peptide for ns4b and might play a role in enhancing viral replication by conferring resistance to lycorine, a suppressor of flavivirus rna synthesis (zou et al., 2009 ) † miller et al., 2006) . ns4b appears also to be involved in er rearrangement and modification during viral replication (westaway et al., 1997) . the ns4b protein of wnv, jev, and denv inhibits type i interferon (ifn-a/b) response by blocking the phosphorylation of stat1 (muñoz-jord an et al., 2003 , 2005 lin et al., 2004; liu et al., 2005; evans & seeger, 2007) . the same is true for kunv and wnv strain ny99, which blocks the activation of stat1/ stat2 and its translocation to the nucleus (liu et al., 2005) . these events disrupt the host immune responses by blocking the ifnalpha/beta/gamma pathways † ns5 7666-10 374, 100 kda, 900 aa cytosol/er/nucleus ns5 is the largest and most conserved among the vbfv proteins. ns5 primarily functions as the rna-dependent rna polymerase (rdrp) through its c-terminal domain (lindenbach et al., 2007) . the ns5 has been crystalized and contains an n-terminal domain with s-adenosyl methionine methyltransferase activity (mtase), and possibly guanylyltransferase (gtase) for rna capping (egloff et al., 2002; malet et al., 2007; yap et al., 2007) . ns5 is also involved in activating other viral proteins, such as ns3 ntpase and rtpase activity in a dose-dependent manner. denv ns5 contains both nuclear localization signal (nls) and nuclear export signal (nes): these facilitate its importation into the nucleus by both importin b1 and the importin a/b complex, and an additional function of the signals could be enhancement of virus replication and hyperphosphorylation of ns5 (johansson et al., 2001; brooks et al., 2002) . for wnv, ns5 is exported from the nucleus in a chromosome region maintenance 1 (crm1)-dependent manner (pryor et al., 2007; rawlinson et al., 2009 ). ns5 is involved in different cellular pathways and has a crucial role in the escape from, or blocking, the interferonalpha/beta (ifn-alpha/beta) signaling pathway. denv ns5 inhibits host tyk2 and stat2 phosphorylation and prevents the activation of jak-stat signaling pathway (mazzon et al., 2009) in addition to the viral rdrp functions of ns5 described for the mbfvs, the tbfv ns5 was the first to be implicated in disrupting innate immune signaling. to suppress critical host responses, lgtv ns5 interacts with ifnar2 and ifngr2, two ifn receptor subunits, and antagonizes ifndependent responses by suppressing jak-stat signal transduction (best et al., 2005; park et al., 2007) . cellular responses targeted at restricting viral replication occur via lysosomal degradation of ns5, ns2b, and ns3. the ns5 of lgtv and tbev interacts with trim79-alpha, an ifn-inducible protein, and induces the degradation (taylor et al., 2011) . the cellular antiviral state is also compromised by tbev ns5 interaction with the scaffold protein scribble (werme et al., 2008) . this complex blocks the phosphorylation of stat1 and disrupts the jak-stat signaling pathway the extreme 3 0 terminal region contains secondary structures and rna elements important for cyclization of the genome and virus viability. the mbfv 3 0 utr can be divided into three regions (markoff, 2003; liu et al., 2009 ): a variable region directly upstream of the stop codon; a second region that is relatively conserved in nucleotide sequences containing hairpin motifs; and a highly conserved core element contains stable sl structures and cyclization motif (villordo & gamarnik, 2009) the 3 0 utr of tbfvs exhibits significant heterogeneity in length and sequence even among closely related strains. the tbfv 3 0 utr can also be divided into a variable region part and an extremely conserved core element (gritsun et al., 1997) , where the length variability of the variable region has been suggested to be related to the number of laboratory passages (mandl et al., 1998; mandl, 2005) . a hypothesis interprets the biological importance of the variable region that influences the replication/translation dynamics in mammalian and tick cells (elvang et al., 2011) . the conserved core element contains highly essential sequential and structural motifs defined as sl1-5 for viral maintenance (mandl et al., 1998; pletnev, 2001) . the pentanucleotide (gagag) motif exposed on the sl4 is strictly conserved among all the flavivirus genomes . other important elements present in the conserved region include the homopurine box, a homopyrimidine box, and cyclization sequences (mandl, 2005; gritsun & gould, 2007a, b) *nucleotide numbers are related to the strain neudoerfl of tbev. † shared function is presumed for both mosquito-and tick-borne flaviviruses. in many cases, no specific studies have been carried out in tbfv. brane (prm/m), and envelope (e), and seven nonstructural proteins: ns1, ns2a, ns2b, ns3, ns4a, ns4b, and ns5 (chambers et al., 1990; ryan et al., 1998; bollati et al., 2010) . the defined functions of these proteins are presented in table 1 . however, the precise function of some of the proteins remains to be elucidated. furthermore, it should be noted that the bulk of the studies have been carried out in mbfv systems, and it is possible that the functions may not be identical in tbfv. recognizing that the vbfvs cycle between vertebrate and arthropod hosts and that both viral and host factors are likely to be involved, it is highly probable that viral persistence is exceedingly complex. the purpose of this review is to evaluate the current literature on flavivirus persistence as well as to suggest ideas for additional research into this interesting and important area. humans are inadvertent targets of vbfv infection (figs 1 and 2), and infection is often associated with debilitating, acute neurological syndromes or hemorrhagic fever. however, there are several lines of evidence suggesting flavivirus persistence in humans (murray et al., 2010; gibney et al., 2011; baty et al., 2012) . the establishment of flavivirus persistence in humans seems to be mainly associated with encephalitic flaviviruses (diamond, 2003) . however, yfv and denv persistence has been described in eukaryotic cells in culture and in successive generations of aedes mosquitoes (lodge et al., 1987; takasaki et al., 2001; farfan-ale et al., 2009) . yfv and denv commonly cause a hemorrhagic illness, jaundice, and dengue shock syndrome, and encephalitis is atypical and extremely rare , and delivered to endosomes, where a ph-dependent fusion of the particles with the endosome membrane occurs (c). subsequent to uncoating, the single-stranded, positive-sense rna genome (d) migrates to the endoplasmic reticulum (er) and is translated (e) as a polyprotein traversing the er membrane several times (f). the polyprotein is cleaved into the viral proteins by viral and cellular proteases, although prm and e remain covalently attached. through the agency of several viral nonstructural proteins and cellular proteins, there is a proliferation of er-derived membranes and the formation of spherules that maintain a pore-like connection to the cytoplasm (g); the viral genome is replicated within these spherules by the viral proteins comprising the replication complex. by an as yet uncharacterized mechanism, progeny genomes are delivered to adjacent er membranes where the capsid protein mediates assembly and inclusion of prm-e into immature virions (h). the immature virions transit the golgi membrane system (i), and as a mild ph change occurs, the cellular enzyme furin cleaves the prm-e linkage (j), allowing the virus particle to assume its final mature stage prior to release from the cell (k). defined roles for the individual viral proteins are enumerated in table 1 . (tomori, 2004; gulati & maheshwari, 2007; varatharaj, 2010) , but persistence cannot be excluded. human infection with the encephalitic viruses almost always occurs following a bite from infected ticks or mosquitoes (figs 1 and 2) . generally, illness tends to be biphasic. the first phase is characterized by a flu-like illness with symptoms such as headache, arthralgia, and malaise. the second phase may present with neurological symptoms, such as mild meningitis to severe encephalitis. survivors of encephalitis may develop persistent neurological sequelae, suggesting neurological tissue damage and/ or viral persistence. neurological tissue damage, such as loss of neurons, has been reported for wnv infection (guarner et al., 2004) . long-term morbidity following tbev infection is known to occur frequently (haglund et al., 1996; haglund & gunther, 2003) . a study conducted in sweden reported chronic postencephalitic syndrome in 36% of patients who had been infected with tbev (haglund et al., 1996) . in up to 60% of patients who develop encephalitis due to wnv infection, neurological and/or neuropsychological symptoms continue to be reported for up to 7 years following the infection (murray et al., 2010; sadek et al., 2010) . the most definitive evidence for viral persistence would be the isolation of viable virus or the demonstration of viral antigens or rna long after the acute illness. several reports describe the isolation of infectious vbfvs from persistently infected humans. for example, the siberian tbev strain za was isolated from an individual who had harbored the virus for 10 years (gritsun et al., 2003) . he later died following 2 years of a progressive form of tbev encephalitis (gritsun et al., 2003) . in addition, isolation of jev from the cerebrospinal fluid was possible for more than 3 weeks following infection in 19% of individuals that had developed encephalitis (ravi et al., 1993) . jev persistence was also demonstrated in peripheral mononuclear cells in infected children in northern india (sharma et al., 1991) . in these children, virus could be isolated by culture 8-9 months after acute infection (sharma et al., 1991) . furthermore, wnv has been transmitted to patients who had received blood transfusions or organ transplants from asymptomatic donors (pealer et al., 2003; montgomery et al., 2006; cdc, 2009) , suggesting viral persistence in the donors as well as stored blood for transfusion. long-term presence of viral nucleic acid is another indicator for persistent infection; however, study results are inconsistent. viral rna can be readily sought using molecular biology techniques, such as rt-pcr and transcription-mediated amplification (tma) (s anchez-seco et al., 2005; muñoz-jord an et al., 2009; patel et al., 2013) . using tma, wnv rna was detectable in the blood of donors for up to 104 days following the index donation prince et al., 2008) . using rt-pcr, wnv rna could also be detected in urine of persistently infected individuals for up to 6.5 years following the acute phase of infection (murray et al., 2010) , although virus could not be isolated from the rna-positive urine samples. in contrast, wnv rna could not be detected after 6 years in urine in another study reported a year later (gibney et al., 2011) . baty and colleagues also failed to detect wnv rna by rt-pcr, but were able to detect viral rna using tma (baty et al., 2012) . similarly for jev, viral rna could not be detected by rt-pcr in the individuals who had anti-jev igm antibodies (zhao et al., 2013) . in the study by gibney et al. (2011) , wnv persistence could not be excluded. even though the results in these studies are somewhat variable, it seems reasonable to conclude that the presence of viral rna can also be taken as an indicator of persistent infection. viral serology may be an additional surrogate measure for vbfv persistence. in general, infection or vaccination leads to a sterilizing immunity, but long-term persistence of anti-vbfv igm antibodies has often been assumed to indicate continued exposure to viral antigens or virus particles (ravi et al., 1993; stiasny et al., 2012) . an exception to this is, of course, denv where antiviral antibody plays a key role in pathogenesis (martina et al., 2009; pierson, 2010) . igm antibodies induced by flaviviruses, such as tbev and wnv, are known to persist in serum and csf for 12 months or more (kapoora et al., 2004; stiasny et al., 2012) . indeed, igm antibodies against tbev persisted for up to 32 months (stiasny et al., 2012) . furthermore, anti-wnv igm antibodies persist in previously exposed blood donors for up 16 months prince et al., 2008) . however, igm antibody persistence does not necessarily correlate with infectious virus persistence. for example, persistent igm antibodies against tbev can be detected in some cases following vaccination without active viral infection (rendi-wagner et al., 2004; stiasny et al., 2012) . for wnv infection, igm antibodies against the nonstructural protein ns5 cannot be used to distinguish between recent/active infection from past infection (prince et al., 2008) . thus, serology may be a less useful marker for viral persistence. a major consideration in vbfv in humans would be identification of sites of viral persistence. as flavivirus persistence seems to be mainly associated with the neurotropic and encephalitogenic viruses, the central nervous system may well be the preferred site for viral persistence. furthermore, this may account for why patients who recover from encephalitis often have prolonged neurological symptoms. however, other studies by murray and colleagues postulated kidneys as a preferred site for the establishment of persistent flavivirus infections following the detection of wnv rna in urine (murray et al., 2010) . certainly, additional work is required to define the true incidence, the biology, and the pathogenic potential in humans of persistent vbfv infections. arthropod hosts play crucial roles in the biology of both tbfv and mbfv and contribute to persistent infection. in nature, ticks are the important arachnid reservoirs of tbfvs. the persistence of the tbfvs in arachnids is well established, and the role of ticks as long-term reservoirs and vectors for the viruses is clear. estimates suggest that ticks transmit about 25% of the known flaviviruses. tbfvs are capable of infecting about 14 tick species, but the ixodid ticks, ixodes scapularis and ixodes ricinus, account for nearly all transmission of tbev to humans. ixodes cookei may also harbor powv and dtv, while the soft-bodied onithodoros tick transmits alkhurma virus (main et al., 1979; charrel et al., 2007) . to transmit virus, these ticks need only 15 min of attachment to the host (crowder et al., 2013) . the virus is present in the tick salivary glands, and saliva constituents enhance infectivity (nuttall & labuda, 2003; girard et al., 2004) . a recent publication revealed that a selected subset of salivary gland genes is expressed when infected ticks feed on na€ ıve mice (mcnally et al., 2012) . ticks acquire the virus while feeding on infected rodents, typically by a process called 'cofeeding' (fig. 1 ; labuda et al., 1993a, b) , and the virus persists throughout several life stages. ticks at the larvae, nymph, and adult life stages can become infected by feeding on infected animals (horizontal transmission) or can transmit virus vertically across instars (transstadial transmission) and through the eggs (transovarial transmission; fig. 1a ; labuda et al., 1993a labuda et al., , 1996 nuttall & labuda, 2003) . indeed, results of a carefully controlled study in our laboratory show that transstadial transmission seems to be very high (mitzel et al., 2007) . the blood meal remains in the midgut for long periods of time, allowing infection of the epithelial cells lining the midgut with subsequent translocation into the hemocoel (nuttall & labuda, 2003) . infection of the midgut cells may be facilitated by the heterophagous nature of ticks (i.e. blood meal digestion is principally an intracellular process). in the open circulatory system, tissues and organs are bathed in hemolymph, which acts as a medium for transporting nutrients, hormones, and immune effector molecules. therefore, the hemolymph likely serves as a viral dissemination medium. tbe virus was found in the esophagus and subesophageal ganglion in dermacentor marginatus larvae and in columnar epidermal cells of dermacentor reticulatus nymphs (nuttall & labuda, 2003) . in d. reticulatus nymphs, tbe virus was demonstrated in epidermal cells and in vacuoles in the region of golgi complexes of salivary gland cells (nosek et al., 1984) . the precise mechanisms by which tbfvs traverse various tissues in the tick and reach the salivary glands remain to be fully elucidated. prevalence surveys indicate that 0.5-5% of ixodid ticks carry the virus in europe, but prevalence of up to 40% has been reported in russia (ustinova et al., 1997) . in north-central usa, up to 4.9% of i. scapularis ticks are infected by powassan or dtv (brackney et al., 2008b; dupuis et al., 2013) . interestingly, in a study carried out in chicago, i. scapularis was found to infest 2.8% of wild birds, which figure prominently in the mbfv cycle ( fig. 1b; hamer et al., 2011) . however, identification of tbfv was not attempted in this study. in brief, tbfv persistence in ticks is well established, and the essential role of ticks in the biology of these viruses is not in doubt. as is the case for tbfvs, the role of mosquitoes in the maintenance of the mbfv cycle is also well characterized. mosquitoes are thought to account for transmitting approximately 50% of more than 70 known flaviviruses (gould, 2001) . diverse mosquito species can be infected by mbfvs. however, the two major mosquito vectors are aedes aegypti (e.g. transmission of yfv and denv) and culex species (e.g. transmission of wnv and jev). adult female mosquitoes become infected when they obtain blood meals from flavivirus-infected animals, and virus replication in the mosquito has been well described (girard et al., 2005; mcgee et al., 2010; colpitts et al., 2012) . girard et al. (2004) used immunohistochemistry to demonstrate that wnv infects epithelial cells of the culex pipiens midgut and that viral antigen can be detected as early as day 2 postinfection. viral antigen staining becomes more intense in the cells of the midgut over time until day 14 to 21 following infection (girard et al., 2004) . using denv-2, virus titer in the midgut peaks to about 9 9 10 3 pfu ml à1 by 10 days postinfection, but declines to about 7.4 9 10 2 pfu ml à1 by day 12 (s anchezvargas et al., 2009 ). dissemination of virus into various tissues occurs at various time points, and the amount of antigen also varies. similar to tbfvs in ticks, the hemolymph serves as a vehicle for viral dissemination. however, virus also spreads in a cell-cell fashion to the muscle of the posterior midgut from 6 to 27 days postinfection (girard et al., 2004) . studies with denv-2 showed that infected mosquitoes mount an rna interference (rnai)-mediated antiviral response, but impairing the vector rnai resulted in increased viral replication (s anchezvargas et al., 2009) . this mechanism could be associated with observations of flavivirus-related rna that persists as dna in mosquitoes (crochu et al., 2004) . as mosquitoes are such efficient vectors for the transmission of the mbfvs, these reports suggest that the viruses have evolved a mechanism of evading the host response to persist in the vector. mosquitoes also acquire and deliver virus horizontally during blood meals and are competent to transmit vertically to progeny by transovarial passage (rosen et al., 1983) . wnv can persist overwinter in mosquitoes that hibernate in cold months. cool temperatures also facilitate persistence of flaviviruses in adult female mosquitoes. for example, st. louis encephalitis virus (slev) survived for more than 100 days of winter in culex quinquefasciatus (kramer & ebel, 2003) . similarly, jev and wnv have been transmitted by mosquitoes that carried the viruses at cold temperatures when exposed to temperature increases equal to ambient levels (kramer & ebel, 2003) . however, at low temperature, mosquitoes are less likely to acquire new infections (colpitts et al., 2012) . at 18°c, low wnv dissemination to the legs of the c. pipiens was observed, and rapid viral dissemination occurred at higher temperatures (colpitts et al., 2012) . furthermore, higher temperatures increase vector population growth rate and the rate of viral evolution in the mosquito (girard et al., 2005) . the prevalence and maintenance of wnv across landscapes is mediated by environmental factors, such as local effects of agriculture on vector and host communities (crowder et al., 2013) . results of investigations carried out in the states of oregon and washington showed that the prevalence of wnv in both c. pipiens and culex tarsalis was similar at 14.5% or 13.5%, respectively (crowder et al., 2013) . however, a study conducted in stratford, connecticut, showed that the c. pipiens was the most dominant mosquito captured in this wnv focus area (anderson et al., 2004) . in the captured mosquitoes, more than 85% of wnv isolations were from the same species, whereas culex salinarius accounted for between 5% and 12% (anderson et al., 2004) . in a similar study conducted in mexico, c. quinquefasciatus was more common at 48.3% and mbfv rna was detected in 70% of the pools (farfan-ale et al., 2009) . it is evident that mosquitoes are important as viral hosts and vectors. furthermore, it can be concluded that the mbfvs have evolved mechanisms of evading the host innate responses to persist in the mbfvs. the principal vertebrate hosts for vbfvs are small mammals, marsupials, and birds (fig. 1) , but the viruses can also infect reptiles (mackenzie et al., 2002 (mackenzie et al., , 2004 steinman et al., 2003; jacobson et al., 2005; root et al., 2005; marschang, 2011) . in most instances, larger animals, such as cervids, goats, and sheep, are incidental hosts. the 1999 outbreak of wnv in humans in new york is thought to be associated with wnv infection in birds (strausbaugh et al., 2001) . the mosquito-borne wesselsbron virus could also be cycled between mosquitoes and birds, but not much is known about the vertebrate host(s). thus, in nature, numerous species are susceptible to vbfvs and might serve as reservoirs or secondary amplifying hosts. in this section, we will survey the literature and the three potential markers for viral persistence: isolation of virus, identification of viral rna or protein, and viral serology. small mammals, particularly rodents, are the principal vertebrate hosts and reservoirs for tbfvs (mansfield et al., 2009; dobler et al., 2012) . in europe, the yellow-necked mouse (apodemus flavicollis) and bank vole (myodes glareolus) are implicated as the most common hosts, and they develop sufficient viremia to infect ticks that feed on them (weidmann et al., 2011; dobler et al., 2012; knap et al., 2012) . in various wild rodent species captured in brandenburg, germany, an average tbev infection rate of 15% was reported (achazi et al., 2011) . in the captured rodents, tbev rna was detected by rt-pcr in the brains and spleens. in north america, deer mice (peromyscus), squirrels, and the striped skunk (mephitis mephitis) are important reservoirs of powv and dtv (main et al., 1979; telford et al., 1997) . based on serological studies, a 6.2% dtv prevalence in the red-backed voles (myodes rutilus) was found in siberia and alaska (deardorff et al., 2013) . dtv in new mexico was serologically prevalent in peromyscus truei and peromyscus maniculatus at 22.2% and 6.0%, respectively (deardorff et al., 2013) . therefore, it seems likely that persistent infection of these small mammals occurs. deer may stabilize and maintain tbfvs at levels that are important for transmission (pugliese et al., 2007; carpi et al., 2009; mcgee et al., 2010; dobler et al., 2012) . while deer are important sources of blood meals for ticks, they develop low virus titer and are therefore not competent in transmission of tbfvs. however, in a broader sense, deer are important in viral persistence because they help sustain tick populations (cagnacci et al., 2012) . the situation for domesticated animals is also less clear than for rodents. dogs, horses, and monkeys can be infected with tbfvs, but case reports in veterinary practice are relatively infrequent (jaenson et al., 2012) . large domesticated animals, such as goats, sheep, and cattle, become viremic for a short while and develop antibodies following infection with tbev, but do not show any specific clinical signs of illness (mansfield et al., 2009; klaus et al., 2012) . however, transmission of tbev via milk from these domesticated animals (fig. 1) has been documented, suggesting that virus may persist following the acute viremia in at least some instances (vereta et al., 1991; caini et al., 2012; hudopisk et al., 2013) . birds and primates are considered to be the primary reservoirs of mbfvs. other mammals are generally accidental hosts, but this may not always be the case, and when viremic, these domestic animals are able to infect mosquitoes. for jev, the major amplifying hosts are birds and pigs, which attain high levels of viremia. they provide a source of infection for the mosquito species that subsequently transmit jev to humans (hukkanen et al., 2006; van den hurk et al., 2009) . jev control has been achieved through vaccination of pigs and humans in korea, japan, and taiwan (igarashi, 2002) while horses are considered dead-end hosts of jev infection due to a very low level of viremia. in geographic regions where pig populations are low, swine may not be important for zoonotic transmission of jev. herons and egrets are important jev-amplifying hosts and source of infection for mosquito species that transmit jev to humans (nemeth et al., 2009) . wild-caught pigeons were shown to have antibodies that persist for up to 15 months. it is not clear whether the antibody persistence is associated with viral persistence. although various bird species do play a prominent role in jev infection, evidence of birds as a source of persistent infection is less certain. in some instances, birds can acquire jev viremia and then fail to develop or lose neutralizing antibodies. birds are also excellent amplifying hosts for wnv, and migratory birds can move the viruses to different areas because they become viremic for several days. near 100% mortality is associated with natural or experimental wnv infection in birds, such as american crows (corvus brachyrhynchos), blue jays (cyanocitta cristata), and greater sage-grouse (centrocercus urophasianus; steele et al., 2000; komar et al., 2003; clark et al., 2006) , but some birds are able to carry the virus for a longer time before they develop antibodies or succumb to disease (mckenzie & goulet, 2010) . for instance, a combined 37% of house sparrows that were either naturally or experimentally infected with wnv tested positive for wnv rna by rt-pcr (wheeler et al., 2012) . in the same study, wheeler et al. (2012) showed that 97% and 100% of wnv-infected house sparrows and finches, respectively, seroconverted following exposure to wnv. birds that develop neutralizing anti-wnv antibodies are protected from reinfection (nemeth et al., 2009 ), suggesting total virus clearance in seroconverted birds, and antibodies against wnv have been found in wild birds, suggesting exposure to wnv. early demonstration of wnv persistence was described in blue-gray pigeons, from which virus was isolated up to 100 days postinfection, and viral antigen could be detected in liver tissue for up to 180 days (ruiz et al., 2010) . american robins (tardus migratorius) are known to be competent reservoirs of wnv and slev (kilpatrick et al., 2006) . wnv persistence was also demonstrated by the detection of viral rna in the presence of antibody for up to 36 weeks in spleens of naturally infected house sparrows and finches (wheeler et al., 2012) . taken together, it seems that there is decent evidence for persistent infection by wnv in some bird species. however, many details about the biology of persistence remain to be studied. antibodies to various flaviviruses, such as slev, powv, jev, and wnv, have been identified in chelonians, snakes, and crocodiles in different geographic locations (steinman et al., 2003; marschang, 2011) , and it is known that alligators and crocodiles can be infected with wnv. however, there are no reports suggesting that reptiles host persistent mbfv infection. in summary, it is apparent that various animals and birds can sponsor persistent vbfv infection. nevertheless, the precise role that these persistent infections play in larger scheme of viral maintenance merits additional study. there are several animal models of persistent mosquito-borne flavivirus infection, including wnv and slev (charlier et al., 2004; kimura et al., 2010) . as shown in table 2 , mice and hamsters have been the main study models. in a nonhuman primate study, wnv was isolated from central nervous system tissues more than 5 months post-intra-cerebral inoculation , suggesting that these species might also be relevant system. persistent wnv has also been studied experimentally in the golden hamster. the clinical outcomes of wnv infection in hamsters vary and depend on animal age and immune competence, viral dose, and route of infection. wnv infection can lead to asymptomatic illness, encephalitis, severe paralysis, and acute death (xiao et al., 2001; morrey et al., 2004; tesh et al., 2005) . adult golden hamsters infected with wnv develop chronic infection, characterized by shedding of virus in urine for up to 8 months (xiao et al., 2001; ding et al., 2005; tonry et al., 2005) . the chronically infected animals exhibit antigens in tubular epithelial cells, interstitial cells, and macrophages of distal renal tubules . interestingly, na€ ıve hamsters inoculated intraperitoneally with wnv-containing urine from persistently infected hamster do not develop clinical disease, but the hamsters become viremic and develop antibody responses (ding et al., 2005) . this suggests possible viral genetic changes, which may facilitate persistent infection, although other explanations cannot be totally excluded. hamsters have also been used as experimental models to study the persistence of slev and are among the natural vertebrate hosts of the banzi flavivirus, a member of the yfv group of flaviviruses (grard et al., 2010) . golden hamsters infected with slev did not develop clinical signs of illness, but they developed viruria and antibodies against slev at 28 days postinfection (siirin et al., 2007) . (appler et al., 2010; pierson & diamond, 2012) : wnv rna persisted in a pantropic manner in 12% of infected mice for up to 6 months. infectious virus could be isolated in 12% of mice for up to 4 months. c3h mice survival rate was lower and 22% when compared to the survival rate of b6 mice, which was 78% macaque rhesus (pogodina et al., 1981) : virus persisted in asymptomatic animals for 5½ months and could be isolated from cerebellum, cerebral subcortical ganglia, lymph nodes, and kidneys golden hamster (mesocricetus auratus; tesh et al., 2005; tonry et al., 2005) : chronic renal infection with persistent shedding of virus for up to 8 months. virus could be recovered by culture, and genotypic and phenotypic changes were identified house sparrow (passer domesticus; nemeth et al., 2009) : infectious virus persisted in tissues for up to 43 days, but was not detectable in sera after 6 days. wnv rna persisted in tissues for up to 65 days slev golden hamster (siirin et al., 2007) : infected animals remained asymptomatic, but virus could be cocultivated in various organs for up to 185 days jev swiss albino mice (mathur et al., 1986a, b) : persistence was demonstrated by reactivation in 41% of congenitally infected pups. in adult mice, viral persistence was shown to last longer (16 weeks) in pregnant mice compared with 4 weeks in nonpregnant mice tick tbev macaque rhesus (pogodina, 1983; pogodina et al., 1984 pogodina et al., , 1981 : monkeys recovered from encephalitis and virus persisted for at least 738 days. in asymptomatic animals, virus persisted for 302 days liv* immunosuppressed guinea pigs (zlotnik et al., 1971) : liv was lethal in young animals, but older animals acquire a nonapparent infection with viral replication in the brain and spleen powv deer mouse (peromyscus leucopus; telford et al., 1997) : not a well-characterized model, but adult mice appear to survive infection *louping ill virus infects sheep. hamsters that become persistently infected with slev shed virus in their urine for up to 185 days postinfection. the shedding of virus in the urine of infected hamsters also suggests that the kidneys could be an organ preferred for viral persistence. c57bl/6j mice maintained a persistent wnv infection in the face of robust neutralizing antibody levels for more than 6 months (appler et al., 2010; stewart et al., 2011) . in this study, infectious virus was recovered in the skin, and viral rna was identified in the skin, as well as the spinal cord and brain (appler et al., 2010) . in contrast, wnv persistence was uncommon in kidneys and rare in the heart (appler et al., 2010) . however, in another study in which c57bl/6j mice were infected with wnv derived from the urine of persistently infected hamsters, the kidney was found to be the preferred organ for viral persistence (saxena et al., 2013) . the hamster-derived wnv was also found to be highly attenuated in both neuroinvasiveness and neurovirulence in infected mice (saxena et al., 2013) . these results also suggest that the virus acquires phenotypic changes to be able to persist. while the inbred laboratory mouse models are patently useful for understanding some aspects of flavivirus persistence, very few natural rodent hosts have been established as experimental animal models. evidence of exposure to wnv has been reported in rodent species, such as rats (rattus), bank voles (m. glareolus), and deer mice (peromyscus; molnar et al., 1976; root et al., 2005; docherty et al., 2006; gomez et al., 2008) . however, infectious virus was only identified in the bank vole, whereas antibodies were detected in all of the species (molnar et al., 1976; root et al., 2005) . although persistence in mammals obviously plays a significant role in tbfv infections, little attention has been devoted to experimental studies of this aspect of tbfv biology. while disease or therapy models have been established for tbfvs (kreil et al., 1997; holbrook et al., 2005; hayasaka et al., 2010; palus et al., 2013) , none of these specifically investigated viral persistence. experiments to determine the susceptibility of several wild and domesticated mammals to powv showed that viremia lasted for 0-3 days in the goat, pig, skunk, red fox, and gray fox (yiang et al., 2013) . in another study, adult deer mice (peromoyscus leucopus) survived challenge with powv without apparent illness, but evidence of viral persistence was not sought (telford et al., 1997) . development of suitable experimental models for persistent tbfv infection would clearly provide useful information about this aspect of these important pathogens. the previous sections have looked at flavivirus persistence in humans, animals, and arthropod vectors, as well as some relevant animal models. in this section, we will survey information related to the initiation and maintenance of persistence. when mammalian cell cultures are acutely infected with tbfv, a legion of general cellular defense and antiviral systems are triggered, as are specific factors designed to limit or restrict virus reproduction. some of these include type i interferon (ifn-a/ifn-b), type iii interferon (ifn-k), mitochondrial activated signaling, and the induction of inflammatory factors, such as interleukins (tam & messner, 1999; madden, 2003; van gerpen, 2003) . for example, the ifn-induced tripartite motif protein, trim79a, has been shown to restrict tbev replication by degrading ns5 (taylor et al., 2011) . the unimpeded deployment of these antiviral factors and systems would lead to cell death. cell death is thought to be mediated primarily through apoptosis (r u zek et al., 2009) , and programmed cell death induced by various tbfvs has been described in neurons, epithelial cells, hepatocytes, kupffer cells, and neuroblastoma cells (ramanathan et al., 2006) . impeding or evading the antiviral response is one characteristic of vbfvs, which plays an important role in viral persistence. during flavivirus infection, ifn production is induced within hours, but viral rna replication complexes are enclosed in vesicles, which may offer protection from recognition by pathogen recognition receptors, thus delaying ifn production (welsch et al., 2009; gillespie et al., 2010; overby et al., 2010; offerdahl et al., 2012; ye et al., 2013) . in addition, some vbfvs can directly affect ifn secretion by inhibiting ifn gene transcription, suppressing ifn signaling or impairing the functions of interferon-stimulated genes (best et al., 2005; robertson et al., 2009; ye et al., 2013) . the humoral and cell-mediated immune responses are also prone to inhibition by vbfvs. studies with wnv in c57bl/6 mice suggest that wnv-specific antibodies correlate with decreased spread to the cns (diamond, 2003) . antibody escape mutants could also evade the t cell recognition (diamond, 2003) , but a precise role of these in viral persistence is yet to be defined. the e protein is thought to be responsible for provoking the cytopathic effect (isaeva et al., 1998; prikhod'ko et al., 2001) . however, the ns3 protease and ns2b-ns3 protease precursor (table 1) are also known to induce apoptosis by binding to caspase 8 (shafee & abubakar, 2003; ramanathan et al., 2006; safronetz et al., 2013) . in addition, ns2a has also been implicated in causing ifn-independent cytopathic effect (chang et al., 1999; melian et al., 2013) . observations in our laboratory (l. mlera, d. offerdahl & m.e. bloom, unpublished results) and those of others (lancaster et al., 1998) indicate that tbfv infection in mammalian cell culture is characterized by an acute phase, which kills most infected cells; however, a small number of cells survive the initial cytolytic phase, and the culture is repopulated with cells almost all of which are infected. clearly, vbfv induces an acutely cytopathic infection in mammalian cells, and thus, the development of a persistent infection implies that cell death factors must be evaded or modulated, although the precise mechanisms are still obscure. the implication of specific viral proteins, domains, or sequences that combat cytolytic cell death has been rather limited, but any viral determinants that limit cell death in the face of acute infection might also enhance the initiation of persistent infection. for example, viral ns4a (table 1) was shown to induce phosphatidylinositol 3-kinase (pi3k)-dependent autophagy and thereby leading to protection from cell death (mclean et al., 2011) . the manipulation of apoptosis by jev, which activates pi3k in infected cells, was also reported (lee et al., 2005) . jev triggers apoptosis during the late stages of infection, and the activation of pi3k is thought to provide protection from early cell death. limited replication implies a low level of viral protein expression and may favor viral persistence. over the years, significant attention has been focused on defective interfering (di) virus particles, which limit replication of the wild-type virus (schmaljohn & blair, 1977; debnath et al., 1991; blitvich et al., 1999) . the di particles represent truncated genomes that can be replicated and encapsidated and compete with wild-type viral particles when they infect cells. in vesicular stomatitis virus (vsv), di particles are thought to modulate virulence (cave et al., 1985) . however, the role of vbfv di particles in persistence is not completely certain. for tbev, the c protein is reported to tolerate internal deletions ranging from 4 to 21 amino acids, and the deletions seem to favor attenuation and immunogenicity (kofler et al., 2002) . di particles of vbfvs, such as murray valley encephalitis (mve), tbev sofjin strain, and wnv, are known to generate truncated ns1 proteins, following infection at high multiplicity of infection (debnath et al., 1991; poidinger et al., 1991; chen et al., 1996; bugrysheva et al., 2001) . persistent infection of vero cells with mve was associated with a truncated ns1, whereas this form of ns1 was not noted in the acute infection (brinton, 1982; lancaster et al., 1998) . the truncated ns1 in mve virus was a result of the presence of di rna, which contained a large internal deletion (lancaster et al., 1998) . in this case, mve di particles reduced the wild-type mve titer by 75-95% (poidinger et al., 1991) . however, a causal role for the mve di particles in maintaining persistent infection was not conclusively demonstrated. studies of the far eastern sofjin strain of the tbev complex identified a 39-kda truncated ns1 in both acutely and persistently infected human kidney rh cells (bugrysheva et al., 2001) . for wnv, naturally occurring di particles interfere with transmission in mosquitoes and minimally impact pathogenesis in mice (pesko et al., 2012) . furthermore, truncated denv rna species, suggesting the presence of di particles, have been identified in acute human infections (li et al., 2011) , but have not been described in other acute vbfv infections in humans. similarly, banzi virus di particles seem to have been generated in resistant c3h/rv mice (smith, 1981) . although di particles and the truncated ns1 may be frequently observed in persistent infections, it is not at all clear that they are independently sufficient for the establishment and maintenance of a persistent infection in vitro or in vivo. stable expression of truncated ns1 failed to render persistent infection with jev, suggesting that truncated ns1 is a consequence rather than a cause for viral persistence (liao et al., 1998) . the role of di particles merits further investigation, but the role of other aspects of virus biology should also be scrutinized. restricted expression of the envelope (e) protein may also favor development of persistence. in kn73 cells that were persistently infected with jev, the e protein was found to be expressed at markedly low levels compared with acutely infected cells (feng et al., 2002) . in these studies, the expression of ns3 was found to be unchanged in the acute and persistent phases of jev infection (feng et al., 2002) . as the e protein is important for pathogenesis and immunity (cdc, 2009), low-level expression could result in immune tolerance and contribute toward viral persistence. in addition to low expression levels, mutations in the e protein of jev, yfv, and wnv might also play a role in vbfv persistence (ding et al., 2005; farfan-ale et al., 2009) . a 138e?k mutation in the e protein of jev and wnv was shown to inhibit cell-cell spread of the virus and to contribute toward the development of a small-plaque phenotype (carson et al., 2006) . while this mutation leads to viral attenuation (carson et al., 2006) and is key in the attenuation of jev sa14-14-2 vaccine (monath et al., 2002) , further elucidation of mutations that may play a role in persistence is required. observations that hamsters and mice infected with mbfvs obtained from urine of other mbfv-infected animals do not suffer severe disease and become persistently infected indicate that the virus is attenuated (rosen et al., 1983; ding et al., 2005) . a number of amino acid-changing mutations in c, e, ns1, ns2a, ns2b, and ns5 were reportedly associated with persistence of wnv in serially passaged hamsters (s anchezvargas et al., 2009) . these mutations are thought to have attenuated the virus (s anchezvargas et al., 2009) . however, the same mutations have not been reported by other groups, suggesting that the mutations may not be specific to development or maintenance of viral persistence. mechanisms not directly involving viral proteins may also play a significant role in persistence. for instance, jev delays the exposure of dsrna to innate sensors and inhibits phosphorylation of irf3 via noncoding viral short flaviviral rna (espada-murao & morita, 2011; chang et al., 2013) . similarly, wnv delays recognition by pathogen recognition receptors by activating irf3 in a rig-i-dependent manner without antagonizing the host ifn response (fredericksen & gale, 2006) . the mechanism of evasion is currently not clear, but membrane-bound vesicles that enclose the rig-i-activating dsrna of tbev have been described (overby et al., 2010) . as the delayed recognition of viral pathogen-associated molecular patterns is only temporary, these manipulations are probably just among the suite of mechanisms deployed for the establishment of viral persistence. the specific viral genes and how they manipulate apoptosis at a cellular level needs further examination. specific host genes that may contribute toward the development of flavivirus persistence in the vertebrate host have not been defined, but there are some suggestions. for instance, the overexpression of the proto-oncogene bcl-2 was reported to prevent apoptosis and promote persistence in jev-infected bhk and cho cells (liao et al., 1998) , suggesting that the control of apoptosis is likely to be implicated. in addition, some inbred laboratory mice strains can carry the 2 0 -5 0 -oligoadenylate synthetase gene, flv r , which confers flavivirus resistance (urosevic et al., 1997) . the animals are productively infected with flaviviruses, but produce low virus titer (brinton & perelygin, 2003; barkhash et al., 2010) . mice that are susceptible to flavivirus infection carry the flv s allele. the flv r -like allele has also been characterized in wild mice and could be a partial explanation of flavivirus persistence in rodents in nature (urosevic et al., 1997) . additional vertebrate genes, apart from the flv, could play a role in varied susceptibilities of different mouse strains to flavivirus infection. this was suggested from recent observations that tbev-infected balbc mice were moderately resistant, sts mice are highly resistant, whereas the balbc/sts recombinant mice were highly sensitive to infection (palus et al., 2013) . immunosuppression may also be a potentiating factor for the establishment of flavivirus persistence in animal hosts. for example, wnv persistence was demonstrated by the detection of wnv rna and immunohistochemistry in brain tissue of an immunosuppressed 57-year-old man 4 months after the initial diagnosis (penn et al., 2006) . the follicular b cell lymphoma, from which he had suffered, may have facilitated, or aggravated, the persistence of wnv. in mice, transient immunosuppression with cyclophosphamide leads to wnv recrudescence (appler et al., 2010) , an observation suggesting that some aspects of the immune system operate to restrict wnv replication during persistence. however, the situation is complicated because wnv is able to persist for up to 16 months in the face of a robust humoral immune response in c57bl/6 mice (appler et al., 2010) . furthermore, wnv persistence was reported in the brains of cd8 + t cell-deficient rodents (shrestha & diamond, 2004) , but the cd8 + t cell deficiency did not affect the antibody response in this mouse model. knowledge about specific immune system components that could facilitate or control viral persistence remains to be characterized. infection by vbfvs of arthropod cells has not received the same degree of study; however, acute infection is not accompanied by the same cytopathic response observed in mammalian cells. in addition, very little is known about how or in what tissues tbfvs persist in the ticks, but the viruses likely evolved mechanisms of modulating or evading the tick immune system over the millenia (robertson et al., 2009) . persistent infection of the i. scapularis cell line ise-6 (munderloh et al., 1994) was readily established and was noncytopathic (offerdahl et al., 2012) . detailed studies of persistent tbfv infection in arthropod cell lines using contemporary techniques and methods are certain to yield useful and interesting information. the mechanism(s) of how mbfvs persist in mosquitoes also remains a suitable topic for investigation. slev persists in the midgut of c. pipiens for hours before infecting the midgut epithelium (whitfield et al., 1973; brackney et al., 2008a) , but this is a short time. interestingly, approximately two-thirds of flavivirus-related sequences were reportedly detected as integrated dsdna form in laboratory-bred and wild aedes mosquitoes (crochu et al., 2004) , although detection of a complete flavivirus genome was not reported. furthermore, the finding of partial flavivirus-like sequences in dna form is not clear. clearly, research into the biology of flavivirus persistence in mosquitoes and ticks has been limited and is worthy of extensive additional research. in summary, it should be apparent that both viral and host factors play a role in the initiation and maintenance of persistent infection at both the cellular and organismal levels. in addition, successful persistence in nature almost certainly depends upon ecological and environmental forces, but these latter factors are wholly beyond the scope of this limited review. multiple rna and dna viruses are known to establish persistence in culture as well as in humans and animals. consideration of several may provide insight, if not direct parallels, useful in the study of biology of persistent vbfv infections. the hepacivirus genus, containing hepatitis c virus (hcv) species, belongs to the flaviviridae family, together with the flavivirus genus under which vbfvs are classified. despite the virus-specific cytotoxic t lymphocytes and antibodies, persistent hcv infections, which are established with high efficiency, are known to occur in humans and animals, such as chimpanzees (main et al., 1979; caini et al., 2012; mcnally et al., 2012) . hcv persistence is associated with various strategies, such as the high genetic variability that facilitates passive immune evasion. in vivo, hcv fails to activate cd4 + t cells, leading to exhaustion of cd8 + t cells. at cellular level, hcv can block interferon induction by blocking rig-i and mitochondrial antiviral signaling (mavs) using its ns3-ns4a protease, which cleaves the ifn promoter-stimulator 1 (gould, 2001; muñoz-jord an et al., 2005; baril et al., 2009; hudopisk et al., 2013; perera-lecoin et al., 2013) . hantavirus infections are another interesting example of viral persistence. hantaviruses are segmented, rna viruses that cause lifelong infections in their reservoir rodent hosts, despite high levels of neutralizing antibodies (botten et al., 2000; meyer & schmaljohn, 2000; easterbrook & klein, 2008) . pathogen recognition receptors, such as rig-i and tlr7, are not elevated in the lungs of infected rats, suggesting that evasion of viral recognition may contribute toward the establishment of a persistent infection. perhaps, the reason for noninduction of rig-i is the fact that hantaviruses do not produce detectable amounts of dsrna (wang et al., 2011) . ifns, such as ifn-b, ifn-k, mxa, and pro-inflammatory chemokines, cytokines, and transcription factor genes are elevated midway in the infection followed by a down-regulation that favors the expression of tgf-b (schountz et al., 2012) . a continued up-regulation of the cytokines could be detrimental to the cell, and the virus would fail to persist. results of a recently established animal model also show that host-adapted snv achieves prolonged and disseminated infection, with no disease in hamsters (safronetz et al., 2013) . therefore, hantaviruses may have evolved mechanisms of manipulating target genes, which establishes persistence when induced. one of the best-studied models of persistent rna virus infection is the rodent-borne arenavirus, lymphocytic choriomeningitis virus (lcmv). in lcmv clone 13 (cl13), a glutamine at position 1079 in the glycoprotein (lysine in the arm 53b strain of lcmv) is important for persistence, but its precise function is unknown (salvato et al., 1991; moskophidis et al., 1995) . the mechanisms of lcmv persistence are linked to the down-regulation of mhc and costimulatory molecules, inflammatory cytokines, as well as virus-induced production of immunosuppressive cytokines (ng et al., 2011) . the inability of cd134-deficient mice to control lcmv infection over time was reported to be a result of cd4 and cd8 responses (boettler et al., 2012) . in addition, ox40 also has a role in the establishment of persistent lmcv infection (boettler et al., 2012) . a recent report showed that ifn blockade of type i ifn signaling results in a cd4 t cell-dependent clearance of lcmv cl13 (teijaro et al., 2013) . this is an interesting observation considering that ifn induction is part of the antiviral response and that its induction can lead to apoptosis. however, the precise mechanism of persistence is not completely clear (easterbrook & klein, 2008) , but it also demonstrates 'clever' viral manipulation of the host system to establish persistence. coxsackievirus infections are another system from which lessons might be drawn. coxsackievirus b3 has been implicated toward the development of certain chronic muscle diseases, such as chronic inflammatory myopathy (lodge et al., 1987; tam & messner, 1999) . coxsackievirus persistence in cell culture can take two forms: (1) an incurable steady state characterized by nonlytic virus infection and (2) an antiviral-curable carrier culture system (brinton, 2013; pierson & kielian, 2013) . while mechanisms of coxsackievirus persistence are not completely understood, down-regulation of the coxsackievirus receptor (car) has been suggested to play a role in viral persistence (varatharaj, 2010) . down-regulation of car in coxsackievirus-infected hl-1 cells occurs rapidly from 60% following three passages to 90% at passage 8 (varatharaj, 2010) . the importance of car down-regulation is emphasized by the fact that in vitro car knockout results in reduced viral replication as well as virus-induced cell lysis (tomori, 2004; gulati & maheshwari, 2007) . these selected examples simply highlight the diversity of possible mechanisms that the vbfv might harness in initiating and maintaining persistence and provide concepts that might be useful to investigate. in the preceding sections, we surveyed a substantial literature with relevance to various aspects of flavivirus persistence. elucidation of how flaviviruses persist in humans could help toward the development of therapeutic interventions that could alleviate morbidity and budgetary burdens associated with neurological sequelae. despite the current knowledge, a relative dearth of knowledge still exists, and additional research is merited on these significant human pathogens. for instance, the definition of specific viral proteins and cellular factors and their interactions in the establishment and maintenance of persistent infection is very limited. as noted, for flaviviruses to persist in infected cells in culture or in vivo, specific host defenses need to be evaded or controlled. the role of ns5 as an interferon antagonist (especially in tbfvs) has been established (best et al., 2005) , but its ifn antagonism in the context of vbfv persistence has not been fully explored. this is also true for mbfv ns4b, which impairs ifn-a/b induction via jak/stat signaling (muñoz-jord an et al., 2005) . furthermore, mutations in ns2a of kunjin virus result in increased ifn levels, suggesting an ifn antagonistic role of ns2a (liu et al., 2004) . intriguing is the fact that vbfvs antagonize ifn even in the cells that will eventually die in the acute phase of infection. as ifn antagonism seems to be important for hcv, interrogating the role of these vbfv proteins to ascertain their role in the establishment of viral persistence will be critical. the specific mammalian host immune responses that are evaded or controlled also need to be identified precisely. although overexpression of bcl-2 in bhk and cho cells resulted in the inhibition of apoptosis and jev persistence, a direct viral effect on bcl-2 was not determined (liao et al., 1998) . it would be useful to understand which, and how, vbfv proteins interact with the bcl-2 pathway. indeed, other host pathways could be involved and need to be elucidated further. the exact correlates of persistent flavivirus infection in the arachnid or insect vector host also need to be determined. the biology of ticks and that of mammals is completely different. for ixodid tick vectors, identifying genetic or biological targets that could play a role in viral persistence is imperative. these vector mechanisms might find application, when known, in efforts to control natural flavivirus persistence cycles in ticks. the mechanism(s) could involve viral genetic changes, such as specific mutations that may render the virus less detectable in infected cells. an important question for transmission and viral evolution dynamics is why arthropod vectors do not appear to be killed by viral infection. the down-regulation of the car, as a coevolutionary development that favors persistence, is intriguing (fechner et al., 2007; pinkert et al., 2011) . for flaviviruses, various receptors have been identified as possible virus-entry mechanisms (smit et al., 2011; perera-lecoin et al., 2013) . investigations into whether there is a down-regulation of flavivirus receptors, as in coxsackievirus persistence, could be useful in defining flavivirus persistence mechanism (s). finally, the establishment of relevant animal models of vbfv persistence will also be crucial for understanding the dynamics of viral persistence and host responses. animals that serve as the natural host reservoirs will be key in developing these models. some models have been established, but may have failed to answer ecologically important question. thus, future work will combine studies encompassing the biology of vbfvs, molecular cell biology, animal models, and eventually virus-host ecology. rodents as sentinels for the prevalence of tick-borne encephalitis virus polypyrimidine-tract-binding protein is a component of the hcv rna replication complex and necessary for rna synthesis long-range rna-rna interactions circularize the dengue virus genome prevalence of west nile virus in tree canopy-inhabiting culex pipiens and associated mosquitoes persistence of west nile virus in the central nervous system and periphery of mice crystal structure of a novel conformational state of the flavivirus ns3 protein: implications for polyprotein processing and viral replication vascular leakage in severe dengue virus infections: a potential role for the nonstructural viral protein ns1 and complement antagonism of the complement component c4 by flavivirus nonstructural protein ns1 mavs dimer is a crucial signaling component of innate immunity and the target of hepatitis c virus ns3/4a protease variability in the 2 0 -5 0 -oligoadenylate synthetase gene cluster is associated with human predisposition to tick-borne encephalitis virus-induced disease complex formation between the ns3 serine-type proteinase of the hepatitis c virus and ns4a and its importance for polyprotein maturation evaluation for west nile virus (wnv) rna in urine of patients within 5 months of wnv infection identification of neutralizing epitopes within structural domain iii of the west nile virus envelope protein molecular determinants of virulence of west nile virus in north america envelope protein glycosylation status influences mouse neuroinvasion phenotype of genetic lineage 1 west nile virus strains inhibition of interferon-stimulated jak-stat signaling by a tick-borne flavivirus and identification of ns5 as an interferon antagonist a polypyrimidine tract facilitates the expression of kaposi's sarcoma-associated herpesvirus vflip through an internal ribosome entry site a novel complex formed between the flavivirus e and ns1 proteins: analysis of its structure and function identification and analysis of truncated and elongated species of the flavivirus ns1 protein ox40 facilitates control of a persistent virus infection structure and functionality in flavivirus ns-proteins: perspectives for drug design dengue neurovirulence in mice: identification of molecular signatures in the e and ns3 helicase domains experimental infection model for sin nombre hantavirus in the deer mouse (peromyscus maniculatus) the effects of midgut serine proteases on dengue virus type 2 infectivity of aedes aegypti stable prevalence of powassan virus in ixodes scapularis in a northern wisconsin focus structure of a flavivirus envelope glycoprotein in its low-ph-induced membrane fusion conformation characterization of west nile virus persistent infections in genetically resistant and susceptible mouse cells. i. generation of defective non plaquing virus particles replication cycle and molecular biology of the west nile virus genetic resistance to flaviviruses the interdomain region of dengue ns5 protein that binds to the viral helicase ns3 contains independently functional importin beta 1 and importin alpha/beta-recognized nuclear localization signals tick-borne encephalitis virus ns1 glycoprotein during acute and persistent infection of cells virus and antibody dynamics in acute west nile virus infection effects of deer density on tick infestation of rodents and the hazard of tick-borne encephalitis. i: empirical assessment tick-borne encephalitis transmitted by unpasteurised cow milk in western hungary prevalence and genetic variability of tick-borne encephalitis virus in host-seeking ixodes ricinus in northern italy long-term clinical and neuropsychological outcomes of west nile virus infection defective interfering virus particles modulate virulence west nile virus transmission via organ transplantation and blood transfusion -louisiana nucleotide changes responsible for loss of neuroinvasiveness in japanese encephalitis virus neutralization-resistant mutants flavivirus genome organization, expression, and replication processing of the yellow fever virus nonstructural polyprotein: a catalytically active ns3 proteinase domain and ns2b are required for cleavages at dibasic sites west nile virus envelope proteins: nucleotide sequence analysis of strains differing in mouse neuroinvasiveness the polypyrimidine tract-binding protein (ptb) is required for efficient replication of hepatitis c virus (hcv) rna membrane permeabilization by small hydrophobic nonstructural proteins of japanese encephalitis virus japanese encephalitis virus non-coding rna inhibits activation of interferon by blocking nuclear translocation of interferon regulatory factor 3 rodent models for the study of therapy against flavivirus infections alkhurma hemorrhagic fever virus in ornithodoros savignyi ticks persistence of japanese encephalitis virus is associated with abnormal expression of the nonstructural protein ns1 in host cells rna-protein interactions: involvement of ns3, ns5, and 3 0 noncoding regions of japanese encephalitis virus genomic rna susceptibility of greater sage-grouse to experimental infection with west nile virus west nile virus: biology, transmission, and human infection sequences of flavivirus-related rna viruses persist in dna form integrated in the genome of aedes spp. mosquitoes west nile virus prevalence across landscapes is mediated by local effects of agriculture on vector and host communities purification and crystallization of dengue and west nile virus ns2b-ns3 complexes interaction between the cellular protein eef1a and the 3 0 -terminal stem-loop of west nile virus genomic rna facilitates viral minus-strand rna synthesis powassan virus in mammals in vitro homotypic and heterotypic interference by defective interfering particles of west nile virus evasion of innate and adaptive immunity by flaviviruses nucleotide and amino acid changes in west nile virus strains exhibiting renal tropism in hamsters epidemiology and distribution of tick-borne encephalitis west nile virus antibody prevalence in wild mammals role of la autoantigen and polypyrimidine tract-binding protein in hcv replication determinants in the envelope e protein and viral rna helicase ns3 that influence the induction of apoptosis in response to infection with dengue type 1 virus isolation of deer tick virus (powassan virus, lineage ii) from ixodes scapularis and detection of antibody in vertebrate hosts sampled in the hudson valley immunological mechanisms mediating hantavirus persistence in rodent reservoirs update on powassan virus: emergence of a north american tick-borne flavivirus structural basis of flavivirus ns1 assembly and antibody recognition an rna cap (nucleoside-2 0 -o-)-methyltransferase in the flavivirus rna polymerase xml: crystal structure and functional characterization interplay of rna elements in the dengue virus 5 0 and 3 0 ends required for viral rna replication the 5 0 and 3 0 downstream aug region elements are required for mosquito-borne flavivirus rna replication west nile virus rna not detected in urine of 40 people tested 6 years after acute west nile virus disease the endoplasmic reticulum provides the membrane platform for biogenesis of the flavivirus replication complex west nile virus dissemination and tissue tropisms in orally infected culex pipiens quinquefasciatus ultrastructural study of west nile virus pathogenesis in culex pipiens quinquefasciatus (diptera: culicidae) poliomyelitis due to west nile virus land use and west nile virus seroprevalence in wild mammals flavivirus infections in humans. els genomics and evolution of aedes-borne flaviviruses direct repeats in the flavivirus 3 0 untranslated region; a strategy for survival in the environment origin and evolution of 3 0 utr of flaviviruses: long direct repeats as a basis for the formation of secondary structures and their significance for virus transmission complete sequence of two tick-borne flaviviruses isolated from siberia and the uk: analysis and significance of the 5 0 and 3 0 -utrs characterization of a siberian virus isolated from a patient with progressive chronic tick-borne encephalitis clinicopathologic study and laboratory diagnosis of 23 cases with west nile virus encephalomyelitis atypical manifestations of dengue secreted dengue virus nonstructural protein ns1 is an atypical barrel-shaped high-density lipoprotein tick-borne encephalitis-pathogenesis, clinical course and long-term follow-up a 10-year follow-up study of tick-borne encephalitis in the stockholm area and a review of the literature: need for a vaccination strategy conserved elements in the 3 0 untranslated region of flavivirus rnas and potential cyclization sequences diverse borrelia burgdorferi strains in a bird-tick cryptic cycle early mortality following intracerebral infection with the oshima strain of tick-borne encephalitis virus in a mouse model structural changes and functional control of the tick-borne encephalitis virus glycoprotein e by the heterodimeric association with protein prm mutational analysis of a neutralization epitope on the dengue type 2 virus (den2) envelope protein: monoclonal antibody resistant den2/den4 chimeras exhibit reduced mouse neurovirulence an animal model for the tickborne flavivirus-omsk hemorrhagic fever virus a single amino acid substitution in envelope protein e of tick-borne encephalitis virus leads to attenuation in the mouse model tick-borne encephalitis associated with consumption of raw goat milk west nile and st louis encephalitis virus antibody seroconversion, prevalence, and persistence in naturally infected pig-tailed macaques (macaca nemestrina) control of japanese encephalitis in japan: immunization of humans and animals, and vector control apoptosis as a mechanism for the cytopathic action of tick-borne encephalitis virus west nile virus infection in farmed american alligators (alligator mississippiensis) in florida why is tick-borne encephalitis increasing? a review of the key factors causing the increasing incidence of human tbe in sweden polypyrimidine tract-binding protein influences negative strand rna synthesis of dengue virus a small region of the dengue virus-encoded rna-dependent rna polymerase, ns5, confers interaction with both the nuclear transport receptor importin-beta and the viral helicase, ns3 flavivirus capsid is a dimeric alpha-helical protein crystal structure of west nile virus envelope glycoprotein reveals viral surface epitopes persistence of west nile virus (wnv) igm antibodies in cerebrospinal fluid from patients with cns disease significance in replication of the terminal nucleotides of the flavivirus genome host heterogeneity dominates west nile virus transmission a single n-linked glycosylation site in the japanese encephalitis virus prm protein is critical for cell type-specific prm protein biogenesis, virus particle release, and pathogenicity in mice flavivirus encephalitis: pathological aspects of mouse and other animal models goats and sheep as sentinels for tick-borne encephalitis (tbe) virus-epidemiological studies in areas endemic and non-endemic for tbe virus in germany patterns of tick-borne encephalitis virus infection in rodents in slovenia capsid protein c of tick-borne encephalitis virus tolerates large internal deletions and is a favorable target for attenuation of virulence functional analysis of the tick-borne encephalitis virus cyclization elements indicates major differences between mosquito-borne and tick-borne flaviviruses experimental infection of north american birds with the new york 1999 strain of west nile virus antibodies protect mice against challenge with tick-borne encephalitis virus (tbev)-infected macrophages structure of dengue virus: implications for flavivirus organization, maturation, and fusion amplification of tick-borne encephalitis virus infection during co-feeding of ticks efficient transmission of tick-borne encephalitis virus between cofeeding ticks importance of localized skin infection in tick-borne encephalitis virus transmission characterization of defective viral rna produced during persistent infection of vero cells with murray valley encephalitis virus common e protein determinants for attenuation of glycosaminoglycan-binding variants of japanese encephalitis and west nile viruses flavivirus activates phosphatidylinositol 3-kinase signaling to block caspase-dependent apoptotic cell death at the early stage of virus infection virulence attenuation of dengue virus due to augmented glycosaminoglycan-binding affinity and restriction in extraneural dissemination towards the design of antiviral inhibitors against flaviviruses: the case for the multifunctional ns3 protein from dengue virus as a target defective interfering viral particles in acute dengue infections antiapoptotic but not antiviral function of human bcl-2 assists establishment of japanese encephalitis virus persistence in cultured cells cleavage at a novel site in the ns4a region by the yellow fever virus ns2b-3 proteinase is a prerequisite for processing at the downstream 4a/ 4b signalase site blocking of the alpha interferon-induced jak-stat signaling pathway by japanese encephalitis virus infection japanese encephalitis virus ns2b-ns3 protease binding to phage-displayed human brain proteins with the domain of trypsin inhibitor and basic region leucine zipper analysis of adaptive mutations in kunjin virus replicon rna reveals a novel role for the flavivirus non-structural protein ns2a in inhibition of beta interferon promoter-driven transcription inhibition of interferon signaling by the new york 99 strain and kunjin subtype of west nile virus involves blockage of stat1 and stat2 activation by nonstructural proteins a single amino acid substitution in the west nile virus non-structural protein ns2a disables its ability to inhibit alpha/ beta interferon induction and attenuates virus virulence in mice cis-acting rna elements in human and animal plus-strand rna viruses structural and functional studies of the promoter element for dengue virus rna replication coxsackievirus b-3 myocarditis acute and chronic forms of the disease caused by different immunopathogenic mechanisms folding and dimerization of tick-borne encephalitis virus envelope proteins prm and e in the endoplasmic reticulum subcellular localization and some biochemical properties of the flavivirus kunjin non-structural proteins ns2a and ns4a the japanese encephalitis serological group of flaviviruses: a brief introduction to the group. japanese encephalitis and west nile viruses emerging flaviviruses: the spread and resurgence of japanese encephalitis, west nile and dengue viruses west nile virus infection and its neurological manifestations powassan virus in ixodes cookei and mustelidae in new england crystal structure of the rna polymerase domain of the west nile virus non-structural protein 5 steps of the tick-borne encephalitis virus replication cycle that affect neuropathogenesis spontaneous and engineered deletions in the 3 0 noncoding region of tick-borne encephalitis virus: construction of highly attenuated mutants of a flavivirus tick-borne encephalitis virus -a review of an emerging zoonosis 0 -and 3 0 -noncoding regions in flavivirus rna viruses infecting reptiles dengue virus pathogenesis: an integrated view ivermectin is a potent inhibitor of flavivirus replication specifically targeting ns3 helicase activity: new prospects for an old drug japanese encephalitis virus latency following congenital infection in mice persistence, latency and reactivation of japanese encephalitis virus infection in mice dengue virus ns5 inhibits interferon-alpha signaling by blocking signal transducer and activator of transcription 2 phosphorylation infection, dissemination, and transmission of a west nile virus green fluorescent protein infectious clone by culex pipiens quinquefasciatus mosquitoes bird community composition linked to human west nile virus cases along the colorado front range flavivirus ns4a-induced autophagy protects cells against death and enhances virus replication the molecular basis of virulence of the encephalitogenic flaviviruses differential salivary gland transcript expression profile in ixodes scapularis nymphs upon feeding or flavivirus infection ns1 0 of flaviviruses in the japanese encephalitis virus serogroup is a product of ribosomal frame shifting and plays a role in viral neuroinvasiveness west nile virus ns2a protein facilitates virus-induced apoptosis independently of interferon response persistent hantavirus infections: characteristics and mechanisms subcellular localization and membrane topology of the dengue virus type 2 non-structural protein 4b the non-structural protein 4a of dengue virus is an integral membrane protein inducing membrane alterations in a 2k-regulated manner spatial and temporal organization of tick-borne encephalitis flavivirus replicated rna in living cells tick-borne flavivirus infection in ixodes scapularis larvae: development of a novel method for synchronous viral infection of ticks a ligand-binding pocket in the dengue virus envelope glycoprotein variable surface epitopes in the crystal structure of dengue virus type 3 envelope glycoprotein studies on the occurrence of tick-borne encephalitis in hungary single mutation in the flavivirus envelope protein hinge region increases neurovirulence for mice and monkeys but decreases viscerotropism for monkeys: relevance to development and safety testing of live, attenuated vaccines transfusion-associated transmission of west nile virus cell death gene expression profile: role of ripk2 in dengue virus-mediated apoptosis nuclear localization of japanese encephalitis virus core protein enhances viral replication modeling hamsters for evaluating west nile virus therapies role of virus and host variables in virus persistence or immunopathological disease caused by a non-cytolytic virus establishment, maintenance and description of cell lines from the tick ixodes scapularis inhibition of interferon signaling by dengue virus inhibition of alpha/ beta interferon signaling by the ns4b protein of flaviviruses highly sensitive detection of dengue virus nucleic acid in samples from clinically ill patients persistent infection with west nile virus years after initial infection persistent west nile virus infection in the house sparrow nuclear localization of dengue virus capsid protein is required for daxx interaction and apoptosis the role of dendritic cells in viral persistence molecular differences between wild-type japanese encephalitis virus strains of high and low mouse neuroinvasiveness the replication and eclipse-phase of the tick-borne encephalitis virus in dermacentor reticulatus dynamics of infection in tick vectors and at the tick-host interface crystal structure of the west nile virus envelope glycoprotein a three-dimensional comparison of tick-borne flavivirus infection in mammalian and tick cell lines jab1 mediates cytoplasmic localization and degradation of west nile virus capsid protein tick-borne encephalitis virus delays interferon induction and hides its double-stranded rna in intracellular membrane vesicles mice with different susceptibility to tick-borne encephalitis virus infection show selective neutralizing antibody response and inflammatory reaction in the central nervous system identification of residues critical for the interferon antagonist function of langat virus ns5 reveals a role for the rna-dependent rna polymerase domain development of one-step quantitative reverse transcription pcr for the rapid detection of flaviviruses transmission of west nile virus through blood transfusion in the united states in 2002 persistent neuroinvasive west nile virus infection in an immunocompromised patient flavivirus entry receptors: an update internally deleted wnv genomes isolated from exotic birds in new mexico: function in cells, mosquitoes, and mice modeling antibody-enhanced dengue virus infection and disease in mice: protection or pathogenesis? degrees of maturity: the complex structure and biology of flaviviruses flaviviruses: braking the entering virus host co-evolution in a persistent coxsackievirus b3 infected cardiomyocyte cell line infectious cdna clone of attenuated langat tick-borne flavivirus (strain e5) and a 3 0 deletion mutant constructed from it exhibit decreased neuroinvasiveness in immunodeficient mice persistence of the tick-borne encephalitis virus and its consequences persistence of tick-borne encephalitis virus in monkeys i features of experimental infection study on west nile virus persistence in monkeys persistence of tick-borne encephalitis virus in monkeys vii some features of the immune response persistent infection of vero cells by the flavivirus murray valley encephalitis virus infection with langat flavivirus or expression of the envelope protein induces apoptotic cell death langat flavivirus protease ns3 binds caspase-8 and induces apoptosis persistence of antibodies to west nile virus nonstructural protein 5 nuclear localization of dengue virus nonstructural protein 5 through its importin alpha/beta-recognized nuclear localization sequences is integral to viral infection seroprevalence study of tick-borne encephalitis, borrelia burgdorferi, dengue and toscana virus in turin province isolation of kyasanur forest disease virus from the insectivorous bat, rhinolophus rouxi and from ornithodoros ticks host cell killing by the west nile virus ns2b-ns3 proteolytic complex: ns3 alone is sufficient to recruit caspase-8-based apoptotic pathway persistence of japanese encephalitis virus in the human nervous system crm1-mediated nuclear export of dengue virus rna polymerase ns5 modulates interleukin-8 induction and virus production persistence of protective immunity following vaccination against tick-borne encephalitislonger than expected the envelope glycoprotein from tick-borne encephalitis virus at 2 a resolution tick-borne flaviviruses: dissecting host immune responses and virus countermeasures regulated cleavages at the west nile virus ns4a-2k-ns4b junctions play a major role in rearranging cytoplasmic membranes and golgi trafficking of the ns4a protein serologic evidence of exposure of wild animals to flaviviruses in the central and eastern united states transovarial transmission of dengue viruses by mosquitoes: aedes albopictus and aedes aegypti local impact of temperature and precipitation on west nile virus infection in culex species mosquitoes in northeast illinois virus-encoded proteinases of the flaviviridae persistent neuropsychological impairment associated with west nile virus infection hamster-adapted sin nombre virus causes disseminated infection and efficiently replicates in pulmonary endothelial cells without signs of disease molecular basis of viral persistence: a single amino acid change in the glycoprotein of lymphocytic choriomeningitis virus is associated with suppression of the antiviral cytotoxic t-lymphocyte response and establishment of persistence a single nucleotide change in the e protein gene of dengue virus 2 mexican strain affects neurovirulence in mice generic rt-nested-pcr for detection of flaviviruses using degenerated primers and internal control followed by sequencing for specific identification dengue virus type 2 infections of aedes aegypti are modulated by the mosquito's rna interference pathway a hamster-derived west nile virus isolate induces persistent renal infection in mice persistent infection of cultured mammalian cells by japanese encephalitis virus kinetics of immune responses in deer mice experimentally infected with sin nombre virus dengue virus type 2 ns3 protease and ns2b-ns3 protease precursor induce apoptosis japanese encephalitis virus latency in peripheral blood lymphocytes and recurrence of infection in children viral and cellular proteins involved in coronavirus replication role of cd8+ t cells in control of west nile virus infection chronic st louis encephalitis virus infection in the golden hamster (mesocricetus auratus) rodenhuis-zybert i & wilschut j (2011) flavivirus cell entry and membrane fusion genetic resistance to lethal flavivirus encephalitis: effect of host age and immune status and route of inoculation on production of interfering banzi virus in vivo poliomyelitis-like illness due to japanese encephalitis virus pathology of fatal west nile virus infections in native and exotic birds during the 1999 outbreak west nile virus infection in crocodiles persistence of virus-specific immune responses in the central nervous system of mice after west nile virus infection quantitative determination of igm antibodies reduces the pitfalls in the serodiagnosis of tick-borne encephalitis west nile encephalitis: an emerging disease in the united states type-and subcomplex-specific neutralizing antibodies against domain iii of dengue virus type 2 envelope protein recognize adjacent epitopes antiplatelet autoantibodies elicited by dengue virus non-structural protein 1 cause thrombocytopenia and mortality in mice electron microscopic study of persistent dengue virus infection: analysis using a cell line persistently infected with dengue-2 virus molecular mechanisms of coxsackievirus persistence in chronic inflammatory myopathy: viral rna persists through formation of a double-stranded complex without associated genomic mutations or evolution trim79alpha, an interferon-stimulated gene product, restricts tick-borne encephalitis virus replication by degrading the viral rna polymerase persistent lcmv infection is controlled by blockade of type i interferon signaling a new tick-borne encephalitis-like virus infecting new england deer ticks persistent west nile virus infection in the golden hamster: studies on its mechanism and possible implications for other flavivirus infections polypyrimidine tract-binding protein (ptb) inhibits hepatitis c virus internal ribosome entry site (hcv ires)-mediated translation, but does not affect hcv replication yellow fever: the recurring plague persistent shedding of west nile virus in urine of experimentally infected hamsters replication enhancer elements within the open reading frame of tick-borne encephalitis virus and their evolution within the flavivirus genus molecular characterization of virus-specific rna produced in the brains of flavivirus-susceptible and -resistant mice after challenge with murray valley encephalitis virus clinical and epidemiological features of tick-borne encephalitis in perm region ecology and geographical expansion of japanese encephalitis virus neurologic sequelae of west nile virus infection encephalitis in the clinical spectrum of dengue infection the transmission of the tick-borne encephalitis virus via cow's milk genome cyclization as strategy for flavivirus rna replication old world hantaviruses do not produce detectable amounts of dsrna in infected cells and the 5 0 termini of their genomic rnas are monophosphorylated relation of genetic phylogeny and geographical distance of tick-borne encephalitis virus in central europe composition and three-dimensional architecture of the dengue virus replication and assembly sites tick-borne encephalitis virus ns5 associates with membrane protein scribble and impairs interferon-stimulated jak-stat signalling ultrastructure of kunjin virus-infected cells: colocalization of ns1 and ns3 with double-stranded rna, and of ns2b with ns3, in virus-induced membrane structures kunjin rna replication and applications of kunjin replicons detection of persistent west nile virus rna in experimentally and naturally infected avian hosts st louis encephalitis virus: an ultrastructural study of infection in a mosquito vector west nile virus infection in the golden hamster (mesocricetus auratus): a model for west nile encephalitis induction of inflammation by west nile virus capsid through the caspase-9 apoptotic pathway west nile virus capsid protein induces p53-mediated apoptosis via the sequestration of hdm2 to the nucleolus japanese encephalitis virus ns2b-ns3 protease induces caspase 3 activation and mitochondria-mediated apoptosis in human medulloblastoma cells crystal structure of the dengue virus rna-dependent rna polymerase catalytic domain at 185-angstrom resolution immune evasion strategies of flaviviruses the ns3 protease and helicase domains of japanese encephalitis virus trigger cell death via caspase dependent and independent pathways structures of immature flavivirus particles west nile virus genome cyclization and rna replication require two pairs of long-distance rna interactions japanese encephalitis virus rna not detected in urine the persistence of louping ill virus in immunosuppressed guinea-pigs a single-amino acid substitution in west nile virus 2k peptide between ns4a and ns4b confers resistance to lycorine, a flavivirus inhibitor this work was supported by the intramural research program, national institute of allergy and infectious diseases, national institutes of health (nih). we wish to acknowledge the able assistance of anita mora and heather murphy for graphic support. danielle offerdahl and jennifer lam provided assistance in manuscript preparation. key: cord-001676-68y733y3 authors: shoemaker, jason e.; fukuyama, satoshi; eisfeld, amie j.; zhao, dongming; kawakami, eiryo; sakabe, saori; maemura, tadashi; gorai, takeo; katsura, hiroaki; muramoto, yukiko; watanabe, shinji; watanabe, tokiko; fuji, ken; matsuoka, yukiko; kitano, hiroaki; kawaoka, yoshihiro title: an ultrasensitive mechanism regulates influenza virus-induced inflammation date: 2015-06-05 journal: plos pathog doi: 10.1371/journal.ppat.1004856 sha: doc_id: 1676 cord_uid: 68y733y3 influenza viruses present major challenges to public health, evident by the 2009 influenza pandemic. highly pathogenic influenza virus infections generally coincide with early, high levels of inflammatory cytokines that some studies have suggested may be regulated in a strain-dependent manner. however, a comprehensive characterization of the complex dynamics of the inflammatory response induced by virulent influenza strains is lacking. here, we applied gene co-expression and nonlinear regression analysis to time-course, microarray data developed from influenza-infected mouse lung to create mathematical models of the host inflammatory response. we found that the dynamics of inflammation-associated gene expression are regulated by an ultrasensitive-like mechanism in which low levels of virus induce minimal gene expression but expression is strongly induced once a threshold virus titer is exceeded. cytokine assays confirmed that the production of several key inflammatory cytokines, such as interleukin 6 and monocyte chemotactic protein 1, exhibit ultrasensitive behavior. a systematic exploration of the pathways regulating the inflammatory-associated gene response suggests that the molecular origins of this ultrasensitive response mechanism lie within the branch of the toll-like receptor pathway that regulates stat1 phosphorylation. this study provides the first evidence of an ultrasensitive mechanism regulating influenza virus-induced inflammation in whole lungs and provides insight into how different virus strains can induce distinct temporal inflammation response profiles. the approach developed here should facilitate the construction of gene regulatory models of other infectious diseases. influenza viruses present major challenges to public health, evident by the 2009 influenza pandemic. highly pathogenic influenza virus infections generally coincide with early, high levels of inflammatory cytokines that some studies have suggested may be regulated in a strain-dependent manner. however, a comprehensive characterization of the complex dynamics of the inflammatory response induced by virulent influenza strains is lacking. here, we applied gene co-expression and nonlinear regression analysis to time-course, microarray data developed from influenza-infected mouse lung to create mathematical models of the host inflammatory response. we found that the dynamics of inflammation-associated gene expression are regulated by an ultrasensitive-like mechanism in which low levels of virus induce minimal gene expression but expression is strongly induced once a threshold virus titer is exceeded. cytokine assays confirmed that the production of several key inflammatory cytokines, such as interleukin 6 and monocyte chemotactic protein 1, exhibit ultrasensitive behavior. a systematic exploration of the pathways regulating the inflammatoryassociated gene response suggests that the molecular origins of this ultrasensitive response mechanism lie within the branch of the toll-like receptor pathway that regulates stat1 phosphorylation. this study provides the first evidence of an ultrasensitive mechanism regulating influenza virus-induced inflammation in whole lungs and provides insight into how different virus strains can induce distinct temporal inflammation response profiles. the approach developed here should facilitate the construction of gene regulatory models of other infectious diseases. invading pathogens induce acute inflammation when molecular signatures are detected by pattern recognition receptors (prrs; e.g., rig-i like receptors [rlrs] and toll-like receptors [tlrs] ) expressed on tissue-resident immune cells and non-immune cell types. prr ligation triggers innate immune responses and leads to the induction of inflammatory and antiviral gene expression, which together function to limit pathogen growth, activate the adaptive immune response, and ultimately resolve the infection [1, 2] . precise regulation of prr-mediated signaling is necessary to both avoid inadvertent tissue damage in response to non-pathogenic stimuli, and to prevent immunopathology resulting from excessive expression of inflammatory molecules. in essence, the ideal inflammatory response must exhibit a balance between appropriate activation against a genuine threat and self-limiting behavior once that threat has been controlled. despite its importance in maintaining normal tissue homeostasis and limiting pathogen-associated diseases, the mechanisms underlying the regulation of this balance are poorly understood. influenza a viruses are recognized by both tlrs and rig-i-like receptors (rlrs) [3] [4] [5] [6] [7] , and some strains are potent inducers of inflammatory and antiviral gene expression. generally, lung tissues infected with pathogenic isolates exhibit high virus titers and robust inflammatory gene expression, as has been documented in in vivo studies with the 1918 spanish influenza virus [8, 9] , highly pathogenic h5n1 avian influenza viruses [10] [11] [12] , and the 2009 h1n1 pandemic influenza virus [13, 14] . in contrast, seasonal influenza viruses typically replicate less efficiently, elicit more restrained inflammatory responses, and are usually not associated with lethal infections. recent evidence has implicated the level of virus replication in infected lung tissues as the primary phenotypic variable driving inflammation and lethal outcomes [15, 16] . other data indicate that influenza viruses that exhibit significant differences in pathogenicity stimulate qualitatively similar host responses that differ primarily at the level of magnitude and kinetics [17] . however, these studies have not revealed the mechanisms that account for the different profile dynamics observed in infections by high and low pathogenic viruses. such information would aid in clarifying not only how some influenza viruses induce lethal disease, but also the general mechanisms that regulate inflammatory balance. to characterize the dynamics of influenza virus-induced inflammation, we developed a novel approach to infer gene regulatory models from dynamic gene expression data. referred to as systems inference microarray analysis, our method builds on current approaches that use co-expression analysis to isolate modules of functional signatures in gene expression data and then extends these methods by fitting the gene expression modules to mathematical equations (models) by using segmented regression analysis. models can be created to look for strain-dependent responses and, unlike traditional differential expression analysis, to predict gene expression under new experimental conditions. by using this method, we set out to determine how influenza viruses that exhibit variable pathogenicity profiles influence the dynamics of the inflammatory response. to characterize the dynamics of the host immune response to specific virus isolates, we infected mice with 10 5 pfu of three virus isolates with distinct pathogenicity profiles and harvested lung tissues at 14 ). an initial inoculation of 10 5 pfu was used as previous studies indicated that a high virus dose was needed to invoke different pathologies in h1n1 and ph1n1-infected mice [13] . as expected, lung virus titers (virus titers determined by plaque assay and reported in plaque forming units [pfu] per gram lung; fig 1) indicated a clear hierarchy of mild, moderate, and severe virus-induced disease. specifically, the h5n1 virus produced the highest lung titers and between days 5 and 7 post-infection, this virus also caused mortality in the animals whose lungs were to be collected on day 7 post-infection (i.e., 'severe' disease). in contrast, all animals infected with the h1n1 or ph1n1 viruses survived the duration of the time course study; however, ph1n1-infected animals were visibly sicker and exhibited higher lung titers relative to those infected with h1n1 at all time points observed 3, 6, 9, 12, 18, 24, 30, 36, 48 , and 60 h and 3, 5, and 7 days), and virus titers in lung tissues were determined by plaque assays in mdck cells. error bars illustrate the standard deviation from the mean. the gray boxes at each time point identify significant pairwise differences between the means of the viruses indicated at the right of the figure panel (significance was determined by anova followed by tukey's honestly significantly different test, p < 0.05). *, three h5n1-infected mice intended for collection on day 7 succumbed to their infections prior to sample collection. after the first 30 h post-infection (i.e., 'moderate' and 'mild' disease, respectively). histopathological analysis of tissue samples collected on days 1, 2, and 5 post-infection (fig 2) also showed that h5n1-infected tissue exhibited the earliest, most severe signs of inflammation and inflammatory immune cell infiltrates followed by ph1n1-infected tissue, whereas h1n1-infected tissue showed mild evidence of inflammation and was most similar to tissue from the control mice (mock-infected mice). we next used co-expression analysis to integrate inflammation-associated gene expression differences between influenza-infected and control lungs into a systems level context. we first asked whether the expression of inflammation-associated genes clustered into modules of coexpressed genes. tissues from the same animals that were used to determine virus growth were used to evaluate changes in global lung transcriptional profiles. a total of 168 microarrays were developed (three per time point for h1n1-infected, ph1n1-infected, h5n1-infected and control mice). one microarray was removed after reviewing replicate quality. after filtering transcripts for minimally confident variation (we required at least one time-matched, infected condition compared with mock-infected absolute fold change 2 and a false discovery rate [fdr]-adjusted p-value < 0.01), the log 2 of the normalized intensity of the retained transcripts (16, 063) for all 167 samples were then clustered by using the weighted gene co-expression network analysis (wgcna) algorithm [18] . in all, 45 distinct co-expression modules were identified (referred to as n1, n2, etc.; s1 table provides the module assignments for all transcripts). to identify the biological role of the host response modules, we performed functional enrichment analysis on each gene module by using david [19] and toppcluster [20] . because each module was comprised of positively and negatively correlated transcripts, we used the module eigengene (i.e., the first principle component of the gene expression matrix) to divide each module into two submodules containing transcripts that were positively or negatively correlated with the parent module's eigengene, denoted as kme+ and kme-(referred to as module membership), respectively. this procedure allowed us to look for biological processes with similar but opposing dynamic responses to the virus infections. functional enrichment analyses were then applied to each submodule by using two bioformatics platforms to ensure robust results. we identified two submodules (n1 kme+ and n2 kme-, referred to as simply n1 and n2 in the remainder of the text) that were enriched for inflammatory response and inflammationassociated pathway signatures by using both bioinformatics platforms, and these two modules became the focus of our study (table 1 summarizes the functional enrichment results for the immune and inflammatory related annotations. the complete enrichment results from toppcluster and david are available in s2 table and s1 and s2 files). the n1 module was uniquely enriched for cytokine activity and type i interferon (ifn) regulating tlr and rlr pathways [21] , as well as transcriptional signatures associated with ifn-regulated activity (i.e., the transcription factor binding sites [tfbs] of irf1, irf7, irf2, isre, and nf-κb). additionally, n1 was the only module that exhibited significant enrichment with a compendium of established ifn-stimulated genes ('isgs'; table 1 ; see methods. the list of isgs is available in s3 file). a more recent study identified 147 ifn stimulated genes in immortalized, human airway epithelial (calu3) cells [22] . of these, 90 mouse homologs were annotated on the microarrays and 70 of the homolog probes were assigned to the n1 module (fisher's exact test; pvalue < 10 -16 ; odds ratio = 36.4), further associating n1 interferon-stimulated gene activity. in contrast, the n2 module was only weakly enriched for some cytokine activity related annotations and not enriched for any of the binding sequences of transcription factors that are members of canonical inflammatory pathways (such as interferons, interferon regulatory factor proteins, or nfκb). instead, it was primarily associated with several annotations related to leukocyte and lymphocyte activity (see summary of toppcluster enrichment results in table 1 ; see s1 and s2 files). further analysis with cten [23] , a platform for associating clustered gene expression data with specific cell types, found n2 to be highly enriched for genes expressed in macrophages in various cellular states (e.g., bone marrow-derived macrophages exposed to lipopolysaccharide [lps]) ( fig 3a; additional details available in s3 table) . the remaining immune-associated submodules (the kme+ n22, n25, n31, and n35 submodules; described in table 1 ) were enriched for several key immune processes such as antigen presentation, and t cell and natural killer (nk) cell activity, but their further assessment would be beyond our focus and the scope of this study. thus, bioinformatics analyses robustly associated the n1 module with inflammation, cytokine production, and type i ifn pathway activity-likely activated in resident lung cells-whereas the n2 module is associated with migration and activation of macrophages in the lung. each module was divided into submodules in which each member gene's expression was positively or negatively correlated with the module's eigengene (denoted kme+ and kme-, respectively). for each module, we provide the number of transcripts assigned to each submodule, the top david annotation clusters for each submodule (parenthesis shows the-log10 of the average enrichment p value for all annotations in the cluster), the top enriched biological processes determined using toppcluster (parenthesis shows the-log10 of the fdr-adjusted p value), the enrichment of established transcription factor binding sites in each submodule (tfbs; parenthesis shows the-log10 of the fdr-adjusted p value; performed using toppcluster), and the enrichment score of a set of ifn-stimulated genes (see methods; enrichment score is the-log10 of the fdr-adjusted p value). doi:10.1371/journal.ppat.1004856.t001 a closer examination of the expression dynamics of each of the inflammation response-associated modules revealed patterns of expression that were consistent with the biological roles predicted by our bioinformatics analyses. we used the scaled difference of the module eigengene to characterize the expression of all genes within each module. we subtracted the mean of the eigengene of the control samples from the eigengene of time-matched, virus-infected dynamics of inflammation-associated co-expression modules. panel (a) shows the enrichment scores (-log 10 of the fdr-adjusted p value) of the genetic signatures of 94 cell types in the n2 module as determined using cten [23] . data corresponding to macrophage signatures in different cellular states are colored red (see s3 table for within the inflammation-associated modules (n1 and n2), the h5n1 virus induced the earliest gene expression changes and the highest peak expression levels, corroborating previous observations that h5n1 viruses are strong inducers of inflammatory and ifn response signaling in vivo [10, 24, 25] . consistent with the prediction that n1 is involved in detecting virus in infected tissues, the n1 module eigengene was the most highly correlated with virus titer (pearson pairwise correlation, ρ = 0.70). module gene expression dynamics further suggested that the n2 module gene is associated with lymphocyte infiltration. exudate macrophages [26] and neutrophil [27] have been identified as factors of severe disease during influenza infection. to associate gene expression dynamics with changes of immune cell counts, a new population of mice were infected with the three influenza viruses, five mice per infection group were sacrificed on days 1, 2, 3 and 7 and the changes in the number of macrophages and neutrophils was assessed (see methods). unlike the previous study by brandes, et al. [27] , strong neutrophil infiltration was not specific to fatal infections but, instead, infiltration of both cell types had a clear hierarchical relationship with the severity of the infection (fig 3d and 3e ). the n2 module eigengene exhibited a lesser correlation to virus titer (ρ = 0.55), but was tightly correlated to macrophage influx into the lung (ρ = 0.90; p-value < 0.01 student's t-test). of the 45 module identified in the studied, n2 had the highest correlation to both macrophage and neutrophil influx (the correlation of macrophage and neutrophil influx and all 45 module eigengenes are provided in s5 table; ρ = 0.80; pvalue < 0.01). the n1 module on the other hand was weakly but significantly correlated with immune cell infiltration (ρ = 0.67 [p-value = 0.03] for neutrophils; ρ = 0.60 [p-value = 0.05] for macrophages), but its eigengene was not the most highly correlated (5 other module eigengenes had a greater absolute correlation). these results further associate n2 with immune cell-specifically macrophage-infiltration, while the sum of the bioinformatics, virus titer correlations and immune cell infiltration evidences associated n1 with inflammation and type i ifn pathway activity. a further advantage of a network approach is that the functional relevance of genes might be inferred from their positions within the co-expression network [28] . we used the module membership (the correlation between the gene's expression and the module eigengene, kme) to isolate potential regulators of the n1 module. among the top intramodular hub genes (i.e., genes with the highest module memberships, see s4 table) , we found mnda, herc6 and cd274 and several interferon regulated, virus replication inhibitory genes such as oas2 and oas3 [29] . herc6 is involved in ubiquitination [30] . mnda is significantly up-regulated in monocytes exposed to interferon α [31] while cd274 is a transmembrane protein expressed on antigen presenting cells and modulates activation of t cells, b cells and myeloid cells. we also observed that interferon stimulated genes tended to have higher intramodular hub rankings, suggesting a regulatory role for interferon (wilcoxon rank sum test, p-value < 10 -12 ). we then considered the module membership rankings of transcription factors known to regulate interferon. of the established interferon regulatory factors that are members of the n1 module (e.g., irf1, irf2, irf7, irf9, stat1, and stat2. nfkb1 and nfkb2 were not assigned to n1), irf1 and irf7 had the highest module memberships (kme = 0.94 and 0.93; ranking = 118 and 225, respectively). irf7 expression was also several orders of magnitude greater than irf1 (s4 table) . together, these findings corroborate our bioinformatics analyses by suggesting that n1 is regulated by interferon, and n1 expression likely results in enhanced cytopatchic effects and regulation of the lymphocyte immune response. network analysis further suggests that irf7 may play a regulatory role upstream of interferon transcription. previous studies have suggested that highly pathogenic influenza virus infections induce an irregular or disproportionate inflammatory response relative to seasonal influenza viruses, and that these differences occur early in the host response [24] . for this reason, we sought to further explore the possibility of isolate-specific or isolate-independent response patterns of the cytokine-associated n1 module. we wanted to infer mathematical relationships that could describe when inflammatory-associated gene expression occurs and what magnitude of expression is expected. by using the eigengene as a representation of the scaled gene expression dynamics, we attempted to infer simple mathematical models that can be related to common signaling mechanisms. surprisingly, when we plotted the n1 sde for each isolate against the corresponding virus titer, we observed a consistent profile for all three viruses; regardless of intrinsic virulence, the fold change in n1 gene expression remained initially low and rapidly increased only after a virus titer of approximately~10 8 pfu/g (of lung) was reached (fig 4a) . following activation, n1 gene expression increased as a function of virus concentration at the same apparent rate for all infection conditions, and more complicated dynamics were observed only during the later phase of the infection when virus clearance was observed (i.e., when the virus titers began to decrease). these observations suggest that ifn-regulated (n1) gene expression was induced by an ultrasensitive response mechanism controlled at the level of the virus titer rather than the virus's intrinsic virulence. ultrasensitive responses characterize the dynamics of several signaling pathways that regulate essential and often toxic biological processes such as the cell cycle [32] and apoptosis [33] . as shown in fig 4b, ultrasensitive responses are typified by an attenuated response to low levels of stimulation but a strong response occurs once a threshold level of stimulus is reached. cooperativity [33] and positive feedback [34] are two mechanisms that produce ultrasensitive responses. to formalize the hypothesis that the inflammatory gene response follows an ultrasensitive response profile, we selected a segmented linear model (slm, defined in fig 4c) to be a simplified representation of the ordinary differential equations normally used to model ultrasensitive responses, and we fit the n1 sde to an slm that was strictly a function of the virus titer. the optimal fit showed a threshold of 10 7.78±0.14 pfu/g is required for n1 module activation to occur, after which the sde's rate of activation (a 2 ) was 0.5±0.07 log 10 (pfu/g) -1 with an intercept (b 2 ) of -3.8 (unitless) (see the methods and s2 fig for additional details) . below this threshold, the model predicted minimal gene expression (a1 = 0.17; b1 = 0.03). the slm goodness of fit on the training data was an adjusted r 2 = 0.72 while an adjusted r 2 = 0.41 was observed when the data was fit to a linear model. a davie's test confirmed that a segmented model was a significantly better fit than a linear correlation model (p-value < 2.2e-16). while the h1n1-infected lung tissue did not exceed an average peak virus titer of 10 7.4 pfu/g (peak titer occurs at 48 hpi in fig 4a) , we observed increased transcriptional activity in h1n1-infected mouse lung tissues after this time point, suggesting either that the actual peak virus titer occurred between 48 hpi and the subsequent time point (60 hpi), or that the model-predicted threshold was slightly over-approximated. we next sought to validate the threshold model by attempting to predict cytokine-associated gene expression in influenza virus-infected lung when only the virus titers are known. for this, we selected the h5n1 virus, which has previously been associated with an excessive illustration of an ultrasensitive response to increasing stimulus. in our model, the response is the change in inflammatory-associated gene expression and the stimulus is the lung virus titers. a segmented linear model (slm) can approximate key aspects of the ultrasensitive response. in this study, the response is gene expression and the stimulus is the concentration of virus in the lung (c) the mathematical definition of an slm. below some threshold virus titer (thr), the scaled eigengene (se) is approximated by a linear model that is a function of the log of the virus titer, the slope (a 1 ), and the intercept (b 1 ). after the threshold virus titer is reached, the slope and intercept parameters change (a 2 and b 2 ) to describe the se after activation. the parameters were fit to the data shown in panel (a) and used to predict the n1 se in newly infected mice. (d) shows a comparison of the h5n1 n1 eigengene from an infection at a dose of 10 5 pfu (as shown in fig 3) and an infection at a dose of 10 3 pfu. panel (e) compares the predicted scaled eigengene based on the slm versus the measured eigengene behavior in mice infected with 10 3 pfu of the h5n1 virus. panel (f) shows gene expression changes for selected constituent genes of the n1 module as a function of virus titer. cytokine response [10] . we infected mice with 10 3 pfu of the h5n1 virus (a 100-fold reduced dose compared with that used in the experiments to fit the model), determined lung virus titers at the same time points used for the initial experiment (s3 fig), and then evaluated the segmented linear model's ability to predict cytokine-associated gene expression. first, we confirmed that the original transcripts assigned to the n1 module were again co-expressed, and thus we used the same transcripts originally assigned to the n1 module to determine the eigengene (see s4 fig for an analysis of the conservation of the n1 module between the two experiments). in this experiment, as expected, the peak average virus titer (10 9.3±0.21 pfu/g) for the 10 3 pfu dose was delayed compared with that for the 10 5 pfu dose (s3 fig, compare to fig 1) . moreover, the sde exhibited an 18-h delay in activation and a 42-h delay in peak expression compared with the 10 5 pfu n1 eigengene (fig 4d) . importantly, based on the virus titers alone, the fitted segmented linear model accurately predicted n1-like sde behavior (r 2 = 0.71 for all time points and r 2 = 0.87 for time points up to peak expression; fig 4e and s5 fig), and this could be further demonstrated at the individual gene level for specific n1-associated transcripts (e.g., herc6, stat1 and irf7; fig 4f) . these observations provide strong evidence that activation of inflammatory-associated gene expression is dictated by a specific virus concentration in infected tissue, and further suggest the novel possibility that the pulmonary innate inflammatory response has a nonlinear, ultrasensitive-like activation profile that promotes tolerance to low concentrations of virus. the dynamics of key inflammatory cytokines are consistent with an ultrasensitive response although transcriptional activation of ifn-stimulated and inflammatory gene expression is a reasonable measure of the effects of inflammation response stimulation, we reasoned that a bona fide ultrasensitive mechanism that regulates this response should be reflected in other aspects of the associated signaling pathway(s). indeed, of the 17 cytokines associated with the n1 module, 15-including key inflammatory proteins, such as interleukin 6 (il-6) and monocyte chemoattractant protein-1 (mcp-1)-were significantly correlated with the n1 module eigengene (pearson's ρ0.5; fdr-adjusted p-value < 0.01; see s6 table) . in addition, when the protein expression levels of these 15 cytokines were plotted against the corresponding titer data, we observed dynamics similar to that of the inflammatory-associated n1 gene module. initially, protein expression was low but strongly increased only after the virus titers exceeded the threshold of~10 8 pfu/gram determined in the gene regulation model (fig 5a) . in contrast, most of the protein levels of the other measured cytokines with transcripts that were not assigned to the n1 module did not show any obvious relationship to the proposed threshold response (s6 fig). the only major exceptions were lif, rantes, and il18 (s6 fig). lif's gene transcript was not annotated on the arrays whereas il18's transcript was not identified as differentially expressed and therefore was not included in the clustering study. the rantes transcript was included in the clustering study and assigned by the wgcna algorithm to n2 although the transcript's correlation to the n1 eigengene suggests it could also have been assigned to the n1 module (pearson correlation = 0.84 and 0.89 to the n1 and n2 eigengene, respectively). some proteins appeared to conform to the threshold model in ph1n1-infected, but not h1n1-or h5n1-infected, mice. these may be cytokines that have strain-dependent responses and do not conform to the model. overall, changes in n1-associated cytokine protein levels in influenza virus-infected mouse lungs were consistent with the proposed virus-titer regulated threshold-mechanism underlying the ifn-mediated response. threshold-like behavior is observed on upstream and downstream components of the ifn signaling pathway. (a) lung tissues from the same infected mice used for the original gene expression analysis were subjected to cytokine array analysis. thirty-two cytokine protein concentrations were measured and log scaled (data points lower than the limit of detection for each protein were set to 0.001 to allow scaling), and the average fold change relative to uninfected, time-matched lung samples was determined. the heat map illustrates protein expression values for the 17 cytokines that had transcripts assigned to the n1 module (a blue-to-yellow scale shows expression levels, as indicated by the color key at the top of the panel; time points in which the change in the cytokine levels were not significant [fdr-adjust p < 0.05] were set to zero); of these, 15 exhibited expression profiles that were highly significantly correlated with the corresponding transcript and the n1 module eigengene (pearson's ρ 0.4 and fdr < 0.01; eotaxin and tnf-α were the only two that did not exhibit a significant, direct correlation). non-n1 cytokines are shown in s6 (fig 5b) . for the h1n1 data, significant levels of pstat1 were observed at 36 hpi which-as noted previously-corresponds to the time immediately after the virus titers in h1n1-infected mice reached their peak (see previous discussion). on the other hand, significant increases in phosphorylated irf3 (pirf3) were observed only in ph1n1 and h5n1 infections, whereas significant increases in ifn-β were observed only in the h5n1 infection. changes in the levels of ifn-β and pirf3 occurred after significant increases in pstat1 and ifn-α occurred. as such, the change in the percentage of pstat1 was more closely correlated to the n1 eigengene than was that of pirf3 (correlation = 0.77±0.06 and 0.67±0.09 respectively), and significant increases in pstat1 corresponded to time points at which the mean virus titer exceeded the threshold level identified in the gene expression analysis (~10 7.78 pfu/g). the greater correlation of ifnα and stat1 activation to the inflammation-associated, n1 module's gene expression dynamics and the enrichment of the n1 module for the irf7 binding sequence (table 1) suggest that the primary driver of the threshold-regulated, inflammatory gene response originates along the irf7 ! ifn-α ! stat1 axis (fig 5c) . our data reveal that the activation of the ifn-associated inflammatory and antiviral response program in influenza-infected mouse lung is characterized by an ultrasensitive response driven by the virus load. the power of the threshold model is illustrated by its ability to accurately predict gene expression in infected mice, and the data further suggest that the molecular basis of threshold behavior originates upstream of ifn-α production. threshold-like and ultrasensitive mechanisms are hypothesized to be necessary for effective management of critical cellular machinery in noisy environments, and are recognized players in the activation of the cell cycle [34] , mitogen-activated protein kinase signaling [35] , and apoptosis [33, 36] . however, while a role for threshold behaviors have been postulated to be essential for filtering noise or errant signaling in complex biomolecular environments [35] , our study is the first to directly link threshold-like behavior to the virus-induced innate immune response. the ultrasensitive response observed in this study provides additional insight into the mechanisms that drive severe pathologies during influenza infection. several works have suggested that viral load is a key determinant of pathology [12, 15] while other works suggest that highly pathogenic influenza viruses induce early, strong inflammatory responses that are independent of the viral load [9, 24, 37] . recently, it was observed that fatal influenza infections in mice map, with titers surpassing the threshold level identified in the slm indicated in red. (b) another set of mice was infected with 10 5 pfu of h1n1, ph1n1, or h5n1 and lung tissues were harvested (3 mice per infection per time point) for immunoblot (stat1, phosphorylated stat1, irf3, and pirf3) or elisa (ifnα, ifn-β) analysis; virus titers were also determined. the heat map indicates the mean percentage of phosphorylated protein (purple) or the mean interferon concentration (pg/ml; green), and virus titers for each time point are indicated below. only significant changes are shown in the heat map (fdr-adjusted pvalue < 0.05 relative to mock-infected samples). titers highlighted in red indicate that the threshold level (as determined in the initial analysis) was exceeded. panel (c) shows a schematic of known canonical pathways that regulate ifn production in response to virus infection. black arrows denote induction whereas red lines denote points at which influenza virus is known to impair these pathways. ifn-α is primarily produced by antigen-presenting cells (apcs) whereas ifn-β is produced in virus-infected, non-apc cells. both ifns can induce stat1 phosphorylation and expression of isgs in responding cells. doi:10.1371/journal.ppat.1004856.g005 ultrasensitive mechanism regulates influenza-induced inflammation coincide with a strong influx of neutrophils in what the author's describe as a viral load-independent, "feedforward" inflammatory circuit [27] . the ultrasensitive response suggested by our study consolidates these hypotheses by suggesting that viral load drives cytokine production (and in turn immune cell infiltration) in a nonlinear manner which is capable of producing states of high and low innate immune responses. characterization of key aspects of the inflammatory response, such as the onset and peak inflammatory gene expression, require a high temporal resolution of the virus growth and host response dynamics; an experimental design that was unique to our study. the ultrasensitive response model does not negate the significance of neutrophil infiltration [27] in determining fatal infections but suggests that viral load drives the high and low innate immune states. the observed threshold may represent the transition to immunopathology; as indicated by the histopathology results (fig 2) . moreover, the influenza virus' ns1 protein is crucial for inhibiting the interferon-mediated antiviral response [38] . the ns1 protein of three viruses used in this study have the sumo1 acceptor site that indicates interferon antagonism capability [39] [40] [41] . additional studies with ns1-mutated viruses and other pathogens may better reveal strain-dependencies for the observed thresholding behavior. the ultrasensitive response further suggests that the innate immune response has a limited capacity to respond to influenza virus infection and supports the development of immunomodulatory therapies. interestingly, after the threshold was exceeded, the rate of activation for inflammatory and interferon-associated gene expression (n1) was conserved for a moderately pathogenic and deadly viruses (fig 4a) . the conserved rate of activation implies that the immune response detects the virus concentration but not the virus growth rate; suggesting the innate immune response is naturally limited in its ability to respond to high growth influenza viruses. additionally, studies in knockout mice indicate that type i ifn-associated pathways are essential for protection during primary infection [42] and that earlier initiation of these pathways coincides with increased survival in mice infected with highly pathogenic isolates [43] . in combination with these studies, the findings here suggest a novel means of protecting high risk groups by treating them with compounds that target the molecular mechanisms responsible for the threshold behavior. lowering the threshold required to induce the cytokine response may be a means of providing protection from severe influenza infection. since these compounds would target host proteins, such treatments would be effective against various influenza virus strains. data from the viruses studied here suggest that post-threshold, inflammatory gene expression primarily reflects interferon-regulated tissue damage, but time-course data from additional highly pathogenic viruses are needed to assess the degree to which interferon activity is associated with virus growth suppression. the a/california/04/09 h1n1 virus (ph1n1) was received from the centers for disease control and prevention. the a/kawasaki/utk-4/09 h1n1 virus (h1n1) served as a reference for a seasonal influenza, whereas a fatal human isolate, a/vietnam/1203/04 h5n1 virus (h5n1), served a highly pathogenic virus. all mouse experience were performed in accordance to the university of tokyo's regulations for animal care and use. these regulations were approved by the animal experiment committee of the institute of medical science, the university of tokyo (approval number: pa10-13). the committee acknowledged and deemed acceptable the legal and ethical responsibilities for the animals, as detailed in the fundamental guidelines for proper conduct of animal experiment and related activities in academic research institutions under the jurisdiction of the ministry of education, culture, sports, science and technology, 2006. all experiments with h5n1 viruses were performed in biosafety level 3 containment laboratories at the university of tokyo, which are approved by the ministry of agriculture, forestry, and fisheries, japan. five-week old c57bl/6j, female mice were obtained from japan slc. for all experiments, mice were anesthetized with isoflurane and intranasally inoculated with either 10 3 or 10 5 pfu of virus. initially, 42 mice were inoculated with 10 5 pfu of the h1n1, ph1n1, or h5n1 virus or mock-infected with pbs (a total of 168 mice). at 14 time points, 3 mice per group were humanely sacrificed and their lungs harvested. the lungs were sectioned and used to assess virus titers (right-upper lobe), cytokine levels (right lower), and the initial gene expression that was used to train the segmented linear model (left-lower). separately, 42 mice were infected with 10 3 pfu of the h5n1, sacrificed at the same 14 time points, and lungs sections obtained as described previously to provide the model validation data. the same inoculation method was used for all mice in this study. the numbers of mice used for flow cytometry, western blot, and interferon protein assay experiments are specific in the corresponding sections. for cytokine and chemokine measurements, mouse lungs were treated with the bio-plex cell lysis kit (bio-rad laboratories, hercules, ca) according to the manufacturer's instructions. concentrations of other cytokines were determined with the bio-plex mouse cytokine 23-plex and 9-plex panels (bio-rad laboratories) array analysis was performed by using the bio-plex protein array system (bio-rad laboratories). virus titers were determined by plaque assay using mdck cells. mouse lung tissues were placed in rnalater (ambion, ca), an rna stabilization reagent and stored at -80°c. all tissues were thawed together and homogenized for 2 minutes at 30 hz using a tissuelyser (qiagen, hilden, germany) as per the manufacturer's instructions. from the homogenized lung tissues, total rna was extracted with the rneasy mini kit (qiagen, hilden, germany) in accordance with the manufacturer's instructions. cy3-labeled crna preparations were hybridized onto agilent-014868 whole mouse genome 4x44k microarrays for 17 h at 65°c. feature extraction software version 7 (agilent technologies) was used for image analysis and data extraction, and takara bio provided whole array quality control metrics. microarray normalization, replicate quality, annotation, and statistical analysis differential expression was assessed by using a linear regression model. by using the limma package [44] 26 version 3.14.1 from bioconductor, probe intensities were background corrected by using the "norm-exponential" method, normalized between arrays (using the quantile method), and averaged over unique probes ids. replicate quality was assessed using hierarchical clustering, resulting in the removal of a single array (the array corresponded to a sample collected at 3 hpi in h5n1-infected mice.) probe intensities were fit to a linear model that compared data from infected samples to time-matched data collected from uninfected mice. probes were annotated by matching to the probe names in the mgug4122a version 2.1 mouse annotation database available from bioconductor. all arrays in this study have been deposited on the geo expression omnibus (gse63786). unsigned co-expression networks were constructed by using the blockwisemodules program from the wgcna package version 1.23.1 [18] in r. the analysis was performed with several different parameterizations to ensure robust clustering. for the results reported in this text, we removed all probes that did not have a confident fold-change greater than 2 (fdr < 0.01) for at least one infected-tissue to control-tissue, time-matched comparison. we then clustered the log 2 of the normalized intensities for all 167 microarrays (corresponding the three samples for each time-point for each infected or control population with the exception of data from h5n1-infected mice at 3 hpi which had two samples). based on the scale-free topology characteristics curve, a power of n = 7 with no reassignment after clustering (reassignthreshold = 0) and a maximum cluster size of 6000 probes was used. we generally observed that allowing gene reassignment between the modules led to poorer clustering based on the distribution the gene's module memberships (the correlation between a gene and the eigengene of the module to which it had been assigned. s10 fig illustrates the distribution of the gene kmes for modules n1 and n2). we then repeated the clustering using different powers (ranging from 7 to 11), allowing different cluster sizes, different subsets of the expression data (e.g., clustering data from each infection separately or together), or relaxing the differential expression condition. in all clusterings performed, the two modules discussed in the text were identifiable. fisher exact tests between each clustering run were used to determine whether the initial modules were significantly conserved under different parameterizations. we also considered if the n1 module would be isolated when using signed versus unsigned network construction. we constructed a signed co-expression network and found that 92% of the kme+ n1 genes are again clustered and confirmed that the gene expression dynamics were maintained (see s11 fig) . toppcluster [20] and david [19] were used for gene ontology and pathway enrichment, and toppcluster was also used for transcription factor binding site enrichment analyses. david uses clusters of related annotations constructed from several annotation databases (e.g., pathway and gene ontology annotations) to determine the function of a set of genes and scores the enrichment by averaging the unadjusted p-values (determined by fisher's exact test) of the annotations within the cluster. toppcluster uses hypergeometric tests to determine the enrichment between a set of genes and gene lists contained in 18 categories (databases) detailed in the toppgene suite [45] . the databases include cis-regulatory motif data [46, 47] , referred to as transcription factor binding sites (tfbs) in the text. both tools were used with their default settings and the gene universe was considered to be all annotated mouse genes. for each module, we considered the enrichment of all genes assigned to the module and the kme+ and kmesubsets. generally, the enrichment analysis of the whole module gene set reiterated the enrichment results of the kme+ and kme-subsets albeit with slightly lower but still significant enrichment. since both tools returned similar go and pathway enrichment results, we summarized the functional and pathway enrichment results in table 1 using the results from david. the enrichment of interferon stimulated genes was determined by using a list of interferon stimulated genes from the interferon stimulated gene database [48] that was downloaded on may 9, 2012 (see s3 file). for each module, all module genes and the kme+ and kme-subsets were tested for enrichment using fisher's exact test in r. the p values were adjusted to control the false discovery rate. cten [23] was used to determine enriched cell signatures in select co-expression modules. the enrichment score reported is the-log10 of the false discovery rate. model fitting and validation was performed in r using the 'segmented' package [49] . five mice per time point per infection were infected with 10 5 pfu of the described virus. five uninfected (naïve) mice served as negative controls. whole lungs were collected from mice, and incubated with collagenase d (roche diagnostics; final concentration: 2 μg/ml) and dnase i (worthington; final concentration: 40 u/ml) for 30 minutes at 37°c. single-cell suspensions were obtained from lungs by grinding tissues through a nylon filter (bd biosciences). red blood cells (rbcs) in a sample were lysed with rbc lysis buffer (sigma). samples were resuspended with pbs containing 2 mm edta and 0.5% bovine serum albumin (bsa), and cell number was determined by using a disposable cell counter (onecell). to block nonspecific binding of antibodies mediated by fc receptors, cells were incubated with purified anti-mouse cd16/32 (fc block, bd biosciences). cells were stained with appropriate combinations of fluorescent antibodies to analyze the population of each immune cell subset. the anti-f4/80 (bm8; ebioscience) antibodies were used. all samples were also incubated with 7-aminoactinomycin d (via-probe, bd biosciences) for dead cell exclusion. data from labeled cells were acquired on a facsaria ii (bd biosciences) and analyzed with flowjo software version 9.3.1 (tree star). three mice per time point per infection group were infected with 10 5 pfu of the described virus. the primary antibodies of mouse anti-stat1 (phospho tyr701) mab (ab29045, abcam), rabbit anti-irf3 (phospho ser396) mab (4947, cell signaling), and mouse anti-βactin (a2228; sigma-aldrich) were used; the secondary antibodies were hrp-conjugated antimouse igg antibody (ge healthcare) and hrp-conjugated anti-rabbit igg antibody (ge healthcare). mouse lungs were collected and homogenized with ripa buffer (thermo scientific, rockford, il, usa) containing proteinase inhibitor (roche, mannheim, germany) and phosphatase inhibitor cocktails (sigma-aldrich, saint louis, missouri, usa). the lysates were then briefly sonicated and centrifuged. each sample was electrophoresed on sodium dodecylsulfate polyacrylamide gels (bio-rad laboratories, hercules, ca, usa) and transferred to a pvdf membrane (millipore, billerica, ma, usa). the membranes were blocked with blocking one (nacalai tesque, kyoto, japan) for 30 min at room temperature, and then were incubated with the primary antibodies overnight at 4°c, followed by the secondary antibodies. they were then washed 3 times with pbs plus tween 20 (pbs-t) for 5 min and incubated with secondary hrp-conjugated antibodies (as described above) for 30 min at room temperature, followed by three washes with pbst. specific proteins were detected by using supersignal west femto maximum sensitivity substrate (thermo scientific, rockford, il, usa). photography and quantification of band intensity were conducted with the versadoc imaging system (bio-rad laboratories, hercules, ca, usa). the quantity of target bands from each sample was standardized by their respective β-actin. three mice per time point per infection group were infected with 10 5 pfu of the described virus. half the lung of each mouse was dissolved in 1 ml of ripa buffer. we measured the interferon-alpha and interferon-beta by using elisa kits (#12100, #42400, pbl assay science, nj, usa) according to the manufacturer's instructions. plates were read at an absorbance of 450 nm using a versa max plate reader (moleculardevices, menlo park, ca). additional gene set overlap tests were performed in r with all of the genes annotated on the array as the reference (background) set. statistical tests to compare means within the western blot, flow cytometry, immune cell count and protein assay data sets were performed in r using the 'multcomp' package [50] . supporting information s1 fig. the scaled difference of the module eigengene. the eigengene is the first principle component (equivalently the first eigenvector) of a matrix of gene expression data. in clustered data in which all genes are highly correlated (as in the case of wgcna), the eigengene is a scaled approximation of how gene expression changes for all genes in a module (i.e. cluster) across experimental conditions. the eigengene of module n1 is shown in (a) for the 56 experimental conditions considered in this study (4 infection conditions and 14 time points). each point represents data from a single animal in each experimental condition (3 animals per condition except for h5n1-infected animals at 3 hpi which had 2; total of 167 samples). while the eigengene illustrates the changes in gene expression of module member genes, it is difficult to interpret the changes with respect to the control data. we therefore scaled the eigengene by subtracting from time-matched data the average of the eigengene from mock-infected animals and then dividing by the highest average eigengene for any experimental condition. the scaled difference of the eigengene (sde) is shown in (b). the sde now represents the fraction of greatest gene expression (i.e., the fraction of the largest log fold change in gene expression observed between all time-matched, infected-to-control samples). for example, the sde peaks in experimental condition 50 (h5n1-infected animals at 30 hpi), corresponding to the condition in which the log fold change was greatest for n1 member genes (see fig 3) . the sde for ph1n1-infected animals at day 3 pi (condition 40) is~0.5. we would expect the log fold change to be approximately half of that observed in h5n1-infected animals at 30 hpi (see the heatmaps in fig 3) . for completeness, we overlay the mean (lines) and the standard deviation to create the segmented linear model to describe the relationship between the n1 module eigengene and the virus titer data, we selected all titer data that occurred prior to and included the peak of the eigengene and then scaled the data such that the eigengene was bound between [0,1]. the model was then fit to the scaled data. panel (a) shows the time points selected as training data from each infection data set (indicated by the orange dots); and panel (b) shows the number of data points available for different ranges of the virus titer (top) and the segmented model's fit to the training data (bottom). in panel (c), we used the residuals (i.e., the difference between the predicted values and the actual values) to compare the slm's accuracy to that of a simple linear model. the line is the running average and the shaded region is the 95% confidence interval of the mean. the mean of the residuals of the segmented model was always near zero for the full range of virus titers and, thus, is a better fit than the linear model. two procedures were applied to determine whether the n1 genes that we identified as co-expressed in the original clustering analysis were also co-expressed in tissue from mice infected with 10 3 pfu of h5n1 virus. (a) compares the correlation between all 1,021 n1 gene transcripts and the eigengene (referred to as the kme) in the original gene expression data (10 5 pfu infection conditions (referred to as '10 5 pfu')) and the 10 3 pfu infection condition (referred to as '10 3 pfu'). more than 90% of the genes exhibited a kme > 0.9. (b) the wgcna algorithm was repeated with all differentially expressed genes (fdr-adjusted p-value < 0.01 and fold change > 2 for at least 1 time point) from the 10 3 pfu h5n1 virus infection, and then the fisher's exact test was used to identify modules enriched for n1 genes. of the modules in the newly constructed co-expression network, only one module was enriched with the n1 transcripts. specifically, of the 826 genes originally assigned to n1, 528 (benjimini-hochberg-adjusted p-value < 2.2e-16) were differentially expressed and assigned to the same module in the 10 3 pfu infection condition, as illustrated by the venn diagram. . the h5n1-10 3 pfu eigengene was constructed using the same set of probes assigned to the n1 module from the 10 5 pfu infection condition and scaled to between [0,1]; and then the segmented linear model trained to the 10 5 pfu h1n1, ph1n1, and h5n1 data was used to predict h5n1-10 3 pfu scaled eigengene values. panel (a) shows a comparison of the predicted (black) and actual (pink arrows depicting the temporal evolution of the gene response) eigengenes, with error bars illustrating the standard deviation of the eigengene and of the log 10 of the virus titer. panel (b) shows how the prediction residuals are distributed over time. for each time point, individual data points (black points) are shown, as well as the average (red points) and standard deviation (gray bars). the greatest deviations occurred at d5 and d7, which was expected as the model was designed only to predict the onset of gene activation and the peak gene expression (peak of the eigengene). on days 5 and 7 post-infection, both the virus titers and gene expression has already peaked and are declining. panel (c) shows how the prediction residuals are distributed across the spectrum of observed virus titers, as compared to a linear model directly fit to the h5n1-10 3 pfu n1 eigengene. individual residuals are indicated by points, and the running average and the 95% confidence intervals are shown by the colored lines and the gray shading, respectively. as for the 10 5 pfu infection condition, the segmented model performed well across the entire virus titer spectrum, and was significantly better than a linear model fitted directly to the data. (tif) s6 fig. protein concentrations of non-n1 module cytokines. as described in the fig 5 leg end, cytokine concentrations were assayed in the lungs of mice infected with 10 5 pfu of h1n1, ph1n1, and h5n1 and compared with those in lung tissues from mock-infected animals. this heat map illustrates protein expression values for non-n1-associated cytokines (14 cytokine expression profiles are shown; il-12(p70)(78) was not detected at any time point and is not included), with a blue-to-yellow scale indicating expression levels (see the key to the right of the panel). the module to which the protein's mrna transcript was assigned during clustering is shown on the right hand side of the heat map (proteins whose gene transcripts were not de are labeled 'na'), and the average virus titers are shown below the heat map (red indicates that titers exceeded the threshold concentration predicted by the segmented linear model). while il-18 and leukemia inhibitory factor (lif) exhibited protein expression patterns consistent with n1 module behavior, the transcripts mapping to these proteins were not differentially expressed and were excluded from the co-expression network construction. (tif) s7 fig. virus titer and immunoblot data from an additional infection experiment with 10 5 pfu of h1n1, ph1n1, or h5n1 virus. as described in the fig 5 legend (panel b) , an additional set of mice was infected with 10 5 pfu of h1n1, ph1n1, or h5n1 virus to determine total and phosphorylated levels of transcription factors by means of immunoblotting. here, virus titers (with standard deviation indicated by gray bars) for each infection condition are shown in panel (a), and immunoblot results are shown in panels (b) and (c). for immunoblot analyses, relative protein concentrations were determined by calculating the ratio of the gray intensity of the measured protein (iκbα, total irf7, total irf3 ['irf3'], phosphorylated irf3 [pirf3'], total stat1 ['stat1'], and phosphorylated stat1 ['pstat1']) relative to the gray intensity of actin (i.e., the loading control; referred to as the relative signal intensity, rsi) in each tissue sample. a linear model was used to compare the mean rsi of each protein at each time point to the mean rsi measured in uninfected animals (referred to as 'naïve'), and a significant difference was defined as having a false discovery rate (fdr) < 0.05. the mean rsi of total irf3, irf7, and iκbα did not significantly deviate from the naïve data, but pstat1, pirf3 and total stat1 significantly differed from the naïve data at several time points (signifithe intramodular correlation (kme or correlation between each transcript and the eigengene) can be used to assess clustering quality. we show boxplots to illustrate the distribution of the kmes for all transcripts belonging to the n1 and n2 modules. (tif) s11 fig. applying signed co-expression network analysis also clusters the n1 kme+ transcripts. the n1 module presented in this work was identified by developing an unsigned co-expression network and then focusing on the kme-and kme+. therefore, the n1 kme+ set of genes should also cluster when developing a signed co-expression network. we confirmed this by repeating the wgcna procedure for signed network (power = 14) . of the n1 kme+ transcripts, 93.2% were assigned to the same cluster in the signed co-expression network (the new module is referred to as n1signed). furthermore, 90.9% if the n1signed transcripts were n1 kme+ transcripts. we then confirmed that the same eigengene dynamics were observed. the scaled difference of the eigengene (sde) of the n1signed module is shown versus time (a) and versus virus titers (b). (tif) s1 file. each of the kme+ submodules, labeled n1, n2,. . .,n45 were analyzed using david bioinformatics tool to determine enriched biological functions. for each module, we report the top enriched annotation clusters as well as various measurements of enrichment (the bonferroni, benjamini, and the false discovery [fdr] adjusted p value. additional information on the enrichment statisticis can be found at david.abcc.ncifcrf.gov (xls) s2 file. each of the kme-submodules, labeled n1, n2,. . .,n45 were analyzed using david bioinformatics tool to determine enriched biological functions. for each module, we report the top enriched annotation clusters as well as various measurements of enrichment (the bonferroni, benjamini, and the false discovery [fdr] adjusted p value. additional information on the enrichment statistics can be found at david.abcc.ncifcrf.gov (xls) s3 file. a list of interferon stimulated genes received from the interferon stimulated gene database. (xlsx) s1 table. the co-expression module to which each transcript was assigned. for each transcript we provide the entrez gene id, the gene symbol, the module it was assigned to and the correlation between the transcript's expression dynamics and the expression dynamics of its assigned eigengene (kme). module n0 is the set of transcripts which the algorithm did not identify as co-expressed. (xlsx) s2 table. enriched annotations identified using toppcluster. each kme+ or kme-submodule was analyzed in toppcluster for enriched domain, go biological process, go molecular function, go cellular component, mouse phenotype, pathway or transcription factor binding site annotations. columns a-d provide information on the annotation, including the category to which the annotation belongs, the database specific annotation id and the full title of the annotation. columns d-at contain the enrichment score for each annotation in each submodule. the enrichment score is the-log10 of the false discovery rate [fdr]-adjusted p value. a threshold enrichment score of 2 (fdr-adjusted p < 0.01) was required to be considered as significantly enriched. (xlsx) s3 table. cten analysis of n2 module. the probes assigned to module n2 were analyzed in cten-a platform for identifying the genomic signatures of select cell type in microarray data. the enrichment score reported for each cell type is the-log10 of the false-discovery adjusted p value. (xlsx) s4 table. a heatmap of the gene expression of the probes assigned to the n1 and n2 modules. arrays were developed from the lungs of mice infected with h1n1, ph1n1, or hpai virus at 14 time points. one array from the hpai infected data set was removed due to quality concerns. for each transcript, we provide annotation information (probe name, systematic name, entrez id, gene symbol, and gene name), identify to which module the transcript was assigned, the kme (the pearson correlation coefficient between the transcripts expression and the eigengene of the module it was assigned to), and the log2 fold change in the transcript's expression across all arrays used the study is illustrated by a heatmap. (xlsx) s5 table. correlation between the changes macrophage and neutrophil cell counts and each module eigengene. (xlsx) s6 table. correlation between changes in cytokine protein levels and cytokine gene transcript levels. (xlsx) regulation of toll-like receptor signaling pathways in innate immune responses immune signaling by rig-i-like receptors cutting edge: influenza a virus activates tlr3-dependent inflammatory and rig-i-dependent antiviral responses in human lung epithelial cells recognition of single-stranded rna viruses by toll-like receptor 7 differential roles of mda5 and rig-i helicases in the recognition of rna viruses rig-i-mediated antiviral responses to single-stranded rna bearing 5'-phosphates 5'-triphosphate rna is the ligand for rig-i aberrant innate immune response in lethal infection of macaques with the 1918 influenza virus genomic analysis of increased host immune and cell death responses induced by 1918 influenza virus lethal dissemination of h5n1 influenza virus is associated with dysregulation of inflammation and lipoxin signaling in a mouse model of infection h5n1 influenza viruses: outbreaks and biological properties fatal outcome of human influenza a (h5n1) is associated with high viral load and hypercytokinemia in vitro and in vivo characterization of new swine-origin h1n1 influenza viruses integrated network analysis reveals a novel role for the cell cycle in 2009 pandemic influenza virus-induced inflammation in macaque lungs h5n1 influenza virus pathogenesis in genetically diverse mice is mediated at the level of viral load viral replication rate regulates clinical outcome and cd8 t cell responses during highly pathogenic h5n1 influenza virus infection in mice specific mutations in h5n1 mainly impact the magnitude and velocity of the host response in mice wgcna: an r package for weighted correlation network analysis resources: expanded annotation database and novel algorithms to better extract biology from large gene lists toppcluster: a multiple gene list feature analyzer for comparative enrichment clustering and network-based dissection of biological systems snapshot: pathways of antiviral innate immunity pathogenic influenza viruses and coronaviruses utilize similar and contrasting approaches to control interferon-stimulated gene responses cten: a web-based platform for identifying enriched cell types from heterogeneous microarray data lethal influenza virus infection in macaques is associated with early dysregulation of inflammatory related genes integrated clinical, pathologic, virologic, and transcriptomic analysis of h5n1 influenza virus-induced viral pneumonia in the rhesus macaque ccr2+ monocyte-derived dendritic cells and exudate macrophages produce influenza-induced pulmonary immune pathology and mortality a systems analysis identifies a feedforward inflammatory circuit leading to lethal influenza infection transcriptomic analysis of autistic brain reveals convergent molecular pathology extracellular 2'-5' oligoadenylate synthetase stimulates rnase l-independent antiviral activity: a novel mechanism of virus-induced innate immunity systematic and quantitative assessment of the ubiquitin-modified proteome the transcriptional landscape of the mammalian genome ultrasensitivity in the regulation of cdc25c by cdk1 bistability in apoptosis: roles of bax, bcl-2, and mitochondrial permeability transition pores ultrasensitivity and positive feedback to promote sharp mitotic entry ultrasensitivity in the mitogen-activated protein kinase cascade identifying fragilities in biochemical networks: robust performance analysis of fas signaling-induced apoptosis the ability of pandemic influenza virus hemagglutinins to induce lower respiratory pathology is associated with decreased surfactant protein d binding influenza a virus lacking the ns1 gene replicates in interferon-deficient systems identification of the non-structural influenza a viral protein ns1a as a bona fide target of the small ubiquitin-like modifier by the use of dicistronic expression constructs modification of nonstructural protein 1 of influenza a virus by sumo1 sumoylation affects the interferon blocking activity of the influenza a nonstructural protein ns1 without affecting its stability or cellular localization myd88 signaling is indispensable for primary influenza a virus infection but dispensable for secondary infection toll-like receptor pre-stimulation protects mice against lethal infection with highly pathogenic influenza viruses linear models and empirical bayes methods for assessing differential expression in microarray experiments toppgene suite for gene list enrichment analysis and candidate gene prioritization systematic discovery of regulatory motifs in human promoters and 3 utrs by comparison of several mammals molecular signatures database (msigdb) 3.0 functional classification of interferon-stimulated genes identified using microarrays segmented: an r package to fit regression models with broken-line relationships simultaneous inference in general parametric models we would like to thank susan watson for editing the manuscript. key: cord-252950-eiphxwmn authors: trouillet-assant, sophie; viel, sebastien; gaymard, alexandre; pons, sylvie; richard, jean-christophe; perret, magali; villard, marine; brengel-pesce, karen; lina, bruno; mezidi, mehdi; bitker, laurent; belot, alexandre title: type i ifn immunoprofiling in covid-19 patients date: 2020-04-29 journal: j allergy clin immunol doi: 10.1016/j.jaci.2020.04.029 sha: doc_id: 252950 cord_uid: eiphxwmn covid patients in icu present a high mortality rate and immunoprofiling reveals heterogeneous ifn-α2 production with about 20% of critically-ill patients unable to produce ifn-α2, highlighting the immune response heterogeneity and opening avenues for targeted therapies. sophie trouillet-assant 1,2* , phd, sebastien viel 2,3,4,5* , pharmd, phd, alexandre gaymard merazga for their excellent work. we thank fabien subtil for his helpful advice for statistical analysis. 41 we also thank the life (lyon immunopathology federation) community for fruitful discussion. 42 capsule summary: 43 covid patients in icu present a high mortality rate and immunoprofiling reveals heterogeneous α2 production with about 20% of critically-ill patients unable to produce ifn-α2, highlighting the 45 immune response heterogeneity and opening avenues for targeted therapies. 46 to the editor, 48 49 severe acute respiratory syndrome coronavirus 2 (sars-cov-2) infection (covid-19) is characterized 50 by a wide spectrum of disease encompassing asymptomatic carriage, mild to severe upper 51 respiratory tract illness that can evolve into respiratory failure or rapidly progressing severe viral 52 pneumonia with acute respiratory distress syndrome (ards). disease severity depends on viral strain 53 and host risk factors have been identified such as age and male gender. in addition, an excessive 54 immune response has been identified in patients showing a cytokine storm associated with ards 1 . 55 various immunosuppressive drugs, including il-6 blockers or jak-stat signaling inhibitors have been 56 suggested for the treatment of sars-cov-2 infection 2 whereas additional clinical trials are evaluating 57 the use of recombinant interferon to foster host antiviral response. (clinicaltrials nct04315948, 58 nct04293887). type i interferons (ifn-i) are major components of the innate immune system and 59 represent critical antiviral molecules 3 . to date, ifn-i response has not been evaluated in covid-19 60 patients and its contribution to the viral control and inflammation is unknown. 61 in this study, we assessed the kinetics of plasma ifn-i in covid-19 patients with a spectrum of 62 severity degree. this study was approved by an ethical committee for biomedical research (comité 63 de protection des personnes hcl). (supplemental material and method of this article online 64 repository). 65 firstly, we explored three patients issued from the first covid cluster diagnosed in france (les 66 contamines, haute savoie, france) in february 2020. we took advantage of the new digital elisa 67 technology single-molecule arrays (simoa) 4 and analyzed the kinetics of plasma inflammatory 68 cytokines. interleukin (il)-6, c-reactive protein (crp) and interferon γ-induced protein 10 (ip-10) 69 were elevated in the two symptomatic patients (pt1, 3) (supplementary figure 1 in the online 70 repository). strikingly, no ifn-α2 was detectable in these two patients. in contrast, il-6, crp and ip-71 elevation of plasmatic ifn-α2 was observed. viral loads were low with no obvious quantitative 73 difference between all three patients. 74 we further explored a larger cohort of 26 critically ill covid patients from one of the intensive care 75 unit (icu) at hospices civils de lyon (lyon, france). of note, all the patients were treated with 76 standard of care and none received antiviral or immunotherapies. considering the first 28 days of 77 infection, more than half of critically ill patients required invasive mechanical ventilation (14/26). we 78 observed that patients demonstrated a peak in ifn-α2 at day 8-10 of symptoms onset corresponding 79 to the viral replication phase, that decreased overtime to low but still detectable ifn-α2 the timing of interferon exposition may be critical to control the virus and avoid 98 immunopathogenesis. channappavanar et al. have shown that delayed ifn-i expression can be 99 detrimental in mice in the context of sars-cov-1 infection 6 . our data suggests that screening 100 patients for ifn production is instrumental to select those who could benefit from early intervention 101 with ifn. following day 10, il-6 remains increased while ifn-α tapered. this kinetics highlight that 102 cytokine inhibitors could be helpful at the second phase of the disease following ifn-i decrease. viral 103 characteristic or individual genetic susceptibility should be explored to understand the defect of ifn-104 α production in some covid patients. some ifn-α2 positive patients also experienced fatal outcome 105 highlighting the multifactorial causes of disease severity. we acknowledge limitations of this study, 106 related to the small number of included patients and the technical limitation for the measurement of 107 ifn-β and ifn-λ, in this proof of concept study. 108 here, we provide new argues for an early intervention with recombinant ifn-α2 and we also 109 highlight the window of opportunity for immunosuppressors at the second phase of the disease, 110 delay between symptom onset and icu admission (days) 7 [1-11] 7[0-15] 0.769 bacterial co-infection during icu stay (n (%)) 3 (60%) 7(33%) diabetes (n (%)) 1 (20%) 3(14%) chronic obstructive pulmonary disease (n (%)) 0 (0%) 3(14%) cardiovascular disease (n (%)) 2(40%) 9 (43%) hypertension (n (%)) 3 (60%) 7 (33%) cancer (n (%)) 1 (20%) 3 (14%) active smokers (n (%)) 0 (0%) 1(5%) mortality at d28 after symptom onset(n(%)) 2 (40%) 8 (38%) 1.000 crp -c-reactive protein, icu -intensive care unit, bmi -body mass index table 1 -clinical characteristics of covid-19 patients in intensive care unit 130 p-value are calculated using mann-whitney test for quantitative values and using fisher-exact test for qualitative ones. a. plasma ifn-α concentrations (fg/ml) were determined by single molecule array (simoa) b.c.d. il-6, crp and ip-10 concentrations were measured using a multiplexed assay with the ella platform. e. viral load is represented as cycle threshold of ip2 rt-qpcr using assay designed by pasteur institut in paris. ifn-interferon ; il-6 -interleukin 6 ; crp -c-reactive protein ; ip-10 -interferon γ-induced protein 10 a. ifn score is a transcriptionnal signature defined by 6 interferon-stimulated gene (isg) quantified using nanostring technology and obtained from paxgene tubes in 4 covid-19 patients. b-d. normal values for healthy volunteers was indicated by grey area. vertical bar indicates median delay between symptom onset and icu admission. concentrations of ifn-γ were quantified in only 16/26 patients because of lack of material. clinical features of patients infected with 114 2019 novel coronavirus in wuhan, china covid-19: 116 consider cytokine storm syndromes and immunosuppression type i interferons (α/β) in immunity 118 and autoimmunity /679 and directive 95/46/ec) and the french data protection law (law n°78-17 on 06/01/1978 and décret n°2019-536 on 29/05/2019), we obtained consent from each patient or his next of kin usa) on plasma samples of covid-19 patients. the assay was based on a 3-step protocol using an hd-1 analyzer (quanterix). il-6, crp and interferon γ-induced protein 10 (ip-10) concentrations were measured using a multiplexed assay with the ella platform (protein simple© ca, usa), according to manufacturer's instructions. plasma il28a/b and il-29 (type iii interferon) have been quantified by elisa (pbl laboratories rna integrity was then evaluated by agilent rna microarray (agilent technologies© data standardization was obtained using the geometric mean of internal control and housekeeping genes count number. interferon score was calculated as previously described 1 . virus quantification load viral load was quantified from nasopharyngeal swabs or endotracheal aspirates. rna extraction was performed by the automated nuclisens® easymag® (biomérieux, marcy l'etoile, france) using manufacturer's instructions. a 25 μl reaction contained 5 μl of rna p-value were calculated using mann-whitney test for quantitative values and using fisher-exact test for qualitative ones comparison of rt-qpcr and nanostring in the measurement of blood interferon response for the diagnosis of type i interferonopathies walzer international center of research in infectiology, lyon university, inserm u1111, cnrs umr 5308, ens, ucbl, lyon, france we explored the first three sars-cov-2 positive patients diagnosed in france (les contamines, france) in february 2020. patient 3 : a high risk contact (a 54-year-old man) initially negative for sars-cov-2 developed fever and cough with respiratory crackles at auscultation on the fifth day of hospital isolation. a bilateral interstitial syndrome at the ct-scan with bilateral ground-glass opacification predominant on the left. sars-cov2 was detected from endotracheal aspirates (eta), all nasopharyngeal swabs were always negative. the daily follow-up revealed a short-lasting excretion with only two successive eta for these three patients, no other respiratory pathogens were detected. these patients did not need oxygenation, nor antibiotics, steroids or antiviral agents. plasma samples and paxgene® tubes were collected from covid-19 patients hospitalized in the university hospital of lyon (hospices civils de lyon), france. diagnosis of covid-19 was confirmed in all patients by rt-pcr.all critically ill patients, admitted to icu, were included in the mir-covid study. this study was registered to the french national data protection agency under the number 20-097 and was approved by an ethical committee for biomedical research (comité de protection des personnes hcl) under the number n°20-41. in agreement with the general data protection regulation (regulation key: cord-023403-jzdrvfvr authors: ahlfors, e.; sveinhaug, m. m.; nango, g.; johansen, c.; lyberg, t. title: proliferation of cells in the oral mucosa, the ear skin and the regional lymph nodes in mice sensitized and elicited with a hapten date: 2008-06-28 journal: scand j immunol doi: 10.1111/j.0300-9475.2004.01423ac.x sha: doc_id: 23403 cord_uid: jzdrvfvr during contact sensitivity reaction, immune cells proliferate. in order to study the histological picture of these proliferation phases, we used a mouse model of contact sensitivity in the oral mucosa and on skin. we also used bromodeoxyuridin (brdu, an analogue to thymidin) that is incorporated into the nucleus during cell replication. the hapten oxazolone (oxa) was used to sensitize and elicit the oral mucosa and/or the ear skin. mice were killed at various times after elicitation, and unsensitized animals were also exposed to the hapten as controls. brdu (25 mg/kg animal) was injected i.p. 2 h before the kill. specimens from the oral mucosa, ear skin and submandibular and auricular lymph nodes were cut and fixed in 4% paraformaldehyde. they were then treated with acid and biotinylated anti‐brdu antibody and developed using abc‐kit and dab. the analyses were performed using a leica light microscope and the computer program analysis. in the oral mucosa, the frequency of proliferating cells were increasing during the observation period, 4–24 h after elicitation, regardless of site of sensitization. the proliferating cells were found mainly in the basal cell layer of the epithelium. similar patterns were found in ear skin. the regional lymph nodes demonstrated a few scattered proliferating cells 4 h after elicitation. after 24 h, these cells were found frequently in the whole lymph node. control animals exhibited considerable less proliferating cells at all times. we conclude that most proliferating cells were found 24 h after elicitation locally at the hapten‐exposed sites (the oral mucosa or the ear skin) as well as in the regional lymph nodes. the nuclear receptor heterodimers of liver x receptors (lxrs) are recently identified as key transcriptional regulators of genes involved in lipid homeostasis and inflammation. lxrs and their ligands are negative regulators of macrophage inflammatory gene expression. multiple sclerosis (ms), a demyelinating disease of the central nervous system of unknown cause, is characterized by recurrent inflammation involving macrophages and their inflammatory mediators. sweden belongs to the countries with a high ms incidence. in italy, incidence is lower, with an exception for sardinia where the incidence is even higher than that in sweden. subjects from sardinia are ethnically more homogeneous and differ from swedes, also regarding genetic background and environment. we studied lxrs and their related molecules of blood mononuclear cells (mncs) from female patients with untreated relapsing-remitting ms from sassari, sardinia and stockholm, sweden. sex-and age-matched healthy controls (hcs) were from both areas. mrna expression was evaluated by real-time pcr. lxr-a was lower (p < 0.05) in ms (mean ae sem: 3.1 ae 0.2; n ¼ 37) compared to hc (3.6 ae 0.1; n ¼ 37). lxr-a was lower in ms from stockholm (2.6 ae 0.2; n ¼ 22) compared to corresponding hc (3.4 ae 0.1; n ¼ 22; p < 0.01) and compared to ms (3.8 ae 0.2; n ¼ 15; p < 0.001) and hc (4 ae 0.2; n ¼ 15; p < 0.001) from sardinia. ms patients from stockholm, but not from sassari, also expressed lower (p < 0.05) lxr-b (à4.1 ae 0.4) compared to corresponding hc (à2.9 ae 0.3). ms from stockholm was associated with higher abca-1 (6.1 ae 0.4 versus 5.0 ae 0.3; p < 0.05) and higher estrogen receptor-b-cx (2.4 ae 0.4 versus 0.8 ae 0.4; p < 0.01) compared to corresponding hc. the hc from sassari had higher androgen receptor (2.9 ae 0.2) compared to ms from sassari (1.4 ae 0.3; p < 0.01), ms (1.3 ae 0.4; p < 0.01) and hc from stockholm (1.2 ae 0.3; p < 0.01). ms from sassari had lower cyclooxygenase-1 compared to corresponding hc (5.1 ae 0.4 versus 6.6 ae 0.3; p < 0.01) and lower prostaglandin-e (à0.03 ae 0.5) compared to the hc (1.4 ae 0.5; p < 0.05) and ms (2.7 ae 0.4; p < 0.05) and hc from stockholm (1.9 ae 0.4, p < 0.001). our findings identify lxrs and their related molecules as being involved in ms from stockholm but not from sassari, while sex hormone receptors seem to be involved in ms in sassari. multiple sclerosis: ifn-b induces cd123 + bdca2 -dendritic cells that produce il-6 and il-10 and have no enhanced type i interferon production y. m. huang, 1 s. adikari, 1 u. båve, 2 a. sanna 1,3 & g. alm 4 dc antigens (bdca) and investigate their ability to produce type i ifn in response to virus stimulation. we show that ifn-b induces development of cd123 þ dc from human blood monocytes, which coexpress bdca4 þ but are negative for bdca2 -, a specific marker for plasmacytoid dc. such ifn-b-modulated dc produce large amounts of il-6 and il-10, but no il-12p40 and have no enhanced ifn-b and ifn-b production. the findings indicate that ifn-bmodulated dc represent a myeloid dc subset with diminished cd11c, bdca-1 and cd1a expression, having potent th2-promoting function but lacking antiviral capacity. the association of psoriasis with throat infections by streptococcus pyogenes suggests a potential antigenic target for the t cells that are known to infiltrate dermis and epidermis of psoriatic skin. streptococcal m protein shares an extensive sequence homology with human epidermal keratins. keratins 16 (k16) and 17 (k17) are mostly absent from uninvolved skin but are upregulated in psoriatic lesions. there is increasing evidence that cd8 þ t cells play an important effector role in psoriasis and m proteinprimed t cells may recognize these shared epitopes in skin via molecular mimicry. to identify candidate epitopes, peptides with sequences from k17 were selected on the basis of predicted binding to hla-cw6 and sequence similarities with m6 protein. matched peptides from the sequence of m6 protein and a set of peptides with poor predicted binding were also selected. cw6 þ individuals with psoriasis and cw6 þ healthy controls, having a family history of psoriasis, were recruited. pbmcs were incubated with the peptide antigens. t-cell activation in the cd4 þ , cd8 þ and later the skin-homing cutaneous lymphocyteassociated antigen (cla)-expressing subset of cd8 þ t cells was evaluated by cd69 expression and intracellular ifn-g accumulation using flow cytometry. we demonstrate that cw6 þ psoriasis patients had significant cd8 þ t-cell ifn-g responses to peptides from k17 and m6 protein selected on the basis of sequence homology and predicted hla-cw*0602 binding. these responses were about 10 times more frequent in the skin-homing cutaneous lymphocyte-associated antigen-expressing (cla þ ) subset of cd8 þ t cells. cd4 þ t cells showed only borderline responses. cd8 þ t cells from cw6 þ nonpsoriatic individuals responded to some m6 peptides but very rarely to k17 peptides, and this also applied to the cla þ cd8 þ subset. these findings indicate that psoriatic individuals have cd8 þ t cells that recognize keratin self-antigens and that epitopes shared by streptococcal m protein and human keratin may be targets for the cd8 þ t cells that infiltrate psoriatic skin lesions. autoantibodies directed against citrulline-containing proteins have an impressive specificity of nearly 100% in ra patients and a suggestive involvement in the pathogenesis. the targeted epitopes are generated by a post-translational modification catalysed by the calcium-dependent enzyme peptidyl arginine deaminase that converts the positively charged arginine to polar but uncharged citrullin. the aim of this study was to analyse the presence of citrulline in the joints at different time points of collagen-induced arthritis in da rats by immunohistochemistry and to investigate how immunogenicity and arthritogenicity was affected by citrullination of rat serum albumin (rsa) and collagen type ii (cii). our results indicate that citrulline could be detected in joints of arthritic animals, first appearance at the onset of disease and increasing as disease progressed into a chronic state. unimmunized animals or time points before clinical signs of arthritis were negative. by morphology, we state that some infiltrating macrophages as well as the cartilage surface stain positive for citrulline, while the major source of citrullinated proteins appears to be fibrin depositions. a specific cit-rsa t-cell response was observed in animals challenged by citrullinated rsa, no response was recorded when rsa was used as a stimulus. the igg analysis reveals not only a response towards the modified protein but also cross-reactivity to native rsa. no t-cell or b-cell response was noted in animals injected with unmodified rsa. cit-cii induced a disease with higher incidence and earlier onset than did the native counterpart. we conclude that, in contrast to the human disease, citrulline does not seem to appear before clinical signs. as inflammation proceeds, citrulline is detected specifically in the joints. all other organs investigated were negative. we also conclude that citrullination of a protein can break tolerance and increase its arthritogenic properties. ectopic germinal centers (gcs) can be detected in the salivary glands of approximately 1/5 of patients with sjögren's syndrome (ss) and appear in both primary and secondary ss. previously, ectopic gc have been associated with increased local autoantibody production. the aim of this study was to determine whether gc in primary sjögren's syndrome (pss) defines a distinct seroimmunological phenotype. retrospectively, a material of 130 haematoxylin and eosin-stained paraffin-embedded tissue sections of minor salivary gland tissue from patients with pss was morphologically screened for the presence of ectopic gc. gc-like lesions were detected in 33/130 (25%) of the pss patients. seventy-two pss patients lacking these structures (gc-) were randomly selected for comparison. focus score was significantly increased in the gc þ patients compared to the gcpatients (p ¼ 0.035). in the gc þ group, 54.5% of the patients presented with anti-ro/ssa compared to 43.7% in the gcgroup. anti-la/ssb was detected in 31.3% of the gc þ patients compared to 25.7% of the gcpatients. sixty-one percentage of gc þ patients presented with increased levels of igg, a nonsignificant difference when compared to 39.4% in the gcpatients (p ¼ 0.089). levels of rf, ana, ena, igm and iga were similar in both patient groups, as were esr and crp. in conclusion, patients with ectopic gc have a higher focus score and more often present with autoantibodies and increased levels of igg compared to pss patients with regular focal infiltration (gc -). our findings may indicate a certain seroimmunological phenotype and warrant for further prospective studies. association between mannose-binding lectin and vascular complications in type 1 diabetes complement activation and inflammation have been suggested in the pathogenesis of diabetic vascular lesions. we investigated serum mannose-binding lectin (mbl) levels and polymorphisms in the mbl gene in type 1 diabetic (t1dm) patients with and without diabetic nephropathy and associated macrovascular complications. polymorphisms in the mbl gene and serum mbl levels were determined in 199 t1dm patients with overt nephropathy and 192 t1dm patients with persistent normoalbuminuria matched for age, sex and duration of diabetes as well as in 100 healthy control subjects. the frequencies of high and low expression mbl genotypes were similar in patients with t1dm and healthy controls. high mbl genotypes were significantly more frequent in diabetic patients with nephropathy than in the normoalbuminuric group, and the risk of having nephropathy, given a high mbl genotype, assessed by odds ratio was 1.52 (1.02-2.27), p ¼ 0.04. median serum mbl concentrations were significantly higher in patients with nephropathy than in patients with normoalbuminuria [2306 mg/l (iqr 753-4867 mg/l) versus 1491 mg/l (iqr 577-2944), p ¼ 0.0003], and even when comparing patients with identical genotypes, serum mbl levels were higher in the nephropathy group than in the normoalbuminuric group. patients with a history of cardiovascular disease had significantly elevated mbl levels independently of nephropathy status [3178 mg/l (iqr 636-5231 mg/l) versus 1741 mg/l (iqr 656-3149 mg/l), p ¼ 0.02]. the differences in mbl levels between patients with and without vascular complications were driven primarily by pronounced differences among carriers of high mbl genotypes (p < 0.0001). our findings suggest that mbl may be involved in the pathogenesis of microvascular and macrovascular complications in type 1 diabetes and that determination of mbl status might be used to identify patients at increased risk of developing these complications. neuroimmunology unit, center for molecular medicine, karolinska institutet, stockholm, sweden. e-mail: judit.wefer@cmm.ki.se dna vaccine coding for the encephalitogenic peptide mog 91-108 protects lew.1av1 from subsequent development of experimental autoimmune encephalomyelitis (eae). protection is associated with a type 1 immune response and is dependent on the presence of cpg dna motifs. the mechanisms underlying the observed reduction of eae development in protected rats have not been fully clarified. we investigated immunological characteristics of lymphocytes after dna vaccinaton and subsequent eae induction. we confirm that protection was not associated with suppression of t1 cells, as transcription of the novel molecule rat t-cell immunoglobulin-and mucindomain-containing molecule (tim-3), reported to be exclusively expressed on differentiated t1 cells, was not altered by dna vaccination. we did not note any clonal deletion upon tolerization, but detected an antigen-specific lymphocyte population upregulating ifng upon recall stimulation 3 weeks after protective dna vaccination. in protected rats, we observed (1) no alterations in antigenspecific th2 or th3 responses, (2) reduced mhc ii expression on splenocytes early after eae induction, (3) antigen-specific upregulation of ifnb upon recall stimulation and (4) reduced il-12rb2 on lymphocytes. we thus demonstrate an association of the protective effect of dna vaccination with expression of ifnb. we are currently investigating the cellular mechanisms behind this ifnb-mediated protection. multiple sclerosis (ms) is an autoimmune condition characterized by degeneration of nerve fibre myelin sheets. a candidate autoantigen, myelin basic protein (mbp), has especially attracted attention. the presence of anti-mbp antibodies is a predictor of definite ms, but their role in the pathogenesis remains obscure. t cells have long been known to play a pivotal role in the pathogenesis of ms. recently, an important role for b cells as autoantigen-presenting cells has been demonstrated in other autoimmune diseases, including rheumatoid arthritis and diabetes. the uptake of mbp by b cells and the presentation of mbp-derive peptides to t helper (th) cells by b cells may be promoted by the formation of complement (c) activating immune complexes (ics) between mbp and natural autoantibodies in healthy individuals and disease-associated anti-mbp antibodies in ms patients, respectively. we have investigated the formation of mbp-containing ic, the binding of mbp to b cells, the mbp-elicited induction of th-cell and b-cell proliferation and the cytokine production in peripheral blood mononuclear cells (pbmcs) from healthy donors grown in the presence of intact or c-inactivated serum from healthy donors or patients with ms. while mbp did not induce measurable proliferation of b cells nor cd4 þ t cells, we observed the production of tnf-a, ifn-g and il-10 by pbmc in response to incubation with mbp in the presence of sera from healthy controls as well as sera from ms patients. by contrast, no production of il-2, il-4 and il-5 was detected. we are currently investigating the capability of ms sera to promote the formation of mbp-containing ic and thereby enhance the cytokine responses, by virtue of elevated anti-mbp contents. the phagolysosomally localized acid sphingomyelinase (asmase) activated by proinflammatory cytokines such as tnf and ifn-g generates the signalling molecule ceramide which in turn results in the activation of proteases like cathepsin d. these characteristics of asmase suggest a possible role of this molecule in the phagocytotic uptake and phagosomal degradation processes of antigens or in antigen presentation. we show here that asmase -/mice fail to eliminate the noncytopathic lymphocytic choriomeningitis (lcm) virus as rapidly as littermate wildtype mice. investigation of the immune response revealed a reduced expansion of cd8 þ t cells. the secretion of ifn-g in response to contact with target cells as well as the cytolytic activity of virus-specific cd8 þ t cells was severely impaired. additionally, both phases of the lcm virus-specific dth response, mediated by cd8 þ and cd4 þ t cells consecutively, were diminished in asmase -/mice. however, the secondary memory response of virus-specific ctl was not altered, and the 614 abstracts ................................................................................................................................................................................................. virus was effectively controlled for at least 3 months by asmase -/mice. in conclusion, the results of this study suggest an involvement of the asmase in the activation, expansion or maturation of virus-specific cd8 þ t cells during the acute infection of mice with the lcm virus. novel markers for alternative activation of macrophages: macrophage galactose-type c-type lectins 1 and 2 in parallel with the th1/th2 dichotomy, macrophages are capable of developing into functionally and molecularly distinct subpopulations, due to differences in, for example cytokine environment and pathological conditions. while the best-studied, classically activated macrophage is induced by type i stimuli such as ifn-g, a type ii cytokine environment antagonizes the classical activation of macrophages and is capable of alternatively activating macrophages. however, molecular markers associated with these type ii cytokine-dependent, alternatively activated macrophages remain scarce. besides the earlier documented markers macrophage mannose receptor and arginase 1, we recently demonstrated that murine alternatively activated macrophages are characterized by increased expression of fizz1 and ym. we now report that expression of the two members of the mouse macrophage galactose-type c-type lectin gene family, termed mmgl1 and mmgl2, is induced in diverse populations of alternatively activated macrophages, including peritoneal macrophages elicited during infection with the protozoan trypanosoma brucei or the helminth taenia crassiceps, and alveolar macrophages elicited in a mouse model of allergic asthma. we also demonstrate that, in vitro, interleukin-4 and interleukin-13 upregulate mmgl1 and mmgl2 expression and that, in vivo, induction of mmgl1 and mmgl2 is dependent on interleukin-4 receptor signalling. moreover, we show that regulation of mgl expression is similar in human monocytes and monocyte-derived macrophages. hence, macrophage galactose-type c-type lectins represent novel markers for both murine and human alternatively activated macrophages; thus, paving the way for further characterization of the phenotype of macrophages occurring in th2 conditions. background: human parvovirus b19 (b19) is a ubiquitous pathogen, normally causing a mild self-limiting disease, but also capable of causing both significant pathology and long-term persistence. the small size and stability of the virus makes it suitable for mapping of the full breath and the kinetics of the cellular immune responses following acute viral infection. methods: five patients with acute primary b19 infection were included in the study and followed consecutively for up to 200 weeks. cellular immune responses were mapped by ifng enzyme-linked immunospot to overlapping peptides spanning the whole b19 genome. results: in all five acutely infected patients, we were able to monitor the kinetics of a strong specific cellular immune reaction. responses peaked at levels of 850-1850 sfc/ million pbmcs, roughly corresponding to 0.3-0.6% b19specific cd8 þ cells circulating in peripheral blood at 10-80 weeks post-infection. the responses in individual patients were directed to three or four different peptide pools, and the specificity was confined to the same cd8 epitopes present in the pools throughout the follow-up period. the majority of responses were directed to the virus nonstructural protein, only two patients showed any response to the capsid proteins, elicited by the same epitope in both cases. conclusion: the cellular immune responses to acute b19 infection are surprisingly narrow in distribution and remain at high levels for up to 80 weeks post-infection. the initial epitope specificity is maintained, and the majority of responses target the virus nonstructural protein, which is not included in vaccine preparations, evaluated against the infection. the relationship between malnutrition and malaria is controversial. on one hand, malaria may cause malnutrition, while on the other, malnutrition itself may modulate susceptibility to the disease. we investigated the association between plasmodium falciparum malaria and malnutrition in a cohort of children living on the coast of kenya. the study involved longitudinal follow-up for clinical malaria episodes and anthropometric measurements at four cross-sectional surveys. we used poisson regression analysis to investigate the association between malaria and nutritional status. compared to baseline (children with a waz or haz score of !à2), the crude incidence rate ratios (irrs) for malaria in children with low haz or waz scores (<à2) during the period prior to assessment were 1.17 (95% ci 0.91-1.50; 0 ¼ 0.21) and 0.94 (0.71-1.25; 0.67), respectively, suggesting no association between malaria and the subsequent development of pem. however, we found that age was acting as an effect modifier in the association between malaria and malnutrition. the irr for malaria in children 0-2 years old who were subsequently characterized as wasted was 1.65 (1.10-2.20; p ¼ 0.01), and a significant overall relationship between malaria and low-haz was found on regression analysis when adjusting for the interaction with age (irr 1.89; 1.01-3.53; p < 0.05). although children living on the coast of kenya continue to suffer clinical episodes of uncomplicated malaria throughout their first decade, the association between malaria and malnutrition appears to be limited to the first 2 years of life. a. astrinidou-vakaloudi, 1 s. xytsas, 1 i. diamanti, 1 h. ioannidis 2 & p. pangidis 2 1 microbiology department of general hospital of thessaloniki 'agios pavlos', thessaloniki, greece, and 2 nefrology, 2 nd ika hospital of thessaloniki, thessaloniki, greece. e-mail: stasa@hol.gr aim: renal dysfunction may influence the colonization of gastric mucosa by urea-splitting bacteria such as helicobacter pylori, by increasing urea concentrations in the gastric juice. our aim was to investigate the prevalence of h. pylori in patients with end-stage renal disease (esrd), receiving long-term haemodialysis treatment. methods: this study included 40 sera from patients with esrd (29 male and 11 female) undergoing periodic haemodialysis; mean time of treatment was 42.6 months. using elisa technique, we investigated the presence of igg and iga antibodies against h. pylori as well as igg caga (antibodies specific for caga(þ) strains of h. pylori). sera from 40 healthy blood donors were used as a control group. results: h. pylori igg antibodies were detected in 32 out of 40 (80%) patients in the dialysis group, while 31/40 (77.5%) tested positive for iga. igg caga antibodies were present in 13 out of 40 (32.5%). prevalence of h. pylori igg, iga and caga igg antibodies in the control group was 33, 7 and 15%, respectively. conclusions: although international data suggest that prevalence of h. pylori infection is the same in esrd patients as in healthy individuals, in our study that seems not to be the case. the higher blood and gastric juice urea levels may be a risk factor (among many others), but more studies are required in order to understand the relation of h. pylori infection in this group of patients. flanders interuniversity institute for biotechnology, department of molecular and cellular interactions, free university of brussels, brussels, and 2 pasteur institute of brussels, mycobacterial immunology, brussels, belgium. e-mail: tgartner@vub.ac.be immunity against tuberculosis (tb), caused by mycobacterium tuberculosis, depends largely on activation and maintenance of strong cell-mediated immune responses involving both cd4 þ and cd8 þ t cells and the ability to respond with th1-type cytokines, particularly ifn-g. recent studies suggested that bcg, the only licensed vaccine against m. tuberculosis, may fail to induce t-cell responses in the lung mucosa and may therefore not protect against pulmonary tb. a decrease in tb mortality may be achieved by enhancing immunity in the lung. the present study evaluated the induction of antigen-specific immunity in the lung by intranasal (i.n.) delivery of the lipoprotein i (opri) from pseudomonas aeruginosa. opri has shown to be a toll-like receptor 2/4 agonist that, when given subcutaneously, induces type-1 immune responses against heterologous antigens. here, a fusion of opri to ag85a of mtb (opri-ag85a) was used as a subunit vaccine in homologous prime-boost immunizations. in addition, opri-ag85a was combined with an ag85a-encoding dna vaccine (ag85a dna) or with bcg in heterologous prime-boost vaccinations. intranasal and parenteral delivery with opri-ag85a elicited comparable t-cell responses in the spleen; in addition, i.n. delivery elicited specific t-cell responses in the lung lymph nodes (llns). intramuscular delivery of ag85a dna induced significant systemic th1 immune responses. intranasal boosting with opri-ag85a enhanced this response and in addition induced an antigen-specific ifn-g response in lln. opri may therefore be an efficient adjuvant for mucosal boosting. we continue to evaluate the protection induced by opri-based prime-boost vaccinations against pulmonary tb. results on the immunogenicity and protection against intravenous mtb h37rv infection will be presented. toll-like receptors (tlrs) are pattern recognition receptors of the innate immune system, which recognize molecular structures on pathogens or cellular stress-associated molecules. tlr-ligand interactions trigger activation of inflammatory signal transduction and expression of genes involved in host defense. in this study, we have examined the requirement for different tlr adaptor molecules in virus-induced chemokine expression and are currently trying to identify the tlr involved. we have found that both a herpesvirus [herpes simplex virus (hsv)] and a paramyxovirus (sendai virus) require a functional genome to induce expression or proinflammatory chemokines in human and murine monocytic cell lines. for both viruses, this is independent of the tlr adaptor molecules trif and mal. however, overexpression of the vaccinia virus-encoded inhibitor of tlr-signalling a52r or dominant-negative myd88 totally inhibited hsv-induced rantes expression but only partially prevented sendai virus from inducing this chemokine. this suggests that hsv-induced rantes expression occurs via a tlr pathways, whereas sendai virus utilizes both tlr-dependent and -independent pathways to stimulate expression of rantes. we are currently trying to identify the tlrs involved. data from these studies will also be presented at the meeting. 2 0 -5 0 -oligoadenylate synthetases are interferon-induced, double-stranded rna-activated antiviral enzymes which are the only proteins known to catalyse 2 0 -specific nucleotidyl transfer. this first crystal structure of a 2 0 -5 0oligoadenylate synthetase reveals a structural conservation with the 3 0 -specific poly(a) polymerase that, coupled with structure-guided mutagenesis, supports a conserved catalytic mechanism for the 2 0 -and 3 0 -specific nucleotidyl transferases. comparison with structures of other superfamily members indicates that the donor substrates are bound by conserved active site features while the acceptor substrates are oriented by nonconserved regions. the 2 0 -5 0oligoadenylate synthetases are activated by viral doublestranded rna in infected cells and initiate a cellular response by synthesizing 2 0 -5 0 -oligoadenylates, that in turn activate rnase l. this crystal structure suggests that activation involves a domain-domain shift and identifies a putative dsrna activation site that is probed by mutagenesis. we demonstrated that this site is required both for the binding of dsrna and for the subsequent activation of oas. this rna-binding site is different from known rna-binding site; rather than forming a defined three-dimensional domain, it is located at the interface of the two major domains in oas. this novel architecture ensures that the dsrna helix can make simultaneously contact with both domains of oas and ensure the subsequent structural rearrangement leading to the activation of oas. our work provides structural insight into cellular recognition of double-stranded rna of viral origin and identifies a novel rna-binding motif. bacteria-specific iga antibodies are efficient opsonins for neutrophils and mononuclear phagocytes, provided that the phagocytes express the fca receptor (cd89). expression of cd89 can be stimulated by inflammatory cytokines, activated complement factors and certain microbial components. in one study, unstimulated phagocytes were able to ingest iga antibody-treated pneumococci, but only in the presence of complement, which was found to be activated by the iga antibodies along the alternative pathway. pneumococci produce iga1 protease that cleaves human iga1, but not iga2, molecules in the hinge region. this leaves iga1 as faba (monovalent) deprived of fca which contains the docking site for cd89. iga1 is the vastly predominant subclass of iga in the upper airways and circulation of humans. aims: to examine the effects of iga1 protease activity and complement on phagocytosis of iga antibody-coated pneumococci by an unstimulated human phagocytic cell line (hl60). materials and methods: iga1 and iga2 monoclonal antibodies to serotype 4 pneumococcal capsular polysaccharide (ps) were generated by heterohybridoma technique involving b cells from human vaccinees. isogenic serotype 4 pneumococci with and without iga1 protease activity, respectively, were obtained after inactivation of the iga gene of the tigr4 strain. opsonophagocytosis was quantitated using the assay described by romero-steiner et al. based on enumeration of surviving bacteria by culture. the integrity of iga molecules was examined by western blotting. results: both iga1 and iga2 antibody to type-4 polysaccharide-induced phagocytosis of iga1 protease-deficient type-4 pneumococci equally well in the absence as in the presence of complement. iga1 antibody to type-4 polysaccharide displayed a fourfold higher opsonophagocytosis titer against iga1 protease deficient compared to homologous wildtype target bacteria. a similar effect of iga1 protease activity of the target bacteria was not observed in a parallel experiment where iga2 antibody to type-4 polysaccharide served as opsonin. iga1 antibody extracted from iga1 protease-producing target bacteria was almost exclusively in the form of faba. conversely, iga1 from protease-deficient bacteria and iga2 from both types of bacteria were intact. conclusions: these results indicate that the iga1 protease activity of s. neumoniae may help the bacteria escape iga1 antibody-mediated opsonophagocytosis. besides, in these experiments, iga-mediated opsonophagocytosis was independent of complement. vitamins e and c have been found to increase the cellular and humeral immunity of pigs. vitamin e deficiency has also been found to predispose pigs to different diseases, e. coli infection is one among them. after weaning, the vitamin e status of pigs often decreases to a critical low level. in this experiment, we studied whether vitamin c supplementation would be a possible feeding strategy to optimize the immune status of weaners. the interaction between vitamin e and c is interesting due to the reported sparing action on vitamin e or synergism between these to vitamins. piglets were weaned at day 28 of age from sows fed increasing dietary vitamin e during lactation, and piglets were during the following 3 weeks fed either a control diet or this diet supplemented with 500 mg stay-c per kg. blood sampling was obtained weekly from day 28 and until day 49 of age. on the same days, one piglet per dietary treatment was killed and alveolar macrophages (am) were harvested. vitamin c supplementation increased the concentration of igm in serum of piglets throughout the weaning period. although the vitamin e concentration in am decreased with increasing age of the piglets, the concentration was numerically higher in piglets of sows fed the high dietary level of vitamin e. however, vitamin c supplementation tended to increase the total am concentration of vitamin e after weaning and increased the proportion of the biologically most active isomer of vitamin e [rrr-(a-tocopherol)] in the am. the eicosanoid synthesis by am was not influenced by the vitamin c supplementation, but the synthesis of leukotriene b4 was decreased 2 weeks after weaning compared to other days of am harvesting. in conclusion, dietary vitamin c supplementation improved the immune responses of piglets after weaning. a whole blood stimulation assay with escherichia coli (o111:b4) endotoxin was established to measure the capacity of dairy cows to produce the proinflammatory cytokine tumour necrosis factor-a (tnf-a) ex vivo. initially, a time-and dose-dependent study was carried out to find the optimal stimulation conditions for the tnf-a response. the tnf-a response peaked between 3 and 4 h at 38.5 c. a dose in the range of 5-10 g of e. coli lipopolysaccharide (lps)/ml whole blood was found to give the maximum tnf-a response. thirty-eight danish-holstein dairy cows were investigated for their tnf-a responsiveness ex vivo in the periparturient period. heparin-stabilized blood samples were collected seven times over a period of 4 months (weeks à3, à1, 2, 3, 5, 9 and 13 around calving) and stimulated with 5 g/ml of e. coli lps. indeed, fluctuations in the tnf-a responsiveness occurred over time. moreover, the mean tnf-a responsiveness of 38 cows was found to be significantly increased (p < 0.001) in the weeks close to calving. however, in the more stabile physiological periods, some cows had a consistently low tnf-a response, whereas others had high a tnf-a response. we are currently investigating whether high and low tnf-a responders to e. coli lps also exist in dairy cows in vivo. moreover, the importance of tnf-a responsiveness ex vivo to dairy cows' susceptibility and clinical response to experimental e. coli infections in the udder is being investigated. coelomic cytolytic factor (ccf) is a 42 kda invertebrate pattern recognition molecule isolated from the coelomic fluid of the earthworm eisenia foetida (oligochaeta, annelida). ccf displays a number of similarities with the mammalian cytokine tumour necrosis factor-a (tnfa) as a result of a shared n,n 0 -diacetylchitobiose lectin-like domain. however, these similarities are solely functional and are not based on any (dna or amino acid) sequence homology, thus suggesting a form of convergent evolution. in particular, the lectin-like domain of tnf-a has been shown to induce membrane depolarization in various mammalian cell types, through interactions with endogenous amiloride-sensitive ion channels. this nonreceptor-mediated activity of tnf-a has been reported to be involved in the resorption of oedema. likewise, the lectin-like domain of ccf also induces membrane depolarization in mammalian cells. here, we show that ccf appears to be able to induce oedema resorption in an alveolar epithelial cell line through its lectin-like domain. this lectin-like domain of ccf interacts (directly or indirectly) with endogenous sodium and/or chloride channels, and not potassium channels, on mammalian cells. additionally, we suggest that the jnk/sapk and erk1/2 pathways are involved in ccf-induced macrophage activation. these results further establish the functional analogy between an invertebrate pattern recognition molecule and a mammalian cytokine and, from a more applied point of view, suggest the possibility of utilizing ccf in the treatment of oedema. release of svegf and svegfr1 from white blood cells and platelets during surgery and stimulation with bacterial antigens introduction: the influence of surgery on release of soluble vascular endothelial growth factor (svegf) and the soluble vascular endothelial growth factor inhibitory receptor 1 (svegfr1) is unknown. we studied the effect of major and minor surgery on potential variations in svegf and svegfr1 concentrations in vivo and on bacterial antigen-induced release of svegf and svegfr1 from whole blood in vitro. methods: sixty-one patients with abdominal diseases undergoing five different surgical procedures were included. blood samples were drawn from anaesthetized patients before and after the operation. white blood cells and platelets were counted, and plasma svegf and svegfr1 was determined by an elisa method. whole blood from each blood sample was stimulated in vitro with bacteria-derived antigens (lps or protein-a) and svegf and svegfr1 levels were subsequently determined in the supernatants. stimulation with isotonic saline served as control assay. neither svegf or svegfr1 in plasma changed during surgery. in vitro stimulation of blood samples with bacteria-derived antigens resulted in a significant increase in svegf (p < 0.0001) and a less pronounced but still significant increase in svegfr1. release of svegf due to stimulation was significantly higher after the operation (nonsignificant), whereas svegfr1 release remained largely unchanged after surgery. correlation between bacterial antigen-induced release of svegf and neutrophile cell count was highly significant (p < 0.0001). there was no correlation between svegf and platelet cell count, and bacterial antigen-induced svegfr1 release did not correlate with counts of neutrophils and platelets. conclusions: plasma svegf and svegfr1 concentrations did not change during surgery. in vitro bacterial stimulation led to increased release of svegf and svegfr1, which was not significantly amplified during surgery and which may be related to number of circulating neutrophils. natural killer cell functions and subsets after in vitro stimulation with il-2 and il-12, with special emphasis on intracellular ifn-g and nk-cell cytotoxicity r. nyboe, 1,2 t. rix, 1,2 j. krog, 1,2 e. tønnesen 1 & m. hokland 2 1 department of anaesthesiology and intensive care, aarhus university hospital, and 2 institute of medical microbiology, and immunology, university of aarhus, aarhus, denmark. e-mail: rnsr@studmed.au.dk materials and methods: isolated cryopreserved human peripheral blood mononuclear cells (pbmcs) were stimulated with il-2 and il-12. this stimulation has previously been shown to activate nk cells. cell cytotoxicity was measured by flow cytometry after incubation with k562 cells. this method was compared to the current standard 51cr release assay. cells were treated with bfa to accumulate ifn-g, stained for surface markers, permeabilized and stained for intracellular ifn-g. flow cytometry was then performed to measure intracellular ifn-g production in pbmc, especially in nk cells. results: we have demonstrated that stimulation with il-2 and il-12 is effective in increasing the number of ifn-gpositive cells. there is a distinct difference between the cd3-cd56dim and the cd3-cd56bright subsets, with a much greater proportion of ifn-g-positive cells in the cd3-cd56bright subset. the effects of stimulation with il-2 and il-12 on cytotoxicity will be presented, as will the relation between ifn-g production and cytotoxicity. in addition, we will present results of these assays applied to porcine cells. discussion: in combination, these tests will address nk cell function by combining cytotoxicity with ifn-g production in nk cell subsets. the results will demonstrate whether this could serve as a useful tool in describing nkcell function, which could be of value in clinical and experimental settings. culture of regulatory t-cell lines from bronchial mucosa t lymphocytes play a major role in many immune responses. in the last decade, special focus has been on the function of th1 and th2 effector cells. now the importance of regulatory cd4 þ cd25 þ t cells in maintenance of the immunological homeostasis emerges. sarcoidosis is a multisystem granulomatous disorder often affecting the lungs. the typical sarcoid granulomas consists of epitheloid cells, macrophages and lymphocytes, mainly cd4 þ t cells of th1 phenotype. we have cultured t cells from bronchial biopsies of patients with sarcoidosis as well as from controls in high levels of interleukin 2 (il-2) and il-4 and demonstrate spontaneously arising cd4 þ cd25 þ populations and high concentrations of il-10 in these cultures. the main difference between cultures of sarcoid origin compared to controls is a very much higher concentration of the inflammatory cytokines il-6 and tnf-a in cultures of sarcoid origin. the effects of hyperbaric exposure on human peripheral blood mononuclear cells, with special emphasis on natural killer cell cytotoxicity and subsets materials and methods: as an experimental physiological stress model, we examined the effects of hyperbaric exposure on peripheral blood mononuclear cells (pbmcs) obtained from venous blood drawn from eight divers during a simulated heliox saturation dive. eight persons working in normobar atmosphere outside the pressurized chamber served as control donors. the spontaneous cytotoxicity of the pbmcs was estimated in a 4 h 51cr-release assay using k562 as nk-sensitive target cells. the pbmcs were characterized, using 4-colour flow cytometry, with special emphasis on the nk-cell subsets. the data were statistically analysed using a multivariate regression model (stata 8.2). p values <0.05 was considered statistically significant. results: the estimated cytotoxicity increased significantly in both the group of divers and control donors during the dive (pdivers < 0.01 and pcontrols < 0.01). although the cytotoxicity increased relatively more (p < 0.01) in the group of divers compared to the group of control donors between day 1 and 2. discussion: the increased cytotoxicity of pbmc estimated in the group of divers indicate that parts of the cellular immune system are affected during the extreme physiological conditions induced during the initial phase of the presented experimental hyperbaric setup. the increase in cytotoxicity observed in the group of control donors could hypothetically reflect the stress level in persons working outside the pressurized chamber during the dive. the interaction with adhesion molecules expressed by vascular endothelium is the first step in lymphocyte infiltration into tissues. at both cutaneous and mucosal sites interleukin-10 (il-10), il-12 and transforming growth factor (tgf)-b are important regulators of chronic inflammatory disease, where cutaneous lymphocyteassociated antigen (cla) and ae integrin (cd103) may be expressed. unlike cla, cd103 is not believed to play a role in tissue-specific homing but may help to retain t cells within epithelial layers. we have previously shown that il-12 alone can together with an unknown cofactor increase the expression of cla. stimulation with streptococcal pyrogenic exotoxin c (spec) increased the expression of cd103 by cd8 þ but not cd4 þ t cells. while il-12 increased superantigen-stimulated expression of cla, this cytokine strongly inhibited the cd103 expres-sion, and a combination of il-12 and tgf-b completely abrogated the induced cd103 expression. conversely, il-10 suppressed cla but increased cd103 expression. these findings indicate that, in addition to suppressing the development of th1-mediated inflammatory responses, il-10 may also inhibit the migration of cd8 þ t cells into the skin while il-12 promotes such migration. thus, the expression of cla and cd103 may be antagonistically regulated by il-10 and il-12, and the balance between these cytokines could influence the t-cell migration of inflammatory cells into epithelial tissues. during contact sensitivity reaction, immune cells proliferate. in order to study the histological picture of these proliferation phases, we used a mouse model of contact sensitivity in the oral mucosa and on skin. we also used bromodeoxyuridin (brdu, an analogue to thymidin) that is incorporated into the nucleus during cell replication. the hapten oxazolone (oxa) was used to sensitize and elicit the oral mucosa and/or the ear skin. mice were killed at various times after elicitation, and unsensitized animals were also exposed to the hapten as controls. brdu (25 mg/ kg animal) was injected i.p. 2 h before the kill. specimens from the oral mucosa, ear skin and submandibular and auricular lymph nodes were cut and fixed in 4% paraformaldehyde. they were then treated with acid and biotinylated anti-brdu antibody and developed using abc-kit and dab. the analyses were performed using a leica light microscope and the computer program analysis. in the oral mucosa, the frequency of proliferating cells were increasing during the observation period, 4-24 h after elicitation, regardless of site of sensitization. the proliferating cells were found mainly in the basal cell layer of the epithelium. similar patterns were found in ear skin. the regional lymph nodes demonstrated a few scattered proliferating cells 4 h after elicitation. after 24 h, these cells were found frequently in the whole lymph node. control animals exhibited considerable less proliferating cells at all times. we conclude that most proliferating cells were found 24 h after elicitation locally at the hapten-exposed sites (the oral mucosa or the ear skin) as well as in the regional lymph nodes. the endogenous nucleoside adenosine is released in excess during inflammation or other metabolic stress and is generally known to deliver tissue protective anti-inflammatory effects. adenosine acts via four adenosine receptors of which the a2a receptor is the predominant form in t cells. adenosine levels are elevated in asthmatic lung, and adenosine can directly induce mast cell degranulation and bronchoconstriction in these patients. instead, the role of anti-inflammatory mechanisms of adenosine on t cells in asthma is unclear. aim: to study the a2a receptor expression in peripheral blood cd4 þ t cells in asthmatic and healthy individuals using flow cytometric and quantitative real-time pcr methods. results: unstimulated cd4 þ cells of asthmatic patients expressed significantly lower levels (p < 0.001) of a2a receptor in protein level (mean percentage of cells positive ae sem: 76.8 ae 1.2, n ¼ 6) compared to healthy individuals (90.4% ae 1.9, n ¼ 4). double staining for cd69 expression showed that stimulation of cd4 þ cells decreased a2a expression in both groups but indicated that the detected lower levels of a2a in unstimulated cells of asthmatics was not due to preactivation in these patients. surprisingly, a2a mrna expression in unstimulated cd4 þ cells was significantly higher (p < 0.05) in asthmatics (n ¼ 28) compared to healthy controls (n ¼ 7). the expression did not correlate with serum total ige levels. conclusions: asthmatic individuals express less a2a adenosine receptor on their peripheral cd4 þ t cells. the higher mrna levels instead may point to a negative feedback regulation in the receptor expression. the role of possibly decreased adenosine-mediated anti-inflammatory effects in asthma pathogenesis require further studies on this t-cell mediated disease. the chronic inflammatory skin disease atopic eczema (ae) affects almost 15% of the population in many countries today. the pathogenesis of ae is not fully understood. a combination of genetic predisposition and environmental factors like microorganisms seems to contribute to the symptoms. the yeast malassezia sympodialis is part of our normal skin micro flora but can act as an allergen and elicit specific ige and t-cell reactivity in patients with ae. recently, we identified a novel major m. sympodialis allergen, designated mala s 11 (22.4 kda), with sequence similarity to the mitochondrial enzyme manganese superoxide dismutase (mnsod). interestingly, mala s 11 has a high degree of homology to human mnsod. the aim of this study was to examine the effects of recombinant mala s 11 on antigen-presenting dendritic cells. monocytederived dendritic cells (mddcs) from healthy blood donors were cultured with or without mala s 11 for different time periods. it was found that the maturation marker cd83 and the costimulatory molecules cd80 and cd86 were upregulated on the mddcs exposed to mala s 11 for 24 h, as demonstrated by flow cytometry. furthermore, coculture of mddcs with mala s 11 for 9 h induced an increased production of the inflammatory cytokines il-6 (200-fold), tnf-a (100-fold) and il-8 (sixfold), as detected by the cytometric bead array (cba) analysis. our results suggest that mala s 11 affects the immune response through dc maturation and production of inflammatory cytokines. the potential cross-reactivity with human mnsod needs to be explored and the exact role of mala s 11 in the pathogenesis of ae assessed in clinical studies involving skin prick and atopy patch tests. allergen-specific immunotherapy (sit) is commonly conducted with allergen extracts adsorbed to aluminium hydroxide (alum). drawbacks linked to the use of alum, such as the formation of granuloma at the site of injection, have led to suggestions of novel allergen carriers. an alternative carrier is 2 mm carbohydrate-based particles (cbps). in mouse, allergen-coupled cbps have been demonstrated to skew the allergen-specific immune response towards a th1-like activity (grönlund et al. immunology, 2002) . we here coupled the recombinant major cat allergen fel d 1 to cbps (cbp-fel d 1) by cyanogen-bromide activation, resulting in covalent binding. the effect of cbp-fel d 1 on monocyte-derived dendritic cells (mddcs) from healthy human blood donors was studied. we found that the majority of the cd1a þ mddcs were capable of taking up fitc-labelled cbp-fel d 1, as demonstrated by flow cytometry and confocal laser scanning microscopy. furthermore, incubation with cbp-fel d 1 resulted in an upregulation of the costimulatory molecule cd86 on the mddcs, which was not observed with fel d 1 or cbps alone. finally, cbp-fel d 1 induced a fivefold increase in the release of the pro-inflammatory cytokine tumour necrosis factor (tnf)-a and a fourfold increase in the release of the chemokine interleukin-8 from mddcs. taken together, the effects cbps possess make them interesting as novel allergen carriers for sit. the cysteine protease der p1 from dust mite of the genus dermatophagoides pteronyssinus is a major type i allergen. about 80% of house dust mite (hdm) allergic individuals are reactive to this protease in standard assays for detection of ige. a curative treatment for atopic allergy is immunotherapy (it) with hdm extracts which are complex mixtures occasionally resulting in anaphylactic reactions. novozymes focuses on developing a recombinant variant of der p1 which exhibit lowered risk of ige-mediated allergic reactions, while maintaining its ability to trigger proper th-cell responses. this may provide a safer alternative for specific it of hdm allergy. a secreted recombinant form of pro-der p 1 expressed by saccharamyces cerevisiae was obtained by fusion of the pro-enzyme to a fungal signal peptide. the n-glycosylation site of der p1 was mutated resulting in a deglycosylated pro-enzyme with a molecular mass of 35 kda. protein purification procedure was developed to obtain nearly pure der p1 protein followed by determination of concentration by active-site-titration with the cysteine protease inhibitor e64. the deglycosylated recombinant pro-der p 1 revealed immunologic similarity to the native der p 1 molecule when compared in basophile histamine release, ige-binding assays and t-cell proliferation assays. by in silico epitope mapping of a modelled 3-dimensional structure of der p1, five putative igg and ige epitopes were predicted. by protein engineering, the predicted epitopes were removed one by one in der p1 and screening for hypoallergenic variants was performed. combining inhaled long-acting b-2 agonist (laba) and inhaled corticosteroid (ics) seems to offer asthma control at a lower dose of ics than achieved by ics alone. fine mapping of t-cell surface markers by flow cytometry offers a detailed status of the individual's inflammatory response. the frequency of mt2 (cd4 þ cd45ra -cd62l þ cd11adim) and mt1 (cd4 þ cd45ra -cd62l -cd11abright) cells in peripheral blood, and their ratio, has been shown to differ predictably in atopics and patients with leprosy, where mt2 correlates with a th2 phenotype and mt1 with a th1 phenotype. stable asthmatics, requiring fluticasone propionate (fp) 750-1000 mg daily or equivalent, were randomized to receive, double-blinded, either seretide 1 [salmeterol and fluticasone propionate (sfc, n ¼ 16)] 50 mg/500 mg bd or fp 500 mg bd (n ¼ 17). if asthma was controlled based on lung function and symptoms at clinic visits every 6 weeks, ics dose was tapered until asthma exacerbated or 0 mg was reached. the frequency and ratio of mt2 and mt1 t cells of the patients was monitored at 6 week intervals. as treatment tapered, the frequency of mt2 cells decreased (p ¼ 0038 from first to final visit), whereas that of mt1 cells increased. the ratio of mt2/mt1 decreased (p ¼ 0049 from first to final visit). in patients receiving laba þ ics, the fall in mt2/mt1 ratio appeared to be more pronounced than in patients receiving ics alone. thus, the mt2 phenotype may be associated with stable asthma, whereas an imminent exacerbation may associate with an increase in the mt1 phenotype. laba may allow for a greater effect of fp on the mt ratio. activation of complement pathways, leading to production of c3a and c5a anaphylatoxins, has been postulated in the pathogenesis of asthma and allergic airway inflammation. the present study was undertaken to investigate the role of mannan-binding lectin (mbl), an initiator of the lectin pathway of complement, in asthma and allergic rhinitis. mbl levels and mbl-induced complement activity were determined in 19 patients of bronchial asthma with allergic rhinitis and 20 unrelated, age-matched controls of indian origin. mbl levels and activity were correlated with percent eosinophilia and percent predicted fev1 values of the patients. association of single nucleotide polymorphisms (snps) in exon 1 and intron 1 of the mbl with the disease, clinical markers, mbl levels and mbl-induced complement activity was analysed using standard statistical tools. significantly higher mbl levels and activity were observed in patients of bronchial asthma with allergic rhinitis as compared to the controls. we identified five snps, of which two, a816g in exon 1 and g1011a in intron 1 of the mbl, were novel. snp g1011a was significantly associated with the disease (p ¼ 0.0024, or ¼ 5.8696, 95% ci: 1.7316 < or < 19.8963). individuals with 'a' allele at position 1011 showed increased mbl levels, activity and disease severity. our results suggest that 'a' allele at position 1011 leading to high mbl levels and complement activity may be contributing to the severity of bronchial asthma and allergic airway inflammation. serum resistance of borrelia burgdorferi strains belonging to the b. afzelii and b. burgdorferi sensu stricto genospecies is dependent on binding of complement inhibitor factor h. we recently reported that factor h binding by b. burgdorferi is due to inducible expression of several approximately 20 kda plasmid-encoded, surface-exposed lipoproteins related to ospe (e.g. erpa, erpp and p21). in addition, a second class of factor h-binding proteins of approximately 27-35 kda has been described. the ospe-related lipoproteins are dramatically induced by b. burgdorferi during transmission from its tick vector into the mammalian host. the induction of ospe-related lipoproteins during mammalian infection may play a key a role in the borrelial evasion of the host's immune system. the goal of the present study was to define the factor h-binding regions of ospe-related proteins using mutagenesis, peptide mapping and surface plasmon resonance analysis (biacore). the combined studies revealed that the c-terminal regions of both human and mouse factor h (scrs 18-20) specifically bind to ospe-related lipoproteins. we also found fhr-1, whose c-terminal scrs 3-5 are homologous to scrs 18-20 of factor h, to bind to ospe. peptide mapping revealed five putative regions (designated i-v) in ospe that could directly interact with factor h. deleting the c-terminal 15 amino acid residues from region v of p21 abolished its ability to bind factor h. at the same time, however, synthetic peptides corresponding to the c-termini of ospe, p21 and erpp did not inhibit factor h binding to ospe. thus, the c-terminal-binding region v appears to be necessary but not sufficient for factor h binding. when a more specific mutation strategy was employed, where single amino acid residues in peptides spanning over the factor h-binding regions were mutated to alanines, we observed that lysines in the factor h-binding regions of ospe were required for factor h binding. the combined data have revealed that key lysine residues in ospe-related lipoproteins and ionic interactions are crucial for factor h interactions. furthermore, binding of ospe to the c-termini of both mouse and human factor h suggests that borrelia spirochetes utilize analogous complement resistance mechanisms in both rodents and man. in borrelia garinii strains, which in in vitro analyses have been found to be sensitive to complement killing, differences in the ospe sequences as well as in the expression of factor h-binding proteins may account for their susceptibility to serum lysis. role of yada, ail and lipopolysaccharide in serum resistance of yersinia enterocolitica serotype o:3 mannan-binding lectin (mbl), l-ficolin and h-ficolin are pattern recognition molecules of the innate immune system. we investigated the ability of these molecules to bind to different serotypes and noncapsulated variants of streptococcus pneumonia and staphylococcus aureus. we found that mbl binds to noncapsulated s. aureus strain (wood) but not any of the examined s. pneumoniae serotypes. l-ficolin binds to some capsulated s. pneumoniae serotypes (11a, 11d and 11f) as well as some capsulated s. aureus serotypes (type-1, -8, -9, -11 and -12). h-ficolin does not bind to any of the examined s. pneumoniae and s. aureus serotypes included in this study but did bind to a strain of aerococcus viridans. when bound to bacteria, mbl and h-ficolin initiated activation of complement factor c4, whereas l-ficolin did not. during this study, quantitative assays for the three proteins were developed and the concentration in 97 plasma samples were determined and the median values were estimated at 0.8 mg of mbl/ml, 3.3 mg of l-ficolin/ml and 18.4 mg of h-ficolin/ ml, respectively. the absence of early complement components (c1, c4 and c2 but not c3) is a predisposing factor for systemic lupus erythematosus (sle). recently, we demonstrated that, in c4-deficient (c4 def.) mice, igm-containing immune complexes (igm-ic) are filtered by the splenic barrier of marginal zone macrophages (mzm), resulting in an increased immune response against antigens within these igm-ic, but this could not be observed in wildtype or c3 def. mice. we hypothesized that splenic cd11b þ mzm play an important role in the induction of autoimmunity, and we therefore analysed their cytokine profile after isolation with the help of magnetic antibody cell sorting. mrna was isolated, and real-time pcr was performed with specific primers for murine ifn-g (ifn-g), interleukin-12 (il-12) and ifn-a (ifn-a). we observe a moderate increase of il-12 and ifn-g mrna in cd11b þ cells of c4 def. mice compared to wildtype cells. surprisingly, the concentration of ifn-a mrna is six times higher in c4 def. mice. preliminary results suggest that mrna in cd11b þ cells of c3 def. mice is even lower than that in wt. six hours following i.v. application of 20 mg of a abstracts 625 .................................................................................................................................................................................................. murine monoclonal igm anti-dsdna antibody, production of il-12, ifn-g and ifn-a mrna is increased in cd11b þ cells of both c4 def. and wt mice. several references described increased levels of inf-a in patients with sle. dendritic cells are discussed as a major source of ifn-a. our observation that c4-deficient, sle-susceptible mice demonstrate an increased spontaneous ifn-a production by splenic cd11b þ marginal zone macrophages could be an early sign and a trigger for the development of sle. this is supported by the fact that the absence of c3 is not a predisposing factor for sle and our observation that c3 def. animals display low levels of ifn-a mrna. 200-400 million people worldwide and represents one of the leading causes for liver cirrhosis and hepatocellular carcinoma. control over the hbv infection is achieved mainly by vaccination with hepatitis b surface antigen (hbsag). hbsag contains n-linked glycosylation side and is recognized by both mbl-a and mbl-c in a cadependent manner. hbsag-mbl complexes activate complement and may thus affect humoural immunity. to investigate the role of mbl in humoural responses to hbsag, we immununized mice that lack both mbl-a and mbl-c proteins with soluble hbsag. it has been shown that deficiencies in other complement components like c1q, c4 and c3 result in decreased antibody responses. however, mbl double ko animals mounted dramatically increased humoural responses. after priming, mbl double kos mounted hbsag-specific igm responses, which were threefold higher than wt controls. after boosting the hbsag, total igg was 10-fold higher in mbl ko than in wt control animals. similar to the response to hbsag, other glycosylated soluble antigens (e.g. invertase) induced better humoural responses in mbl double ko animals, suggesting that mbl plays an important role in a negative feedback regulation of adaptive immunity. reconstitution experiments with rmbl partially rescued the ko phenotype. we propose that the clearance of glycoprotein antigens in mbl ko is handled differently from the wt, resulting in better stimulation of humoural responses. alternatively, glycoprotein-ag-mbl-rich complexes inhibit b-cell responsiveness via putative mbl receptors. the complement system is an important part of the innate immune system. the activation of complement proceeds through three different pathways that converge in the generation of c3-activating enzyme complexes. complement activation via the lectin pathway is initiated when recognition molecules, mannan-binding lectin (mbl) or ficolin, bind to carbohydrate structures characteristic for microbial surfaces. in the circulation, mbl and ficolins are found in association with three structurally related mblassociated serine proteases (masp)-1, -2 and -3 and a small, nonenzymatic component, map19. masp-2 has been shown to elicit complement activation through the sequential proteolytic cleavage of c4 and c2 upon binding of mbl/masp-2 complexes to microbial surfaces. we have recently uncovered a polymorphism in the masp-2/map19 gene in a patient shown to be deficient in the lectin pathway of complement activation. the polymorphism results in a single amino acid substitution in the n-terminal part of the masp-2 protein. recombinant wildtype masp-2 and masp-2 containing the amino acid substitution in question was produced, and the ability to activate complement was studied. the mutation had a profound impact on masp-2 function, resulting in the lack of complement activation through the lectin pathway. elisa-based experiments showed that the mutation leads to the impairment of complement activation through influencing the binding of masp-2 to mbl or ficolins. deficiencies in the lectin pathway of complement activation have so far been accounted for only by lack of functional mbl. the mutation described above is the first defect described affecting both activation through mbl and the ficolins. .................................................................................................................................................................................................. th1, th2 and treg cell balance. dcs are present in the gut mucosa and may thus be target for modulation by gut microbes, including ingested probiotics. here, we tested the hypothesis that species of lactic acid bacteria, important members of the gut flora, differentially activate dc. a large panel of human gut-derived lactobacillus and bifidobacterium spp. was screened for dc-polarizing capacity by exposing bone marrow-derived murine dc to lethally irradiated bacteria. cytokines in culture supernatants and dc-surface maturation markers were analysed. substantial differences were found among strains in the capacity to induce interleukin-12 (il-12) and tumour necrosis factor (tnf)-a, while the differences for il-10 and il-6 were less pronounced. bifidobacteria tended to be weak il-12 and tnf-a inducers, while both strong and weak il-12 inducers were found among the strains of lactobacillus. remarkably, strains weak in il-12 induction inhibited il-12 and tnf-a production induced by an otherwise strong cytokine-inducing strain of lactobacillus casei, while il-10 production remained unaltered. selected strains were tested for induction of dc maturation markers. those lactobacilli with greatest capacity to induce il-12 were most effective in upregulating surface mhc class ii and cd86. moreover, l. casei-induced upregulation of cd86 was reduced in the presence of a weak il-12inducing l. reuteri. in conclusion, human lactobacillus and bifidobacterium spp. polarize differentially dc maturation. thus, the potential exists for th1/th2/treg-driving capacities of the gut dc to be modulated according to composition of gut flora including ingested probiotics. the intestinal micro flora is indispensable in developing and maintaining homeostasis of the gut-associated immune system. evidence indicates that lactic acid bacteria (lab), e.g. lactobacilli and bifidobacteria, have beneficial effects on the host. established health effects include increased gut maturation, antagonisms towards pathogens and immune modulation. the objective of this study is to evaluate the immunomodulating properties of a range of lab of human origin. as dendritic cells (dcs) play a pivotal role in the balance between tolerance and immunity to commensal microorganisms, in vitro-generated immature dcs serve as a suitable model for studying the immunomodulating effects of lab. human immature dcs were generated in vitro from monocytes and exposed to lethally uv-irradiated lab. the effect of various species of lab on dcs in direct contact was evaluated. furthermore, the maturation pattern of dcs separated from the bacteria by an epithelial cell layer (caco-2 cells), which should mimic the intestinal environment, was studied. cytokine secretion (il-12, il-10 and tnf-a) and upregulation of maturation surface markers on dcs (cd83 and cd86) was measured. different lab induced diverse cytokine responses. some strains were strong il-12 and tnf-a inducers and others weak. all strains induced il-10. different lab also differentially modulated expression of cd83 and cd86 on dcs. although some variation in the response to lab of dcs generated from different blood donors was observed, general differences in the effect of the various lab was revealed. experiments with the dc caco-2 coculture system are ongoing. different species of lab differentially affect dc maturation; this suggets that the gut flora plays a pivotal role in polarization of the immune response. natural killer (nk) cells are cells of the nonspecific immune system lysing altered self-cells. a noncytolytic subset of nk cells may serve a regulatory role by secreting cytokines. bacteria translocating across the gastrointestinal mucosa are presumed to gain access to nk cells, as consumption of certain lactic acid bacteria has been shown to increase in vivo nk cytotoxicity. here, we investigated how human gut flora-derived lactobacilli affect nk cells in vitro, by measuring proliferation and ifn-g production of human nk cells upon bacterial stimulation. cd3 -cd56 þ nk cells were isolated from buffy coats by negative isolation using non-nk lineage-specific antibodies and magnetic beads. nk cells were incubated with 10mg/ml uv-inactivated bacteria or 10mg/ml phytohemagglutinin (pha) for 4 days. proliferation was assessed by incorporation of radioactive thymidine into nk-cell dna. the ifn-g concentration was measured by elisa. incubation of nk cells with a lactobacillus acidophilus strain increased the proliferation of the nk cells and induced ifn-g production, both to levels comparable to pha stimulation. the proliferative response was further enhanced with autologous monocytes present, probably because cytokines, secreted by monocytes having engulfed bacteria, stimulated the nk cells. in contrast, a lactobacillus paracasei strain caused the nk cells to proliferate only in the presence of monocytes. these results demonstrate that various strains of lactobacilli have the capacity to activate nk cells in vitro, in a monocyte-dependent or -independent way. hence, the encounter of nk cells with lactic acid bacteria will affect nk-cell activation. such activation of nk cells may potentially skew an on-going or subsequent immune response towards a th1 response. lactobacilli are nonpathogenic gram-positive inhabitants of the normal human intestine known for their healthpromoting effects. in our earlier work, it is shown that human monoclonal antibody isolated from sera of a patient with waldenstrom macroglobulinaemia possess innate antibody characteristics and binds to lactic acid bacteria. according to the immune network model, immunization with this bacteria could induce the perturbations in immune system that might result in production of anti-lactobacillus antibodies, human monoclonal antibody like (ab1) and anti-idiotypic antibody (ab2). in this study, balb/c mice were immunized with two doses of bacteria lactobacillus acidophilus in complete and incomplete freund's adjuvant and phosphate-buffered saline (pbs), respectively. seven days after the last immunization, sera from immunized mice were collected and the presence of lactobacillus-specific ab1 and ab2 were determined by elisas. in the sera of immunized mice, antibodies specific to bacteria lactobacillus acidophilus were shown. the concentration of lactobacillus-specific antibodies was higher in the sera of hyperimmunized mice (mice immunized with 1 mg of igm dj) than in sera of mice immunized with 100 times lower doses of immunogen (0.01 mg per doses). moreover, ab1 and ab2 antibodies were detected in the sera of lactobacillus-hyperimmunized mice. in this study, we have shown the idiotypic network interactions in mice immunized with bacteria lactobacillus acidophilus. the normal gastrointestinal flora is crucial for the maturation of the acquired immunity via effects on antigenpresenting cells (apcs). here, we have investigated how two types of apcs, monocytes and dendritic cells (dcs), react to different bacterial strains typical of the commensal intestinal flora. purified monocytes and monocyte-derived dcs were stimulated with uv-inactivated gram-positive (lactobacillus plantarum and bifidobacterium adolescentis) and gram-negative (escherichia coli and veillonella parvula) bacterial strains. monocytes produced higher levels of il-12p70 and tnf, as detected by elisa, in response to l. plantarum than to e. coli and v. parvula. in contrast, dcs secreted high amounts of il-12p70, tnf, il-6 and il-10 in response to e. coli and v. parvula but were practically unresponsive to l. plantarum and b. adolescentis. the lack of response to the gram-positive strains correlated with a lower surface expression of toll-like reseptor 2 (tlr2) on dcs compared to monocytes. the surface expression of tlr4 on dcs was undetectable when analysed by flow cytometry, but blocking this receptor decreased the tnf production in response to v. parvula, indicating that low tlr4 expression on dcs is sufficient to mount an inflammatory response to gram-negative bacteria. ifn-g increased the expression of tlr4 on dcs and also potentiated the cytokine response to gram-negative bacteria. our results indicate that, when monocytes differentiate into dcs, their ability to respond to different commensal bacteria dramatically changes, thereby becoming unresponsive to probiotic gram-positive bacteria. these results may have important implications for the capacity of different groups of commensal bacteria to regulate mucosal and systemic immunity. probiotic bacteria, e.g. lactobacillus spp., may improve diseases such as chronic inflammatory bowel disease. we examined cytokine production and phenotypic change after in vitro stimulation of t cells from healthy volunteers using different probiotic strains. methods: t cells were cultured from colonic biopsies from eight healthy volunteers (agnholt and kaltoft, exp clin immunogenet 2001; 18:213-25) , and dendritic cells were matured from their peripheral blood mononuclear cells. t-cell cultures were stimulated with autologous bacterial sonicate or strains of lactobacillus spp., with and without the addition of dendritic cells. cytokine levels (tnf-a, ifn-g, il-10 and gm-csf) and phenotype (cd3, cd4, cd25 and cd69) were measured on day 4. results: lactobacillus spp. induced higher productions of tnf-a and il-10 than did autologous bacteria. in presence of dendritic cells, the production of all cytokines increased. however, the increases of ifn-g and tnf-a were more pronounced in wells with autologous bacteria than in wells with lactobacillus spp. the addition of dendritic cells upregulated cd25 expression without simultaneous upregulation of cd69. the upregulation was pronounced after stimulation with lactobacillus rhamnosus gg compared with autologous bacteria and other lactobacilli. discussion: in presence of dendritic cells, autologous bacteria induced inflammatory cytokines, while probiotics mainly induced regulatory cytokines. lactobacillus rhamnosus gg induced a regulatory phenotype (cd25 þ ), in part mediated by dendritic cells. future studies will address whether this shift to a cd25 þ phenotype represents a differentiation into competent regulatory t cells. in a clinical context, such cells might be used for treatment of inflammatory diseases. protein microarrays will play a key role in the postgenomic era and offer a unique possibility to perform highthroughput global proteome analysis. a chip can be printed with thousands of protein probes (e.g. antibodies), the biological sample added (e.g. a proteome) and any binding detected. we aim to develop protein microarrays based on human recombinant scfv antibody fragments for global proteome analysis. the concept of comparing proteomic maps of healthy versus diseased samples will allow diseasespecific proteins to be detected. in fact, antibody microarrays will allow us to perform comparative proteome analysis on any sample format in a species-independent manner, as long as a proteome can be isolated. however, the complexity of proteomes, containing several thousands of different proteins, is a problem. here, we have designed antibody microarrays targeting the water-soluble fraction of a proteome. to this end, an anticytokine antibody array was developed and human dendritic cells (aeactivation) was used as model system. the results showed that our antibody microarrays could be used to examine the cytokine profile in complex samples. furthermore, we have taken the first steps towards comparing our results with those of other technologies on both the protein and gene level. due to the complexity of the model proteome, we also examined the possibility to prefractionate the proteome in a simple one-step procedure (based on size) prior to the labelling step. in more detail, the sample proteome was fractionated into two fractions using membrane devices with different molecular weight cut-offs. the results showed that the fractionation considerably enhanced the assay sensitivity allowing cytokines in the pg/ml range to be readily detectable. the immunomodulatory effect of heat shock protein 70: immunization with a dna construct based on the malarial antigen fused with a fragment of hsp 70 primes for a th-1 type of response finding an appropriate adjuvant for human vaccination is crucial. heat shock proteins (hsps) act as adjuvants when coadministered with peptide antigens or given as fusion proteins. however, there is a potential risk of autoimmunity when using the complete molecules, because hsps are evolutionary conserved. to overcome this, we first evaluated the adjuvant effect against two different antigens of a less-conserved fraction of plasmodium falciparum hsp70 (pf70c) and compared it to the whole hsp70 molecule from trypanosoma cruzi (tchsp70). we found that pf70c exhibited similar adjuvant properties as the whole molecule. we later evaluated the adjuvant potential of pf70c against the malarial antigen eb200 in a chimeric dna construct. no appreciable levels of eb200-specific abstracts 629 .................................................................................................................................................................................................. antibodies were detected in mice immunized only with the dna constructs. however, dna primed the immune system, because subsequent challenge with the corresponding recombinant fusion proteins elicited a strong th-1 antibody response. in contrast, no priming effect was observed for ex vivo ifn-g production but stimulation with the hsp-chimeric fusion protein induced a stronger secretion of ifn-g in vitro than other proteins used. these results indicate that the use of hsps is promising in the design of new vaccines. high-throughput proteomics on antibody-based microarrays: the importance of probe and surface design in analogy to dna microarrays, protein microarrays offer a new distinct possibility to perform sensitive highthroughput global proteome analysis. however, the development of the protein microarray technology will place high demands upon the design of both probes and solid supports. the analysis of thousands of heterogeneous proteins on a single microarray requires the use of uniform probes, such as antibodies, directly designed for protein microarray applications. we have recently generated a human recombinant single-chain fv antibody library, genetically constructed around one framework, the ncoder-library, containing 2 â 1010 clones. single framework antibody fragments (sinfabs) selected from this library were successfully applied as probes for microarrays providing sensitive detection in the 600 attomol (mass spectrometry) and the 300 zeptomole range (fluorescence). however, the choice of framework is critical. we have shown that the selected ncoder framework displayed excellent functional on-chip stability and arrayed dehydrated probes retained their activity for several months. furthermore, we have addressed the issues of biocompatibility of the solid support and immobilization strategies for our microarray setup. an in-house-designed substrate, macroporous silicon coated with nitrocellulose (map3-nc7), displayed properties equal to, or better than, those of five commercially available supports used as reference surfaces. we have also evaluated different coupling strategies, such as adsorption, covalent coupling, diffusion and affinity coupling. using a novel affinity tag, the double-(his)6-tag, we increased the binding efficiency of sinfab-molecules to ni2 þ -coated solid supports, thereby allowing nonpurified probes to be directly applied. the mannan-binding lectin (mbl) pathway is part of the innate immune system providing a first line of defence against infections. mbl and ficolins circulate in complexes with mbl-associated serine proteases (masp-1, -2 and -3). after recognition of a microorganism by mbl, activation of the complement system occurs. masp-1 and masp-3 share five domains (making up the so-called a-chain), whereas they have unique protease domains (b-chains). before the identification of masp-3, an assay for masp was presented, based on antibodies against the a-chain of masp-1. with the new knowledge of the three masps, and the sharing of domains by masp-1 and masp-3, assays specific for the protease domains have to be constructed, if one wishes to measure the proteins individually. we present an assay for quantifying total masp-3 in plasma and serum samples. the assay is a sandwich-type assay using as catching antibody a monoclonal antibody against the common a-chain of masp-1/3 and a developing secondary antibody against the c-terminal part of the protease domain of masp-3. we have used this assay for estimating the normal concentration of the protein as well as the concentration in patients and also for characterizing by gel permeation chromatography the masp-3 protein in serum. inducible costimulator ligand (icosl) is a costimulatory molecule related to b7.1 (cd80) and b7.2 (cd86). b cells, monocytes, dendritic cells and endothelial cells express icosl. inducible costimulator (icos) interacts with icosl, and this interaction leads to signals involved in isotype switching and the development of immunological memory. hitherto, no polymorphisms of this gene have been described. the aim of this study was to reveal variation of the icosl gene in normal individuals. all eight exons, except exon 1, were sequenced with flanking introns in 10 healthy blood donors. eight single nucleotide polymorphisms (snps) and two length polymorphisms were found. one of the snps was found in the coding regions of the gene. the base involved was located in exon 3 and caused a conservative amino acid change from valine (gtt) to isoleucine (att). three individuals were heterozygous g/a for the exon polymorphism, while the remaining seven individuals were homozygous for the wildtype g/g. exon 3 encodes the immunoglobulin variable (igv)-like domain of the molecule which is situated outside the cell. this means that the amino acid could be critical for the stability of the molecule or could constitute part of the binding site for icos. the results form the basis for further experiments to find possible associations of the alleles to diseases caused by immune dysregulation. especially, the exon 3 variant is interesting and could play a role for the development of immunological diseases. besides, it would be interesting to see whether both exon 3 alleles are expressed or only the wildtype allele is functional. interferon-a/b (ifn-a/b) is increasingly used as antiviral and immunomodulatory therapies. unfortunately, bioavailability varies with ifn species and mode of administration, and all ifn species are potentially immunogenic. assays for antiviral activity (ifn) and antiviral neutralization (antibodies, nab) have been used for some time to monitor patients on ifn biologicals. these assays require laborious titrations making them unsuitable for large-scale clinical use. myxovirus a (mxa) is a resistance gtpbinding protein that is specifically induced by treatment with type 1 ifns. for example, ifn-b-induced mxa in blood leucocytes has been used as a biomarker in ifn-btreated patients with multiple sclerosis. however, the degree of specificity of mxa in this regard is unclear, and measurements of mxa protein and/or mrna are not yet suitable for routine clinical use. in an attempt to find new and better reporter genes (and, hopefully, genes and gene products with proven specificity for ifn-a and -b), microarray screenings with u133a genechips (affymetrix) were carried using human blood leucocytes and the human lung carcinoma cell line a549. we studied the simultaneous expression of 22,000 transcripts before and after exposure to human recombinant ifn-a and ifn-b and other antiviral and immunomodulatory cytokines. the results will be presented at the conference. interferon-a/b (ifn-a/b) is increasingly used as antiviral and immunomodulatory therapies. unfortunately, bioavailability varies with ifn species and mode of administration, and all ifn species are potentially immunogenic. assays for antiviral activity (ifn) and antiviral neutralization (antibodies, nab) have been used for some time to monitor patients on ifn biologicals. these assays require laborious titrations making them unsuitable for large-scale clinical use. our laboratories have therefore modified the antiviral assays for ifn bioactivity and nab, so that they are suitable for large-scale screening in specialized laboratories. the read-out is survival of a subcloned a549 cell line in the presence of an otherwise lethal amount of virus. thus, survival increases in the presence of type 1 ifn and decreases in the presence of nab against the ifn added to the cells. mxa is induced by type 1 ifn and can be used for measuring the nab activity. in another assay, the mxa level in the a549 cell line is measured. in an attempt to find a new and better reporter gene for type 1 ifn than mxa and genes specific for either ifn-a or -b, a micro array screen was carried using the u133a chip from affymetrix. the expression of 22,000 genes can be studied simultaneous with this technology. the results will be presented at the conference. in our laboratory, we have developed a database system, which we believe is of immediate interest to the general scientific community. the database represents a computerbased replacement for the laboratory notebooks used in the majority of research laboratories worldwide. in addition, the database provides an effective tool for organizing and managing laboratory information at all levels, spanning from managing and revising standard operating procedures and producing documentation of research activities to keeping track of data and conclusions. using the commercially available database toolkit software filemaker pro, we have developed a relational database solution for management of laboratory information. the system consists of a hierarchy of five interrelated databases, each pertaining to a separate type of information, namely, overall project information, information relating to individual experiment setups, documentation of daily research activity, generated data and descriptions of standard operating procedures. like other databases, each individual database consists of a number of records, each comprised of a set of fields in which information is entered. in each record, a certain field is reserved to specify the relation of the record to a record in another database at a higher level. thus, the database is essentially five databases linked by a hierarchy of one-to-many relations, organizing information in a folder-like structure. importantly, the database system allows multiple users to access and edit records simultaneously, and the data entered in one database immediately becomes accessible through the other databases. the limitations of laboratory notebooks are apparent when looking for information, which is dispersed throughout one or more notebooks, or possibly on loose sheets of paper or printouts 'somewhere'. the often complicated process of gathering laboratory data or results when writing grant applications or research papers is made considerably easier with the database system. thus, the database solution presented should be broadly attractive to researchers, irrespective of their scientific discipline. an effective sars vaccine is likely to include components that can induce specific cytotoxic t-cell (ctl) responses. the specificities of such responses are governed by hlarestricted presentation of sars-derived peptide epitopes. exact knowledge of how the immune system handles protein antigens would allow for the identification of such linear sequences directly from genomic/proteomic sequence information. the latter was recently established when a causative coronavirus (sars cov) was isolated and full-length sequenced. here, we have combined advanced bioinformatics and high-throughput immunology to perform an hla supertype, genome-wide scan for sars-specific cytotoxic t cell epitopes. the scan includes all nine human hla supertypes in total covering >99% of all major human populations. for each hla supertype, we have selected the 15 top candidates for test in biochemical-binding assays. at this time (approximately 6 months after the genome was established), we have tested the majority of the hla supertypes and identified almost 100 potential vaccine candidates. these should be further validated in sars survivors and used for vaccine formulation. we suggest that immunobioinformatics may become a fast and valuable tool in rational vaccine design. rationale: major histocompatibility complex class i (mhc i) molecules monitor the protein content of the cell by binding small derived peptides and presenting them to cytotoxic cd8 þ t cells. the goal of the human mhc project is to predict the binding strength of any given peptide/mhc complex. this prediction allows the design of peptide-based vaccines. the prediction requires representative binding data from mhc alleles from all the nine hla supertypes. here, we describe the genetic construction, protein production and purification as well as the establishment-binding assays for two recombinant mhc supertype alleles, hla-b*1501 and hla-b*5801. methods: using the quikchange multisite directed mutagenesis kit (stratagene), codon-optimized genes encoding hla-b*1501 and hla-b*5801 are created. the two mhc i molecules are fermented and purified by ion exchange chromatography, hydrophobic interaction chromatography and size exclusion chromatography. the binding (kd) of natural t-cell epitopes, as well as predicted peptide ligands, is described by radioactive immunoassays (rias) and enzyme-linked immunosorbent assays (elisas). the mhc molecules are biotinylated during expression. results: the expression of mhc i resulted in multiple disulfide bond isomers, which are separated by hydrophobic interaction chromatography and used in subsequent binding studies resulting in the determination of kd for various peptide ligands ranging from strong binders we have previously demonstrated that bioinformatics tools such as artificial neural networks (anns) are capable of performing pathogen-, genome-and hlawide predictions of peptide-hla interactions. these tools may therefore enable a fast and rational approach to epitope identification and thereby assist in the development of vaccines and immunotherapy. a crucial step in the generation of such bioinformatics tools is the selection of data representing the event in question (in casu peptide-hla interaction). this is particularly important when it is difficult and expensive to obtain data. herein, we demonstrate the importance in selecting information-rich data and we develop a computational method, query-bycommittee, which can perform a global identification of such information-rich data in an unbiased and automated manner. furthermore, we demonstrate how this method can be applied to an efficient iterative development strategy for these bioinformatics tools. methods: a large panel of binding affinities of peptides binding to hla a*0204 was measured by a radioimmunoassay (ria). this data was used to develop multiple first generation anns, which formed a virtual committee. this committee was used to screen (or 'queried') for peptides, where the anns agreed ('low-qbc'), or disagreed ('high-qbc'), on their hla-binding potential. seventeen low-qbc peptides and 17 high-qbc peptides were synthesized and tested. the high-or low-qbc data were added to the original data, and new high-or low-qbc second generation anns were developed, respectively. this procedure was repeated 40 times. the high-qbc-enriched ann performed significantly better than the low-qbc-enriched ann in 37 of the 40 tests. conclusion: these results demonstrate that high-qbcenriched networks perform better than low-qbc-enriched networks in selecting informative data for developing peptide-mhc-binding predictors. this improvement in selecting data is not due to differences in network training performance but due to the difference in information content in the high-qbc experiment and in the low-qbc experiment. finally, it should be noted that this strategy could be used in many contexts where generation of data is difficult and costly. interleukin-18 (il-18), a pro-inflammatory cytokine that is produced by both lymphoid and nonlymphoid cells, has a critical role in modulation of innate and adaptive immunity. its primary function in stimulation of ifn-g production and stimulation of nk-cell-cytotoxic activities makes this cytokine a candidate for cancer immunotherapy. in oral cavity, this cytokine is produced by oral epithelia and carcinoma cells and is related to tumour regression in nude mice bearing salivary adenocarcinoma. however, direct effects of this cytokine on oral cancer cells have not been elucidated. in this project, we investigated il-18 effect on an oral carcinoma (kb) cell line. with rt-pcr technique, kb-cell line was found to express il-18 receptors (il-18ra and il-18rb), indicating that this oral carcinoma line is a target for il-18 study. we showed that recombinant human il-18 inhibited kb-cell proliferation by 17% at concentration of 100 ng/ml (p < 0.05), whereas ldh release by these cells in treatment group and control groups was comparable, indicating that il-18 suppression of cell proliferation was not mediated by the induction of cell death. to further address this hypothesis, we found that il-18 treatment did not induce apoptotic cell death, as studied by dna laddering and tunel assays. in addition, expression pattern of cell death-controlling genes (bcl-2 and bax) was not altered by this cytokine. findings in these studies indicated that suppression of kb-cell proliferation may be attributed to control of cell cycle, growth arrest or induction of cell differentiation. the data presented in this project could provide an insight of how cancer cell directly responds to il-18, as this cytokine is an important regulator of anticancer mechanisms. aloe emodin (ae) is a naturally occurring compound with wide spectrum of biological properties, including antimicrobial, vasorelaxant, immunosuppressive and anticancer actions. this anthraquinone induces apoptosis in several tumour cell lines with special affinity to tumours of neuroectodermal origin. high amounts of nitric oxide (no) released by activated macrophages induce tumour cell death. therefore, we explored the capacity of ae to modulate no-mediated antitumour response in vitro. interestingly, while ae markedly suppressed no release from macrophages alone, it significantly potentiated no production in cocultures of macrophages and c6 cells, after 48 h of cultivation. accordingly, the viability of c6 cells cocultivated with macrophages was reduced in the presence of ae. moreover, the observed ae-imposed potentiation of no production in macrophages was closely related to macrophage culture cell density. according to these data, we proposed that no modulator capacity of ae strongly depended on intercellular contact, indicating that macrophage antitumour response was not compromised but even potentiated by ae. immunotherapy represents an attractive fourth-modality therapeutic approach, especially in the light of the shortcomings of conventional surgery, radiation and chemotherapies in the management of metastatic cancer. to this end, a large number of peptide antigens derived from taa have been applied in immunotherapeutic trials for the treatment of various malignancies, e.g. cancers of the breast, prostate and kidney, in addition to haematological cancers. in some cases the response rates have been impressive and no adverse autoimmunity have been observed. a major strategic difficulty associated with these trials relates to the choice of best-suited peptide antigens. the vast majority of the antigens described thus far is not vital for survival and growth of the tumour cells, and immunoselection of antigen-loss variants may therefore prove to be an additional obstacle for the clinical applicability of most of the known peptide epitopes. in this respect, the development of acquired antigen loss during immunotherapy has been demonstrated in several cases. obviously, the development of loss-variant tumour cells implies that these cells acquire a pronounced growth advantage and are left unaffected by further treatment. ideally, target antigens should be derived from proteins required for survival and growth of tumour cells, as antigens with these characteristics would not be inflicted by the development of loss-variant tumour cells. in this respect, several inhibitors of apoptosis proteins (iaps) are universally expressed among tumours and play an important role in tumour cell escape from apoptosis. we have characterized spontaneous t-cell reactivity against iapderived peptides in cancer patients. from the iap survivin, we have characterized peptides restricted to the class i molecules hla-a1, a2, a3, a11, b7 and b35. furthermore, we have demonstrated that survivin-specific t cells infiltrate metastatic lesions and that isolated survivinspecific ctls are capable of killing hla-matched tumour cells. survivin-derived peptides are now in clinical trial, and continued work in our lab has demonstrated that other iaps are targets for spontaneous t-cell reactivity in cancer patients. we previously reported that in mice with large progressing t-cell lymphoma tumours, dysfunctions in the antitumour ctl activity occur, associated with an accumulation of splenic arginase-producing myeloid suppressor cells (mscs). in this study, we first demonstrate that both the presence and the activation state of these msc depends on tumour evolution. while in tumour regressors hardly any arginase-producing msc can be found, both the amount and the arginase activity of this population expands from early over late progressors. this gradual induction of mscs is paralleled by an increasing suppression of ctl activity and th1, but not th2, cytokine production. upon analysing the molecular repertoire of msc in vitro, we found, besides arginase1, a well-established marker for alternatively activated myeloid cells or m2, a strong upregulation of fizz1 and ym, two additional recently identified markers for m2. further evaluation of molecular markers by microarray analysis in msc yielded genes involved in wound healing (e.g. coagulation factor xiiia), anti-inflammation (e.g. selenoprotein p), immunomodulation (e.g. pd-l2) and fat and sugar metabolism (e.g. leptin receptor). of note, many of these genes are regulated by type 2 cytokines (il-4, il-13 and il-10) and are therefore rather m2 associated. overall, our data provide new markers for msc in cancer and further establish their m2 activation state. study. only sp-a showed a significant expression in normal mucosa which was downregulated in crc. as the absolute signal level was below the noise threshold, these results have to be interpreted with caution and require confirmation by direct measurenment of the proteins. our results suggest that there is no major role for the human collectins in colorectal cancer. tetramerization is visualized by sds-page. conclusion: an effective method for the production of highly pure mhc i molecules has been applied to hla-b*1501 and hla-b*5801, and ria and elisa binding assays for those alleles have been established background: proliferation, differentiation and apoptosis are essential processes in the normal functions of the mammary epithelium. the hypothesis examined in this study is that the transcription factor bcl-6 is critically important not only for regulating b-cell growth and development but also for mammary epithelial apoptosis. methodology: twenty breast cancer cases and 31 healthy controls were used to investigate whether bcl-6 protein in involved in breast cancer (grade iii). full length bcl-6 cdna was retrovirally transduced into eph-4 cell line. we then used flow cytometry of brdurd-stained cells to investigate the cell-cycle duration of the control and transduced cell lines. tunel was used as a marker of apoptosis to find out differences in the frequencies of apoptotic cells in the control and transduced cell lines. finally, immunohistochemistry staining was performed to detect bcl-6 in breast cancer (iii). results: restoration of bcl-6 into eph-4 cells not only inhibits apoptosis but also prolongs the cell cycle and results in increased cell size and protein content. the results also indicated that the cell-cycle time of bcl-6-transduced eph-4 cells is prolonged by about 3 h, presumably as a result of the action of bcl-6 at the bcl-6 at the g1/s transition. we found differences in the frequencies of viable and apoptotic cells in cultures of the parent eph-4 cells, control-transduced eph-4 cells and bcl-6-transduced eph-4 cells. consistently, we demonstrated that bcl-6 is expressed in 90% of high grade of breast carcinoma, which is considered as the most aggressive of tumours. conclusion: together, these results suggest that bcl-6 is likely to be involved in mammary gland development and carcinogenesis. inflammatory cytokines have a critical role in modulation of both innate and adaptive immunity in response to foreign antigen. they also play an important role in anticancer immunity. for example, they can promote cell-mediated immunity against cancer cells. with their immunostimulatory effects, these cytokines are being tested for cancer treatment in the form of dna vaccine or adjuvant or therapeutic cytokines. direct effect of these cytokines on cancer cell, however, is still unclear. in this project, we investigated whether il-1( and il-18 can modulate cancer cell proliferation. we employed a simple nonradioactive proliferation (mtt) assay and detection of lactate dehydrogenase (ldh) to test the effect of these recombinant human cytokines on various cancer cell lines, including breast cancer cell line (mcf-7), oral carcinoma cell line (kb), colon cancer cell line (caco-2) and choriocarcinoma cell line (jar). cytokines used in this study had both inhibitory and stimulatory effect on cell proliferation. findings in this project could provide an insight of cancer cell response to these cytokines and this could lead to a consideration on using cytokine as immunotherapy for cancer treatment.capacity of ae to modulate nitric oxide production depended on intercellular contact donor t cells are involved in the antitumour effects observed after bmt. thus, patients receiving t-celldepleted bmt have a higher risk of leukaemia relapse compared to patients receiving nonmanipulated bmt, and patients experiencing graft-versus-host disease (gvhd) have a lower risk of disease relapse than patients who do not experience gvhd. although the importance of donor t cells for the curative action of bmt has been established, the exact mechanisms and molecules involved in this graft-versus-tumour effect remain largely unknown. in a recently initiated project, we have conducted a longitudinal study of t-cell clonotypes in patients who received peripheral blood stem cell grafts after nonmyeloablative conditioning. peripheral blood samples were obtained sequentially after transplant, and the mononuclear cells (mncs) were isolated and cryopreserved. cd8 þ t cells were isolated from the mncs by use of immunomagnetic beads or facs and analysed for the presence of clonally expanded cells by t-cell receptor clonotype mapping based on rt-pcr and denaturing gradient gel electrophoresis (dgge). using this gel-based methodology, clonally expanded t cells were monitored after transplant and compared to the clinical data of the patients. the preliminary results demonstrates the presence of clonally expanded cd8 þ t cells at all time points analysed. furthermore, a number of clonotypes persisted for more than 6 months, and other clonotypes emerged during this period. the appearance of newly emerged clonotypes which coincided with clinical gvhd could indicate a role for these t cells in the pathogenesis of gvhd. background: deficiency of the mannan-binding lectin (mbl) pathway of innate immunity leads to increased susceptibility to infections. in patients with colorectal cancer, postoperative infection is associated with poor prognosis. the aim of the present study was to evaluate (1) the relation between the mbl pathway and postoperative infectious complications and survival of patients resected for colorectal cancer and (2) the role of mbl as acute phase reactant compared to crp. methods: preoperative mbl concentration, mbl/mblassociated serine protease (masp) activity and crp were determined in serum from 611 patients and 150 healthy controls. the patients were observed for 8 years. postoperative infections, recurrence and survival were recorded. results: the mbl pathway components were increased in the patients (p < 0.0001) compared to healthy controls. low mbl levels were predictive of pneumonia (p ¼ 0.01), and pneumonia (n ¼ 87) was associated with poor survival (p ¼ 0.003, hr ¼ 1.5, 95% ci 1.1-1.9). mbl and mbl/ masp activity could not predict postoperative overall infections. mbl showed no correlation (spearman's r ¼ 0.02, 95% ci à0.06-0.10) with crp. conclusions: low preoperative mbl levels are predictive of pneumonia, which is associated with poorer survival. mbl concentration and mbl/masp activity was not predictive of other postoperative infections or long-term prognosis. mbl apparently is not a surrogate measure of crp. department of surgery, university hospital of erlangen, erlangen, germany. e-mail: michael.siassi@rzmail.uni-erlangen.de introduction: the human collectins, mannan-binding lectin (mbl), surfactant protein-a (sp-a) and surfactantprotein-d (sp-d) play a central role in the innate immune system. immunological responses to malignant transformation of epithelial cells gained increasing interest recently. a former study could demonstrate binding of mbl to certain colorectal carcinoma (crc) cell lines in vitro. we therefore examined the expression of human collectins in normal colon mucosa and in colorectal carcinomas. materials and methods: colon samples from 20 crc patients and 10 normal mucosa samples were collected immediately after surgery. the tissue was microdissected and rna isolated (qiagen, rneasy-kit). gene expression profiles were analysed using gene-chips (affymetrix, hg-u133). we analysed the data for the expression of mbl, its associated serine proteases mannan-binding lectinassociated serine protease 1/2 (masp 1/2), sp-a and sp-d. the signal intensity of the genes of interest was compared using the mann-whitney u-test. results: the expression of human collectins in normal human colon mucosa was generally low. only the expression of sp-a and masp-2 reached the noise threshold of 250 signals. these genes were significantly downregulated in crc specimens. the expression of the other proteins showed no difference in normal mucosa and crc. conclusion: as demonstrated before, the expression of human collectins in normal colon was low in this being the first lymph node to receive drainage from the tumour area, the sentinel node offers a unique possibility to obtain tumour-reactive lymphocytes. we investigated antitumour immune responses in sentinel nodes from patients with bladder cancer, by assaying tumour-specific proliferation and tcr vb repertoires. during tumour surgery, sentinel lymph nodes were identified by peritumoural injection of blue dye. fresh specimens of tumour, sentinel and nonsentinel lymph nodes were obtained, and single-cell suspensions were prepared. cells were assayed for reactivity against autologous tumour extract in [ 3 h]-thymidine incorporation assays and characterized by flow cytometry. parallel analyses of the expression of vb gene families were performed with padlock probes, linear oligonucleotides which upon target recognition can be converted to circular molecules by a ligase. probes were reacted with cdna prepared from magnetically separated cd4 þ cells, and the tcr repertoire was determined by hybridizing the products to oligonucleotide microarrays. dose-dependent proliferation in response to tumour extract could be detected in sentinel lymph nodes. common clonal expansions were detected among tumourinfiltrating lymphocytes and in sentinel lymph nodes. nonsentinel lymph nodes displayed a divergent tcr vb repertoire. these results indicate an ongoing immune response against tumour antigens in sentinel nodes, draining urinary bladder cancer. identification of sentinel lymph nodes makes it possible to obtain tumour-reactive lymphocytes for use in adoptive immunotherapy. key: cord-011438-imbpgsub authors: zhang, yun; xu, zhichao; cao, yongchang title: host–virus interaction: how host cells defend against influenza a virus infection date: 2020-03-29 journal: viruses doi: 10.3390/v12040376 sha: doc_id: 11438 cord_uid: imbpgsub influenza a viruses (iavs) are highly contagious pathogens infecting human and numerous animals. the viruses cause millions of infection cases and thousands of deaths every year, thus making iavs a continual threat to global health. upon iav infection, host innate immune system is triggered and activated to restrict virus replication and clear pathogens. subsequently, host adaptive immunity is involved in specific virus clearance. on the other hand, to achieve a successful infection, iavs also apply multiple strategies to avoid be detected and eliminated by the host immunity. in the current review, we present a general description on recent work regarding different host cells and molecules facilitating antiviral defenses against iav infection and how iavs antagonize host immune responses. influenza a virus (iav) can infect a wide range of warm-blooded animals, including birds, pigs, horses, and humans. in humans, the viruses cause respiratory disease and be transmitted by inhalation of virus-containing dust particles or aerosols [1] . severe iav infection can cause lung inflammation and acute respiratory distress syndrome (ards), which may lead to mortality. thus, causing many influenza epidemics and pandemics, iav has been a threat to public health for decades [2] . the virus is an enveloped, segmented, negative-strand rna virus, belonging to the orthomyxoviriae family. the eight viral gene segments encode as many as 18 proteins. besides polymerase basic 1 (pb1), pb1-n40, pb1-f2, pb2, polymerase acid (pa), hemagglutinin (ha), nucleoprotein (np), neuraminidase (na), matrix 1 (m1), matrix 2 (m2), nonstructural protein 1 (ns1) and ns2 (also known as nuclear export protein, nep), new viral proteins were recently uncovered, such as pb2-s1 [3] , pa-x (product of ribosomal frameshifting) [4] , pa-related proteins pa-n155 and pa-n182 [5] , m42 [6] , and ns3 [7] . ha, na, and m2 proteins constitute surface of the iav virion, where ha is the most abundant surface protein. according to the genetic and antigenic diversity of the ha and na proteins, iavs were divided into 18 ha and 11 na subtypes. h17n10 and h18n11 subtypes were recently identified in bats [8, 9] . ha is a type i glycosylated protein, which is responsible for virus entry to host cell. functional ha protein is a homotrimer structurally composed of a stem region and a globular head region in each monomer. the head region bearing n-acetylneuraminic acid (sialic acid, sa) binding pocket is critical for receptor attachment, and contains most antigenic determinants. the stem region undergoing conformational changes is responsible for low ph-triggered membrane fusion [10] , and plays an important role in cross protection against heterosubtypic iav infection [11] . n38 glycan at this region iavs can infect a broad spectrum of host species, including both wild and domestic birds, as well as many mammalian species. the virus is capable of interspecies transmission to new species. however, no interspecies transmission of the bat iavs has been reported so far [54] . furthermore, the high frequency of mutations and recombination increases the risk of iav adaptation in humans. besides three pandemic subtypes (h1n1, h2n2, and h3n2), other subtypes, including h5n1, h5n6, h7n7, h7n9, n9n2, and h10n8 could cross the species barrier and cause human infections [55] [56] [57] [58] . several effect factors are essential in iav host switch events, including the receptor-binding properties of ha [16] , as well as cellular receptors [59] [60] [61] [62] . long and his colleagues summarized the role of host factors in iavs adaption to humans, and the review is recommended here for further reading [63] . noteworthy is the fact that most phylogenetically diverse iavs with different origins could successfully replicate in swine [64] . since pigs have both sa α-2,6 and sa α-2,3 galactose receptors [65] , they can serve as a suitable mixing reservoir for both human and avian iavs, thus raising global concern on periodic zoonotic infections. take the emergence of influenza a (h1n1) pdm09 (ph1n1) and influenza a (h3n2) a/canada/1158/2006 strains for instance, both strains are swine-origin iavs and were the consequence of adaption and reassortment of several swine lineages [66, 67] . furthermore, some genes of these strains originated from avian iavs [68] . with the development of gene sequencing technology, machine learning (ml) facilitated with large genomic datasets are used in prediction about sequence changes in newly invaded viruses from other animal hosts, take the "batch-learning self-organizing map (blsom)" method for instance [69] . ml is also applied in characterization of distinct host tropism protein signatures [70] , and prediction of amino acid changes for interspecies transmission [71] . these studies provided measures in identification of potential high-risk strains. in addition, the nucleotides and dinucleotide compositions of viruses play important roles in prediction of viral host species [72] . combining gene sequencing technology viruses 2020, 12, 376 4 of 23 and ml methods, researchers applied large iav genomic datasets to analyze species selection bias of iav mono-/dinucleotide composition and predict human-adaptive swine or avian iavs [73, 74] . the application of multi-disciplinary subjects would provide useful information for prediction of pandemic influenza. in general, the life cycle of the iav is generally divided into four steps: virus entry into the host cell, transcription and replication of the viral genome, assembly, and virus budding. though alveolar epithelial cell is the primary target cell for iavs, different iav subtypes have different patterns of viral attachment (pva). for human iavs, alveolar type ii epithelial cells, as well as immune cells such as alveolar macrophages and dendritic cells, are major target cells for an established infection [75, 76] . two seasonal iavs and pandemic h1n1 virus, preferred to attach to ciliated epithelial cells and goblet cells in the upper respiratory tract (urt), and avian iavs, take h5n1 for instance, attached seldom to these cells [77] . in the lower respiratory tract (lrt), human iav h1n1 and h3n2 attached to more cell types than avian iav h5n1, a highly pathogenic avian iav (hpaiv) strain. however, h5n1 could bind to type ii pneumocytes [78] . considering the fact that metabolism in the type ii pneumocytes is quite active, infection of hpaivs is more likely to cause severe pneumonia [78] . other research on low pathogenic avian iavs (lpaivs), which generally do not cause severe pneumonia, showed that these viruses usually attach to human submucosal gland cells, thus can be cleared by the mucus [79] . iav infection starts from recognition of sa by ha protein, though in vitro research claimed that these n-linked glycans were not essential for virus entry [80] . the cleavage of ha precursor protein ha0 into ha1 (containing receptor binding domain) and ha2 (containing fusion peptide) in low ph environment during ha transport is critical for virion internalization [81] . some research showed that type ii transmembrane serine protease such as transmembrane protease serine 2 (tmprss2), human airway trypsin-like protease (hat), transmembrane protease serine 4 (tmprss4), homo sapiens serine protease desc1 and homo sapiens transmembrane protease, serine 13 (mspl) can cleave human and avian iav ha proteins at an arginine residue [82] . in addition, for avian iavs, ha0 of hpaivs can be cleaved by subtilisin-like protease, while that of lpaivs is cleaved by trypsin-like proteases [83] or thrombin [84] . therefore, in avian iavs, the cleavage sites are considered to be the major determinants for virus virulence [85] , and rna folding in the cleavage region could be an important factor for virulence determination [86, 87] . proteins in the vrnp complex contain different nuclear localization signals (nlss), thus helping the vrnp complex to enter the host cell nucleus via active transport, take the crm1-dependent pathway for instance [88] . the acidic environment of the endosome also activates m2 ion channel, hence acidifies the viral core, resulting in entrance of vrnp complex into the host cell [34] . replication of viral genome does not require a primer but a full-length complementary rna (crna), which is essential for the newly formed vrnp complex. the viral rna polymerases first bind to the 3 end and the 5 end of the segmented viral rna and crna, respectively, then start replication with the help of the 5 cap of host pre-mrnas via a pb1-pb2-mediated "cap snatching" mechanism [27, 89] . the conserved segment-specific nucleotides at the 3 and 5 ends of the viral genome could modulate genome expression and replication during infection [90] . in addition, dephosphorylation at a specific position of the h1n1 ns1 protein results in attenuated virus replication [91] . mature viral mrnas are transported to the cytoplasm by a "daisy-chain" complex and translated subsequently [88, 92] . new synthesis of ha occurs on the rough endoplasmic reticulum (er). glycosylation and palmitoylation of the protein are completed later in the golgi [93, 94] . after synthesis and maturation of na and m2 proteins, the trans-golgi network (tgn), together with coat protein i (copi) complex and gtpase rab proteins, transport the newly synthesized ha, na, and m2 proteins to the apical plasma membrane (pm). these proteins then assemble with viral genomic segments. the virions are finally closed and m1 and m2 proteins mediate virion budding from the apical side of the viruses 2020, 12, 376 5 of 23 cells [28, [95] [96] [97] [98] . na protein cleavages the sa residues, which allows the virions to be released from the plasma membrane [99] . since iav has a relatively small genome, host machinery is required in order to accomplish the viral life cycle. to uncover host dependency factors that are necessary for iav replication, numerous large-scale rna interference (rnai) screens and genome-wide crispr/cas 9 screen were performed [29, [100] [101] [102] [103] . for instance, son dna binding protein was important for iav virion trafficking in an early infection stage and cdc-like kinase 1 facilitated aiv replication [29] . usp47 facilitated viral entry, whereas tnfsf12 (april) and tnfsf12-tnfsf13 (twepril) helped with viral replication [101] . using genome-wide crispr/cas9 screen, several genes of sialic acid biosynthesis and related glycosylation pathways were involved with h5n1 infection [102] , and wdr7, ccdc115, and tmem199 were essential for viral entry and regulation of v-type atpase assembly [103] . furthermore, single-cell transcriptome sequencing (rna-seq) was applied to explore host-virus interactions, revealing a correlation between defective viral genomes and virus-induced host transcriptional programs [104] . these data provide valuable information for developing host-targeted therapeutics. host immune system functions immediately after detection of the virus. host mucosal immune system (mis), induced after virus invasion, serves as the first line to prevent iav from adhering to the susceptible cells. in the urt, mucosal response is induced in the naso-associated lymphoid tissues (nalt), while in the lrt, it occurs in bronchus-associated lymphoid tissues (balt). host innate immunity, including phagocytic cells, interferons (ifns), proinflammatory cytokines, etc., applies multiple mechanisms in defending iav infection [105] . host adaptive immunity, mediated by b lymphocytes and t lymphocytes, together with other immune mechanisms, reacts specifically to neutralize and eliminate the virus. on the other hand, to establish a successful infection, iavs also employ a plethora of strategies to avoid being detected or being cleared by the host immunity. notable strategies include regulation of ifn signaling [106] , inhibition of cytokine expressions [107, 108] , modulation of apoptosis [109] [110] [111] , interference of autophagy [112] , and effects on antibody production [113] . the iav-host immunity interaction was summarized by several reviews [114, 115] . upon detection of infection, innate effector cells, including natural killer (nk) cells, neutrophils, and dendritic cells (dcs), etc., are recruited to the infected sites. nk cells are large granular lymphocytes, making up 10% of the resident lymphocytes in the lung. after recruitment from the blood, nk cells interact with dcs and macrophages to secret various cytokines and restrict infection via lysis of the iav-infected cells. the lysis process is mediated by interaction between nk receptors p46 (most nkp46) and iav ha protein expressed by the infected cell [116, 117] . interestingly, liver nk cells other than lung nk cells possessed a memory phenotype to protect mice against subsequent iav infection, though the lung nk cells are important in control of primary iav infection [118] . however, nk cells are also shown to exacerbate iav pathology, since depletion of nk cells led to increased resistance to high dose h1n1 infection in mice [119, 120] . the contribution of nk cells to anti-iav defense in mouse models was later shown to be strain and dose dependent. in addition, the host genetic background also played an important role [121] . neutrophils are key innate immune cells recruited to infection sites by cellular migration through vascular endothelium. they function in clearance of pathogens via phagocytosis, producing extracellular traps, and degranulation [122] . in addition, they also regulate adaptive immunity via guiding influenza specific cd8 + t cells to the infection sites [123] . the function of dendritic cells (dcs) is to monitor invading pathogens. after iav infection, the conventional dcs migrate from lung to lymph nodes through interaction between ccr7 and its ligand, and present antigens to t cells [124, 125] . one study based on a mouse model showed that, during iav infection, immature and mature dcs were specialized in iav ha processing, since both types of dcs could present one epitope of h1n1 ha (ha amino acids 107-119), whereas another epitope (ha amino acids 302-313) could only be processed by mature dcs [126] . the complex role of dcs in initiation of robust immunity against iav infection is reviewed by waithman and mintern [127] . t cells and b cells are critical components in adaptive immunity against iav infection. cd8 + t cells differentiate into cytotoxic t lymphocytes (ctls) and defend iav infection via producing cytokines and effector molecules, and cytotoxic effects (i.e., lysis) of infected cells mediated by mhc class i. cd4 + t cells target iav-infected epithelial cells through binding with mhc class ii molecules and contribute to b cell activation thus consequently promote antibody production. the activation of t cells and b cells in iav infection will be exposited in section 3.5. the reaction of innate immunity is nonspecific. it is triggered by recognition of pathogen associated molecular patterns (pamps) via host pathogen recognition receptors (prrs). toll-like receptors (tlrs), retinoic acid-inducible gene-i proteins (rig-i), and nod-like receptors are common prrs, the activation of which leads to activation of innate immune signaling and further production of cytokines as well as other antiviral molecules. toll-like receptors are responsible for sensing pathogens at cell membranes, endosomes, and lysosome [128] . tlr3 and tlr7 are shown to be involved in iav detection at endosomes [105] . tlr3 recognizes double stranded rna (dsrna) which may be released by cellular stress and cell death [105] and unidentified rna structures in phagocytosed cells infected with iavs [129] . in macrophages and dendritic cells, tlr3 interacted with tir-domain-containing adapter, then activated the serine-threonine kinase iκkε (ikkε) and tank binding kinase 1 (tbk1) to phosphorylate interferon regulatory factor 3 (irf3), the process of which further led to expression of ifn-β [130] . in addition, an over-reacting tlr3 activation promoted iav pathogenesis, which could be reduced by a single-stranded oligonucleotide (sson) functioning as a tlr3 inhibitor, resulting in restrained viral loads both in vitro and in vivo [131] . tlr7 recognizes single stranded rna (ssrna). in plasmacytoid dendritic cells (pdcs), after activation of tlr7 during iav infection, irf7 or nuclear factor kappa-light-chain-enhancer of activated b cells (nf-κb) were activated via myeloid differentiation factor 88 (myd88) to induce type i ifns [132] . in avian macrophages, activation of tlr7 produced pro-inflammatory molecules such as interleukin (il)-1β [133] . in addition, in mouse models, tlr7 played an important role in activation of nk cells [134] . it was also shown to be involved in development of adaptive immunity to prevent iav infection [135, 136] . rig-i recognizes ssrnas and transcriptional products of iavs, which triggers activation of the caspase activation and recruitment domains (cards) via dephosphorylation or ubiquitination by e3 ligases, resulting in activation of transcription factors including irfs and nf-κb [137] . otub1 played an essential role in regulation of rig-i [138] . in addition, melanoma differentiation-associated gene 5 (mda5) was also involved in sensing transcriptional products of iavs in the cytoplasm [139] . for nod-like receptor family, pyrin domain containing 3 (nlrp3) and nlr apoptosis inhibitory protein 5 were activated after iav infection [140] . iav m2 ion channel and pb1-f2 were involved in activation of nlrp3 inflammasome and stimulate il-1β secretion subsequently [141, 142] . the role of the nlrp3 inflammasome in regulation of anti-iav responses is discussed in detail by sarvestani and his colleagues [143] . delayed oseltamivir and sirolimus combined treatment could suppress nlrp3 inflammasome mediated secretion of il-1β and il-18, resulting in attenuation of h1n1-induced lung injury [144] . after detecting viral components, transcription factors including nf-κb and irfs are activated, leading to transcription of ifns and pro-inflammatory cytokines. ifns bind to receptors, resulting in upregulation of multiple interferon-stimulated genes (isgs) [145] . it is well known that type i ifns viruses 2020, 12, 376 7 of 23 (ifn-α and ifn-β) and type iii ifns (ifn-λ 1-4) play critical roles in antiviral responses. mice failed to restrict non-pathogenic iav when both type i and type iii ifn receptors were knocked out [146] . the expressed ifns consequentially bind to different receptors. type i ifns interact with ifn-α/β receptors (ifnar), whereas type iii ifns interact with ifn-λ receptors (ifnlr). janus kinase-signal transducer and activator of transcription (jak-stat) signaling pathway is then activated, resulting in transcription of numerous ifn-stimulated genes (isgs) [147, 148] . though ifn-λs share many characteristics such as expression patterns, signaling pathways, etc. with type i ifns, they are the first ifns produced at the infected epithelial sites to block virus spread [149] . furthermore, ifn-λs served an important role in programming dcs to direct effective t cell immunity against iav infection [150] . isgs encode various antiviral proteins functioning in different ways to defend iav infection. for instance, mxa gtpase from the mx family could retain viral genome from entry to the cytoplasm via blocking the function of iav np. in addition, in vitro research found that avian iavs were more sensitive to mxa than human iavs [151, 152] . cholesterol 25-hydroxylase (ch25h) were identified to block iav entry via altering the cellular membrane properties to interfere with viral fusion, and amplified the activation of immune cells [153] . guanylate-binding protein 3 (gbp3) of ifn-inducible gtpases inhibited iav replication via binding to the viral polymerase complex [154] . members of the tripartite motif-containing (trim) family are also involved in cellular anti-iav processes. for instance, trim14 could interact with iav np for ubiquitination and proteasomal degradation, thus restricting iav replication in a type i ifn and nf-κb independent manner [155] . trim22 degraded iav np via polyubiquitination, thus resulting in inhibition of iav infection [156] . trim 25 regulated the re-localization of rig-i and was responsible for rig-i ubiquitination as well as rig-i-mediated ifn production [157] . trim32 recognized iav pb1 protein and reduced its polymerase activity [158] . trim41 targeted np for ubiquitination and degradation in vitro [159] . for further reading on other isgs, several reviews regarding ifn responses during iav infection are recommended here [160, 161] . a general description of activation of innate immunity and ifn signaling pathway after iav infection is illustrated in figure 1 . signal transducer and activator of transcription (jak-stat) signaling pathway is then activated, resulting in transcription of numerous ifn-stimulated genes (isgs) [147, 148] . though ifn-λs share many characteristics such as expression patterns, signaling pathways, etc. with type i ifns, they are the first ifns produced at the infected epithelial sites to block virus spread [149] . furthermore, ifnλs served an important role in programming dcs to direct effective t cell immunity against iav infection [150] . isgs encode various antiviral proteins functioning in different ways to defend iav infection. for instance, mxa gtpase from the mx family could retain viral genome from entry to the cytoplasm via blocking the function of iav np. in addition, in vitro research found that avian iavs were more sensitive to mxa than human iavs [151, 152] . cholesterol 25-hydroxylase (ch25h) were identified to block iav entry via altering the cellular membrane properties to interfere with viral fusion, and amplified the activation of immune cells [153] . guanylate-binding protein 3 (gbp3) of ifn-inducible gtpases inhibited iav replication via binding to the viral polymerase complex [154] . members of the tripartite motif-containing (trim) family are also involved in cellular anti-iav processes. for instance, trim14 could interact with iav np for ubiquitination and proteasomal degradation, thus restricting iav replication in a type i ifn and nf-κb independent manner [155] . trim22 degraded iav np via polyubiquitination, thus resulting in inhibition of iav infection [156] . trim 25 regulated the re-localization of rig-i and was responsible for rig-i ubiquitination as well as rig-i-mediated ifn production [157] . trim32 recognized iav pb1 protein and reduced its polymerase activity [158] . trim in order to counter ifn-stimulated antiviral proteins, iav viral proteins apply multiple strategies. for instance, ha protein was shown to trigger ubiquitination of ifnar to attenuate the type i ifn signaling pathway [162] . the follow-up work showed that poly (adp-ribose) polymerase 1 (parp1) functions as an interacting partner of ha protein to mediate the ha-induced ifnar degradation [163] . ns1 is the most important ifns antagonist protein via mechanisms including inhibition of the trim25-mediated rig-i ubiquitination, suppression of protein kinase r (pkr), in order to counter ifn-stimulated antiviral proteins, iav viral proteins apply multiple strategies. for instance, ha protein was shown to trigger ubiquitination of ifnar to attenuate the type i ifn signaling pathway [162] . the follow-up work showed that poly (adp-ribose) polymerase 1 (parp1) functions as an interacting partner of ha protein to mediate the ha-induced ifnar degradation [163] . ns1 is the most important ifns antagonist protein via mechanisms including inhibition of the trim25-mediated rig-i ubiquitination, suppression of protein kinase r (pkr), phosphorylation of iκb kinases (ikk) α and β in the nf-κb pathway, interruption of the phosphorylation of stat1, stat2, and stat3 [39, 115] , and degradation of otub1 [138] . phosphorylation of ns1 is crucial for its function of antagonizing ifn-β expression, since dephosphorylation at position 73 and 83 of the protein induced a high level of ifn-β [91] . non-structural protein pb1-f2, identified from a+1 open reading frame (orf) of pb1 gene segment [164] , is multifunctional in deregulation of type i interferon [165, 166] . it counteracted rlr-mediated activation of ifn pathway not only by targeting mitochondrial mavs [115, 165, 167] , but also by binding to the dead-box helicase ddx3 to induce proteasome-dependent degradation [166] . furthermore, pb1-f2 interacted with mitochondrial tu translation elongation factor (tufm) to mediate formation of autophagosome, thus inducing complete mitophagy, which is critical for mavs degradation [167] . novel pa-x protein could also modulate innate immune responses. a review regarding the function of ns1 and pa-x proteins in antagonizing host innate immunity is recommended here [114] . though autophagy is essential for cellular metabolism and homeostasis, it also plays important roles in innate immune responses against pathogen infection. for cellular homeostasis, the mtor pathway is one of the most conserved autophagic pathways. the mtor complex 1 (mtorc1) negatively regulates the ulk1 kinase activity, thus affecting the autophagy induction [168] . c-jun n-terminal protein kinase 1 (jnk1) disrupts the bcl-2/beclin-1 complex through phosphorylation, thus regulating the autophagy induction [169, 170] . jnk1 is also reported to upregulate beclin-1 expression through phosphorylation of transcription factor c-jun in vitro [171] . in contrast to the autophagic pathways for cellular metabolism and homeostasis, less is known about autophagosome formation after iav infection [172] . to restrict infection of multiple viruses including iavs, trim23 is essential to mediate autophagy via its ring e3 ligase and adp-ribosylation factor (arf) gtpase activity [173] . beclin-1 and tufm-regulated autophagy also inhibited iav replication [112] . in hela cells and a549 cells, iav infection activated jnk1 to induce autophagosome formation and tgf-β-activated kinase 1 might contribute to the process [174, 175] . furthermore, autophagy was involved in maintaining memory b cells to counteract iav infection [176] . iav also utilizes autophagy to complete its life cycle. ns1 protein is proposed to suppress jnk1-mediated autophagy induction [174] . m2 could also block autophagosome maturation and mediate microtubule-associated protein 1 light chain 3 (lc3)-bound membrane redistribution, thus allowing filamentous budding of iav [177] [178] [179] . circ-gatad2a (gata zinc finger domain containing 2a), induced by iav infection, could inhibit autophagy and promote iav replication [180] . for a comprehensive reading on iav-induced apoptosis, a review is recommended here [181] . upon detection of iavs, dcs trigger production of ifns and cytokines, which in turns assist maturation of the dcs into antigen presenting cells (apcs), and initiate t cell immune responses. through the activation of ag-bearing dcs, naïve cd4 + t cells differentiate into th1, th2, th7, regulatory t cells (treg cells), follicular helper t cells, and killer cells. th1 and follicular helper t cells are the most abundant cd4 + t helper cells. they can secret antiviral cytokines, regulate cd8 + t cell differentiation, promote b cell activation, and maintain immunological memory [182, 183] . th17 cells induced pulmonary pathogenesis and could decrease mortality of iav-infected mice [184, 185] . in addition, γδ t cells, expanding in the late stage of iav infection with a t cell receptor (tcr)-independent viruses 2020, 12, 376 9 of 23 manner, could efficient eliminate iav-infected airway epithelial cells, resulting in lower viral titers [186] . new surrogate markers cd49d and cd11a were used to explore the kinetics of iav-specific cd4 t cells responses, revealing endogenous cd4 t cell response to primary iav infection is predominantly composed of t-bet+ cells [187] . cd8 + t cells are major components for virus clearance in adaptive immunity. after activated by dcs, cd8 + t cells undergo rapid expansion, differentiation, and migration to the infected sites. in general, to establish effective primary cytotoxic t lymphocyte (ctl) responses, cd4 + t cells play an essential role, with a mouse model as an exception [188] . ctls produce cytotoxic granules containing perforin and granzymes (gra and grb) to induce apoptosis and interrupt iav replication [189] . in addition, ctls produce cytokines, such as tnf, fasl, and trail, which recruit death receptors to induce apoptosis [190] . in addition, il16 deficiency enhanced the th1 and ctl responses upon iav infection [191] . furthermore, as cd8 + cells could last for two years in murine models, iav-specific memory ctls reacted specific to epitopes in conserved iav proteins [192] . in the nasal epithelia, they could prevent the spread of the virus from the urt to the lung [193] . to establish memory cd8 + t cells, autophagy plays an important role [194] , while the function of cd4 + t cells in memory ctl responses is "context-dependent". a recent study showed that cd4+ t cells promoted iav-specific ctl memory at the initial priming stage of viral infection [195] . grant and her colleagues summarized and discussed the importance of cd8 + t cell immunity against iavs [192] , and this review is recommended for further reading. with the help of cd40 ligand (cd40l), cd4 + cells contribute to b cell activation [183] . with the help of memory t cells, naïve b cells could reduce morbidity and promote recovery on heterosubtypic infection [196] . for different types of antibodies, igg could inhibit pathogenesis, while iga functions in blocking iav transmission [197] . in addition, iav-specific antibody-dependent cell-mediated cytotoxicity (cdcc) also plays a role in cross-protection against iav infection. a general description of adaptive immunity against primary iav infection is illustrated in figure 2 . antigenic shift and drift, resulting in reassorted and mutated ha and/or na, are responsible for aiv escaping from host immunity [50] [51] [52] . furthermore, additional glycosylation on h5 ha could also induce virus escape from neutralizing antibodies [198] . apoptosis represents programmed single cell death that occurs in cell physiological remodeling, cell proliferation, or immune response to invading pathogens [199] . besides prototypical changes, cells undergoing apoptosis can be detected through dna and biochemical assays, take the tunel and in situ end-labeling (isel) techniques for instance. two primary pathways are involved in activation of apoptosis: the intrinsic or mitochondrial pathway, and the extrinsic or death receptor pathway. the intrinsic pathway is also known as "the mitochondrial pathway", which operates in response to various intracellular stress. several factors such as nitric oxide (no), cytochrome c, and second mitochondria-derived activator of caspases (smac) can activate this pathway, and the key player of this pathway is proteins in the bcl-2 family, which are activated by stress signals and then release apoptotic factors via destabilizing the mitochondrial membrane [200, 201] , resulting in release of mitochondrial cytochrome c. cytochrome c then binds to apoptosis protease activating factor-1 (apaf-1) and forms a complex with pro-caspase 9 (then cleaved into caspase 9), the function of which is to cleave its effector pro-caspase 3 [202] . in addition, smac, localizing in the cytosol, could initiate activation of caspase 9 via blocking the activity of iap [203] . the extrinsic pathway is regulated by extracellular ligands acting on transmembrane "death receptors": the first apoptosis signal (fas) receptor-fas ligand (fasr/fasl) and the tnf-αtnf receptor 1 (tnfα/tnfr1) [199] . in the fasr/fasl model, fas ligand binds to its receptor fasr [204] , forming the death-inducing signaling complex (disc) with pro-caspase 8, resulting in activation of caspase 8 and downstream activation of other caspases (caspase-3, caspase-6, and caspase-7) [199] . in the tnfα/tnfr1 pathway, tnfr1-associated death domain protein (tradd) is activated after binding of tnfα to tnfr1, leading to recruitment of fadd and receptor interacting protein (rip) [205] . fadd then associates with pro-caspase 8 to form the disc, resulting in activation of caspase 8 and apoptosis. could prevent the spread of the virus from the urt to the lung [193] . to establish memory cd8 + t cells, autophagy plays an important role [194] , while the function of cd4 + t cells in memory ctl responses is "context-dependent". a recent study showed that cd4+ t cells promoted iav-specific ctl memory at the initial priming stage of viral infection [195] . grant and her colleagues summarized and discussed the importance of cd8 + t cell immunity against iavs [192] , and this review is recommended for further reading. with the help of cd40 ligand (cd40l), cd4 + cells contribute to b cell activation [183] . with the help of memory t cells, naïve b cells could reduce morbidity and promote recovery on heterosubtypic infection [196] . for different types of antibodies, igg could inhibit pathogenesis, while iga functions in blockin during iav infection, viruses modulate host apoptotic responses in a time-dependent manner [206] . for instance, in order to earn enough time for replication and virion formation, iav inhibited apoptosis via upregulating the anti-apoptotic phophoinositide-3-kinase-protein kinase b (pi3k-akt) pathway at the beginning of infection. however, in the later phase of infection, the virus suppressed this pathway to upregulate the pro-apoptotic p53 pathway, thus allowing successful release of virions [207] . several viral proteins are involved in regulation of host apoptosis. np protein induces host apoptosis to favor viral replication through interaction with ring finger 43 (rnf43) [208] , apoptotic inhibitor 5 (api5) [209] , or clusterin [111] . pb1-f2 also induced apoptosis and promoted viral replication through dysregulating mitochondrial potential [206] . furthermore, m1 promoted apoptosis by binding to heat shock protein 70, thus activating caspase and the subsequent apoptosis [210] . in addition, ns1 expression was reported to induce apoptosis in mdck and hela cells [211] . however, mutant iav lacking the ns1 gene could induce apoptosis in cultured cells [212] . the function of ns1 in inhibiting apoptosis may be explained by its ability to inhibit type i ifn [213, 214] . these data demonstrate sophisticated mechanisms of iav in regulating host apoptosis. furthermore, the role of these viral proteins in apoptosis suggests that these proteins may present suitable targets for anti-iav therapies. a comprehensive review on influenza a virus-induced apoptosis discussed by ampomah and lim is recommended here [181] . in addition, recent in vitro research found that apoptosis was induced at early iav infection stage, while later the cell death pathway was shifted to pyroptosis. the switch process was promoted by the type i ifn-mediated jak-stat signaling pathway through expression of the bcl-xl gene [215] . during iav infection, multiple immune systems coordinate together to protect the host. accordingly, viruses antagonize the immune system through multiple measures to establish a successful infection. considering the high frequencies in genome mutations and recombination, vaccination is the most effective way to defend against the viruses via inducing cross-protective antibodies and/or enhancing immune responses. several studies in vaccine development have tried to enhance host immune responses. for instance, vaccine candidate containing ha targeted to chemokine receptor (porcine mip1α) was shown to enhance t cell responses, resulting in a strong and cross-reactive cellular immunity in vaccinated pigs [216] . another example is an attempt to intranasally administer a polyanhydride nano vaccine (iav-nanovax), which could promote robust lung-resident germinal center (gc) b cells with lung-localized iav-specific antibody responses as well as lung-resident memory cd4 + and cd8 + t cell responses [217] . for anti-iav drugs, currently, na inhibitors (relenza tm and tamiflu tm ) are applied clinically as anti-influenza drugs [218] . these drugs inhibit the activity of na by preventing viral budding [21] . in addition, cap-dependent endonuclease inhibitor (baloxavir marboxil) targeting pa is also applied against influenza a and b virus infection [219] . our progressing understanding of the iav life cycle of the virus and iav-host interaction could contribute to anti-influenza drug design. since the recognition of ha protein to sa linked glycoproteins is the first step in iav infection, effective blocking of the interaction between viral ha and sa receptor serves as a favorable target in drug design [220, 221] . favipiravir, a nucleotide analogue that selectively inhibits the rna-dependent rna polymerase, is licensed in japan to be applied against emerging influenza viruses resistant to other antivirals [222, 223] . oleanolic acid (oa), a kind of pentacyclic triterpene natural product, and its analogues, as well as its derivatives, were shown to bind to ha, thus blocking the attachment of iavs to mdck cells [224] [225] [226] . pvf-tet is a peptide-based ha inhibitor, which was shown to sequester ha into amphisome (fusion of late endosome with autophagosome) and protected mice from the lethal iav infection [227] . new effective drugs targeting the polymerase would be a promising strategy against iav infection, since they would directly reduce or eliminate viral replication. numerous sites, including the cap-binding site [228] , the endonuclease [229, 230] , and pa-pb1 inter-subunit interface [231] can serve as potential targeting sites for new drug design. coumarin compounds, including eleutheroside b 1 , isofraxidin, fraxin, esculetin, fraxetin, and scoparone, were investigated for their antiviral and anti-inflammatory activities against influenza virus in vitro [31] . other candidates, such as naproxen, a non-steroidal anti-inflammatory drug, was shown to target np protein at residues f209 and y148, thus antagonizes the crm1-mediated nuclear export of np. it is suggested to have a broad-spectrum anti-influenza activity [232] . verdinexor (kpt-335), a novel orally bioavailable drug, blocks crm1-mediated nuclear export of np and repress nf-κb activation, thus reducing cytokine production and eliminating virus-associated immunopathology [233] . for further reading on candidate anti-iv therapeutics, a review summarized by davidson is recommended here [234] . with the increasing knowledge obtained through massive investigations on host immunity against iav infection, promoting host immune responses not limited to antibody enhancement would have good prospects not only for vaccine design, but also for development of novel antiviral agents. author contributions: manuscript preparation, y.z.; revision, z.x.; supervision, y.c.; funding acquisition, y.z. all authors read and approved the final version of the manuscript. funding: this study was supported by the "zhujiang talent program" overseas youth talent introduction program (post-doctoral program) and doctoral initiative project of natural science foundation of guangdong province (18zxxt49). the authors declare that they have no financial and personal relationships with other people or organizations that can influence the work. there is no professional or other personal interest of any nature or kind in any product, service and/or company that could be construed as influencing the position presented in this review. they do not have any commercial or associative interest that represents conflicts of interest in connection with the work submitted. influenza virus aerosols in the air and their infectiousness influenza: the once and future pandemic identification of a novel viral protein expressed from the pb2 segment of influenza a virus an overlapping protein-coding region in influenza a virus segment 3 modulates the host response identification of novel influenza a virus proteins translated from pa mrna identification of a novel splice variant form of the influenza a virus m2 ion channel with an antigenically distinct ectodomain adaptive mutation in influenza a virus non-structural gene is linked to host switching and induces a novel protein by alternative splicing a distinct lineage of influenza a virus from bats new world bats harbor diverse influenza a viruses epidemiology, evolution, and pathogenesis of h7n9 influenza viruses in five epidemic waves since 2013 in china a stable trimeric influenza hemagglutinin stem as a broadly protective immunogen glycan repositioning of influenza hemagglutinin stem facilitates the elicitation of protective cross-group antibody responses early alterations of the receptor-binding properties of h1, h2, and h3 avian influenza virus hemagglutinins after their introduction into mammals influenza hemagglutinin and neuraminidase membrane glycoproteins receptor binding profiles of avian influenza virus hemagglutinin subtypes on human cells as a predictor of pandemic potential role of receptor binding specificity in influenza a virus transmission and pathogenesis structures and receptor binding of hemagglutinins from human-infecting h7n9 influenza viruses avian-to-human receptor-binding adaptation of avian h7n9 influenza virus hemagglutinin influenza a penetrates host mucus by cleaving sialic acids with neuraminidase modified sialic acids on mucus and erythrocytes inhibit influenza a ha and na functions influenza neuraminidase. influenza other respir comparative thermostability analysis of zoonotic and human influenza virus a and b neuraminidase neuraminidase as an influenza vaccine antigen: a low hanging fruit, ready for picking to improve vaccine effectiveness structural basis of protection against h7n9 influenza virus by human anti-n9 neuraminidase antibodies structure of influenza virus ribonucleoprotein complexes and their packaging into virions at the centre: influenza a virus ribonucleoproteins influenza virus rna polymerase: insights into the mechanisms of viral rna synthesis human host factors required for influenza virus replication genome-wide rnai screen identifies human host factors crucial for influenza virus replication the nucleolar protein lyar facilitates ribonucleoprotein assembly of influenza a virus inhibition viral rnp and anti-inflammatory activity of coumarins against influenza virus sequences of mrnas derived from genome rna segment 7 of influenza virus: colinear and interrupted mrnas code for overlapping proteins crystal structures of influenza a virus matrix protein m1: variations on a theme the m2 proton channels of influenza a and b viruses influenza a virus m2 ion channel activity is essential for efficient replication in tissue culture the influenza a virus matrix protein 2 undergoes retrograde transport from the endoplasmic reticulum into the cytoplasm and bypasses cytoplasmic proteasomal degradation regulation of the extent of splicing of influenza virus ns1 mrna: role of the rates of splicing and of the nucleocytoplasmic transport of ns1 mrna influenza a virus in vitro transcription: roles of ns1 and np proteins in regulating rna synthesis influenza virus non-structural protein ns1: interferon antagonism and beyond the multifunctional ns1 protein of influenza a viruses recognition of viruses by cytoplasmic sensors rig-i detects viral genomic rna during negative-strand rna virus infection structural basis for influenza virus ns1 protein block of mrna nuclear export changes in rna secondary structure affect ns1 protein expression during early stage influenza virus infection structure and function of the influenza a virus non-structural protein 1 the influenza virus nep (ns2 protein) mediates the nuclear export of viral ribonucleoproteins interaction of the influenza virus nucleoprotein with the cellular crm1-mediated nuclear export pathway a second crm1-dependent nuclear export signal in the influenza a virus ns2 protein contributes to the nuclear export of viral ribonucleoproteins chd3 facilitates vrnp nuclear export by interacting with nes1 of influenza a virus ns2 investigational hemagglutinin-targeted influenza virus inhibitors an overview of the epidemiology and emergence of influenza a infection in humans over time influenza vaccine: the challenge of antigenic drift effector t cells control lung inflammation during acute influenza virus infection by producing il-10 novel insights into bat influenza a viruses human infection with an avian h9n2 influenza a virus in hong kong in 2003 clinical and epidemiological characteristics of a fatal case of avian influenza a h10n8 virus infection: a descriptive study human infection with a novel avian influenza a (h5n6) virus human infection with a novel avian-origin influenza a (h7n9) virus enabling the 'host jump': structural determinants of receptor-binding specificity in influenza a viruses three mutations switch h7n9 influenza to human-type receptor specificity structure and receptor specificity of the hemagglutinin from an h5n1 influenza virus influenza a virus hemagglutinin-neuraminidase-receptor balance: preserving virus motility host and viral determinants of influenza a virus species specificity history and epidemiology of swine influenza in europe receptor binding and membrane fusion in virus entry: the influenza hemagglutinin origins and evolutionary genomics of the 2009 swine-origin h1n1 influenza a epidemic swine influenza (h3n2) infection in a child and possible community transmission avian influenza virus transmission to mammals novel bioinformatics strategies for prediction of directional sequence changes in influenza virus genomes and for surveillance of potentially hazardous strains distinct host tropism protein signatures to identify possible zoonotic influenza a viruses scoring amino acid mutations to predict avian-to-human transmission of avian influenza viruses dinucleotide composition in animal rna viruses is shaped more by virus family than by host species machine learning methods for predicting human-adaptive influenza a viruses based on viral nucleotide compositions predicting reservoir hosts and arthropod vectors from evolutionary signatures in rna virus genomes influenza a viruses target type ii pneumocytes in the human lung emerging cellular targets for influenza antiviral agents seasonal and pandemic human influenza viruses attach better to human upper respiratory tract epithelium than avian influenza viruses human and avian influenza viruses target different cells in the lower respiratory tract of humans and other mammals avian influenza and sialic acid receptors: more than meets the eye? influenza a virus entry into cells lacking sialylated n-glycans avian influenza among waterfowl hunters and wildlife professionals tmprss2: a potential target for treatment of influenza virus and coronavirus infections cleavage activation of the human-adapted influenza virus subtypes by matriptase reveals both subtype and strain specificities a novel neuraminidase-dependent hemagglutinin cleavage mechanism enables the systemic spread of an h7n6 avian influenza virus proteolytic activation of the 1918 influenza virus hemagglutinin from low to high pathogenicity-characterization of h7n7 avian influenza viruses in two epidemiologically linked outbreaks subtype-specific structural constraints in the evolution of influenza a virus hemagglutinin genes nuclear traffic of influenza virus proteins and ribonucleoprotein complexes the cap-snatching endonuclease of influenza virus polymerase resides in the pa subunit mutations of the segment-specific nucleotides at the 3 end of influenza virus ns segment control viral replication the tyrosine 73 and serine 83 dephosphorylation of h1n1 swine influenza virus ns1 protein attenuates virus replication and induces high levels of beta interferon crystal structure of the m1 protein-binding domain of the influenza a virus nuclear export protein (nep/ns2) n-linked glycosylation of the hemagglutinin protein influences virulence and antigenicity of the 1918 pandemic and seasonal h1n1 influenza a viruses site-specific s-acylation of influenza virus hemagglutinin: the location of the acylation site relative to the membrane border is the decisive factor for attachment of stearate influenza a virus hemagglutinin and neuraminidase mutually accelerate their apical targeting through clustering of lipid rafts apical trafficking pathways of influenza a virus ha and na via rab17-and rab23-positive compartments a rab11-and microtubule-dependent mechanism for cytoplasmic transport of influenza a virus viral rna influenza virus assembly and budding characterization of temperature sensitive influenza virus mutants defective in neuraminidase uncovering the global host cell requirements for influenza virus replication via rnai screening. microbes infect knockdown of specific host factors protects against influenza virus-induced cell death genome-wide crispr/cas9 screen identifies host factors essential for influenza virus replication genome-wide crispr screen identifies host dependency factors for influenza a virus infection cell-to-cell variation in defective virus expression and effects on host responses during influenza virus infection innate immunity to influenza virus infection innate immune evasion strategies of influenza viruses the role of il-10 in regulating immunity to persistent viral infections formyl peptide receptor 2 is regulated by rna mimics and viruses through an ifn-β-stat3-dependent pathway influenza a virus enhances its propagation through the modulation of annexin-a1 dependent endosomal trafficking and apoptosis influenza virus induces apoptosis via bad-mediated mitochondrial dysregulation influenza a virus nucleoprotein induces apoptosis in human airway epithelial cells: implications of a novel interaction between nucleoprotein and host protein clusterin autophagy induction regulates influenza virus replication in a time-dependent manner antigen-specific b-cell receptor sensitizes b cells to infection by influenza virus modulation of innate immune responses by the influenza a ns1 and pa-x proteins host immune response to influenza a virus infection nkp46 o-glycan sequences that are involved in the interaction with hemagglutinin type 1 of influenza virus evasion of natural killer cells by influenza virus respiratory influenza virus infection induces memory-like liver nk cells in mice critical role of natural killer cells in lung immunopathology during influenza infection in mice nk cells exacerbate the pathology of influenza virus infection in mice swift and strong nk cell responses protect 129 mice against high-dose influenza virus infection a role for neutrophils in viral respiratory disease. front. immunol. 2017, 8, 550 neutrophil trails guide influenza-specific cd8+ t cells in the airways clearance of influenza virus from the lung depends on migratory langerin+cd11b-but not plasmacytoid dendritic cells induction of tolerance to innocuous inhaled antigen relies on a ccr7-dependent dendritic cell-mediated antigen transport to the bronchial lymph node activation of dendritic cells alters the mechanism of mhc class ii antigen presentation to cd4 t cells dendritic cells and influenza a virus infection toll-like receptor adaptor molecules enhance dna-raised adaptive immune responses against influenza and tumors through activation of innate immunity toll-like receptor 3 promotes cross-priming to virus-infected cells cutting edge: influenza a virus activates tlr3-dependent inflammatory and rig-i-dependent antiviral responses in human lung epithelial cells a single-stranded oligonucleotide inhibits toll-like receptor 3 activation and reduces influenza a (h1n1) recognition of single-stranded rna viruses by toll-like receptor 7 antiviral response elicited against avian influenza virus infection following activation of toll-like receptor (tlr)7 signaling pathway is attributable to interleukin (il)-1β production respiratory influenza a virus infection triggers local and systemic natural killer cell activation via toll-like receptor 7 tlr7 recognition is dispensable for influenza virus a infection but important for the induction of hemagglutinin-specific antibodies in response to the 2009 pandemic split vaccine in mice toll-like receptor 7 is required for effective adaptive immune responses that prevent persistent virus infection viral rna detection by rig-i-like receptors otub1 is a key regulator of rig-i-dependent immune signaling and is targeted for proteasomal degradation by influenza a ns1 rig-i-mediated antiviral responses to single-stranded rna bearing 5 -phosphates nod proteins: regulators of inflammation in health and disease influenza virus activates inflammasomes via its intracellular m2 ion channel activation of the nlrp3 inflammasome by iav virulence protein pb1-f2 contributes to severe pathophysiology and disease the role of the nlrp3 inflammasome in regulation of antiviral responses to influenza a virus infection delayed oseltamivir plus sirolimus treatment attenuates h1n1 virus-induced severe lung injury correlated with repressed nlrp3 inflammasome activation and inflammatory cell infiltration interferons and viruses: an interplay between induction, signalling, antiviral responses and virus countermeasures interferon-lambda contributes to innate immunity of mice against influenza a virus but not against hepatotropic viruses interferon-stimulated genes: a complex web of host defenses type i and type iii interferons drive redundant amplification loops to induce a transcriptional signature in influenza-infected airway epithelia interferon-λ mediates non-redundant front-line antiviral protection against influenza virus infection without compromising host fitness interferon-λ modulates dendritic cells to facilitate t cell immunity during infection with influenza a virus the human interferon-induced mxa protein inhibits early stages of influenza a virus infection by retaining the incoming viral genome in the cytoplasm pandemic influenza a viruses escape from restriction by human mxa through adaptive mutations in the nucleoprotein 25-hydroxycholesterol acts as an amplifier of inflammatory signaling a new splice variant of the human guanylate-binding protein 3 mediates anti-influenza activity through inhibition of viral transcription and replication inhibition of influenza a virus replication by trim14 via its multifaceted protein-protein interaction with trim22 inhibits influenza a virus infection by targeting the viral nucleoprotein for degradation trim25 ring-finger e3 ubiquitin ligase is essential for rig-i-mediated antiviral activity trim32 senses and restricts influenza a virus by ubiquitination of pb1 polymerase trim41-mediated ubiquitination of nucleoprotein limits influenza a virus infection influenza virus infection, interferon response, viral counter-response, and apoptosis influenza virus activation of the interferon system hemagglutinin of influenza a virus antagonizes type i interferon (ifn) responses by inducing degradation of type i ifn receptor 1 parp1 enhances influenza a virus propagation by facilitating degradation of host type i interferon receptor a novel influenza a virus mitochondrial protein that induces cell death influenza virus protein pb1-f2 inhibits the induction of type i interferon by binding to mavs and decreasing mitochondrial membrane potential co-degradation of interferon signaling factor ddx3 by pb1-f2 as a basis for high virulence of 1918 pandemic influenza influenza a virus protein pb1-f2 impairs innate immunity by inducing mitophagy nutrient-dependent mtorc1 association with the ulk1-atg13-fip200 complex required for autophagy connecting endoplasmic reticulum stress to autophagy through ire1/jnk/beclin-1 in breast cancer cells reactive oxygen species-mediated c-jun nh2-terminal kinase activation contributes to hepatitis b virus x protein-induced autophagy via regulation of the beclin-1/bcl-2 interaction the pivotal role of c-jun nh2-terminal kinase-mediated beclin 1 expression during anticancer agents-induced autophagy in cancer cells the regulation of autophagy by influenza a virus trim23 mediates virus-induced autophagy via activation of tbk1 influenza a virus ns1 protein suppresses jnk1-dependent autophagosome formation mediated by rab11a recycling endosomes role of tgf-β-activated kinase 1 (tak1) activation in h5n1 influenza a virus-induced c-jun terminal kinase activation and virus replication essential role for autophagy in the maintenance of immunological memory against influenza infection matrix protein 2 of influenza a virus blocks autophagosome fusion with lysosomes a lir motif in influenza a virus m2 is required for virion stability proton channel activity of influenza a virus matrix protein 2 contributes to autophagy arrest circular rna gatad2a promotes h1n1 replication through inhibiting autophagy influenza a virus-induced apoptosis and virus propagation antigen-specific and non-specific cd4+ t cell recruitment and proliferation during influenza infection expanding roles for cd4+ t cells in immunity to viruses il-17-induced pulmonary pathogenesis during respiratory viral infection and exacerbation of allergic disease imbalanced pro-and anti-th17 responses (il-17/granulocyte colony-stimulating factor) predict fatal outcome in 2009 pandemic influenza ketogenic diet activates protective γδ t cell responses against influenza virus infection kinetics and phenotype of the cd4 t cell response to influenza virus infections t cell mediated immunity to influenza: mechanisms of viral control non-cytotoxic antiviral activities of granzymes in the context of the immune antiviral state pulmonary immunity to viruses il16 deficiency enhances th1 and cytotoxic t lymphocyte response against influenza a virus infection human influenza viruses and cd8 + t cell responses resident memory cd8+t cells in the upper respiratory tract prevent pulmonary influenza virus infection autophagy is a critical regulator of memory cd8+ t cell formation cd4+t help promotes influenza virus-specific cd8+t cell memory by limiting metabolic dysfunction b cells promote resistance to heterosubtypic strains of influenza via multiple mechanisms1 recombinant iga is sufficient to prevent influenza virus transmission in guinea pigs addition of n-glycosylation sites on the globular head of the h5 hemagglutinin induces the escape of highly pathogenic avian influenza a h5n1 viruses from vaccine-induced immunity apoptosis: a review of programmed cell death bcl-2 gene family in endocrine pathology: a review nitric oxide: no apoptosis or turning it on? promotion of caspase activation by caspase-9-mediated feedback amplification of mitochondrial damage smac, a mitochondrial protein that promotes cytochrome c-dependent caspase activation by eliminating iap inhibition the many roles of fas receptor signaling in the immune system the fas signaling pathway: more than a paradigm viral control of mitochondrial apoptosis control of apoptosis in influenza virus-infected cells by up-regulation of akt and p53 signaling the nucleoprotein of influenza a virus induces p53 signaling and apoptosis via attenuation of host ubiquitin ligase rnf43 nucleoprotein of influenza a virus negatively impacts antiapoptotic protein api5 to enhance e2f1-dependent apoptosis and virus replication cell death regulation during influenza a virus infection by matrix (m1) protein: a model of viral control over the cellular survival pathway influenza virus ns1 protein induces apoptosis in cultured cells loss of function of the influenza a virus ns1 protein promotes apoptosis but this is not due to a failure to activate phosphatidylinositol 3-kinase (pi3k) pathogenic potential of interferon αβ in acute influenza infection ifnλ is a potent anti-influenza therapeutic without the inflammatory side effects of ifnα treatment influenza a virus infection triggers pyroptosis and apoptosis of respiratory epithelial cells through the type i interferon signaling pathway in a mutually exclusive manner targeting of ha to chemokine receptors induces strong and cross-reactive t cell responses after dna vaccination in pigs polyanhydride nanovaccine induces robust pulmonary b and t cell immunity and confers protection against homologous and heterologous influenza a virus infections influenza neuraminidase inhibitors: antiviral action and mechanisms of resistance. influenza other respir. viruses baloxavir marboxil: the new influenza drug on the market emerging antiviral strategies to interfere with influenza virus entry discovery of the first series of small molecule h5n1 entry inhibitors favipiravir (t-705), a novel viral rna polymerase inhibitor favipiravir (t-705), a broad spectrum inhibitor of viral rna polymerase synthesis, structure activity relationship and anti-influenza a virus evaluation of oleanolic acid-linear amino derivatives design, synthesis of oleanolic acid-saccharide conjugates using click chemistry methodology and study of their anti-influenza activity design, synthesis and biological evaluation of amino acids-oleanolic acid conjugates as influenza virus inhibitors the inducible amphisome isolates viral hemagglutinin and defends against influenza a virus infection discovery of a novel, first-in-class, orally bioavailable azaindole inhibitor (vx-787) of influenza pb2 a novel endonuclease inhibitor exhibits broad-spectrum anti-influenza virus activityin vitro identification and characterization of influenza variants resistant to a viral endonuclease inhibitor polymerase acidic protein-basic protein 1 (pa-pb1) protein-protein interaction as a target for next-generation anti-influenza therapeutics naproxen exhibits broad anti-influenza virus activity in mice by impeding viral nucleoprotein nuclear export verdinexor targeting of crm1 is a promising therapeutic approach against rsv and influenza viruses treating influenza infection, from now and into the future key: cord-023443-pvz7dll9 authors: nan title: abstracts for the scandinavian society for immunology 35th annual meeting and 20th summer school date: 2004-06-02 journal: scand j immunol doi: 10.1111/j.1365-3083.2004.01423.x sha: doc_id: 23443 cord_uid: pvz7dll9 nan the nuclear receptor heterodimers of liver x receptors (lxrs) are recently identified as key transcriptional regulators of genes involved in lipid homeostasis and inflammation. lxrs and their ligands are negative regulators of macrophage inflammatory gene expression. multiple sclerosis (ms), a demyelinating disease of the central nervous system of unknown cause, is characterized by recurrent inflammation involving macrophages and their inflammatory mediators. sweden belongs to the countries with a high ms incidence. in italy, incidence is lower, with an exception for sardinia where the incidence is even higher than that in sweden. subjects from sardinia are ethnically more homogeneous and differ from swedes, also regarding genetic background and environment. we studied lxrs and their related molecules of blood mononuclear cells (mncs) from female patients with untreated relapsing-remitting ms from sassari, sardinia and stockholm, sweden. sex-and age-matched healthy controls (hcs) were from both areas. mrna expression was evaluated by real-time pcr. lxr-a was lower (p < 0.05) in ms (mean ae sem: 3.1 ae 0.2; n ¼ 37) compared to hc (3.6 ae 0.1; n ¼ 37). lxr-a was lower in ms from stockholm (2.6 ae 0.2; n ¼ 22) compared to corresponding hc (3.4 ae 0.1; n ¼ 22; p < 0.01) and compared to ms (3.8 ae 0.2; n ¼ 15; p < 0.001) and hc (4 ae 0.2; n ¼ 15; p < 0.001) from sardinia. ms patients from stockholm, but not from sassari, also expressed lower (p < 0.05) lxr-b (à4.1 ae 0.4) compared to corresponding hc (à2.9 ae 0.3). ms from stockholm was associated with higher abca-1 (6.1 ae 0.4 versus 5.0 ae 0.3; p < 0.05) and higher estrogen receptor-b-cx (2.4 ae 0.4 versus 0.8 ae 0.4; p < 0.01) compared to corresponding hc. the hc from sassari had higher androgen receptor (2.9 ae 0.2) compared to ms from sassari (1.4 ae 0.3; p < 0.01), ms (1.3 ae 0.4; p < 0.01) and hc from stockholm (1.2 ae 0.3; p < 0.01). ms from sassari had lower cyclooxygenase-1 compared to corresponding hc (5.1 ae 0.4 versus 6.6 ae 0.3; p < 0.01) and lower prostaglandin-e (à0.03 ae 0.5) compared to the hc (1.4 ae 0.5; p < 0.05) and ms (2.7 ae 0.4; p < 0.05) and hc from stockholm (1.9 ae 0.4, p < 0.001). our findings identify lxrs and their related molecules as being involved in ms from stockholm but not from sassari, while sex hormone receptors seem to be involved in ms in sassari. multiple sclerosis: ifn-b induces cd123 + bdca2 -dendritic cells that produce il-6 and il-10 and have no enhanced type i interferon production y. m. huang, 1 s. adikari, 1 u. båve, 2 a. sanna 1,3 & g. alm 4 dc antigens (bdca) and investigate their ability to produce type i ifn in response to virus stimulation. we show that ifn-b induces development of cd123 þ dc from human blood monocytes, which coexpress bdca4 þ but are negative for bdca2 -, a specific marker for plasmacytoid dc. such ifn-b-modulated dc produce large amounts of il-6 and il-10, but no il-12p40 and have no enhanced ifn-b and ifn-b production. the findings indicate that ifn-bmodulated dc represent a myeloid dc subset with diminished cd11c, bdca-1 and cd1a expression, having potent th2-promoting function but lacking antiviral capacity. the association of psoriasis with throat infections by streptococcus pyogenes suggests a potential antigenic target for the t cells that are known to infiltrate dermis and epidermis of psoriatic skin. streptococcal m protein shares an extensive sequence homology with human epidermal keratins. keratins 16 (k16) and 17 (k17) are mostly absent from uninvolved skin but are upregulated in psoriatic lesions. there is increasing evidence that cd8 þ t cells play an important effector role in psoriasis and m proteinprimed t cells may recognize these shared epitopes in skin via molecular mimicry. to identify candidate epitopes, peptides with sequences from k17 were selected on the basis of predicted binding to hla-cw6 and sequence similarities with m6 protein. matched peptides from the sequence of m6 protein and a set of peptides with poor predicted binding were also selected. cw6 þ individuals with psoriasis and cw6 þ healthy controls, having a family history of psoriasis, were recruited. pbmcs were incubated with the peptide antigens. t-cell activation in the cd4 þ , cd8 þ and later the skin-homing cutaneous lymphocyteassociated antigen (cla)-expressing subset of cd8 þ t cells was evaluated by cd69 expression and intracellular ifn-g accumulation using flow cytometry. we demonstrate that cw6 þ psoriasis patients had significant cd8 þ t-cell ifn-g responses to peptides from k17 and m6 protein selected on the basis of sequence homology and predicted hla-cw*0602 binding. these responses were about 10 times more frequent in the skin-homing cutaneous lymphocyte-associated antigen-expressing (cla þ ) subset of cd8 þ t cells. cd4 þ t cells showed only borderline responses. cd8 þ t cells from cw6 þ nonpsoriatic individuals responded to some m6 peptides but very rarely to k17 peptides, and this also applied to the cla þ cd8 þ subset. these findings indicate that psoriatic individuals have cd8 þ t cells that recognize keratin self-antigens and that epitopes shared by streptococcal m protein and human keratin may be targets for the cd8 þ t cells that infiltrate psoriatic skin lesions. autoantibodies directed against citrulline-containing proteins have an impressive specificity of nearly 100% in ra patients and a suggestive involvement in the pathogenesis. the targeted epitopes are generated by a post-translational modification catalysed by the calcium-dependent enzyme peptidyl arginine deaminase that converts the positively charged arginine to polar but uncharged citrullin. the aim of this study was to analyse the presence of citrulline in the joints at different time points of collagen-induced arthritis in da rats by immunohistochemistry and to investigate how immunogenicity and arthritogenicity was affected by citrullination of rat serum albumin (rsa) and collagen type ii (cii). our results indicate that citrulline could be detected in joints of arthritic animals, first appearance at the onset of disease and increasing as disease progressed into a chronic state. unimmunized animals or time points before clinical signs of arthritis were negative. by morphology, we state that some infiltrating macrophages as well as the cartilage surface stain positive for citrulline, while the major source of citrullinated proteins appears to be fibrin depositions. a specific cit-rsa t-cell response was observed in animals challenged by citrullinated rsa, no response was recorded when rsa was used as a stimulus. the igg analysis reveals not only a response towards the modified protein but also cross-reactivity to native rsa. no t-cell or b-cell response was noted in animals injected with unmodified rsa. cit-cii induced a disease with higher incidence and earlier onset than did the native counterpart. we conclude that, in contrast to the human disease, citrulline does not seem to appear before clinical signs. as inflammation proceeds, citrulline is detected specifically in the joints. all other organs investigated were negative. we also conclude that citrullination of a protein can break tolerance and increase its arthritogenic properties. ectopic germinal centers (gcs) can be detected in the salivary glands of approximately 1/5 of patients with sjögren's syndrome (ss) and appear in both primary and secondary ss. previously, ectopic gc have been associated with increased local autoantibody production. the aim of this study was to determine whether gc in primary sjögren's syndrome (pss) defines a distinct seroimmunological phenotype. retrospectively, a material of 130 haematoxylin and eosin-stained paraffin-embedded tissue sections of minor salivary gland tissue from patients with pss was morphologically screened for the presence of ectopic gc. gc-like lesions were detected in 33/130 (25%) of the pss patients. seventy-two pss patients lacking these structures (gc-) were randomly selected for comparison. focus score was significantly increased in the gc þ patients compared to the gcpatients (p ¼ 0.035). in the gc þ group, 54.5% of the patients presented with anti-ro/ssa compared to 43.7% in the gcgroup. anti-la/ssb was detected in 31.3% of the gc þ patients compared to 25.7% of the gcpatients. sixty-one percentage of gc þ patients presented with increased levels of igg, a nonsignificant difference when compared to 39.4% in the gcpatients (p ¼ 0.089). levels of rf, ana, ena, igm and iga were similar in both patient groups, as were esr and crp. in conclusion, patients with ectopic gc have a higher focus score and more often present with autoantibodies and increased levels of igg compared to pss patients with regular focal infiltration (gc -). our findings may indicate a certain seroimmunological phenotype and warrant for further prospective studies. association between mannose-binding lectin and vascular complications in type 1 diabetes complement activation and inflammation have been suggested in the pathogenesis of diabetic vascular lesions. we investigated serum mannose-binding lectin (mbl) levels and polymorphisms in the mbl gene in type 1 diabetic (t1dm) patients with and without diabetic nephropathy and associated macrovascular complications. polymorphisms in the mbl gene and serum mbl levels were determined in 199 t1dm patients with overt nephropathy and 192 t1dm patients with persistent normoalbuminuria matched for age, sex and duration of diabetes as well as in 100 healthy control subjects. the frequencies of high and low expression mbl genotypes were similar in patients with t1dm and healthy controls. high mbl genotypes were significantly more frequent in diabetic patients with nephropathy than in the normoalbuminuric group, and the risk of having nephropathy, given a high mbl genotype, assessed by odds ratio was 1.52 (1.02-2.27), p ¼ 0.04. median serum mbl concentrations were significantly higher in patients with nephropathy than in patients with normoalbuminuria [2306 mg/l (iqr 753-4867 mg/l) versus 1491 mg/l (iqr 577-2944), p ¼ 0.0003], and even when comparing patients with identical genotypes, serum mbl levels were higher in the nephropathy group than in the normoalbuminuric group. patients with a history of cardiovascular disease had significantly elevated mbl levels independently of nephropathy status [3178 mg/l (iqr 636-5231 mg/l) versus 1741 mg/l (iqr 656-3149 mg/l), p ¼ 0.02]. the differences in mbl levels between patients with and without vascular complications were driven primarily by pronounced differences among carriers of high mbl genotypes (p < 0.0001). our findings suggest that mbl may be involved in the pathogenesis of microvascular and macrovascular complications in type 1 diabetes and that determination of mbl status might be used to identify patients at increased risk of developing these complications. neuroimmunology unit, center for molecular medicine, karolinska institutet, stockholm, sweden. e-mail: judit.wefer@cmm.ki.se dna vaccine coding for the encephalitogenic peptide mog 91-108 protects lew.1av1 from subsequent development of experimental autoimmune encephalomyelitis (eae). protection is associated with a type 1 immune response and is dependent on the presence of cpg dna motifs. the mechanisms underlying the observed reduction of eae development in protected rats have not been fully clarified. we investigated immunological characteristics of lymphocytes after dna vaccinaton and subsequent eae induction. we confirm that protection was not associated with suppression of t1 cells, as transcription of the novel molecule rat t-cell immunoglobulin-and mucindomain-containing molecule (tim-3), reported to be exclusively expressed on differentiated t1 cells, was not altered by dna vaccination. we did not note any clonal deletion upon tolerization, but detected an antigen-specific lymphocyte population upregulating ifng upon recall stimulation 3 weeks after protective dna vaccination. in protected rats, we observed (1) no alterations in antigenspecific th2 or th3 responses, (2) reduced mhc ii expression on splenocytes early after eae induction, (3) antigen-specific upregulation of ifnb upon recall stimulation and (4) reduced il-12rb2 on lymphocytes. we thus demonstrate an association of the protective effect of dna vaccination with expression of ifnb. we are currently investigating the cellular mechanisms behind this ifnb-mediated protection. multiple sclerosis (ms) is an autoimmune condition characterized by degeneration of nerve fibre myelin sheets. a candidate autoantigen, myelin basic protein (mbp), has especially attracted attention. the presence of anti-mbp antibodies is a predictor of definite ms, but their role in the pathogenesis remains obscure. t cells have long been known to play a pivotal role in the pathogenesis of ms. recently, an important role for b cells as autoantigen-presenting cells has been demonstrated in other autoimmune diseases, including rheumatoid arthritis and diabetes. the uptake of mbp by b cells and the presentation of mbp-derive peptides to t helper (th) cells by b cells may be promoted by the formation of complement (c) activating immune complexes (ics) between mbp and natural autoantibodies in healthy individuals and disease-associated anti-mbp antibodies in ms patients, respectively. we have investigated the formation of mbp-containing ic, the binding of mbp to b cells, the mbp-elicited induction of th-cell and b-cell proliferation and the cytokine production in peripheral blood mononuclear cells (pbmcs) from healthy donors grown in the presence of intact or c-inactivated serum from healthy donors or patients with ms. while mbp did not induce measurable proliferation of b cells nor cd4 þ t cells, we observed the production of tnf-a, ifn-g and il-10 by pbmc in response to incubation with mbp in the presence of sera from healthy controls as well as sera from ms patients. by contrast, no production of il-2, il-4 and il-5 was detected. we are currently investigating the capability of ms sera to promote the formation of mbp-containing ic and thereby enhance the cytokine responses, by virtue of elevated anti-mbp contents. the phagolysosomally localized acid sphingomyelinase (asmase) activated by proinflammatory cytokines such as tnf and ifn-g generates the signalling molecule ceramide which in turn results in the activation of proteases like cathepsin d. these characteristics of asmase suggest a possible role of this molecule in the phagocytotic uptake and phagosomal degradation processes of antigens or in antigen presentation. we show here that asmase -/mice fail to eliminate the noncytopathic lymphocytic choriomeningitis (lcm) virus as rapidly as littermate wildtype mice. investigation of the immune response revealed a reduced expansion of cd8 þ t cells. the secretion of ifn-g in response to contact with target cells as well as the cytolytic activity of virus-specific cd8 þ t cells was severely impaired. additionally, both phases of the lcm virus-specific dth response, mediated by cd8 þ and cd4 þ t cells consecutively, were diminished in asmase -/mice. however, the secondary memory response of virus-specific ctl was not altered, and the 614 abstracts ................................................................................................................................................................................................. virus was effectively controlled for at least 3 months by asmase -/mice. in conclusion, the results of this study suggest an involvement of the asmase in the activation, expansion or maturation of virus-specific cd8 þ t cells during the acute infection of mice with the lcm virus. novel markers for alternative activation of macrophages: macrophage galactose-type c-type lectins 1 and 2 in parallel with the th1/th2 dichotomy, macrophages are capable of developing into functionally and molecularly distinct subpopulations, due to differences in, for example cytokine environment and pathological conditions. while the best-studied, classically activated macrophage is induced by type i stimuli such as ifn-g, a type ii cytokine environment antagonizes the classical activation of macrophages and is capable of alternatively activating macrophages. however, molecular markers associated with these type ii cytokine-dependent, alternatively activated macrophages remain scarce. besides the earlier documented markers macrophage mannose receptor and arginase 1, we recently demonstrated that murine alternatively activated macrophages are characterized by increased expression of fizz1 and ym. we now report that expression of the two members of the mouse macrophage galactose-type c-type lectin gene family, termed mmgl1 and mmgl2, is induced in diverse populations of alternatively activated macrophages, including peritoneal macrophages elicited during infection with the protozoan trypanosoma brucei or the helminth taenia crassiceps, and alveolar macrophages elicited in a mouse model of allergic asthma. we also demonstrate that, in vitro, interleukin-4 and interleukin-13 upregulate mmgl1 and mmgl2 expression and that, in vivo, induction of mmgl1 and mmgl2 is dependent on interleukin-4 receptor signalling. moreover, we show that regulation of mgl expression is similar in human monocytes and monocyte-derived macrophages. hence, macrophage galactose-type c-type lectins represent novel markers for both murine and human alternatively activated macrophages; thus, paving the way for further characterization of the phenotype of macrophages occurring in th2 conditions. background: human parvovirus b19 (b19) is a ubiquitous pathogen, normally causing a mild self-limiting disease, but also capable of causing both significant pathology and long-term persistence. the small size and stability of the virus makes it suitable for mapping of the full breath and the kinetics of the cellular immune responses following acute viral infection. methods: five patients with acute primary b19 infection were included in the study and followed consecutively for up to 200 weeks. cellular immune responses were mapped by ifng enzyme-linked immunospot to overlapping peptides spanning the whole b19 genome. results: in all five acutely infected patients, we were able to monitor the kinetics of a strong specific cellular immune reaction. responses peaked at levels of 850-1850 sfc/ million pbmcs, roughly corresponding to 0.3-0.6% b19specific cd8 þ cells circulating in peripheral blood at 10-80 weeks post-infection. the responses in individual patients were directed to three or four different peptide pools, and the specificity was confined to the same cd8 epitopes present in the pools throughout the follow-up period. the majority of responses were directed to the virus nonstructural protein, only two patients showed any response to the capsid proteins, elicited by the same epitope in both cases. conclusion: the cellular immune responses to acute b19 infection are surprisingly narrow in distribution and remain at high levels for up to 80 weeks post-infection. the initial epitope specificity is maintained, and the majority of responses target the virus nonstructural protein, which is not included in vaccine preparations, evaluated against the infection. the relationship between malnutrition and malaria is controversial. on one hand, malaria may cause malnutrition, while on the other, malnutrition itself may modulate susceptibility to the disease. we investigated the association between plasmodium falciparum malaria and malnutrition in a cohort of children living on the coast of kenya. the study involved longitudinal follow-up for clinical malaria episodes and anthropometric measurements at four cross-sectional surveys. we used poisson regression analysis to investigate the association between malaria and nutritional status. compared to baseline (children with a waz or haz score of !à2), the crude incidence rate ratios (irrs) for malaria in children with low haz or waz scores (<à2) during the period prior to assessment were 1.17 (95% ci 0.91-1.50; 0 ¼ 0.21) and 0.94 (0.71-1.25; 0.67), respectively, suggesting no association between malaria and the subsequent development of pem. however, we found that age was acting as an effect modifier in the association between malaria and malnutrition. the irr for malaria in children 0-2 years old who were subsequently characterized as wasted was 1.65 (1.10-2.20; p ¼ 0.01), and a significant overall relationship between malaria and low-haz was found on regression analysis when adjusting for the interaction with age (irr 1.89; 1.01-3.53; p < 0.05). although children living on the coast of kenya continue to suffer clinical episodes of uncomplicated malaria throughout their first decade, the association between malaria and malnutrition appears to be limited to the first 2 years of life. a. astrinidou-vakaloudi, 1 s. xytsas, 1 i. diamanti, 1 h. ioannidis 2 & p. pangidis 2 1 microbiology department of general hospital of thessaloniki 'agios pavlos', thessaloniki, greece, and 2 nefrology, 2 nd ika hospital of thessaloniki, thessaloniki, greece. e-mail: stasa@hol.gr aim: renal dysfunction may influence the colonization of gastric mucosa by urea-splitting bacteria such as helicobacter pylori, by increasing urea concentrations in the gastric juice. our aim was to investigate the prevalence of h. pylori in patients with end-stage renal disease (esrd), receiving long-term haemodialysis treatment. methods: this study included 40 sera from patients with esrd (29 male and 11 female) undergoing periodic haemodialysis; mean time of treatment was 42.6 months. using elisa technique, we investigated the presence of igg and iga antibodies against h. pylori as well as igg caga (antibodies specific for caga(þ) strains of h. pylori). sera from 40 healthy blood donors were used as a control group. results: h. pylori igg antibodies were detected in 32 out of 40 (80%) patients in the dialysis group, while 31/40 (77.5%) tested positive for iga. igg caga antibodies were present in 13 out of 40 (32.5%). prevalence of h. pylori igg, iga and caga igg antibodies in the control group was 33, 7 and 15%, respectively. conclusions: although international data suggest that prevalence of h. pylori infection is the same in esrd patients as in healthy individuals, in our study that seems not to be the case. the higher blood and gastric juice urea levels may be a risk factor (among many others), but more studies are required in order to understand the relation of h. pylori infection in this group of patients. flanders interuniversity institute for biotechnology, department of molecular and cellular interactions, free university of brussels, brussels, and 2 pasteur institute of brussels, mycobacterial immunology, brussels, belgium. e-mail: tgartner@vub.ac.be immunity against tuberculosis (tb), caused by mycobacterium tuberculosis, depends largely on activation and maintenance of strong cell-mediated immune responses involving both cd4 þ and cd8 þ t cells and the ability to respond with th1-type cytokines, particularly ifn-g. recent studies suggested that bcg, the only licensed vaccine against m. tuberculosis, may fail to induce t-cell responses in the lung mucosa and may therefore not protect against pulmonary tb. a decrease in tb mortality may be achieved by enhancing immunity in the lung. the present study evaluated the induction of antigen-specific immunity in the lung by intranasal (i.n.) delivery of the lipoprotein i (opri) from pseudomonas aeruginosa. opri has shown to be a toll-like receptor 2/4 agonist that, when given subcutaneously, induces type-1 immune responses against heterologous antigens. here, a fusion of opri to ag85a of mtb (opri-ag85a) was used as a subunit vaccine in homologous prime-boost immunizations. in addition, opri-ag85a was combined with an ag85a-encoding dna vaccine (ag85a dna) or with bcg in heterologous prime-boost vaccinations. intranasal and parenteral delivery with opri-ag85a elicited comparable t-cell responses in the spleen; in addition, i.n. delivery elicited specific t-cell responses in the lung lymph nodes (llns). intramuscular delivery of ag85a dna induced significant systemic th1 immune responses. intranasal boosting with opri-ag85a enhanced this response and in addition induced an antigen-specific ifn-g response in lln. opri may therefore be an efficient adjuvant for mucosal boosting. we continue to evaluate the protection induced by opri-based prime-boost vaccinations against pulmonary tb. results on the immunogenicity and protection against intravenous mtb h37rv infection will be presented. toll-like receptors (tlrs) are pattern recognition receptors of the innate immune system, which recognize molecular structures on pathogens or cellular stress-associated molecules. tlr-ligand interactions trigger activation of inflammatory signal transduction and expression of genes involved in host defense. in this study, we have examined the requirement for different tlr adaptor molecules in virus-induced chemokine expression and are currently trying to identify the tlr involved. we have found that both a herpesvirus [herpes simplex virus (hsv)] and a paramyxovirus (sendai virus) require a functional genome to induce expression or proinflammatory chemokines in human and murine monocytic cell lines. for both viruses, this is independent of the tlr adaptor molecules trif and mal. however, overexpression of the vaccinia virus-encoded inhibitor of tlr-signalling a52r or dominant-negative myd88 totally inhibited hsv-induced rantes expression but only partially prevented sendai virus from inducing this chemokine. this suggests that hsv-induced rantes expression occurs via a tlr pathways, whereas sendai virus utilizes both tlr-dependent and -independent pathways to stimulate expression of rantes. we are currently trying to identify the tlrs involved. data from these studies will also be presented at the meeting. 2 0 -5 0 -oligoadenylate synthetases are interferon-induced, double-stranded rna-activated antiviral enzymes which are the only proteins known to catalyse 2 0 -specific nucleotidyl transfer. this first crystal structure of a 2 0 -5 0oligoadenylate synthetase reveals a structural conservation with the 3 0 -specific poly(a) polymerase that, coupled with structure-guided mutagenesis, supports a conserved catalytic mechanism for the 2 0 -and 3 0 -specific nucleotidyl transferases. comparison with structures of other superfamily members indicates that the donor substrates are bound by conserved active site features while the acceptor substrates are oriented by nonconserved regions. the 2 0 -5 0oligoadenylate synthetases are activated by viral doublestranded rna in infected cells and initiate a cellular response by synthesizing 2 0 -5 0 -oligoadenylates, that in turn activate rnase l. this crystal structure suggests that activation involves a domain-domain shift and identifies a putative dsrna activation site that is probed by mutagenesis. we demonstrated that this site is required both for the binding of dsrna and for the subsequent activation of oas. this rna-binding site is different from known rna-binding site; rather than forming a defined three-dimensional domain, it is located at the interface of the two major domains in oas. this novel architecture ensures that the dsrna helix can make simultaneously contact with both domains of oas and ensure the subsequent structural rearrangement leading to the activation of oas. our work provides structural insight into cellular recognition of double-stranded rna of viral origin and identifies a novel rna-binding motif. bacteria-specific iga antibodies are efficient opsonins for neutrophils and mononuclear phagocytes, provided that the phagocytes express the fca receptor (cd89). expression of cd89 can be stimulated by inflammatory cytokines, activated complement factors and certain microbial components. in one study, unstimulated phagocytes were able to ingest iga antibody-treated pneumococci, but only in the presence of complement, which was found to be activated by the iga antibodies along the alternative pathway. pneumococci produce iga1 protease that cleaves human iga1, but not iga2, molecules in the hinge region. this leaves iga1 as faba (monovalent) deprived of fca which contains the docking site for cd89. iga1 is the vastly predominant subclass of iga in the upper airways and circulation of humans. aims: to examine the effects of iga1 protease activity and complement on phagocytosis of iga antibody-coated pneumococci by an unstimulated human phagocytic cell line (hl60). materials and methods: iga1 and iga2 monoclonal antibodies to serotype 4 pneumococcal capsular polysaccharide (ps) were generated by heterohybridoma technique involving b cells from human vaccinees. isogenic serotype 4 pneumococci with and without iga1 protease activity, respectively, were obtained after inactivation of the iga gene of the tigr4 strain. opsonophagocytosis was quantitated using the assay described by romero-steiner et al. based on enumeration of surviving bacteria by culture. the integrity of iga molecules was examined by western blotting. results: both iga1 and iga2 antibody to type-4 polysaccharide-induced phagocytosis of iga1 protease-deficient type-4 pneumococci equally well in the absence as in the presence of complement. iga1 antibody to type-4 polysaccharide displayed a fourfold higher opsonophagocytosis titer against iga1 protease deficient compared to homologous wildtype target bacteria. a similar effect of iga1 protease activity of the target bacteria was not observed in a parallel experiment where iga2 antibody to type-4 polysaccharide served as opsonin. iga1 antibody extracted from iga1 protease-producing target bacteria was almost exclusively in the form of faba. conversely, iga1 from protease-deficient bacteria and iga2 from both types of bacteria were intact. conclusions: these results indicate that the iga1 protease activity of s. neumoniae may help the bacteria escape iga1 antibody-mediated opsonophagocytosis. besides, in these experiments, iga-mediated opsonophagocytosis was independent of complement. vitamins e and c have been found to increase the cellular and humeral immunity of pigs. vitamin e deficiency has also been found to predispose pigs to different diseases, e. coli infection is one among them. after weaning, the vitamin e status of pigs often decreases to a critical low level. in this experiment, we studied whether vitamin c supplementation would be a possible feeding strategy to optimize the immune status of weaners. the interaction between vitamin e and c is interesting due to the reported sparing action on vitamin e or synergism between these to vitamins. piglets were weaned at day 28 of age from sows fed increasing dietary vitamin e during lactation, and piglets were during the following 3 weeks fed either a control diet or this diet supplemented with 500 mg stay-c per kg. blood sampling was obtained weekly from day 28 and until day 49 of age. on the same days, one piglet per dietary treatment was killed and alveolar macrophages (am) were harvested. vitamin c supplementation increased the concentration of igm in serum of piglets throughout the weaning period. although the vitamin e concentration in am decreased with increasing age of the piglets, the concentration was numerically higher in piglets of sows fed the high dietary level of vitamin e. however, vitamin c supplementation tended to increase the total am concentration of vitamin e after weaning and increased the proportion of the biologically most active isomer of vitamin e [rrr-(a-tocopherol)] in the am. the eicosanoid synthesis by am was not influenced by the vitamin c supplementation, but the synthesis of leukotriene b4 was decreased 2 weeks after weaning compared to other days of am harvesting. in conclusion, dietary vitamin c supplementation improved the immune responses of piglets after weaning. a whole blood stimulation assay with escherichia coli (o111:b4) endotoxin was established to measure the capacity of dairy cows to produce the proinflammatory cytokine tumour necrosis factor-a (tnf-a) ex vivo. initially, a time-and dose-dependent study was carried out to find the optimal stimulation conditions for the tnf-a response. the tnf-a response peaked between 3 and 4 h at 38.5 c. a dose in the range of 5-10 g of e. coli lipopolysaccharide (lps)/ml whole blood was found to give the maximum tnf-a response. thirty-eight danish-holstein dairy cows were investigated for their tnf-a responsiveness ex vivo in the periparturient period. heparin-stabilized blood samples were collected seven times over a period of 4 months (weeks à3, à1, 2, 3, 5, 9 and 13 around calving) and stimulated with 5 g/ml of e. coli lps. indeed, fluctuations in the tnf-a responsiveness occurred over time. moreover, the mean tnf-a responsiveness of 38 cows was found to be significantly increased (p < 0.001) in the weeks close to calving. however, in the more stabile physiological periods, some cows had a consistently low tnf-a response, whereas others had high a tnf-a response. we are currently investigating whether high and low tnf-a responders to e. coli lps also exist in dairy cows in vivo. moreover, the importance of tnf-a responsiveness ex vivo to dairy cows' susceptibility and clinical response to experimental e. coli infections in the udder is being investigated. coelomic cytolytic factor (ccf) is a 42 kda invertebrate pattern recognition molecule isolated from the coelomic fluid of the earthworm eisenia foetida (oligochaeta, annelida). ccf displays a number of similarities with the mammalian cytokine tumour necrosis factor-a (tnfa) as a result of a shared n,n 0 -diacetylchitobiose lectin-like domain. however, these similarities are solely functional and are not based on any (dna or amino acid) sequence homology, thus suggesting a form of convergent evolution. in particular, the lectin-like domain of tnf-a has been shown to induce membrane depolarization in various mammalian cell types, through interactions with endogenous amiloride-sensitive ion channels. this nonreceptor-mediated activity of tnf-a has been reported to be involved in the resorption of oedema. likewise, the lectin-like domain of ccf also induces membrane depolarization in mammalian cells. here, we show that ccf appears to be able to induce oedema resorption in an alveolar epithelial cell line through its lectin-like domain. this lectin-like domain of ccf interacts (directly or indirectly) with endogenous sodium and/or chloride channels, and not potassium channels, on mammalian cells. additionally, we suggest that the jnk/sapk and erk1/2 pathways are involved in ccf-induced macrophage activation. these results further establish the functional analogy between an invertebrate pattern recognition molecule and a mammalian cytokine and, from a more applied point of view, suggest the possibility of utilizing ccf in the treatment of oedema. release of svegf and svegfr1 from white blood cells and platelets during surgery and stimulation with bacterial antigens introduction: the influence of surgery on release of soluble vascular endothelial growth factor (svegf) and the soluble vascular endothelial growth factor inhibitory receptor 1 (svegfr1) is unknown. we studied the effect of major and minor surgery on potential variations in svegf and svegfr1 concentrations in vivo and on bacterial antigen-induced release of svegf and svegfr1 from whole blood in vitro. methods: sixty-one patients with abdominal diseases undergoing five different surgical procedures were included. blood samples were drawn from anaesthetized patients before and after the operation. white blood cells and platelets were counted, and plasma svegf and svegfr1 was determined by an elisa method. whole blood from each blood sample was stimulated in vitro with bacteria-derived antigens (lps or protein-a) and svegf and svegfr1 levels were subsequently determined in the supernatants. stimulation with isotonic saline served as control assay. neither svegf or svegfr1 in plasma changed during surgery. in vitro stimulation of blood samples with bacteria-derived antigens resulted in a significant increase in svegf (p < 0.0001) and a less pronounced but still significant increase in svegfr1. release of svegf due to stimulation was significantly higher after the operation (nonsignificant), whereas svegfr1 release remained largely unchanged after surgery. correlation between bacterial antigen-induced release of svegf and neutrophile cell count was highly significant (p < 0.0001). there was no correlation between svegf and platelet cell count, and bacterial antigen-induced svegfr1 release did not correlate with counts of neutrophils and platelets. conclusions: plasma svegf and svegfr1 concentrations did not change during surgery. in vitro bacterial stimulation led to increased release of svegf and svegfr1, which was not significantly amplified during surgery and which may be related to number of circulating neutrophils. natural killer cell functions and subsets after in vitro stimulation with il-2 and il-12, with special emphasis on intracellular ifn-g and nk-cell cytotoxicity r. nyboe, 1,2 t. rix, 1,2 j. krog, 1,2 e. tønnesen 1 & m. hokland 2 1 department of anaesthesiology and intensive care, aarhus university hospital, and 2 institute of medical microbiology, and immunology, university of aarhus, aarhus, denmark. e-mail: rnsr@studmed.au.dk materials and methods: isolated cryopreserved human peripheral blood mononuclear cells (pbmcs) were stimulated with il-2 and il-12. this stimulation has previously been shown to activate nk cells. cell cytotoxicity was measured by flow cytometry after incubation with k562 cells. this method was compared to the current standard 51cr release assay. cells were treated with bfa to accumulate ifn-g, stained for surface markers, permeabilized and stained for intracellular ifn-g. flow cytometry was then performed to measure intracellular ifn-g production in pbmc, especially in nk cells. results: we have demonstrated that stimulation with il-2 and il-12 is effective in increasing the number of ifn-gpositive cells. there is a distinct difference between the cd3-cd56dim and the cd3-cd56bright subsets, with a much greater proportion of ifn-g-positive cells in the cd3-cd56bright subset. the effects of stimulation with il-2 and il-12 on cytotoxicity will be presented, as will the relation between ifn-g production and cytotoxicity. in addition, we will present results of these assays applied to porcine cells. discussion: in combination, these tests will address nk cell function by combining cytotoxicity with ifn-g production in nk cell subsets. the results will demonstrate whether this could serve as a useful tool in describing nkcell function, which could be of value in clinical and experimental settings. culture of regulatory t-cell lines from bronchial mucosa t lymphocytes play a major role in many immune responses. in the last decade, special focus has been on the function of th1 and th2 effector cells. now the importance of regulatory cd4 þ cd25 þ t cells in maintenance of the immunological homeostasis emerges. sarcoidosis is a multisystem granulomatous disorder often affecting the lungs. the typical sarcoid granulomas consists of epitheloid cells, macrophages and lymphocytes, mainly cd4 þ t cells of th1 phenotype. we have cultured t cells from bronchial biopsies of patients with sarcoidosis as well as from controls in high levels of interleukin 2 (il-2) and il-4 and demonstrate spontaneously arising cd4 þ cd25 þ populations and high concentrations of il-10 in these cultures. the main difference between cultures of sarcoid origin compared to controls is a very much higher concentration of the inflammatory cytokines il-6 and tnf-a in cultures of sarcoid origin. the effects of hyperbaric exposure on human peripheral blood mononuclear cells, with special emphasis on natural killer cell cytotoxicity and subsets materials and methods: as an experimental physiological stress model, we examined the effects of hyperbaric exposure on peripheral blood mononuclear cells (pbmcs) obtained from venous blood drawn from eight divers during a simulated heliox saturation dive. eight persons working in normobar atmosphere outside the pressurized chamber served as control donors. the spontaneous cytotoxicity of the pbmcs was estimated in a 4 h 51cr-release assay using k562 as nk-sensitive target cells. the pbmcs were characterized, using 4-colour flow cytometry, with special emphasis on the nk-cell subsets. the data were statistically analysed using a multivariate regression model (stata 8.2). p values <0.05 was considered statistically significant. results: the estimated cytotoxicity increased significantly in both the group of divers and control donors during the dive (pdivers < 0.01 and pcontrols < 0.01). although the cytotoxicity increased relatively more (p < 0.01) in the group of divers compared to the group of control donors between day 1 and 2. discussion: the increased cytotoxicity of pbmc estimated in the group of divers indicate that parts of the cellular immune system are affected during the extreme physiological conditions induced during the initial phase of the presented experimental hyperbaric setup. the increase in cytotoxicity observed in the group of control donors could hypothetically reflect the stress level in persons working outside the pressurized chamber during the dive. the interaction with adhesion molecules expressed by vascular endothelium is the first step in lymphocyte infiltration into tissues. at both cutaneous and mucosal sites interleukin-10 (il-10), il-12 and transforming growth factor (tgf)-b are important regulators of chronic inflammatory disease, where cutaneous lymphocyteassociated antigen (cla) and ae integrin (cd103) may be expressed. unlike cla, cd103 is not believed to play a role in tissue-specific homing but may help to retain t cells within epithelial layers. we have previously shown that il-12 alone can together with an unknown cofactor increase the expression of cla. stimulation with streptococcal pyrogenic exotoxin c (spec) increased the expression of cd103 by cd8 þ but not cd4 þ t cells. while il-12 increased superantigen-stimulated expression of cla, this cytokine strongly inhibited the cd103 expres-sion, and a combination of il-12 and tgf-b completely abrogated the induced cd103 expression. conversely, il-10 suppressed cla but increased cd103 expression. these findings indicate that, in addition to suppressing the development of th1-mediated inflammatory responses, il-10 may also inhibit the migration of cd8 þ t cells into the skin while il-12 promotes such migration. thus, the expression of cla and cd103 may be antagonistically regulated by il-10 and il-12, and the balance between these cytokines could influence the t-cell migration of inflammatory cells into epithelial tissues. during contact sensitivity reaction, immune cells proliferate. in order to study the histological picture of these proliferation phases, we used a mouse model of contact sensitivity in the oral mucosa and on skin. we also used bromodeoxyuridin (brdu, an analogue to thymidin) that is incorporated into the nucleus during cell replication. the hapten oxazolone (oxa) was used to sensitize and elicit the oral mucosa and/or the ear skin. mice were killed at various times after elicitation, and unsensitized animals were also exposed to the hapten as controls. brdu (25 mg/ kg animal) was injected i.p. 2 h before the kill. specimens from the oral mucosa, ear skin and submandibular and auricular lymph nodes were cut and fixed in 4% paraformaldehyde. they were then treated with acid and biotinylated anti-brdu antibody and developed using abc-kit and dab. the analyses were performed using a leica light microscope and the computer program analysis. in the oral mucosa, the frequency of proliferating cells were increasing during the observation period, 4-24 h after elicitation, regardless of site of sensitization. the proliferating cells were found mainly in the basal cell layer of the epithelium. similar patterns were found in ear skin. the regional lymph nodes demonstrated a few scattered proliferating cells 4 h after elicitation. after 24 h, these cells were found frequently in the whole lymph node. control animals exhibited considerable less proliferating cells at all times. we conclude that most proliferating cells were found 24 h after elicitation locally at the hapten-exposed sites (the oral mucosa or the ear skin) as well as in the regional lymph nodes. the endogenous nucleoside adenosine is released in excess during inflammation or other metabolic stress and is generally known to deliver tissue protective anti-inflammatory effects. adenosine acts via four adenosine receptors of which the a2a receptor is the predominant form in t cells. adenosine levels are elevated in asthmatic lung, and adenosine can directly induce mast cell degranulation and bronchoconstriction in these patients. instead, the role of anti-inflammatory mechanisms of adenosine on t cells in asthma is unclear. aim: to study the a2a receptor expression in peripheral blood cd4 þ t cells in asthmatic and healthy individuals using flow cytometric and quantitative real-time pcr methods. results: unstimulated cd4 þ cells of asthmatic patients expressed significantly lower levels (p < 0.001) of a2a receptor in protein level (mean percentage of cells positive ae sem: 76.8 ae 1.2, n ¼ 6) compared to healthy individuals (90.4% ae 1.9, n ¼ 4). double staining for cd69 expression showed that stimulation of cd4 þ cells decreased a2a expression in both groups but indicated that the detected lower levels of a2a in unstimulated cells of asthmatics was not due to preactivation in these patients. surprisingly, a2a mrna expression in unstimulated cd4 þ cells was significantly higher (p < 0.05) in asthmatics (n ¼ 28) compared to healthy controls (n ¼ 7). the expression did not correlate with serum total ige levels. conclusions: asthmatic individuals express less a2a adenosine receptor on their peripheral cd4 þ t cells. the higher mrna levels instead may point to a negative feedback regulation in the receptor expression. the role of possibly decreased adenosine-mediated anti-inflammatory effects in asthma pathogenesis require further studies on this t-cell mediated disease. the chronic inflammatory skin disease atopic eczema (ae) affects almost 15% of the population in many countries today. the pathogenesis of ae is not fully understood. a combination of genetic predisposition and environmental factors like microorganisms seems to contribute to the symptoms. the yeast malassezia sympodialis is part of our normal skin micro flora but can act as an allergen and elicit specific ige and t-cell reactivity in patients with ae. recently, we identified a novel major m. sympodialis allergen, designated mala s 11 (22.4 kda), with sequence similarity to the mitochondrial enzyme manganese superoxide dismutase (mnsod). interestingly, mala s 11 has a high degree of homology to human mnsod. the aim of this study was to examine the effects of recombinant mala s 11 on antigen-presenting dendritic cells. monocytederived dendritic cells (mddcs) from healthy blood donors were cultured with or without mala s 11 for different time periods. it was found that the maturation marker cd83 and the costimulatory molecules cd80 and cd86 were upregulated on the mddcs exposed to mala s 11 for 24 h, as demonstrated by flow cytometry. furthermore, coculture of mddcs with mala s 11 for 9 h induced an increased production of the inflammatory cytokines il-6 (200-fold), tnf-a (100-fold) and il-8 (sixfold), as detected by the cytometric bead array (cba) analysis. our results suggest that mala s 11 affects the immune response through dc maturation and production of inflammatory cytokines. the potential cross-reactivity with human mnsod needs to be explored and the exact role of mala s 11 in the pathogenesis of ae assessed in clinical studies involving skin prick and atopy patch tests. allergen-specific immunotherapy (sit) is commonly conducted with allergen extracts adsorbed to aluminium hydroxide (alum). drawbacks linked to the use of alum, such as the formation of granuloma at the site of injection, have led to suggestions of novel allergen carriers. an alternative carrier is 2 mm carbohydrate-based particles (cbps). in mouse, allergen-coupled cbps have been demonstrated to skew the allergen-specific immune response towards a th1-like activity (grönlund et al. immunology, 2002) . we here coupled the recombinant major cat allergen fel d 1 to cbps (cbp-fel d 1) by cyanogen-bromide activation, resulting in covalent binding. the effect of cbp-fel d 1 on monocyte-derived dendritic cells (mddcs) from healthy human blood donors was studied. we found that the majority of the cd1a þ mddcs were capable of taking up fitc-labelled cbp-fel d 1, as demonstrated by flow cytometry and confocal laser scanning microscopy. furthermore, incubation with cbp-fel d 1 resulted in an upregulation of the costimulatory molecule cd86 on the mddcs, which was not observed with fel d 1 or cbps alone. finally, cbp-fel d 1 induced a fivefold increase in the release of the pro-inflammatory cytokine tumour necrosis factor (tnf)-a and a fourfold increase in the release of the chemokine interleukin-8 from mddcs. taken together, the effects cbps possess make them interesting as novel allergen carriers for sit. the cysteine protease der p1 from dust mite of the genus dermatophagoides pteronyssinus is a major type i allergen. about 80% of house dust mite (hdm) allergic individuals are reactive to this protease in standard assays for detection of ige. a curative treatment for atopic allergy is immunotherapy (it) with hdm extracts which are complex mixtures occasionally resulting in anaphylactic reactions. novozymes focuses on developing a recombinant variant of der p1 which exhibit lowered risk of ige-mediated allergic reactions, while maintaining its ability to trigger proper th-cell responses. this may provide a safer alternative for specific it of hdm allergy. a secreted recombinant form of pro-der p 1 expressed by saccharamyces cerevisiae was obtained by fusion of the pro-enzyme to a fungal signal peptide. the n-glycosylation site of der p1 was mutated resulting in a deglycosylated pro-enzyme with a molecular mass of 35 kda. protein purification procedure was developed to obtain nearly pure der p1 protein followed by determination of concentration by active-site-titration with the cysteine protease inhibitor e64. the deglycosylated recombinant pro-der p 1 revealed immunologic similarity to the native der p 1 molecule when compared in basophile histamine release, ige-binding assays and t-cell proliferation assays. by in silico epitope mapping of a modelled 3-dimensional structure of der p1, five putative igg and ige epitopes were predicted. by protein engineering, the predicted epitopes were removed one by one in der p1 and screening for hypoallergenic variants was performed. combining inhaled long-acting b-2 agonist (laba) and inhaled corticosteroid (ics) seems to offer asthma control at a lower dose of ics than achieved by ics alone. fine mapping of t-cell surface markers by flow cytometry offers a detailed status of the individual's inflammatory response. the frequency of mt2 (cd4 þ cd45ra -cd62l þ cd11adim) and mt1 (cd4 þ cd45ra -cd62l -cd11abright) cells in peripheral blood, and their ratio, has been shown to differ predictably in atopics and patients with leprosy, where mt2 correlates with a th2 phenotype and mt1 with a th1 phenotype. stable asthmatics, requiring fluticasone propionate (fp) 750-1000 mg daily or equivalent, were randomized to receive, double-blinded, either seretide 1 [salmeterol and fluticasone propionate (sfc, n ¼ 16)] 50 mg/500 mg bd or fp 500 mg bd (n ¼ 17). if asthma was controlled based on lung function and symptoms at clinic visits every 6 weeks, ics dose was tapered until asthma exacerbated or 0 mg was reached. the frequency and ratio of mt2 and mt1 t cells of the patients was monitored at 6 week intervals. as treatment tapered, the frequency of mt2 cells decreased (p ¼ 0038 from first to final visit), whereas that of mt1 cells increased. the ratio of mt2/mt1 decreased (p ¼ 0049 from first to final visit). in patients receiving laba þ ics, the fall in mt2/mt1 ratio appeared to be more pronounced than in patients receiving ics alone. thus, the mt2 phenotype may be associated with stable asthma, whereas an imminent exacerbation may associate with an increase in the mt1 phenotype. laba may allow for a greater effect of fp on the mt ratio. activation of complement pathways, leading to production of c3a and c5a anaphylatoxins, has been postulated in the pathogenesis of asthma and allergic airway inflammation. the present study was undertaken to investigate the role of mannan-binding lectin (mbl), an initiator of the lectin pathway of complement, in asthma and allergic rhinitis. mbl levels and mbl-induced complement activity were determined in 19 patients of bronchial asthma with allergic rhinitis and 20 unrelated, age-matched controls of indian origin. mbl levels and activity were correlated with percent eosinophilia and percent predicted fev1 values of the patients. association of single nucleotide polymorphisms (snps) in exon 1 and intron 1 of the mbl with the disease, clinical markers, mbl levels and mbl-induced complement activity was analysed using standard statistical tools. significantly higher mbl levels and activity were observed in patients of bronchial asthma with allergic rhinitis as compared to the controls. we identified five snps, of which two, a816g in exon 1 and g1011a in intron 1 of the mbl, were novel. snp g1011a was significantly associated with the disease (p ¼ 0.0024, or ¼ 5.8696, 95% ci: 1.7316 < or < 19.8963). individuals with 'a' allele at position 1011 showed increased mbl levels, activity and disease severity. our results suggest that 'a' allele at position 1011 leading to high mbl levels and complement activity may be contributing to the severity of bronchial asthma and allergic airway inflammation. serum resistance of borrelia burgdorferi strains belonging to the b. afzelii and b. burgdorferi sensu stricto genospecies is dependent on binding of complement inhibitor factor h. we recently reported that factor h binding by b. burgdorferi is due to inducible expression of several approximately 20 kda plasmid-encoded, surface-exposed lipoproteins related to ospe (e.g. erpa, erpp and p21). in addition, a second class of factor h-binding proteins of approximately 27-35 kda has been described. the ospe-related lipoproteins are dramatically induced by b. burgdorferi during transmission from its tick vector into the mammalian host. the induction of ospe-related lipoproteins during mammalian infection may play a key a role in the borrelial evasion of the host's immune system. the goal of the present study was to define the factor h-binding regions of ospe-related proteins using mutagenesis, peptide mapping and surface plasmon resonance analysis (biacore). the combined studies revealed that the c-terminal regions of both human and mouse factor h (scrs 18-20) specifically bind to ospe-related lipoproteins. we also found fhr-1, whose c-terminal scrs 3-5 are homologous to scrs 18-20 of factor h, to bind to ospe. peptide mapping revealed five putative regions (designated i-v) in ospe that could directly interact with factor h. deleting the c-terminal 15 amino acid residues from region v of p21 abolished its ability to bind factor h. at the same time, however, synthetic peptides corresponding to the c-termini of ospe, p21 and erpp did not inhibit factor h binding to ospe. thus, the c-terminal-binding region v appears to be necessary but not sufficient for factor h binding. when a more specific mutation strategy was employed, where single amino acid residues in peptides spanning over the factor h-binding regions were mutated to alanines, we observed that lysines in the factor h-binding regions of ospe were required for factor h binding. the combined data have revealed that key lysine residues in ospe-related lipoproteins and ionic interactions are crucial for factor h interactions. furthermore, binding of ospe to the c-termini of both mouse and human factor h suggests that borrelia spirochetes utilize analogous complement resistance mechanisms in both rodents and man. in borrelia garinii strains, which in in vitro analyses have been found to be sensitive to complement killing, differences in the ospe sequences as well as in the expression of factor h-binding proteins may account for their susceptibility to serum lysis. role of yada, ail and lipopolysaccharide in serum resistance of yersinia enterocolitica serotype o:3 mannan-binding lectin (mbl), l-ficolin and h-ficolin are pattern recognition molecules of the innate immune system. we investigated the ability of these molecules to bind to different serotypes and noncapsulated variants of streptococcus pneumonia and staphylococcus aureus. we found that mbl binds to noncapsulated s. aureus strain (wood) but not any of the examined s. pneumoniae serotypes. l-ficolin binds to some capsulated s. pneumoniae serotypes (11a, 11d and 11f) as well as some capsulated s. aureus serotypes (type-1, -8, -9, -11 and -12). h-ficolin does not bind to any of the examined s. pneumoniae and s. aureus serotypes included in this study but did bind to a strain of aerococcus viridans. when bound to bacteria, mbl and h-ficolin initiated activation of complement factor c4, whereas l-ficolin did not. during this study, quantitative assays for the three proteins were developed and the concentration in 97 plasma samples were determined and the median values were estimated at 0.8 mg of mbl/ml, 3.3 mg of l-ficolin/ml and 18.4 mg of h-ficolin/ ml, respectively. the absence of early complement components (c1, c4 and c2 but not c3) is a predisposing factor for systemic lupus erythematosus (sle). recently, we demonstrated that, in c4-deficient (c4 def.) mice, igm-containing immune complexes (igm-ic) are filtered by the splenic barrier of marginal zone macrophages (mzm), resulting in an increased immune response against antigens within these igm-ic, but this could not be observed in wildtype or c3 def. mice. we hypothesized that splenic cd11b þ mzm play an important role in the induction of autoimmunity, and we therefore analysed their cytokine profile after isolation with the help of magnetic antibody cell sorting. mrna was isolated, and real-time pcr was performed with specific primers for murine ifn-g (ifn-g), interleukin-12 (il-12) and ifn-a (ifn-a). we observe a moderate increase of il-12 and ifn-g mrna in cd11b þ cells of c4 def. mice compared to wildtype cells. surprisingly, the concentration of ifn-a mrna is six times higher in c4 def. mice. preliminary results suggest that mrna in cd11b þ cells of c3 def. mice is even lower than that in wt. six hours following i.v. application of 20 mg of a abstracts 625 .................................................................................................................................................................................................. murine monoclonal igm anti-dsdna antibody, production of il-12, ifn-g and ifn-a mrna is increased in cd11b þ cells of both c4 def. and wt mice. several references described increased levels of inf-a in patients with sle. dendritic cells are discussed as a major source of ifn-a. our observation that c4-deficient, sle-susceptible mice demonstrate an increased spontaneous ifn-a production by splenic cd11b þ marginal zone macrophages could be an early sign and a trigger for the development of sle. this is supported by the fact that the absence of c3 is not a predisposing factor for sle and our observation that c3 def. animals display low levels of ifn-a mrna. 200-400 million people worldwide and represents one of the leading causes for liver cirrhosis and hepatocellular carcinoma. control over the hbv infection is achieved mainly by vaccination with hepatitis b surface antigen (hbsag). hbsag contains n-linked glycosylation side and is recognized by both mbl-a and mbl-c in a cadependent manner. hbsag-mbl complexes activate complement and may thus affect humoural immunity. to investigate the role of mbl in humoural responses to hbsag, we immununized mice that lack both mbl-a and mbl-c proteins with soluble hbsag. it has been shown that deficiencies in other complement components like c1q, c4 and c3 result in decreased antibody responses. however, mbl double ko animals mounted dramatically increased humoural responses. after priming, mbl double kos mounted hbsag-specific igm responses, which were threefold higher than wt controls. after boosting the hbsag, total igg was 10-fold higher in mbl ko than in wt control animals. similar to the response to hbsag, other glycosylated soluble antigens (e.g. invertase) induced better humoural responses in mbl double ko animals, suggesting that mbl plays an important role in a negative feedback regulation of adaptive immunity. reconstitution experiments with rmbl partially rescued the ko phenotype. we propose that the clearance of glycoprotein antigens in mbl ko is handled differently from the wt, resulting in better stimulation of humoural responses. alternatively, glycoprotein-ag-mbl-rich complexes inhibit b-cell responsiveness via putative mbl receptors. the complement system is an important part of the innate immune system. the activation of complement proceeds through three different pathways that converge in the generation of c3-activating enzyme complexes. complement activation via the lectin pathway is initiated when recognition molecules, mannan-binding lectin (mbl) or ficolin, bind to carbohydrate structures characteristic for microbial surfaces. in the circulation, mbl and ficolins are found in association with three structurally related mblassociated serine proteases (masp)-1, -2 and -3 and a small, nonenzymatic component, map19. masp-2 has been shown to elicit complement activation through the sequential proteolytic cleavage of c4 and c2 upon binding of mbl/masp-2 complexes to microbial surfaces. we have recently uncovered a polymorphism in the masp-2/map19 gene in a patient shown to be deficient in the lectin pathway of complement activation. the polymorphism results in a single amino acid substitution in the n-terminal part of the masp-2 protein. recombinant wildtype masp-2 and masp-2 containing the amino acid substitution in question was produced, and the ability to activate complement was studied. the mutation had a profound impact on masp-2 function, resulting in the lack of complement activation through the lectin pathway. elisa-based experiments showed that the mutation leads to the impairment of complement activation through influencing the binding of masp-2 to mbl or ficolins. deficiencies in the lectin pathway of complement activation have so far been accounted for only by lack of functional mbl. the mutation described above is the first defect described affecting both activation through mbl and the ficolins. .................................................................................................................................................................................................. th1, th2 and treg cell balance. dcs are present in the gut mucosa and may thus be target for modulation by gut microbes, including ingested probiotics. here, we tested the hypothesis that species of lactic acid bacteria, important members of the gut flora, differentially activate dc. a large panel of human gut-derived lactobacillus and bifidobacterium spp. was screened for dc-polarizing capacity by exposing bone marrow-derived murine dc to lethally irradiated bacteria. cytokines in culture supernatants and dc-surface maturation markers were analysed. substantial differences were found among strains in the capacity to induce interleukin-12 (il-12) and tumour necrosis factor (tnf)-a, while the differences for il-10 and il-6 were less pronounced. bifidobacteria tended to be weak il-12 and tnf-a inducers, while both strong and weak il-12 inducers were found among the strains of lactobacillus. remarkably, strains weak in il-12 induction inhibited il-12 and tnf-a production induced by an otherwise strong cytokine-inducing strain of lactobacillus casei, while il-10 production remained unaltered. selected strains were tested for induction of dc maturation markers. those lactobacilli with greatest capacity to induce il-12 were most effective in upregulating surface mhc class ii and cd86. moreover, l. casei-induced upregulation of cd86 was reduced in the presence of a weak il-12inducing l. reuteri. in conclusion, human lactobacillus and bifidobacterium spp. polarize differentially dc maturation. thus, the potential exists for th1/th2/treg-driving capacities of the gut dc to be modulated according to composition of gut flora including ingested probiotics. the intestinal micro flora is indispensable in developing and maintaining homeostasis of the gut-associated immune system. evidence indicates that lactic acid bacteria (lab), e.g. lactobacilli and bifidobacteria, have beneficial effects on the host. established health effects include increased gut maturation, antagonisms towards pathogens and immune modulation. the objective of this study is to evaluate the immunomodulating properties of a range of lab of human origin. as dendritic cells (dcs) play a pivotal role in the balance between tolerance and immunity to commensal microorganisms, in vitro-generated immature dcs serve as a suitable model for studying the immunomodulating effects of lab. human immature dcs were generated in vitro from monocytes and exposed to lethally uv-irradiated lab. the effect of various species of lab on dcs in direct contact was evaluated. furthermore, the maturation pattern of dcs separated from the bacteria by an epithelial cell layer (caco-2 cells), which should mimic the intestinal environment, was studied. cytokine secretion (il-12, il-10 and tnf-a) and upregulation of maturation surface markers on dcs (cd83 and cd86) was measured. different lab induced diverse cytokine responses. some strains were strong il-12 and tnf-a inducers and others weak. all strains induced il-10. different lab also differentially modulated expression of cd83 and cd86 on dcs. although some variation in the response to lab of dcs generated from different blood donors was observed, general differences in the effect of the various lab was revealed. experiments with the dc caco-2 coculture system are ongoing. different species of lab differentially affect dc maturation; this suggets that the gut flora plays a pivotal role in polarization of the immune response. natural killer (nk) cells are cells of the nonspecific immune system lysing altered self-cells. a noncytolytic subset of nk cells may serve a regulatory role by secreting cytokines. bacteria translocating across the gastrointestinal mucosa are presumed to gain access to nk cells, as consumption of certain lactic acid bacteria has been shown to increase in vivo nk cytotoxicity. here, we investigated how human gut flora-derived lactobacilli affect nk cells in vitro, by measuring proliferation and ifn-g production of human nk cells upon bacterial stimulation. cd3 -cd56 þ nk cells were isolated from buffy coats by negative isolation using non-nk lineage-specific antibodies and magnetic beads. nk cells were incubated with 10mg/ml uv-inactivated bacteria or 10mg/ml phytohemagglutinin (pha) for 4 days. proliferation was assessed by incorporation of radioactive thymidine into nk-cell dna. the ifn-g concentration was measured by elisa. incubation of nk cells with a lactobacillus acidophilus strain increased the proliferation of the nk cells and induced ifn-g production, both to levels comparable to pha stimulation. the proliferative response was further enhanced with autologous monocytes present, probably because cytokines, secreted by monocytes having engulfed bacteria, stimulated the nk cells. in contrast, a lactobacillus paracasei strain caused the nk cells to proliferate only in the presence of monocytes. these results demonstrate that various strains of lactobacilli have the capacity to activate nk cells in vitro, in a monocyte-dependent or -independent way. hence, the encounter of nk cells with lactic acid bacteria will affect nk-cell activation. such activation of nk cells may potentially skew an on-going or subsequent immune response towards a th1 response. lactobacilli are nonpathogenic gram-positive inhabitants of the normal human intestine known for their healthpromoting effects. in our earlier work, it is shown that human monoclonal antibody isolated from sera of a patient with waldenstrom macroglobulinaemia possess innate antibody characteristics and binds to lactic acid bacteria. according to the immune network model, immunization with this bacteria could induce the perturbations in immune system that might result in production of anti-lactobacillus antibodies, human monoclonal antibody like (ab1) and anti-idiotypic antibody (ab2). in this study, balb/c mice were immunized with two doses of bacteria lactobacillus acidophilus in complete and incomplete freund's adjuvant and phosphate-buffered saline (pbs), respectively. seven days after the last immunization, sera from immunized mice were collected and the presence of lactobacillus-specific ab1 and ab2 were determined by elisas. in the sera of immunized mice, antibodies specific to bacteria lactobacillus acidophilus were shown. the concentration of lactobacillus-specific antibodies was higher in the sera of hyperimmunized mice (mice immunized with 1 mg of igm dj) than in sera of mice immunized with 100 times lower doses of immunogen (0.01 mg per doses). moreover, ab1 and ab2 antibodies were detected in the sera of lactobacillus-hyperimmunized mice. in this study, we have shown the idiotypic network interactions in mice immunized with bacteria lactobacillus acidophilus. the normal gastrointestinal flora is crucial for the maturation of the acquired immunity via effects on antigenpresenting cells (apcs). here, we have investigated how two types of apcs, monocytes and dendritic cells (dcs), react to different bacterial strains typical of the commensal intestinal flora. purified monocytes and monocyte-derived dcs were stimulated with uv-inactivated gram-positive (lactobacillus plantarum and bifidobacterium adolescentis) and gram-negative (escherichia coli and veillonella parvula) bacterial strains. monocytes produced higher levels of il-12p70 and tnf, as detected by elisa, in response to l. plantarum than to e. coli and v. parvula. in contrast, dcs secreted high amounts of il-12p70, tnf, il-6 and il-10 in response to e. coli and v. parvula but were practically unresponsive to l. plantarum and b. adolescentis. the lack of response to the gram-positive strains correlated with a lower surface expression of toll-like reseptor 2 (tlr2) on dcs compared to monocytes. the surface expression of tlr4 on dcs was undetectable when analysed by flow cytometry, but blocking this receptor decreased the tnf production in response to v. parvula, indicating that low tlr4 expression on dcs is sufficient to mount an inflammatory response to gram-negative bacteria. ifn-g increased the expression of tlr4 on dcs and also potentiated the cytokine response to gram-negative bacteria. our results indicate that, when monocytes differentiate into dcs, their ability to respond to different commensal bacteria dramatically changes, thereby becoming unresponsive to probiotic gram-positive bacteria. these results may have important implications for the capacity of different groups of commensal bacteria to regulate mucosal and systemic immunity. probiotic bacteria, e.g. lactobacillus spp., may improve diseases such as chronic inflammatory bowel disease. we examined cytokine production and phenotypic change after in vitro stimulation of t cells from healthy volunteers using different probiotic strains. methods: t cells were cultured from colonic biopsies from eight healthy volunteers (agnholt and kaltoft, exp clin immunogenet 2001; 18:213-25) , and dendritic cells were matured from their peripheral blood mononuclear cells. t-cell cultures were stimulated with autologous bacterial sonicate or strains of lactobacillus spp., with and without the addition of dendritic cells. cytokine levels (tnf-a, ifn-g, il-10 and gm-csf) and phenotype (cd3, cd4, cd25 and cd69) were measured on day 4. results: lactobacillus spp. induced higher productions of tnf-a and il-10 than did autologous bacteria. in presence of dendritic cells, the production of all cytokines increased. however, the increases of ifn-g and tnf-a were more pronounced in wells with autologous bacteria than in wells with lactobacillus spp. the addition of dendritic cells upregulated cd25 expression without simultaneous upregulation of cd69. the upregulation was pronounced after stimulation with lactobacillus rhamnosus gg compared with autologous bacteria and other lactobacilli. discussion: in presence of dendritic cells, autologous bacteria induced inflammatory cytokines, while probiotics mainly induced regulatory cytokines. lactobacillus rhamnosus gg induced a regulatory phenotype (cd25 þ ), in part mediated by dendritic cells. future studies will address whether this shift to a cd25 þ phenotype represents a differentiation into competent regulatory t cells. in a clinical context, such cells might be used for treatment of inflammatory diseases. protein microarrays will play a key role in the postgenomic era and offer a unique possibility to perform highthroughput global proteome analysis. a chip can be printed with thousands of protein probes (e.g. antibodies), the biological sample added (e.g. a proteome) and any binding detected. we aim to develop protein microarrays based on human recombinant scfv antibody fragments for global proteome analysis. the concept of comparing proteomic maps of healthy versus diseased samples will allow diseasespecific proteins to be detected. in fact, antibody microarrays will allow us to perform comparative proteome analysis on any sample format in a species-independent manner, as long as a proteome can be isolated. however, the complexity of proteomes, containing several thousands of different proteins, is a problem. here, we have designed antibody microarrays targeting the water-soluble fraction of a proteome. to this end, an anticytokine antibody array was developed and human dendritic cells (aeactivation) was used as model system. the results showed that our antibody microarrays could be used to examine the cytokine profile in complex samples. furthermore, we have taken the first steps towards comparing our results with those of other technologies on both the protein and gene level. due to the complexity of the model proteome, we also examined the possibility to prefractionate the proteome in a simple one-step procedure (based on size) prior to the labelling step. in more detail, the sample proteome was fractionated into two fractions using membrane devices with different molecular weight cut-offs. the results showed that the fractionation considerably enhanced the assay sensitivity allowing cytokines in the pg/ml range to be readily detectable. the immunomodulatory effect of heat shock protein 70: immunization with a dna construct based on the malarial antigen fused with a fragment of hsp 70 primes for a th-1 type of response finding an appropriate adjuvant for human vaccination is crucial. heat shock proteins (hsps) act as adjuvants when coadministered with peptide antigens or given as fusion proteins. however, there is a potential risk of autoimmunity when using the complete molecules, because hsps are evolutionary conserved. to overcome this, we first evaluated the adjuvant effect against two different antigens of a less-conserved fraction of plasmodium falciparum hsp70 (pf70c) and compared it to the whole hsp70 molecule from trypanosoma cruzi (tchsp70). we found that pf70c exhibited similar adjuvant properties as the whole molecule. we later evaluated the adjuvant potential of pf70c against the malarial antigen eb200 in a chimeric dna construct. no appreciable levels of eb200-specific abstracts 629 .................................................................................................................................................................................................. antibodies were detected in mice immunized only with the dna constructs. however, dna primed the immune system, because subsequent challenge with the corresponding recombinant fusion proteins elicited a strong th-1 antibody response. in contrast, no priming effect was observed for ex vivo ifn-g production but stimulation with the hsp-chimeric fusion protein induced a stronger secretion of ifn-g in vitro than other proteins used. these results indicate that the use of hsps is promising in the design of new vaccines. high-throughput proteomics on antibody-based microarrays: the importance of probe and surface design in analogy to dna microarrays, protein microarrays offer a new distinct possibility to perform sensitive highthroughput global proteome analysis. however, the development of the protein microarray technology will place high demands upon the design of both probes and solid supports. the analysis of thousands of heterogeneous proteins on a single microarray requires the use of uniform probes, such as antibodies, directly designed for protein microarray applications. we have recently generated a human recombinant single-chain fv antibody library, genetically constructed around one framework, the ncoder-library, containing 2 â 1010 clones. single framework antibody fragments (sinfabs) selected from this library were successfully applied as probes for microarrays providing sensitive detection in the 600 attomol (mass spectrometry) and the 300 zeptomole range (fluorescence). however, the choice of framework is critical. we have shown that the selected ncoder framework displayed excellent functional on-chip stability and arrayed dehydrated probes retained their activity for several months. furthermore, we have addressed the issues of biocompatibility of the solid support and immobilization strategies for our microarray setup. an in-house-designed substrate, macroporous silicon coated with nitrocellulose (map3-nc7), displayed properties equal to, or better than, those of five commercially available supports used as reference surfaces. we have also evaluated different coupling strategies, such as adsorption, covalent coupling, diffusion and affinity coupling. using a novel affinity tag, the double-(his)6-tag, we increased the binding efficiency of sinfab-molecules to ni2 þ -coated solid supports, thereby allowing nonpurified probes to be directly applied. the mannan-binding lectin (mbl) pathway is part of the innate immune system providing a first line of defence against infections. mbl and ficolins circulate in complexes with mbl-associated serine proteases (masp-1, -2 and -3). after recognition of a microorganism by mbl, activation of the complement system occurs. masp-1 and masp-3 share five domains (making up the so-called a-chain), whereas they have unique protease domains (b-chains). before the identification of masp-3, an assay for masp was presented, based on antibodies against the a-chain of masp-1. with the new knowledge of the three masps, and the sharing of domains by masp-1 and masp-3, assays specific for the protease domains have to be constructed, if one wishes to measure the proteins individually. we present an assay for quantifying total masp-3 in plasma and serum samples. the assay is a sandwich-type assay using as catching antibody a monoclonal antibody against the common a-chain of masp-1/3 and a developing secondary antibody against the c-terminal part of the protease domain of masp-3. we have used this assay for estimating the normal concentration of the protein as well as the concentration in patients and also for characterizing by gel permeation chromatography the masp-3 protein in serum. inducible costimulator ligand (icosl) is a costimulatory molecule related to b7.1 (cd80) and b7.2 (cd86). b cells, monocytes, dendritic cells and endothelial cells express icosl. inducible costimulator (icos) interacts with icosl, and this interaction leads to signals involved in isotype switching and the development of immunological memory. hitherto, no polymorphisms of this gene have been described. the aim of this study was to reveal variation of the icosl gene in normal individuals. all eight exons, except exon 1, were sequenced with flanking introns in 10 healthy blood donors. eight single nucleotide polymorphisms (snps) and two length polymorphisms were found. one of the snps was found in the coding regions of the gene. the base involved was located in exon 3 and caused a conservative amino acid change from valine (gtt) to isoleucine (att). three individuals were heterozygous g/a for the exon polymorphism, while the remaining seven individuals were homozygous for the wildtype g/g. exon 3 encodes the immunoglobulin variable (igv)-like domain of the molecule which is situated outside the cell. this means that the amino acid could be critical for the stability of the molecule or could constitute part of the binding site for icos. the results form the basis for further experiments to find possible associations of the alleles to diseases caused by immune dysregulation. especially, the exon 3 variant is interesting and could play a role for the development of immunological diseases. besides, it would be interesting to see whether both exon 3 alleles are expressed or only the wildtype allele is functional. interferon-a/b (ifn-a/b) is increasingly used as antiviral and immunomodulatory therapies. unfortunately, bioavailability varies with ifn species and mode of administration, and all ifn species are potentially immunogenic. assays for antiviral activity (ifn) and antiviral neutralization (antibodies, nab) have been used for some time to monitor patients on ifn biologicals. these assays require laborious titrations making them unsuitable for large-scale clinical use. myxovirus a (mxa) is a resistance gtpbinding protein that is specifically induced by treatment with type 1 ifns. for example, ifn-b-induced mxa in blood leucocytes has been used as a biomarker in ifn-btreated patients with multiple sclerosis. however, the degree of specificity of mxa in this regard is unclear, and measurements of mxa protein and/or mrna are not yet suitable for routine clinical use. in an attempt to find new and better reporter genes (and, hopefully, genes and gene products with proven specificity for ifn-a and -b), microarray screenings with u133a genechips (affymetrix) were carried using human blood leucocytes and the human lung carcinoma cell line a549. we studied the simultaneous expression of 22,000 transcripts before and after exposure to human recombinant ifn-a and ifn-b and other antiviral and immunomodulatory cytokines. the results will be presented at the conference. interferon-a/b (ifn-a/b) is increasingly used as antiviral and immunomodulatory therapies. unfortunately, bioavailability varies with ifn species and mode of administration, and all ifn species are potentially immunogenic. assays for antiviral activity (ifn) and antiviral neutralization (antibodies, nab) have been used for some time to monitor patients on ifn biologicals. these assays require laborious titrations making them unsuitable for large-scale clinical use. our laboratories have therefore modified the antiviral assays for ifn bioactivity and nab, so that they are suitable for large-scale screening in specialized laboratories. the read-out is survival of a subcloned a549 cell line in the presence of an otherwise lethal amount of virus. thus, survival increases in the presence of type 1 ifn and decreases in the presence of nab against the ifn added to the cells. mxa is induced by type 1 ifn and can be used for measuring the nab activity. in another assay, the mxa level in the a549 cell line is measured. in an attempt to find a new and better reporter gene for type 1 ifn than mxa and genes specific for either ifn-a or -b, a micro array screen was carried using the u133a chip from affymetrix. the expression of 22,000 genes can be studied simultaneous with this technology. the results will be presented at the conference. in our laboratory, we have developed a database system, which we believe is of immediate interest to the general scientific community. the database represents a computerbased replacement for the laboratory notebooks used in the majority of research laboratories worldwide. in addition, the database provides an effective tool for organizing and managing laboratory information at all levels, spanning from managing and revising standard operating procedures and producing documentation of research activities to keeping track of data and conclusions. using the commercially available database toolkit software filemaker pro, we have developed a relational database solution for management of laboratory information. the system consists of a hierarchy of five interrelated databases, each pertaining to a separate type of information, namely, overall project information, information relating to individual experiment setups, documentation of daily research activity, generated data and descriptions of standard operating procedures. like other databases, each individual database consists of a number of records, each comprised of a set of fields in which information is entered. in each record, a certain field is reserved to specify the relation of the record to a record in another database at a higher level. thus, the database is essentially five databases linked by a hierarchy of one-to-many relations, organizing information in a folder-like structure. importantly, the database system allows multiple users to access and edit records simultaneously, and the data entered in one database immediately becomes accessible through the other databases. the limitations of laboratory notebooks are apparent when looking for information, which is dispersed throughout one or more notebooks, or possibly on loose sheets of paper or printouts 'somewhere'. the often complicated process of gathering laboratory data or results when writing grant applications or research papers is made considerably easier with the database system. thus, the database solution presented should be broadly attractive to researchers, irrespective of their scientific discipline. an effective sars vaccine is likely to include components that can induce specific cytotoxic t-cell (ctl) responses. the specificities of such responses are governed by hlarestricted presentation of sars-derived peptide epitopes. exact knowledge of how the immune system handles protein antigens would allow for the identification of such linear sequences directly from genomic/proteomic sequence information. the latter was recently established when a causative coronavirus (sars cov) was isolated and full-length sequenced. here, we have combined advanced bioinformatics and high-throughput immunology to perform an hla supertype, genome-wide scan for sars-specific cytotoxic t cell epitopes. the scan includes all nine human hla supertypes in total covering >99% of all major human populations. for each hla supertype, we have selected the 15 top candidates for test in biochemical-binding assays. at this time (approximately 6 months after the genome was established), we have tested the majority of the hla supertypes and identified almost 100 potential vaccine candidates. these should be further validated in sars survivors and used for vaccine formulation. we suggest that immunobioinformatics may become a fast and valuable tool in rational vaccine design. rationale: major histocompatibility complex class i (mhc i) molecules monitor the protein content of the cell by binding small derived peptides and presenting them to cytotoxic cd8 þ t cells. the goal of the human mhc project is to predict the binding strength of any given peptide/mhc complex. this prediction allows the design of peptide-based vaccines. the prediction requires representative binding data from mhc alleles from all the nine hla supertypes. here, we describe the genetic construction, protein production and purification as well as the establishment-binding assays for two recombinant mhc supertype alleles, hla-b*1501 and hla-b*5801. methods: using the quikchange multisite directed mutagenesis kit (stratagene), codon-optimized genes encoding hla-b*1501 and hla-b*5801 are created. the two mhc i molecules are fermented and purified by ion exchange chromatography, hydrophobic interaction chromatography and size exclusion chromatography. the binding (kd) of natural t-cell epitopes, as well as predicted peptide ligands, is described by radioactive immunoassays (rias) and enzyme-linked immunosorbent assays (elisas). the mhc molecules are biotinylated during expression. results: the expression of mhc i resulted in multiple disulfide bond isomers, which are separated by hydrophobic interaction chromatography and used in subsequent binding studies resulting in the determination of kd for various peptide ligands ranging from strong binders we have previously demonstrated that bioinformatics tools such as artificial neural networks (anns) are capable of performing pathogen-, genome-and hlawide predictions of peptide-hla interactions. these tools may therefore enable a fast and rational approach to epitope identification and thereby assist in the development of vaccines and immunotherapy. a crucial step in the generation of such bioinformatics tools is the selection of data representing the event in question (in casu peptide-hla interaction). this is particularly important when it is difficult and expensive to obtain data. herein, we demonstrate the importance in selecting information-rich data and we develop a computational method, query-bycommittee, which can perform a global identification of such information-rich data in an unbiased and automated manner. furthermore, we demonstrate how this method can be applied to an efficient iterative development strategy for these bioinformatics tools. methods: a large panel of binding affinities of peptides binding to hla a*0204 was measured by a radioimmunoassay (ria). this data was used to develop multiple first generation anns, which formed a virtual committee. this committee was used to screen (or 'queried') for peptides, where the anns agreed ('low-qbc'), or disagreed ('high-qbc'), on their hla-binding potential. seventeen low-qbc peptides and 17 high-qbc peptides were synthesized and tested. the high-or low-qbc data were added to the original data, and new high-or low-qbc second generation anns were developed, respectively. this procedure was repeated 40 times. the high-qbc-enriched ann performed significantly better than the low-qbc-enriched ann in 37 of the 40 tests. conclusion: these results demonstrate that high-qbcenriched networks perform better than low-qbc-enriched networks in selecting informative data for developing peptide-mhc-binding predictors. this improvement in selecting data is not due to differences in network training performance but due to the difference in information content in the high-qbc experiment and in the low-qbc experiment. finally, it should be noted that this strategy could be used in many contexts where generation of data is difficult and costly. interleukin-18 (il-18), a pro-inflammatory cytokine that is produced by both lymphoid and nonlymphoid cells, has a critical role in modulation of innate and adaptive immunity. its primary function in stimulation of ifn-g production and stimulation of nk-cell-cytotoxic activities makes this cytokine a candidate for cancer immunotherapy. in oral cavity, this cytokine is produced by oral epithelia and carcinoma cells and is related to tumour regression in nude mice bearing salivary adenocarcinoma. however, direct effects of this cytokine on oral cancer cells have not been elucidated. in this project, we investigated il-18 effect on an oral carcinoma (kb) cell line. with rt-pcr technique, kb-cell line was found to express il-18 receptors (il-18ra and il-18rb), indicating that this oral carcinoma line is a target for il-18 study. we showed that recombinant human il-18 inhibited kb-cell proliferation by 17% at concentration of 100 ng/ml (p < 0.05), whereas ldh release by these cells in treatment group and control groups was comparable, indicating that il-18 suppression of cell proliferation was not mediated by the induction of cell death. to further address this hypothesis, we found that il-18 treatment did not induce apoptotic cell death, as studied by dna laddering and tunel assays. in addition, expression pattern of cell death-controlling genes (bcl-2 and bax) was not altered by this cytokine. findings in these studies indicated that suppression of kb-cell proliferation may be attributed to control of cell cycle, growth arrest or induction of cell differentiation. the data presented in this project could provide an insight of how cancer cell directly responds to il-18, as this cytokine is an important regulator of anticancer mechanisms. aloe emodin (ae) is a naturally occurring compound with wide spectrum of biological properties, including antimicrobial, vasorelaxant, immunosuppressive and anticancer actions. this anthraquinone induces apoptosis in several tumour cell lines with special affinity to tumours of neuroectodermal origin. high amounts of nitric oxide (no) released by activated macrophages induce tumour cell death. therefore, we explored the capacity of ae to modulate no-mediated antitumour response in vitro. interestingly, while ae markedly suppressed no release from macrophages alone, it significantly potentiated no production in cocultures of macrophages and c6 cells, after 48 h of cultivation. accordingly, the viability of c6 cells cocultivated with macrophages was reduced in the presence of ae. moreover, the observed ae-imposed potentiation of no production in macrophages was closely related to macrophage culture cell density. according to these data, we proposed that no modulator capacity of ae strongly depended on intercellular contact, indicating that macrophage antitumour response was not compromised but even potentiated by ae. immunotherapy represents an attractive fourth-modality therapeutic approach, especially in the light of the shortcomings of conventional surgery, radiation and chemotherapies in the management of metastatic cancer. to this end, a large number of peptide antigens derived from taa have been applied in immunotherapeutic trials for the treatment of various malignancies, e.g. cancers of the breast, prostate and kidney, in addition to haematological cancers. in some cases the response rates have been impressive and no adverse autoimmunity have been observed. a major strategic difficulty associated with these trials relates to the choice of best-suited peptide antigens. the vast majority of the antigens described thus far is not vital for survival and growth of the tumour cells, and immunoselection of antigen-loss variants may therefore prove to be an additional obstacle for the clinical applicability of most of the known peptide epitopes. in this respect, the development of acquired antigen loss during immunotherapy has been demonstrated in several cases. obviously, the development of loss-variant tumour cells implies that these cells acquire a pronounced growth advantage and are left unaffected by further treatment. ideally, target antigens should be derived from proteins required for survival and growth of tumour cells, as antigens with these characteristics would not be inflicted by the development of loss-variant tumour cells. in this respect, several inhibitors of apoptosis proteins (iaps) are universally expressed among tumours and play an important role in tumour cell escape from apoptosis. we have characterized spontaneous t-cell reactivity against iapderived peptides in cancer patients. from the iap survivin, we have characterized peptides restricted to the class i molecules hla-a1, a2, a3, a11, b7 and b35. furthermore, we have demonstrated that survivin-specific t cells infiltrate metastatic lesions and that isolated survivinspecific ctls are capable of killing hla-matched tumour cells. survivin-derived peptides are now in clinical trial, and continued work in our lab has demonstrated that other iaps are targets for spontaneous t-cell reactivity in cancer patients. we previously reported that in mice with large progressing t-cell lymphoma tumours, dysfunctions in the antitumour ctl activity occur, associated with an accumulation of splenic arginase-producing myeloid suppressor cells (mscs). in this study, we first demonstrate that both the presence and the activation state of these msc depends on tumour evolution. while in tumour regressors hardly any arginase-producing msc can be found, both the amount and the arginase activity of this population expands from early over late progressors. this gradual induction of mscs is paralleled by an increasing suppression of ctl activity and th1, but not th2, cytokine production. upon analysing the molecular repertoire of msc in vitro, we found, besides arginase1, a well-established marker for alternatively activated myeloid cells or m2, a strong upregulation of fizz1 and ym, two additional recently identified markers for m2. further evaluation of molecular markers by microarray analysis in msc yielded genes involved in wound healing (e.g. coagulation factor xiiia), anti-inflammation (e.g. selenoprotein p), immunomodulation (e.g. pd-l2) and fat and sugar metabolism (e.g. leptin receptor). of note, many of these genes are regulated by type 2 cytokines (il-4, il-13 and il-10) and are therefore rather m2 associated. overall, our data provide new markers for msc in cancer and further establish their m2 activation state. study. only sp-a showed a significant expression in normal mucosa which was downregulated in crc. as the absolute signal level was below the noise threshold, these results have to be interpreted with caution and require confirmation by direct measurenment of the proteins. our results suggest that there is no major role for the human collectins in colorectal cancer. tetramerization is visualized by sds-page. conclusion: an effective method for the production of highly pure mhc i molecules has been applied to hla-b*1501 and hla-b*5801, and ria and elisa binding assays for those alleles have been established background: proliferation, differentiation and apoptosis are essential processes in the normal functions of the mammary epithelium. the hypothesis examined in this study is that the transcription factor bcl-6 is critically important not only for regulating b-cell growth and development but also for mammary epithelial apoptosis. methodology: twenty breast cancer cases and 31 healthy controls were used to investigate whether bcl-6 protein in involved in breast cancer (grade iii). full length bcl-6 cdna was retrovirally transduced into eph-4 cell line. we then used flow cytometry of brdurd-stained cells to investigate the cell-cycle duration of the control and transduced cell lines. tunel was used as a marker of apoptosis to find out differences in the frequencies of apoptotic cells in the control and transduced cell lines. finally, immunohistochemistry staining was performed to detect bcl-6 in breast cancer (iii). results: restoration of bcl-6 into eph-4 cells not only inhibits apoptosis but also prolongs the cell cycle and results in increased cell size and protein content. the results also indicated that the cell-cycle time of bcl-6-transduced eph-4 cells is prolonged by about 3 h, presumably as a result of the action of bcl-6 at the bcl-6 at the g1/s transition. we found differences in the frequencies of viable and apoptotic cells in cultures of the parent eph-4 cells, control-transduced eph-4 cells and bcl-6-transduced eph-4 cells. consistently, we demonstrated that bcl-6 is expressed in 90% of high grade of breast carcinoma, which is considered as the most aggressive of tumours. conclusion: together, these results suggest that bcl-6 is likely to be involved in mammary gland development and carcinogenesis. inflammatory cytokines have a critical role in modulation of both innate and adaptive immunity in response to foreign antigen. they also play an important role in anticancer immunity. for example, they can promote cell-mediated immunity against cancer cells. with their immunostimulatory effects, these cytokines are being tested for cancer treatment in the form of dna vaccine or adjuvant or therapeutic cytokines. direct effect of these cytokines on cancer cell, however, is still unclear. in this project, we investigated whether il-1( and il-18 can modulate cancer cell proliferation. we employed a simple nonradioactive proliferation (mtt) assay and detection of lactate dehydrogenase (ldh) to test the effect of these recombinant human cytokines on various cancer cell lines, including breast cancer cell line (mcf-7), oral carcinoma cell line (kb), colon cancer cell line (caco-2) and choriocarcinoma cell line (jar). cytokines used in this study had both inhibitory and stimulatory effect on cell proliferation. findings in this project could provide an insight of cancer cell response to these cytokines and this could lead to a consideration on using cytokine as immunotherapy for cancer treatment.capacity of ae to modulate nitric oxide production depended on intercellular contact donor t cells are involved in the antitumour effects observed after bmt. thus, patients receiving t-celldepleted bmt have a higher risk of leukaemia relapse compared to patients receiving nonmanipulated bmt, and patients experiencing graft-versus-host disease (gvhd) have a lower risk of disease relapse than patients who do not experience gvhd. although the importance of donor t cells for the curative action of bmt has been established, the exact mechanisms and molecules involved in this graft-versus-tumour effect remain largely unknown. in a recently initiated project, we have conducted a longitudinal study of t-cell clonotypes in patients who received peripheral blood stem cell grafts after nonmyeloablative conditioning. peripheral blood samples were obtained sequentially after transplant, and the mononuclear cells (mncs) were isolated and cryopreserved. cd8 þ t cells were isolated from the mncs by use of immunomagnetic beads or facs and analysed for the presence of clonally expanded cells by t-cell receptor clonotype mapping based on rt-pcr and denaturing gradient gel electrophoresis (dgge). using this gel-based methodology, clonally expanded t cells were monitored after transplant and compared to the clinical data of the patients. the preliminary results demonstrates the presence of clonally expanded cd8 þ t cells at all time points analysed. furthermore, a number of clonotypes persisted for more than 6 months, and other clonotypes emerged during this period. the appearance of newly emerged clonotypes which coincided with clinical gvhd could indicate a role for these t cells in the pathogenesis of gvhd. background: deficiency of the mannan-binding lectin (mbl) pathway of innate immunity leads to increased susceptibility to infections. in patients with colorectal cancer, postoperative infection is associated with poor prognosis. the aim of the present study was to evaluate (1) the relation between the mbl pathway and postoperative infectious complications and survival of patients resected for colorectal cancer and (2) the role of mbl as acute phase reactant compared to crp. methods: preoperative mbl concentration, mbl/mblassociated serine protease (masp) activity and crp were determined in serum from 611 patients and 150 healthy controls. the patients were observed for 8 years. postoperative infections, recurrence and survival were recorded. results: the mbl pathway components were increased in the patients (p < 0.0001) compared to healthy controls. low mbl levels were predictive of pneumonia (p ¼ 0.01), and pneumonia (n ¼ 87) was associated with poor survival (p ¼ 0.003, hr ¼ 1.5, 95% ci 1.1-1.9). mbl and mbl/ masp activity could not predict postoperative overall infections. mbl showed no correlation (spearman's r ¼ 0.02, 95% ci à0.06-0.10) with crp. conclusions: low preoperative mbl levels are predictive of pneumonia, which is associated with poorer survival. mbl concentration and mbl/masp activity was not predictive of other postoperative infections or long-term prognosis. mbl apparently is not a surrogate measure of crp. department of surgery, university hospital of erlangen, erlangen, germany. e-mail: michael.siassi@rzmail.uni-erlangen.de introduction: the human collectins, mannan-binding lectin (mbl), surfactant protein-a (sp-a) and surfactantprotein-d (sp-d) play a central role in the innate immune system. immunological responses to malignant transformation of epithelial cells gained increasing interest recently. a former study could demonstrate binding of mbl to certain colorectal carcinoma (crc) cell lines in vitro. we therefore examined the expression of human collectins in normal colon mucosa and in colorectal carcinomas. materials and methods: colon samples from 20 crc patients and 10 normal mucosa samples were collected immediately after surgery. the tissue was microdissected and rna isolated (qiagen, rneasy-kit). gene expression profiles were analysed using gene-chips (affymetrix, hg-u133). we analysed the data for the expression of mbl, its associated serine proteases mannan-binding lectinassociated serine protease 1/2 (masp 1/2), sp-a and sp-d. the signal intensity of the genes of interest was compared using the mann-whitney u-test. results: the expression of human collectins in normal human colon mucosa was generally low. only the expression of sp-a and masp-2 reached the noise threshold of 250 signals. these genes were significantly downregulated in crc specimens. the expression of the other proteins showed no difference in normal mucosa and crc. conclusion: as demonstrated before, the expression of human collectins in normal colon was low in this being the first lymph node to receive drainage from the tumour area, the sentinel node offers a unique possibility to obtain tumour-reactive lymphocytes. we investigated antitumour immune responses in sentinel nodes from patients with bladder cancer, by assaying tumour-specific proliferation and tcr vb repertoires. during tumour surgery, sentinel lymph nodes were identified by peritumoural injection of blue dye. fresh specimens of tumour, sentinel and nonsentinel lymph nodes were obtained, and single-cell suspensions were prepared. cells were assayed for reactivity against autologous tumour extract in [ 3 h]-thymidine incorporation assays and characterized by flow cytometry. parallel analyses of the expression of vb gene families were performed with padlock probes, linear oligonucleotides which upon target recognition can be converted to circular molecules by a ligase. probes were reacted with cdna prepared from magnetically separated cd4 þ cells, and the tcr repertoire was determined by hybridizing the products to oligonucleotide microarrays. dose-dependent proliferation in response to tumour extract could be detected in sentinel lymph nodes. common clonal expansions were detected among tumourinfiltrating lymphocytes and in sentinel lymph nodes. nonsentinel lymph nodes displayed a divergent tcr vb repertoire. these results indicate an ongoing immune response against tumour antigens in sentinel nodes, draining urinary bladder cancer. identification of sentinel lymph nodes makes it possible to obtain tumour-reactive lymphocytes for use in adoptive immunotherapy. key: cord-003130-p2h8p5bm authors: lindqvist, richard; upadhyay, arunkumar; överby, anna k. title: tick-borne flaviviruses and the type i interferon response date: 2018-06-21 journal: viruses doi: 10.3390/v10070340 sha: doc_id: 3130 cord_uid: p2h8p5bm flaviviruses are globally distributed pathogens causing millions of human infections every year. flaviviruses are arthropod-borne viruses and are mainly transmitted by either ticks or mosquitoes. mosquito-borne flaviviruses and their interactions with the innate immune response have been well-studied and reviewed extensively, thus this review will discuss tick-borne flaviviruses and their interactions with the host innate immune response. tick-borne flaviviruses (tbfv), flaviviridae family, includes many pathogens causing severe human disease, ranging from mild fever to encephalitis and hemorrhagic fever. there are more than 70 viruses in the genus flavivirus, and they are transmitted by arthropods such as mosquitoes (dengue virus (denv), japanese encephalitis virus (jev) and west nile virus (wnv), yellow fever virus (yfv), and zika virus (zikv) and ticks (tick-borne encephalitis virus (tbev), langat virus (lgtv), kyasanur forest disease virus (kfdv), omsk hemorrhagic fever virus (ohfv), powassan virus (powv), and louping-ill virus (liv)) [1] [2] [3] [4] [5] . among the tbfvs, tbev, powv, and liv are encephalitic, whereas ohfv and kfdv are hemorrhagic viruses. there are general features that distinguish tbfv from mosquito-borne. one such feature is that tick-borne viruses tend to persist in certain stable foci whereas mosquito-borne viruses may suddenly emerge and they can rapidly spread to new areas and continents causing large epidemics [6] [7] [8] [9] [10] [11] . mosquito-borne flaviviruses are transmitted horizontally from mosquito to vertebrate to mosquito, and for some viruses, humans act as the amplifying host [12] [13] [14] . mosquitoes have a short life compared to ticks, as it can take several years for a tick to develop from egg to adult [15] . ticks also only take one blood meal at each of their life stages, larvae, nymph, and adult, this means that tick-borne viruses need to be maintained in infected ticks during a very long time [16] . thus, replication in ticks needs to be lower compared to mosquitoes, which have higher viral turnover and a faster generation cycle, this also contributes to the high mutation rate in mosquito-borne flaviviruses, and relative stable genomes of tbfvs [17, 18] . infection of tick-borne flaviviruses usually cause a short viremia in humans and larger vertebrates. this route of transmission has been described as less important in tick-borne flavivirus infection [19] , instead tbev has been shown to be transmitted by viremic rodents and among co-feeding ticks without viremia [20] . humans act as dead-end hosts since they do not produce high enough viremia to transmit the virus to new ticks. according to phylogenetic differences, tbev has been divided into three different subtypes, european, siberian, and far eastern. the european subtype is mainly transmitted by ixodes ricinus, whereas the siberian and far eastern subtypes are primarily transmitted by ixodes persulcatus [21, 22] . tbev is found in central, eastern, and northern europe and asia ( figure 1 ) and correlates with the presence of infected ticks. ixodes ricinus is found throughout europe, whereas ixodes persulcatus is found in eastern europe in the west, and china and japan in the east [23] . tbev is considered one of the most important arboviruses in central and eastern european countries and in russia, with about 13,000 estimated human cases annually [24] . in fact, over the last decade there has been an approximately 300% increase in the number of tbe cases in europe [25] , and tbev is currently spreading into new regions in france, sweden, norway, and italy [26] [27] [28] [29] . this increase is thought to be due to growth in population and spread of ticks, which is promoted by factors including climate change, social and political change, and changes in the land use [30, 31] . the increased expansion in europe also poses an increased risk for the population engaged in outdoor activities. tbev is a zoonotic disease and the natural cycle of tbev is dependent and maintained in a complex cycle involving ticks as the vector and reservoir of the virus and small rodents as hosts for ticks [32] . humans are not part of the natural transmission cycle of tbev and are the incidental host when infected by a bite from an infected tick [33] . transmission through consumption of unpasteurized milk has also been reported for tbev [34, 35] , as well as transmission via solid organ transplant [36] . during the tick bite, the virus is inoculated into the skin of the vertebrate host. the initial replication is believed to occur locally in the dendritic cells. this is followed by infection of the draining lymph nodes, resulting in the primary viremia and subsequent infection of the peripheral tissues, where further replication maintains the viremia for several days [37] [38] [39] [40] . the disease course of tbev is biphasic; the initial phase is characterized by flu-like symptoms and is followed by a second phase involving cns infection, with meningitis, encephalitis, or meningoencephalitis [41] [42] [43] . the mortality rate of tbev varies from 1 to 20% depending on the subtype, in which the european tbev subtype has shown lower mortality rates compared to the siberian and far eastern [23, 40, 44] . among the patients that experience neuroinvasive tbev infection, approximately 25-40% of the survivors suffer from long lasting neurological sequelae [45, 46] . no antivirals are available for treatment of tbev infection but there is an effective vaccine [25, 47] . lgtv, which is closely related to tbev, is found in south east asia and russia [17] . lgtv has not been associated with human disease under natural infections although it shares 84% sequence identity with tbev [17] . because of its avirulence in humans and close similarity to tbev, lgtv is often used as a model virus for tbev under biosafety level-2 conditions. powv is found in russia and north america, and is the only tbfv present in america ( figure 1 ) [48] . it is transmitted by ixodes scapularis, ixodes cookei, and several other ixodes tick species, to small and medium size mammals, whereas humans are accidental dead-end hosts. milk-borne powv transmission might also be possible since powv virus has been found to be secreted in milk under experimental settings [49] . although not much is known about powv pathogenesis, recent studies in mice have found that tick saliva was important to enhance powv transmission and the outcome of disease [50] . furthermore, it has been demonstrated that powv infects macrophages and fibroblasts in the skin, shortly after the tick bite, also, other unidentified cells were shown to be infected [51] . interestingly, macrophages were found to be the primary target for powv in the spleen [52] , and in the cns, which is the main target site for powv infection, neurons have been shown to be the primary target for powv in mice and humans [52, 53] . during the last 10 years there has been an increase of powv in the usa with approximately 100 reported cases [54, 55] . the recent rise in incidence could be due to increased surveillance and diagnosis of powv, or it may represent a true emergence of the disease in endemic areas, or both [55] . the incubation period ranges from 1 week to 1 month. the symptoms of powv infection may include fever, headache, vomiting, weakness, confusion, seizures, and memory loss with a case fatality rate of 10% [54] . approximately half of the survivors experience permanent neurological symptoms, such as recurrent headaches, muscle wasting, and memory problems (https://www.cdc.gov). there are no antiviral treatments or vaccines available against powv. liv is mainly distributed in the uk and ireland, but it has also been detected in sheep in norway and on the bornholm island in denmark ( figure 1 ). liv is most commonly known as pathogen of sheep and red grouse although humans can also get infected [17, 56] . animals develop a febrile disease, which can progress to fatal encephalitis [57] . like tbev, the vector of liv is the tick species ixodes ricinus [57] . ticks transmit the virus to animals, however, natural exposure to humans is rare [33] . instead humans that are exposed to infected animals such as veterinarians, farmers, butchers, and abattoir workers, as well as laboratory scientists have acquired liv infection [58] [59] [60] [61] . in between the years of 1934 and 1991, 31 human cases of liv was described [62] . in humans, liv causes a disease that closely resembles tbev, with initial flu-like symptoms which can progress to severe neurological disease. distinct but closely related viruses are also found in spain (spanish sheep encephalomyelitis virus), turkey (turkish sheep encephalitis virus), and greece (greek goat encephalitis virus) [33] . ohfv distribution is restricted to western siberia ( figure 1 ) [17] . the main vector of ohfv is the meadow tick, dermacentor reticulatus, which can also transmit the virus to humans. however, humans are mainly infected after contact with infected muskrats (ondatra zibethicus) which are very sensitive to the infection and often succumb to the infection [63] . muskrats develop high viremia which can last for several weeks. human infection occurs through contact with urine, feces, and blood [63] . secretion of ohfv in unpasteurized goat milk has been reported but no milk-borne outbreaks have been observed [63] . the exact number of annual cases are uncertain because of misdiagnoses and unreported cases, but 165 cases were reported between 1988 and 1997 [63] . ohfv may cause a biphasic disease; the initial phase is characterized by high fever, bleeding from the nose, mouth, and uterus. thirty to fifty percent of the cases experience a second phase characterized by high fever and reappearance of the symptoms from the initial phase. case fatality rates range from 0.5 to 2.5% [63] . no antiviral treatments are available against ohfv, instead treatment is focused on supportive care to minimize hemorrhage and other complications [17] . kfdv is only found in india (figure 1 ), although a similar genetic variant, alkhurma hemorrhagic fever virus (ahfv) has been detected in saudi arabia [17, 47] . kfdv is mainly transmitted by ticks belonging to the genus hemaphysalis but other tick genera have also been shown to be able to transmit kfdv [64, 65] . during hemorrhagic kfdv infection, the initial phase is similar to tbev infection with fever and flu-like symptoms, but it may also include bleeding from the nose, mouth or gastrointestinal tract [65, 66] . the second phase, which is experienced by 1-20% of the cases, includes neurological symptoms such as headache, mental disturbance, and tremor, however, no evidence of meninges or encephalitis have been found [65] . there are about 400-500 reported annual cases of kfdv [47] , with case fatality rates ranging from 3 to 10% [17, 65] . in 1990 a kfdv vaccine was developed, although it provided some protection, due to low efficacy, the number of cases still increased from 1999 to 2012 [67] [68] [69] . treatment of kfdv is limited to supportive care [47] . tbfvs are enveloped viruses around 50 nm in diameter. the envelope carries two surface proteins, the envelope (e) protein and the membrane (m) protein. the latter is derived from a precursor protein, prm. the nucleocapsid (nc) lies inside the viral envelope and consists of multiple copies of capsid (c) protein and the viral genome. the tbfv genomes are single stranded, positive-sense rna of approximately 11,000 nucleotides. it has a 5 -cap with a single open reading frame (orf). the orf is flanked by 5 and 3 untranslated regions (utrs). the viral protein is encoded by the orf as a single polyprotein, which is co-and post-translationally cleaved by cellular and viral proteases into individual viral proteins (three structural and seven non-structural). the polyprotein is arranged in the order 5 -c-prm-e-ns1-ns2a-ns2b-ns3-ns4a-ns4b-ns5-3 [70, 71] . the first step of virus replication starts when the virus binds to its receptor and is taken up into the cell by receptor-mediated endocytosis. the attachment is mediated by viral e protein and entry receptor on the host cell. the entry receptor for tbfvs has not been identified, but attachment to heparan sulphate and glycosaminoglycan, which are present in abundance on many cell types of both vertebrates and ticks, is predicted to play a role during binding and entry [72] . once the virus has entered the cell, it is transported to endosomes, where the acidic environment of these vesicles leads to reorganization and conformational change of the e protein, resulting in the fusion of the viral and endosomal membrane and the release of the viral capsid into the cytoplasm [73] [74] [75] . the viral rna (vrna) also functions as mrna, associating with ribosomes to produce the polyprotein. the transmembrane domain of the viral protein is recognized as a signal peptide and recruits the vrna/ribosomes/nascent polypeptide complex to the er membrane, where it is co-translationally translocated into the er membrane [76, 77] . the nascent polypeptide is then processed by the cellular and viral proteases into structural and non-structural (ns) viral proteins. some of the viral proteins such as ns2b, ns4a, and ns4b integrate and alter the membrane of the er to form a membrane vesicular structure, with a small pore connecting the interior of the vesicle to the cytoplasm [78] [79] [80] [81] [82] . these vesicles are the site for the formation of replication complexes (rc) and rna replication [80, 82, 83] . following rna replication, genomic rna is proposed to exit the vesicle through the vesicular pore and is packaged by c proteins into the nc on the cytoplasmic side of the er membrane [82, 84, 85] . during the process of budding of nc from the cytoplasm into the er, it acquires the lipid envelope along with e and prm proteins, which are associated with the er membrane. the mechanism that ensures efficient incorporation of nc into the er membrane with the e and prm protein is poorly understood. following budding into the er lumen, the immature virions are transported through the cellular secretory pathways in a copi and copii dependent manner [86] to the extracellular medium. the immature virion has heterodimers of e and prm that completely cover the lipid bilayer to form a spiky proteinaceous coat. during the transport through the golgi, the e protein is glycosylated [87] . the acidic environment of the golgi induces conformational changes in the e and prm proteins, which exposes a cleavage site on prm. cleavage of prm by the cellular protease furin results in the formation of a mature virion [88, 89] . mature virions are then released by exocytosis, which completes the viral life cycle [90] [91] [92] (figure 2 ). in arthropods, such as mosquitos and ticks, rna interferences (rnai) are the most important antiviral defense and tick cells have been shown to mount rnai responses against lgtv and tbev [93] . proteins involved in the rnai mediated response such as argonaute 30 (ago30), ago 16 and dicer (dcr) 90 were subsequently identified as inhibitors of lgtv [93] . furthermore rna-seq and mass spectrometric analysis revealed that when challenged by tbev infection, tick cells upregulated genes involved in immunity and metabolism, whereas genes involved in cellular stress were downregulated [94] . using gene silencing approaches, this study confirmed the antiviral effect of ago 30 and dcr 90 in tick cells. furthermore, novel antiviral genes such as complement factor h, heat shock protein (hsp) 70 and 90 and trypsin was found to inhibit lgtv in tick cells [94] . taken together, studies so far have identified activation and antiviral actions of the rnai mediated response in tick cells as well as other inhibitory proteins such as hsp70, hsp90, and trypsin. the innate immune response provides the first line of defense against viral infections. this defense includes physical and chemical barriers such as the skin and mucous membranes and the acidity of the stomach. they serve as an initial barrier that protects the host from infection. once these barriers have been breached, the innate immune response relies on a set of germline-encoded receptors, known as pathogen recognition receptors (prrs), which recognize pathogen specific molecular patterns that are sensed as a danger signal by the host cell [95, 96] . engagement of prrs results in the activation of several defense mechanisms including, production of interferon (ifn), induction of phagocytosis, cytokines, chemokines, antimicrobial peptides, antiviral proteins, as well as activation of leukocytes and t-cells. furthermore, the innate immune response harbors cellular components, such as dendritic cells, macrophages, and natural killer (nk) cells [97] . in 1957 isaacs and lindenmann discovered ifn as a secreted factor that interfered with viral replication [98] . in the early pioneer work on ifn in the fifties and sixties, tbev served as a model system, and tbev was shown to induce ifn after infection and was also sensitive to pretreatment of ifns [80, [99] [100] [101] [102] . pretreatment of ifn was also found to inhibit kfdv, ohfv, and powv titers in a549 cells (adenocarcinomic human alveolar basal epithelial cells) [103] , thus ifn pretreatment induced broad spectrum inhibition of tbfvs. ifn has also been found to be strongly induced in the brains of powv infected peromyscus lecuopus, which are known as a natural host of the virus [104] . similarly, ifn was found to be induced in sheep infected with liv [105] , and ifn-stimulated genes were induced in brains of kfdv infected mice, indicating an activated ifn response [106] . three distinct classes of ifn, type i (ifn α and β), type ii (ifnγ), and type iii ifn (ifnλ) have been described. the expression of the ifnλ-receptors and ifnγ are restricted, but the type i ifns can be expressed by most cell types and their receptor, the interferon-α/β receptor (ifnar) is expressed on all nucleated cells [107] . there are 17 different type i ifns in humans; these cytokines are produced by cells upon recognition of pathogen-associated molecular patterns, which are foreign to the host cell [95, 108] , and mediates antiviral activity by autocrine and paracrine signaling through ifnar [109, 110] . signaling through ifnar induces an antiviral state by the expression of hundreds of interferon stimulated genes (isgs) [111, 112] . the host cell detects invading pathogens through prrs, which recognize foreign molecular patterns that are generated during infection. the particular prr involved in the recognition of the pathogen depends on the infecting virus [108] . in flavivirus infection, the most important prrs are toll-like receptor (tlr)3, tlr7, tlr8, retinoic acid-inducible gene i (rig-i), and melanoma differentiation-associated protein-5 (mda-5). tlr3 recognizes double stranded (ds) rna, which is formed as an intermediate during flavivirus replication [113, 114] . tlr7 and 8, which are located in the endosomes, recognize single stranded (ss) rnas, such as the genomic rna of flaviviruses [95, [115] [116] [117] . rig-i and mda-5 recognize viral rna in the cytoplasm of infected cells; rig-i recognizes short dsrna and 5 triphosphorylated and 5 diphosphorylated ssrna, whereas mda-5 recognizes long dsrna [118] [119] [120] . upon recognition, the different prr will recruit distinct adaptor molecules. tlr3 recruits tir-domain-containing adapter-inducing interferon-β (trif) [121] , tlr7 and 8 recruit myeloid differentiation primary response 88 (myd88) [122] , whereas the rig-i-like helicases, rig-i and mda-5, recruit interferon-beta promoter stimulator-1 (ips-1) (also known as mitochondrial antiviral-signaling protein (mavs), virus-induced signaling adapter (visa) and card adapter-inducing ifn-beta (cardif)) [123] [124] [125] [126] . ligation of prrs and downstream recruitment of the adapter molecules result in the activation of transcription factors nf-κb and interferon regulatory factor (irf) 3 and irf7, which translocate to the nucleus and induces the expression and subsequent secretion of type i ifn [127] . although the innate antiviral response has been well-studied in mosquito-borne flavivirus infection, the responses to tbfvs are less investigated, and most studies have used tbev which therefore will be the main focus of the remainder of this review. in tbev infection, ifnβ induction has been shown to correlate with amount of intracellular viral rna, and the ips-1 pathway has been shown to protect mice from lethal lgtv infection and prolong survival after tbev infection [80, 128] . absence of ips-1 resulted in a lower systemic ifnα response, which correlated with higher viral replication in the peripheral tissues [128] . furthermore, ips-1 was shown to be of particular importance for the ifn production locally within the brain, where ips-1 −/− mice had lower induction of ifnβ within the olfactory bulb despite higher viral burdens [128] . furthermore, ifnβ induction was shown to be completely dependent on ips-1 and irf3 in mouse embryonic fibroblasts and viral recognition through the ips-1-pathway was shown to be dependent on rig-i and not mda-5 in human osteosarcoma cells (u2os) [80, 83] . interestingly, rig-i has been shown to co-localize with stress granules (sg) during tbev infection [129] . sg contains ribonucleoprotein aggregates with translationally stalled mrnas, 40s ribosomes, and several rna-binding proteins. sg function to prevent the generation of defective proteins [130] . sg are induced during tbev infection and sg components tia-1/tiar was found to bind viral rna and inhibit viral translation [131] . little is known about the role of the tlr7/8-myd88 pathway in tick-borne flavivirus infection; however, tlr7 was shown to suppress lgtv replication within neurons of the brain although it did not affect pathogenesis [132] . what we are aware of; no animal experiments have shown any significance of the tlr3-trif pathway in tick-borne flavivirus infection. however, several studies have looked at the prevalence of polymorphisms in the tlr3 gene in tbe patients [133] [134] [135] [136] . in particular, one polymorphism has been investigated the t allele in rs3775291. this polymorphism has been shown to reduce tlr3 signaling by about 30% [137] . although the data regarding tlr3 is somewhat conflicting, grygorczuk et al. hypothesized that a functional tlr3 facilitates the onset of neurological disease [135] by supporting the penetration through the blood brain barrier, but has a protective effect during the established cns infection [134] . the differences between studies might also be connected to the different subtypes of the tbev strain and the genetic background of the studied populations [134] . taken together, studies so far have demonstrated the importance of the rig-i-like-ips-1 pathway in tick-borne flavivirus infection, whereas the role of the tlr pathways remains unclear. after secretion, type i ifn signals in an autocrine and paracrine manner by binding to the heterodimeric ifnar, which consists of two subunits, ifnar1 and ifnar2 [138] . these subunits are associated with janus activated kinases (jak) localized in the cytoplasm; ifnar1 interacts with tyrosine kinase-2 (tyk2), whereas ifnar2 interacts with jak1. ligation of ifnar results in activation of tyk2 and jak1, which subsequently phosphorylate signal transducers and activators of transcription (stat)-1 and stat-2. phosphorylated stat-1 and stat-2 then form a heterodimer, which associates with irf9 to form ifn-stimulated gene factor-3 (isgf3) that binds to the ifn-stimulated response element (isre) in the promoter of many isgs to enhance the transcription of several hundreds of ifn-stimulated genes [110, 139, 140] . together, these isgs act to coordinate an antiviral response that is able to inhibit almost any step in the viral life cycle [140] . the ifn response in vivo in mice is very important to protect mice from lethal infection with lgtv. mice lacking ifnar all succumbed within 6 days of infection, whereas 80% of wt mice (6-8-week-old) survived the infection. interestingly, all ifnβ −/− mice survived the infection, demonstrating that the ifnαs can compensate for loss of ifnβ in lgtv infection. furthermore, ifnar was shown to be a critical determinant of lgtv tropism as lgtv rna was found in all organs in the absence of ifnar, whereas in wt, only low viral burdens can be detected in the olfactory bulb [99, 128, 141] . using transgenic mice, it was further shown that the ifn response was needed both in the peripheral tissue, as well as locally in the cns, in order to clear lgtv infection [99, 100] . within the cns, lgtv mainly infected neurons [99, 128] , which is also the case in lethal tbev infection of humans [142] . although neurons remain the main target cell of tbev, astrogliosis have been shown in post mortem human brains [142, 143] . in vitro studies on primary cortical astrocytes show a fast up regulation and secretion of ifn after tbev infection, which is able to protect neighboring astrocytes and neurons from infection already 6 and 3 h post infection, respectively [100] . astrocytes have been shown to be resistant to tbev-induced cytopathic effects [144, 145] , and it was later shown that ifnar expression protected the astrocytes from the virus-induced cytopathic effects [100] . pretreatment of cells with ifns strongly inhibits growth of most tbfvs in cell culture [100, 103, [146] [147] [148] [149] and this is due to the upregulation and concerted actions of several hundreds of isgs. only a few isgs have been identified to play a role in tbev and lgtv infection [86, 141, 147, 149, 150] . one of them, the 2 -5 -oligoadenylate synthetase (2 -5 -oas) (oas) is activated by double-stranded rna, leading to the oas protein polymerization into 2 -5 -linked oligoadenylates (2-5as) [151, 152] . these 2-5as activate rnase l, resulting in the degradation of viral rna [153] . several polymorphisms in the oas genes have been shown to correlate with severe forms of tbe in patients [154] . also, the murine isoform oas1b, which is often lacking in inbred mice strains, confers strain dependent resistance against neurovirulence from far eastern tbev [155] . two other isgs which have been shown to be antivirally active against tbev are virus inhibitory protein endoplasmic reticulum associated interferon inducible (viperin) and tripartite motif-79α (trim79α). the trim proteins are a family of proteins able to mediate antiviral activity against many different viruses [156] . the rodent specific trim79α was identified to interact with lgtv ns5 in a yeast two-hybrid screen. trim79α expression inhibited viral infection of lgtv and tbev by mediating lysosomal dependent degradation of ns5. interestingly this mechanism was quite specific to tick-borne flaviviruses as the mosquito borne wnv was not inhibited by trim79α nor did trim79α interact with ns5 of wnv [147] . viperin is highly conserved in evolution and was first identified as an ifn-inducible protein with antiviral activity against human cytomegalovirus in 2001 [157] . over the last 17 years, viperin has gained lot of attention and was shown to exhibit broad antiviral activity [158] . viperin is also known as one of the most highly upregulated genes after viral infection [146, 159] . within the family of flaviviridae, viperin has been shown to inhibit several members, such as wnv [160] , denv [161] , zikv [162] , hepatitis c virus [163] , and tbev [86, 141, 149, 150] . viperin is an iron sulphur protein with three domains; a n-terminus amphipathic alpha-helix which mediates the intracellular localization to the er, a s-adenosylmethionine (sam) radical domain [158, 164, 165] homologous to a family of proteins that use sam as a cofactor [166] , and a highly conserved c-terminal domain, important for iron sulphur (fe/s) maturation [149, 167] . although viperin is able to inhibit several different viruses, the mechanism of action, the important motif in viperin and the step of viral life cycle inhibited differs for different viruses [158] . viperin interacts with many viral and host factors for its antiviral function. tbev replication is strongly inhibited by viperin, our research shows that viperin has no effect on the binding or entry of tbev. however, viperin targets genome replication, packaging, and release of tbev (figure 3 ) [86, 149, 150] . viperin specifically targets the plus-sense rna synthesis with no significant effect on the negative-sense rna of tbev during genomic replication [149] . viperin's fe/s maturation is dependent on ciao1 [149, 167] . the fe/s cluster and a functional sam domain of viperin is essential to inhibit the synthesis of the plus-sense rna of tbev [149] . however, the target for the radical sam activity important for the antiviral activity of tbev is currently unknown. the structure of viperin was recently characterized [168] , and based on similarities to other radical sam enzymes, several different hypothesis have been put forward regarding the substrate of viperin and its antiviral activity [168] [169] [170] . however, none of these have shown importance in the context of mammalian viral infection. although the exact mode of action against tbev is not well understood, recent data indicates that viperin interacts with several viral proteins; both structural prm and e and non-structural ns2a, ns2b, and ns3. these interactions lead to a viperin-ns3 dependent degradation of viral proteins. the degradation of tbev ns3 was shown to be proteasome dependent [150] (figure 3) . interestingly, ifn treatment induces an increase of tbev capsid particles and this effect was found to be dependent on viperin. viperin mediated this effect by interacting, via its n-terminus, with the cellular protein golgi brefeldin a resistant guanine nucleotide exchange factor 1 (gbf1) [86] . gbf1 is a key protein in the cellular secretory pathway and essential in the life cycle of many rna viruses, which utilize vesicular trafficking in their replication cycle and assembly process [171] [172] [173] [174] (figure 3 ). viperin targets the ns3 protein for proteasomal degradation which inhibits the synthesis of + strand rna. furthermore, ns3 interacts with e, ns2a, and ns2b and these proteins are degraded by viperin in a ns3-dependent manner. viperin also interferes with particle assembly by inducing secretion of c particles in a copii-dependent manner, independent of copi. viperin mediates this effect by interacting and sequestering gbf1. red arrow = secretory pathway, blue arrow = "?" vesicular transport via unknown pathway. although viperin has been demonstrated to be antiviral active against many different viruses in vitro, few studies have investigated viperin's role in vivo. in tbfv infection, viperin was shown to control lgtv dissemination and replication in the brain after intraperitoneal administration, and viperin promoted survival after intracranial infection [141] . interestingly, viperin has been shown to be expressed in the brain at the basal state [128] , with high basal expression in primary astrocytes [100, 141] . viperin's role within the brain during neurotropic lgtv infection was further mapped to certain brain regions, as viperin inhibited viral replication in the olfactory bulb and cerebrum, but not in the cerebellum or brainstem. this correlates very well with tbev infection since viperin strongly inhibited tbev replication in primary neurons and astrocytes from the cerebrum, but not in granular cell neurons isolated from the cerebellum. interestingly, cortical neurons were completely dependent on viperin for ifn-mediated inhibition of tbev, whereas in astrocytes, in which the ifn-mediated antiviral activities are strongly dependent on viperin, other isgs could partly compensate for loss of viperin [141] . taken together, viperin has been shown to be an isg that strongly inhibits tick-borne flaviviruses in vivo and in vitro. this strong antiviral effect on tbev is mediated by targeting the virus at multiple steps of the life cycle. interestingly, viperin targeting the synthesis of plus-sense rna is dependent on the sam domain or the c-terminal domain, while the n-terminal domain of viperin is responsible for interfering with virus assembly and release. in order to establish an infection, a pathogen needs to overcome or evade the innate immune response. to breach the very first line of defense, the skin-tbfvs use the tick to deliver the virus through the skin via tick saliva. furthermore, tick saliva contains immunomodulatory compounds that enhance viral transmission and dissemination [175, 176] . although there are several mechanisms in which mosquito-borne flaviviruses actively suppress the induction of type i ifn, no such mechanism has been identified in tick-borne flaviviruses so far [177] . instead, tbev, like other flaviviruses, utilizes a passive evasion mechanism in which the virus hides its dsrna intermediates in vesicular structures inside the er membranes, and thus delaying the recognition by the cytosolic rig-i like receptors and subsequent irf3 phosphorylation and ifn induction ( figure 4a ) [80, 81, 83] . the most conserved inhibition of the type i ifn system within the flaviviridae family is the antagonism of ifnar signaling carried out by ns5; this mechanism is conserved between several mosquito and tick-borne flaviviruses [148, [177] [178] [179] [180] [181] . in lgtv infection, ns5 was shown to inhibit the jak-stat pathway and ns5 was shown to interact with the ifnar receptor [148] . similarly, it was shown that ns5 of tbev interacts with scribble (hscrib) which mediates ns5 localization to the plasma membrane and this interaction enables ns5 to inhibit type i and type ii ifn mediated jak-stat signaling [178] . knockdown of hscrib altered ns5 cellular localization and reversed the inhibition of the jak-stat signaling [178] . further studies revealed that ns5 of tbev inhibited the cell surface expression of ifnar1 by binding to prolidase (pepd) [182] . pepd is a peptidase that is needed for ifnar1 maturation and subsequent cell surface expression. ns5 binding of pepd prevented maturation of complex n-linked oligosaccharides on ifnar1, which in turn disrupted its surface expression ( figure 4b ) [179, 182] . in kfdv infection, ifn treatment failed to reduce viral titers when added after infection, this effect was also found to be mediated by the ns5 proteins antagonism [180, 183] . during tbfv infection, a subgenomic noncoding rna is formed, called subgenomic flavivirus rna (sfrna) [93, 184, 185] . it is produced as a product of incomplete degradation of genomic viral rna by cellular 5 -3 exoribonuclease xrn1 [184] . the ability to produce sfrna in wnv was shown to be needed for efficient viral growth in vitro and for pathogenicity in mice [184] . interestingly, denv sfrna was found to bind to trim25 to inhibit rig-i-induced type i interferon expression in huh-7 cells [186] . furthermore, denv and wnv sfrna was found to suppress the rnai response in both mammalian and insect cells [187] . similarly, in tbev infection, sfrna has been demonstrated to inhibit the antiviral rnai response in tick cells [93] . pepd is needed for maturation and subsequent transport of ifnar1 to the plasma membrane. ifnar1 and ifnar2 heterodimer on plasma membrane can be activated by ifnα/β which leads to the signaling cascade and phosphorylation and translocation of stat1/2-irf9 into the nucleus and upregulation of isgs. right panel: ns5 interferes with ifn signaling. ns5 protein interacts with pepd thus preventing ifnar1 plasma membrane localization (red t). ns5 also prevents stat1 phosphorylation (red t). arrows indicate protein transport. even though recent studies have shed light on the role of innate immunity during tick-borne flavivirus infection, much remains unknown. for example, the role of the tlr-trif and tlr-myd88 pathway in pathogenesis and viral recognition. furthermore, most studies were performed using lgtv and tbev, and although they are closely related to powv, liv, kfdv, and ohfv, their interactions with the innate immune response might differ. although ifn has been shown to strongly control viral tropism and pathogenesis of tick-borne flaviviruses, few antiviral isgs have been identified. viperin has been shown to be the most important isg in cortical neurons, however, other isgs that target tbev expressed in astrocytes and granular cell neurons are yet to be identified and very little is known about the early response against the hemorrhagic tbfvs. we also know very little about how the innate immune response regulates the neuroinvasion of neurotropic tbfv, and the specific interactions between the tick vector and the different viruses. emergence and spreading potential of zika virus dengue virus: a global human threat: review of literature west nile virus: a re-emerging pathogen revisited alphaviruses (togaviridae) and flaviviruses (flaviviridae) hepatitis c virus: virology and life cycle characteristics of a nidus of tick-borne encephalitis in the construction zone of the krasnoyarsk hydroelectric station and development of measures for protection of workers against ticks; preliminary report stable prevalence of powassan virus in ixodes scapularis in a northern wisconsin focus a global perspective on the epidemiology of west nile virus zika virus: new clinical syndromes and its emergence in the western hemisphere west nile virus and its emergence in the united states of america flavivirus encephalitis the changing epidemiology of yellow fever and dengue yellow fever: a disease that has yet to be conquered modes of transmission of zika virus ticks and tickborne bacterial diseases in humans: an emerging infectious threat short report: duration of tick attachment required for transmission of powassan virus by deer ticks zoonotic tick-borne flaviviruses molecular characterization of tick-virus interactions dynamics of infection in tick vectors and at the tick-host interface non-viraemic transmission of tick-borne encephalitis virus: a mechanism for arbovirus survival in nature sequence analysis and genetic classification of tick-borne encephalitis viruses from europe and asia tick-borne flaviviruses tick-borne encephalitis epidemiology and distribution of tick-borne encephalitis tick-borne encephalitis 2010: epidemiology, risk areas, and virus strains in europe and asia-an overview. ticks tick borne dis distribution of ixodes ricinus ticks and prevalence of tick-borne encephalitis virus among questing ticks in the arctic circle region of northern norway persistent viremia and urine shedding of tick-borne encephalitis virus in an infected immunosuppressed patient from a new epidemic cluster in north-eastern italy why is tick-borne encephalitis increasing? a review of the key factors causing the increasing incidence of human tbe in sweden a new hot spot for tick-borne encephalitis (tbe): a marked increase of tbe cases in france in 2016 to what extent has climate change contributed to the recent epidemiology of tick-borne diseases? socio-economic factors in the differential upsurge of tick-borne encephalitis in central and eastern europe survival strategy of tick-borne encephalitis virus: cellular basis and environmental determinants tick-borne encephalitis virus-a review of an emerging zoonosis an outbreak of tick-borne encephalitis associated with raw goat milk and cheese consumption, croatia avsic-zupanc, t. tick-borne encephalitis associated with consumption of raw goat milk, slovenia a cluster of fatal tick-borne encephalitis virus infection in organ transplant setting tick-borne flaviviruses: dissecting host immune responses and virus countermeasures langerhans cells migrate to local lymph nodes following cutaneous infection with an arbovirus the togaviridae and flaviviridae tick-borne encephalitis: pathogenesis and clinical implications the clinical and epidemiological profile of tick-borne encephalitis in southern germany 1994-1998: a prospective study of 656 patients tickborne encephalitis in an area of high endemicity in lithuania: disease severity and long-term prognosis human tick-borne encephalitis and characterization of virus from biting tick tick-borne encephalitis virus and the immune response of the mammalian host neurological complications of tick borne encephalitis: the experience of 89 patients studied and literature review tick-borne encephalitis-pathogenesis, clinical course and long-term follow-up. vaccine tick-borne viruses: a review from the perspective of therapeutic approaches experimental milk-borne transmission of powassan virus in the goat tick saliva enhances powassan virus transmission to the host, influencing its dissemination and the course of disease immune cell targets of infection at the tick-skin interface during powassan virus transmission spinal cord ventral horns and lymphoid organ involvement in powassan virus infection in a mouse model powassan encephalitis: a case report with neuropathology and literature review update on powassan virus: emergence of a north american tick-borne flavivirus powassan virus: an emerging arbovirus of public health concern in north america. vector borne zoonotic dis louping ill virus in the uk: a review of the hosts, transmission and ecological consequences of control louping ill virus: an endemic tick-borne disease of great britain louping ill in man louping-ill meningo-encephalitis; a further case and a serological survey occupational infections in the edinburgh abattoir laboratory infections with louping-ill virus louping ill in man: a forgotten disease omsk haemorrhagic fever kyasanur forest disease. viii. isolation of kyasanur forest disease virus from naturally infected ticks of the genus haemaphysalis kyasanur forest disease clinical, clinicopathologic, and hematologic features of kyasanur forest disease kyasanur forest disease: an epidemiological view in india field evaluation of formalin inactivated kyasanur forest disease virus tissue culture vaccine in three districts of karnataka state coverage and effectiveness of kyasanur forest disease (kfd) vaccine in karnataka, south india flavivirus rna transactions from viral entry to genome replication insect-specific flaviviruses: a systematic review of their discovery, host range, mode of transmission, superinfection exclusion potential and genomic organization role of heparan sulfate for attachment and entry of tick-borne encephalitis virus molecular mechanisms involved in the early steps of flavivirus cell entry. microbes infect rodenhuis-zybert, i.; wilschut, j. flavivirus cell entry and membrane fusion flaviviral replication complex: coordination between rna synthesis and 5 -rna capping de novo translation initiation on membrane-bound ribosomes as a mechanism for localization of cytosolic protein mrnas to the endoplasmic reticulum a three-dimensional comparison of tick-borne flavivirus infection in mammalian and tick cell lines the endoplasmic reticulum provides the membrane platform for biogenesis of the flavivirus replication complex tick-borne encephalitis virus delays interferon induction and hides its double-stranded rna in intracellular membrane vesicles hiding from intracellular pattern recognition receptors, a passive strategy of flavivirus immune evasion three-dimensional architecture of tick-borne encephalitis virus replication sites and trafficking of the replicated rna formation of membrane-defined compartments by tick-borne encephalitis virus contributes to the early delay in interferon signaling spatial and temporal organization of tick-borne encephalitis flavivirus replicated rna in living cells visual detection of flavivirus rna in living cells viperin targets flavivirus virulence by inducing assembly of non-infectious capsid particles n-linked glycan in tick-borne encephalitis virus envelope protein affects viral secretion in mammalian cells, but not in tick cells structure of dengue virus: implications for flavivirus organization, maturation, and fusion structure of the immature dengue virus at low ph primes proteolytic maturation cleavage of protein prm is necessary for infection of bhk-21 cells by tick-borne encephalitis virus proteolytic activation of tick-borne encephalitis virus by furin assembly and maturation of the flavivirus kunjin virus appear to occur in the rough endoplasmic reticulum and along the secretory pathway, respectively induction and suppression of tick cell antiviral rnai responses by tick-borne flaviviruses ixodes scapularis and ixodes ricinus tick cell lines respond to infection with tick-borne encephalitis virus: transcriptomic and proteomic analysis pathogen recognition and innate immunity innate immunity and pathogen-host interaction toll-like receptor control of the adaptive immune responses virus interference. i. the interferon type i interferon protects mice from fatal neurotropic infection with langat virus by systemic and local antiviral responses fast type i interferon response protects astrocytes from flavivirus infection and virus-induced cytopathic effects the role of interferon in tick-borne encephalitis virus-infected l cells. i. acute infection an interferon-like substance released from tickborne encephalitis virus-infected chick embryo fibroblast cells inhibitors of the tick-borne, hemorrhagic fever-associated flaviviruses peromyscus leucopus mouse brain transcriptome response to powassan virus infection innate and adaptive immune responses to tick-borne flavivirus infection in sheep kyasanur forest disease virus infection in mice is associated with higher morbidity and mortality than infection with the closely related alkhurma hemorrhagic fever virus the interferons and their receptors-distribution and regulation recognition of viruses by innate immunity complex modulation of cell type-specific signaling in response to type i interferons how cells respond to interferons functional classification of interferon-stimulated genes identified using microarrays identification of genes differentially regulated by interferon alpha, beta, or gamma using oligonucleotide arrays pattern recognition receptors and inflammation recognition of double-stranded rna and activation of nf-kappab by toll-like receptor 3 flavivirus activation of plasmacytoid dendritic cells delineates key elements of tlr7 signaling beyond endosomal recognition recognition of single-stranded rna viruses by toll-like receptor 7 species-specific recognition of single-stranded rna via toll-like receptor 7 and 8 the rna helicase rig-i has an essential function in double-stranded rna-induced innate antiviral responses 5 -triphosphate rna is the ligand for rig-i differential recognition of double-stranded rna by rig-i-like receptors in antiviral immunity role of adaptor trif in the myd88-independent toll-like receptor signaling pathway interferon-alpha induction through toll-like receptors involves a direct interaction of irf7 with myd88 and traf6 identification and characterization of mavs, a mitochondrial antiviral signaling protein that activates nf-kappab and irf 3 ips-1, an adaptor triggering rig-i-and mda5-mediated type i interferon induction visa is an adapter protein required for virus-triggered ifn-beta signaling cardif is an adaptor protein in the rig-i antiviral pathway and is targeted by hepatitis c virus master regulators of signalling by toll-like receptors and cytosolic pattern-recognition receptors type i interferon response in olfactory bulb, the site of tick-borne flavivirus accumulation, is primarily regulated by ips-1 the host cell response to tick-borne encephalitis virus antiviral innate immunity and stress granule responses the stress granule component tia-1 binds tick-borne encephalitis virus rna and is recruited to perinuclear sites of viral replication to inhibit viral translation toll-like receptor 7 suppresses virus replication in neurons but does not affect viral pathogenesis in a mouse model of langat virus infection association of single nucleotide polymorphism rs3775291 in the coding region of the tlr3 gene with predisposition to tick-borne encephalitis in a russian population the increased concentration of macrophage migration inhibitory factor in serum and cerebrospinal fluid of patients with tick-borne encephalitis a functional toll-like receptor 3 gene (tlr3) may be a risk factor for tick-borne encephalitis virus (tbev) infection polymorphisms in chemokine receptor 5 and toll-like receptor 3 genes are risk factors for clinical tick-borne encephalitis in the lithuanian population effects of single nucleotide polymorphisms on toll-like receptor 3 activity and expression in cultured cells mechanisms of type-i-and type-ii-interferon-mediated signalling a diverse range of gene products are effectors of the type i interferon antiviral response interferon-stimulated genes: roles in viral pathogenesis cell-type-and region-specific restriction of neurotropic flavivirus infection by viperin visualization of central european tick-borne encephalitis infection in fatal human cases contribution to the histology of tick-borne encephalitis infection and injury of human astrocytes by tick-borne encephalitis virus tick-borne encephalitis virus infects rat astrocytes but does not affect their viability analysis of tick-borne encephalitis virus-induced host responses in human cells of neuronal origin and interferon-mediated protection trim79alpha, an interferon-stimulated gene product, restricts tick-borne encephalitis virus replication by degrading the viral rna polymerase inhibition of interferon-stimulated jak-stat signaling by a tick-borne flavivirus and identification of ns5 as an interferon antagonist viperin is an iron-sulfur protein that inhibits genome synthesis of tick-borne encephalitis virus via radical sam domain activity viperin restricts zika virus and tick-borne encephalitis virus replication by targeting ns3 for proteasomal degradation the human 2 ,5 -oligoadenylate synthetase family: interferon-induced proteins with unique enzymatic properties the nature of the catalytic domain of 2 -5 -oligoadenylate synthetases antiviral actions of interferons variability in the 2 -5 -oligoadenylate synthetase gene cluster is associated with human predisposition to tick-borne encephalitis virus-induced disease susceptibility to flavivirus-specific antiviral response of oas1b affects the neurovirulence of the far-eastern subtype of tick-borne encephalitis virus trim family proteins: retroviral restriction and antiviral defence viperin (cig5), an ifn-inducible antiviral protein directly induced by human cytomegalovirus the role of viperin in the innate antiviral response differential innate immune response programs in neuronal subtypes determine susceptibility to infection in the brain by positive-stranded rna viruses the interferon-inducible gene viperin restricts west nile virus pathogenesis viperin is induced following dengue virus type-2 (denv-2) infection and has anti-viral actions requiring the c-terminal end of viperin viperin is an important host restriction factor in control of zika virus infection the antiviral protein viperin inhibits hepatitis c virus replication via interaction with nonstructural protein 5a viperin: a multifunctional, interferon-inducible protein that regulates virus replication the interferon inducible gene: viperin structural characterization reveals that viperin is a radical s-adenosyl-l-methionine (sam) enzyme cellular requirements for iron-sulfur cluster insertion into the antiviral radical sam protein viperin structural studies of viperin, an antiviral radical sam enzyme a unifying view of the broad-spectrum antiviral activity of rsad2 (viperin) based on its radical-sam chemistry a b12-dependent radical sam enzyme involved in oxetanocin a biosynthesis quantitative proteomic analysis of host-virus interactions reveals a role for golgi brefeldin a resistance factor 1 (gbf1) in dengue infection identification of class ii adp-ribosylation factors as cellular factors required for hepatitis c virus replication a kinome-wide small interfering rna screen identifies proviral and antiviral host factors in severe acute respiratory syndrome coronavirus replication, including double-stranded rna-activated protein kinase and early secretory pathway proteins role of the gtpase rab1b in ebolavirus particle formation enhancement of tick-borne encephalitis virus transmission by tick salivary gland extracts tick salivary compounds: their role in modulation of host defences and pathogen transmission innate immune evasion mediated by flaviviridae non-structural proteins tick-borne encephalitis virus ns5 associates with membrane protein scribble and impairs interferon-stimulated jak-stat signalling the many faces of the flavivirus ns5 protein in antagonism of type i interferon signaling the generation of a reverse genetics system for kyasanur forest disease virus and the ability to antagonize the induction of the antiviral state in vitro identification of residues critical for the interferon antagonist function of langat virus ns5 reveals a role for the rna-dependent rna polymerase domain flavivirus antagonism of type i interferon signaling reveals prolidase as a regulator of ifnar1 surface expression limited effects of type i interferons on kyasanur forest disease virus in cell culture a highly structured, nuclease-resistant, noncoding rna produced by flaviviruses is required for pathogenicity noncoding subgenomic flavivirus rna: multiple functions in west nile virus pathogenesis and modulation of host responses dengue subgenomic rna binds trim25 to inhibit interferon expression for epidemiological fitness noncoding flavivirus rna displays rna interference suppressor activity in insect and mammalian cells we would like to acknowledge yong-dae gwon for constructing figure 1 , and harry tracy for critically reading the final version of the manuscript. the authors declare no conflict of interest. key: cord-000125-uvf5qzfd authors: kenworthy, rachael; lambert, diana; yang, feng; wang, nan; chen, zihong; zhu, haizhen; zhu, fanxiu; liu, chen; li, kui; tang, hengli title: short-hairpin rnas delivered by lentiviral vector transduction trigger rig-i-mediated ifn activation date: 2009-09-03 journal: nucleic acids res doi: 10.1093/nar/gkp714 sha: doc_id: 125 cord_uid: uvf5qzfd activation of the type i interferon (ifn) pathway by small interfering rna (sirna) is a major contributor to the off-target effects of rna interference in mammalian cells. while ifn induction complicates gene function studies, immunostimulation by sirnas may be beneficial in certain therapeutic settings. various forms of sirna, meeting different compositional and structural requirements, have been reported to trigger ifn activation. the consensus is that intracellularly expressed short-hairpin rnas (shrnas) are less prone to ifn activation because they are not detected by the cell-surface receptors. in particular, lentiviral vector-mediated transduction of shrnas has been reported to avoid ifn response. here we identify a shrna that potently activates the ifn pathway in human cells in a sequenceand 5′-triphosphate-dependent manner. in addition to suppressing its intended mrna target, expression of the shrna results in dimerization of interferon regulatory factor-3, activation of ifn promoters and secretion of biologically active ifns into the extracellular medium. delivery by lentiviral vector transduction did not avoid ifn activation by this and another, unrelated shrna. we also demonstrated that retinoic-acid-inducible gene i, and not melanoma differentiation associated gene 5 or toll-like receptor 3, is the cytoplasmic sensor for intracellularly expressed shrnas that trigger ifn activation. a specific double-stranded rna (dsrna) structure, $21-22 bp dsrna with 3 0 overhangs, plays a critical role in initiating both microrna (mirna)-and small interfering rna (sirna)-mediated gene silencing, as it is the structure recognized by the rna interference (rnai) machinery, the rna-induced silencing complex (risc) (1) (2) (3) . except for preformed sirna duplexes of $21 bp, the risc-loaded small rnas are generated by a ribonuclease (rnase) iii-like enzyme that is found in virtually all eukaryotic organisms. this enzyme, aptly named dicer for its ability to cleave a variety of larger (>30 bp) dsrna molecules into the $21 bp dsrna with a characteristic 3 0 overhang of 2 nt, is a multidomain rna-binding protein and itself a component of risc. the primary sequence of the rnas is not important in risc formation, and rnai can suppress virtually any target as long as rules of sequence complementarities between the small rna and the target rna are satisfied. dsrnas are also a type of pathogen-associated molecular pattern (pamp) that are detected by cellular innate immunity sensors named pattern recognition receptors (prrs) (4) . the interaction between a pamp and a prr triggers activation of the interferon (ifn) pathway in mammalian cells, which significantly changes the gene-expression profile in the cells and contributes to the well-documented off-target effect of rnai. ifn induction is especially problematic in antiviral studies employing rnai, where the antiviral effect of ifn must be distinguished from that of rnai. typical ifn-inducing structure patterns include dsrna of certain length, single-stranded rna (ssrna) containing 5 0 -triphosphates (5 0 -ppp), the dsrna analogue polyinosinic-polycytidylic acid (poly i:c), and certain dsdna molecules. these rna patterns are generally believed to possess 'non-self' properties to allow the cell to recognize foreign (often viral) rnas specifically. various forms of sirna duplexes have been reported to trigger ifn induction both in vitro and in vivo (5) (6) (7) (8) (9) , probably through the cell surface-and/or endosomeexpressed toll-like receptors (tlrs), including tlr3 and tlr7 (6, 8, 9) . short-hairpin rnas (shrnas) expressed from a dna plasmid have also been shown to activate ifn (10) . the double-stranded form of these rnas is below the size limit of the stem-loop rnas that can be detected by the rna-activated protein kinase (pkr) (11) and is probably detected by other cytoplasmic prrs. two cytoplasmic rna helicases, retinoic-acid-inducible gene i (rig-i) and melanoma differentiation associated gene 5 (mda5), signal to the ifn-b promoter when activated by specific rna structures (12) (13) (14) . although both prrs signal through the mitochondrial antiviral signaling protein mavs/cardif/visa/ips-1 (15) (16) (17) (18) , studies of ligand specificity suggest that rig-i and mda5 are parallel sensors with overlapping substrates. for example, although both prrs are activated by poly i:c in cell culture systems (12, (19) (20) (21) (22) (23) , mda5 appears to be more important in mediating the poly i:c response in vivo (13, 14) . in addition, rig-i can bind and respond to ssrnas bearing 5 0 -ppp, whereas mda5 is not activated by 5 0 -ppp-containing rna (24, 25) . finally, several cytosolic sensors for dsdna has been recently reported (26) (27) (28) (29) (30) (31) . nevertheless, current data on what constitutes effective substrates for either prr are incomplete and sometimes controversial. here we report for the first time that shrnas delivered by lentiviral transduction triggered ifn activation and that rig-i and mavs, but not mda5 or tlr3, mediated the ifn activation triggered by intracellularly expressed shrna, which could activate both ifn-a and ifn-b promoters. ifn activation depended on sequence, a 5 0 -ppp and correct processing of the rna hairpin by dicer; it was independent of promoter choice, presence of blunt ends, route of delivery and rnai potency. gs5 and lh86 cells have been described earlier (32, 33) . huh-7 and 293ft cells were maintained in dmem supplemented with 10% fbs. we used the following antibodies: anti-cypa (biomol, plymouth meeting, pa, usa); anti-cypb (afenity bioreagents, rockford, il, usa); anti-ku80, anti-flag and anti-actin (sigma-aldrich, st louis, mo, usa); anti-ifn stimulate gene (isg)15 (rockland immunochemicals, gilbertsville, pa, usa); anti-ns5a (virogen, watertown, ma, usa) and anti-ns3 (in-house). gsb1 and h801 cells have been described earlier (34) . poly i : c was purchased from sigma-aldrich, and synthetic hairpin rna was purchased from integrated dna technologies (coralville, ia, usa). synthetic sirna was purchased from ambion (austin, tx, usa). protein contents of cell lysate were quantified with the bio-rad dc protein assay (bio-rad, hercules, ca, usa), and an equal amount of total protein was loaded in each lane. samples for irf-3 dimerization assay were run on a polyacrylamide gel under non-denaturing conditions (35) . other samples were denatured and separated by sodium dodecyl sulfate polyacrylamide gelelectrophoresis (sds-page). proteins were then transferred onto a nitrocellulose membrane and stained with the appropriate antibodies with the snap i.d. tm system (millipore, worcester, ma, usa) according to the manufacturer's instructions. for luciferase assays, cells were seeded to a confluency of 50%, and for all other assays, cells were seeded to a confluency of 30%. the next day, transfections of dna plasmids and synthetic rnas were performed with lipofectamine tm 2000 (invitrogen, carlsbad, ca, usa) according to the manufacturer's instructions. plasmids pgl3-ifna1, pgl3-ifnb, prl-tk, pcmv-flag-irf-3 and pcr3.1-irf-7a have been described earlier (36) . shrnas were expressed from a human immunodeeciency virus (hiv)-based lentiviral vector (32, 37) , and sh-pcaf was constructed on the basis of a previously reported sequence (38) . plasmid sh-b971/h1 was constructed by cloning of the dna fragment encoding the sh-b971 rna into psilencer 3.0-h1 (ambion, austin, tx, usa) according to the manufacturer's instructions. the rig-i and tlr3 constructs have been described (39, 40) . the rig-i c construct encodes flag-tagged, c-terminal 707 aa of human rig-i cloned into a bicistronic expression vector modified from pbicep-cmv-1 (sigma-aldrich, st louis, mo, usa), in which the cmv promoter was replaced with the elongation-factor-1 promoter. the mda5, mda5-c constructs were kindly provided by fujita (12) . hcv genotype 2a ns3-4a protease was expressed from the pcmv-3tag-1a plasmid (stratagene, la jolla, ca, usa). 293ft cells were seeded in 24-well plates and were transfected 16 h later with 400 ng of a shrna expression vector, 40 ng of pgl3-ifna1 or pgl3-ifnb, 20 ng of prl-tk and 50 ng of pcr3.1-irf-7a. cells were collected 48 h after transfection. luciferase assays were performed with the dual-glo õ luciferase assay system reagents (promega, madison, wi) and luminescence quantified with a modulus microplate reader (turner biosystems, sunnyvale, ca, usa). ratios of firefly luciferase (from the pgl3 vectors) to renilla luciferase (from the prl-tk vector) were calculated, and that of the sh-b971 sample was normalized to 100%. sequences of shrna are shown in table 1 . lentiviral vector production and transduction were performed as described earlier (37) . viral vectors were pelleted by ultracentrifugation at 50 000g at 4 c for 3 h and resuspended in a volume of pbs that was 1% of the original medium volume. the titers of the concentrated vectors were then measured with a p24 elisa kit (zeptometrix, buffalo, ny, usa). real-time reverse transcription pcr (rt-pcr) was performed as described earlier (32) . the primers used were oas1 forward, 5 0 -agg tgg taa agg gtg gct cc-3 0 and oas1 reverse 5 0 -aca acc agg tca gcg tca gat-3 0 ; rig-i forward 5 0 -gag gca gag gaa gag caa gag g-3 0 and rig-i reverse 5 0 -cgc ctt cag aca tgg gac gaa g-3 0 ; gapdh forward 5 0 -tca ctg cca ccc aga aga ctg-3 0 and gapdh reverse 5 0 -gga tga cct tgc cca cag c-3 0 . the primers for hcv detection were 5 0 -cgc tca atg cct gga gat ttg-3 0 and 5 0 -gca ctc gca agc acc cta tc-3 0 . for flow cytometry, gs5 cells were fixed 48 h after treatment in a solution of 2% paraformaldehyde and analyzed with a facscanto flow cytometer (bd biosciences, san jose, ca, usa). mean gfp intensity was plotted, and that of the sh-ntc sample was normalized to 100%. total rna from transiently transfected 293ft cells was extracted with rna stat-60 (tel-test, friendswood, tx, usa) and separated on a 7.5% urea polyacrylamide gel. the transfer of rna onto nitrocellulose membrane and hybridization were performed according to standard molecular biology protocols. the probe for detecting the expression of sh-b971 and its variants was a synthetic dna oligomer corresponding to the bottom strand of sh-b971. radioactive labeling of the probe was performed with an end-labeling protocol with t7 polynucleotide kinase (ambion, austin, tx, usa). the exposure and detection of the radioactive signal was performed with a typhoon imager (ge healthcare, piscataway, nj, usa) with quantity one software (bio-rad, hercules, ca, usa). a short-hairpin rna directed at cypb induces ifn production in human embryonic kidney cells to investigate the potential role of the cyclophilins (cyps) in hcv replication (41), we delivered several shrnas directed at mrnas of three cyps into hcv replicon cells by means of a lentiviral vector, using a murine u6 promoter to drive the expression of the shrna ( figure 1a ) (37) . we observed a discrepancy between two anti-cypb shrnas (b971 and b710) in their relative efficiency in knocking down cypb expression and in suppressing hcv. lentiviral vector sh-b971 was less efficient in knocking down cypb expression but potently inhibited hcv ns5a expression in a human hepatoma cell line containing replicating hcv rna ( figure 1b , left). viral inhibition was independent of cypb knockdown, as control medium from transfected 293ft cells that did not contain any lentiviral vector particles, generated by omission of the packaging plasmids during transfection, also inhibited hcv replication ( figure 1b , right) without affecting cypb expression. the fast kinetics of viral inhibition (complete inhibition with 48 h, data not shown) was also more consistent with ifn than with rnai-based inhibition. the presence of ifn in the lentiviral vector preparation of sh-b971 was confirmed by strong induction of 2 0 -5 0 -oligoadenylate synthetase 1 (oas1), a classic ifn-induced gene, in both naı¨ve huh-7 and the hcv replicon cell line (gs5) treated with the medium ( figure 1c ). in addition, hcv replication in an ifn-resistant hcv replicon cell line (h801), in contrast to that in a wildtype replicon cell line (gsb1) (34), was not inhibited by the sh-b971 medium ( figure 1d ), suggesting the lack of additional viral inhibiting agents in the sh-b971 medium. expression of sh-b971 in 293ft cells also induced dimerization of irf-3, confirming the activation of the ifn production pathway in these transfected cells ( figure 1e ). finally, sh-b971 was able to activate both ifn-a and ifn-b promoters, although the activation of the ifn-a promoter required coexpression of irf-7, which is normally expressed at very low levels in 293-based cells ( figure 1f ). these results demonstrate that sh-b971 is a potent activator of irf-3 and irf-7, master regulators of ifn expression in human cells. we next investigated the role of the different viral/ exogenous rna sensors, rig-i, mda5 and tlr3, in sh-b971-triggered ifn production. mammalian expression plasmids encoding each of these proteins, as well as the dominant negative (dn) mutants of rig-i and mda5, were transfected into 293ft cells with shrnas and an ifn-b promoter reporter construct. the signaling to ifn-b promoter and the expression of the prr proteins were then examined 48 h after transfection. in the absence of sensor proteins, the sh-b971 increased activation of the ifn-b promoter by 2.6-fold ( figure 2a ). coexpression of mda5 or tlr3 did not increase or decrease sh-b971's ability to activate ifn-b promoter relatively to the negative control shrna (sh-ntc), but in the presence of rig-i coexpression, the induction of ifn-b promoter by sh-b971 was increased to $30-fold. moreover, ectopic expression of a dn mutant of rig-i (rig-i c), but not that of mda5 (mda5-c), completely abrogated ifn promoter activation by sh-b971. with the exception of tlr3, which required prolonged exposure of the western blot to be detected, the cytoplasmic sensors and their mutants were expressed at comparable levels ( figure 2b ). moreover, activation of irf-3 ( figure 1e ) and ifn promoters ( figure 1f ) in 293ft cells, which do not contain a functional tlr3 signaling pathway (42) , indicates that tlr3 plays a negligible role, if any, in ifn induction by sh-b971. the combination of sh-b971 and rig-i produced the highest level of ifn-b promoter activity, which were confirmed by western blotting showing that endogenous isg15 induction was only detectable in cells cotransfected with sh-b971 and wild-type rig-i ( figure 2b ). to confirm further that biologically active ifn was released from these cells, we applied the culture medium of the transfected 293ft cells to an hcv replicon cell line (gs5) in which ns5a-gfp expression is used for monitoring viral rna replication (43) . hcv replication in this cell line is extremely sensitive to ifn, and the effect of the cytokine can be readily measured as the change in the mean gfp intensity of the treated cells. as shown in figure 2c , culture medium from sh-b971 efficiently suppressed hcv replication, resulting in a decrease in figure 1 . a small-hairpin rna directed at cypb induces ifn production in human embryonic kidney cells. (a) sequence of sh-b971, which was expressed from a self-inactivating human immunodeficiency virus (hiv) vector with a murine u6 promoter (59) . (b) inhibition of hcv expression by culture media of sh-b971-transfected 293ft cells. gs5 cells were treated with culture supernatant taken from 293ft cells transfected with various shrna plasmids with (left) or without (right) the packaging plasmids overnight. cells were then cultured in fresh media for an additional 6 days before being lysed for western blotting. (c) oas1 induction by culture supernatant from 293ft cells transfected with sh-b971. huh 7 and gs5 cells were treated with culture supernatant from 293ft cells transfected with either sh-luc or sh-b971 for 24 h before rna extraction and real-time rt-pcr analysis. oas1 rna level was normalized to that of gapdh rna. (d) transfected culture media failed to suppress hcv replication in an ifn-resistant cell line. hcv replicon cells were cultured as described earlier (34) and then treated with the indicated culture medium from transfected 293ft cells. hcv rna was analyzed with real-time rt-pcr. (e) irf-3 dimerization in response to sh-b971 expression. flag-irf-3 was cotransfected with a shrna into 293ft cells. cells were lysed 24 h after transfection, and total cell lysate was separated on a polyacrylamide gel under non-denaturing conditions, transferred and stained with an anti-flag antibody. (f) ifn-a and ifn-b promoter activation by sh-b971 expression. sh-ntc, sh-c454 (an shrna directed at cypc), or sh-b971 was cotransfected along with luciferase reporter plasmids with or without irf-7. the ratios of firefly luciferase readings to renilla luciferase readings were plotted. the ns5a-gfp intensity within 48 h of treatment. cotransfecting wild-type rig-i produced a medium with stronger inhibition, whereas the rig-c drastically suppressed the antiviral effect of the medium. finally, real-time rt-pcr analysis revealed that sh-b971, but not the negative control shrna, strongly activated expression of endogenous rig-i, a well-characterized isg whose induction requires paracrine/autocrine action of ifn (44, 45) . as expected, poly i : c activated rig-i expression in the same assay ( figure 2d ). these results, taken together, show that rig-i is the cellular sensor that mediates the ifn induction by sh-b971. the majority of the shrnas that we use in the lab do not activate rig-i expression and ifn signaling despite having essentially the same structure as sh-b971, so we wanted to determine whether the sequence of sh-b971 is distinctive enough to trigger the production of ifn. we first tested a synthetic sirna duplex with the same target sequence as sh-b971. this sirna (si-b971-syn) should resemble the final dicer product of sh-b971 except for the 5 0 -ends. the synthetic sirna contains 5 0 -oh groups, whereas the dicer products probably figure 3a ) while failing to activate ifn production, as measured by the gfp-hcv assay ( figure 3b ). to determine whether the sequence of the intact hairpin rna before dicer cleavage is sufficient to trigger ifn, we tested a synthetic shrna (sh-b971-syn) that had exactly the same sequence as the predicted intracellular sh-b971 transcript generated by the u6 promoter. again, the 5 0 -end of the synthetic sh-b971 had a 5 0 -oh group instead of any phosphate. sh-b971-syn behaved similarly to si-b971-syn in that it knocked down cypb expression without activating ifn response (figure 3) . these results suggest that the 5 0 -end status of sh-b971 is important for ifn activation, consistent with the previously finding that a 5 0 -triphosphate is required for rig-i activation (24, 25) . to determine the contribution of the individual residues of the sh-b971 sequence, we introduced a series of point mutations into the shrna and tested them for ifn induction. we changed the first nucleotide from a to g, c, or t while maintaining base-pairing between nucleotides +1 and +47. these mutant shrnas lacked the ability to activate ifn production (table 1) . changing the +1 nucleotide to g while leaving the +47 nucleotide intact also abolished ifn activation by the shrna (a1/g), as did the reciprocal mutation u47/c. the importance of the first nucleotide was further confirmed by the inability of sh-b971+1 to activate ifn. the target of sh-b971+1 was shifted 1 nt downstream on the cypb mrna, producing an shrna starting with a g at the +1 position. the presence of an a at the +1 position was not, however, sufficient to render a shrna competent for ifn activation, as replacing the first nucleotide of the sh-ntc with an a did not generate an ifn-inducing shrna (ntc-a and ntc+1). these results indicate that a protruding/unpaired a at the end of the hairpin or the rna duplex, a potential result of 'breathing' at the end of the dsrna, is not sufficient to trigger ifn induction as previously suggested (38) . two point mutations located farther into the stem structure of the shrna (9g9 and b18a1) also reduced its ability to induce ifn even though the base-pairing was perfectly maintained in these mutants. finally, replacing the 9-nt hairpin loop with a 7-nt loop that had been previously shown to abolish shrna-mediated rnai (loop a mutant) (46) eliminated sh-b971's ability to induce ifn, suggesting the importance of rna processing in the induction. to determine whether the inability of the mutant shrnas to induce ifn was due to lower expression levels, we performed northern blotting analysis of the shrna expression on the wild-type and two mutants. the mutants a1/g and loop a were chosen because their final sirna products have exactly the same sequence as that of the wild-type sh-b971 and can thus be detected with the same efficiency by the same probe. although sh-a/g and sh-loop a were clearly unable to activate ifn-b promoter ( figure 4a ), they were both expressed at levels comparable to those of the wild-type sh-b971 product ( figure 4b) . interestingly, the final sirna product of sh-loop a was slightly smaller than those of sh-b971 and sh-a1/g, suggesting that cleavage did occur and perhaps occurred one or 2 nt into the stem to compensate for the shorter loop. blunt-ended sirna has been previously reported to be stronger inducers of ifn than the sirnas with overhangs (47) . indeed, a previously reported ifn-inducing shrna, sh-pcaf (p300/creb-binding protein-associated factor), contains a blunt end (38) and was more potent in activating ifn than sh-b971 ( figure 5a ), which is predicted to form an overhang of 2-3 ts at each end of the final sirna. we therefore constructed a version of the sh-b971 that would be blunt at the end that is not processed by dicer by adding two extra as to the 5 0 -end of the shrna. this modification (blunt sh-b971) did not increase the ability of sh-b971 to activate ifn-b promoter ( figure 5a ). we confirmed, in two independent experiments, that ifn induction by sh-pcaf was also mediated by rig-i. first, cotransfection of dn rig-i resulted a 50-to 100-fold inhibition of ifn induction by sh-pcaf ( figure 5b ), whereas wild-type rig-i increased ifn induction by several fold in the same assay. second, when hcv ns3-4a protease, which cleaves mavs, thereby blocking the rig-i pathway, was coexpressed with either sh-b971 or sh-pcaf, ifn induction by these shrnas were severely compromised ( figure 5c ), further substantiating a role of the rig-i and mavs pathway in mediating ifn induction by both the blunt-ended sh-pcaf and the sh-b971 with overhang. the proper expression of ns3-4a protease was confirmed by western blotting ( figure 5d ). to assess the contribution of the promoter choice in ifn activation by intracellular expressed shrna, we expressed sh-b971 from another commonly used pol iii promoter, the human h1 promoter. both the original, mu6-driven sh-b971 and the h1-driven sh-b971 activated ifn-b promoter ( figure 6a ) and resulted in secretion of ifn into the transfected cell-culture media, which in turn suppressed hcv replication ( figure 6b ). proper expression of the sirna ( figure 6c ) and the subsequent knockdown of cypb expression ( figure 6d ) all appeared normal for sh-b971 expressed from the h1 promoter plasmid, which has a backbone different from that of our lentiviral vector carrying the mu6 promoter. these data suggest that ifn induction by sh-b971 is not restricted to a particular promoter or expression construct. further supporting this conclusion was the observation that the expression cassette by itself, removed and isolated from the lentiviral plasmid by restriction digestion, could also activate ifn production in transfected 293ft cells (data not shown). to this point, all the ifn induction experiments were done with transient transfection of dna vectors and it was possible that certain features of the double-stranded plasmid dna are responsible for ifn induction. we first tried to address this point by transfecting just the shrnaexpressing cassette, generated either by pcr or restriction enzyme digestion, into 293ft cells and confirming that these fragments of $200 bp were sufficient to trigger ifn induction (supplementary figure s1) . to definitively rule out any contribution by dsdna, we used a lentiviral transduction system which has been suggested to express shrnas that can escape detection by prrs and ifn activation (48) . we produced lentiviral particles containing shrnas from 293ft cells using standard . sh-b971 expressed from an h1 promoter triggers ifn activation. sh-b971 expressed from an h1 promoter was capable of (a) activating ifn-b promoter and (b) triggering ifn production to inhibit hcv replication in gs5 cells. (c) intracellular levels of u6-and h1-driven sh-b971 products. rna extraction and northern blotting were performed as described in figure 4b . (d) knockdown of cypb expression by sh-b971 expressed from an h1 promoter. methods, centrifuged them to separate the vectors from the ifn-containing media, and then used them to infect naı¨ve 293ft cells ( figure 7a ). both sh-b971 and sh-pcaf vectors induced ifn production when delivered as concentrated lentiviral particles, measured both by hcv suppression ( figure 7b ) and by oas induction ( figure 7c ) in huh-7 cells. to rule out the possibility that residual ifn in the concentrated viral particles was responsible for these results, we added 100 u/ml ifn to the negative control vector sample before the concentration step. this preparation, designated sh-ntc*, was not able to trigger ifn production in naı¨ve 293ft cells, suggesting that the concentration step effectively removed the soluble ifn from the viral particle pellet. proper knockdown of the sirna target of sh-b971 was confirmed by this route of shrna delivery ( figure 7d ). to prove definitively that ifn induction by the shrnas was mediated by the lentiviral infection route, we tested the effect of an inhibitor of hiv reverse transcriptase, nevirapine, on ifn induction by sh-b971 and sh-pcaf. as shown in figure 7e , inclusion of nevirapine at the time of transduction effectively blocked the ability of both shrnas to induce ifn in the transduced cells, suggesting the importance of the reverse transcription step in the expression of the shrnas delivered by the lentiviruses. to determine whether lentiviral vector-delivered shrna can trigger ifn induction in cells other than 293ft cells, we transduced a human hepatoma cell line, lh86, which has been reported to produce ifn upon viral infection (33) , and examined ifn induction in these cells. culture medium from lh86 cells transduced with sh-pcaf contained biologically active ifn, which suppressed hcv replication in gs5 cells ( figure 7f ), indicating that the ability of shrnas delivered by lentivirus to induce ifn response was not limited to 293ft cells. it has been reported that certain chemically synthesized and phage polymerase in vitro transcribed sirnas can non-specifically induce ifn responses and produce offtarget effect via various prrs, including tlrs. however, the induction of ifn response by shrnas and its underlying mechanisms have not been as well studied. the actual number of shrnas that are capable of triggering ifn response will certainly be larger than the few that have been reported in the literature, yet very little is known about the unique characteristics of the select shrnas and the pathway that they use to activate ifn production. the present study identifies rig-i, but not mda5 or tlr3, as the mediator for activation of ifn responses by two shrnas that are distinct in sequence and structure but both capable of ifn induction in human cells. this was demonstrated by induction of irf-3 dimerization, activation of ifn promoters, induction of endogenous isgs (isg15, oas and rig-i), and secretion of ifn, all of which depended on rig-i and its downstream adaptor, mavs. in addition, we show that delivery of these shrnas via lentiviral transduction does not reduce their ifn-inducing capacity, indicating that the ability of lentiviral vector transduction to avoid ifn induction by shrnas, as reported previously (48), may not be universally applicable to all the shrnas. specific recognition of dsrnas or ssrnas bearing 5 0 -triphosphates by rig-i is presumably determined mostly by structural features other than the nucleotide sequence of the rna. yet ifn activation by sh-b971 exhibited a stringent dependence on specific nucleotides at multiple positions of the shrna. an aa dinucleotide at the beginning of the u6 transcript has previously been suggested to result in aberrant transcription, and preserving a c/g sequence at positions à1/+1 suggested to avert ifn induction (38) . we indeed observed a strict requirement for an adenylate at the +1 position of sh-b971 for rig-i recognition and ifn activation, but we observed no difference in expression levels or the apparent sizes of the sh-b971 rnas bearing either an a or a g at the +1 position. furthermore, mutations introduced elsewhere in the shrna also abolished or diminished sh-b971's ability to activate ifn, suggesting additional sequence requirement for efficient rig-i recognition and ifn triggering. despite these results, because we were not successfully in cloning and sequencing the vectorexpressed sirna, we cannot exclude the possibility that the adenylate at the +1 position interferes with transcription and that the resultant abnormal transcript contributes to ifn induction. interestingly, the loop a mutant, which contains a predicted loop of 7 nt, generated a sirna duplex inside the cells that is slightly smaller than that of the shrnas with a wild-type hairpin loop, suggesting the processing by dicer into the stem, perhaps fulfilling the requirement of a length of 9 nt for the hairpin loop (46) . this mutant form of sh-b971 was not, however, able to trigger ifn activation. despite the abilities of both sh-b971 and sh-pcaf to activate the rig-i pathway, the two shrnas are unrelated in sequence. two short stretches of sirna sequences, guccuuccaa and ugugu, that have been previously defined as ifn-or cytokine-activating motifs (8, 9) are not found in either sh-b971 or sh-pcaf. any common sequence motifs of ifn-activating shrnas, if any, remain to be defined. the two shrnas also differ in that one is predicted to contain one blunt end and the other two ends with overhangs. these results suggest that, although blunt ends may increase sirna's ability to be recognized by rig-i (47), they are not required for ifn activation by an endogenously expressed shrna. the best-characterized rna structure motif recognized by rig-i is the 5 0 -ppp, which is absent from virtually all the cellular rnas as a result of either 5 0 -capping or internal cleavage before their appearance in the cytoplasm. a synthetic shrna that has the same sequence as sh-b971 but lacks the 5 0 -ppp failed to induce ifn, suggesting the 5 0 -end status of the intracellularly expressed sh-b971 contributes to ifn activation. whether or not the 5 0 -end of an shrna is capped has not been investigated. murine u6 rna does not contain the trimethylguanosine cap that is present on mrnas and other u small nuclear rnas; instead it contains a g-monomethyl phosphate cap at its 5 0 -end (49) . capping of heterologous transcripts produced from the mu6 promoter, however, requires a stem loop at the 5 0 -end of the transcript and an auauac sequence immediately after (50) . most shrnas, including sh-b971 and sh-pcaf, would not meet these requirements and thus should contain unmodified 5 0 -ppp. similarly, no evidence of a cap structure for h1 transcripts could be found in the literature. we attempted to express sh-b971 using a mirna expression cassette and the pol ii promoter (51) . the primary transcript generated with this construct would be capped at 5 0 -end by a trimethylguanosine cap and the final sirna duplex would bear a monophosphate at the 5 0 -ends of both strands because of drosha and dicer cleavage. this version of the sh-b971 vector was much weaker in its ability to trigger ifn activation. unfortunately the intracellular expression of the rna duplex was also much weaker and barely detectable by northern blotting. in addition, no knockdown of the target cypb mrna was seen with this mirna-based sh-b971 (data not shown). as a result, whether sh-b971, if expressed at higher level from this construct, could effectively activate ifn remains unclear. so far as we know, ours is the first report of ifn activation in the target cells by shrnas delivered by lentiviral transduction. a previous report of ifn induction by lentiviral vector-expressed shrna only examined the ifn generated in the vector-producing cells, which then up-regulated ifn-stimulated genes in the transduced cells (10) . the distinction is important as lentiviral vectors used in a gene-therapy setting will likely be purified and free of any ifn that has been generated during the vector preparation step, but ifn activation in the target cells would pose a more serious concern. our data suggest the importance of screening shrnas for ifn induction in the transduced cells in vitro before largescale studies. an hiv reverse transcriptase inhibitor efficiently blocked ifn production by both sh-b971 and sh-pcaf when delivered by transduction, indicating the virion-encapsulated rna was not able to trigger ifn activation. in this respect, it is interesting to note that positive-stranded rna viruses, which produce dsrna intermediates in the cytoplasm during replication (52) (53) (54) (55) , often replicate in membrane enclosed vesicles (56) , this sequestration of viral dsrna in membranous structures may shield the rna from the cytoplasmic prrs and contribute to a successful infection. ifn-induction and rnai by shrnas appear to be independent functions of the same rna (57). our results also showed that ifn-induction by sh-b971 is independent of its ability to suppress target mrna expression through rnai. on the other hand, it might be possible to screen for duel functional sirnas that confer therapeutic benefits by both rnai and immunostimulation (58) . for example, sirnas that target either viral genomes or cellular cofactors of the viruses can be screened for their ability to trigger ifn activation in hopes of find 'super sirnas' with increased efficacy against ifn-sensitive viruses. an rna-directed nuclease mediates post-transcriptional gene silencing in drosophila cells single-stranded antisense sirnas guide target rna cleavage in rnai human risc couples microrna biogenesis and posttranscriptional gene silencing pathogen recognition and innate immunity activation of the interferon system by short-interfering rnas small interfering rnas mediate sequence-independent gene suppression and induce immune activation by signaling through toll-like receptor 3 interferon induction by sirnas and ssrnas synthesized by phage polymerase sequence-specific potent induction of ifn-alpha by short interfering rna in plasmacytoid dendritic cells through tlr7 sequence-dependent stimulation of the mammalian innate immune response by synthetic sirna induction of an interferon response by rnai vectors in mammalian cells 0 -triphosphate-dependent activation of pkr by rnas with short stem-loops the rna helicase rig-i has an essential function in doublestranded rna-induced innate antiviral responses differential roles of mda5 and rig-i helicases in the recognition of rna viruses essential role of mda-5 in type i ifn responses to polyriboinosinic : polyribocytidylic acid and encephalomyocarditis picornavirus ips-1, an adaptor triggering rig-i-and mda5-mediated type i interferon induction cardif is an adaptor protein in the rig-i antiviral pathway and is targeted by hepatitis c virus identification and characterization of mavs, a mitochondrial antiviral signaling protein that activates nf-kappab and irf 3 visa is an adapter protein required for virus-triggered ifn-beta signaling the v proteins of paramyxoviruses bind the ifn-inducible rna helicase, mda-5, and inhibit its activation of the ifn-beta promoter shared and unique functions of the dexd/h-box helicases rig-i, mda5, and lgp2 in antiviral innate immunity polyinosinicpolycytidylic acid induces the expression of gro-alpha in beas-2b cells ebola virus vp35 protein binds double-stranded rna and inhibits alpha/beta interferon production induced by rig-i signaling expression of ip-10/cxcl10 is upregulated by double-stranded rna in beas-2b bronchial epithelial cells rig-i-mediated antiviral responses to single-stranded rna bearing 5 0 -phosphates aim2 recognizes cytosolic dsdna and forms a caspase-1-activating inflammasome with asc aim2 activates the inflammasome and cell death in response to cytoplasmic dna an orthogonal proteomic-genomic screen identifies aim2 as a cytoplasmic dna sensor for the inflammasome hin-200 proteins regulate caspase activation in response to foreign cytoplasmic dna dai (dlm-1/zbp1) is a cytosolic dna sensor and an activator of innate immune response rna polymerase iii detects cytosolic dna and induces type i interferons through the rig-i pathway characterization of hepatitis c virus subgenomic replicon resistance to cyclosporine in vitro hepatitis c virus triggers apoptosis of a newly developed hepatoma cell line through antiviral defense system defective jak-stat activation in hepatoma cells is associated with hepatitis c viral ifn-alpha resistance induction of irf-3/-7 kinase and nf-kappab in response to double-stranded rna and virus infection: common and unique pathways a kaposi's sarcoma-associated herpesviral protein inhibits virus-mediated induction of type i interferon by blocking irf-7 phosphorylation and nuclear accumulation identification of cellular cofactors for human immunodeficiency virus replication via a ribozyme-based genomics approach determinants of interferonstimulated gene induction by rnai vectors gb virus b disrupts rig-i signaling by ns3/4a-mediated cleavage of the adaptor protein mavs distinct poly(i-c) and virus-activated signaling pathways leading to interferon-beta production in hepatocytes cyclophilin a is an essential cofactor for hepatitis c virus infection and the principal mediator of cyclosporine resistance in vitro recognition of double-stranded rna and activation of nf-kappab by toll-like receptor 3 effect of cell growth on hepatitis c virus (hcv) replication and a mechanism of cell confluencebased inhibition of hcv rna and protein expression antitumor nk activation induced by the toll-like receptor 3-ticam-1 (trif) pathway in myeloid dendritic cells central role of interferon regulatory factor-1 (irf-1) in controlling retinoic acid inducible gene-i (rig-i) expression a system for stable expression of short interfering rnas in mammalian cells a structural basis for discriminating between self and nonself double-stranded rnas in mammalian cells stable expression of shrnas in human cd34+ progenitor cells can avoid induction of interferon responses to sirnas in vitro gamma-monomethyl phosphate: a cap structure in spliceosomal u6 small nuclear rna capping of mammalian u6 small nuclear rna in vitro is directed by a conserved stem-loop and auauac sequence: conversion of a noncapped rna into a capped rna a lentiviral microrna-based system for single-copy polymerase ii-regulated rna interference in mammalian cells visualization of double-stranded rna in cells supporting hepatitis c virus rna replication double-stranded rna is produced by positive-strand rna viruses and dna viruses but not in detectable amounts by negative-strand rna viruses subcellular localization and membrane topology of the dengue virus type 2 non-structural protein 4b sars-coronavirus replication is supported by a reticulovesicular network of modified endoplasmic reticulum seeking membranes: positive-strand rna virus replication complexes sirna and isrna: two edges of one sword 0 -triphosphate-sirna: turning gene silencing and rig-i activation against melanoma design of hiv vectors for efficient gene delivery into human hematopoietic cells the authors thank dr andre irsigler and dr jason robotham for technical assistance and dr anne b. thistle for proofreading the manuscript. supplementary data are available at nar online.conflict of interest statement. none declared. key: cord-256293-5lpe8hg2 authors: kageyama, y.; aida, k.; kawauchi, k.; morimoto, m.; ebisui, t.; akiyama, t.; nakamura, t. title: jinhua qinggan granule, a chinese herbal medicine against covid-19, induces rapid changes in the plasma levels of il-6 and ifn-î³ date: 2020-06-12 journal: nan doi: 10.1101/2020.06.08.20124453 sha: doc_id: 256293 cord_uid: 5lpe8hg2 background: currently, effective vaccines or specific therapeutic agents against covid-19 are not available. however, in china, traditional chinese herbal medicines have provided therapeutic benefit to patients with covid-19. jinhua qinggan granule (jhqgg) is a chinese multi-herbal formula previously developed for the treatment of h1n1 influenza and has been encouraged for patients clinically suspected of covid-19 during medical observation. however, the immunological mechanism for the efficacy of jhqgg has not been confirmed. objectives: we thus examined whether the administration of jhqgg affects hematological and immunological measures in healthy individuals. method: we enrolled 18 healthy volunteers, all of whom tested negative for antibodies to sars-cov-2. peripheral blood samples were collected 1 h after oral administration of jhqgg and subjected to hematological, biochemical, and cytokine tests. results: jhqgg rapidly induced a significant decrease in the plasma level of il-6 and an increase in the plasma level of ifn-{gamma}. conclusions: our finding suggests that the therapeutic efficacy of jhqgg against covid-19 is, in part, associated with its rapid immunomodulatory activity. there have been no specific vaccines or drugs proven to be clinically effective against covid-19. however, in china, the majority of covid-19 patients have been treated with a combination of traditional chinese and modern western medicine [1] [2] [3] . the use of several chinese herbal formulas is encouraged for the treatment of covid-19 in the latest version of the diagnosis and treatment protocol released by the national health commission of china. one of them is jinhua qinggan granule (jhqgg), which was formulated specifically for the symptoms of h1n1 influenza [4] . previous preclinical studies showed that jhqgg reduces pulmonary lesions and mortality in mice infected with the h1n1 influenza virus [4] . a clinical study also demonstrated that jhqgg reduces the duration of fever and alleviates respiratory symptoms of patients with h1n1 influenza [4] . on the basis of its therapeutic efficacy for influenza, jhqgg has been recommended for patients clinically suspected of covid-19 during medical observation. in a randomized controlled trial using mild cases in wuhan, combined administration of jhqgg with western medicine significantly ameliorated respiratory symptoms and relieved psychological anxiety compared with the administration of western medicine alone [5] [6] [7] . however, the pharmacological mechanism underlying the efficacy of jhqgg has not been confirmed. we thus examined whether jhqgg administration affects hematological and immunological measures in healthy individuals. this is an open-label, single-arm study to obtain a clue to the pharmacological action of jhqgg. we enrolled a total of 18 healthy volunteers (5 males, 13 females; ages 22-58 years; mean age [sd], 33.8 [10.7] years), all of whom tested negative for igm and igg antibodies to sars-cov-2. individuals were excluded if they had current infectious, inflammatory, or immune-related diseases. jhqgg was kindly provided by hugh wang, juxiechang (beijing) pharmaceutical co., ltd. the subjects were instructed to take a packet (5 g) orally 40 min after lunch. this dose is known to be effective for the treatment of influenza [8] . peripheral blood samples were obtained 1 h after the administration. hematological and biochemical tests were outsourced to srl, inc. (tokyo, japan). plasma all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june 12, 2020. . https://doi.org/10.1101/2020.06.08.20124453 doi: medrxiv preprint cytokines were quantified using v-plex proinflammatory panel 1 human kit (meso scale diagnostics) and human il-18 elisa kit (abcam). all collected data (n = 18) were subjected to the statistical analysis using a two-tailed paired t-test. hematocrit and mean corpuscular volume changed marginally within the normal ranges (table 1) , but there were no significant differences in other measures of complete blood count and blood biochemistry between pre-and post-administration. notably, in blood cytokine analysis, the plasma levels of il-6 and ifn-γ were significantly decreased and increased, respectively, compared with those in pre-administration (il-6, 2.75 vs. 1.63, 95% ci: -1.90 to -0.326, p = 0.00837; ifn-γ, 6.20 vs. 7.17, 95% ci: 0.193-1.73, p = 0.0172). the plasma il-6 was decreased in 14 (77.8%) out of 18 subjects, whereas the plasma ifn-γ was increased in 13 (72.2%) out of 18 subjects (fig. 1) . we also found a relatively large decrease in the il-18 level; however, the difference was not statistically significant (266 vs. 193, 95% ci: -154-7.69, p = 0.0732). dysregulated cytokine production is a hallmark of patients with covid-19. elevated blood levels of il-2, soluble il-2 receptor (sil-2r), il-4, il-6, il-10, and tnf-α have been observed especially in severe cases [9] [10] [11] [12] . in particular, il-6 plays pivotal roles in the exacerbation of covid-19. evidence is accumulating that an aberrant increase in il-6 leads to complex immune dysregulation caused by sars-cov-2 infection. the blood il-6 level is positively correlated with the severity and mortality in covid-19 patients [13] [14] [15] . ifn-γ is a central mediator of antiviral immunity with an ability to directly interfere with viral replication. in addition, ifn-γ is indirectly involved in viral clearance through potentiating the action of ifn-α/β, activating th1-dependent immune responses, and enhancing the mhc class i pathway [16] . compared to healthy individuals, covid-19 patients have significantly lower numbers of cd4 + t, cd8 + t, and nk cells with reduced capacity to produce ifn-γ, which causes the slightly decreased expression of ifn-γ [10, 17] . notably, the reduced cytotoxic potential of cd4 + t, cd8 + t, and nk cells is il-6-dependent [17] and severe covid-19 cases have a higher il-6/ifn-γ ratio than moderate cases [18] . in conclusion, jhqgg can down-and up-regulate the plasma levels of il-6 and ifn-γ, respectively, immediately after oral administration. the rapid immunomodulatory effects of jhqgg may be able to remedy the immune dysregulation observed in covid-19 patients all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june 12, 2020. . https://doi.org/10.1101/2020.06.08.20124453 doi: medrxiv preprint 4 and thus provide therapeutic benefit to mild to severe cases as well as asymptomatic or suspected cases. we thank hugh wang [juxiechang (beijing) pharmaceutical co., ltd.] for generously providing us with jhqgg. this study was carried out in accordance with the code of ethics of the world medical association (declaration of helsinki). all procedures were approved by the ethics committees of the takanawa clinic (approval number: 2020-2). a signed informed consent form was obtained from each participant prior to inclusion in this study. all experimental procedures and data analyses were conducted by investigators who were blinded to the subjects' clinical information using a de-identified dataset. this study has been registered on under the trial number umin000040407. this research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. ebisui contributed to the conception and design of the study, contributed to data acquisition, analysis, and interpretation, and critically revised the manuscript for important intellectual content. tetsu akiyama contributed to the conception and design of the study and critically revised the manuscript for important intellectual content. tsutomu nakamura contributed to the conception and design of the study, contributed to data analysis and interpretation, and drafted the manuscript. all authors gave final approval of the submitted version of the manuscript and agree to be accountable for all aspects of the work. all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june 12, 2020. 12. cao x. covid-19: immunopathology and its implications for therapy. nat rev all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june 12, 2020. . https://doi.org/10.1101/2020.06.08.20124453 doi: medrxiv preprint all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june 12, 2020. . https://doi.org/10.1101/2020.06.08.20124453 doi: medrxiv preprint high-density lipoprotein; ldl: low-density lipoprotein; crp: c-reactive protein; ifn: interferon; il: interleukin; tnf-α: tumor necrosis factor-α; sd: standard deviation; ci: confidence interval. *p < 0.05, **p < 0.01. all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june 12, 2020. pre post all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june 12, 2020. . https://doi.org/10.1101/2020.06.08.20124453 doi: medrxiv preprint traditional chinese medicine for covid-19 treatment traditional chinese medicine in the treatment of patients infected with 2019-new coronavirus (sars-cov-2): a review and perspective the important role of polysaccharides from a traditional chinese medicine-lung cleansing and detoxifying decoction against the covid-19 pandemic complementary and alternative medicine is expected to make greater contribution in controlling the prevalence of influenza herbal medicine for the treatment of coronavirus disease 2019 (covid-19): a systematic review and meta-analysis of randomized controlled trials efficacy and safety of integrated traditional chinese and western medicine for corona virus disease 2019 (covid-19): a systematic review and meta-analysis the clinical benefits of chinese patent medicines against covid-19 based on current evidence treating influenza patients of windheat affecting fei syndrome by jinhua qinggan granule: a double-blinded randomized control trial. zhongguo zhong xi yi jie he za zhi clinical features of patients infected with 2019 novel coronavirus in wuhan clinical and immunological features of severe and moderate coronavirus disease 2019 complex immune dysregulation in covid-19 patients with severe respiratory failure imbalanced host response to sars-cov-2 drives development of covid-19 viral and host factors related to the clinical outcome of covid-19 molecular mechanisms of ifn-γ to up-regulate mhc class i antigen processing and presentation impaired immune cell cytotoxicity in severe covid-19 is il-6 dependent high il-6/ifn-γ ratio could be associated with severe disease in covid-19 patients key: cord-000725-rafwlw0t authors: hindinger, claudia; bergmann, cornelia c.; hinton, david r.; phares, timothy w.; parra, gabriel i.; hussain, shabbir; savarin, carine; atkinson, roscoe d.; stohlman, stephen a. title: ifn-γ signaling to astrocytes protects from autoimmune mediated neurological disability date: 2012-07-27 journal: plos one doi: 10.1371/journal.pone.0042088 sha: doc_id: 725 cord_uid: rafwlw0t demyelination and axonal degeneration are determinants of progressive neurological disability in patients with multiple sclerosis (ms). cells resident within the central nervous system (cns) are active participants in development, progression and subsequent control of autoimmune disease; however, their individual contributions are not well understood. astrocytes, the most abundant cns cell type, are highly sensitive to environmental cues and are implicated in both detrimental and protective outcomes during autoimmune demyelination. experimental autoimmune encephalomyelitis (eae) was induced in transgenic mice expressing signaling defective dominant-negative interferon gamma (ifn-γ) receptors on astrocytes to determine the influence of inflammation on astrocyte activity. inhibition of ifn-γ signaling to astrocytes did not influence disease incidence, onset, initial progression of symptoms, blood brain barrier (bbb) integrity or the composition of the acute cns inflammatory response. nevertheless, increased demyelination at peak acute disease in the absence of ifn-γ signaling to astrocytes correlated with sustained clinical symptoms. following peak disease, diminished clinical remission, increased mortality and sustained astrocyte activation within the gray matter demonstrate a critical role of ifn-γ signaling to astrocytes in neuroprotection. diminished disease remission was associated with escalating demyelination, axonal degeneration and sustained inflammation. the cns infiltrating leukocyte composition was not altered; however, decreased il-10 and il-27 correlated with sustained disease. these data indicate that astrocytes play a critical role in limiting cns autoimmune disease dependent upon a neuroprotective signaling pathway mediated by engagement of ifn-γ receptors. cns resident cells are targets of autoimmune mediated damage but also active participants in disease development, progression and control [1, 2] . however, their contributions to neuroprotection and regulation by inflammatory mediators are not well defined. cns insults, including autoimmune disease, initiate rapid astrocyte activation characterized by cellular hypertrophy and increased intermediate filament glial fibrillary acidic protein (gfap) expression [3] [4] [5] . astrocytes form a physical barrier surrounding areas of inflammation initially limiting bystander tissue damage [6, 7] . however, this barrier subsequently impedes axonal regeneration contributing to sustained disability [3, 5, 8] . innate and adaptive pro-inflammatory astrocyte functions include production of pro-inflammatory cytokines, reactive oxygen species, chemokines, and matrix metalloproteinases [3] [4] [5] . by contrast, secretion of anti-inflammatory cytokines and scavengers of reactive oxygen species, as well as inhibition of both microglial activation and tumor necrosis factor (tnf) secretion, all support an astrocyte mediated anti-inflammatory function [3] [4] [5] . therefore, astrocyte activation constitutes a ubiquitous, yet heteroge-neous response associated with both promoting and inhibiting cns repair [3] [4] [5] . ms and eae are both associated with t cells secreting ifn-c (th1 cells) and il-17 (th17 cells) which play complex, not fully understood roles in disease initiation and progression [2, 9] . in vivo and in vitro evidence indicates that suppression of encephalitogenic t cell proliferation within the cns during eae and activation of anti-inflammatory programs are in part mediated via astrocytes [4, 5] . similar to astrocytes, ifn-c mediates both proand anti-inflammatory functions during autoimmune disease [10] . early ifn-c induced effects are pro-inflammatory; ifn-c facilitates inflammatory cell access, shapes their composition, increases major histocompatibility complex (mhc) expression, contributes to macrophage and microglia activation, and initiates oligodendrocyte death [11] . similarly, ifn-c mediated protection during eae is also multifaceted [12] [13] [14] [15] . it acts as a negative regulator of neutrophil accumulation, th17 cell activation, il-1r signaling, protease secretion, and chemokine activity. it also inhibits proinflammatory cytokine secretion via induction of suppressor of cytokine secretion (socs) proteins, facilitates t cell apoptosis and protects oligodendrocytes via inducing endoplasmic reticulum (er) stress responses [10, 11, 16, 17] . this highlights the critical role of a single mediator in both promoting disease but also limiting inflammatory mediated damage required to initiate repair cascades. based on the gatekeeper functions of astrocytes and the diverse biological effects of ifn-c, we set out to determine how ifn-c signaling specifically to astrocytes influences cns autoimmune disease. the results demonstrate that ifn-c signaling to astrocytes had no profound effects on initial disease progression, but played an essential protective role during the transition from acute to chronic disease. clinical remission induced by ifn-c signaling to astrocytes coincided with reduced demyelination, axonal degeneration, and astrocyte activation. the ifn-c receptor is expressed ubiquitously; however, these data reveal astrocytes as the primary target and mediator of the well established antiinflammatory activity of ifn-c within the cns. to understand the role of ifn-c signaling to astrocytes during the pathogenesis of cns autoimmune disease, eae was induced in transgenic mice expressing a signaling deficient dominant negative ifn-c receptor 1 specifically on astrocytes (gfapcr1d mice) [18] . peripheral activation of self reactive t cells in gfapcr1d mice was similar to wt mice (data not shown). this is consistent with both the cns restricted transgene expression in the gfapcr1d mice as well as the similar t cell activation following peripheral immunization with a non-self antigen [18] . following immunization the incidence of disease, initiation of clinical symptoms, and initial symptom progression were unaltered by the inability of astrocytes to respond to ifn-c ( fig. 1a ; table 1 ). in addition, neither immunized group exhibited clinical symptoms of atypical eae associated with the absence of ifn-c [19] . however, clinical symptoms in gfapcr1d mice began to diverge from wt mice at , day 12 post immunization (p.i.) prior to the peak of clinical disease. in both groups clinical disease peaked at , day 14 p.i., but severity was increased from a score of 3.1 in wt mice to 4.0 in the gfapcr1d group ( fig. 1a ; table 1 ). gfapcr1d and wt mice were compared at the peak of acute disease to determine if ifn-c signaling altered astrocyte activation or cns inflammation. astrocyte hypertrophy and gfap expression ( fig. 1b) were similar in both groups, indicating no overt effects of ifn-c on initial astrocyte reactivity. furthermore, neither the extent of inflammation nor the anatomic distribution of inflammatory cells was altered (fig. 1b) . flow cytometry confirmed no difference in the overall extent of cd45 hi inflammatory cells recruited into the cns ( fig. 2a) . furthermore, percentages of cd4 + t cells ( fig. 2a) , cd8 + t cells and macrophages within the infiltrates were also similar (fig. 3) . in contrast to the association between increased eae severity and neutrophil accumulation in the global absence of ifn-c [14, 15] , only a small percentage of neutrophils were identified in the cns of both groups by flow cytometry (fig. 3) ; their low presence was confirmed by the inability to identify cells with the characteristic morphology of neutrophils in the brain by histopathology (fig. 1b) . the absence of increased neutrophils in the cns of gfapcr1d mice during acute disease suggested that clinical disease was aggravated by a mechanism distinct from global ifn-c deficiency. in addition to the similar frequency of cd4 + t cells in the brains of the two groups ( fig. 2a) , myelin oligodendrocyte glycoprotein (mog) reactive cd4 + t cells secreting ifn-c were also similar ( fig. 2b ). by contrast, mog specific cd4 + t cells secreting il-17 ( fig. 2b ) and foxp3 + regulatory cd4 + t cells (tregs) were decreased in gfapcr1d mice compared to wt mice (fig. 2c) . reduced th17 and tregs may be attributed to increased ifn-c [10, 16] . alternatively, as ifn-c is protective in eae [12] [13] [14] [15] , increased disease severity may reflect reduced bioavailable ifn-c due to sequestration of ifn-c binding to the dominant negative receptor. however, measurement of ifn-c in cell free supernatants derived from dissociated brains at the peak of acute disease demonstrated protein levels of 2.160.2 ng/brain in wt mice versus 3.660.5 ng/brain in gfapcr1d mice (n = 3; p,0.05). as overall frequencies of mog reactive cd4 + t cells secreting ifn-c were similar, increased ifn-c in the brains of gfapcr1d mice suggested enhanced secretion at the cellular level. although a contribution of nk or cd8 t cells could not be excluded, these potential sources of ifn-c were unlikely due to their low frequencies (nk ,5% and cd8 + t cells , 5%, see fig. 3 ) and their equivalent frequencies in the wt and gfapcr1d mice. furthermore, reduced th17 cell and treg frequencies were consistent with suppression of these populations due to elevated ifn-c [10, 16, 20] . inflammation in spinal cords from the gfapcr1d mice was also similar to wt controls at the peak of clinical symptoms (fig. 4) . although numbers and distribution of cd4 + t cells were also similar (4.661.3/mm 2 in gfapcr1d mice vs. 3.860.1/ mm 2 in wt mice; fig. s1 ), spinal cords of gfapcr1d mice exhibited a ,2-fold increase in demyelination (fig. 4) . areas of demyelination encompassed 7.361.0% of spinal cord white matter in gfapcr1d mice versus 3.860.9% in wt mice (p#0.01). furthermore, the increase in demyelination was associated with a prominent loss of axons in gfapcr1d mice compared to controls (fig. 4) . despite elevated demyelination and axonal loss in the absence of ifn-c signaling to astrocytes, spinal cords showed no evidence of differential astrocyte activation by either immunohistochemistry (fig. 4 ), or differences in gfap mrna expression during the peak of acute disease (fig. 5 ). although ccl5, il-1 and tnf mrna expression were increased in the spinal cords of the gfapcr1d mice, no differences in ifn-c, inos, cxcl10 or il-6 mrna were consistent with similar inflammation (fig. 5) . these data suggest that the initial disease pathogenesis, reflected by an increased demyelination in spinal cords, but not brain, during ascending paralysis is dampened by ifn-c signaling to astrocytes. subsequent to peak disease severity the clinical symptoms in wt mice began a modest decline by day 16 p.i. (fig. 1a ). by contrast, gfapcr1d mice not only exhibited increased severity of clinical symptoms following day 14 p.i., but the continued disease escalation was associated with increased mortality ( fig. 1a ; table 1 ). sustained morbidity and significant mortality in gfapcr1d mice after day 30 p.i. implied a critical role for ifn-c induced astrocyte signaling in neuroprotection and limiting disability. a similar absence of clinical remission was found in a preliminary experiment comparing eae sjl mice carrying the gfapcr1d gene (data not shown). these data suggest that astrocyte responses to ifn-c are protective, irrespective of genetic background. escalating disease in gfapcr1d mice coincided with focal areas of intense inflammation in spinal cords, which contrasted with the more diffuse inflammation in wt mice (fig. 6 ). demyelination was also increased with myelin loss encompassing 5.761.8% of spinal cord area in gfapcr1d mice versus 2.361.4% in wt mice (p#0.05) at day 35 p.i. (fig. 6 ). the demyelinated areas further exhibited enhanced axonal damage in gfapcr1d mice (fig. 6) , supporting a correlation between enhanced tissue damage, sustained morbidity and increased mortality ( fig. 1a ; table 1 ). astrocyte activation associated with areas of myelin loss is a prominent finding in the cns of both patients with ms and rodents with eae. although demyelination was increased in the cns of gfapcr1d mice, the extent of astrocyte activation associated with spinal cord white matter lesions was similar in both groups ( fig. 6 ; ,60 gfap + cells/mm 2 ). by contrast, the frequency of activated astrocytes in spinal cord grey matter areas that were not associated with demyelinated lesions, was increased in gfapcr1d mice with 101.5623.0 gfap + activated astrocytes/mm 2 versus 25.5615.5 in wt mice (p,0.001) (fig. 7) . a similar increase in astrocyte activation within grey matter distal to white matter lesions was also detected in gfapcr1d sjl mice during chronic eae (data not shown). flow cytometric analysis during chronic disease revealed an ,4fold increase in cd45 hi inflammatory cells confirming increased inflammation in the absence of ifn-c signaling to astrocytes (fig. 8a ). similar to the acute disease, relative percentages of neutrophils, macrophages, cd4 + and cd8 + t cells were all similar (fig. 3) . furthermore, no differences in expression of activation markers on cns derived cd4 + t cells, including cd69, cd122, cd127, fas, fasl, icos and cd152 were evident between the groups (data not shown). the percentages of mog reactive th1 cells were also not altered in the cns of gfapcr1d relative to wt mice (fig. 8b ) and the decreased percentage of potentially destructive th17 cells identified during acute disease (fig 2) , was also sustained during chronic disease (fig. 8b ). equivalent expression of il-7, il-23 and tgf-b mrna (fig. 8c ) suggested that the decrease in th17 cells was dependent upon an increase in ifn-c and not related to a defect in activation or maintenance signals [20] . increased inflammation and sustained mhc class ii expression on microglia (fig. 8a ) further suggested that ifn-c signaling to astrocytes down regulates inflammation universally without altering the composition of the cns infiltrates. this concept is supported by sustained composition of cns infiltrates during inflammation induced astrocyte apoptosis [21] . the inability to restrain ongoing inflammation during eae in gfapcr1d mice was evident at multiple levels. astrocyte activation was sustained consistent with increased expression of gfap mrna. ccl2, ccl5 and cxcl10 mrna levels were increased (fig. 8c ). the expression of mrna encoding potentially destructive immune mediators including il-1, il-6, inos, and tnf were all increased (fig. 8c ). ifn-c was ,2-fold higher in the cns of gfapcr1d mice (fig. 9) . lastly, the level of the antiinflammatory cytokine il-10 was reduced ( fig. 9 ), suggesting limited availability of il-10 may be a key in prolonging astrocyte activation and inflammation [22] . a possible link between reduced il-10 and the inability of ifn-c signaling to astrocytes is provided by the capacity of ifn-c to induce il-27 in astrocytes [23] , thereby promoting il-10 production. indeed il-27 in the cns of the gfapcr1d mice was significantly reduced compared to wt mice during chronic disease (fig. 9 ). by contrast, il-12 ( fig. 9) , an indirect suppressor of cns inflammation by promoting ifn-c production [24] , was similar in the cns of both gfapcr1d and wt mice. astrocytes derived from gfapcr1d and wt mice were stimulated with ifn-c to confirm ifn-c dependent il-27 secretion by astrocytes [25] . while ifn-c induced il-27 secretion by astrocytes from wt mice, il-27 secretion was significantly reduced in cultures derived from gfapcr1d mice (fig. 10) . these data support the possibility that ifn-c mediated il-27 secretion by astrocytes regulates eae effector t cell function, inflammation and tissue destruction via induction of il-10; however, this does not exclude the possibility that the inability of astrocytes to respond to ifn-c could directly or indirectly dysregulate a variety of other immunomodulatory functions [4, 5, 17] . in the eae model of ms, ifn-c functions as a proinflammatory cytokine in the early stages of disease [10, 11, 26 ], yet it also assumes a prominent anti-inflammatory role during the transition to remission [10, [12] [13] [14] [15] . protection has been attributed to a variety of potentially interrelated mechanisms including limiting neutrophil accumulation, th17 cell activation, il-1r signaling, matrix metalloproteinase secretion, pro-inflammatory cytokine secretion and chemokine activity; in addition ifn-c facilitates t cell apoptosis and protects oligodendrocytes from death via an er stress response [10, 11, 17] . the activities of astrocytes during autoimmunity also range from pro-to antiinflammatory [3] [4] [5] . however, to what extent a direct action of ifn-c on astrocytes contributes to inhibitory mechanism is unclear. the data herein demonstrate that among the multiple early functions of astrocytes imposed by innate responses are largely pro-inflammatory during eae [3, 6, 7, 26] . for example, astrocytes contribute to the loss of bbb integrity via secretion of reactive oxygen species, chemokines and pro-inflammatory cytokines [3] [4] [5] . this pro-inflammatory milieu is associated with initial axonal damage prior to accumulation of self reactive t cells [27] , which further promotes immune mediated damage. blocking the pro-inflammatory transcription factor nf-kb in astrocytes improves recovery during chronic eae [28, 29] . supporting an initial pro-inflammatory role of astrocytes dependent on innate, not ifn-c responsiveness, inhibition of ifn-c signaling to astrocytes did not influence eae onset or incidence, initial disease progression, astrocyte activation, or bbb integrity as indicated by similar entry of inflammatory cells into the brain. by contrast, an inflammation dampening effect of ifn-c became evident during the onset of disease remission. astrocytes limit ongoing inflammation and pathogenic processes at several levels. they not only facilitate repair by inhibiting inflammatory cell entry into the cns parenchyma [6, 7, 30] , but also down regulate t cell effector function and proliferation [4, 5, 21] . for example, eae in gfap deficient mice results in more severe and widespread inflammation [31] . in addition, t cell-astrocyte interactions as well as astrocyte secretion of an unidentified soluble product, distinct from nitric oxide, prostaglandins, or tryptophan metabolism, suppress t cell proliferation [4, 5] . t cell proliferation was also not inhibited via defective il-2 release, despite the suggestion that t cell-astrocyte interactions facilitate secretion of anti-inflammatory cytokines [4, 5] . lastly, astrocytes may limit inflammation by triggering apoptosis in t cells [32] . nevertheless, the role of ifn-c signaling in these potentially anti-inflammatory, protective mechanisms is not clear. elevated ifn-c in the cns of gfapcr1d mice during both acute and chronic disease excluded limited ifn-c due to sequestration by the transgenic receptor as a mediator of increased clinical severity and mortality. indeed, enhanced ifn-c production may reflect an attempt to compensate for the loss of ifn-c dependent astrocyte mediated control of inflammation [33] . sustained expression of pro-inflammatory cytokines, particularly il-6 and tnf, thus represents a primary mechanism underlying ongoing tissue destruction in gfapcr1d mice. il-6 is known to mediate neurological dysfunction [34] and astrocytes are the predominant source of il-6 during cns autoimmune disease. furthermore, its secretion is down regulated by ifn-c induced socs activity [17] . on the other hand, sustained tnf implicated dysregulated activation of microglia or macrophages via increased ifn-c or il-6, as tnf is predominantly secreted by activated cns macrophages and microglia during eae [35, 36] . however, the down regulation of microglia activation, including tnf secretion, following interaction with activated astrocytes [37] questions this notion. while both il-6 and tnf may thus contribute to sustained pathological changes, the source of tnf remains unclear. similarly, astrocytes are a potential source of the ifn-c induced chemokine cxcl10 during eae, one of the chemokines controlling t cell recruitment [38] . however, the increased expression of cxcl10 coupled with the inability of the astrocytes in the gfapcr1d mice to respond to ifn-c suggests altered microglia activation and secretion of cxcl10 [38] or activation of cxcl10 transcription via an independent signaling pathway mediated by tnf or type 1 interferons [39, 40] . importantly, pathology further correlated with decreases in both tregs and il-10, but not with increased antigen specific th17 cells, although astrocytes are critical targets of il-17 [41] . although it is possible that the increased ifn-c in the cns influenced peripheral activation of antigen specific th17 cells, previous data demonstrated no evidence for expression of the transgene in peripheral organs, including lymphoid organs [18] . il-10, secreted by a variety of cells types including cd4 + t cells, cd8 + t cells and tregs [42] , inhibits both acute and chronic eae [43, 44] . the decrease in this anti-inflammatory cytokine suggests that the induction of il-27 secretion is a critical aspect of astrocyte responses to ifn-c, thus promoting il-10 secretion by t cells [23, 25] . however, our data is unable to distinguish the contribution of paracrine versus autocrine ifn-c induced signals to astrocytes on il-10 production, as il-10 also regulates astrocyte activation [22] , and can itself be secreted by astrocytes to exert autologous functions [45] . in addition to mediating an imbalance of protective versus detrimental cytokines, our results demonstrate that ifn-c signaling to astrocytes limits the extent of astrocyte activation during chronic eae, specifically in grey matter. activated astrocytes, especially within and adjacent to areas of demyelination are a prominent feature of white matter plaques associated with both chronic ms and eae [3] [4] [5] . astrocytes protect neurons and oligodendroglia, but also form glial scars which inhibit regeneration after neuronal injury [3, 5, 8, 20] . the absence of reactive astrocytes increases axonal regeneration after injury [46] , consistent with the concept that limiting astrogliosis is critical for axonal regeneration after neuronal injury. while the significance of sustained astrocyte activation in grey matter tracks is unclear in our model, it is consistent with increased axonal damage, and suggests possible damage to axons outside the lesions, which may not be manifested by histological analysis. limiting inflammation following either infection or during an autoimmune attack is a prerequisite for the initiation of repair. this is critically important within the cns which has limited regenerative capacity. in summary, our data identify astrocytes as prominent targets underlying ifn-c mediated suppression of chronic cns inflammation [19] . the data further provide a link between sustained inflammatory responses, enhanced demyelination and axonal degeneration associated with loss of neurological function during eae and chronic progressive ms. although the inability of astrocytes to respond to ifn-c did not alter disease in the brain, engagement of the ifn-c receptor on astrocytes in spinal cord limits demyelination and functions in a neuroprotective capacity. current therapies for ms are primarily focused on antiinflammatory and immunomodulatory approaches and have been partially successful in treating acute episodes. the identification of astrocytes as critical responders and mediators of ifn-c signaling in limiting cns autoimmune disease may provide insights into new approaches to limit long term progression to disability. brains and spinal cords from perfused mice were homogenized separately as previously described [47, 48] . homogenates were centrifuged at 4506g for 7 min at 4uc. supernatants were stored at -80uc for cytokine determination (see below). cells were resuspended in 30% percoll (amersham biosciences, piscataway, nj) and isolated by centrifugation (8006g for 30 min at 4uc) onto 70% percoll cushions. non-specific binding was inhibited by incubation with anti-cd16/cd32 (2.4g2; bd biosciences, san diego, ca) and a 10% mixture of normal goat, human, mouse and rat serum for 10 min on ice. fitc, pe, percp, and apc conjugates with monoclonal antibodies (mab) used to identify and quantify microglia and inflammatory cells were: cd4 (gk1.5), cd8a (53-6.7), cd45 (30-f11), mhc class ii (2g9), ly6g (1a8) (bd biosciences), and f4/80 (serotec, raleigh, nc). microglia and inflammatory cells were distinguished based on differential cd45 expression. cd4 + t cells were identified as cd45 hi cd4 + , cd8 + t cells as cd45 hi cd8 + , macrophages as cd45 hi f4/80 + and neutrophils as cd45 hi ly6g + mhc class ii -. mog specific induction of cytokines was determined by stimulation of 1610 6 cns cells with 20 mg/ml peptide for 6 h at 37uc with golgistop (bd biosciences) added for the last 4 h. intracellular cytokines were detected with fitc-anti-ifn-c (clone xmg1.2; bd biosciences) and pe-anti-il-17 (clone tc11-18h10; bd biosciences). intracellular foxp3 was detected by staining for cell surface markers, followed by permeabilization with fixation/ permeabilization reagent (ebioscience, san diego, ca) and incubation with pe-labeled anti-foxp3 (fjk-16s; ebioscience). cells were analyzed on a facscalibur flow cytometer (bd biosciences) using cellquest pro software (bd biosciences). data was analyzed using flowjo (7.6.1) software (tree star inc., ashland, or). cytokines were determined by elisa using antibody pairs and recombinant cytokine standards from bd bioscience. il-27 was measured using quantikine mouse il-27p28 immunoassays (r&d systems inc., minneapolis, mn). following anesthesia, mice were perfused with pbs (ph 7.2). brains and spinal cords were fixed with clark's solution (75% ethanol and 25% glacial acetic acid), and embedded in paraffin. spinal cords were divided into 6 sections prior to embedding, corresponding to cervical, thoracic and lumbar levels. cross sections (6 mm), were stained with either hematoxylin and eosin (h&e) or luxol fast blue (lfb). immunoperoxidase staining was used to identify activated astrocytes with anti-gfap (abcam, cambridge, ma) and axonal integrity with smi-31 and smi-32 mab (sternberger monoclonals inc., lutherville, md) followed by visualization using vectastain abc kit (vector laboratories, burlingame, ca) and 3,39-diaminobenzidine (sigma-aldrich). sections from at least 3 separate experiments containing at least 3 mice per group were reviewed in a blinded manner. numbers of gfap + cells in spinal cord were determined in 15 non-overlapping 406 fields (0.2 mm 2 ) in white matter and gray matter. stained spinal cord sections of all 6 levels on individual glass slides were scanned (406) and digitally imaged at high resolution with an aperio scanscope (vista, ca). aperio software was used to quantify areas of demyelination within the white matter tracks of each of the 6 sections per individual mouse. for analysis of cd4 + t cells spinal cords were embedded in tissue-tek o.c.t. (andwin scientific, tryon, nc), flash frozen in liquid nitrogen and stored at 280uc. blocks were warmed to 220uc prior to cutting 10 mm sections by cryostat. following fixation with acetone for 2 min at 4uc, non-specific antibody binding was blocked with cyto q background buster (innovex biosciences, richmond, ca) for 15 min. sections were stained with anti-cd4 (l3t4) antibody (vector laboratories) diluted in cyto q immuno diluent (innovex biosciences) followed by biotinylated rabbit anti-rat, peroxidase abc reagent and visualized with novared substrate (all from vector laboratories). sections were counter stained with hematoxylin to visualize the nuclei. spinal cord sections were scanned (406) and digitally imaged at high resolution with an aperio scanscope. mixed glial cultures (,70% astrocytes) were established from neonatal gfapcr1d and wt mice as previously described [47] . il-27 secretion was determined 48 h after rifn-c (100 ng/ml) stimulation. frozen tissues were homogenized in trizol (invitrogen, carlsbad, ca) and cdna prepared as described [47, 48] . quantitative real-time pcr was performed using 4 ml of cdna and sybr green master mix (applied biosystems, foster city, ca) in duplicate on a 7500 fast real-time pcr system (applied biosystems). expression levels were normalized to ubiquitin or gapdh using the following formula: statistical significance was determined by two-tailed student's t test. a value of p,0.05 was considered statistically significant. figure s1 cd4 + t cell recruitment into the spinal cord during acute eae. cd4 + t cells were visualized in 10 mm frozen sections of spinal cords from wt and gfapcr1d tg mice at day 18 p.i. immunoperoxidase stain (novared chromogen with hematoxylin counterstain). scale bars = 50 microns. (tif) multiple sclerosis: a complicated picture of autoimmunity the immunology of multiple sclerosis astrocytes: biology and pathology astrocytes in the tempest of multiple sclerosis astrocytes-friends or foes in multiple sclerosis leukocyte infiltration, neuronal degeneration, and neurite outgrowth after ablation of scar-forming, reactive astrocytes in adult transgenic mice reactive astrocytes form scar-like perivascular barriers to leukocytes during adaptive immune inflammation of the cns regeneration beyond the glial scar autoimmune t cell responses in the central nervous system how interferon-gamma keeps autoimmune diseases in check the integrated stress response prevents demyelination by protecting oligodendrocytes against immune-mediated damage intrathecal delivery of ifn-gamma protects c57bl/6 mice from chronicprogressive experimental autoimmune encephalomyelitis by increasing apoptosis of central nervous system-infiltrating lymphocytes interferon-gamma confers resistance to experimental allergic encephalomyelitis mice with a disrupted ifn-gamma gene are susceptible to the induction of experimental autoimmune encephalomyelitis (eae) ifn-gamma plays a critical down-regulatory role in the induction and effector phase of myelin oligodendrocyte glycoprotein-induced autoimmune encephalomyelitis cross-regulation of signaling pathways by interferongamma: implications for immune responses and autoimmune diseases socs1 and socs3 in the control of cns immunity astrocyte expression of a dominant-negative interferon-gamma receptor regional cns responses to ifn-gamma determine lesion localization patterns during eae pathogenesis empowering t helper 17 cells in autoimmunity gp130-dependent astrocyte survival is critical for the control of autoimmune central nervous system inflammation attenuation of astroglial reactivity by interleukin-10 suppressive effect of il-27 on encephalitogenic th17 cells and the effector phase of experimental autoimmune encephalomyelitis early administration of il-12 suppresses eae through induction of interferon-gamma suppression of autoimmune inflammation of the central nervous system by interleukin 10 secreted by interleukin 27-stimulated t cells astrocytes are active players in cerebral innate immunity astrocyte-associated axonal damage in pre-onset stages of experimental autoimmune encephalomyelitis inhibition of transcription factor nf-kappab in the central nervous system ameliorates autoimmune encephalomyelitis in mice transgenic inhibition of astroglial nf-kappa b improves functional outcome in experimental autoimmune encephalomyelitis by suppressing chronic central nervous system inflammation reactive astrocytes protect tissue and preserve function after spinal cord injury experimental autoimmune encephalomyelitis in mice lacking glial fibrillary acidic protein is characterized by a more severe clinical course and an infiltrative central nervous system lesion apoptosis of inflammatory cells in immune control of the nervous system: role of glia ifn-gamma shapes immune invasion of the central nervous system via regulation of chemokines neurologic disease induced in transgenic mice by cerebral overexpression of interleukin 6 increased production of interferon gamma and tumor necrosis factor precedes clinical manifestation in multiple sclerosis: do cytokines trigger off exacerbations? tnfr1 signalling is critical for the development of demyelination and the limitation of t-cell responses during immune-mediated cns disease il-12 production by central nervous system microglia is inhibited by astrocytes chemokines and glial cells: a complex network in the central nervous system tnfa and ifnc synergistically enhance transcriptional activation of cxcl10 in human airway smooth muscle cells via stat-1, nf-kb, and the transcriptional coactivator creb-binding protein concomitant detection of ifna signature and activated monocyte/dendritic cell precursors in the peripheral blood of ifna-treated subjects at early times after repeated local cytokine treatments astrocyterestricted ablation of interleukin-17-induced act1-mediated signaling ameliorates autoimmune encephalomyelitis regulation and functions of the il-10 family of cytokines in inflammation and disease il-10 is critical in the regulation of autoimmune encephalomyelitis as demonstrated by studies of il-10-and il-4-deficient and transgenic mice central nervous system expression of il-10 inhibits autoimmune encephalomyelitis multiple sclerosis: cytokine receptors on oligodendrocytes predict innate regulation axonal plasticity and functional recovery after spinal cord injury in mice deficient in both glial fibrillary acidic protein and vimentin genes inhibition of interferon-gamma signaling in oligodendroglia delays coronavirus clearance without altering demyelination distinct regulation of mhc molecule expression on astrocytes and microglia during viral encephalomyelitis we thank bruce trapp for helpful discussions, wenqiang wei, eric barron and ernesto barron for assistance with the histopathology and jeffrey tarcy, kate stenson and maria ramirez for assistance with mouse husbandry and immunological assays. key: cord-048485-b8xb1f12 authors: hulst, marcel; kerstens, hinri; de wit, agnes; smits, mari; van der meulen, jan; niewold, theo title: early transcriptional response in the jejunum of germ-free piglets after oral infection with virulent rotavirus date: 2008-06-04 journal: arch virol doi: 10.1007/s00705-008-0118-6 sha: doc_id: 48485 cord_uid: b8xb1f12 germ-free piglets were orally infected with virulent rotavirus to collect jejunal mucosal scrapings at 12 and 18 hours post infection (two piglets per time point). ifn-gamma mrna expression was stimulated in the mucosa of all four infected piglets, indicating that they all responded to the rotavirus infection. rna pools prepared from two infected piglets were used to compare whole mucosal gene expression at 12 and 18 hpi to expression in uninfected germ-free piglets (n = 3) using a porcine intestinal cdna microarray. microarray analysis identified 13 down-regulated and 17 up-regulated genes. northern blot analysis of a selected group of genes confirmed the data of the microarray. genes were functionally clustered in interferon-regulated genes, proliferation/differentiation genes, apoptosis genes, cytoskeleton genes, signal transduction genes, and enterocyte digestive, absorptive, and transport genes. down-regulation of the transport gene cluster reflected in part the loss of rotavirus-infected enterocytes from the villous tips. data mining suggested that several genes were regulated in loweror mid-villus immature enterocytes and goblet cells, probably to support repair of the damaged epithelial cell layer at the villous tips. furthermore, up-regulation was observed for ifn-γ induced guanylate binding protein 2, a protein that effectively inhibited vsv and emcv replication in vitro (arch virol 150:1213–1220, 2005). this protein may play a role in the small intestine’s innate defense against enteric viruses like rotavirus. electronic supplementary material: the online version of this article (doi:10.1007/s00705-008-0118-6) contains supplementary material, which is available to authorized users. with an estimated death rate of more than 400,000 per year, mainly affecting children less than 5 years of age in developing countries, rotavirus is recognized as one of the major infectious diseases of the gastrointestinal tract [38] . rotaviruses are members of the family reoviridae, viruses with segmented double-stranded rna genomes [17] . in the small intestine, mature enterocytes near the top of the villi are the primary target cells for virus replication [29] . replication in these cells provokes numerous intraand extracellular pathological changes that inevitably lead to disruption of the absorptive and digestive functions of the small intestine, and consequently, to malabsorption and diarrhea. these changes include destruction of enterocyte brush borders, enterocyte vacuolization, loss and destruction of enterocytes, villus blunting and atrophy, thinning of the intestinal wall, and crypt hyperplasia (for comprehensive reviews, see [29, 39] ). however, the nature and severity of histopathological alterations in vivo can be quite different depending on the species and virulence of the rotavirus strain. there is no clear correlation between these alterations and manifestation of clinical symptoms. a systemic inflammatory response can be absent, and rotavirus infections can be asymptomatic [29, 39] . this suggests that the interplay between host and viral factors is important for determining the course of this disease. for electronic supplementary material the online version of this article (doi:10.1007/s00705-008-0118-6) contains supplementary material, which is available to authorized users. instance, rotavirus nsp4 acts as an enterotoxin that induces diarrhea in mice in the absence of rotavirus replication [5] . nsp4 affects ca 2+ and electrolyte homeostasis in an auto-and paracrine fashion in both rotavirus-infected and uninfected intestinal cells [41] . nsp4 increases ca 2+ permeability of the er and plasma membrane, resulting in an increased ca 2+ concentration in the cytosol ([ca 2+ ] cyt ), causing derailment of numerous ca 2+ -dependent cellular processes [40] . in uninfected enterocytes and crypt cells this rise in [ca 2+ ] cyt is induced by binding of exogenous nsp4 to an apical receptor that modulates the plc-ip 3 pathway [16] . the higher [ca 2+ ] cyt triggers laminal secretion of peptides and amines by uninfected enterocytes, and luminal cland h 2 o secretion by crypt cells [28, 39] . in infected enterocytes, the rise in [ca 2+ ] cyt is believed to be independent of plc modulation [41] , and this rise perturbs cytoskeleton and tight junction integrity, which ultimately leads to cell lysis [9, 10, 26, 37] . in vitro studies with cell lines, mainly derived from colon, have contributed significantly toward understanding the pathogenesis of rotavirus on a molecular level. however, the intestinal mucosa consists of a diversity of specialized cell types in different states of differentiation. presumably, all these different types of cells respond differently to environmental changes, and accordingly to changes in their neighboring cells. therefore, the regulation of genes responsible for these complex phenotypic responses in vivo may not be detected by challenging single types of cultured cells with rotavirus. to address this issue, we studied the early transcriptional response in jejunal mucosa of 3-week-old, just-weaned piglets after oral infection with virulent group a rotavirus. to assign measured responses exclusively to rotavirus, we performed these experiments in germ-free piglets. differential expression patterns of uninfected versus infected jejunum were recorded 12 and 6 h before severe diarrhea was expected, using a homemade pig intestinal cdna microarray [34] . the biological significance of elevated or reduced expression of these genes for rotavirus pathogenesis is discussed. seven germ-free piglets (groot yorkshire 9 [cofok 9 large white]) were obtained by caesarean section and housed in isolators, fed with sterilized condensed milk till the age of 14 days and thereafter with pelleted feed (sterilized by x-ray radiation) and water ad lib. on day 21, three of the seven piglets were transported to the necropsy room and served as uninfected control piglets. the four remaining pigs were orally infected with virus suspension diluted in a total volume of 5 ml pbs and containing 2 9 10 7 rotavirus particles (as determined by negative-stain semi-quantitative electron microscopy) of strain rv277 [45] . the virus suspension was prepared from the contents of the small and large intestine of a rotavirus-infected gnotobiotic piglet [32] . the above applied oral dose caused severe diarrhea from 24 hpi (hours post infection) in 3-week-old gnotobiotic piglets [32] . infected piglets were housed in their isolators under the same conditions as described above for another period of 12 (two piglets) or 18 h (two piglets) before they were transported to the necropsy room. immediately after arrival in the necropsy room, 10 ml of edta blood for hematological analysis was collected from the jugular vein. subsequently, animals were killed by barbiturate overdose and their intestines were taken out. the jejunum was opened and rinsed with cold saline, and 10 cm of mucosa in the middle of the jejunum was scraped off with a glass slide, frozen in liquid nitrogen, and kept at -70°c until rna and dna extraction. an adjacent part of the collected jejunum was fixed in 4% formaldehyde and used to determine the villus height and crypt depth. villus and crypt dimensions were determined on hematoxylin-eosinstained 5-lm tissue sections [34] . during the experiment, fecal samples were collected at 0, 12 and 18 hpi from the rectum for determination of the percent dry matter [18] . fecal samples were tested for the presence or absence of rotavirus by elisa [33] . the germ-free status of each piglet was confirmed by analyzing throat saliva and feces samples, collected on days 6, 12 and 19, and on the day of slaughter, for the presence of microorganisms. isolation of rna and dna from 1 g of frozen mucosal scrapings, total rna (dnasefree) was isolated using trizol ò reagent (invitrogen) as described recently [34] . the yield per gram of tissue and the purity of the rna were calculated from measurement of the extinction at 260 and 280 nm. the integrity of all rna samples was checked by analyzing 5 lg of rna on a denaturizing 1% (w/v) agarose gel. after ethidium bromide staining, the gel was scanned to calculate the 28s/18s peak ratio (volume 28s over volume 18s) for each preparation. rna with a ratio [2 was considered of adequate quality to be used for real-time pcr and microarray analysis. a part of the isolated rna was used to prepare rna pools for microarray analysis. a control pool was prepared by mixing equal amounts of rna isolated from the jejunum of the three uninfected piglets (n = 3). the same was done for the two infected piglets slaughtered at 12 h and for the two piglets slaughtered at 18 h. after gentle homogenization in lysis buffer, dna was extracted from 0.5 g of frozen mucosal scrapings, and 4 lg of purified dna was analyzed on a 0.9% agarose gel [22] . the relative concentrations of interferon-gamma (ifn-c) and ornithine decarboxylase antizyme 1 (oaz1) mrna in all rna samples was determined by real-time pcr. two hundred ng of total rna was reverse transcribed in a standard rt reaction using superscript ii reverse transcriptase (invitrogen) and pd(n) 6 primers. ifn-c cdna in these rt reactions was quantified using labeled light-cycler probes (roche diagnostics) as described [15] and expressed as pg/ll control plasmid. a 20-mer forward primer (5 0 -gacccgacgcttgcttcatg-3 0 ) and a 19mer reverse primer (5 0 -gagtgagcgtttatttgcac-3 0 ), generating a cdna fragment homolog to nucleotide 702-895 of the human oaz1 mrna reference sequence (gi:34486089), were used to quantify oaz1 cdna using cybergreen as label in a standard lightcycler reaction. the relative concentration of oaz1 mrna was calculated by extrapolation on a standard curve prepared from dilutions of an rt reaction prepared from a reference rna sample [34] . the quantity of 18s rrna in each rna sample was determined using the above described rt reactions by real-time pcr [15] and used to normalize the ifn-c and oaz1 concentrations. the quantity of 18s rrna showed no essential differences among all individual rna samples (average concentration ± sd; 4.95 ± 0.82 lg/ll of control plasmid). the same collection of pig probes (ests) used in earlier studies [34, 35] were spotted in triplicate on corning ul-tragaps slides. briefly, this collection consisted of 2,928 probes prepared from jejunal mucosal scrapings collected from 4-week-(672) and 12-week-(2,256) old pigs, probes coding for porcine cytokines (ifn-c, tnf-a, gmcsf, il-2, 4, 6, 8, and 10) and lung surfactant proteins sftpa and sftpd, and 110 marc1 and marc2 probes (porcine ests) homolog to trefoils, collectins, defensins, and glycosyltransferases [34] . a list of the probes already sequenced/ annotated is accessible on the website of arch virol (gene list hulst et al. pdf). dual-color (cy3-cy5) hybridization of slides was performed using the rna micromax tsa labeling and detection kit (perkinelmer) as described earlier [34] . messenger rna levels in both infected pools (12 and 18 hpi) were independently compared to the expression levels in the control (uninfected) pool. for each comparison, a dye swap was performed. in addition, a control hybridization experiment was performed in which a microarray slide was simultaneously hybridized with cy3labeled control rna and cy5-labeled control rna. scanning of slides, processing of raw images, creation of data reports, data-normalization and statistical analysis were performed as described by niewold et al. [34] with minor modifications. briefly, probes were considered to be differentially expressed when at least four of the six data points (spots) on both dye swap slides hybridized with a ratio of 3.6-fold (m = [log 2 (cy3/cy5)] \ -1.85 or [1.85) or more (3.6 is considered significant according to the manufacturer of the tsa kit) and were identified by significant analysis of microarrays (sam) [43] with a median false discovery rate (fdr or q value) of \5%. equal amounts of total rna (5 lg) were separated on a denaturizing 1% (w/v) agarose gel. after several washes with rnase-free water, the gel was blotted on hybond-n membranes (amersham), and blots were hybridized with 32 p-labeled dna fragments homolog to the mrna in question, in the same manner as was described in an earlier study [34] . after post-hybridization washes, the blots were scanned using a storm phosphor-imager (molecular dynamics, sunnyvale, california, usa). four 3-week-old germ-free piglets were orally infected with a dose of rotavirus that caused severe diarrhea from 24 hpi in 3-week-old gnotobiotic piglets [32] . for practical reasons, three uninfected germ-free piglets were slaughtered at the zero time point (mock, see table 1 ). in order to isolate highquality rna from jejunal mucosal scrapings, infected piglets were slaughtered 12 and 18 hpi. thus, 12 and 6 h before severe diarrhea would have been induced. in three of the four infected animals, rotavirus was detected in their feces. determination of the percent dry matter showed that only the fecal samples collected 18 hpi (piglets 65 and 67) had a significantly lower (pasty) consistency [18] . this indicated that not all of the piglets developed diarrhea before 12 hpi, and that the two piglets slaughtered 18 hpi did not develop the severe form of diarrhea normally observed at 24 hpi [32] . in jejunal tissue sections prepared from these two 18-h piglets, villus length was decreased to two-thirds of the average length measured in corresponding sections prepared from the three control piglets. no significant differences in crypt depths were observed between infected and control animals. for both piglets that were slaughtered at 18 h, these results indicated that the orally applied rotavirus reached the transcriptional response to rotavirus 1313 jejunum and induced the desired limited (not severe) pathological symptoms. in addition, the lower concentration of lymphocytes in the blood indicated that the animals were effectively infected with rotavirus [47] . in the feces of piglet 63, slaughtered at 12 h, no rotavirus could be detected. moreover, only a small decrease in villus length (20%) was observed for this piglet and its 12-h replicate. to find additional evidence whether the jejunum of piglet 63 was effectively challenged with rotavirus, the level of ifn-c mrna in jejunal mucosal scrapings was measured by real-time pcr (fig. 1 ). in rna samples isolated from the three uninfected pigs, hardly any ifn-c mrna could be detected. in contrast, the ifn-c mrna levels in scrapings of all infected piglets were up to 50-fold higher than in uninfected piglets, whereas oaz1 mrna levels were nearly equal for all rna samples. this indicated that the jejunum of all piglets, including piglet 63, responded to the orally applied rotavirus challenge. despite the loss of cells from the tip of the villi, the amount of rna (table 1 ) isolated from all infected piglets was comparable to that of uninfected piglets. on an ethidium-bromide-stained agarose gel, no degradation of rna was visible for any of the extracted rna samples (see also fig. 2a ). in addition, 28s/18s peak ratios were [2 for all these samples (table 1 ). these results showed that scrapings collected from all piglets yielded high-quality rna suitable for real-time pcr and microarray analysis. no random (necrotic) and/or fragmentized (apoptotic) dna was visible after gel electrophoreses of dna samples extracted from any of the piglets, indicating that the majority of cells imbedded in the epithelial layers of all the piglets were not apoptotic or necrotic (results not shown). mid-jejunal mucosal gene expression analysis was performed using a homemade pig cdna small intestinal microarray [34] . in two separate hybridization experiments, mrna expression levels in an uninfected rna pool (n = 3) were compared to expression levels in rna pools prepared from 12-and 18-h infected piglets (both n = 2). for both comparisons, dye swaps were performed. probes that hybridized differentially with a ratio (fc; infected over uninfected) of .28 or [3.6 in both slides of the dye-swap and that were identified with the lowest possible false-discovery rate (fdr; based on sam, [43] ), i.e., 0.35% for the 18-h comparison and 4.6% for the 12-h comparison, were selected for further analysis. raising of the fdr to a maximum of 10% did not identify additional probes with a fc \ 0.28 or [3.6 in either comparison. selected probes were sequenced and annotated after blastn or blastx analysis (when not yet annotated). for each differential expressed probe, the mean fc calculated from the two dye-swap slides is presented in table 2 . when cy3-and cy5-labeled cdna was prepared from the same uninfected rna pool and simultaneously hybridized on the array, none of the probes that hybridized differentially in the 12-and 18-h comparisons hybridized differentially (results not shown). six out of the nine probes that hybridized with a fc of 3.6-fold or more in the 12-h comparison (panel ''higher in infected'') also hybridized significantly more strongly in the 18-h comparison. for three of these probes (r14-r16), the ratio of differential expression further increased with time. in contrast, only one probe (r1) hybridized significantly less strongly at both time points. based on literature search and data mining, a tentative function was assigned for the genes identified by blast analysis (see table 2 ). in addition, the fc (infected over uninfected) of genes which were also found to be regulated in a previous study by cuadras et al. [13] , i.e., 16 h after infection of human intestinal epithelial caco-2 cells with rotavirus live virus vaccine rvv, is provided in parentheses in table 2 after the annotations. probes coding for ifn-c, tnf-a, gm-csf, and il-2, 4, 6, 8, and 10 were spotted on the array. however, the fluorescence intensity was not higher than the background threshold for any of these probes, indicating that mrna concentrations of these cytokines (including ifn-c) in infected and uninfected mucosal scrapings were too low to detect by microarray analysis, probably due to the relatively low percentage of cytokine-producing (immune) cells present in intestinal mucosa [6] . northern blots (nb) loaded with equal amounts of rna from each of the piglets were hybridized with p 32 -labeled cdna probes homologous to six differentially expressed mrnas and to three mrnas that were not identified as differentially expressed. for all nine mrnas, the length of the transcript(s) detected on blots were comparable to the length of porcine or human mrna reference sequences posted in the ncbi databank or reported in the literature. in accordance with array data, nb analysis showed that expression levels of gbp-2, krt20, sat, mgam, and casp3 mrnas were significantly higher in both 18-hinfected piglets than in uninfected piglets. for all of these mrnas, hybridization intensities for piglet 65 were nearly equal to that of piglet 67. gbp-2, krt20, sat, and mgam mrna expression was also up-regulated in infected piglet 63, slaughtered at 12 hpi. this indicated that the response to rotavirus infection in this 12-h piglet was comparable to the response observed in both 18-h piglets. however, no significant up-regulation of these mrnas was observed in the other piglet slaughtered at 12 h (64), indicating that this piglet responded differently to the rotavirus infection than its 12-h replicate and the two piglets slaughtered at 18 h. in accordance with array data, nb analysis showed that the expression level of ifabp2 mrna was significantly lower in 18-h-infected piglets than in uninfected piglets. for mrnas that showed no significant differential expression on the arrays (glutathione-s-transferase, calbindin-d, and aldolase-b), no large differences in hybridization intensities were observed between uninfected piglets and the two piglets slaughtered 12 hpi and one of the piglets slaughtered 18 hpi (65). however, significantly lower hybridization intensities were observed for calbindin-d and aldolase-b mrnas for piglet 67 than for its 18-h replicate. using microarray analysis, we detected a set of genes that are differently expressed in rotavirus-infected jejunal mucosa compared to uninfected mucosa. for nine mrnas, expression levels in individual piglets were analyzed by nb. these analysis confirmed the array data. in addition, nb analysis showed that one piglet slaughtered at 12 hpi responded quite similarly to rotavirus infection as both 18h piglets did, whereas its 12-h replicate did not, despite the fact that this latter piglet also showed an ifn-c mrna response. in addition, 7 out of the 12 genes differentially expressed at 12 hpi also reacted at 18 hpi. these results indicated that three out of four infected piglets responded quite analogously. because we used a limited number of germ-free piglets per time point and measured responses in a mixed population of cells, we imposed stringent criteria for selection of genes ([3.6-fold up-or down-regulation and a false-discovery ratio of less than 5%). using this approach, we minimized the chance of selecting genes hybridizing differentially solely due to inter-animal variation in gene expression and/or cell composition. however, such stringent selection criteria could have excluded the detection of more rotavirus-regulated genes, especially of genes regulated exclusively in specific types of cells that are present in low quantities in the jejunal mucosa. the different responsiveness of one of the 12-h piglets, however, obliged us to interpret our overall results carefully, especially, concerning the five genes that reacted solely at 12 hpi (txn, frk, dppc-2, if144, and actb; see table 2 ). nevertheless, data mining revealed relevant relationships between these five genes, 18 h response genes, and processes known to be important for rotavirus pathogenesis. cuardras et al. [13] measured the transcriptional response in the human enterocyte cell line caco-2, 1, 6, 12 and 16 hpi with rhesus rotavirus live vaccine. four genes up-regulated in our experiments (gbp-2, sat, mgam, ppa1) were also up-regulated 16 hpi in caco-2 cells. recently, aich et al. [1] profiled the transcriptional response in surgically prepared jejunal loops from 1-dayold colostrum-deprived calves after 18 h of perfusion with bovine rotavirus (brv). several genes for which we detected more than 3.6-fold up-or down-regulation (txn, nadh5, sglt1, actb, sat, casp3, and ppa1) were also present on the cdna array they used (ncbi geo acc. number gpl325). none of these genes showed a differential expression of twofold or more in their study. the different route of administration and virulence of the strain used, the digestive differences between the jejunum of omnivores and herbivores, and, in the case of the study of cuardras et al., the various specialized cell-types present in the jejunum of living animals versus cultured colon-derived caco-2 cells are probably responsible for the poor correspondence between these three studies. based on relevant literature and functional information in databanks, we assigned a function and a possible type(s) of cell(s) responsible for expression for most of the genes on our list (fig. 3) . in this hypothetical model, information from existing models dealing with the pathogenesis of rotavirus infection [29, 39, 40] and the development and maintenance of the small intestinal epithelium [20] were used to fit in our data. possible functions of these genes in relation to processes and pathways known to be important for rotavirus pathogenesis are discussed below. measurements of villus length indicated that considerable numbers of epithelial cells were lost from the tip of the villi, including (infected) mature enterocytes. in part, down-regulation of genes involved in transport of ions and nutrients over the membranes of mature enterocytes, like meprin a, sglt1, and ifabp2, may be a direct result of this loss. in another part, replication of rotavirus in enterocytes imbedded in the epithelial layer could have down-regulated transcription of these genes. this may also be the case for other down-regulated genes from our list, especially for genes detected only at 18 hpi (r4-r13, table 2 ). in addition to down-regulation of sglt1, ifabp2, and meprin a, we observed up-regulation of two other genes that may affect the absorptive and digestive function of the intestine: a gene coding for a protein carrying a ca 2+ -permeable cation channel cd20/ige fc receptor subunit b domain (ms4a2) and a gene homolog to a bacterial oligo/dipeptide permease (dppc-2). it is tempting to link up-regulation of ms4a2 directly to nsp4induced enhancement of ca 2+ permeability of the plasma and er membranes in intestinal epithelial cells [29, 40] . likewise, up-regulation of the dppc-2 homolog may be related to enhanced laminal secretion of peptides and amines by uninfected epithelial cells, a process believed to be triggered by raised [ca 2+ ] cyt [40] . characterization of these porcine transcripts/proteins is needed to provide further insight in the role of these genes in rotavirus pathogenesis. the same applies for the tmem106b gene. this gene showed the highest level of up-regulation. so far, only a duf1356 protein domain with unknown function has been predicted in tmem106b. recently, it was reported that rotavirus infection in infant mice induced apoptosis in vivo [7] . although dna analysis showed that the majority of cells present in infected mucosal scrapings were not apoptotic, we observed upregulation of the apoptosis effector protein casp3. this suggests that programmed cell death in the epithelial layer was stimulated by rotavirus infection. nb analysis detected a considerable level of casp3 mrna expression in uninfected mucosal scrapings. this constitutive expression of casp3 is, most likely, related to the process of maintenance of the absorptive status of the intestinal epithelial layer. a process in which mature enterocytes continually die due to apoptosis and are replaced by differentiating cells migrating from surrounding crypts to the tip of the villi [20] . in contrast to mature enterocytes, an in vivo study in mice showed that goblet cells are largely spared from apoptosis in rotavirus-infected mice [8] . moreover, migration of goblet cells from the crypt to the tips of the villi was stimulated in these mice. we found up-regulation of the goblet cell marker gene krt20 [51] and the lower and mid-villus immature enterocyte marker gene mgam [42] . this could indicate that transcriptional activity in both of these cell types was promoted. stimulation of apoptosis in rotavirus-infected enterocytes and higher proliferation/differentiating activity in goblet cells and immature enterocytes could be a coordinated response of the jejunum to remove infected enterocytes and overlay villus tips with fresh enterocytes, goblet cells, and mucus layer. we did not detect genes on our array that were directly associated with cell-cycle progression/arrest. however, down-regulation of the nuclear kinase frk (an antagonist of cell proliferation) and tms4f20 may be associated indirectly with this process. in humans, tms4f20 is strongly homologous to tm4sf4, a protein that reduced the ability of the crypt cell line ht29 to proliferate [48] . several other genes that may play a role in cell death and repair were regulated. sat was up-regulated, and txn and prr13 were down-regulated. for this later protein, reduced expression in cells was correlated with increased sensitivity to taxane-induced cell death, a caspase-independent process characterized by the polymerization of tubulins to extraordinarily stable microtubules [27, 30] . txn is the major carrier of redox potential in cells, and it is crucial for the defence of cells against oxidative-stressmediated apoptosis. txn also regulates gene expression by increasing binding of redox-sensitive transcription factors like p53 [44] , nf-jb [25] , and the nrf-2/polyaminetable 2 . information about the paneth cell marker thoc4 is provided in reference [24] transcriptional response to rotavirus 1319 inhibited sat expression [23] . therefore, up-regulation of sat gene expression at 18 hpi may be directly related to down-regulation of txn at 12 hpi. sat is a rate-limiting enzyme in spermine/spermidine metabolism. acetylation of these polyamines by sat promoted their degradation and excretion [46] . recently, it was reported that depletion of polyamines suppresses apoptosis in normal intestinal epithelial cells by akt-kinase-mediated inhibition of casp3 activity [50] . nb analysis showed that the increase in sat mrna expression coincided with the goblet cell marker krt20. therefore, it would be interesting to determine whether sat expression in goblet cells can be stimulated by rotavirus infection and whether it plays a role in protecting these cells from apoptosis [8] . interestingly, it was recently demonstrated that rna viruses can directly modulate polyamine metabolism by regulation of sat transcription and splicing [36] . a most interesting gene that we found more than tenfold up-regulated codes for an as yet not-well-characterized hypothetical human protein (loc646627) carrying a phospholipase a2 inhibitor domain (pla2-inh). the magnitude and kinetics of up-regulation of this gene corresponded exactly with gcnt3, suggesting that this gene was expressed in the same types of cells as gcnt3, most likely mucus-producing goblet cells and/or differentiating (immature) enterocytes. the amino acid sequence translated from our pla2-inh est showed an overall amino acid identity of 56%, and all cysteines aligned perfectly with cysteines of the human loc646627 protein and of other proteins that bear a typical pla2-inh domain. pla2s comprise a diverse family of cytosolic and secreted enzymes that hydrolyze membrane phospholipids to free fatty acids. they play an important role in many exogenous and intracellular processes, ranging from fatty acid metabolism and lysis of membranes to the synthesis of arachidonic acid (a-acid), an essential precursor for the production of inflammatory mediators such as eicosanoids. secreted pla2s are calcium-dependent enzymes. cytosolic pla2s (cpla2) can also be calcium-independent. a moderate increase in [ca 2+ ] cyt mediated translocation of calcium-dependent cpla2 to intracellular membranes where it hydrolyses phospholipids to a-acid [12] . a similar effect was observed after activation of the ms4a2 calcium-permeable cation channel (up-regulated here at 18 hpi, see above) on the surface of mast cells [14] . perhaps, enhanced expression of a pla2-inh in our study may be a countermeasure of specific intestinal epithelial cells to normalize and/or inhibit pla2 enzyme activity in response to extra-and intracellular changes in [ca 2+ ] evoked by rotavirus replication, either to protect specific cells from extra-and intracellular membrane-damage or to negatively regulate a-acid production. with respect to the latter process, we observed down-regulation of the enzymes cyp2c39 and acsl3, which both utilize a-acid acid as substrate. interestingly, the capsid protein of parvovirus possesses pla2 activity [49] , and hmcv particles carry a cell-derived pla2 activity [2] . for both viruses, pla2 activity appeared to be essential for infectivity. our results showed that ifn-c mrna expression in the jejunum of infected piglets peaked around 12 hpi and tended to decline beyond this time point (see fig. 1 ). this suggests that ifn-c was produced for a short period. recently, aich et al. [1] measured the mrna expression levels of several cytokines in jejunal loops perfused for 18 h with brv. however, they observed no ifn-c mrna response. because our orally administered rotavirus needed time to reach the jejunum, our 18 h infection period represents a shorter period than 18 h of perfusion. after 18 h of perfusion, expression of ifn-c mrna may have dropped to normal levels. interestingly, they did detect a rotavirus-induced il-6 (alias ifn-b 2) mrna response at 18 hpi [1] . recent studies showed that the interplay between ifn-gamma and il-6 controls the influx and clearance of neutrophils and, subsequently, the transition to a more sustainable influx of mononuclear cells during acute inflammation [21, 31] . therefore, it would be interesting to study which immune cells produce ifn-c and il-6 and whether an orchestrated action of these cells regulates an influx of vital immune cells in the jejunum after rotavirus infection. the ifn-c-inducible gbp-2 gene was up-regulated 18 hpi. overexpression of gbp-1 and gbp-2 in hela cells and nih 3t3 cells abrogated the cytopathogenic effect mediated by vsv and emcv, respectively, by an unknown mechanism [3, 11] . furthermore, it was shown that expression of murine gbp-2 in nih 3t3 cells neutralized the cytotoxic effect of the taxane drug paclitaxel [4] . this drug specifically stimulates polymerization of tubulins to extraordinarily stable microtubules. these stable microtubules interfere with the function of normal microtubule filaments and inevitably induce cell death [4] . the reduced expression of prr13 we observed (discussed above) may be an indication that the formation of extraordinarily stable microtubules in intestinal epithelial cells actually takes place in response to rotavirus infection. in fact, several studies have shown that rotavirus infection induces disorganization of the cytoskeleton network and microtubule filaments in enterocytes [9, 10, 26] . moreover, we also observed the up-regulation of the ifn-a/b-inducible gene ifi44, a cytosolic protein associated with microtubular structures, and the cytoskeleton gene actb. therefore, enhanced expression of gbp-2 in specific intestinal epithelial cells could contribute to a cellular mechanism(s) that impairs and/or prevents disorganization of the microtubule filaments. further in vivo studies are needed to determine whether the genes identified in this study are representative for an intestine with a normal microflora. if so, more focussed studies involving in situ hybridization and immuno-histology may specify where along the crypt-villus axis and in which type of epithelial (or immune) cells elevated or reduced expression of these genes is induced. comparative analysis of innate immune responses following infection of newborn calves with bovine rotavirus and bovine coronavirus human cytomegalovirus carries a cell-derived phospholipase a2 required for infectivity interferon-induced guanylate binding protein-1 (gbp-1) mediates an antiviral effect against vesicular stomatitis virus and encephalomyocarditis virus the interferoninduced gtpase, mgbp-2, confers resistance to paclitaxelinduced cytotoxicity without inhibiting multinucleation agedependent diarrhea induced by a rotavirus nonstructural glycoprotein intrahepatic gene expression during chronic hepatitis c virus infection in chimpanzees changes in small intestinal homeostasis, morphology, and gene expression during rotavirus infection of infant mice homeostasis and function of goblet cells during rotavirus infection in mice rotavirus infection induces an increase in intracellular calcium concentration in human intestinal epithelial cells: role in microvillar actin alteration rotavirus infection induces cytoskeleton disorganization in human intestinal epithelial cells: implication of an increase in intracellular calcium concentration inhibition of vsv and emcv replication by the interferon-induced gtpase, mgbp-2: differential requirement for wild-type gtp binding domain ca 2+ influx through crac channels activates cytosolic phospholipase a2, leukotriene c4 secretion, and expression of c-fos through erk-dependent and -independent pathways in mast cells gene expression pattern in caco-2 cells following rotavirus infection phosphorylation and activation of ca(2+)-sensitive cytosolic phospholipase a2 in mcii mast cells mediated by highaffinity fc receptor for ige age, gender and litter-related variation in t-lymphocyte cytokine production in young pigs the rotavirus enterotoxin nsp4 mobilizes intracellular calcium in human intestinal cells by stimulating phospholipase c-mediated inositol 1, 4, 5-trisphosphate production rotaviruses and their replication transmission of f4 + e coli in groups of early weaned piglets molecular cloning of spermidine/spermine n1-acetyltransferase from the periimplantation porcine uterus by messenger ribonucleic acid differential display: temporal and conceptus-modulated gene expression twists and turns in the development and maintenance of the mammalian small intestine epithelium il-6 and its soluble receptor orchestrate a temporal switch in the pattern of leukocyte recruitment seen during acute inflammation inactivation of the rnase activity of glycoprotein e rns of classical swine fever virus results in a cytopathogenic virus increased thioredoxin-1 inhibits ssat expression in mcf-7 human breast cancer cells inclusion of linseed and linseed expeller meal in piglet diets affects intestinal gene expression profiles regulatory role for a novel human thioredoxin peroxidase in nf-kappab activation rotavirus infection reduces sucrase-isomaltase expression in human intestinal epithelial cells by perturbing protein targeting and organization of microvillar cytoskeleton txr1: a transcriptional regulator of thrombospondin-1 that modulates cellular sensitivity to taxanes role of the enteric nervous system in the fluid and electrolyte secretion of rotavirus diarrhea pathogenesis of rotavirus diarrhea mechanisms of cd47-induced caspase-independent cell death in normal and leukemic cells: link between phosphatidylserine exposure and cytoskeleton organization interplay between ifn-gamma and il-6 signaling governs neutrophil trafficking and apoptosis during acute inflammation state university of utrecht 33. nabuurs mj, hoogendoorn a, van zijderveld-van bemmel a (1996) effect of supplementary feeding during the sucking period on net absorption from the small intestine of weaned pigs development of a porcine small intestinal cdna micro-array: characterization and functional analysis of the response to enterotoxigenic e. coli the early transcriptional response of pig small intestinal mucosa to invasion by salmonella enterica serovar typhimurium dt104 induction of alternatively spliced spermidine/spermine n1-acetyltransferase mrna in the human kidney cells infected by venezuelan equine encephalitis and tickborne encephalitis viruses rotavirus-induced structural and functional alterations in tight junctions of polarized intestinal caco-2 cell monolayers global illness and deaths caused by rotavirus disease in children pathogenesis of intestinal and systemic rotavirus infection role of ca 2+ in the replication and pathogenesis of rotavirus and other viral infections the rotavirus nonstructural glycoprotein nsp4 mobilizes ca 2+ from the endoplasmic reticulum sucrase-isomaltase gene expression along the crypt-villus axis of human small intestine is regulated at level of mrna abundance significance analysis of microarrays applied to the ionizing radiation response thioredoxindependent redox regulation of p53-mediated p21 activation intestinal permeability in pigs during rotavirus infection effects of conditional overexpression of spermidine/spermine n1-acetyltransferase on polyamine pool dynamics, cell growth, and sensitivity to polyamine analogs rotavirus infection alters peripheral t-cell homeostasis in children with acute diarrhea a tetraspan membrane glycoprotein produced in the human intestinal epithelium and liver that can regulate cell density-dependent proliferation a viral phospholipase a2 is required for parvovirus infectivity akt kinase activation blocks apoptosis in intestinal epithelial cells by inhibiting caspase-3 after polyamine depletion keratin 20 serine 13 phosphorylation is a stress and intestinal goblet cell marker acknowledgments the authors would like to thank arie hoogendoorn for his assistance in the animal experiment.open access this article is distributed under the terms of the creative commons attribution noncommercial license which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited. key: cord-255013-njpuc475 authors: he, xiaocui; korytář, tomáš; zhu, yaqing; pikula, jiří; bandouchova, hana; zukal, jan; köllner, bernd title: establishment of myotis myotis cell lines model for investigation of host-pathogen interaction in a natural host for emerging viruses date: 2014-10-08 journal: plos one doi: 10.1371/journal.pone.0109795 sha: doc_id: 255013 cord_uid: njpuc475 bats are found to be the natural reservoirs for many emerging viruses. in most cases, severe clinical signs caused by such virus infections are normally not seen in bats. this indicates differences in the virus-host interactions and underlines the necessity to develop natural host related models to study these phenomena. due to the strict protection of european bat species, immortalized cell lines are the only alternative to investigate the innate anti-virus immune mechanisms. here, we report about the establishment and functional characterization of myotis myotis derived cell lines from different tissues: brain (mmbr), tonsil (mmto), peritoneal cavity (mmpca), nasal epithelium (mmnep) and nervus olfactorius (mmnol) after immortalization by sv 40 large t antigen. the usefulness of these cell lines to study antiviral responses has been confirmed by analysis of their susceptibility to lyssavirus infection and the mrna patterns of immune-relevant genes after poly i:c stimulation. performed experiments indicated varying susceptibility to lyssavirus infection with mmbr being considerably less susceptible than the other cell lines. further investigation demonstrated a strong activation of interferon mediated antiviral response in mmbr contributing to its resistance. the pattern recognition receptors: rig-i and mda5 were highly up-regulated during rabies virus infection in mmbr, suggesting their involvement in promotion of antiviral responses. the presence of cd14 and cd68 in mmbr suggested mmbr cells are microglia-like cells which play a key role in host defense against infections in the central nervous system (cns). thus the expression pattern of mmbr combined with the observed limitation of lyssavirus replication underpin a protective mechanism of the cns controlling the lyssavirus infection. overall, the established cell lines are important tools to analyze antiviral innate immunity in m. myotis against neurotropic virus infections and present a valuable tool for a broad spectrum of future investigations in cellular biology of m. myotis. bats belong to one of the most abundant, diverse and widely distributed mammalian groups. in the order of chiroptera which is divided into two suborders megachiroptera and microchiroptera, a total of 1,240 species have been yet described [1] . bats evolved early and changed very little over the past 52 million years [2] . their wide distribution and migratory behaviour favour bats as vectors for viruses and raise concerns over their role in zoonotic diseases [3] [4] [5] . among the large number of viruses detected in bats, some like hendra virus, nipah virus, severe acute respiratory syndrome coronavirus (sars), ebola virus, west nile virus were reported to be zoonotic [4, [6] [7] [8] [9] [10] [11] . also 13 of the 15 lyssaviruses, except mokola virus and ikoma lyssavirus, were detected in bats. in north america bats host rabv, whereas in europe european bat lyssavirus type 1 and 2 (eblv-1 and eblv-2) are found in different bat species [12, 13] . annually, there are approximately 55,000 human deaths caused by rabies, especially in the developing countries of asia and africa [14] . despite most human rabies deaths are associated with dog rabies, some of them can be directly linked to the contact with bats, such as 8 out of 226 human rabies cases were of bat origin in the americas in 1983 and a few human cases caused by eblvs were reported in europe to date [15] [16] [17] [18] . although bat associated viruses can cause severe diseases in various mammals, they seem to be less pathogenic for bats [3, [19] [20] [21] [22] [23] [24] [25] . after experimental infection with hendra or nipah virus, bats showed no clinical disease, while guinea pigs succumbed to the same dose of virus [21, 22] . similar situation was also observed in hendra virus infection in horses and bats [24] . lyssaviruses are the only viruses that were reported to cause clinical disease in bats [26] . however, only a small proportion of bats develop clinical symptoms after experimental infection [25, 27] . this indicates a critical difference in the development of viral disease between bats and other mammals and requires profound investigation of bat immunology and host-virus interactions. since all of 52 identified european bats species are endangered and strictly protected, the use of animal trials for the investigation of immune mechanisms in bats is not possible. thus, development of stable cell lines for in vitro studies derived from european bat species is desirable. so far, several bat cell lines were reported in previous studies, but most of them were established from non-european bats, like tb1-lu from tadarida brasiliensis, mvi/it from myotis velifer incautus, and several primary immortalized cell lines from pteropus alecto, carollia perspicillata, eidolon helvum and rousettus aegyptiacus [28] [29] [30] [31] . viral infection studies have been carried out in the fruit bat cell lines to investigate the susceptibility, infection kinetics of henipavirus as well as the host innate immunity [28, 32] . however, the susceptibility to lyssavirus has not yet been examined in these cell lines. additionally, except for a brain cell line from eptesicus serotinus employed to investigate the type i interferon (ifn) response after lyssavirus infection [33] , the use of a bat cell line as a tool for studies into lyssavirus infection in its natural reservoir host is rare. a broader variety of bat cell lines, particularly european bat cell lines from tissues of immune relevance, is therefore urgently in demand for lyssavirus-host studies. in this study, we established different cell lines from the european bat m. myotis, evaluated their susceptibility to eblv-1, eblv-2 and rabv infection and investigated innate immune gene responses after the polyinosinic:polycytidylic acid (poly i:c) stimulation. the established m. myotis cell lines present a valuable in vitro model to study the interactions between lyssaviruses and their natural host, and to shed light on the mechanisms of resistance in bat's central nervous system (cns). ethical approval for all of the capturing and sampling were confirmed by the competent authorities in the respective federal republic of germany and czech republic. the czech academy of sciences ethics committee reviewed and approved the animal use protocol no. 169/2011 in compliance with law no. 312/ a single m. myotis male was captured in sloupsko-sosuvske caves of the moravian karst (czech republic, coordinates 49u 249 40.880 and 16u 449 20.540). the bat was kept to minimize stress and handling between capture and euthanasia in a clean plastic box with soft mesh to enable roosting under temperature of hibernation torpor of 6uc and transferred to our laboratory at veterinary and pharmaceutical sciences brno (czech republic) within a day. it was anesthetized to insensitiveness using isofluranum (isofluran, piramal healthcare, uk), and then transcription pcr (rt-pcr) using sv40t specific primers [35] . the protein expression was controlled by the immunofluorescence and western blot as described below. briefly, cells were first fixed with 3% paraformaldehyde and permeabilized with 0.5% triton x. after washing with pbs, cells were stained with mouse anti-sv40t monoclonal antibody (santa cruz biotechnology) and goat anti-mouse igg alexa fluor (invitrogen) as second antibody and visualized by fluorescence microscope. for western blot, the same mouse antibody was used as primary antibody and bound antibody was detected with goat anti-mouse igg peroxidase (sigma). images were developed using the ecl kit (thermo scientific pierce) according to the manufacturer's instructions. to confirm the identity of the established m. myotis cell lines derived from brain (cerebrum) (designated mmbr), tonsil (mmto), peritoneal cavity (mmpca), nasal epithelium (mmnep) and nervus olfactorius (mmnol), a m. myotis-specific pcr was developed. an nadh dehydrogenase subunit 1 (nd1) gene (genbank accession number: dq915043) from m. myotis was used as a species specific molecular marker. the genomic dna from different cell lines was isolated by dneasy blood & tissue kit (qiagen). the concentration and purity of genomic dna were determined by nanodrop (thermo). pcr was performed using a specific primer pair nd1-f and nd1-r (table 1) and genomic dna as a template by gotaq flexi dna polymerase (promega) to get the nd1 fragments. pcr products were cloned into pcr2.1 vector (invitrogen) and transformed into e. coli competent cells. plasmids were extracted from positive clones and sequenced by applied biosystems 3130 genetic analyzer (life technologies) at the friedrich-loeffler-institute, germany. to evaluate the ifn response of m. myotis cell lines and the induction of ifn mediated signaling, poly i:c was used to stimulate the cells. different cell lines were seeded in 24-well plates at a density ranging from 1.2 to 2610 5 cells/well, and cultured as described above. around 20 hours after seeding, cells were transfected with poly i:c (sigma) at a concentration of 10 mg/ ml by lipofectamine 2000 (invitrogen) following the manufacturer's instructions. twenty four hours post stimulation, cells were harvested into rlt buffer (qiagen) for rna extraction by an rneasy mini kit (qiagen). early after immortalization, the third passage immortalized cell lines were used to check the infectivity of rabv. cells were infected with rabv (european fox isolate, fused with green fluorescent protein, gfp) at a moi of 10. twenty four hours post infection (hpi), infected cells were fixed and permeabilized as described above and visualized by fluorescence microscope. mmbr and mmto cells that were infected with a serial moi of 0.01, 0.1, and 1.0 were harvested at 24 hpi and used for rna extraction. to confirm the infectivity in later passaged cells, different immortalized cell lines of more than 15 passages were infected with lyssaviruses rabv, eblv-1 (e. serotinus isolate) and eblv-2 (m. daubentonii isolate) at a moi of 0.1. the infected cells were cultured as described above. cells were collected for rna extraction at 24 hpi and quantitative real-time pcr (qrt-pcr) was performed on the cfx96 touchdetection system (bio-rad) using sensifast sybr one-step kit (bioline) according to the protocol. immunofluorescence analysis was performed on fixed cells using fitc conjugated anti-rabies monoclonal antibody (sifin) at 72 hpi as described before [36] . to further confirm the susceptibility, mmbr and mmnol cell lines were infected with eblv-1 at a moi of 0.01 to set the sensitivity at a ct value of 22 for the inoculation dose. the viral supernatant was either changed or not changed with fresh medium at 1 hpi, and viral replication levels were measured by qrt-pcr over 72 hpi. qrt-pcr was introduced to measure the mrna expression levels of immune related molecules in response to poly i:c stimulation and virus infection. the selected molecules include ifn induced genes: ifn stimulated gene 56 (isg56), isg43, myxovirus resistance 1 (mx1) and ifn induced protein with tetratricopeptide repeats 3 (ifit3), and pattern recognition receptors (prrs): toll-like receptor 3 (tlr3), retinoic acidinducible gene 1 (rig-1) and melanoma differentiation-associated protein 5 (mda+5). all of these primers were designed based the sequence resources from our own un-published sequence database and public databases of bat species. the softwares for primers design include primer premier 5, online tools: http://bioinfo.ut. ee/primer3-0.4.0/ and http://www.ncbi.nlm.nih.gov/tools/ primer-blast/. primers of target genes and internal control bactin were listed in table 1 . qrt-pcr was performed on the cfx96 touchdetection system (bio-rad) using sensifast sybr one-step kit (bioline) according to the protocol. to assess the specificity of the pcr amplification, a melting curve analysis was performed at the end of the reaction. the relative expression levels of targets were calculated by 2 2ddct method [37] . molecular characterization of the mmbrbecause the mmbr is derived from the cns, the target of fatal infections by lyssaviruses, a further characterization of cell type of mmbr is desired to improve the understanding of the antiviral defense in the cns. the expressions of cluster of differentiation (cd) 68, a marker for cells of macrophage lineage [38] , and cd14, a marker expressed in activated microglia [39] , were investigated by rt-pcr in different cell lines. specific primers for cd14, cd68 and internal control b-actin were listed in table 1 . the rt-pcr was prepared according to the instructions of the one-step rt-pcr kit (qiagen). all data were presented as means 6 s.d. statistical significant differences were analysed by one-way anova using the spss software package. five m. myotis cell lines brain (mmbr), tonsil (mmto), peritoneal cavity (mmpca), nasal epithelium (mmnep) and nervus olfactorius (mmnol) were successfully established by transformation with sv40t gene integrating into the chromosomal dna. varying cell morphologies were observed in the cell lines, with mmbr, mmto, mmnep and mmnol being fibroblastic-like, and mmpca being epithelial-like (fig. 1a) . the mrna expression of sv40t antigen was detected in all cell lines (fig. 1b) . protein level expression was confirmed in four of the five cell lines by immunofluorescence microscopy and western blot, respectively ( fig. 1c and d) . in mmto, the protein level of sv40 t antigen was under detectable because the transcriptional level is significantly low determined by rt-pcr (fig. 1b) . after immortalization, all five cell lines grew for more than 30 passages. the identity of the cell lines was validated by a m. myotis specific pcr using nd1 gene as a molecular marker. a predicted 515-bp fragment was obtained from genomic dna of each cell line, and further confirmed by sequencing. as the first step towards the characterization of the innate immune competence of different cell lines, the permanent or inducible expression of molecules involved in cell autonomous responses was examined. the prrs: tlr3, rig-1 and mda5, display a various distribution pattern in different cell lines (fig. 2) . of note, mmbr has the lowest levels of tlr3, rig-1 and mda5 (fig. 2) . for tlr3, about 10-fold higher mrna levels (p,0.05) were observed in mmto, mmpca, mmnep and mmnol compared to mmbr, respectively, while for mda5 about 30-fold (mmto; mmnep) or about 6-fold (mmpca; mmnol) (p,0.05) higher expression levels were measured (fig. 2) . additionally, more than 200 times higher expression levels of rig-1 were shown in other cell lines compared to mmbr (p,0.05) (fig. 2) . further investigation focused on the expression of tlr3, isg56, isg43 and mx1 induced by the poly i:c stimulation (fig. 3) . the obtained results indicate a 4-fold in mmbr, mmnep and mmnol and 8-fold in mmpca (p,0.05) increase in the tlr3 expression, whilst no change in mmto (fig. 3a) . all of the ifn induced genes were up-regulated to different extents in different cell lines (fig. 3b, c, d and e) . in detail, isg56 expression increased from 19-fold in mmbr to as high as more than 9000-fold in mmnol (p,0.05) (fig. 3b) . the expression of isg43 ranged from 10 to 145 times more and mx1 from 2 to 100 times more in mmto and mmbr, respectively ( fig. 3c and d) . ifit3 was upregulated from 12 to 420 times more in mmto and mmnol, respectively (p,0.05) (fig. 3e ). being a natural reservoir species, the main advantage of the permanent m. myotis cell lines is their susceptibility to lyssavirus infection. at an early stage of immortalization, cell lines displayed a significant susceptibility to rabv (moi of 10 at 24 hpi) as demonstrated by the infection with gfp fused rabv. notably, mmbr exhibited considerably lower viral load compared to the other cell lines (fig. 4) . later, all passaged immortalized cell lines showed susceptibility to eblv-1, eblv-2 and rabv in a different extent (fig. 5a and b) . generally, the mmbr cell line presented lower sensitivity to all three lyssaviruses (moi of 0.1) than the other four cell lines measured by qrt-pcr at 24 hpi (fig. 5a) , and monitored by immunofluorescence at 72 hpi (fig. 5b) . thus, the susceptibility could be ordered as mmnol and mmnep fully susceptible with a very high replication rate, mmpca and mmto susceptible with a much less viral replication of eblv-1 and 2, mmbr susceptible for eblv-1 and rabv with a very low viral replication and just single infected cells after eblv-2 infection (fig. 5b) . the different susceptibility of the cell lines to lyssavirus infection was further confirmed by the growth kinetics of eblv-1 in two representative models: mmbr, much less susceptible and mmnol, highly susceptible (fig. 5c ). to further evaluate the cell line models for study of the different susceptibility between mmbr and other cell lines, mrna expressions of prrs and ifn induced genes were investigated in mmto and mmbr after rabv infection (moi 0.01 to 1.0). the expression of all three prrs remained mostly unchanged in mmto, while it was significantly regulated in mmbr with 2-fold increased expression of tlr3, about 25-fold increased expression of rig-1 and mda5 at moi of 1.0 (p,0.05) (fig. 6a) . a comparable expression pattern was observed for the isg56, isg43, mx1 and ifit3, which was nearly not up-regulated in mmto but displayed a dose dependent increase in mmbr along with the increase of moi, especially for isg56 and ifit3 (fig. 6b ). isg56 mrna level increased from 6 to 513 times, ifit3 from 2 to 85 times in the infected mmbr (p,0.05). microglia are macrophage-like cells that are resident immune effector cells in the cns [39] . they are activated in response to infection or injury and play a central role in immune surveillance and host defense [39] . the rt-pcr results showed that cd14 and cd68 are expressed only in mmbr but not in the other four cell lines (fig. 7) . this suggested mmbr is a microglia-derived cell line. cell autonomous and innate immune mechanisms are the first line defenses against viral infections. this is mediated mainly by the prrs and the machinery of the ifns and ifn induced effector molecules [40] [41] [42] . viral pathogens like lyssaviruses developed evasive strategies to escape these host defenses by counteracting the ifn mediated immune responses [43] . co-evolution of the lyssaviral evading and bat's protective mechanisms resulted in an optimal balance, which protect bats as the 'natural host' from severe clinical symptoms or death. bats, which changed very little over past 52 million years, illustrate this phenomenon very well by the resistance to lethal diseases caused by viruses in other mammals [11, [21] [22] [23] [24] .to understand the specificity of hostpathogen interactions in 'natural host' like bats, studies in bats have to be performed. however, due to the strict protection of the endangered european bat species, in vitro models have to be used. in this study, we successfully established five m. myotis cell lines derived from neural and immune related tissues. to ensure the suitability of these cell lines to analyze virus-host cell interaction, the susceptibility to the infection as well as the presence of corresponding defensive pathways have to be confirmed. first, the existence of the viral sensors tlr3, rig-1 and mda5 in these permanent cell lines suggests a capacity of these cell lines to sense a broad range of rna viruses. the increased expression of dsrna receptor tlr3 and ifn induced genes isg56, isg43, mx1 and ifit3 after stimulation with poly i:c mimicking a viral infection indicates that these cell lines can be used as effective in vitro models to study the bat's innate immune responses to virus infection [32, 44] . furthermore, to serve as valuable models would be a varying susceptibility of such cell lines to infection by lyssaviruses. in the present study, different susceptibility observed in different m. myotis cell lines using eblv-1, eblv-2 and rabv might be related to the different capacity of the cell lines to produce antiviral mediators and control the infection. moreover, the strong difference in the susceptibility to rabv infection between mmbr and other cell lines provides a unique opportunity for comparative investigations of cell autonomous and innate immune mechanisms in a reservoir host. in addition to the lyssaviruses, the other member from the rhabdoviridae family, like vesicular stomatitis virus (vsv) can also be investigated by using these models in the future studies. preliminary results indicate a correlation between the observed varying susceptibility and the ability to up-regulate the prrs and the ifn induced genes. emerging evidences have shown that prrs play pivotal roles in antiviral immunity in the cns [45] . in the brain derived cell line mmbr, the high up-regulations of rig-1 and mda5 revealed activation of rig-i-like receptor pathway during rabv infection. as previously reported, rig-1 is a major prr to induce ifn in the rabv infected cells, and mda5 may function to sustain the ifn induction [46] . the increased expressions of ifn induced genes: isg56, isg43, mx1 and ifit3 in mmbr indicate that the production of ifn was induced by activated rig-1 and mda5. in contrast, the low expression level of tlr3 implies a vague involvement of tlr3 in anti-rabv infection immunity or resistance. it was shown that tlr3 participated in and benefited the rabv pathogenesis in human neuron cells [47] . however, the roles of tlr3 during rabv infection in bats need further investigations. importantly, the significant expression patterns of prrs observed in presented cell line models provide an access to this issue in vitro. to reach a successful infection, the viruses must overcome the barriers of innate immune system. it was reported that ifn production and signaling pathways were antagonized in p. alecto cell lines under henipavirus infection [32] . similarly, a recent study showed limited expressions of type i ifns and ifn induced genes during lyssaviruses infection in an e. serotinus brain cell line [33] . a correlation between the low viral load and high expression levels of ifn induced genes in mmbr contrasts to the high viral load and a silent expression pattern of antiviral effectors in mmto, providing an evidence of a countermeasure to ifn system by lyssavirus in the peripheral tissue versus a protective mechanism to infection in the brain tissue of bats. microglial cells are one of the major cell populations in the brain tissue. additionally, comparing to neurons, they can be infected by different rabv strains to a lesser extent [48, 49] . the presence of cd14 and cd68 as well as the anti-lyssavirus responses in mmbr support a microglia-like feature of mmbr in the cns. it was reported that a mouse microglia cell line can activate strong innate immunity during rabv infection [50] . the robust immune responses of the microglia-like mmbr demonstrated a critical role of microglia in the anti-rabies defense in bat's cns. in addition to the function of microglia, the clearance of infected viruses in the cns requires systematical responses through the complex interactions of different brain resident cells. herein, the establishment and identification of a microglia-like cell model is a first step towards understanding of the complex reactions of cns in response to lyssavirus infection in the reservoir species. overall, this preliminary study using established cell lines implies that immune mechanisms that control the virus replication are present in the cns of bats. it seems that the ability to control the pathogenic rabv replication via ifn system in the cns contributes to the asymptomatic outcome in bats. in conclusion, the established immortalized cell lines from the european bat m. myotis displaying a variable susceptibility to different lyssaviruses will serve as a useful model to study virus-host interactions and antiviral resistance mechanisms in the 'natural' lyssavirus host. this study provides a preliminary insight into the antiviral innate immunity correlated to cns against neurotropic viruses infection in bats. microbat paraphyly and the convergent evolution of a key innovation in old world rhinolophoid microbats primitive early eocene bat from wyoming and the evolution of flight and echolocation bats: important reservoir hosts of emerging viruses severe acute respiratory syndrome coronavirus-like virus in chinese horseshoe bats emerging viruses: coming in on a wrinkled wing and a prayer emerging encephalitogenic viruses: lyssaviruses and henipaviruses transmitted by frugivorous bats virology. what links bats to emerging infectious diseases? bats are natural reservoirs of sars-like coronaviruses coronavirus antibodies in african bat species a review of studies on animal reservoirs of the sars coronavirus fruit bats as reservoirs of ebola virus molecular epidemiology of bat lyssaviruses in europe novel lyssavirus in bat re-evaluating the burden of rabies in africa and asia human rabies of bat origin in europe human rabies due to lyssavirus infection of bat origin public health concerns in bat rabies across europe. euro surveillance : bulletin europeen sur les maladies transmissibles fatal human rabies caused by european bat lyssavirus type 2a infection in scotland emerging diseases in chiroptera: why bats? passive surveillance (1987 to 2004) of united kingdom bats for european bat lyssaviruses experimental nipah virus infection in pteropid bats (pteropus poliocephalus) experimental hendra virus infectionin pregnant guinea-pigs and fruit bats (pteropus poliocephalus) antiviral immune responses of bats: a review transmission studies of hendra virus (equine morbillivirus) in fruit bats, horses and cats experimental study of european bat lyssavirus type-2 infection in daubenton's bats (myotis daubentonii) bats and viruses: friend or foe? pathogenesis studies with australian bat lyssavirus in grey-headed flying foxes (pteropus poliocephalus) establishment, immortalisation and characterisation of pteropid bat cell lines type i interferon reaction to viral infection in interferon-competent, immortalized cell lines from the african fruit bat eidolon helvum establishment of cell line from embryonic tissue of pipistrellus ceylonicus bat species from india & its susceptibility to different viruses bat airway epithelial cells: a novel tool for the study of zoonotic viruses interferon production and signaling pathways are antagonized during henipavirus infection of fruit bat cell lines anti-lyssaviral activity of interferons kappa and omega from the serotine bat, eptesicus serotinus white-nose syndrome fungus: a generalist pathogen of hibernating bats standardized detection of simian virus 40 by real-time quantitative polymerase chain reaction in pediatric malignancies enhanced passive bat rabies surveillance in indigenous bat species from germany -a retrospective study analysis of relative gene expression data using real-time quantitative pcr and the 2(-delta delta c(t)) method molecular cloning of cd68, a human macrophage marker related to lysosomal glycoproteins role of microglia in central nervous system infections interferon signalling network in innate defence induction and function of type i and iii interferon in response to viral infection regulation of the type i ifn induction: a current view rhabdovirus evasion of the interferon system type iii ifns in pteropid bats: differential expression patterns provide evidence for distinct roles in antiviral immunity innate antiviral signalling in the central nervous system interferon in rabies virus infection tolllike receptor 3 (tlr3) plays a major role in the formation of rabies virus negri bodies rabies virus-induced activation of mitogen-activated protein kinase and nf-kappab signaling pathways regulates expression of cxc and cc chemokine ligands in microglia rabies viruses infect primary cultures of murine, feline, and human microglia and astrocytes global gene expression changes in bv2 microglial cell line during rabies virus infection we would like to thank dr. thomas müller and dr. conrad m. freuling for the eblv-1 and eblv-2 strains, dr. stefan finke for the rabv strain, matthias lenk for the prsvag1 plasmid, and dr. miroslav kovarik from the administration of the moravian karst protected landscape area (nature conservation agency of the czech republic) for cooperation in obtaining the m. myotis specimen. key: cord-034467-jh9msz1c authors: olagoke, olusola; quigley, bonnie l.; timms, peter title: koalas vaccinated against koala retrovirus respond by producing increased levels of interferon-gamma date: 2020-10-31 journal: virol j doi: 10.1186/s12985-020-01442-7 sha: doc_id: 34467 cord_uid: jh9msz1c koala retrovirus (korv) is believed to be in an active state of endogenization into the koala genome. korv is present as both an endogenous and exogenous infection in all koalas in northern australia. korv has been linked to koala pathologies including neoplasia and increased susceptibility to chlamydia. a korv vaccine recently trialled in 10 northern koalas improved antibody response and reduced viral load. this communication reports the expression of key immune genes underlining the innate and adaptive immune response to vaccination in these northern koalas. the results showed that prior to vaccination, il-8 was expressed at the highest levels, with at least 200-fold greater expression compared to other cytokines, while cd8 mrna expression was significantly higher than cd4 mrna expression level. interferon-γ was up-regulated at both 4and 8-weeks post-vaccination while il-8 was down-regulated at 8-weeks post-vaccination. korv is currently undergoing endogenization into the koala genome, thus offering an exciting opportunity to study retroviral endogenization in real time [1, 2] . nine korv subtypes (korv-a to -i) have been identified with the subtypes a and b being the most studied [3] . all northern koalas (queensland and new south wales) are thought to be harbour at least korv-a, a korv subtype which is both endogenous and infectious in these koala populations. korv infection has been linked to immune alterations, with infected southern koalas shown to have altered immune profiles [4] and korv-b infected northern koalas shown to possess levels of immune dysregulation [5] . korv has also been linked to increased susceptibility to diseases in koalas [6] . we recently described antibody production and reduction in viral load following vaccination in koalas harbouring endogenous korv [7] . briefly, koalas were vaccinated with a recombinant korv env protein along with a triadjuvant of poly i:c, polyphosphazine and host defence peptide 1002. each animal received the vaccine subcutaneously on day zero with a booster dose delivered four weeks after. the vaccine was shown to lead to a significant increase in serum anti-korv igg levels and neutralising antibody titres. the antibodies were shown to be cross-reactive against exogenous korv subtypes. in the present study, we examined the expression of important koala cytokines, immune markers and host restrictions factors to determine their pre-and post-vaccination levels in northern koalas harbouring endogenous korv. to understand the expression profiles of key immune genes associated with vaccine response, we compared the pre-vaccination levels to 4-and 8-weeks post-vaccination levels. we chose to analyse the gene expression changes in a non-antigen stimulated model. genes targeted included nine cytokines (ccl4l, interleukin open access *correspondence: oolagoke@usc.edu.au genecology research centre, university of the sunshine coast, sunshine coast, qld, australia (il)-1β, il-4, il-6, il-8, il-10, il-17a, il-18 and interferon gamma (ifn-γ), four host restriction factors (bst2, isg15, rsad2 and trim1) and two t-cell markers (cd4 and cd8β). total rna was purified from 200 µl whole koala blood using trizol ® ls (thermofisher), as per manufacturer's instructions. rna was treated with the turbo dna-free kit (thermofisher) before target gene transcripts were quantified using a custom nanostring probe panel (systems biology and data science, griffith university, australia). five koala housekeeping genes (actb, gapdh, hmg20a, nckap1l and stx12) were included in the panel for normalization and quality control. normalized transcripts were quantified pre-vaccination and converted to fold-change for post-vaccination analysis. expression levels of the target genes prior to vaccination were examined to gain an insight into the baseline immune genes expression of koalas harbouring endogenous korv. il-8 was by far the most abundant cytokine mrna expressed in these korv infected koalas (fig. 1a) , with over 200-fold higher expression compared to other cytokines. both il-6 and il-1β were the next most abundantly expressed cytokines and they had comparable expression levels (fig. 1a ). the remaining cytokines tested (ccl4l, il-10, il-17a, il-18, il-4 and ifn-γ) had low, but detectable levels in all 10 koalas investigated (fig. 1a) . additionally, all four host restriction factor genes (bst2, isg15, rsad2 and trim1) were expressed at quantifiable levels in all 10 koalas (fig. 1b) . finally, cd4 gene expression levels were significantly lower than cd8β gene expression levels (~ 10 folds) (fig. 1c) . after vaccination, ifn-γ had the most pronounced expression change, with significant up-regulation at 4-weeks (log 2 fold change = 0.4; p = 0.006) and continued up-regulation at 8-weeks (log 2 fold change = 0.8; p = 0.046) post-vaccination (fig. 2a) . there were also a small but statistically significant down-regulation of il-17a at 4-weeks post-vaccination (log 2 fold change = − 0.4; p = 0.039) and il-8 by 8-weeks post-vaccination (log 2 fold change = − 0.3; p = 0.012) (fig. 2a) . there were no significant changes in the mrna expression of the other cytokines tested (ccl4l, il-1β, il-10, il-18, il-4, and il-6) at either 4-or 8-weeks post-vaccination, data not shown. in addition, none of the host restriction factors tested (bst2, isg15, rsad2 and trim1) had significant changes in their expression either at 4-or 8-weeks post-vaccination when compared to prevaccination levels. interestingly, the post-vaccination expression of cd4 mrna remained largely unchanged relative to pre-vaccination levels, while cd8β mrna was slightly upregulated at 4-weeks post-vaccination (log 2 fold change = 0.4; p = 0.003) (fig. 2b) . in this cohort of korv vaccinated and endogenously infected koalas, a small but significant increase in the expression of ifn-γ at both 4-and 8-weeks post-vaccination was observed, compared to pre-vaccination levels. while the biological impact of such sustained up-regulation is not fully known yet, a recent study showed that korv-positive southern (victorian) koalas had significantly lowered ifn-γ levels compared to korv-negative koalas [4] . the authors of that study argued that such decreased ifn-γ levels may increase susceptibility to chlamydia and other opportunistic infections in korvpositive koalas. this work is the first to report an increase in ifn-γ in response to korv vaccination and, as such, suggests a means to counteract korv-associated reduction in ifn-γ levels in koalas. the antiviral properties of ifn-γ have been well documented. for instance, longlasting porcine circovirus vaccine efficacy was shown to be sustained, in part, by ifn-γ producing cells [8] . ifn-γ has also been linked to b-cell differentiation into igg production [9] . the increase in ifn-γ detected in this work may offer insight into the sustained anti-korv igg production and reduction in viral load reported in koalas following vaccination [7] . a possible mechanism for the observed increase in ifn-γ levels post-vaccination may be the inclusion of poly[di(sodium carboxylatoethylphenoxy)phosphazene] (pcep) as an adjuvant component in the korv vaccine. pcep has been previously shown to induce a strong ifn-γ response and enhance antigenspecific immune response in mice following vaccination [10] . several host restriction factors able to interfere with multiple stages of the viral life cycle have been identified in humans and animals [11] . our study investigated the expression of four host restriction factors in koalas harbouring endogenous korv: bst2, isg15, rsad2 and trim1. all four genes were found to be expressed at quantifiable levels. surprisingly, bst2, isg15 and rsad2 were expressed at relatively high levels, suggesting that these restriction factors may have biological importance in koalas harbouring endogenous korv. although there was not a vaccine-induced up-regulation in these host restriction genes, the detection and investigation of these genes nonetheless present a new line of research into innate antiviral mechanisms for koalas. the role of korv in modulating the cd4:cd8 ratio in koalas has been previously investigated. a study of captive koalas harbouring endogenous korv reported a cd4:cd8 expression median ratio of 2.1 (range: 0.1-6.3) [12] . surprisingly, all koalas in the current study had very low levels of cd4 mrna expression when compared to cd8β mrna expression. this observation was not improved by vaccination. when compared to the normal range in humans (1.5-2.5) [13] , the levels observed in this work suggest an abnormally low cd4 expression in koalas. unfortunately, the lack of koala-specific reagents prevented further investigation into whether the reduced cd4 mrna expression directly translated into fewer cd4 t cells. however, a positive association between cd4 molecule and cd4 mrna expression has been previously shown in human studies [14] . as such, these results could suggest an ongoing korv-associated immunosuppression, possibly through the preferential loss of cd4 t cells in korv-infected koalas, similar to what is seen in cats infected with feline leukemia virus and feline immunodeficiency virus [15] . finally, il-8 was by far the most expressed cytokine detected in tested koalas. increased il-8 expression has been implicated in several pathologies including leukemia [16] . high levels of il-8 is also associated with progression to disease in people infected with hiv-1, when compared to those who were able to maintain natural control of the infection [17, 18] . the extremely high level of il-8 seen in these korv infected koalas is highly interesting, as it may be an important biomarker for underlying conditions that may be korv-associated. in these endogenously infected koalas, korv vaccination led to a small but significant decrease in il-8 mrna expression. a recent study showed that reduction in il-8 levels was required for chlamydia clearance in korvinfected koalas [19] . if further studies can conclusively link il-8 to koala pathologies, then the role of il-8 inhibitors, such as through vaccination, should be further explored. this work described increased ifn-γ mrna expression following korv vaccination in koalas harbouring endogenous korv. a very high expression level of il-8 mrna prior to vaccination, followed by a slight but significant decrease post-vaccination, was also observed. finally, this work provides new insight into possible mechanisms for korv-associated immunosuppression in korv infected koalas. korv: koala retrovirus; il: interleukin; ifn: interferon. retroviral invasion of the koala genome long-read genome sequence assembly provides insight into ongoing retroviral invasion of the koala germline helping koalas battle disease-recent advances in chlamydia and koala retrovirus (korv) disease understanding and treatment in koalas altered immune parameters associated with koala retrovirus (korv) and chlamydial infection in free ranging victorian koalas (phascolarctos cinereus) altered immune cytokine expression associated with korv b infection and season in captive koalas an exogenous retrovirus isolated from koalas with malignant neoplasias in a us zoo therapeutic vaccination of koalas harbouring endogenous koala retrovirus (korv) improves antibody responses and reduces circulating viral load memory t cell proliferative responses and ifn-γ productivity sustain long-lasting efficacy of a cap-based pcv2 vaccine upon pcv2 natural infection and associated disease b cells responses and cytokine production are regulated by their immune microenvironment poly[di(sodium carboxylatoethylphenoxy)phosphazene] (pcep) is a potent enhancer of mixed th1/th2 immune responses in mice immunized with influenza virus antigens restriction factors: from intrinsic viral restriction to shaping cellular immunity against hiv-1 expression profiles of the immune genes cd4, cd8β, ifnγ, il-4, il-6 and il-10 in mitogen-stimulated koala lymphocytes (phascolarctos cinereus) by qrt-pcr imbalance in the game of t cells: what can the cd4/cd8 t-cell ratio tell us about hiv and health? plospathog correlation between the expression of cd4 and the level of cd4 mrna in human b-cell lines clinical aspects of feline retroviruses: a review plasma interleukin 8 level predicts for survival in chronic lymphocytic leukaemia host factor predictors in long-term nonprogressors hiv-1 infected with distinct viral clades il-8 alterations in hiv-1 infected children with disease progression antibiotic treatment of chlamydia-induced cystitis in the koala is linked to expression of key inflammatory genes in reactive oxygen pathways publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations the authors would like to thank the many groups that have significantly supported the overall koala vaccine development work, including the departments of transport and main roads, and environment and heritage protection of the queensland government, the moreton bay rail project team, moreton bay regional council, city of gold coast, redland city council, lone pine koala sanctuary, endeavour veterinary ecology, koala action inc., friends of koala (lismore), australia zoo wildlife hospital, staff and volunteers at the adelaide koala and wildlife hospital, as well as all koala rescue groups, zoos south australia, and vaccine and infectious diseases organization (vido), canada. we would also like to thank dr nic west and dr jelena vider (systems biology and data science, griffith university, australia) for help with nanostring experimentation. this work was financially supported by the australian research council linkage grant lp150100046 received by pt. the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. the data that support the findings of this study are available from the authors on request. all animal work reported in this study was approved by university of the sunshine coast animal ethics committee (ana18138). this study was conducted in compliance with approved institutional guidelines. not applicable. the authors declare no competing interests.received: 8 july 2020 accepted: 27 october 2020 key: cord-001868-utsvsja0 authors: uematsu, takayuki; iizasa, ei’ichi; kobayashi, noritada; yoshida, hiroki; hara, hiromitsu title: loss of card9-mediated innate activation attenuates severe influenza pneumonia without compromising host viral immunity date: 2015-12-02 journal: sci rep doi: 10.1038/srep17577 sha: doc_id: 1868 cord_uid: utsvsja0 influenza virus (ifv) infection is a common cause of severe viral pneumonia associated with acute respiratory distress syndrome (ards), which is difficult to control with general immunosuppressive therapy including corticosteroids due to the unfavorable effect on viral replication. studies have suggested that the excessive activation of the innate immunity by ifv is responsible for severe pathologies. in this study, we focused on card9, a signaling adaptor known to regulate innate immune activation through multiple innate sensor proteins, and investigated its role in anti-ifv defense and lung pathogenesis in a mouse model recapitulating severe influenza pneumonia with ards. we found that influenza pneumonia was dramatically attenuated in card9-deficient mice, which showed improved mortality with reduced inflammatory cytokines and chemokines in the infected lungs. however, viral clearance, type-i interferon production, and the development of anti-viral b and t cell immunity were not compromised by card9 deficiency. syk or card9-deficient dcs but not macrophages showed impaired cytokine but not type-i interferon production in response to ifv in vitro, indicating a possible role for the syk-card9 pathway in dcs in excessive inflammation of ifv-infected lungs. therefore, inhibition of this pathway is an ideal therapeutic target for severe influenza pneumonia without affecting viral clearance. the host's innate immune system including macrophages and dendritic cells (dcs) to secrete inflammatory cytokines/chemokines, such as il-6, tnf, cxcl1 and cxcl10 13, 14 , which play crucial roles in the pathogenesis of ards 10, [15] [16] [17] . therefore, inhibition of cytokine/chemokine production via targeting the host's innate immune system may lead to the development of effective treatment options for ards. caspase recruitment domain family member 9 (card9) is an adaptor protein that delivers nf-κ b activating signals through multiple innate sensor proteins, such as a wide array of c-type lectin (clr) and immunoglobulin (ig)-superfamily receptors that are coupled to immunoreceptor tyrosine-based activation motifs (itams) [18] [19] [20] , as well as cytoplasmic rna sensors, such as rig-i and mda5, and the dna sensor rad50 21, 22 . several lines of evidence have implied a role for card9-mediated innate activation pathways in influenza pathogenesis and immunity. the clr dendritic cell-specific icam-3 grabbing non-integrin (dc-sign), which has a cytoplasmic itam-like sequence, was shown to bind influenza virus a 23, 24 . additionally, card9 was found to be required for the production of inflammatory cytokines after infection with rna viruses, such as vesicular stomatitis virus and encephalomyocarditis virus, by regulating nf-κ b signaling through rig-i and mda5 21 . however, the roles of card9 in influenza pneumonia, as well as in protection against ifv are yet to be elucidated. in this study, we demonstrate a role for card9-mediated innate activation in influenza pathogenesis and immunity. our results showed that the card9 pathway was involved in fatal influenza pneumonia mediated by inflammatory cytokine/chemokine production, whereas it was dispensable for type-i interferon production as well as the development of anti-viral acquired immunity. therefore, inhibition of this pathway may represent an ideal target for the treatment of severe influenza pneumonia without affecting viral clearance. adapted ifv a/pr/8/34 strain (pr8) shows similar lung pathology to human ards 10-12 , we intratracheally infected wild-type (wt: c57bl/6) and card9 -/mice with a lethal dose (10 4 pfu/mouse) of pr8 to determine whether card9-mediated innate immune responses contributed to severe influenza pneumonia. we observed that pr8-infected wt mice appeared visibly ill with ruffled fur and reduced oral intake during the 6 to 10 days after infection, whereas card9 -/mice appeared more active than wt mice. consistent with their activity, the final survival rate up to day 21 after infection was dramatically improved in card9 -/mice (~80%) as compared with wt mice (~40%; fig. 1a ). histopathological analysis of ifv-infected lungs on days 4 and 8 revealed that lung inflammation, which was the most obvious on day 8 in wt mice, was much less severe in card9 -/mice (fig. 1b) . given that inflammatory cytokines/chemokines were linked to lung damage in severe influenza pneumonia [10] [11] [12] [15] [16] [17] 25 , we analyzed infiltrated cells and cytokine/chemokine levels in bronchoalveolar lavage fluid (balf) collected from the ifv-infected lungs of wt and card9 -/mice. consistent with the pathological data, the number of cd45 + balf cells were significantly fewer in the lungs of card9 −/− mice than in those of wt mice on day 8, with a marked decrease in the numbers of t cells and neutrophils ( fig. 1c ) that were the two main infiltrated subpopulations at this time point (supplementary fig. s1 ). however, the numbers of b cells, nk cells, macrophages, cdcs, and pdcs were not significantly affected by the card9 deficiency. the balf levels of inflammatory cytokines and chemokines, i.e., il-6, tnf-α , ccl3/mip-1α , cxcl1/kc, and cxcl10/ip-10, which have been reported to contribute to lung pathology 10, 13, 14 , peaked on day 4 and were considerably lower in the card9 −/− mice than in the wt mice (fig. 1d ). however, no reduction in cytokine/chemokine levels was evident on day 8, indicating that a card9-mediated innate response controls cytokine/chemokine production at an early time point after ifv infection and influences subsequent inflammatory cell recruitment and lung pathology at a later time point. collectively, the loss of card9 attenuated severe influenza pneumonia and improved host mortality. card9 deficiency does not compromise anti-viral protective immunity. activation of innate immunity in response to ifv regulates anti-viral protective immunity 26 . to evaluate the impact of card9 deficiency on anti-ifv protection, we first analyzed the viral burden in the ifv-infected lungs of wt and card9 -/mice. notably, we found that card9 deficiency did not increase virus titers and the amount of viral rna in the lungs, but instead significantly accelerated viral clearance in the later stage (day 8) of infection ( fig. 2a) . type-i interferons (ifn-α /β ) are the primary factors that induce host resistance to ifv 26, 27 . in contrast to cytokine/chemokine production, card9 deficiency did not significantly affect ifn-α /β production in ifv-infected lungs (fig. 2b) . the type ii interferon ifn-γ is not essential for viral clearance 28 , but its presence during ifv infection ameliorates the severity of inflammation and lung injury 29 . ifn-γ production was significantly more in the lungs of card9 −/− mice than in those of wt mice on day 8 but not on day 4 (fig. 2b) ; thus, this increase might contribute to the improvement of lung pathology in card9 −/− mice. moreover, the expression of ip-10 encoded by an ifn-responsive gene was significantly impaired in card9 −/− mice on day 4 (fig. 1d) , albeit no significant reduction was seen in either type i or type ii interferons, suggesting that synergistic signals of interferons and various cytokines/chemokines are required for the higher expression of ip-10 at an early phase of infection. next, we examined the induction of virus-specific cd8 + t cells using the h-2d b tetramer coupled with a viral nuclear protein (np)-derived peptide (np 366-374 ) 30, 31 . the percentages of np-specific cd8 + t cells in the draining lymph nodes of the lungs on day 8 were comparable between wt and card9 −/− mice (fig. 2c) . to evaluate the impact of card9 deficiency on acquired immunity to ifv, wt and card9 −/− mice were immunized with a sub-lethal dose (1/10 of ld 50 ) of the pr8 strain and rechallenged with a high lethal dose (100 ld 50 ) of pr8 after 4 weeks. we found that card9 deficiency did not alter viral clearance after the challenge (fig. 2d ). next, we assessed the induction of humoral and cellular acquired immunity to ifv in card9 -/mice. we re-infected wt and card9 -/mice on day 21 after their first infection (representing the second infection) and analyzed the production of virus-specific antibodies as well as the development of virus-specific cd8 + t cells on day 14 after the second infection. we found that the card9 deficiency affected neither the production of virus-specific igg in the serum nor virus-specific iga in the lung mucosa (fig. 2e) . additionally, card9 deficiency did not affect the ifn-γ response by splenic cd8 + t cells specific to a np antigen epitope np 366-374 (fig. 2f) . collectively, the card9 pathway is dispensable for the induction of protective immunity against both the primary and secondary ifv infections, and its deficiency even improves primary viral clearance upon lethal-dose infections probably because of improvement in lung damage and elevated ifn-γ production. card9 deficiency impairs inflammatory cytokine, but not type-i interferon, production by dcs. because macrophages and dcs are known to be an early source of inflammatory cytokines and type-i interferons in pulmonary ifv infection 13,14 , we examined whether card9 was required for their production by myeloid cells in response to ifv. thioglycolate-induced peritoneal macrophages (tg-mfs), bone marrow-derived macrophages (bmmfs), alveolar macrophages (amfs), conventional dcs (cdcs), or flt3 ligand-induced plasmacytoid dcs (flt3l-dcs) prepared from wt or card9 -/mice were brought into contact with pr8 in vitro and the production of il-6, tnf-α , and ifn-α /β was measured. il-6 and tnf-α produced by tg-mfs (fig. 3a) or bmmfs (fig. 3b ) and those mrnas produced by amfs (fig. 3c ) in response to pr8 were not affected by card9 deficiency. as previously reported 18, 19 , card9 was required for a cytokine response to ox-zymosan through dectin-1-syk but dispensable for the response to lps through tlr4-myd88 in cdcs ( supplementary fig. s2a ). we found that card9 -/-cdcs (fig. 3d ) or flt3l-dcs (fig. 3e ) produced significantly lower levels of il-6 and tnf-α than wt cells in response to pr8. in contrast, ifn-α /β production was not compromised in card9 -/-cdcs or flt3l-dcs. these findings suggest that card9 deficiency primarily affected dcs but not mfs regarding the production of inflammatory cytokines but not ifn-α /β in response to ifv. it has been reported that the card9-ips-1 pathway mediates the cytokine response to rna viruses through rlhs 21 , while card9 has been found to transmit signals from myeloid itam-coupled receptors via syk [18] [19] [20] . thus, we evaluated the contributions of these pathways. ips-1-deficient (ips-1 -/-) cdcs and myd88-deficient (myd88 -/-) flt3l-dcs produced significantly lower levels ifn-α /β than wt cells in response to pr8 (fig. 3d ,e), consistent with previous findings showing that the ifn-α /β response to rna viruses is mediated mainly through rig-i/ips-1 in cdcs, and through tlr7/myd88 in pdcs 32, 33 . in addition, il-6 and tnf-α production was impaired in ips-1 -/-cdcs and myd88 -/-flt3l-dcs. lower levels of cytokine production were also observed in myd88 -/-cdcs, likely due to impaired activation through several tlrs (i.e. tlr3, 7, 8 and 9) that sense virus nucleic acids 34, 35 . we found that inducible syk-deficient (syk del/del ) cdcs and flt3l-dcs produced significantly lower levels of inflammatory cytokines than wt cells similar to the responses of card9 -/-cdcs and flt3l-dcs, whereas the syk deficiency did not affect cytokine production in response to lps (supplementary fig. s2a ). consistent with these findings, treatment of wt cdcs (fig. 4a ) and flt3l-dcs (fig. 4b) with the syk inhibitor bay61-3606 reduced the production of inflammatory cytokines but not type-i interferons in response to pr8 in a dose-dependent manner. the treatment with bay61-3606 reduced the cytokine response to ox-zymosan but not to lps, indicating that the effect of the inhibitor on cytokine expression depends on the stimulus but cannot be attributed to its general effect on cytokine gene expression ( supplementary fig. s2b ). overall, these results suggest that the attenuation of influenza pneumonia by card9 deficiency was likely attributable to the reduced cytokine/chemokine production by pulmonary dcs through the syk-card9 pathway in response to ifv. we have shown that card9-mediated activation of the innate immune system exacerbates influenza pneumonia in mice. card9 deficiency resulted in the reduction of inflammatory cytokine/chemokines and the infiltration of inflammatory cells in ifv-infected lungs, resulting in improved mouse mortality rates. given the pathological similarity between the mouse intratracheal infection model and influenza-associated ards in human 10-12 , we postulate that the card9 pathway contributes to the exacerbation of human influenza pneumonia. although the syk-card9-mediated innate immune balf were analyzed on day 0 and 14 after the second infection. (f) ifv-specific adaptive t cell response. splenocytes from wt and card9 -/mice (n = 6 per group) 14 days after infection as in (e) were stimulated in vitro with ifv nucleoprotein peptide (np 366-374 ) for 3 days. the culture supernatants were analyzed for ifn-γ production. data are presented as mean ± sd of triplicates. data are representative of three independent experiments. *p < 0.05 by student's t-test. scientific reports | 5:17577 | doi: 10.1038/srep17577 response is crucial for anti-fungal acquired immunity [36] [37] [38] [39] , our data showed that card9 was dispensable for anti-ifv immunity, as demonstrated by the findings that card9 deficiency did not alter viral burden, the elevation of ifn-α /β , or the induction of anti-viral adaptive t and b cell responses in the ifv-infected mice. thus, other innate mechanisms independent of the card9 pathway may play a more dominant role in protective immunity against ifv. innate immune sensors for ifv and its downstream signaling pathway have been well illustrated 26, 40, 41 . in our study, card9 deficiency resulted in a selective reduction in inflammatory cytokines, but not ifn-α /β production, by both cdcs and flt3l-dcs in response to ifv. this pattern of impairment was observed in ips-1 -/-cdcs, and was consistent with results from previous reports indicating that card9 selectively regulates the cytokine response through rig-i-ips-i in cdcs 21 . additionally, we found that syk del/del and syk inhibitor-treated cdcs showed the same pattern of impairment as card9 -/or ips-1 -/-cdcs. on the other hand, in flt3l-dcs known to release large amounts of inflammatory cytokines and type-i interferons upon recognition of ifv 32,33 , ips-1 deficiency affected neither cytokine or type-i interferon responses. however, syk deficiency in flt3l-dcs, similar to that in cdcs, resulted in a selective reduction in inflammatory cytokines but not ifn-α /β . thus, it is likely that the syk-card9 pathway controls detrimental cytokine production by pulmonary dcs upon acute influenza infection. alternatively, because card9 deficiency impairs cytokine production by dcs following stimulation with several tlr ligands 18 , it may be important to examine whether the syk-card9 pathway is involved in the tlr7-meditated response to ifv. the involvement of the syk-card9 pathway implies the presence of itam-coupled receptors that sense ifv and stimulate cytokine production by dcs. it has been reported that an interaction between dc-sign, which possesses an itam-like "hemitam" motif, and the carbohydrate of ifv hemagglutinin induces maturation of dcs and promotes endocytosis of the virus 42 , indicating the signal-activating capacity of dc-sign upon virus binding. however, it is unclear whether the dc-sign hemitam is capable of transmitting sufficient signals to produce inflammatory cytokines. with regard to other viruses, recognition of the dengue virus by the dap12-associated clr clec5a results in increased vascular permeability due to the overproduction of tnf-α , resulting in fatal outcomes 43 . however, its ability to recognize the ifv remains unknown. thus, it is worth examining whether these clrs are involved in the cytokine response to ifv. it is also conceivable that damage-associated molecular patterns (damps), generated due to lung damage by ifv infection, may be ligands for some itam-coupled receptors. indeed, the fcrγ -associated clr mincle was shown to recognize sap130, a component of the ribonucloprotein released from dead cells 44 . in conclusion, card9-mediated innate immune activation in pulmonary dcs acts to exacerbate severe influenza pneumonia, but is dispensable for host protection against ifv. thus, inhibiting the card9 pathway may represent a promising therapeutic target for the control of pivp without affecting viral clearance. mice. card9 -/-, syk flox/flox , ips-1 -/and myd88 -/mice have been previously described 18, [45] [46] [47] . these mice were backcrossed at least 8 times onto c57bl/6 mice. c57bl/6 mice were purchased from clea japan, inc. (tokyo, japan). the animals were housed in specific pathogen-free conditions. all experiments were approved by the institutional animal care and use committee for kitasato university medical center and animals were treated in accordance with the regulations for animal experiments in kitasato university. all surgeries were performed under ketamine hydrochloride/xylazine anesthesia, and all efforts were made to minimize suffering. antibodies and reagents. fluorescein isothiocyanate (fitc)-conjugated anti-f4/80 (clone bm8), fitc-conjugated anti-cd45r/b220 (clone ra3-6b2), fitc-conjugated cd19 (clone 6d5), phycoerythrin (pe)-conjugated anti-ly-6g (clone 1a8), pe-conjugated anti-siglech (clone 551), biotin-conjugated anti-nk1.1 (clone pk136), biotin-conjugated anti-bst2/pdca-1 (clone 927), peridinin chlorophyll protein/cy5.5 (percp/cy5.5)-conjugated anti-cd45 (clone 30-f11), phycoerythrin-cy7 (pc7)-conjugated anti-cd3ε (clone 145-2c11) and pc7-conjugated anti-cd11c (clone n418) monoclonal antibodies were purchased from biolegend (san diego, ca). fitc-conjugated anti-cd8 (clone kt15) monoclonal antibodies, pe-conjugated h-2d b tetramer coupled with a viral np-derived asnenmetm peptide (np 366-374 : asnenmetm), h-2d b -restricted influenza np 366-374 peptide were purchased from mbl (nagoya, japan). syk inhibitor iv (bay 61-3606) was purchased from merck millipore (billerica, ma). lps from escherichia coli 0111:b4 was purchased from sigma-aldrich (st. louis, mo). naclo-oxidized zymosan was prepared as described 48 . ifv infection in mice. c57bl/6 and card9 -/mice were anesthetized and infected with 10 4 plaque forming units (pfu) (unless otherwise indicated) of a mouse-adapted ifv (a/pr/8/34 strain: h1n1 isotype, kindly provided by the kitasato institute, tokyo, japan) by intratracheal administration as described 10-12 . histology. whole lungs were collected at 0, 4, and 8 days after ifv infection. paraffin embedding and hematoxylin and eosin staining of tissues were performed using standard methodologies. . bal was carried out as described previously 25, 49 . in brief, tracheas of mice were cannulated with 1.2-mm diameter polyethylene catheters. lungs were instilled with 1 ml of pre-warmed pbs containing 5 mm edta, followed by the retrieval of lavage fluid aliquots. cells in the bal fluid (balf) were counted after red blood cell lysis and subjected to flow cytometric analysis. the supernatants of the balf were subjected to multi cytokine/chemokine expression analysis and cytokine elisa. cytomix cytokine bead assay (bender medsystems, vienna, austria), with the exception of ifn-β , which was measured by a verikine tm mouse interferon-β elisa kit (pbl interferonsource, piscataway, nj). cell culture supernatants from splenocytes were assayed using a specific elisa kit for ifn-γ (biolegend). cell culture supernatants from tg-mfs, bmmfs, cdcs, and flt3l-dcs were assayed using specific elisa kits for il-6, tnf-α (biolegend), ifn-α and ifn-β (pbl interferonsource). all measurements were performed in triplicate. for quantitative pcr (qpcr) analysis for cytokine mrnas, total rna was extracted from cells with reliaprep rna cell miniprep system (promega, madison, wi) and cdna was synthesized with primescript rt reagent kit (takara bio, shiga, japan) according to the manufacturer's instructions. qpcr was performed with the applied biosystems 7900ht fast real time pcr system (life technologies). primer sequences were as follows: mouse il6, forword, 5′-cca ctt cac aag tcg gag gct ta -3′, and reverse, 5′-gca agt gca tca tcg ttg ttc ata c -3′; and tnf forword, 5′-gtt cta tgg ccc aga ccc tca c -3′, and reverse, 5′-ggc acc act agt tgg ttg tct ttg -3′. viral copy numbers. mice were euthanized by intraperitoneal administration of sodium pentobarbital at 0, 4, or 8 days after ifv infection. lung tissues were homogenized using a gentlemacs dissociator (miltenyi biotec) and rna was extracted with an isogen ii rna extraction kit (nippon gene, tokyo, japan). reverse transcription was conducted with the uni-12 primer (5′-agc aaa agc agg -3′) 50 and qpcr was preformed with primers specific for np (forward: 5′-gat tgg tgg aat tgg acg at -3′; reverse: 5′-aga gca cca ttc tct cta tt -3′) using the applied biosystems 7900ht fast real time pcr system (life technologies, carlsbad, ca). the standard calibration curve for qpcr was obtained by stepwise dilution of the cloned np gene fragment with a known copy number. ifv rechallenge. wt and card9 -/mice were left uninfected or infected intratracheally with sublethal dose (10 3 pfu/mouse: 1/10 ld 50 ) of pr8. twenty-eight days after the first infection, mice were challenged with a high lethal dose (10 6 pfu/mouse: 100 ld 50 ) of pr8. two days postchallenge, virus titers in the lungs were measured in a plaque assay in mdck cells. antigen-specific b cell responses. b cell-mediated humoral responses were measured as virion-specific immunoglobulin production by elisa, as previously described 32 . briefly, 96-well elisa plates (corning) were coated with ultrasonicated influenza virion (a/pr/8/34 strain) at 5 × 10 6 pfu/ ml in a carbonate buffer (ph 9.6), and incubated overnight at 4 °c. plates were then washed with pbs containing 0.05% tween 20 (wako pure chemical industries). serum and balf collected from mice at day 14 after the secondary infection were serially diluted with pbs/tween 20 containing 5% skim milk, applied onto the virion-coated plates, and incubated for 2 h at room temperature. after washing, goat anti-mouse total igg or iga conjugated to horseradish peroxidase (jackson immunoresearch, baltimore pike, pa) was applied and incubated for 2 h at room temperature. after washing, the plates were stained with a tmb substrate set (biolegend). the reaction was terminated with 1 m h 2 so 4 (wako pure chemical industries) and the absorbance was measured. antigen-specific t cell responses. ifv-specific t cell responses were measured as viral np-specific ifn-γ secretion by splenocytes, as described previously 32, 51 . briefly, 5 × 10 5 splenocytes extracted from mice 14-days after the secondary infection were seeded on 96-well cell culture plates (corning) and then stimulated with 10 μ g/ml of np 366-374 peptides. after 3 days of culture, supernatants were collected and analyzed for ifn-γ production by elisa (biolegend). tg-mfs were prepared as described 36 . amfs were isolated from balf of wt or card9 -/mice. bmmfs, cdcs, or flt3l-dcs were prepared by culturing bone marrow cells for 5-8 days with rpmi1640 medium (wako pure chemical industries) supplemented with 10% fetal bovine serum (life technologies) and antibiotics (100 iu/ml penicillin and 100 μ g/ml streptomycin; sigma-aldrich) containing m-csf (25 ng/ml, peprotech, rocky hill, nj), gm-csf (20 ng/ml, peprotech) or human flt3-ligand (100 ng/ml, peprotech), respectively. the flt3l-dcs derived from the culture contain constantly 10-13% of pdca-1 + siglech + plasmacytoid dcs (pdcs) irrespective of deficiency in syk, card9, ips-1 or myd88. for syk deletion in vitro, cultured cells derived from syk flox/flox mice were incubated with 0.6 μ m of the active metabolite of tamoxifen, 4-hydroxytamoxifen (sigma-aldrich) for 3 d. for stimulation of mfs/dcs of, 1 × 10 5 cells were seeded on 24-well culture plates (corning) and incubated overnight, followed by replacement of 200 μ l of serum-free medium containing 10 6 pfu (multiplicity of infection [m.o.i] = 10) of pr8. after 1 h of incubation, unabsorbed viruses were removed and the cells were incubated for a further 24 h in serum-containing medium. dcs were also stimulated with lps (100 ng/ml) or naclo-oxidized zymosan (10 μ g/ml)