key: cord-304718-w469n0o8 authors: wang, yan; yan, jiangwei; shi, yuling; li, ping; liu, chuanxuan; ma, qingjun; yang, ruifu; wang, xiaoyi; zhu, lina; yang, xiao; cao, cheng title: lack of association between polymorphisms of masp2 and susceptibility to sars coronavirus infection date: 2009-05-01 journal: bmc infect dis doi: 10.1186/1471-2334-9-51 sha: doc_id: 304718 cord_uid: w469n0o8 background: the pathogenesis of severe acute respiratory disease syndrome (sars) is not fully understood. one case-control study has reported an association between susceptibility to sars and mannan-binding lectin (mbl) in china. as the downstream protein of mbl, variants of the mbl-associated serine protease-2 (masp2) gene may be associated with sars coronavirus (sars-cov) infection in the same population. methods: thirty individuals with sars were chosen for analysis of masp2 polymorphisms by means of pcr direct sequencing. tag single nucleotide polymorphisms (tagsnps) were chosen using pairwise tagging algorithms. the frequencies of four tag snps (rs12711521, rs2261695, rs2273346 and rs7548659) were ascertained in 376 sars patients and 523 control subjects, using the beckman snpstream ultra high throughput genotyping platform. results: there is no significant association between alleles or genotypes of the masp2 tagsnp and susceptibility to sars-cov in both beijing and guangzhou populations. diplotype (rs2273346 and rs12711521)were analyzed for association with susceptibility to sars, no statistically significant evidence of association was observed. the beijing and guangzhou sample groups were homogeneous regarding demographic and genetic parameters, a joined analysis also showed no statistically significant evidence of association. conclusion: our data do not suggest a role for masp2 polymorphisms in sars susceptibility in northern and southern china. severe acute respiratory disease syndrome (sars), a new and highly infectious disease that is caused by a previously undescribed coronavirus in humans, has created a major public health threat in many countries [1] [2] [3] . progress has been made in understanding sars coronavirus (sars-cov) and the epidemiology, clinical manifestations, laboratory findings and radiological features of this disease have all been studied in detail [4, 5] . however, its pathogenesis is still not fully understood. it has been reported that diabetes mellitus and heart disease are risk factors for adverse prognosis of sars [6] , however, little is known about the contribution of genetic factors. sars has been found to have a profoundly adverse effect on the immune system [7] . variation in host immunity may be one of the important factors that determine susceptibility to sars. a few case-control studies have reported an association between sars susceptibility and human leucocyte antigen (hla) and mbl [8] [9] [10] [11] . deficiency of mbl, a key component of the innate immune system, has been detected in sars patients. such a deficiency may increase susceptibility to sars infection. three papers reported the association of hla and susceptibility to sars [8] [9] [10] . of these, two reported the association of hla with susceptibility and resistance to the development of sars [8, 9] . in a more recent paper, no statistical significance was found with the susceptibility and severity of the disease [10] . mbl is a member of the collectin family and plays an important role in innate immunity [12, 13] . mbl and ficolins distinguish between self, non-self and altered-self by recognizing patterns of ligands on the surface of microorganisms or aberrant cells. when this happens, mbl-associated serine protease-2 (masp-2) is activated and cleaves complement factors to initiate the antibody-independent pathway of the complement system, thus starting inflammatory reactions. mbl-associated serine proteases interact with mbl via the collagenous region of larger mbl oligomers. four related proteins derived from two genes have been reported; namely masp1, its alternative splicing variant masp3, and masp2 with its alternative splicing variant map19 [14] [15] [16] . masp2 activates the complement system by cleaving complement proteins c4 and c2. masp2 is an essential component of the lectin pathway of complement activation. masp2 deficiency is observed because of genetic polymorphisms. it is known that a masp2 polymorphism, namely d120g has a functional effect on the protein, and does not allow the formation of an active mbl-masp complex. this variant has been described for the first time in a patient with an inherited deficiency of masp2, who was characterized by augmented susceptibility to infection and development of immunological disease. with regard to sars-cov infection, the codon 54 variant of the mbl gene has been shown to be associated with infection susceptibility but not with disease severity [11] . as the downstream protein of mbl, variants of the masp2 gene may be associated with sars-cov infection. to examine the hypothesis that polymorphisms of the masp2 gene in sars patients are genetic factors that influence infection susceptibility, we studied masp2 gene polymorphisms in dna from two groups of chinese sars patients, and compared these with normal blood donors from the same region. this study was performed with the approval of the ethical committee of the chinese human genome center. a total of 376 sars patients, who were diagnosed based on who criteria, were recruited during the 2003 outbreak. all sars patients selected for study were unrelated and were shown to be seropositive by anti-sars-n protein elisa and immunofluorecent assay (ifa). the specificity of the elisa assay is more than 99% [17, 18] . of these, 272 were from the wards at xiaotangshan hospital, beijing, china, and the remaining 104 were from the eighth people's hospital of guangzhou in southern china. a total of 523 age, sex, and ethnicity-matched healthy, genetically unrelated and seronegative (confirmed by anti-sars n protein antibody elisa and sars antibody ifa) adults were recruited as control subjects. we extracted genomic dna from peripheral blood leukocytes of affected individuals and controls using the qiaamp dna mini kit (qiagen, valencia, ca). dna was quantified using standardized fluorometric reading on a du 650 spectrophotometer (beckman coulter, fullerton, ca). each sample was diluted to a final concentration of 10 ng/ ul. genomic dna from 30 individuals with sars was chosen for analysis of masp2 gene polymorphisms. the sample included 60 chromosomes, which provided a 95% confidence level to detect alleles with a frequency >5% [19] . with a candidate-gene strategy, we screened for polymorphisms in all exons (~2.0 kb), 5'-and 3'-flanking regions (lengths of 2.5 kb), untranslated regions (~0.3 kb), and about 2.5 kb intronic sequences. dna sequence spanning the masp2 gene was obtained from the national center for biotechnology information (ncbi) website (available from http://www.ncbi.nlm.nih.gov/; nt_021937.18). primers were designed using the premier program (primer premier 5). pcr primer sequences see additional file 1. dna samples from the 30 individuals were amplified and purified. the pcr reaction volume was 50 μl, containing 10-50 ng of dna,10 pmol of each forward and reverse primer, 0.2 mmol/l of each dntp,0.3 u of dna polymerase(tiangen biotec co, beijing, china),10 mmol/l of tris-hcl, 1.5 mmol/l of mgcl 2 , 50 mmol/l of kcl, and 0.1% triton x-100. amplication was carried out in a geneamp pcr system 9700(abi) with cycle parameters of 5 min 94°c (initial denaturation), 32 rounds of 94°c 30 s, 54-55°c 30 s, and 72°c 45 s, and a final extention for 5 min at 72°c. pcr products were sequenced using an abi prism dye terminator sequencing kit with amplitaq gold dna polymerase (applied biosystems division/perkin-elmer, foster city, ca) and loaded onto an abi 3100 sequencer. polymorphism candidates were identified by the polyphred program. (p. green, personal communication) snp genotypes were verified by manual evaluation of the individual sequence traces. genotyping was completed using snpstream ultra high throughput genotyping system (beckman coulter, fullerton, ca) according to the manufacture's instructions. briefly, the method combines solution-phase multiplex single nucleotide extension (sne) with a solid-phase sorting of labeled sne primers by hybridization to a chip that contains 384 4 × 4 arrays of 12 oligo-nucleotide tags and 4 oligo-nucleotides for positive and negative controls. each sne primer contained 1 of the 20 oligonucleotide tags at its 5' end, and the sne reactions were performed in 12-plex. the microarray plate was imaged by the snpscope reader (beckman coulter, fullerton, ca). the two-color system allowed the detection of the snp by comparing signals from the two fluorescent dyes. the image signals were then transferred to genotyping software that translated the images of the arrays into genotype calls. the error rate based upon sequencing for 10% of the snps examined in the present study was 0-1.2%. the exact test was calculated to evaluate if the population was in hardy-weinberg equilibrium by the markov chain method, taking p < 0.05 as the cutoff for assessing significance. the tagsnps were chosen on a pairwise basis and linkage disequilibrium (ld) calculation was performed on the confidence interval basis using haploview 3.2 software. genotype and alleles frequencies for each polymorphism were determined by gene counting. diplotypes of each individual were inferred using the algorithm developed by stephens et al. (2001) (phase). the chi square test was used to determine whether allele frequencies differed between sars cases and controls. binary logistic regression was used to analyze the association between a single locus and sars susceptibility, adjusted for sex and age status, and odds ratio and 95% ci were used to measure strength of association in a genetic risk association study. these statistical analyses were implemented in statistical package for the social sciences 13.0 software (spss inc, chicago, illinois, usa). demographic characteristics are shown in table 1 . the age and sex distribution of the patients and controls were not significantly different, which indicated that the matching of controls to cases was adequate. screening masp2 for polymorphisms sequencing of the 11 exons of masp2, the 5' and 3' regions of the gene, and some intronic sequences in 30 individuals with sars identified 17 polymorphisms ( table 2 ). eleven of the snps have been published in the dbsnp database http://www.ncbi.nlm.nih.gov/snp/ index.html. allele and genotype frequencies were consistent with those expected under hardy-weinberg equilibrium. nine snps (allele frequency >5%) were chosen with haploview for assessment. the snps were contained in two blocks of ld (fig. 1) , as defined by lewontin's| d'|. snps including rs7548659, rs2273347 and rs6695096 were located outside of the defined ld blocks. four tag-snps were chosen with the pairwise tagging algorithm implemented in the tagger program of haploview: rs7548659, rs12711521, rs2273346 and rs2261695 (r 2 threshold was 0.8). genotype frequencies of the tag-snp except rs2261695 in other populations that have been published in the hapmap database were shown in table 3 genotype data and diplotype (rs2273346 and rs12711521) were analyzed for association with susceptibility to sars, in beijing and guangzhou populations, using binary logistic regression for overall genotypic association (tables 4 and 5 ). no statistically significant evidence of association was observed. the beijing and guangzhou sample groups were homogeneous regarding demographic and genetic parameters, so a joined analysis was done, no statistically significant evidence of association was observed (table 6 ). possible contribution of host genetic factors to the susceptibility and outcome of sars-cov infection has been investigated through several association studies [20] [21] [22] [23] , mbl deficiency because of polymorphisms in the mbl gene has been shown to be involved in sars-cov infection. as the downstream protein of mbl, improper masp activity can interfere with complement functions [24] . there is an association between the genetics of masp2 and its serum level [24] [25] [26] [27] . thiel et al. [24] have analyzed the mutation of p.156_159-dupchnh and snps p.r99q, p.r118c, p.d120g, p.p126l and p.v377a in four populations: africans from zambia, hong kong chinese, brazilian amerindians and danish caucasians. p.156_159-dupchnh was only found in chinese (gene frequency 0.26%), associated with low levels of masp2, and p.d120g was found only in caucasians. p.p126l and p.r99q were present in africans and amerindians only. masp2 levels were reduced in individuals with p.v377a (rs2273346). therefore, we chose 30 individuals with sars from beijing for analysis of masp2 gene polymorphisms. the sample included 60 chromosomes, which provided a 95% confidence level to detect alleles with a frequency >5%. however, we only observed the snp rs2273346 (p.v377a) among those mentioned in the study of thiel et al. we analyzed four tagsnps in the masp2 gene in two different sars patients and controls, searching for a possible genotype-phenotype correction, but no statistically significant difference was found for any polymorphisms between the different groups genotyped, while the possible role of the rare variants is to be determined. our analysis indicates that masp2 polymorphisms is not directly related to sars-cov susceptibility in northern and southern chinese. the authors declare that they have no competing interests. a two snp diplotypes span the genomic region of exon 9 (rs2273346 and rs12711521). a two snp diplotypes span the genomic region of exon 9 (rs2273346 and rs12711521). zln have contributed to genotyping; cc, lcx and mqj performed the data analyses, prepared the manuscript and supervised this study. all authors contribute to writing of the final manuscript. all authors read and approved the final manuscript. coronavirus as a possible cause of severe acute respiratory syndrome a novel coronavirus associated with severe acute respiratory syndrome identification of a novel coronavirus in patients with severe acute respiratory syndrome short term outcome and risk factors for adverse clinical outcomes in adults with severe acute respiratory syndrome(sars) world health organization: consensus document on theepidemiology of severe acute respiratory syndrome (sars) clinical features and short-term outcomes of 144 patients with sars in the greater toronto area pathogenetic mechanisms of severe acute respiratory syndrome association of hla class i with severe acute respiratory syndrome coronavirus infection association of human-leukocyteantigen class i (b*0703) and class ii (drb1*0301) genotypes with susceptibility and resistance to the development of severe acute respiratory syndrome lack of association between hla-a, -b and-drb1 alleles and thedevelopment of sars: a cohort of 95 sars-recovered individuals in a population of guangdong southern china influence of fcgammariia and mbl polymorphisms on severe acute respiratory syndrome. tissue antigens association between mannan-binding lectin genepolymorphisms and susceptibility to severe acute respiratory syndromecoronavirus infection a newcomplementdependent bactericidal factor found in nonimmune mouse sera: specific binding to polysaccharide of ra chemotype salmonella masp-3 and its association with distinct complexes of the mannan-binding lectin complement activation pathway a second serine protease associated with mannan-binding lectin that activates complement two constituents of the initiation complex of the mannan-binding lectin activation pathway of complement are encoded by a single structural gene profile of antibodies to the nucleocapsid protein of the severe acute respiratory syndrome (sars)-associated coronavirus in probable sars patients diagnosis of severe acute respiratory syndrome (sars) by detection of sars coronavirus nucleocapsid antibodies in an antigencapturing enzyme-linked immunosorbent assay variation is the spice of life ace1 polymorphism and progression of sars the association of rantes polymorphism with severe acute respiratory syndrome in hong kong and beijing chinese association of sars susceptibility with single nucleic acid polymorphisms of oas1 and mxa genes: a case-control study homozygous l-sign (clec4m) plays a protective role in sars coronavirus infection deficiency of mannanbinding lectin associated serine protease-2 due to missense polymorphisms mannan-binding-lectin-associated serine proteases, characteristics and disease associations horcajada. novel masp2 variants detected among north african and sub-saharan individuals genetic influences on mannan-binding lectin (mbl) and mannan-binding lectin associated serine protease-2 (masp-2) activity primers used for discovery of masp2 polymorphisms. click here for file [http://www.biomedcentral.com/content/supplementary/1471-2334-9-51-s1.doc] the pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2334/9/51/prepub key: cord-007375-hqmyund4 authors: tang, yi-wei; li, haijing; wu, huiyun; shyr, yu; edwards, kathryn m. title: host single-nucleotide polymorphisms and altered responses to inactivated influenza vaccine date: 2007-10-01 journal: j infect dis doi: 10.1086/521370 sha: doc_id: 7375 cord_uid: hqmyund4 we analyzed the relationship between host gene polymorphisms and responses in recipients of inactivated influenza vaccine, who were classified into poor, normal, or adverse response groups. the frequency of the mannose-binding lectin-2 codon 54 allele was significantly different among the 3 types of responders, with a decreased odds ratio for the development of poor or adverse responses (p = .033). there was no statistical relationship between responses and either tumor necrosis factor-α or interleukin (il)-10 promoter polymorphisms among the 3 response groups. when poor and normal responses were combined, the -1082 a allele in the il-10 promoter conferred a significantly decreased risk of the development of adverse responses (p = .041). these data indicate that host polymorphisms play a role in determining responses to influenza vaccine. populations, occasional serious adverse events, in addition to local pain and tenderness at the injection site, have been noted [2] . vaccine responses are determined not only by the chemical nature of the antigen and the manner in which it is delivered but also by environmental factors and host genetic factors. identifying the predictors of individual variability in immune responses to vaccination and the factors that contribute to an increased risk for adverse reactions would enhance our understanding of vaccine responses. it has been demonstrated that host polymorphisms affect the development and progression of certain diseases and disorders by either encoding altered gene products or causing changes in transcriptional regulation. the mannose-binding lectin (mbl)-2 gene encodes a calcium-dependent protein that plays an important role in innate immunity; circulating mbl-2 levels are largely the result of several single-nucleotide polymorphisms (snps) in the exon 1 gene and promoter region. variant mbl-2 alleles have been associated with increased susceptibility to several infections [3] [4] [5] . tumor necrosis factor (tnf)-a and interleukin (il)-10 are 2 important cytokines that are associated with the regulation of cellular immune responses. several polymorphisms in their promoter regions have been shown to directly affect their gene transcription and are associated with the development and progression of autoimmune and infectious diseases [6, 7] . furthermore, the imbalance in th1/th2 cytokine production may contribute to the adverse responses induced by vaccines when natural infection occurs [8] . the present study focused on 8 host snps in 3 immunogenetic genes: codons 52 (rs503078), 54 (rs1800450), and 57 (rs1800451) in the mbl-2 exon 1 gene; ϫ238 (rs36152) and ϫ308 (rs1800629) in the tnf-a promoter region; and ϫ592 (rs1800872), ϫ819 (rs1800871), and ϫ1082 (rs1800896) in the il-10 promoter region. using immune responses and adverse events assessed in an earlier influenza vaccine trial that enrolled 5210 subjects [9] , we defined poor and normal immune responses and the presence or absence of adverse events as the variables of interest. archived serum specimens were retrieved, genomic dna was extracted, and 8 snp alleles were determined by either (1) polymerase chain reaction (pcr) followed by restriction fragment-length polymorphism (rflp) analysis or (2) real-time allele-discrimination taqman pcr. subjects who experienced either poor influenza virus-specific antibody responses or adverse events to vaccines were compared with those who had brisker immune responses and no adverse reactions. subjects, materials, and methods. a large number of healthy volunteers had been enrolled in a national institutes of health-funded, double-blind randomized controlled trial at vanderbilt university to compare the safety, immunogenicity, and efficacy of standard trivalent inactivated influenza vaccine (h1n1, h3n2, and b) with experimental bivalent cold-adapted live attenuated vaccine (h1n1 and h3n2) in the 1990s [9] . only recipients of the inactivated vaccine were included in the present study (institutional review board approval number 990325). the recipients were categorized on the basis of their vaccine-induced responses into the following groups: (1) poor responders, defined as vaccine recipients with a prevaccination hemagglutination-inhibition (hi) antibody titer of !1:16 who achieved a !4-fold increase in hi titer to both the h1n1 and h3n2 vaccine components after the first vaccination; (2) adverse responders, defined as vaccine recipients with a temperature у38.3њc after the first vaccination; and (3) normal responders, defined as vaccine recipients with a у4-fold increase in hi antibody titer to both the h1n1 and h3n3 vaccine components and a temperature of р38.3њc after the first vaccination. all subjects categorized as poor and adverse responders who had archived serum specimens available were included in the present study. approximately the same number of subjects was selected from normal responders, who were matched for age, sex, and race to the poor and adverse responders. genomic dna was extracted from serum by use of a qiaamp blood kit (qiagen), in accordance with the manufacturer's instructions [5] . dna extracted from 200 ml of serum ranged from 150 to 1000 ng (on the basis of measurement of optical density at 260 nm), and ∼20 ng of dna was used in pcr amplification. pcr was used to amplify a 119-bp region of exon 1 that contains codons 52, 54, and 57 and to detect and determine the alleles in these codons. polymorphisms were determined by rflp analysis through the digestion of pcr products with the restriction enzymes bani, mboii, and mlui (new england biolabs), followed by separation on 4% gel with ethidium bromide staining [10] . a real-time allele-discrimination pcr assay was used to detect and discriminate alleles ϫ238 and ϫ308 snp in the tnf-a promoter region and ϫ592, ϫ819, and ϫ1082 snp in the il-10 promoter region, by use of the abi prism 7700 sequence detection system (applied biosystems). pcr amplification was performed by denaturation at 95њc for 10 min, followed by 40 cycles of denaturation at 92њc for 15 s, and then annealing and extension at 62њc for 1 min. after pcr, the genotype of each sample was attributed automatically by measuring allele-specific fluorescence with the abi prism 7900 sequence detection system, by use of the sds software for allelic discrimination (version 2.2.2; applied biosystems). nucleotide sequences of primers and fluorophore taqman mgb probes were designed using primer express software (version 1.5; applied biosystems) (for tnf-a ϫ238, 5 -aaatcagtcagtggcccagaa-3 , 5 -tcattcaaccagc-ggaaaact-3 , 5 -fam-ctccctgctccgatt-mgb-3 , and 5 -vic-ctccctgctctgatt-mgb-3 ; for tnf-a ϫ308, 5 -gaaatggaggcaataggttttgag-3 , 5 -gtaggaccct-ggaggctgaac-3 , 5 -fam-ccgtccccatgcc-mgb-3 , and 5 -vic-ccgtcctcatgcc-mgb-3 ; for il-10 ϫ592, 5 -agctgaagaggtggaaacatgtg-3 , 5 -caagcagcccdifferences in age, sex, and race among the poor, normal, and adverse responders were examined using epiinfo software (version 6; centers for disease control and prevention). because very few subjects were determined to be homozygous for all 8 tested snps, all alleles with heterozygous and homozygous mutations were combined for statistical analysis. a multinomial logistic regression was performed for a global test in which the 3 response groups were analyzed simultaneously to obtain an overall p value for the 3 groups. unconditional univariate logistic regression was used to test the association between host polymorphisms and varied vaccine-induced responses as well as to estimate odds ratios (ors) and 95% confidence intervals (cis). the wild type of the snps was treated as the reference category for the or calculation. the probability of the logistic regression was modeled for an abnormal response (either poor or adverse) for mbl-2 and for an adverse response for tnfa and il-10. all p values presented in this report are 2-sided, and was considered to indicate statistical significance. p р .05 all of the calculations were done using sas software (version 9.1; sas institute). results. vaccinees enrolled in the parent vaccine trial who had received inactivated influenza vaccine and who fell into 1 of the 3 above-defined response categories were re-reviewed. all poor and adverse responders with available archived serum specimens were included. approximately the same number of subjects was selected for the normal response group, who were matched by age, sex, and race with the other 2 groups. a total of 298 subjects were included in the present study-99, 101, and 98 in the poor, normal, and adverse response groups, respectively. no significant differences were noted in age ( , , , and mean ‫ע‬ sd 30.6 ‫ע‬ 14.2 30.9 ‫ע‬ 14.3 32.3 ‫ע‬ 14.9 years), sex (proportion male, 45.5%, 48.5%, and 49.0%), or race (proportion white, 94.9%, 92.1%, and 92.9%) among the poor, normal, and adverse response groups, respectively. we first analyzed the 3 snps in the mbl-2 exon 1 gene; the frequency of each allele in codons 52, 54, and 57 is listed in table 1. the differences in each allele among the poor, normal, and adverse responders were analyzed by multinomial logistic regression. there were no significant differences in codon 52 and 57 alleles; however, a significant difference in allele frequency in codon 54 was detected ( ; ). an two host snps in the tnf-a (ϫ238 and ϫ308) and 3 in the il-10 (ϫ592, ϫ819, and ϫ1082) promoter regions were determined by real-time allele-discrimination taqman pcr. the multinomial logistic regression analysis did not discern any significant differences in allele frequency among the poor, normal, and adverse responders (table 2) . we then focused on testing our hypothesis that imbalanced th1/th2 cytokine production is associated with an adverse response, indicated by a temperature у38.4њc. we combined the subjects in the poor and normal response groups and compared them with adverse responders by unconditional univariate logistic regression analysis. in comparison to the poor/normal response group, the gra polymorphism in the il-10 promoter ϫ1082 allele indicated a significantly decreased risk for the development of adverse responses (or, 0.558 [95% ci, 0.319-0.976]) (table 2) . these data suggest that il-10 promoter polymorphisms may be associated with adverse systemic responses to influenza vaccine. discussion. in humans, the immune response to vaccination is heterogeneous despite the use of a constant formulation, route of administration, and dosage. although the majority of vaccinees generate brisk immune responses with no adverse reactions, ∼5% experience either hyporesponsiveness or adverse events [2] . the present study explored the possibility that host gene polymorphisms influence inactivated influenza vaccineinduced immune responses by comparing the frequencies of 8 snps in the mbl-2 gene and in the tnf-a and il-10 promoter regions among different groups. we found a significant difference in allele frequency in the mbl-2 codon 54 among the poor, normal, and adverse responders, suggesting that the allele polymorphism is independently associated with poor and adverse responses to influenza vaccination. mbl-2 is a member of the collectin family and is important in the initiation of the lectin pathway of complement activation and in opsonization [11] . the variant alleles in the mbl-2 gene are associated with mbl-2 deficiency, especially in individuals homozygous for the variant alleles [3] . host polymorphisms can affect the development and progression of certain diseases and disorders by encoding altered gene products, resulting in poor immune responses. the variant codon 54 allele is more prevalent than codons 52 and 57 in white populations. individuals with allele a in codon 54 demonstrate an even lower mbl-2 protein concentration than individuals with the 52 t allele, and the mbl-2 protein produced is incapable of activating the classic complement pathway [12] . of the 3 polymorphisms within the mbl-2 gene, codon 54 has independently been found to be associated with increased susceptibility to infection [4, 13] . when natural disease occurs after receipt of inactivated vaccine, th1/th2 cytokine imbalances have been demonstrated for respiratory syncytial virus infection and measles [8] . inflammatory responses must be finely tuned: too strong a response produces adverse events after vaccination, whereas too weak a response attenuates the immune responses. the th1-like tnfa is a potent immunomodulator and proinflammatory cytokine that has been implicated in the pathogenesis and development of various infectious diseases. in contrast, the th2-like il-10 is a potent anti-inflammatory cytokine that plays a role in down-regulating cell-mediated and cytotoxic inflammatory responses. several polymorphisms in the promoter regions of the genes for these 2 cytokines have been reported that affect the transcriptional regulation of the 2 genes. il-10 responses to influenza vaccination have been reported to vary significantly among age groups and vaccines [8] . we hypothesized that snps in promoter regions result in imbalanced th1/th2 cytokine production, which leads to the occurrence of adverse events after the administration of inactivated influenza vaccine. although a multivariate analysis did not show that the allele frequencies of 5 snps in the tnf-a and il-10 promoter regions among the poor, normal, and adverse response groups altered responses, a specific analysis between adverse and nonadverse (poor or normal) responses indicated that the ϫ1082 gra polymorphism in the il-10 promoter region conferred a significantly decreased risk for the development of adverse responses. previous studies have indicated that the gra snp at ϫ1082 is important in il-10 regulation; homozygous individuals (g/g) had higher il-10 expression after in vitro stimulation [11] . the snp at ϫ1082 has been associated with increased susceptibility of infection, severity of illness, organ dysfunction, and mortality [14, 15] . these findings support the present data by suggesting that the ϫ1082 allele polymorphism in the il-10 promoter region may be associated with adverse responses induced by influenza vaccine. by use of genetic sequencing of the human genome, scientists are relating polymorphisms in various host gene alleles to variable host immune responses to infectious agents and vaccines. because most previous vaccine trials have focused on humoral antibody responses, archival serum specimens are available for study. several earlier studies as well as the present one have demonstrated that substantial quantities of human genomic dna are present in such clinical samples as serum and cerebrospinal fluid [5] . with appropriate approval by institutional review boards, retrospective studies using the large quantity of archived serum specimens from previous vaccine trials and prospective studies can be conducted to assess many genetic factors. there were several limitations to the present study, including (1) the small quantities of dna available from serum specimens, (2) only 1 adverse event (fever) was assessed, and (3) a small number of host snps and not the entire genome were assessed. in spite of these limitations; however, this study suggests that additional studies should be conducted to confirm these findings. influenza vaccination and reduction in hospitalizations for cardiac disease and stroke among the elderly guillain-barre syndrome and the 1978-1979 influenza vaccine association of mannose-binding lectin gene heterogeneity with severity of lung disease and survival in cystic fibrosis mannose-binding lectin in severe acute respiratory syndrome coronavirus infection analysis of candidate-host immunogenetic determinants in herpes simplex virus-associated mollaret's meningitis variation in the tnf-alpha promoter region associated with susceptibility to cerebral malaria association of tnf2, a tnf-alpha promoter polymorphism, with septic shock susceptibility and mortality: a multicenter study responses to influenza vaccination in different t-cell subsets: a comparison of healthy young and older adults a randomized controlled trial of cold-adapted and inactivated vaccines for the prevention of influenza a disease mannose-binding lectin (mbl) deficiency: variant alleles in a midwestern population of the united states hutchinson iv. an investigation of polymorphism in the interleukin-10 gene promoter distinct and overlapping functions of allelic forms of human mannose binding protein mannose-binding lectin gene polymorphism is a modulating factor in repeated respiratory infections association of il-10 polymorphism with severity of illness in community acquired pneumonia pneumococcal septic shock is associated with the interleukin-10-1082 gene promoter polymorphism we thank jennifer doersam, jamie rickmyre, sandra yoder, shufang meng, jody peters, amondrea blackman, pam palmer, sharon tollefson, and william dupont for their excellent technical assistance and barney graham, david persing, and bonnie lafleur for their helpful suggestions and review of the research proposal and/or manuscript. key: cord-292581-6ipzvryb authors: alagarasu, kalichamy; bachal, rupali v; bhagat, asha b; shah, paresh s; dayaraj, cecilia title: elevated levels of vitamin d and deficiency of mannose binding lectin in dengue hemorrhagic fever date: 2012-05-04 journal: virol j doi: 10.1186/1743-422x-9-86 sha: doc_id: 292581 cord_uid: 6ipzvryb background: altered plasma concentrations of vitamin d and mannose binding lectin (mbl), components of innate immunity, have been shown to be associated with the pathogenesis of viral infections. the objective of the present study was to find out whether plasma concentrations of mbl and vitamin d are different in patients with dengue fever (df) and dengue hemorrhagic fever (dhf). the results: the plasma concentrations of vitamin d and mbl were assessed in 48 df cases, 45 dhf cases and 20 apparently healthy controls using elisa based methods. vitamin d concentrations were found to be higher among both df and dhf cases as compared to healthy controls (p < 0.005 and p < 0.001). vitamin d concentrations were not different between df and dhf cases. when the dengue cases were classified into primary and secondary infections, secondary dhf cases had significantly higher concentrations of vitamin d as compared to secondary df cases (p < 0.050). mbl concentrations were not significantly different between healthy controls and dengue cases. mbl concentrations were observed to be lower in dhf cases as compared to df cases (p < 0.050). although mbl levels were not different df and dhf cases based on immune status, the percentage of primary dhf cases (50%) having mbl levels lower than 500 ng/ml were less compared to primary df cases (p = 0.038). conclusions: the present study suggests that higher concentrations of vitamin d might be associated with secondary dhf while deficiency of mbl may be associated with primary dhf. dengue, caused by dengue virus (denv), constitutes a public health emergency of international concern. denv infection in humans results in a spectrum of outcomes ranging from asymptomatic to undifferentiated fever, mild form of the disease namely dengue fever (df) to severe forms including dengue hemorrhagic fever (dhf) and dengue shock syndrome (dss) that may be fatal [1] . the outcome of denv infection is determined by multiple factors including viral virulence, host genetics and host immune responses [2] . among the various components of host immune responses, t cells, antibodies, cytokine storm and complement factors contribute to the pathogenesis of dengue [2] . epidemiological studies have shown an association between dhf/dss and secondary denv infection. preexisting antibodies and cross reactive t cell responses induced by the primary infection is believed to exacerbate the disease during secondary infection [3, 4] . proinflammatory cytokines namely interleukin-8 (il-8), tumor necrosis factor-α and interferon-γ and anti inflammatory cytokine il-10 also contribute to dengue disease pathogenesis [5] [6] [7] [8] [9] [10] [11] . activation of t cells, antibodies and cytokines are influenced by various immunomodulators. increase or decrease in the levels of these immunomodulators influences the outcome of viral infections [12] . vitamin d is a potent immunomodulator affecting both innate and adaptive immune responses. vitamin d binds to vitamin d receptor (vdr), translocates to the nucleus and influences gene expression. vitamin d enhances the phagocytic capacity of macrophages and induces antimicrobial peptide gene expression contributing to innate immune responses [13] . vitamin d inhibits t-helper 1 (th1) cell and cytotoxic t cell responses. it decreases b-cell proliferation, plasma-cell differentiation and igg secretion [14] . vitamin d also enhances th2 cytokine and il-10 responses [15, 16] . vitamin d deficiency increases the risk of cancer, tuberculosis, as well as influenza and human immunodeficiency virus infection [12, 17, 18] . vitamin d has been reported to influence the expression of denv receptors in immune cells [19] [20] [21] . a study from vietnam has shown the association of vitamin d receptor gene polymorphisms with susceptibility to dhf [22] . one of the major pathways of complement activation is initiated by binding of the virus to mannose binding lectin (mbl). mbl is a pattern recognition molecule that recognizes specific sugar molecules present on the surface of microorganisms including denv [23, 24] . point mutations in the mbl2 gene lead to reduced concentrations of functional oligomers. genetically determined variation in serum concentrations of mbl has been shown to influence the susceptibility to infectious, autoimmune and cardiovascular diseases [23] . alleles of mbl2 gene that are associated with higher concentrations of functional mbl, have been shown to be associated with thrombocytopenia in dengue infected patients [25] . mbl concentrations were also found to be increased in acute samples of dhf cases as compared to df cases [26] . since mbl and vitamin d are known to influence innate and adaptive immune responses and denv pathogenesis is immune mediated, we hypothesized that altered levels of plasma vitamin d and mbl might be associated with dengue disease severity. therefore, we investigated the levels of plasma vitamin d and mbl in dengue infected patients in the context of disease severity and immune status. among the 93 patients included in the study, based on the df/dhf defining criteria of the world health organization (who) [27] , 48 had df and 45 had dhf. males were over represented in both df and dhf patients. the male to female ratio in df was 2:0.76 and in dhf, it was 2:1. demographic and clinical characteristics of patients were given in table 1 . the median age of dhf cases (23.0 years) was significantly lower than that of df cases (29.5 years) (p = 0.034). the number of primary cases with df (42.5%) was higher than the number of primary cases with dhf (18.6%) (p = 0.026). when the presence of clinical symptoms was compared between df and dhf cases, the presence of fever with chills, headache, myalgia, arthralgia, retro orbital pain and rash were reported equally in df and dhf cases. presence of nausea/vomiting and abdominal pain was represented by the dhf cases. thrombocytopenia was significantly over represented in dhf cases as compared to df cases (p < 0.001). the median count of platelets was significantly lower in dhf cases (p = 0.020) ( table 1) . among the 45 dhf cases, gastrointestinal bleeding, manifested by melena or hematemesis was reported in 21 (46.7%) cases, hematuria was observed in six (13.3%), gum bleeding in seven (15.6%), conjunctival hemorrhage in one (2.2%) and epistaxis in one (2.2%). plasma leakage was observed in 9 (20%) patients either as ascites (n = 5) and/or as pleural effusion (n = 4). shock/hypotension was observed in five patients (11.1%). no fatality was observed in the cases included in the study. plasma 25-hydroxy vitamin d 3 (vitamin d) concentrations were investigated in 45 df cases, 38 dhf cases and 20 healthy controls. vitamin d concentrations were found to be significantly higher in df and dhf cases as compared to healthy controls (healthy controls vs. df cases p < 0.005; healthy controls vs. dhf cases p < 0.001). among df and dhf cases, the vitamin d concentrations were found to be higher in dhf cases, though, the difference was not statistically significant (p > 0.050) ( figure 1 ). the sample size has a power of 0.94 and 0.80 to detect the differences observed between dhf and healthy controls and between df and healthy controls respectively. when the patients were grouped based on immune status and disease severity, secondary dhf cases had significantly higher concentrations of vitamin d as compared to secondary df cases (p < 0.050). the sample size has a power of 0.79 to detect the differences observed between secondary df and secondary dhf cases. vitamin d concentrations were not significantly different between primary df and primary dhf cases (p > 0.050). when comparisons were made between primary df and secondary df or primary dhf and secondary dhf, vitamin d levels were not different (p > 0.050) ( figure 2 ). plasma mbl concentrations were assessed in 48 df cases, 45 dhf cases and 20 healthy controls. mbl concentrations were not significantly different between healthy controls and df or dhf cases (p > 0.050). when df and dhf cases were compared, significantly lower concentrations of mbl were observed in dhf cases (p < 0.050) (figure 3 ). the sample size has a power of 0.52 to detect the observed differences between dhf and df cases. the sample size is underpowered to detect the differences (effect size of less than 0.5) observed between df and healthy controls or dhf and healthy controls. however, the sample size has a power of above 0.80 to detect an effect size of 0.8 and above. when the patients were classified into primary and secondary cases and compared, irrespective of df or dhf, mbl concentrations were not different (p > 0.050). mbl concentrations were lower in primary dhf cases as compared to primary df cases though not statistically significant. mbl levels were not different between secondary df and secondary dhf ( figure 4 ). in the present study, plasma concentrations of vitamin d and mbl were investigated in dengue patients from pune, maharashtra, western india. pune is endemic to dengue with 600-800 cases occurring annually. all four serotypes have been reported to be circulating in pune [28] . the demographic and clinical characteristics reported in the present study were similar to that reported in our earlier study [5] . investigation of vitamin d concentrations revealed significantly higher concentrations of vitamin d in dengue patients (both df and dhf) as compared to apparently healthy controls suggesting that higher concentrations of vitamin d might be associated with symptomatic disease. further analysis revealed that the association was more evident in secondary dhf. it has been shown that vitamin d induces expression of dendritic cell specific intercellular adhesion molecule 3 grabbing non integrin (dc-sign), the primary receptor for denv entry into immature dendritic cells [19] . it has also been shown that calcitriol (active form of vitamin d) increases the expression of fcγ receptors on human monocytic cell lines and monocyte derived dendritic cells [20, 21] . the increased concentrations of vitamin d, in denv infected cases with secondary infection, might enhance viral entry through increased expression of fcγ receptors leading to higher viral load, uncontrolled inflammatory responses and subsequent development of dhf. vitamin d is also known to suppress th1 cytokines and enhance il-10 production by peripheral blood mononuclear cells in response to microbial antigens [14] [15] [16] . since il-10 is known to play a role in dengue disease pathogenesis [11] , it is possible that vitamin d could also contribute to disease pathogenesis through altered il-10 response. since the effect of vitamin d is also dependent on single nucleotide polymorphisms in the vdr gene [29] , the influence of vitamin d on dengue in the context of host genetics needs to be investigated. analysis of circulating concentrations of mbl in dengue cases and healthy controls revealed no significant difference between the two groups suggesting that the mbl mediated pathway of complement activation might be inhibited or may not be induced during denv infection. deficiency of mbl has been reported in patients infected with other rna viruses such as crimean-congo hemorrhagic fever virus, respiratory syncytial virus and severe acute respiratory syndrome (sars) corona virus [30] [31] [32] . interaction between complement components and non structural protein 1 (ns1) of flaviviruses has been reported to inhibit classical and lectin pathways of complement activation [33] . the present study revealed significantly lower levels of mbl in dhf cases suggesting that reduced activation of mbl mediated complement pathway might be associated with dhf. mbl is known to interact with n-linked glycans of structural proteins of denv and neutralize the virus by blocking viral fusion. in experimentally infected mice, mbl dependent intravascular clearance of denv has also been reported [24] . therefore, it is possible that mbl deficiency might have led to decreased activation of mbl mediated pathway of complement and reduced intravascular clearance of denv leading to higher viral load. higher viral load has been shown to be associated with dhf in several studies [34, 35] . in the present study, deficiency of mbl was more evident in cases with primary dhf. the association of mbl deficiency with dhf in primary infection also suggests that the protective effects of mbl and the innate immune responses are more important during primary infection which could be overshadowed by presence of cross reactive complement fixing antibodies during secondary infection [36] . in contrast to the present study, a study from brazil has reported higher concentrations of mbl among dhf cases [7] . a higher mbl concentration might also lead to increased inflammation through enhancement of the production of pro-inflammatory cytokines. increased concentration of factor d and decreased concentration of factor h have been reported in dhf cases suggesting that imbalance in the regulation of factors h and d of the alternative pathway of complement activation is associated with dhf [26] . since mbl concentrations are dependent on the presence of mutations in the structural and promoter regions of mbl2 gene, it is possible that variant alleles of mbl2 gene polymorphisms might be associated with dhf. a case control study from brazil has shown the association of wild type alleles of the mbl2 gene with thrombocytopenia in dengue patients [25] . further case control studies are needed to confirm the phenotypic effects of mbl2 gene polymorphisms in dengue patients with primary infection from india. although, the present study is sufficiently powered to detect intermediate to large effect size, with regard to mbl, the study is underpowered and further studies with larger sample size are needed to confirm the preliminary associations. the present study suggests that higher concentrations of vitamin d are associated with secondary dhf. this association may be related to the inducing effect of vitamin d on fcγ receptor expression which might subsequently lead to higher viral load in dengue cases with secondary infection and hence development of dhf. the study also suggests that mbl deficiency is associated with primary dhf. this association might be related to the reduced activation of the mbl pathway of complement leading to higher viral load in dengue cases with primary infection. this is the first study that correlates the concentrations of vitamin d and mbl with immune status of dengue cases. blood samples from patients with dengue like illness were referred to the national institute of virology for diagnosis. samples were transported on ice and plasma was separated and aliquoted. one aliquot was used for dengue specific igm elisa and leftover aliquots were stored in -80°c. a total number of 93 samples, which were positive for dengue specific igm elisa, were included in the study. all the samples were collected during the seasonal outbreak in pune in 2009. clinical presentations of the patients recorded by the clinicians were used to classify the patients. the patients were classified into those with df and those with dhf. patients with fever, headache, myalgia, retro-orbital pain, and rash were defined as df. dhf patients were categorized by the presence of at least two of the dhf defining criteria of the who [27] : hemorrhagic tendencies/manifestations, thrombocytopenia, and evidence of plasma leakage. samples from 20 apparently healthy blood donors were also used in the study. this study was approved by the national institute of virology human ethics committee. waiver of the informed consent was granted by the committee on the basis of "use of leftover specimens after clinical investigation" under the indian council of medical research guidelines 2006. the in-house national institute of virology (niv) igm capture elisa kit was used to detect denv-specific igm. a known positive (p) and a known negative (n) serum control were used in every test. a sample showing p/n ratio >2.1 times the optical density was considered positive. the igg capture elisa (e-den02g, panbio, windsor, australia) was used to classify the cases into primary or secondary denv infection. igg levels of >22 units (defined by the manufacturers) indicated secondary infection. 25-hydroxyvitamin d 3 (vitamin d) was quantitated in the plasma samples using an enzyme immunoassay kit (ids ltd, uk) according to the manufacturer's protocol. calibrators and controls were used in each assay. the percent binding (b/b%) of each calibrator, control and unknown samples were calculated by the following formula: b/b % = mean absorbance/mean absorbance for '0' calibrator x100. a calibration curve with b/b% and vitamin d concentrations were used to find out the concentrations of vitamin d in unknown samples in nm/l. the detection limit of the kit is 5 nm/l. estimation of plasma mbl concentrations was done using the mbl oligomer elisa kit (bioporto diagnostics, denmark) according to the manufacturer's instructions. calibrators were used in each assay and a calibration curve was constructed by plotting the mean absorbance values for each calibrator on the y-axis against the corresponding mbl concentrations in ng/ml on the x-axis. the mbl concentration of each diluted plasma sample was then found by locating the point on the curve corresponding to the absorbance value of each diluted plasma sample and reading its corresponding concentration in ng/ml from the x-axis. the concentration of mbl in the undiluted plasma sample is calculated by multiplying this result by the sample dilution factor. the detection limit of the kit is 2 ng/ml. using the statcalc program (epi info version 6.0.4, cdc, atlanta, ga, july 1996), the chi-square test with yates correction or fisher exact test (when any cell value was less than 5) was performed to examine differences in demographic and clinical characteristics of the dengue patients. age and platelet counts were compared using mann-whitney u test. concentrations of mbl and 25 [oh]d in plasma samples were compared between study groups using kruskal-wallis test with dunn's multiple comparison for selected groups. the p values from dunn's multiple comparison for selected groups were provided. since mbl levels varies depending on the presence of polymorphisms in the mbl2 gene, categorization of study subjects based on mbl deficiency (defined by a cutoff value of <500 ng/ml) was done and compared between study groups using the chi-square test with yates correction or fisher exact test. this cutoff value (<500 ng/ml) has been earlier shown to be a reliable predictor of low producing mbl2 genotypes using receiver operating characteristic analysis with individual data from 1642 healthy subjects from 4 studies [37] . all statistical analysis was performed using graphpad prism (version 4). a two tailed p value less than 0.05 was considered significant. power calculations were performed using the software g*power version 3.1.3. the achieved power for significant results were calculated using the wilcoxon-mann-whitney test (two groups) option available in 't' test family of the software. the sample size, level of significance and effect size were provided as input. for calculating the effect size, the software uses mean values with standard deviation of the two groups [38] . the authors declare that they have no competing interests govt of india: zoonosis division, national institute of communicable diseases dengue virus pathogenesis: an integrated view t lymphocyte response to heterologus secondary dengue virus infections antibodies determine virulence in dengue clinical findings and pro-inflammatory cytokines in dengue patients in western india: a facility-based study elevated levels of il-8 in dengue hemorrhagic fever detection of circulant tumor necrosis factor-alpha, soluble tumor necrosis factor p75 and interferon-gamma in brazilian patients with dengue fever and dengue hemorrhagic fever multiplex cytokine profile from dengue patients: mip-1beta and ifn-gamma as predictive factors for severity 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 elevated plasma interleukin-10 levels in acute dengue correlate with disease severity vitamin d and the antiviral state cutting edge: vitamin d-mediated human antimicrobial activity against mycobacterium tuberculosis is dependent on the induction of cathelicidin vitamin effects on the immune system: vitamins a and d take centre stage a: 1alpha, 25-dihydroxyvitamin d3 has a direct effect on naive cd4(+) t cells to enhance the development of th2 cells narayanan pr: 1, 25 dihydroxyvitamin d3 modulated cytokine response in pulmonary tuberculosis vitamin d and prevention of colorectal cancer low serum vitamin d levels and tuberculosis: a systematic review and meta-analysis immunophenotype of vitamin d receptor polymorphism associated to risk of hiv-1 infection and rate of disease progression zheng m: 1 alpha, 25-dihydroxyvitamin d3 and its analogues modulate the phagocytosis of human monocyte-derived dendritic cells differentiation of a human monocytic cell line by 1,25-dihydroxyvitamin d3 (calcitriol): a morphologic, phenotypic, and functional analysis susceptibility to dengue hemorrhagic fever in vietnam: evidence of an association with variation in the vitamin d receptor and fc gamma receptor iia genes the role of mannose binding lectin in innate immunity direct complement restriction of flavivirus infection requires glycan recognition by mannose-binding lectin mbl2 gene polymorphisms protect against development of thrombocytopenia associated with severe dengue phenotype alternative complement pathway deregulation is correlated with dengue severity world health organisation: prevention and control of dengue and dengue haemorrhagic fever: comprehensive guidelines.: who regional publication detection of dengue-4 virus in pune, western india after an absence of 30 years-its association with two severe cases high-dose vitamin d3 during intensive-phase antimicrobial treatment of pulmonary tuberculosis: a double-blind randomised controlled trial serum levels of mannan-binding lectin in patients with crimean-congo hemorrhagic fever. vector borne zoonotic dis serum mannose-binding lectin levels are linked with respiratory syncytial virus (rsv) disease mannose-binding lectin in severe acute respiratory syndrome coronavirus infection antagonism of the complement component c4 by flavivirus nonstructural protein ns1 differing influences of virus burden and immune activation on disease severity in secondary dengue-3 virus infections dengue viremia titer, antibody response pattern, and virus serotype correlate with disease severity complement-mediated neutralization of dengue virus requires mannose-binding lectin low serum mannose binding lectin levels increases the risk of death due to pneumococcal infection a flexible statistical power analysis program for the social, behavioral, and biomedical sciences elevated levels of vitamin d and deficiency of mannose binding lectin in dengue hemorrhagic fever we thank anand singh and kakade m for their help in sample collection, performing igm elisa and storage of samples. we thank mr atul walimbe for his help in performing power calculations. the authors wish to thank the indian council of medical research for providing the funds and the director, national institute of virology for the support. submit your next manuscript to biomed central and take full advantage of: key: cord-000524-5y9kfyk9 authors: ling, man to; tu, wenwei; han, yan; mao, huawei; chong, wai po; guan, jing; liu, ming; lam, kwok tai; law, helen k. w.; peiris, j. s. malik; takahashi, k.; lau, yu lung title: mannose-binding lectin contributes to deleterious inflammatory response in pandemic h1n1 and avian h9n2 infection date: 2011-11-11 journal: the journal of infectious diseases doi: 10.1093/infdis/jir691 sha: doc_id: 524 cord_uid: 5y9kfyk9 background. mannose-binding lectin (mbl) is a pattern-recognition molecule, which functions as a first line of host defense. pandemic h1n1 (pdmh1n1) influenza a virus caused massive infection in 2009 and currently circulates worldwide. avian influenza a h9n2 (h9n2/g1) virus has infected humans and has the potential to be the next pandemic virus. antiviral function and immunomodulatory role of mbl in pdmh1n1 and h9n2/g1 virus infection have not been investigated. methods. in this study, mbl wild-type (wt) and mbl knockout (ko) murine models were used to examine the role of mbl in pdmh1n1 and h9n2/g1 virus infection. results. our study demonstrated that in vitro, mbl binds to pdmh1n1 and h9n2/g1 viruses, likely via the carbohydrate recognition domain of mbl. wild-type mice developed more severe disease, as evidenced by a greater weight loss than mbl ko mice during influenza virus infection. furthermore, mbl wt mice had enhanced production of proinflammatory cytokines and chemokines compared with mbl ko mice, suggesting that mbl could upregulate inflammatory responses that may potentially worsen pdmh1n1 and h9n2/g1 virus infections. conclusions. our study provided the first in vivo evidence that mbl may be a risk factor during pdmh1n1 and h9n2/g1 infection by upregulating proinflammatory response. the pandemic h1n1 2009 (pdmh1n1) influenza a virus has spread globally since the outbreak first started in mexico in 2009. as of august 2010, the virus had already caused more than 18 449 deaths in at least 214 countries worldwide [1] . the world health organization officially announced the step-down of pdmh1n1 to postpandemic phase on 10 august 2010, and the virus was expected to circulate as a seasonal virus in the human population thereafter [2] . gene segments of the pdmh1n1 virus are derived from multiple lineages, including the eurasian swine, the classical swine, and the triple reassortant swine lineages. multiple genetic reassortment events of viral components have taken place and thus gave rise to this novel pandemic virus [3] . clinical symptoms of pdmh1n1 infection are usually mild, possibly due to the cross-protection offered by memory cytotoxic t lymphocytes established from previous exposure to seasonal influenza [4] . h9n2 avian influenza virus (h9n2/g1) is widely prevalent among poultry in various eurasian regions, including mainland china and hong kong. in 1999 and 2003, h9n2 influenza was reported in 3 children in hong kong. all 3 of them developed relatively mild symptoms and recovered within a week [5, 6] . the 6 internal genes of h9n2/g1 virus were found to be related to the highly pathogenic avian influenza virus a/hong kong/483/97 (h5n1). although the h9n2/g1 virus was found mainly in poultry, it has the ability to transmit across species, and with its genetic similarity to the highly pathogenic h5n1, it also poses a threat of becoming pandemic [7] . mannose-binding lectin (mbl) is a serum protein primarily produced by the liver. it belongs to the collectin family that comprises the collagen-like domain and the carbohydrate recognition domain (crd). mbl functions as a key patternrecognition molecule recognizing a wide range of pathogens [8, 9] . lectin pathway activation [10] and opsonophagocytosis are triggered upon mbl binding to pathogens [11] . while the mbl gene is highly polymorphic in humans, clinical association studies have demonstrated that mbl deficiency was associated with increased susceptibility to certain infections [12, 13] . the antiviral role of mbl in influenza virus infection remains controversial. previous studies suggested that mbl demonstrates in vitro anti-influenza virus function, including inhibition of viral hemagglutination and direct neutralization of the virus either in a complement dependent or independent manner [14] [15] [16] . however, other studies have shown that the antiviral function of mbl may vary among different strains of influenza viruses, depending on the number of potential glycosylation sites on the viral hemagglutinin (ha) globular domain [17, 18] . influenza virus-infected epithelial cells and macrophages can initiate a cellspecific response that includes the transcription and release of proinflammatory cytokines and chemokines [19, 20] . although some studies have indicated that mbl may regulate proinflammatory cytokine and chemokine release from phagocytes in response to bacterial stimulation [21, 22] , little is known about its immunomodulatory role in influenza [23] . in the present study, we investigated whether mbl could display any in vitro or in vivo antiviral function toward pdmh1n1 and h9n2/g1 viruses, as well as whether it could modulate the inflammatory response upon infection by these two strains of influenza virus. influenza virus a/california/04/2009 (pdmh1n1) were propagated in embryonated chicken eggs and purified by ultracentrifugation with minor modification of our previous work [4, 24] . influenza virus a/quail/hong kong/g1/97 (h9n2/g1) was grown in madin-darby canine kidney (mdck) cells with modified eagle's medium (invitrogen) containing 2 lg/ml n-p-tosyl-l-phenylalanine chloromethyl ketone (tpck)treated trypsin (sigma-aldrich). virus stocks were purified by adsorption to and elution from turkey red blood cells and stored at 280°c until use as previously described [25] . the determination of virus titer was performed by titrating virus in mdck cells, with daily observation of cytopathic effect and confirmation by hemagglutination assay. the tissue culture infective dose affecting 50% of the cultures (tcid 50 ) was calculated by the reed-muench formula. ultraviolet (uv)-irradiated virus was prepared by irradiation with energy of 0.2 j in a uv crosslinker as described previously [26] . the binding assay was performed as described previously [12] . in brief, 96-well flat-bottom polystyrene plates (corning-costar) were precoated with 100 ll/well of 10 2 , 10 3 ,10 4 , and 10 5 tcid 50 uv-irradiated influenza viruses or phosphate-buffered saline (pbs). after incubation at room temperature overnight, wells were blocked for 2 hours at room temperature with 1% bovine serum albumin (bsa) in pbs with 0.05% sodium azide. different concentrations of recombinant human mbl (rhmbl) (0, 0.5, 2, 6, or 8 lg/ml), which was kindly provided by dr k. takahashi (laboratory of developmental immunology, harvard department of pediatrics, massachusetts general hospital, boston), were added and incubated overnight at 4°c. then 100 ll of 0.2 lg/ml biotinylated monoclonal anti-mbl antibody (hyb131-01, antibody shop) diluted in pbs with 1% bsa was added into each well. bound antibody was detected by using horseradish peroxidase-conjugated streptavidin and tetramethybenzidine substrate solution (r&d systems). the binding of mbl to influenza virus was evaluated by the absolute absorbance values measured at 450 nm (a 450 ). breeding pairs of mbl wild-type (wt) and mbl knockout (ko) mice on c57b6/j were provided by dr takahashi [27] . they were maintained under specific pathogen-free conditions in the animal facilities of the laboratory animal unit, the university of hong kong. female mice were used at 6-10 weeks of age. they were anesthetized and inoculated intranasally with 30 ll of 10 3 tcid 50 pdmh1n1 virus, 10 5 tcid 50 h9n2/g1 virus, or pbs at day 0. virus-infected or mocktreated mice were weighed daily. all animal care and experiments were conducted in accordance with the committee on the use of live animals in teaching and research guidelines of the university of hong kong. virus-infected or mock-treated mice were sacrificed at days 3, 7, and 14 after infection. the lungs were harvested and homogenized by a tissue homogenizer (omni international). the homogenates were centrifuged at 2500 rpm for 10 minutes at 4°c. supernatants were used for virus titer determination and cytokine detection. expression levels of interleukin (il) 1a, il-2, il-4, il-6, il-10, tumor necrosis factor (tnf) a, interferon (ifn) c, macrophage inflammatory proteins (mip)-1a, mip-1b, monocyte chemotactic protein (mcp)-1, mcp-3, and regulated upon activation, normal t-cell expressed, and secreted (rantes) in the lung homogenates were quantitatively determined by flow cytometrybased immunoassay (mouse th1/th2 cytokine 10plex and mouse chemokines 6plex flowcytomix multiplex, bender medsystems) according to the manufacturer's protocol. interleukin 1b and keratinocyte chemoattractant (kc) simplex were purchased separately (bender medsystems). in brief, lung homogenates were prepared and processed accordingly. the samples were acquired on a bd lsrii (bd bioscience), and the amount of cytokine (ng/ml) was calculated by flowcytomix pro 2.3 software (bender medsystems). virus-infected or mock-treated mice were sacrificed at the indicated time point for histopathologic analysis. the lung tissues were fixed in 10% formalin and embedded in paraffin. fivemicrometer-thick, paraffin-embedded sections were cut and stained with hematoxylin and eosin (h&e) to analyze histological lesions. histopathologic score of lung tissues was examined by a board-certified pathologist blinded to the exposure status. lung inflammatory changes were graded using a semiquantitative scoring system based on the following parameters: peribronchiolar and bronchial infiltrates, bronchiolar and bronchial luminal exudates, perivascular infiltrates, parenchymal pneumonia, and edema, as previously described [28] . each parameter was graded on a scale of 0-4 with 0 as absent, 1 as slight, 2 as mild, 3 as moderate, and 4 as severe. the total lung inflammation score was expressed as the sum of the scores for each parameter. the degree of cell infiltration was independently scored on an increasing scale of 0-3 with 0 as no cells, 1 as few cells, 2 as moderate influx of cells, and 3 as extensive influx of cells [29] . data were expressed as mean (standard error of the mean). unpaired student t test in graphpad prism 5.0 software (graphpad) was used for statistical analysis. a p value ,.05 was considered significant. rhmbl binds both pdmh1n1 and h9n2/gi viruses a microtiter capture assay demonstrated that mbl could bind to pdmh1n1 and h9n2/g1 in vitro ( figure 1 ). the mbl-virus binding occurred in a dose-dependent manner ( figure 1a and 1d). similarly, increased amount of virus could also result in increased binding by mbl ( figure 1b and 1e) . mbl utilizes the crd to recognize pathogens in a calcium-dependent manner [30] . further addition of ethylenediaminetetraacetic acid (edta) in the assay inhibited the binding of mbl to both strains of influenza virus ( figure 1c and 1f) , suggesting that the binding occurred through the crd of mbl. wild-type and mbl ko mice, 6-10 weeks of age, were infected intranasally with 30 ll of 10 3 tcid 50 pdmh1n1 virus or 10 5 tcid 50 h9n2/g1 virus. the viral dosage chosen for the experiment was previously demonstrated to be sublethal (data not shown). mice were inoculated with 30 ll of pbs as the mock treatment. no mice died throughout the 14-day experiment. the body weight of mice, which was a physiological value indicating infection progress and the health of the animals, was recorded daily. mice receiving mock treatment did not lose body weight, demonstrating the absence of potential harmful effects due to anesthetics and intranasal inoculation (figure 2a ). both strains of influenza virus could successfully infect mbl wt and mbl ko mice as evidenced by the significant weight loss in these mice after virus infection. compared to mbl ko mice, mbl wt mice had more weight loss upon virus infection. for pdmh1n1 virus infection, mbl wt mice showed a significantly greater body weight drop on days 3-12 compared with mbl ko mice ( figure 2b ). the mean peak body weight loss observed in the mbl wt mice and mbl ko mice were -23.51% and -17.38%, respectively. for h9n2/g1 virus infection, mbl wt mice only showed a significantly greater weight loss on day 8 when compared with mbl ko mice ( figure 2c ). although similar mean peak weight loss was observed between the mbl wt mice and mbl ko mice after h9n2/g1 virus infection, mbl wt mice recovered more slowly than the ko mice. collectively, these data suggested that the presence of mbl caused a more severe infection by the pdmh1n1 and h9n2/g1 viruses. wild-type and mbl ko mice infected with pdmh1n1 or h9n2/g1 virus were sacrificed on days 3, 7, and 14 after infection. virus titers in lung homogenates were determined by tcid 50 . as shown in figure 3 , these 2 strains of influenza virus were detectable in the lung homogenates collected from mbl wt and ko mice on day 3 and day 7, confirming viral lung infection. on day 14, titers for both strains of virus were undetectable, which was consistent with the regain of body weight by mbl wt and mbl ko mice and suggested recovery from the infection. for pdmh1n1 virus infection, there was no significant difference in the lung virus titer between mbl ko and mbl wt mice on days 3 and 7 after infection ( figure 3a ). in contrast, significantly less virus titer was detected in mbl ko mice on day 7 but not on day 3 after h9n2/g1 virus infection compared to that in mbl wt mice ( figure 3b ). to further investigate whether mbl would modify the inflammatory response upon pdmh1n1 and h9n2/g1 virus infection, a panel of 14 proinflammatory cytokines and chemokines were examined in the lung homogenates collected from the infected mbl wt and mbl ko mice. the simultaneous profiling of the cytokines and chemokines was examined by using bead-based suspension array, which could allow the sensitive and specific detection of these proteins in the available amount of lung homogenates. as shown in figures 4 and 5 , upon pdmh1n1 and h9n2/g1 virus infection, inflammatory response assayed by cytokines and chemokines production was triggered in both mbl wt and mbl ko mice. except for il-2, of which the level remained constantly low during the course of experiment, the kinetics of individual proteins were similar in that they were readily detectable on day 3, reached the highest level on day 7, and declined on day 14. strikingly, mbl ko mice had reduced inflammatory responses during infection. among the 14 cytokines examined, the majority of them showed significantly lower amounts in mbl ko mice lung homogenates than in mbl wt mice, including il-1a, il-1b, il-6, il-10, tnf-a, ifn-c ( figures 4a and 5a) , kc, mip-1a, mip-1b, mcp-1, mcp-3 and rantes (figures 4b and 5b) . these results suggested that mbl upregulates the inflammatory response to influenza virus infection, resulting in elevated production of proinflammatory cytokines and chemokines in the mbl wt mice as compared with the mbl ko mice. to further confirm the severe inflammatory response in the mbl wt mice compared with mbl ko mice, lung sections were stained with h&e for histological analysis to evaluate inflammation-associated lung damage caused by pdmh1n1 and h9n2/g1 influenza virus infection. in the histological sections of mbl wt mice, more severe lung inflammation and more cell infiltration were observed when compared to that of mbl ko mice on day 7 ( figure 6 ). consistent with our cytokine and chemokine data, the pulmonary histological analysis suggested that the mbl wt mice had a more severe inflammatory response upon pdmh1n1 and h9n2/g1 virus infection. mannose-binding lectin is a pattern-recognition molecule, which provides first line of host defense. accumulating evidence has suggested that mbl exhibits in vitro anti-influenza virus properties by direct neutralization, inhibiting influenza virus hemagglutination, binding to the influenza virus as an opsonin, and activating the complement system through the lectin pathway [14, 16, 23] . however, these properties vary among different virus strains and subtypes [17, 18] . in this study, we focused on the pandemic influenza a h1n1 virus and avian influenza a h9n2/g1 virus, which are of potential threat to the global community. we demonstrated that despite these 2 strains of viruses being bound by rhmbl via the crd of mbl at the physiological level, they infected both mbl wt and mbl ko mice effectively. our results are consistent with a recent in vitro study by job et al [18] , in which mbl was found to bind to pdmh1n1 fairly in vitro but the virus was resistant to the antiviral activity of mbl. the number and position of potential glycosylation sites on the viral ha globular domain determine the binding affinity between mbl and the virus. even though mbl can physically bind to the virus, the binding may be insufficient for executing any antiviral function. arguably, chang et al [23] recently reported that mbl deficiency increases susceptibility to infection with influenza a virus philippine 82 h3n2 (phil82), which is a human strain. we reconcile with the suggestion that mbl effects would differ depending on strains of influenza a virus and thus mbl causes variable antiviral activities and host responses. the degree of glycosylation on the globular head of the ha molecule is believed to be essential for mbl to exhibit its antiviral properties. for phil82 virus, the high-mannose oligosaccharide at residue 165 of the ha molecule has already been shown to be crucial for the neutralization by mbl [31] . although pdmh1n1 virus contains a single potential glycosylation site at the base of the ha globular head (asn 104 ), it lacks potential glycosylation sites on the globular head region of ha (asn 142 , asn 144 , asn 172 , asn 177 , and asn 179 ) [18] . to our knowledge, binding of mbl on h9n2/g1 virus is not well documented in the literature. therefore, we analyzed the potential glycosylation sites on ha of h9n2/g1 virus based on an in silico approach as suggested by job et al [18] . the ha sequence data was retrieved from genbank (aaf00706.1) and we used netnglyc 1.0 server to predict the number of potential glycosylation sites. we found that there was no potential glycosylation sites near residue 165 of the ha molecule of h9n2/g1 virus. we speculate that as a result of the absence of potential glycosylation sites near the receptor-binding domain of the ha globular head of both pdmh1n1 virus and h9n2/g1 virus, mbl fails to interfere with the viral binding to target cells despite its ability to bind the virus. this can adequately explain the discrepancy between the present in vivo data and chang's study [23] . in this study, mbl wt mice were found to have a more severe disease in terms of greater weight loss and worse lung pathology than mbl ko mice during either pdmh1n1 or h9n2/g1 virus infection. this suggests that mbl may contribute to the disease severity seen in the mbl wt mice. to elucidate the mechanisms, we investigated the immune response, such as production of cytokines and chemokines at various time points during the infection. most cytokines and chemokines were detected with similar kinetics, with the peak on day 7 following influenza virus infection in both mbl wt and mbl ko mice. these data suggest that the most critical phase of influenza infection occurs around day 7 after infection, and this is interestingly, we found that mbl contributed to a more severe proinflammatory response by increasing the production of several proinflammatory cytokines, such as il-1a, il-1b, il-6, tnf-a, and ifn-c. interleukin 1a and il-1b are multifunctional proinflammatory cytokines produced readily by influenzainfected leukocytes. they are capable of inducing fever, anorexia, and weight loss [32] . enhanced production of these cytokines can contribute to the acute lung immunopathology after influenza virus infection in mice [33] and induce gene expression of other cytokines like il-6 and tnf-a [34, 35] . despite the abundance of il-6 following the influenza virus infection, in vivo studies showed that it does not contribute significantly to the pathogenesis of influenza virus infection because the mortality and morbidity observed in mice infected with h5n1 are comparable in both mbl wt and il-6 deficient mice [36, 37] . tumor necrosis factor a is readily produced by influenza virusinfected leukocytes and can activate macrophages, stimulate dendritic cell maturation and neutrophils, further enhance the inflammatory response, and activate efficient antigen presentation system in the infected site [20, 38] . excessive production of tnf-a causes tissue injury, hemorrhagic shock, and death in mice [39, 40] . interferon c is also an important proinflammatory cytokine that has different functions, including the activation of macrophages, differentiation of th1 from t cells, enhancement of antigen presentation, and expression of the chemokine gene [41, 42] . these proinflammatory cytokines are commonly found in the acute-phase response to influenza virus infection and may induce immunity but also cause damage to the host tissue [43] . these cytokines were also increased in our infected murine lung, with significantly higher levels in mbl wt mice than in mbl ko mice on day 7, coinciding with body weight loss and lung histological findings. interleukin 10, an antiinflammatory cytokine, was also significantly higher in mbl wt mice than in mbl ko mice on day 7. interleukin 10 deficiency was reportedly protective in high-dose influenza virus infection [44] , implying that increased il-10 may be deleterious to the host. as a consequence of such an overwhelming ''cytokine storm'' [45, 46] , the mbl wt mice were found to have a worse disease course than in the mbl ko mice, including greater body weight loss and more severe lung inflammation. in addition, we also found that most chemokines, including kc, mip-1a, mip-1b, mcp-1, and mcp-3, were elevated in mbl wt mice compared with the mbl ko mice in both pdmh1n1 and g1/97 virus infection. influenza virus-infected macrophages produced large amounts of these chemokines in vitro [25, 47, 48] . functionally, these chemokines are important mediators for immune cell activation and chemotactic factors, which recruit leukocytes to the infected sites [49] . this may help account for our histological observation that more inflammatory cell infiltration was observed in mbl wt mice than in mbl ko mice. the role of mbl in modulating immune responses has also been observed in staphylococcus aureus infection. it was shown that mbl amplifies the host immune response during s. aureus infection by cooperating with toll-like receptors 2 and 6 and augments the production of proinflammatory cytokines and chemokines [50] . the observation from our present study prompted us to further investigate whether mbl may also cooperate with other pattern-recognition receptors and thus further amplify the host response during influenza virus infection. in conclusion, we have shown for the first time that mbl is a risk factor leading to a more severe pdmh1n1 and h9n2/g1 virus infection by upregulating proinflammatory responses. financial support. this work was supported in part by the national world health organization. pandemic (h1n1) world health organization. pandemic (h1n1) 2009dbriefing note 23 antigenic and genetic characteristics of swine-origin 2009 a(h1n1) influenza viruses circulating in humans cytotoxic t lymphocytes established by seasonal human influenza cross-react against 2009 pandemic h1n1 influenza virus human infection with an avian h9n2 influenza a virus in hong kong in 2003 human infection with influenza h9n2 h9n2 influenza viruses possessing h5n1-like internal genomes continue to circulate in poultry in southeastern china phylogenetic perspectives in innate immunity the mannosebinding lectin: a prototypic pattern recognition molecule a second serine protease associated with mannan-binding lectin that activates complement the human mannose-binding protein functions as an opsonin mannose-binding lectin in severe acute respiratory syndrome coronavirus infection association of mutations in mannose binding protein gene with childhood infection in consecutive hospital series human mannose-binding protein functions as an opsonin for influenza a viruses complementdependent neutralization of influenza virus by a serum mannosebinding lectin human mannan-binding lectin inhibits the infection of influenza a virus without complement collectin-mediated antiviral host defense of the lung: evidence from influenza virus infection of mice pandemic h1n1 influenza a viruses are resistant to the antiviral activities of innate immune proteins of the collectin and pentraxin superfamilies defense against influenza a virus infection: essential role of the chemokine system julkunen i. tnf-alpha and ifn-alpha enhance influenza-a-virus-induced chemokine gene expression in human a549 lung epithelial cells mannose-binding lectin regulates the inflammatory response of human professional phagocytes to neisseria meningitidis serogroup b mannose-binding lectin recognizes peptidoglycan via the n-acetyl glucosamine moiety, and inhibits ligand-induced proinflammatory effect and promotes chemokine production by macrophages lack of the pattern recognition molecule mannose-binding lectin increases susceptibility to influenza a virus infection influenza virus directly infects human natural killer cells and induces cell apoptosis differential expression of chemokines and their receptors in adult and neonatal macrophages infected with human or avian influenza viruses inhibition of human natural killer cell activity by influenza virions and hemagglutinin mannose-binding lectin-deficient mice are susceptible to infection with staphylococcus aureus p58(ipk): a novel ''cihd'' member of the host innate defense response against pathogenic virus infection the aminobisphosphonate pamidronate controls influenza pathogenesis by expanding a {gamma}{delta} t cell population in humanized mice mannose-binding proteins in human serum: identification of mannose-specific immunoglobulins and a calcium-dependent lectin, of broader carbohydrate specificity, secreted by hepatocytes changes in the hemagglutinin molecule of influenza type a (h3n2) virus associated with increased virulence for mice role of interleukin-1 in infectious diseases interleukin-1 is responsible for acute lung immunopathology but increases survival of respiratory influenza virus infection inflammasomes as mediators of immunity against influenza virus biologic basis for interleukin-1 in disease inhibition of the cytokine response does not protect against lethal h5n1 influenza infection role of host cytokine responses in the pathogenesis of avian h5n1 influenza viruses in mice inflammatory responses in influenza a virus infection shock and tissue injury induced by recombinant human cachectin anti-cachectin/tnf monoclonal antibodies prevent septic shock during lethal bacteraemia clinical aspects and cytokine response in severe h1n1 influenza a virus infection molecular pathogenesis of influenza a virus infection and virusinduced regulation of cytokine gene expression local and systemic cytokine responses during experimental human influenza a virus infection. relation to symptom formation and host defense il-10 deficiency unleashes an influenza-specific th17 response and enhances survival against highdose challenge lung pathology in fatal novel human influenza a (h1n1) infection th1 and th17 hypercytokinemia as early host response signature in severe pandemic influenza induction of proinflammatory cytokines in human macrophages by influenza a (h5n1) viruses: a mechanism for the unusual severity of human disease? comparison of proinflammatory cytokine expression and cellular signal transduction in human macrophages infected with different influenza a viruses chemokines-chemotactic cytokines that mediate inflammation mannosebinding lectin enhances toll-like receptors 2 and 6 signaling from the phagosome key: cord-282433-p6jl9gxf authors: tu, xinyi; chong, wai po; zhai, yun; zhang, hongxing; zhang, fang; wang, shixin; liu, wei; wei, maoti; siu, nora ho on; yang, hao; yang, wanling; cao, wuchun; lau, yu lung; he, fuchu; zhou, gangqiao title: functional polymorphisms of the ccl2 and mbl genes cumulatively increase susceptibility to severe acute respiratory syndrome coronavirus infection date: 2015-03-27 journal: j infect doi: 10.1016/j.jinf.2015.03.006 sha: doc_id: 282433 cord_uid: p6jl9gxf objectives: to assess associations between the functional polymorphisms g-2518a at the chemokine (c–c motif) ligand 2 gene (ccl2) and mannose binding lectin (mbl) codon 54 variant (a/b) and susceptibility to sars. methods: we genotyped the ccl2 g-2518a and mbl codon 54 variant (a/b) in 4 case–control populations of chinese descent, totally consisting of 932 patients with sars and 982 control subjects. results: both the high-ccl2-producing gg genotype and the low-mbl-producing b allele were consistently associated with increased risks of sars-cov infection in all 4 case–control populations (joint p = 1.6 × 10(−4) and 4.9 × 10(−8), for ccl2 and mbl respectively), with no interaction between polymorphisms could be detected. furthermore, all the 4 case–control studies demonstrated a cumulative effect on risk of sars-cov infection for the combination of polymorphisms (joint p = 1.3 × 10(−10)). however, tests using the area under the curve (auc) indicated that at this stage, the polymorphisms were unlikely to be appropriate for risk prediction testing because of low auc values (all <66%). additionally, no association was observed between the polymorphisms and severity of sars. conclusions: the ccl2 g-2518a and mbl codon 54 variant have a significantly cumulative effect on increased risk of sars-cov infection. functional polymorphisms of the ccl2 and mbl genes cumulatively increase susceptibility to severe acute respiratory syndrome coronavirus infection introduction severe acute respiratory syndrome (sars) is a newly emerged infectious disease of humans caused by a novel coronavirus (cov), the sars-cov. 1 pathogenesis of sars is complex and host genetic background is considered to be one of the factors in determining susceptibility to, and outcome of sars. 2 previous reports have shown that polymorphisms of several putatively important genes affect an individual's susceptibility to sars-cov infection or disease severity of sars. 3à10 in particular, our previous two independent association studies have implicated that a functional polymorphism at codon 54 in exon 1 (rs1800450, g230a, denoted as a/b variant) of mannose binding lectin (mbl), which encodes a protein belonging to the family of collectin and plays a critical role in the innate immune response, conferred a significantly increased susceptibility to sars-cov infection. 3, 9 however, on the basis of the fact that the susceptibility to infectious disease is determined at different functional levels of innate and adaptive immunity, 11 we hypothesize that an unknown number of other unidentified genes are likely to mediate the susceptibility to sars, including sars-cov infection and disease severity. chemokines play important role in cells trafficking during immune responses. among the chemokine family, the chemokine (cec motif) ligand 2 (ccl2), also designated as monocyte chemoattractant protein-1 (mcp-1), is known as a potent chemoattractant for monocytes and macrophages, and is considered to be involved in several diseases characterized by intense macrophage infiltration. 12 ccl2 influences both innate immunity, through effects on monocytes and macrophages, and adaptive immunity, through control of t helper cell polarization. 13 in the case of sars, our and other studies have shown that ccl2 was one of the earliest and most prominent chemokines upregulated in either lung epithelial cells or monocyte derived dendritic cells infected with sars-cov. 14, 15 the upregulation of ccl2 mediate the migration of monocytes and macrophages, which were the infiltrating cells indeed observed in the lung tissues of patients with sars. 16 notably, the overexpression of ccl2 was consistently detected in plasma of patients with sars in several independent studies. 17à19 furthermore, after treatment with corticosteroid, which is an effective cytokine modulator and has a beneficial effect on sars patients, the level of plasma ccl2 was reduced significantly from 5 to 8 days. 17 additionally, it has also been reported that the higher level of serum ccl2 in patients is correlated with more advanced disease severity of sars. 17 in the lungs of balb/c mice, the pdzbinding motif of recombinant sars-cov envelope protein is a determinant of viral pathogenesis and induces the deleterious exacerbated immune response including increased expression of ccl2. 20 on the basis of the above relevance of the ccl2 in the pathogenesis of sars, we hypothesize that the ccl2 may be the excellent biologic candidate susceptibility gene for sars. it is expected that the genetic variation within ccl2 could contribute to inter-individual differences in susceptibility to, and outcome of sars. recently, a functional single nucleotide polymorphism (snp) (rs1024611, g-2518a) in the distal regulatory region of the ccl2 at position à2518 relative to the transcription start site has been well characterized. 21 compared with the ccl2 -2518a allele, the à2518g allele conferred a greater ccl2 transcriptional activity mediated by differential protein-dna interactions, an increased ccl2 protein production in vitro and in vivo, and an enhanced leukocyte trafficking to tissues. 21à23 furthermore, prevalence of the high-ccl2-producing à2518g allele has been shown to be associated with increased susceptibility or severity of infectious diseases, including hiv-1 infection and aids dementia, 21,24 hcv infection, 22 hbv clearance, 25 hcmv reactivation, 26 and pulmonary tuberculosis. 27 the role of this functional polymorphism in sars, however, has never been specifically evaluated. in this study, we therefore investigated whether the functional polymorphism g-2518a in the ccl2 gene have any bearing on the sars. to this end, we genotyped the ccl2 g-2518a polymorphism in 4 independent caseecontrol populations of chinese descent, totally including 932 patients with sars and 982 control subjects. we also reassessed the mbl codon 54 variant (a/b) in susceptibility to sars-cov infection in the present study. furthermore, we investigated whether a combination of these two functional polymorphisms of ccl2 and mbl would have a cumulative effect on risk of sars-cov infection. four independent caseecontrol populations of chinese ancestry were included in the present study. the details about enrollment criterion and demographic characteristics of these populations had been described previously, 3, 4, 9 and were also provided in the supplementary materials and methods and supplementary the polymorphism ccl2 g-2518a (rs1024611) was genotyped by polymerase chain reaction-restriction fragment length polymorphism (pcr-rflp) analysis. 28 the sequences of primers and conditions of pcr and enzyme digestion were provided in the supplementary materials and methods. the mbl codon 54 a/b variant (rs1800450, g230a) was genotyped by using pcr direct sequencing as described previously. 9 this polymorphism had been investigated in beijing community population and hong kong population in our previous studies 3, 9 ; therefore, the present study genotyped this polymorphism only in beijing health care worker (hcw) and tianjin population. the results of snp genotyping were tested for deviations from hardyeweinberg equilibrium (hwe) by using the markov chain method implemented in the genepop software (available at: http://wbiomed.curtin.edu.au/ genepop/). associations between snps and risk of sars were assessed by use of logistic regression analyses in spss software (version 9.0; spss inc., chicago, il). for ccl2 g-2518a, the reference category is the combination of the heterozygous genotype ag and the homozygous minor genotype aa, while for mbl codon 54 variant (a/b) the reference category is the homozygous major genotype aa. the odds ratios (ors) and 95% confidential intervals (cis) were calculated and adjusted for potential confounders including age (continuous; years), sex (categorical; female or male) and populations (categorical; beijing community, beijing hcw, tianjin, or hong kong), where it was appropriate. the meta-analysis of data generated from multiple stages was conducted to estimate pooled genetic effects using fix-effect model based on mantelehaenszel method. we calculated cochran's q statistic to test for betweengroup heterogeneity and the i 2 statistic to quantify the proportion of the total variation due to heterogeneity. the heterogeneity was considered significant for p < .05. for there was no significant heterogeneity for multiple populations, we further analyzed the pooled data by logistic regression with adjustment for potential confounders. the ccl2 gene resides on chromosome 17q11.2-q12, while the mbl gene resides on chromosome 10q11.2, meaning that ccl2 g-2518a and mbl codon 54 variant (a/b) are not in linkage equilibrium with each other. to further test whether or not a snp is dependent on the other, independence test was performed for a snp with adjustment for the other and potential confounders. logistic regression with the use of an interaction term was also performed to investigate potential geneegene interaction between two snps. for there was no significant interaction detected, we then tested the cumulative effects of snps on sars by counting the number of at-risk genotypes associated with sars for these two snps in each subject. the odds ratio for patients carrying any combination of one or two atrisk genotypes was estimated by comparing them with those carrying none of the at-risk genotypes by logisticregression analysis. the cochranearmitage trend test was used to assess the disease risk upon the increasing number of at-risk genotypes. the population attributable fractions (pafs) were estimated for the at-risk genotypes of ccl2 g-2518a and mbl codon 54 variant (a/b) with the use of the formula paf z f(or e 1)/[1 þ f(or e 1)], where f is the prevalence of at-risk genotype associated with sars and or is used in the place of relative risk. 29 the paf value indicates percentage of the increase in the risk of developing sars attributed to at-risk genotypes. the specificity and sensitivity of the regression model were calculated by constructing receiver-operatingcharacteristic (roc) curves, and then statistics for the area under the curve (auc) were calculated to estimate the ability of models to distinguish case subjects from control subjects. the values for auc range from 50% (no caseecontrol discrimination) to 100% (perfect discrimination of cases and controls). an association was considered significant at a p value of less than .05 in individual populations, and with more stringent threshold in the pooled samples based on the bonferroni correction for multiple testing of two snps. all statistical tests were two-sided. all analyses were performed using the spss (version 9.0, spss inc., chicago, il, usa) and stata 9.2 software (statacorp lp, college station, tx, usa). initially, we estimated the effect of ccl2 g-2518a on susceptibility to sars-cov infection in beijing community population consisted of 352 patients with sars and 392 controls. the observed genotype frequencies for this polymorphism conformed to the hwe in both patients and controls (p > .05). on the basis of logistic regression analysis with adjustment for age and sex, the subjects carrying the gg genotype had a significantly increased susceptibility to sars-cov infection, compared with ones carrying the à2518a allele (i.e., ag plus aa genotype) (or 1.41, 95% ci, 1.03 to 1.92, p z .031; table 1 ). to test for replication of the association, we then genotyped the g-2518a in three additional caseecontrol sample sets. the genotype distributions of all groups did not deviate from the hwe. again, there was an excess of gg genotype in patients than in controls in beijing hcw (p z .025), tianjin (p z .038) and hong kong population (p z .010), respectively. meta-analysis on pooled data from all the four chinese populations provided unequivocal evidence for a relationship between ccl2 g-2518a and susceptibility to sars-cov infection (fig. 1 ). or associated with gg genotype was 1.58 (95% ci, 1.28 to 1.96, p z 2.3 â 10 à5 , p heterogeneity z .43, i 2 z 0%; fig. 1a ). given there was no significant heterogeneity for the four chinese populations, we further analyzed the pooled data by logistic regression, and a strong support for an association between the g-2518a and susceptibility to sars-cov infection was observed (p z 1.6 â 10 à4 ), with the or being 1.48 (95% ci, 1.21e1.82) for the at-risk gg genotype, compared with the ag plus aa genotype. on the basis of the ors combined with the frequencies of gg genotype, pafs were estimated to account for 12.8e53.1% of sars cases in the 4 individual populations, and 14.6% in the overall population (table 1) . we also reassessed the association between sars and the codon 54 variant (a/b) in mbl, which were previously reported to be associated with sars in both beijing community and hong kong population (table 1 ). 3, 9 again, we replicated that the subjects bearing the variant b allele (bb plus ab genotype), which is associated with low serum mbl, 30 have a significantly increased risk of sars-cov infection in both beijing hcw (p z .019) and tianjin population (p z .012), compared with those homozygous for the wild-type a allele. combined analysis of the 4 caseecontrol populations by meta-analysis yielded a stronger support for the association (or 1.82, 95% ci, 1.47 to 2.25, p z 5.3 â 10 à8 , p heterogeneity z .50, i 2 z 0%; fig. 1b ). logistic regression gave a similar result (or 1.79, 95% ci, 1.45 to 2.21, p z 4.9 â 10 à8 ; table 1 ). the pafs were 18.8e53.3% for the 4 individual populations, and the pooled paf was 21.9% for the overall population (table 1) . we further evaluated whether the effects for the ccl2 g-2518a and mbl codon 54 variant (a/b) on risk of sarsfig. 1c) . the joint pafs for the combination of at-risk genotypes of ccl2 and mbl were 29.1e41.0% in the 4 individual populations, and the joint paf was 28.5% in the pooled population (table 2) . we next calculated statistics for auc to estimate the ability of each of three models to distinguish case subjects from control subjects. in the four individual populations, the auc was 53.2e61.0 for model 1 (sex, age, and the number of at-risk genotypes at ccl2), 53.9 to 63.6 for model 2 (sex, age, and the number of at-risk genotypes at mbl), and 55.7 to 65.8 for model 3 (sex, age, region, and the number of at-risk genotypes at both ccl2 and mbl) with all p values equal or less than .10 ( supplementary fig. 1 ). in the pooled population, the auc was 53.7 (95% ci, 51.1 to 56.3; p z 4.8 â 10 à3 ) for model 1, 55.5 (95% ci, 52.9 to 58.1; p z 2.9 â 10 à5 ) for model 2, and 57.0 (95% ci, 54.5 to 59.6; p z 1.0 â 10 à7 ) for model 3 ( supplementary fig. 1) . we also assessed whether there was an association between the ccl2 g-2518a and mbl codon 54 variant (a/ b) and severity of sars, but found that the two snps were associated with the severity of sars neither individually nor jointly (supplementary table 3 ). this study includes 932 patients with sars totally, which accounts for about 12% of the sars cases worldwide during the sars outbreak, being so far the largest cohort in the field of genetic association studies of sars. additionally, given whether the patients were infected in the hospital or community is a potential confounding factor, we matched the cases and controls for this factor in each caseecontrol series. furthermore, we performed statistical adjustment for age and sex to minimize other potential biases. taken together, the large size of the investigation, the consistency of the observations in 4 independent caseecontrol series and the low p values distinguish our study from previous studies investigating the influence of different other polymorphisms on the development of sars, and strengthen the association between the ccl2 g-2518a and mbl codon 54 variant (a/b) and susceptibility to sars-cov infection. to our best knowledge, this is the first report that functional polymorphisms of the ccl2 and mbl genes cumulatively increase susceptibility to sars-cov infection, confirming the initial hypothesis that these genes may play a role in the pathogenesis of this disorder. several previous studies have consistently demonstrated that the carriers of the à2518g allele confer up-regulated transcriptional activity, higher ccl2 mrna and protein levels in vitro and in vivo, and more infiltration of leukocytes into tissues in comparison with a carriers. 21à23 therefore, one might expect that the individuals who carry the at-risk gg genotype, and thus have higher expression of ccl2 and successively more attraction of monocytes and macrophages, may have an increased susceptibility to sars-cov infection. this hypothesis is biologically plausible. previous studies have consistently reported an overexpression of ccl2 in the lung tissues and sera of sars patients. 17e19 the pulmonary tissues of sars patients were also well characterized by pronounced infiltration of monocytes and macrophages. 17, 31, 32 furthermore, sars-cov was shown to infect the monocyte-derived macrophages in vitro 15, 33 and, to be readily detectable in macrophages in lungs and other target organs of sars patients. 31, 32 based on these observations, gu et al. have argued that the sars virus infects resident, infiltrating, and circulating immune cells, such as macrophages and monocytes; and then, these infected circulating cells may carry sars-cov to various target organs as manifested by widespread dissemination. 31, 32 notably, this kind of spread of infection by infected phagocytes (so-called "trojan horses effect") has been implicated before in the infection of hiv, measles virus and other intracellular microorganisms. 34, 35 considering the known pathophysiologic role of the ccl2 in these disorders, these examples further suggest the high-ccl2-producing gg genotype may be the at-risk genotype conferring increased susceptibility to sars-cov infection. consistent with our previous findings, 3, 9 this study further confirmed the significant association between the low-mbl-producing b allele and increased risk of sars-cov infection in two additional independent sample sets, suggesting that mbl deficiency may be a susceptibility factor for the acquisition of sars. the independent confirmation of associations of these two snps with sars-cov infection supports the validity of genetic association studies in complex diseases. this finding promoted us to investigate the role of geneegene interaction in the genotype-to-phenotype relationship of sars-cov infection. however, no consistent significant genetic interaction between these two snps on risk of sars-cov infection was observed across all caseecontrol populations and in the pooled samples. furthermore, no functional studies reported for the interactive effect of mbl and ccl2 on the susceptibility to sars-cov infection. several previous studies have implicated that mbl could increase the secretion of ccl2 from human monocytic u937 cell lines, peripheral blood mononuclear cells and umbilical vein endothelial cells mediated by a variety of bacterial microbes. 36, 37 whether or not there exit biological interactions between mbl and ccl2 under the condition of sars-cov infection remains to be investigated in future studies. it has to be noted that many chemokines not only take part in the process of viral infection, but are also involved in cell damage and organ dysfunction. indeed, several lines of evidence have indicated that ccl2 secretion is upregulated during the development of sars and correlates with intensifying immuno-mediated damage to the lungs and other target organs, resulting in acute lung injury and, subsequently, multi-organ dysfunction. 38 therefore, we speculate that the high-ccl2-producing gg genotype may enhance the inflammation and associate with more severe clinical outcomes of sars. however, we did not find a genetic association between ccl2 polymorphism and admission to intensive care units or deaths due to sars. this result may be due to the limited number of patients with severe sars or died from sars in the present study. for example, as for disease severity this study has merely powers of 14.6% (two-sided test of significance, a z 0.05) to detect ors of >1.66 in beijing community, and 9.5% to detect ors of <0.73 in hong kong population, for carriers of the gg genotype relative to the carriers of the aa plus ag genotypes at the position à2518 of the ccl2 gene (supplementary table 3 ). in contrast, as for sars-cov infection this study has powers of 56.2% to detect ors of >1.41, and 82.7% to detect ors of >1.57, in beijing community and hong kong populations, respectively, for the same snp (table 1) . it certainly warrants confirmation in additional studies with larger collections of sars patients. the allele and genotype frequencies of the ccl2 g-2518a polymorphism vary with ethnicity (according to the hapmap project database). indeed, in this study with 982 control subjects, we found that the frequency of the à2518g allele and gg genotype was 52.0% and 27.0%, compared with around 34.7% and 12.0%, respectively, among caucasians from the iceland. 39 therefore, although the highly significant associations between ccl2 g-2518a and susceptibility to sars-cov infection were biologically plausible, and strengthened by our 4 independent caseecontrol studies, it remains interesting to investigate that whether there exist population-specific differences for this polymorphism to sars susceptibility between chinese and europeans. interestingly, a significant cumulative effect on the susceptibility to sars-cov infection was observed for the combination of two functional snps in ccl2 and mbl across the four independent caseecontrol series (p trend z 1.3 â 10 à10 in the pooled samples). we estimated that individuals who have both of the at-risk genotypes have an odds ratio of 2.88 for sars-cov infection (table 2) . therefore, pafs can be calculated to be 14.6% for ccl2, 21.9% for mbl, and 28.5% for both in the pooled samples, indicating that, for example, 28.5% of the increase in the risk of sars-cov infection can be attributed to carrying 1 or 2 at-risk genotypes of the two polymorphisms. if the associations are real, then at-risk genotypes of the two polymorphisms are associated with a low to moderate fraction of sars-cov infection among chinese, suggesting that there exist unrevealed genes conferring susceptibility to such an infection. in fact, previous reports have shown that polymorphisms of several genes affect an individual's susceptibility to sars-cov infection or disease severity of sars, 3à10 although most of these susceptibility genes need to be confirmed in independent sample sets. in addition, it is notable that at this stage, ccl2 g-2518a and mbl codon 54 variant (a/b) are unlikely to be appropriate for risk prediction testing individually or in combination, because of low aucs (53.7% for ccl2, 55.5% for mbl, and 57.0% for both) in pooled population. however, as further susceptibility polymorphisms are identified and confirmed, and interaction effects among such polymorphisms together with other risk factors are taken into account, prediction of the susceptibility of sars-cov infection may become more accurate and clinically usable. this study has several potential limitations. first, the small sample size of subjects homozygous for the minor allele b of mbl codon 54 variant (a/b) did not allow calculation of separate ors for the bb genotype (the genotype with the greatest risk). second, the small sample sizes likely meant that there was low power to detect genetic interaction between polymorphisms. third, the small sample sizes of the two new caseecontrol populations (beijing hcw and tianjin) meant that the effect estimates for these groups were larger in magnitude and had wider cis (that is, they were less stable as shown in table 1 and fig. 1 ) than the effect estimates generated from the other two larger caseecontrol populations. last, there was no adjustment for co-morbid conditions (such as chronic diseases) that could affect susceptibility to sars. in summary, our findings indicate that individuals who are genetically predisposed to produce greater amounts of ccl2 protein seem to be more susceptible to sars-cov infection. we also confirmed our previous findings that low-mbl-producing allele is a susceptibility factor for the acquisition of sars. furthermore, a combination of ccl2 and mbl polymorphisms has a stronger genetic association with the susceptibility to sars-cov infection. however, 1) no interaction between polymorphisms could be detected; 2) no association was observed between the polymorphisms and severity of sars; and 3) although there were statistically significant associations between the two polymorphisms and sars-cov infection, tests using the auc curve do not indicate that the at-risk genotypes have clinical usefulness (that is, the at-risk genotypes cannot discriminate cases from controls). since both the ccl2 and mbl are important components in innate immunity system, our findings further support the hypothesis that variability in the innate immune response can be involved in susceptibility to sars-cov infection during the vulnerable period before the production of specific antibodies. 9 sars-associated coronavirus pathogenesis of severe acute respiratory syndrome mannose-binding lectin in severe acute respiratory syndrome coronavirus infection lack 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heart study effect of genetic variants of ccr2 and ccl2 on the natural history of hiv-1 infection: ccl2-2518gg is overrepresented in a cohort of spanish hiv-1-infected subjects association of common promoter polymorphisms of mcp1 with hepatitis b virus clearance polymorphisms in the genes encoding chemokine receptor 5, interleukin-10, and monocyte chemoattractant protein 1 contribute to cytomegalovirus reactivation and disease after allogeneic stem cell transplantation a functional promoter polymorphism in monocyte chemoattractant protein-1 is associated with increased susceptibility to pulmonary tuberculosis association of cytokine and chemokine gene polymorphisms with severe acute respiratory syndrome the use of attributable fraction in the design and interpretation of epidemiologic studies mannose-binding lectin deficiencyerevisited multiple organ infection and the pathogenesis of sars molecular pathology in the lungs of severe acute respiratory syndrome patients sars-coronavirus replication in human peripheral monocytes/macrophages mechanisms of microbial traversal of the blood-brain barrier hiv infection and dementia mannose-binding lectin without the aid of its associated serine proteases alters lipopolysaccharide-mediated cytokine/chemokine secretion from human endothelial cells c1q and mbl, components of the innate immune system, influence monocyte cytokine expression the immunobiology of sars* examination of genetic effects of polymorphisms in the mcp-1 and ccr2 genes on mi in the icelandic population no potential conflict of interest relevant to this article was reported. supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.jinf.2015.03.006. key: cord-256769-flfycl7i authors: stoermer, kristina a.; morrison, thomas e. title: complement and viral pathogenesis date: 2011-03-01 journal: virology doi: 10.1016/j.virol.2010.12.045 sha: doc_id: 256769 cord_uid: flfycl7i the complement system functions as an immune surveillance system that rapidly responds to infection. activation of the complement system by specific recognition pathways triggers a protease cascade, generating cleavage products that function to eliminate pathogens, regulate inflammatory responses, and shape adaptive immune responses. however, when dysregulated, these powerful functions can become destructive and the complement system has been implicated as a pathogenic effector in numerous diseases, including infectious diseases. this review highlights recent discoveries that have identified critical roles for the complement system in the pathogenesis of viral infection. have been identified, such as interaction of c1q with the c-type lectin sign-r1 expressed on macrophages (kang et al., 2006) , c5 activation by thrombin (huber-lang et al., 2006) , and the properdin-activated pathway (kemper et al., 2010) . the classical pathway is primarily activated by igm and certain igg isotypes bound to antigen. these immune complexes interact with the complement component c1q. c1q binding leads to the activation of two serine proteases associated with c1q, c1r and c1s. c1s cleaves c4 into c4a and c4b resulting in the exposure of a reactive thioester that allows covalent attachment of c4b on surfaces. c2 binds c4b and is also cleaved by c1s to form the classical pathway c3 convertase (c4bc2a). c3 convertases cleave c3 to amplify complement activation and lead to the generation of ligands for a variety of complement receptors. the lectin pathway is initiated by pattern recognition receptors such as mannose-binding lectin (mbl) and the ficolins (fig. 1) . mbl and ficolins contain carbohydrate-recognition domains that recognize carbohydrate patterns on the surfaces of cells or invading microorganisms. mbl and the ficolins are in a complex with enzymes known as mbl-associated serine proteases (masps). similar to c1s, masp-2 activates the complement system by cleaving both c4 and c2 to form the c4bc2a c3 convertase. the alternative pathway is activated by spontaneous hydrolysis of c3 (c3-h 2 o) (fig. 1) . this pathway also functions as an amplification loop for the cleavage of c3 initially triggered by other mechanisms. c3-h 2 o or c3b bound to target surfaces are bound by the protease factor b (fb). factor d (fd) is a serine protease that cleaves c3-h 2 o or c3b-bound fb, resulting in the generation of bb and formation of the alternative pathway c3 convertase (c3bbb). both the classical and alternative convertases function to cleave c3 to c3a and c3b. similar to c4, cleavage of c3 exposes a reactive thioester bond in c3b that allows for the covalent attachment of c3b to target surfaces. in addition, c3b can bind to either the classical or alternative c3 convertases resulting in a change of the substrate specificity of the convertases from c3 to c5. these c5 convertases cleave c5 to c5a and c5b. release of c5b promotes assembly of the c5b-c9 membrane attack complex (mac) which can directly lyse pathogens or pathogen-infected cells. the anaphylatoxins c3a and c5a interact with specific receptors to promote chemotaxis and regulate effector functions of cells of both the innate and adaptive immune response. a variety of soluble and membrane-associated proteins regulate complement activation. factor h (fh), c4b-binding protein (c4bp), and c1 inhibitor (c1-inh) are soluble proteins that regulate activation of the complement system. fh promotes the dissociation of c3bbb convertases and acts as a cofactor for factor i (fi)-mediated cleavage of c3b. c4bp functions similar to fh in that it promotes dissociation of c4bc2a convertases and acts as a cofactor for fi-mediated cleavage of c4b. the c1-inh is a serine protease inhibitor that inactivates both the c1q-associated proteases of the classical pathway (c1r and c1s) as well as the masps of the lectin pathway. membrane-associated regulators of complement activation include decay accelerating factor (daf/cd55), membrane cofactor protein (mcp/cd46), complement receptor 1 (cr1/cd35), cd59, and crry (rodent-specific). these proteins function to destabilize both the classical and alternative complement convertases, act as cofactors for fi-mediated cleavage of c3b and c4b, and prevent assembly of the mac on cell surfaces. the complement system is increasingly recognized as a mediator of protection or pathology in a variety of viral infections. furthermore, the continued identification of novel mechanisms of viral antagonism of complement highlight the important role this system has in viral pathogenesis. here, we review recent studies of viral interactions with a variety of components of the complement system and emphasize fig. 1 . schematic of the complement system. complement is activated by three major pathways. the classical pathway is primarily activated when c1q interacts with igm and certain igg isotypes bound to antigen. c1q-associated c1s cleaves c4 and c2 to form the classical pathway (cp) c3 convertase (c4bc2a). the lectin pathway (lp) is initiated by carbohydrate pattern recognition receptors such as mannose-binding lectin (mbl) and the ficolins (f) which are in a complex with enzymes known as mbl-associated serine proteases (masps). masp-2 activates the complement system by cleaving both c4 and c2 to form the c4bc2a c3 convertase. the alternative pathway (ap) is activated by spontaneous hydrolysis of c3 (c3-h 2 o). this pathway also functions as an amplification loop for the cleavage of c3 initially triggered by other mechanisms. c3-h 2 o or c3b bound to target surfaces are bound by the protease factor b (fb). factor d (fd) is a serine protease that cleaves c3-h 2 o or c3b-bound fb, resulting in the generation of bb and formation of the alternative pathway c3 convertase (c3bbb). both the classical and alternative convertases function to cleave c3 to c3a and c3b. cleavage of c3 exposes a reactive thioester bond in c3b that allows for the covalent attachment of c3b to target surfaces. in addition, c3b can bind to either the classical or alternative c3 convertases resulting in a change of the substrate specificity of the convertases from c3 to c5. these c5 convertases cleave c5 to c5a and c5b. release of c5b promotes assembly of the c5b-c9 membrane attack complex (mac) which can directly lyse pathogens or pathogen-infected cells. c3b is further cleaved by factor i (fi), a function enhanced by factor h (fh), to generate degradation products such as ic3b and c3dg. c3b and its degradation products interact with cellular receptors to regulate effector functions such as phagocytosis and b cell activation. the anaphylatoxins c3a and c5a interact with specific receptors to promote chemotaxis and regulate effector functions of cells of both the innate and adaptive immune response. those findings that describe how these interactions impact the development of virus-induced disease. perhaps the best evidence that complement has an important role in the outcome of virus infection is the identification of specific mechanisms evolved by viruses to evade the complement system (fig. 2 ). one mechanism employed by viruses is to directly encode proteins that have structural and functional homology to host proteins that function as regulators of complement activation. gammaherpesviruses, including kaposi's sarcoma-associated herpesvirus, herpesvirus saimiri, and murine γ-herpesvirus 68 (γhv68) encode homologs of complement regulatory proteins (albrecht and fleckenstein, 1992; mullick et al., 2003a; spiller et al., 2003; virgin et al., 1997) . the γhv68 regulator of complement activation (rca) protein is expressed on the surfaces of infected cells and is detected in supernatants of γhv68infected cells, thus it has both membrane-associated and soluble forms (kapadia et al., 1999) . recombinant γhv68 rca protein and supernatants from γhv68-infected cells blocked c3 deposition on zymosan examples of viral evasion of the complement system. a.) virally encoded proteins allow viruses to evade complement-mediated destruction. human astrovirus (hastv) coat protein (cp) binds mbl and c1q, inhibiting the activation of both the lectin and classical pathways. influenza a matrix (m1) protein also binds c1q. flavivirus nonstructural protein 1 (ns1) binds c4 and c1s, leading to enhanced cleavage of c4 to c4b, as well as factor h (fh), leading to increased cofactor activity of fh for factor i (fi)-mediated cleavage of c3b into ic3b. additionally, membrane-bound flavivirus ns1 decreases deposition of c3b and the mac on cell surfaces. the murine gammaherpesvirus-68 (γhv68) regulator of complement protein (rca) blocks c3 deposition, whereas the hsv-1 glycoprotein (gc) binds c3b and blocks the binding of properdin and c5 to c3b. the variola virus inhibitor of complement enzymes (spice), the vaccinia virus complement control protein (vcp), the monkeypox virus inhibitor of complement enzymes (mopice), and the ectromelia virus inhibitor of complement enzymes (emice) function as cofactors for fi-mediated cleavage of c3b by binding to c3b and c4b. viruses, with their corresponding proteins that interfere with the complement cascade in parentheses, are indicated in red. b.) some viruses recruit host complement regulatory proteins into their virions. human immunodeficiency virus-1 (hiv-1), human t-lymphotropic virus-1 (htlv-1), and human cytomegalovirus (hcmv) incorporate the host complement control proteins cd55/daf and cd59 into their virions, while simian virus 5 (sv5) and mumps virus (muv) recruit cd46/mcp into their virions. physiological complement regulatory proteins are shown in green. viruses that incorporate these regulatory proteins into their virions are indicated in red. beads by both the classical and alternative activation pathways (kapadia et al., 1999) . to directly test the role of the γhv68 rca protein in pathogenesis, kapadia et al. generated a γhv68 deleted for the rca protein and evaluated the outcome of infection in both wildtype and c3-deficient mice (kapadia et al., 2002) . deletion of the γhv68 rca protein resulted in decreased virulence following intracranial inoculation of weanling mice or intraperitoneal inoculation of adult ifn-γ −/− mice, models of acute γhv68-induced meningoencephalitis and γhv68 persistent infection, respectively. genetic deletion of c3 in the host was able to restore virulence of the rca protein-deleted virus in the model of acute meningoencephalitis and enhanced its persistent replication, confirming that rca protein interaction with the host complement system is a critical regulator of γhv68 pathogenesis. interestingly, although wild-type mice had a reduced number of latently infected cells compared to c3 −/− mice, indicating that complement regulates the establishment of γhv68 latent infection, this effect was not counteracted by the γhv68 rca protein (kapadia et al., 2002) . poxviruses, such as variola virus, vaccinia virus, monkeypox virus, and ectromelia virus, also encode complement regulatory proteins that have structural and functional homology to host encoded regulators of the complement pathway (mullick et al., 2003b) ( fig. 2a ). the variola virus inhibitor of complement enzymes (spice), the vaccinia virus complement control protein (vcp), the monkeypox virus inhibitor of complement enzymes (mopice), and the ectromelia virus inhibitor of complement enzymes (emice) all bind to c3b, as well as c4b, and function as cofactors for fi-mediated cleavage of c3b moulton et al., 2010; rosengard et al., 2002; sahu et al., 1998) . a vcp-deleted vaccinia virus produced smaller skin lesions in rabbits compared to wild-type vaccinia virus (isaacs et al., 1992) and pathogenesis studies in cynomolgus monkeys with different strains of monkeypox virus revealed that mopice is deleted from the less virulent strains (chen et al., 2005) . furthermore, spice is a much more potent inhibitor of human complement compared to vcp and mopice, correlating with the increased virulence of variola virus in humans compared to vaccinia virus and monkeypox virus rosengard et al., 2002) . taken together, these studies and the findings that multiple components of the complement pathway are required for mice to survive ectromelia virus infection (discussed below), indicate that complement activation and viral evasion of the complement system are critical determinants of poxvirus pathogenesis. in contrast to herpesviruses and poxviruses, flaviviruses encode a protein that antagonizes the complement system despite the lack of any sequence homology to known regulators of the complement system. the nonstructural protein 1 (ns1) encoded by flaviviruses is a glycosylated protein detected within infected cells, on cell surfaces, and secreted from infected cells (alcon-lepoder et al., 2006) . ns1 accumulates in the serum of dengue virus-infected individuals and high circulating levels are associated with severe disease (avirutnan et al., 2006; libraty et al., 2002) . in an attempt to purify wnv ns1 from cell supernatants, chung et al. observed that fh copurified with ns1 (chung et al., 2006) . soluble wnv ns1 was found to enhance the cofactor activity of fh for fi-mediated cleavage of c3b to ic3b, while cell surface-associated ns1 decreased deposition of c3b and the c5b-c9 membrane attack complex on cell surfaces ( fig. 2a) . flavivirus ns1 also binds to c4 and c1s (avirutnan et al., 2010) ( fig. 2a) . these activities enhanced the cleavage of c4 to c4b and resulted in reduced activity of the classical c3 convertase (c4b2a) and reduced c4b and c3b deposition on cell surfaces (avirutnan et al., 2010) , providing an additional mechanism by which flaviviruses can evade complement-dependent neutralization. soluble ns1 has also been reported to bind the complement inhibitory factor clusterin, which normally inhibits the formation of the c5b-c9 membrane attack complex (kurosu et al., 2007) , however, a functional consequence of this interaction has not been reported. many other viruses employ a similar complement evasion strategy by encoding proteins that bind and inhibit or sequester complement components. for example, the coat protein of human astrovirus type 1 (hastv-1) suppresses complement activation by binding c1q, functionally displacing the protease tetramer, and thus inhibiting classical pathway activation (bonaparte et al., 2008; hair et al., 2010) ( fig. 2a) . this was further demonstrated for serotypes 2 and 4 (bonaparte et al., 2008) . the astrovirus coat protein also bound mbl and inhibited mannan-mediated activation of the lectin pathway (hair et al., 2010) . as discussed below, c1q enhances the neutralizing and hemagglutination inhibition activity of anti-influenza antibodies. experimental evidence suggests that the matrix (m1) protein of influenza a virus has evolved to counteract this host response, as m1 prevents complement-mediated neutralization of influenza virus in vitro by binding c1q and blocking the interaction between c1q and igg (zhang et al., 2009 ). herpes simplex virus 1 (hsv-1) encodes several immune modulators, including glycoprotein c (gc), which inhibits activation of the complement cascade by binding c3 and c3 fragments (friedman et al., 1984; fries et al., 1986; kostavasili et al., 1997; tal-singer et al., 1991) and by blocking binding of properdin and c5 to c3b (fries et al., 1986; hung et al., 1994; kostavasili et al., 1997) . these effects are mediated by two distinct domains in gc: one domain blocks properdin and c5 binding to c3b, and the other directly binds c3 and c3 fragments (hung et al., 1994) . in vitro, gc protects hsv-infected cells from complement-mediated lysis (harris et al., 1990 ) and cell-free virus from complement-mediated neutralization (friedman et al., 1996; hidaka et al., 1991) . it was further demonstrated that natural igm antibody binds and neutralizes hsv-1 and hsv-2 gc-null viruses via a c1q-, c3-, and c5-dependent mechanism (hook et al., 2006) . studies in animal models have confirmed the importance of gc-mediated complement inhibition in hsv-1 pathogenesis. when inoculated intravaginally into wild-type guinea pigs, but not c3-deficient guinea pigs, a gc-null hsv-1 replicated less efficiently and caused significantly less severe vaginitis compared to wild-type hsv-1 (lubinski et al., 1998) . the c3-dependent attenuated phenotype of gc-null hsv-1 was confirmed by inoculating wild-type and c3-deficient mice via skin scratch and evaluating hsv-1-induced zosteriform disease (lubinski et al., 2002 (lubinski et al., , 1998 . further studies with this murine model demonstrated that both domains of gc that modulate complement activation are critical virulence factors; however, hsv-1 specifically lacking the c3 binding domain was more attenuated compared to hsv-1 specifically lacking the c5/ properdin inhibitory domain (lubinski et al., 1999) . importantly, the gc mutant viruses were as virulent as wild-type virus in c3-deficient mice, indicating that the gc interactions with the complement system regulate hsv-1 pathogenesis (lubinski et al., 1999) . most recently, the knowledge gained from these studies was utilized to improve vaccine efficacy against hsv-1 infection. awasthi and colleagues demonstrated that combined immunization with the hsv-1 glycoprotein d (gd), a potent immunogen, and gc prevented hsv-1 evasion from complement, due to the development of an anti-gc antibody response, and enhanced the protection provided by gd immunization (awasthi et al., 2009) . a number of viruses evade the complement system by recruiting host complement regulatory proteins into their virions (fig. 2b ). for instance, human immunodeficiency virus-1 (hiv-1), human t-lymphotropic virus-1 (htlv-1), and human cytomegalovirus (hcmv) incorporate the complement control proteins cd55/daf and cd59 into their virions, thus protecting virions from complement-mediated destruction spear et al., 1995) . simian virus 5 (sv5) and mumps virus (muv), both paramyxoviruses, can be neutralized in a c3-dependent manner resulting in virion aggregation and virion lysis, respectively (johnson et al., 2008) . further studies revealed that both sv5 and muv virions contained cd46/mcp, a membrane-associated protein that has cofactor activity for fi-mediated cleavage of c3b and c4b. the incorporation of cd46 into sv5 and muv conferred virion-associated cofactor activity and increased resistance of sv5 and muv to complement-dependent neutralization ). thus, these viruses have evolved to usurp host complement regulatory proteins to avoid complement-mediated destruction. mannose binding lectin (mbl) is a c-type lectin that plays an important role in innate immunity by binding to carbohydrates on a wide range of pathogens (fujita, 2002) . recognition of carbohydrates is mediated via a c-terminal carbohydrate recognition domain. once bound, mbl can activate complement, due to its association with masps, or act directly as an opsonin. polymorphisms in the promoter and structural regions of the human mbl2 gene affect mbl oligomer formation and circulating levels of the protein (madsen et al., 1995) . due to these different mutations, humans exhibit a 1000-fold variation in circulating mbl levels that occur with varying frequencies in different populations. recently, a number of studies have demonstrated that direct interactions between mbl and virus particles can neutralize infection. mbl has been found to bind directly to virions from a number of different virus families, including human immunodeficiency virus (hiv), severe acute respiratory syndrome coronavirus (sars-cov), ebola virus, dengue virus (denv), and west nile virus (wnv) (fig. 3) . the finding that the hiv envelope glycoprotein, gp120, is modified with high mannose oligosaccharides led researchers to test the potential of hiv and gp120 to function as ligands for mbl. mbl was shown to bind directly to hiv-infected cells and recombinant gp120 (ezekowitz et al., 1989) . the importance of these interactions was demonstrated by experiments showing mbl could inhibit infection of specific target cells by cell culture-derived hiv. although primary isolates of hiv bound to mbl (saifuddin et al., 2000) , mbl binding inefficiently inhibited infection of peripheral blood mononuclear cells (ying et al., 2004) . however, mbl binding did lead to efficient opsonization and uptake of hiv by mononuclear phagocytes (ying et al., 2004) . despite these findings, the role of mbl in hiv pathogenesis is still unclear. different studies investigating the association between mbl levels and hiv infection have found no association, increased susceptibility among individuals with low mbl levels, and increased susceptibility among individuals with high mbl levels (ji et al., 2005a) . however, this area of research has led to an effort to identify lectins that interact with hiv and inhibit infection as a novel therapeutic approach to prevent hiv infection and disease (alexandre et al., 2010; hoorelbeke et al., 2010; huskens et al., 2010) . mbl has also been reported to bind to sars-cov or retroviral particles pseudotyped with the sars-cov spike glycoprotein (sars-s) (ip et al., 2005; zhou et al., 2010) (fig. 3) . similarly, mbl binds efficiently to retroviral particles pseudotyped with ebola virus or marburg virus glycoproteins (ji et al., 2005a) (fig. 3) . importantly, all of these studies showed direct mbl-mediated neutralization of virus infection (ip et al., 2005; ji et al., 2005b) . mbl binding to sars-s was dependent on a single n-linked glycosylation site in sars-s (n330) (zhou et al., 2010) . mbl binding blocked the interaction of sars-s with dc-sign, previously identified to interact with sars-s (yang et al., 2004) , but not with the major sars-cov receptor angiotensinconverting enzyme 2 (ace2) (li et al., 2003) . serum levels of mbl were reported to be significantly lower in patients with sars than in control patients; there was, however, no association between mbl genotypes and mortality related to sars (ip et al., 2005) . a second study also reported that mbl gene polymorphisms associated with reduced mbl levels were significantly associated with susceptibility to sars-cov infection (zhang et al., 2005) . however, a third study reported that mbl genotypes and allele frequencies do not influence the outcome of infection with sars-cov (yuan et al., 2005) . finally, studies showed that mbl binds n-linked glycans on the structural proteins of wnv and denv, resulting in neutralization through a c3-and c4-dependent mechanism that occurred, in part, by blocking viral fusion . these findings indicated that mbl opsonization was not sufficient for neutralization, but rather deposition of c3 or c4 onto virions was also required. for wnv, neutralization occurred only with virus produced in insect cells; for denv, neutralization occurred with insect and mammalian cellderived virus . experiments in mice demonstrated an accelerated intravascular clearance of denv or of a wnv mutant with two n-linked glycans on its e protein, but not wild-type wnv, that was mbl-dependent . the complement system enhances humoral immunity by a number of different mechanisms. complement regulates effector functions of both natural and immune antibodies, complement component c3 and its receptors participate in the capture and transport of antigen to the b cell compartments of secondary lymphoid tissue (gonzalez et al., 2010) , and complement receptor 2 (cr2, cd21) and complement receptor 1 (cr1, cd35) expression by follicular dendritic cells function to retain antigen in the lymphoid follicles, which is required for the generation of a normal humoral immune response (fang et al., 1998) . on b lymphocytes, cr2 forms a complex with other proteins, such as cd19, to activate signal transduction pathways that regulate b cell activation. coligation of the b cell receptor and cr2, which binds the cleavage products ic3b, c3dg, and c3d covalently attached to antigen, lowers the threshold of b cell activation (bradbury et al., 1992; hebell et al., 1991; heyman et al., 1990) . in fact, linking c3d to a model antigen generated a fusion protein that was 100-1000 fold more immunogenic (dempsey et al., 1996) . numerous studies have established a critical role for the complement system in regulating humoral immunity to virus infection. below, we highlight recent findings with a particular emphasis on studies that have investigated these pathways in the context of viral pathogenesis. natural antibody and complement neutralize virus. the humoral immunity of naïve individuals consists primarily of natural igm antibodies (ochsenbein and zinkernagel, 2000) . natural igm is polyreactive and it is thought that endogenous antigens drive the generation of natural igm. igm, which exists in circulation primarily as a pentamer, has a 1000-fold greater binding affinity for c1q compared with igg, making it a potent activator of the complement system (ehrenstein and notley, 2010) . initial studies revealed the presence of natural igm specific for various viral pathogens, including lymphocytic choriomeningitis virus (lcmv), vesicular stomatitis virus (vsv), and vaccinia virus (vv), in the sera of naïve mice. by reconstituting rag-1 −/− mice with sera from naïve mice, researchers demonstrated that natural antibodies protected mice from both vsv dissemination and disease (ochsenbein et al., 1999a) . earlier studies had shown that a natural igm antibody to a vsv antigen forms an immune complex that activates c1 and initiates complement activation via the classical pathway at the viral surface, thus neutralizing vsv (beebe and cooper, 1981) . this neutralization was associated with c3b deposition on the viral envelope that likely interferes with vsv attachment to susceptible cells. natural igm and components of the classical activation pathway have also been shown to neutralize influenza virus (jayasekera et al., 2007) . interestingly, rather than resulting in lysis of virions, these interactions resulted in coating and aggregation of virus particles. to test the significance of these interactions on influenza pathogenesis, rag-1 −/− mice were reconstituted with natural igm and then challenged with the mouse-adapted pr8 strain of influenza. reconstitution prolonged mouse survival following influenza challenge, suggesting that natural igm and complement also protect the host from influenza infection. furthermore, both complement and igm were required for the development of protective immunity against influenza virus after immunization (fernandez gonzalez et al., 2008) . similar to influenza virus, sera from naïve mice neutralizes ectromelia virus by a mechanism dependent on both complement and natural antibodies (moulton et al., 2008) . further analyses revealed that opsonization of ectromelia virus particles with c3b and c4b was the primary mediator of neutralization. consistent with these findings, ectromelia virus dissemination was more efficient and viral loads in tissues were greater in mice deficient in c3. in mortality studies, the genetic deficiency in the complement components c3, c4, or fb resulted in a higher mortality rate compared to wild-type mice. in addition, reconstitution of b cell-deficient μmt mice with sera containing natural antibodies significantly increased survival following inoculation of ectromelia virus. in sum, these studies have revealed the important role of natural antibodies and complement in protection from virus infection. complement-mediated enhancement of b lymphocyte responses protects from virus-induced disease. the study of humoral responses to herpesvirus infections has yielded important insights into complement-mediated regulation of b cell function. mice deficient in c3, c4, or cr2 (which in mice encodes both cr1 and cr2) had significantly diminished igg responses following herpes simplex virus-1 (hsv-1) infection, suggesting that activation of the classical pathway plays a key role in regulating the antibody response to hsv-1 infection (da costa et al., 1999) . interestingly, c3-deficient mice reconstituted with bone marrow from wild-type mice developed normal antibody responses following hsv-1 infection, while wild-type mice reconstituted with bone marrow from c3-deficient mice had significantly diminished igg responses (verschoor et al., 2003 (verschoor et al., , 2001 . these experiments indicated that c3 derived from bone marrow cells was essential to enhance b cell activation in response to hsv-1 infection and were some of the first studies to describe an important function for complement factors synthesized locally by a non-hepatic cellular source. in contrast to hsv-1 infection, igg responses to vsv infection, which is neurotropic in mice and can cause paralysis and death, were similar in c3 −/− mice compared to wild-type mice (ochsenbein et al., 1999b) . instead, researchers discovered that complement components c3 and c4, but not cr2, were critical for initial anti-vsv igm responses. further experiments suggested that complement was critical for targeting vsv antigens to marginal zone macrophages in lymphoid tissues to stimulate b cells and igm production. c3 −/− mice, but not cr2 −/− mice, were more susceptible to lethal vsv infection, suggesting that the depressed igm responses were critical to limit vsv spread and replication in the central nervous system. unlike vsv infection, the genetic absence of c3 or cr2 resulted in increased wnv-induced mortality, earlier wnv entry into the central nervous system, and greater viral loads of wnv in the brains of mice, suggesting complement-mediated regulation of b cell responses was critical to control wnv dissemination, replication, and disease (mehlhop et al., 2005) . in fact, c3 and cr1/cr2 were required for normal anti-wnv igm and igg responses in mice and passive transfer of immune serum protected c3 −/− mice from lethal wnv infection. in further studies, antibody responses to wnv were found to be normal in mice deficient in fb and fd, components of the alternative complement activation pathway (mehlhop and diamond, 2006) . these findings were consistent with previous findings in which igg responses to a t-dependent antigen were normal in fb −/− mice (matsumoto et al., 1997) and indicated that components of the alternative complement activation pathway are not required for the development of an anti-wnv antibody response. in contrast, components of the classical and lectin complement activation pathways, c4 and c1q, were required for normal humoral responses to wnv infection (mehlhop and diamond, 2006) . interestingly, c4, but not c1q, was required for normal anti-wnv igm responses, whereas both c4 and c1q were required for normal anti-wnv igg responses. these findings suggest that distinct complement pathways regulate igm vs. igg responses, at least in the context of a viral infection. importantly, similar to c3 and cr2 deficient mice, c4-and c1qdeficient mice were much more susceptible to lethal wnv-infection, providing further evidence that the host's complement system is critical to limit the severity of wnv disease (mehlhop and diamond, 2006) . c1q regulates anti-viral antibody effector mechanisms. a number of recent studies have identified a critical role for complement, and in particular the complement component c1q, in the regulation of antiviral antibody effector mechanisms. researchers investigating the serum-specific enhancement of influenza virus hemagglutinin (ha)specific monoclonal antibodies discovered that c1q could enhance both neutralization activity and hemagglutination inhibition (hi) activity (feng et al., 2002) . c1q more strongly influenced hi activity, and this enhancement did not require c3, suggesting that the effects were independent of c1q-mediated complement activation. c1qmediated enhancement of hi activity and neutralizing activity was dependent on the antibody isotype and epitope specificity (feng et al., 2002; mozdzanowska et al., 2006) . antibody-dependent enhancement (ade) of infection has been described for multiple viruses . in the case of dengue viruses, ade is associated with more severe disease (balsitis et al., 2010; kyle and harris, 2008; zellweger et al., 2010) . utilizing in vitro assays designed to measure ade of dengue virus infection, researchers discovered that the presence of fresh serum could completely abolish ade (mehlhop et al., 2007; yamanaka et al., 2008) . further analyses demonstrated that the inhibitory effect of fresh serum on ade was c1q-dependent and antibody isotypespecific (mehlhop et al., 2007; yamanaka et al., 2008) . however, in separate studies the suppression of ade by fresh serum was found to be c3-dependent or c3-independent, thus the extent to which other complement components participate in mediating these effects is unclear. in addition to suppressing ade, c1q was shown to enhance the neutralizing activity of anti-wnv antibodies (mehlhop et al., 2009) . mechanistic studies revealed that c1q reduced the number of antibodies required to bind a wnv virion and neutralize infectivity. interestingly, c1q reduced the number of antibodies required for neutralization to levels below the minimum number required for ade. importantly, the ability of an anti-wnv antibody to protect from lethal infection was decreased in c1q −/− mice compared to wild-type mice, indicating that c1q enhancement of neutralizing activity regulates wnv pathogenesis. in contrast to the effect of c1q on ade of flavivirus infection, ade of ebola zaire virus infection is c1qdependent . the glycoprotein of the reston strain of ebola virus, which is less pathogenic in humans, induces less enhancing activity compared to the zaire strain, suggesting that ade may also play a role in ebola virus pathogenesis. regulation of t cells by the complement system. increasing evidence from various experimental systems indicates that complement regulates cd4 + and cd8 + t cell activation and effector functions (dunkelberger and song, 2010; kemper and atkinson, 2007) . numerous complement receptors and membrane complement regulatory proteins can be expressed by antigen-presenting cells (apcs) and t cells. engagement of these receptors on apcs regulates apc effector functions, such as chemokine and cytokine gene expression, and modulation of apc function influences t cell activation during antigen presentation. direct action of complement on t cells is less well understood. ligation of cr1 on t cells has been shown to inhibit t cell proliferation (wagner et al., 2006) , while the c5a receptor (c5ar) has been shown to regulate t cell trafficking (tsuji et al., 2000) . in addition, several membrane complement regulatory proteins have been linked to t cell regulation. for example, cd46, which binds c3b and c4b and serves as a cofactor for their proteolytic inactivation, was found to regulate the proliferation and effector functions of cd4 + and cd8 + t cells (marie et al., 2002) . critically, complement regulates the effector function of activated t cells by regulating the development of th1, th2, and th17 helper cells. for example, c3ar-deficient mice were protected against th2-dependent airway hypereactivity and this has been linked to both decreased th2 cell responses and enhance-ment of il-17 producing helper t cells (drouin et al., 2001; lajoie et al., 2010) . in contrast c5 provides protection against airway hypereactivity by regulating th17 cytokine production (lajoie et al., 2010) . finally, evidence suggests that complement also regulates the termination of t cell responses by inducing the development of regulatory t cells (kemper et al., 2003) . utilizing mouse models, researchers discovered that c3 was required for normal t cell responses to influenza virus, lymphocytic choriomeningitis virus (lcmv) infection, and friend virus (fv) infection (banki et al., 2010; kopf et al., 2002; suresh et al., 2003) . in vitro, dendritic cells exposed to complement-opsonized fv or hiv were better inducers of cd8 + t cell activation (banki et al., 2010) . these effects were also observed with complement opsonized fv in mice. furthermore, c3-deficient mice had reduced numbers of fv-specific cd8 + t cells and higher numbers of infected spleen cells compared to wild-type controls, suggesting complement-mediated regulation of t cell responses functions to control fv replication (banki et al., 2010) . similarly, mice deficient in c3 had delayed clearance of influenza virus and increased titers in the lungs. these findings correlated with a dramatic decrease in the recruitment of virusspecific cd4 + and cd8 + t cells producing ifn-γ to the lung of influenza virus-infected c3 −/− mice. following either lcmv or influenza virus infection, c3 was required for normal priming of cd4 + and cd8 + t cells and mice deficient in cr1 and cr2 (cr2 −/− mice) did not show any of these defects, indicating that c3 regulates t cell responses in a cr1/cr2-independent manner (kopf et al., 2002; suresh et al., 2003) . in lcmv-infected mice, the effects of c3 on cd8 + t cell priming and expansion were epitopedependent and influenced by the mouse genetic background, suggesting that c3 may regulate epitope selection. subsequent experiments indicated that the c5ar may have an important role in the regulation of t cell responses to influenza virus infection as treatment of mice with a c5ar antagonist reduced the number of influenza virus-specific cd8 + t cells in both the lungs and lymphoid tissue (kim et al., 2004) . studies utilizing a mouse model of wnv infection and disease identified distinct complement pathways that regulate antiviral t cell responses (mehlhop and diamond, 2006) . mice deficient in fb were found to be highly susceptible to lethal wnv infection, despite normal anti-wnv antibody responses. instead, fb −/− mice, but not c1q −/− mice, had reduced cd8 + t cell responses in the spleen and impaired trafficking of cd4 + and cd8 + t cells to the cns. similar findings were observed in wnv-inoculated c4 −/− mice, suggesting that both the lectin and alternative complement activation pathways contribute to regulation of t cell functions. thus, similar to fv, influenza virus, and lcmv infections, complement activation was required for normal t cell priming and trafficking following wnv infection. the role of complement regulatory proteins in regulating t cell responses to viral infection has also been investigated. mice deficient in decay accelerating factor (daf; cd55), which regulates formation and inactivation of the c3 and c5 convertases, had an increased expansion of cd8 + t cells with greater killing capacity following lcmv infection and this correlated with lower viral titers. these enhanced cd8 + t cell responses were reversed in mice deficient for daf and c3 (daf-1 −/− ; c3 −/− ) or daf and the c5ar (daf-1 −/− ; c5ar −/− ). in contrast, following vaccinia virus infection, cd4 + , but not cd8 + , t cell responses were enhanced in mice deficient in cd59a, which blocks formation of the membrane attack complex (longhi et al., 2005) . taken together, these studies suggest that membrane complement regulators regulate both cd4 + and cd8 + t cell immunity. however, cd4 + t cell responses following vaccinia virus infection were enhanced in both cd59a −/− and cd59a −/− ; c3 −/− mice, indicating that the effects of cd59a on t cell responses were likely independent of complement activation, at least following vaccinia virus infection. hepatitis c virus (hcv) appears to exploit the complement system to establish persistence. hcv core protein is the first protein expressed during the early phase of hcv infection and free hcv core particles are found in the blood of hcv-infected patients (maillard et al., 2001) . by evaluating various vaccinia virus (vv)/ hcv recombinants in mice, large et al. discovered that a recombinant vv expressing the hcv core protein produced a lethal infection in mice and suppressed the vv-specific cd8 + t cell response (large et al., 1999) . using a yeast two-hybrid approach, kittlesen and colleagues identified that the hcv core protein bound the gc1q receptor (gc1qr) specific for the globular heads of the c1q protein (kittlesen et al., 2000) (fig. 3) . binding of gc1qr by the hcv core protein inhibited human peripheral blood t cell proliferation in standard one-way mixed lymphocyte reactions (mlr) or following mitogen stimulation (kittlesen et al., 2000) , a similar effect to the binding of c1q, the natural ligand for the gc1qr. this suppression was reversed by the addition of anti-gc1qr or anti-core antibodies in the t-cell proliferation assay (kittlesen et al., 2000) . more specifically, binding of hcv core protein to gc1qr on activated human peripheral blood t cells decreased il-2 and ifn-γ production and decreased il-2r and cd69 expression (yao et al., 2001) . a recent clinical study investigated the impact of hcv infection on t cell responses in acute as compared to resolved versus chronic infection (cummings et al., 2009) . during the acute phase of hcv infection, the frequency of gc1qr + cd4 + t cells increased compared to healthy controls. six months later, the frequency of gc1qr + cd4 + t cells remained elevated in chronic patients compared to that in resolved patients. chronic patients also had higher levels of circulating hcv core protein. tcr stimulation increased the frequency of gc1qr + cd4 + t cells, resulting in core-induced inhibition of t cell responses in both resolved and chronic patients. these results suggest that hcv infection expands gc1qr + cd4 + t cells, increasing the susceptibility to core-mediated immune dysregulation (cummings et al., 2009) . the gc1qr is also expressed by other immune cells, such as dendritic cells (dcs) and b cells. dcs isolated from patients chronically infected with hcv display a reduced capacity to induce t cell activation and to produce th1 cytokines (auffermann-gretzinger et al., 2001; bain et al., 2001; dolganiuc et al., 2003; kakumu et al., 2000; kanto et al., 1999 kanto et al., , 2004 . waggoner and colleagues demonstrated that binding of the hcv core protein to gc1qr on human monocytederived dcs (mddcs) inhibited tlr-induced il-12 production but not the production of other tlr-induced cytokines (waggoner et al., 2007) . in addition, hcv core protein engagement of gc1qr on mddcs promoted the production of th2 cytokines such as il-4 by cocultured cd4 + t cells. these results suggest that engagement of gc1qr on dcs by hcv core limits the induction of th1 responses (waggoner et al., 2007) . in contrast to the effects on t cells and dcs, hcv core protein binding to gc1qr on b cells leads to b cell activation and proliferation: increased costimulatory and chemokine receptor expression, cell proliferation, igm and igg production, and stat1 phosphorylation, and down-regulation of socs-1 expression (yao et al., 2008) . these data suggest that hcv exploits the complement system to dysregulate the host immune response via various mechanisms in order to establish persistence. to further investigate the role of the hcv core protein in hcv pathogenesis, transgenic mice were developed with tetracycline-regulated conditional hcv core protein expression (chang et al., 2009) . these mice develop liver inflammation, steatosis, and fibrosis. microarray analyses of inflamed liver showed induction of many components of both the complement and coagulation pathways, and administration of cd55 (daf) decreased hepatic inflammation, suggesting that the hcv core and the complement system contribute to hepatic inflammation associated with hcv infection. cryoglobulinemia is a systemic vasculitis that damages small and medium-sized arteries and veins of the skin, kidneys, and peripheral nerves (dammacco et al., 2001) , and evidence suggests that the deposition of immune complexes on the vessel wall activates complement and mediates this damage. mixed cryoglobulinemia (mc), which involves multiple immunoglobulin isotypes, is observed in 10-70% of hepatitis c virus (hcv)-infected patients and is associated with increased duration of hcv infection (charles and dustin, 2009 ). in hcv-positive patients with mc, circulating, nonenveloped hcv core protein was detected in cryoprecipitable immune complexes (sansonno et al., 2003) . as discussed above, the hcv core protein interacts with the gc1qr. the gc1qr can be proteolytically cleaved and released from the cell surface (kittlesen et al., 2000) . studies with hcv-positive mc patients revealed that mc following hcv infection correlated with significantly higher levels of circulating gc1qr compared to hcv-infected patients without mc (sansonno et al., 2009 ). in addition, higher serum gc1qr levels negatively correlated with circulating levels of the c4d fragment, which was instead sequestered in the vascular bed from skin biopsies of mc patients, indicative of complement activation in the vascular bed (sansonno et al., 2009) . these data suggest that following hcv infection, dysregulated shedding of gc1qr molecules contributes to vascular cryoglobulin-induced damage via a complement-mediated pathway (sansonno et al., 2009 ). hcv has also been linked to pulmonary diseases such as asthma and chronic obstructive pulmonary disease (copd) (moorman et al., 2005a) . in in vitro studies using normal human lung fibroblasts, moorman and colleagues found that the hcv core protein induced il-8 mrna and protein expression via gc1qr (moorman et al., 2005b) . taken together, these studies suggest that the hcv core protein interaction with the gc1q receptor of the complement system contributes to the pathogenesis of multiple diseases associated with hcv infection. further investigation into the proinflammatory role of the hcv core protein may provide a clearer understanding of the pathogenesis of hcv-associated diseases. chronic hcv infection is characterized by persistent complement activation, and can lead to liver fibrosis despite antiviral therapies. by interbreeding fibrosis-susceptible and fibrosis-resistant strains of mice, c5 was identified to be associated with hepatic fibrosis, and small molecule inhibitors of the c5a receptor had antifibrotic effects in vivo (hillebrandt et al., 2002) . polymorphisms of the human gene c5 were associated with advanced liver fibrosis, as compared with mild fibrosis, in chronic hcv infection (hillebrandt et al., 2005) (fig. 3) . moreover, the at-risk c5 haplotype in humans was associated with high serum c5 levels (hillebrandt et al., 2005) . these data suggest that c5 has a causal role in non-hcv and hcv-associated hepatic fibrosis across species and that c5-targeted therapeutics could be a beneficial treatment strategy. although denv ns1 can inhibit activation of the complement system (avirutnan et al., 2010) , evidence suggests that complement activation may contribute to increased disease severity following denv infection (fig. 3) . dengue hemorrhagic fever (dhf) and dengue shock syndrome (dss) are serious clinical conditions that typically occur after a second dengue infection by a different viral serotype or after a primary infection in infants born to dengue-immune mothers. lower levels of c3, c4, and fb, and higher levels of c3 cleavage products, were detected in severely ill dhf patients and correlated with signs of shock, suggesting that complement activation contributes to the more severe forms of dengue virus-induced disease (bokisch et al., 1973; churdboonchart et al., 1983; nascimento et al., 2009 ). in addition, dhf patients have been found to have higher levels of fd, which cleaves fb to yield the active (c3bbb) c3 convertase, and lower levels of fh, which inactivates the (c3bbb) c3 convertase, compared to patients with df (nascimento et al., 2009) . other studies have shown that plasma levels of sns1 and products of complement activation, including those with a known vascular effect such as c3a, c5a, and the terminal sc5b-9 complement complex, were present at higher levels in dhf patients before plasma leakage took place (avirutnan et al., 2006) , further supporting the hypothesis that complement activation is involved in the development of severe disease. comparison of global gene expression profiles in peripheral blood mononuclear cells (pbmcs) of patients with df or dhf discovered that the complement inhibitor cd59 was more strongly up-regulated in pbmcs from df patients than in pbmcs from dhf patients. furthermore, the wild-type mbl genotype, but not mbl genotypes associated with reduced mbl levels, was associated with the development of dengue-related thrombocytopenia and more severe disease (acioli-santos et al., 2008) . this is interesting given the recent findings that mbl binds denv resulting in complement activation and virus neutralization . these studies implicate the complement system in the pathogenesis of the more severe forms of dengue virus associated disease, dhf/dss; however, the mechanisms by which the complement system contributes to disease severity are not well understood. arthritogenic alphaviruses, including ross river virus (rrv) and chikungunya virus, are mosquito-borne viruses that cause debilitating musculoskeletal inflammation and pain in humans. evidence of complement activation was detected in synovial fluid from rrvinfected patients (morrison et al., 2007) . similarly, in a mouse model of rrv-induced disease, complement activation products were detected in inflamed joint and muscle tissues of wild-type mice (morrison et al., 2007) . rrv-infected c3-deficient mice exhibited less severe destruction of skeletal muscle tissue and less severe disease signs, indicating an important role for complement in rrv pathogenesis (fig. 3) . mice deficient in cr3 (cd11b −/− ) also developed less severe tissue damage and disease signs following rrv infection (morrison et al., 2008) (fig. 3) . interestingly, neither c3 nor cr3 deficiency prevented recruitment of inflammatory leukocytes to musculoskeletal tissues, suggesting that c3 and cr3 may act downstream of inflammatory cell invasion to promote tissue damage during rrv infection (morrison et al., 2007 (morrison et al., , 2008 . the genetic absence of c3 and cr3 significantly diminished rrv-induced expression of s100a9, s100a8, and il-6 within inflamed skeletal muscle tissue, indicating that the induction was regulated, at least in part, by cr3 interaction with its c3-derived ligand ic3b (morrison et al., 2008) . furthermore, mice deficient in mbl (mbl-dko) also developed less severe rrv-induced disease and these mice had reduced mbl and c3 deposition on tissues (unpublished results), providing evidence that rrv infection leads to complement activation via the lectin-dependent activation pathway. taken together, these studies suggest that interfering with the complement cascade and/or complement receptor signaling may represent a useful route for therapeutic intervention of rrv-induced disease. finally, recent evidence suggests that the complement system may contribute to seizures induced by virus infection. infection of wildtype mice with theiler's murine encephalomyelitis virus (tmev) results in acute seizures (libbey et al., 2008 (libbey et al., , 2010 . in contrast, c3 −/− mice developed far fewer seizures following tmev infection which correlated with reduced numbers of activated microglia and macrophages in the hippocampus and dentate gyrus as well as decreased neuronal cell loss (libbey et al., 2010) (fig. 3) . interestingly, depletion of systemic complement with cobra venom factor did not impact tmev-induced seizures, suggesting that locally produced complement within the central nervous system is sufficient to enhance tmev-induced seizures (libbey et al., 2010) . it is clear that the complement system is a critical determinant of the outcome of infection by a variety of different viruses. our understanding of the mechanisms by which complement protects from virus-induced disease has improved dramatically. research in this area will not only continue to contribute to our knowledge of viral pathogenesis, but will continue to provide insight into the regulation of immune responses, and lead to improved therapeutic and vaccine approaches for both viral and non-viral pathogens. perhaps less well understood are the mechanisms by which complement functions as a pathogenic effector in some virus-induced diseases. further progress towards identifying the signals and pathways that lead to complement activation, which are not understood for many viruses, particularly in vivo, and a deeper understanding of the impact of complement activation on host immune responses to viral infection may shed light. for example, recent studies have identified critical roles for the complement system in the regulation of th17 cells (lajoie et al., 2010) , regulation of myeloid-derived suppressor cells (markiewski et al., 2008) , and the induction of autophagy (joubert et al., 2009) , all of which have been linked to virus-induced disease. in addition, the complement system is intricately linked with other pathways that play important roles in viral pathogenesis, such as the toll-like receptor signaling network and the coagulation system markiewski et al., 2007) . thus, continued investigation of the role of complement in viral pathogenesis will provide important insights into virus-host interactions and strategies to prevent or treat virus-induced disease. mbl2 gene polymorphisms protect against development of thrombocytopenia associated with severe dengue phenotype new member of the multigene family of complement control proteins in herpesvirus saimiri secretion of flaviviral non-structural protein ns1: from diagnosis to pathogenesis mannose-rich glycosylation patterns on hiv-1 subtype c gp120 and sensitivity to the lectins, 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complement and natural antibody are required in the long-term memory response to influenza virus glycoprotein c of herpes simplex virus 1 acts as a receptor for the c3b complement component on infected cells immune evasion properties of herpes simplex virus type 1 glycoprotein gc glycoprotein c of herpes simplex virus 1 is an inhibitor of the complement cascade direct complement restriction of flavivirus infection requires glycan recognition by mannose-binding lectin evolution of the lectin-complement pathway and its role in innate immunity complement-dependent transport of antigen into b cell follicles complement driven by conformational changes human astrovirus coat protein binds c1q and mbl and inhibits the classical and lectin pathways of complement activation crosstalk pathways between toll-like receptors and the complement system glycoprotein c of herpes simplex virus type 1 prevents complement-mediated cell lysis and virus neutralization suppression of the immune response by a soluble complement receptor of b lymphocytes in vivo inhibition of the antibody response by a complement receptor-specific monoclonal antibody glycoprotein c of herpes simplex virus type 1 is essential for the virus to evade antibody-independent complement-mediated virus inactivation and lysis of virus-infected cells genome-wide analysis of hepatic fibrosis in inbred mice identifies the susceptibility locus hfib1 on chromosome 15 complement factor 5 is a quantitative trait gene that modifies liver fibrogenesis in mice and humans the complement system as a therapeutic target in autoimmunity herpes simplex virus type 1 and 2 glycoprotein c prevents complement-mediated neutralization induced by natural immunoglobulin m antibody actinohivin, a broadly neutralizing prokaryotic lectin, inhibits hiv-1 infection by specifically targeting high-mannosetype glycans on the gp120 envelope generation of c5a in the absence of c3: a new complement activation pathway the interaction of glycoprotein c of herpes simplex virus types 1 and 2 with the alternative complement pathway microvirin, a novel alpha(1, 2)-mannose-specific lectin isolated from microcystis aeruginosa, has anti-hiv-1 activity comparable with that of cyanovirin-n but a much higher safety profile mannose-binding lectin in severe acute respiratory syndrome coronavirus infection vaccinia virus complement-control protein prevents antibody-dependent complement-enhanced neutralization of infectivity and contributes to virulence natural antibody and complement mediate neutralization of influenza virus in the absence of prior immunity mannose binding lectin (mbl) and hiv mannose-binding lectin binds to ebola and marburg envelope glycoproteins, resulting in blocking of virus interaction with dc-sign and complement-mediated virus neutralization differential mechanisms of complementmediated neutralization of the closely related paramyxoviruses simian virus 5 and mumps virus the paramyxoviruses simian virus 5 and mumps virus recruit host cell cd46 to evade complement-mediated neutralization autophagy induction by the pathogen receptor cd46 decreased function of peripheral blood dendritic cells in patients with hepatocellular carcinoma with hepatitis b and c virus infection a dominant complement fixation pathway for pneumococcal polysaccharides initiated by sign-r1 interacting with c1q impaired allostimulatory capacity of peripheral blood dendritic cells recovered from hepatitis c virus-infected individuals reduced numbers and impaired ability of myeloid and plasmacytoid dendritic cells to polarize t helper cells in chronic hepatitis c virus infection murine gammaherpesvirus 68 encodes a functional regulator of complement activation critical role of complement and viral evasion of complement in acute, persistent, and latent gamma-herpesvirus infection t-cell regulation: with complements from innate immunity activation of human cd4 + cells with cd3 and cd46 induces a t-regulatory cell 1 phenotype properdin: emerging roles of a patternrecognition molecule complement c5a receptor is essential for the optimal generation of antiviral cd8 + t cell responses interaction between complement receptor gc1qr and hepatitis c virus core protein inhibits t-lymphocyte proliferation complement component c3 promotes t-cell priming and lung migration to control acute influenza virus infection mechanism of complement inactivation by glycoprotein c of herpes simplex virus secreted complement regulatory protein clusterin interacts with dengue virus nonstructural protein 1 global spread and persistence of dengue complement-mediated regulation of the il-17a axis is a central genetic determinant of the severity of experimental allergic asthma suppression of host immune response by the core protein of hepatitis c virus: possible implications for hepatitis c virus persistence angiotensinconverting enzyme 2 is a functional receptor for the sars coronavirus seizures following picornavirus infection role for complement in the development of seizures following acute viral infection high circulating levels of the dengue virus nonstructural protein ns1 early in dengue illness correlate with the development of dengue hemorrhagic fever structure and regulatory profile of the monkeypox inhibitor of complement: comparison to homologs in vaccinia and variola and evidence for dimer formation cutting edge: murine cd59a modulates antiviral cd4 + t cell activity in a complementindependent manner herpes simplex virus type 1 glycoprotein gc mediates immune evasion in vivo in vivo role of complement-interacting domains of herpes simplex virus type 1 glycoprotein gc herpes simplex virus type 1 evades the effects of antibody and complement in vivo interplay between promoter and structural gene variants control basal serum level of mannan-binding protein nonenveloped nucleocapsids of hepatitis c virus in the serum of infected patients linking innate and acquired immunity: divergent role of cd46 cytoplasmic domains in t cell induced inflammation complement and coagulation: strangers or partners in crime? modulation of the antitumor immune response by complement abrogation of the alternative complement pathway by targeted deletion of murine factor b protective immune responses against west nile virus are primed by distinct complement activation pathways complement activation is required for induction of a protective antibody response against west nile virus infection complement protein c1q inhibits antibody-dependent enhancement of flavivirus infection in an igg subclass-specific manner complement protein c1q reduces the stoichiometric threshold for antibody-mediated neutralization of west nile virus hepatitis c virus and the lung: implications for therapy induction of p38-and gc1qr-dependent il-8 expression in pulmonary fibroblasts by soluble hepatitis c core protein complement contributes to inflammatory tissue destruction in a mouse model of ross river virus-induced disease complement receptor 3 promotes severe ross river virus-induced disease surviving mousepox infection requires the complement system ectromelia virus inhibitor of complement enzymes protects intracellular mature virus and infected cells from mouse complement enhancement of neutralizing activity of influenza virus-specific antibodies by serum components kaposi's sarcomaassociated herpesvirus (human herpesvirus 8) open reading frame 4 protein (kaposica) is a functional homolog of complement control proteins herpes and pox viral complement control proteins: 'the mask of self alternative complement pathway deregulation is correlated with dengue severity natural antibodies and complement link innate and acquired immunity control of early viral and bacterial distribution and disease by natural antibodies protective t cell-independent antiviral antibody responses are dependent on complement complement: a key system for immune surveillance and homeostasis variola virus immune evasion design: expression of a highly efficient inhibitor of human complement interaction of vaccinia virus complement control protein with human complement proteins: factor i-mediated degradation of c3b to ic3b1 inactivates the alternative complement pathway role of virion-associated glycosylphosphatidylinositol-linked proteins cd55 and cd59 in complement resistance of cell line-derived and primary isolates of hiv-1 interaction of mannose-binding lectin with primary isolates of human immunodeficiency virus type 1 non-enveloped hcv core protein as constitutive antigen of cold-precipitable immune complexes in type ii mixed cryoglobulinaemia role of the receptor for the globular domain of c1q protein in the pathogenesis of hepatitis c virus-related cryoglobulin vascular damage host cell-derived complement control proteins cd55 and cd59 are incorporated into the virions of two unrelated enveloped viruses. human t cell leukemia/lymphoma virus type i (htlv-i) and human cytomegalovirus (hcmv) complement regulation by kaposi's sarcoma-associated herpesvirus orf4 protein complement component 3 is required for optimal expansion of cd8 t cells during a systemic viral infection antibody-dependent enhancement of viral infection: molecular mechanisms and in vivo implications antibody-dependent enhancement of ebola virus infection herpes simplex virus glycoprotein c is a receptor for complement component ic3b early local generation of c5a initiates the elicitation of contact sensitivity by leading to early t cell recruitment cutting edge: myeloid complement c3 enhances the humoral response to peripheral viral infection myeloid c3 determines induction of humoral responses to peripheral herpes simplex virus infection complete sequence and genomic analysis of murine gammaherpesvirus 68 hcv core protein interaction with gc1q receptor inhibits th1 differentiation of cd4 + t cells via suppression of dendritic cell il-12 production the complement receptor 1, cr1 (cd35), mediates inhibitory signals in human t-lymphocytes infection-enhancing and -neutralizing activities of mouse monoclonal antibodies against dengue type 2 and 4 viruses are controlled by complement levels ph-dependent entry of severe acute respiratory syndrome coronavirus is mediated by the spike glycoprotein and enhanced by dendritic cell transfer through dc-sign hepatitis c virus core protein inhibits human t lymphocyte responses by a complement-dependent regulatory pathway differential regulation of socs-1 signalling in b and t lymphocytes by hepatitis c virus core protein interaction of mannose-binding lectin with hiv type 1 is sufficient for virus opsonization but not neutralization influence of fcgammariia and mbl polymorphisms on severe acute respiratory syndrome enhanced infection of liver sinusoidal endothelial cells in a mouse model of antibody-induced severe dengue disease association between mannose-binding lectin gene polymorphisms and susceptibility to severe acute respiratory syndrome coronavirus infection influenza a virus m1 blocks the classical complement pathway through interacting with c1qa a single asparagine-linked glycosylation site of the severe acute respiratory syndrome coronavirus spike glycoprotein facilitates inhibition by mannose-binding lectin through multiple mechanisms complement regulators and inhibitory proteins an enormous body of work has contributed to the knowledge of the complement system and viral interactions with the complement system. with that said, we acknowledge the research that was not specifically mentioned in this review. we thank michelle davis for her essential and outstanding contributions to the figures in this review. this work was supported by nih/niaid research grant k22 ai079163 awarded to t.e.m. k.a.s. was supported by the predoctoral nih institutional training grant t32 ai052066. key: cord-017438-x12d2ewc authors: gupta, anita title: mbl deficiency as risk of infection and autoimmunity date: 2012-03-20 journal: animal lectins: form, function and clinical applications doi: 10.1007/978-3-7091-1065-2_42 sha: doc_id: 17438 cord_uid: x12d2ewc in pathogen recognition by c-type lectins, several levels of complexity can be distinguished; these might modulate the immune response in different ways. firstly, the pathogen-associated molecular pattern repertoire expressed at the microbial surface determines the interactions with specific receptors (fig. 42.1). secondly, each immune cell type possesses a specific set of pathogen-recognition receptors. thirdly, changes in the cell-surface distribution of c-type lectins regulate carbohydrate binding by modulating receptor affinity for different ligands. crosstalk between these receptors results in a network of multimolecular complexes, adding a further level of complexity in pathogen recognition (cambi and figdor 2005; thiel et al. 2006) (see 10.1007/978-3-7091-1065-2_23). mbl deficiency is genetically determined and predisposes to recurrent infections and chronic inflammatory diseases. mbl deficiency has been implicated in susceptibility and course of viral, bacterial, fungal, and protozoan infection. more than 10% of the general population may, depending on definition, be classified as mbl deficient, underlining the redundancy of the immune system. mbl-disease association studies have been a fruitful area of research, which implicates a role for mbl in infective, inflammatory and autoimmune disease processes. mbl deficiency predisposes both to infection by extra-cellular pathogens and to autoimmune disease. in pathogen recognition by c-type lectins, several levels of complexity can be distinguished; these might modulate the immune response in different ways. firstly, the pathogenassociated molecular pattern repertoire expressed at the microbial surface determines the interactions with specific receptors (fig. 42 .1). secondly, each immune cell type possesses a specific set of pathogen-recognition receptors. thirdly, changes in the cell-surface distribution of c-type lectins regulate carbohydrate binding by modulating receptor affinity for different ligands. crosstalk between these receptors results in a network of multimolecular complexes, adding a further level of complexity in pathogen recognition (cambi and figdor 2005; jack et al. 2001; thiel et al. 2006 ) (see chap. 23 ). mbl deficiency is genetically determined and predisposes to recurrent infections and chronic inflammatory diseases. mbl deficiency has been implicated in susceptibility and course of viral, bacterial, fungal, and protozoan infection. more than 10% of the general population may, depending on definition, be classified as mbl deficient, underlining the redundancy of the immune system. mbl-disease association studies have been a fruitful area of research, which implicates a role for mbl in infective, inflammatory and autoimmune disease processes. mbl deficiency predisposes both to infection by extra-cellular pathogens and to autoimmune disease. mannose-binding lectin is a c-type serum lectin and is primarily produced by the liver (bouwman et al. 2005) in response to infection, and is part of many other factors termed "http://en. wikipedia.org/wiki/acute_phase_protein" \o "acute phase protein" acute phase proteins. mbl is made up of 96-kda structural units, which in turn are composed of three identical 32-kda primary subunits. the subunits consist of an n-terminal cross-linking region, a collagenlike domain, and a c-terminal carbohydrate-recognition domain (crd) (chap. 23; turner and hamvas 2000) . circulating mbl is composed of higher-order oligomeric structures, which include dimers, trimers, tetramers, pentamers, and hexamers of the structural homotrimeric unit. the oligomeric configuration of the structural units allows the mbl molecule to have multiple crds, facilitating multivalent ligand binding. each crd of mbl is structurally identical and is able to bind a range of oligosaccharides including n-acetylglucosamine d-mannose, nacetylmannosamine, and l-fucose (turner 1996) . although the various sugars are bound with different affinities, the cluster-like array of multiple binding sites allows activation of complement to be most effective. mbl is considered to play a major role in innate defense against pathogens, involving recognition of arrays of mblbinding carbohydrates on microbial surfaces. however, recent studies have shown that mbl is also involved in the recognition of self-targets, such as apoptotic and necrotic cells (nauta et al. 2003) . in plasma, mbl is associated with mbl-associated serine proteases (masps). currently, three masps have been identified, masp-1, masp-2, and masp-3 (chap. 23). mbl belongs to the class of collectins in the c-type lectin superfamily, whose function appears to be pattern recognition in the first line of defense in the pre-immune host. mbl recognizes carbohydrate patterns, found on the surface of a large number of pathogenic micro-organisms, including bacteria, viruses, protozoa and fungi. mannose-binding lectin binds to the repeating sugar arrays on surfaces of pathogens through multiple lectin domains and, following binding, is able to activate the complement system via an associated serum protease, masp-2. importantly, mbl activates the complement system through a distinctive third pathway, independent of g.s. gupta both antibody and the c1 complex (table 42 .1). the mbl binds to neutral carbohydrates on microbial surfaces and recognises carbohydrates such as mannose, glucose, l-fucose, n-acetyl-mannosamine (mannac) , and n-acetyl-glucosamine (glcnac). oligomerisation of mbl enables high avidity binding to repetitive carbohydrate ligands, such as those present on microbial surfaces, including e. coli, klebisella aerogenes, neisseria meningitides, staphylococcus aureus, s. pneumonia, a. fumigatus and c. albicans (c/r kerrigan and brown 2009) . however, there is also a great variation in the binding of mbl to various organisms; candida albicans, b-haemolytic group a streptococci and staphylococcus aureus bind with high affinity, while clostridium sp. pseudomonas aeruginosa, staphylococcus epidermidis, b-haemolytic streptococci and streptococcus pneumoniae exhibit low or no binding (santos et al. 2001) . it is also observed that some organisms (e.g. klebsiella sp. and escherichia coli) show a variable pattern of binding. later, it was shown that the absence of sialic acid from the lipooligosaccharide (los) of neisseria meningitidis serogroup b, serogroup c and neisseria gonorrhoeae permits mbl binding and presence of sialic acid on los results in poor or no mbl binding. in a similar study on salmonella sp, it was found that mbl binds to rough chemotype but exhibits low or no binding with smooth chemotype (ambrosio and de messias-reason 2005) . in order to activate the complement system after mbl binds to its target (for example, mannose on the surface of a bacterium), the masp protein functions to cleave the blood protein c4 into c4a and c4b. the c4b fragments can then bind to the surface of the bacterium, and initiate the formation of a c3 convertase. the subsequent complement cascade, catalyzed by c3 convertase, results in creating a membrane attack complex, which causes the lysis of the pathogen to which mbl is bound (worthley et al. 2005 (worthley et al. , 2006 (fig. 42.1 ). being an important component of innate immunity, acting as an ante-antibody and/or as a disease modifier, mbl is thought to influence disorders as diverse as meningococcal disease, rheumatoid arthritis, cystic fibrosis and recurrent miscarriage. vulvovaginal candidiasis is a yeast infection of vulva and vagina; millions of women suffer from vulvovaginal candidiasis 5 worldwide. women bearing mbl variant allele are at a higher risk for vulvovaginal candidiasis syndrome 343. the cervicovaginal lavage (cvl) mbl levels and gene mutation frequency were both higher in women suffering from vulvovaginal candidiasis than in controls (liu et al. 2006b ). on the other hand, mbl levels were low (0.30 ng/ml) in women with recurrent vulvovaginal candidiasis and were associated with a high gene mutation frequency compared to controls (1.28 ng/ml) (ip and lau 2004) . parenteral administration of mbl increased resistance (pellis et al. 2005) . the autologous function for mbl, perhaps, is to perform a regulatory role within the immune system. the mbl interacts with human peripheral blood cells such as b lymphocytes and natural killer cells. the mbl is capable of binding to differently glycosylated ligands on several autologous cell types via its carbohydrate-recognition domain. it was speculated that this could have functional significance at extravascular sites, but perhaps only in individuals possessing mbl genotypes conferring mbl sufficiency (downing et al. 2003 (downing et al. , 2005 . nevertheless, mbl genotyping of various populations has led to the suggestion that there may be some biological advantage associated with absence of the protein. in addition, the protein also modulates disease severity, at least in part through a complex, dose-dependent influence on cytokine production. moreover, there appears to be a genetic balance in which individuals generally benefit from high levels of the protein. these findings suggest that the concept of mbl as a protein involved solely in first line defense is an over-simplification and the protein should rather be viewed as having a range of activities including disease modulation (turner and hamvas 2000; dommett et al. 2006; worthley et al. 2006) . the mechanisms and signaling pathways involved in such processes remain to be elucidated. though the deficiency of mbl is associated with increased susceptibility to infections, roos et al. (2004) indicated that antibody-mediated classical pathway activation can compensate for impaired target opsonization via mbl pathway in mbl-deficient individuals. lack of mbl may be most relevant in the context of a co-existing secondary immune deficiency. replacement therapy appears promising. the development of a recombinant product should permit the extension of mbl therapy to randomized clinical trials of sufficient size to provide clear evidence about the physiological significance of this intriguing glycoprotein. the mbl has attracted great interest as a potential candidate for passive immunotherapy to prevent infection (gupta et al. 2008 ). the single point mutations in exon 1 of human mbl-2 gene appear to impair the generation of functional oligomers leading to the secondary structural abnormalities of the collagenous triple helix and a failure to form biologically functional higher order oligomers. such deficiencies of the functional protein are common in certain populations, e.g. in sub-saharan africa, but virtually absent in others, e.g. indigenous australians. there is an increased incidence of infections in individuals with such mutations and an association with the autoimmune disorders such as sle and rheumatoid arthritis. thus, the mbl is a potential candidate for passive immunotherapy to prevent infection. 42.2.1.1 polymorphism in mbl gene is associated in exon 1 at codon 52, 54, and 57 the concentration of mbl in plasma is determined genetically, primarily by the genetic polymorphism of the first exon of the structural gene and promoter region. the gene encoding mbl, mbl2 (mbl1 is a pseudogene), is located on long arm chromosome 10q11.2-q21 and contains four exons. a number of single nucleotide polymorphisms (snps) have been characterized in the gene. exon 1 harbours three missense snps, giving rise to amino acid exchanges in the first part of the collagenous region. two of these snps: gly 54 asp, named 'b' and gly 57 glu, named 'c' exchange glycine with an acetic amino acid. the third (arg 52 cys, 'd') introduces a cysteine in the collagen region (the residue numbers includes the leader sequence of 20 residues) ( fig. 42 .2). the wild type is denoted 'a'. heterozygous individuals for d, b, and c mutations have a substantial decrease in mbl serum concentration, whereas mbl is undetectable in the serum of homozygous individuals. three structural mutations within exon 1 at codons 52, 54, and 57 have been invariably referred to as the d, b and c variants. in addition to these three variant structural alleles, the promoter region also shows a number of snps as well, some of which influences the expression of mbl. the three snps in the coding region of the mbl2 gene those are associated with abnormal polymerization of the mbl molecule, decreased serum concentrations of mbl and strongly impaired function. these mbl snps are associated with increased susceptibility to infections, especially in immunecompromised persons, as well as with accelerated progression of chronic diseases. normal serum levels of mbl range from 800 to 1,000 ng/ml in healthy caucasians, however, wide variations can occur due to point mutations in codons 52, 54 and 57 of exon 1 and in the promotor region of the mbl gene (turner 2003) . separate point mutations in the collagenous domain of human mbl are associated with immunedeficiency, caused by reduced complement activation by the variant mbls as well as by lower serum mbl concentrations. mbl deficiency with b mutation is associated with 26% of caucasian populations (steffensen et al. 2000) . in a cohort of 236 australian blood donors, 5 mbl promoter and coding snps were genotyped. significant associations were found between both coding and promoter polymorphisms and mbl antigenic and functional levels (minchinton et al. 2002) . point mutations in exon 1 at codons 52, 54 and 57 and a promoter polymorphism at à221 bp of mbl gene were associated with increased susceptibility to various infectious diseases (steffensen et al. 2003; roos et al. 2006) . the codon 54 mutation was frequent in both a british caucasian and a hong kong chinese population. the replacement of glycine-54 with an aspartic acid residue disrupts the fifth gly-xaa-yaa repeat in the collagen-like domain of each 32 kda mbp peptide chain and prevents the formation of the normal triple helix (sumiya et al. 1991) . super et al. (1992) suggested that this genotype occurs in 5% of the population and encodes a functional protein. super et al. (1992) also suggested that the gly 54 asp allele does not account for a deficiency state, but instead suggested that mbp may have two predominant allelic forms that have overlapping function and differ only in their ability to activate the classical pathway of complement. of 123 healthy danish individuals investigated, 93 were homozygous (743.6%) for ggc, 28 heterozygous (22.8%), and 2 homozygous for gac (1.6%). the gene frequency of the gac allele was found to be 0.13. dna sequencing of the cloned exon 1 from one gac homozygous individual revealed no other substitution. the median mbp concentration in the group containing the gac allele was 43.4 times lower than in the ggc homozygous group. however, the range of mbp in plasma was wide and overlapping between the groups. mbp protein was detected in both the gac homozygotes. this study suggested that the gac allele is able to produce a functional mbp protein which may be detected in serum at low concentrations (garred et al. 1992 ). the point mutation (gga to gaa) involving codon 57 of exon 1 has been reported in gambians from west africa. in the gambians the codon 57 mutation was remarkably common whereas the codon 54 mutation was very rare. in contrast, the codon 54 mutation was frequent in both the british caucasian and the hong kong chinese population. it was predicted that both homozygous and heterozygous individuals would have profoundly reduced serum levels of the protein and this was confirmed by immunoassay complement activation through lectin pathway. these two mutations have arisen independently because of the divergence of african and non-african populations. codon 52 polymorphism increases risk of premature delivery: mbl2 gene polymorphisms are associated with an increased risk of neonatal infections. a relation between the maternal mbl2 genotype and the risk of premature delivery has been indicated. bodamer et al. (2006) suggested that the frequency of the codon 52 polymorphism was higher in the pre-term group compared to the term group (10.8% vs. 4.9%), while the frequency of codon 54 polymorphism was equal in both groups (11.3% vs. 11.8%). data suggest that the fetal mbl2 genotype might be an additional genetic factor contributing to the risk of premature delivery. in the 5 0 -untranslated region (position þ4, p/q variant) of the mbl2 gene. three base substitutions in exon 1 in codons 52 (d), 54 (b) and 57 (c) result in amino-acid changes (arginine to cysteine, glycine to aspartic acid and glycine to glutamic acid, respectively) and decreased level and function of mbl. the normal allele is named a proteases (reprinted by permission from macmillan publishers ltd: genes immun. garred et al. # 2006) inflammatory response syndrome are associated with increased prevalence of positive bacterial cultures and sepsis but not with altered prevalence of septic shock or decreased 28day survival. furthermore, cd14 snps were associated with gram-negative bacteria and toll-like receptor-2 with grampositive bacteria, whereas mbl was not associated with a particular organism class. the prevalence of the codon 54 mutation of the mbl gene in patients having repeated respiratory infections as well as the prevalence of the mbl mutant genotype among patients with diffuse panbronchiolitis was further supported (gomi et al. 2004 ). mellitus: insulin resistance is a feature of gestational diabetes mellitus (gdm). a genetic predisposition to a proinflammatory state could favor the appearance of gdm during pregnancy. an association has been found between g54d and gdm. gdm patients carrying the g54d mutation require insulin therapy more frequently and have heavier infants than gdm women homozygous for the wild-type allele. an inverse correlation in gdm patients between neonatal weight and plasma mbl levels has been reported. thus, pregnant women bearing the g54d mbl allele have a greater risk for developing gdm and having heavier infants (megia et al. 2004) . mbl genotypes in acute lymphoblastic leukemia: epidemiological studies show that acute lymphoblastic leukemia (all) can be induced by interactions between the immune system and early childhood infections. since, certain types of childhood acute lymphoblastic leukemia develop as a multiple step process involving both pre-and postnatal genetic events, the mbl may play a critical role in the immune response in early childhood before specific immune protection develops. schmiegelow et al. (2002) indicated that low-level mbl genotypes is associated with an increased risk of childhood all, particularly with early age at onset. childhood all may often be initiated in utero. the prenatal origin of childhood leukemia was ascertened in children with b-precursor acute lymphoblastic leukemia carrying the chromosomal translocation t(12;21), the most common subtype of all childhood all. study provided evidence that the development of t(12;21) bprecursor all may be initiated in utero. however, age at leukemia may be inversely correlated with the burden of cells with leukemia clonal markers, i.e. leukemia predisposed cells at birth (hjalgrim et al. 2002) . mbl genotypes in viral hepatitis: the prevalence of mutations in mbl gene was assessed in patients of hepatitis b. a mutation in codon 52 of the mbp gene was present in two (11%) of 19 caucasian patients with acute hepatitis b and nine (27%) of 33 caucasian patients with chronic hepatitis b, compared with four (4%) of 98 caucasian controls. by contrast the prevalence of the mutation was similar in asian patients with chronic hepatitis b and in asian controls (one [5%] of 20 vs. two [2%] of 117). mutations in codon 54 and codon 57 were found in similar proportions of patients and controls. these findings showed in caucasian, but not asian, patients an association of the codon 52 mutation of the mbl gene with persistent hepatitis b virus (hbv) infection. they suggest an important role for this gene, or a gene in linkage disequilibrium with mbl, in determining outcome after hbv infection in adult but not neonatal life (thomas et al. 1996) . bellamy et al. (1998) investigated the association between variant mbp alleles and malaria, tuberculosis, and hbv in adults and children in gambia. of the 2,041 gambians screened for mbp mutations, 944 (46%) were homozygous for the wild-type allele, 922 (45%) were carriers of a single variant allele, and 175 (8.6%) possessed 2 mutant alleles. the most common mutation in africansthe codon 57 variant allele -was weakly associated with resistance to tuberculosis in both patients and controls. although mbp deficiency may predispose to recurrent infections, this study failed to provide evidence that such a deficiency is a major risk factor for infectious diseases (bellamy et al. 1998) . the hepatitis c virus (hcv) envelope glycoprotein e2 binds the dc-sign and the related liver endothelial cell lectin through high-mannose n-glycans. several highmannose n-glycans in a structurally defined cluster on e2 bind to several subunits of the oligomeric lectin crd and represent a strategy by which hcv targets to and concentrates in the liver and infects dendritic cells (lozach et al. 2003) . to determine the relevance of mbl polymorphism to hepatitis c virus infection, matsushita et al. (1998) determined the mbl genotypes in 159 hepatitis c virusinfected chronic hepatitis patients and 218 healthy controls in japan by looking at 4 polymorphic loci: 2 (h/l and x/y) within the promoter region and 2 (p/q and a/b) within exon-1 of the mbl gene. it indicated that the mbl-related innate immune system plays an important role in elimination of hepatitis c virus during interferon therapy (matsushita et al. 1998 ). matsushita et al. (2001) demonstrated that the mbl genotype can be significantly associated with increased risk for to primary biliary cirrhosis (pbc), and that increased production of mbl plays a critical role in immunopathogenesis. masp-2 auto-activation in mutated mbl the mutations gly25!asp and gly 28 !glu disrupt the disulfide-bonding arrangement of the protein and cause at least a fivefold increase in the half-time of secretion of mbp compared with wild-type rat serum mbp. a similar phenotype, including a threefold increase in the half-time of secretion, disruption of the disulfide bonding arrangement, and inefficient complement fixation, was observed when nearby glucosylgalactosyl hydroxylysine residues at positions 27 and 30 were replaced with arginine residues. the results suggest that defective secretion resulting from structural changes in the collagen-like domain is likely to be a contributory factor for mbp immunodeficiency (heise et al. 2000) . to investigate the molecular defects associated with heterozygosity, rat serum mbp polypeptides (mbp-a: 56% identical in sequence to human mbp) and rat mbp polypeptides containing mutations associated with human immunodeficiency were co-expressed in mammalian expression system. the resulting proteins are secreted almost exclusively as heterooligomers that were defective in activating the complement cascade. functional defects were caused by structural changes to the n-terminal collagenous and cysteine-rich domains of mbp, disrupting interactions with associated serine proteases. these mutations demonstrated how a snp gives rise to the molecular defects that lead to the disease phenotype in heterozygous individuals (wallis 2002) . wallis et al. (2005) analyzed the molecular and functional defects associated with two variant proteins of lectin pathway. mutations gly 25 !asp and gly 28 !glu created comparable structural changes in rat mbl but the g28e variant activated complement >10-fold less efficiently than the g25d variant, which in turn had approximately sevenfold lower activity than wild-type mbl. analysis of mutant mbl-masp-2 complexes formed from recombinant proteins showed that reduced complement activation by both mutant mbls was due to failure of activation of masp-2 efficiently on binding to a mannancoated surface. disruption of mbl-masp-2 interactions as well as to changes in oligomeric structure and reduced binding to carbohydrate ligands compared with wild-type mbl probably account for the intermediate phenotype of the g25d variant. however, carbohydrate binding and -masp-2 activation are ostensibly completely decoupled in complexes assembled from the g28e mutant, such that the rate of masp-2 activation is no greater than the basal rate of zymogen masp-2 autoactivation. analogous molecular defects in human mbl probably combine to create the mutant phenotypes of immunodeficient individuals (wallis et al. 2005) . since it is difficult to evaluate mbl in patients blood on the only basis of protein contents, or in combination with mbl genotyping due to possible association of altered oligomeric state of mbl, dumestre-perard et al. (2002) purified mbl from human plasma and showed the presence of mbl in two different oligomeric forms. data on the specific activity of these forms showed that the higher oligomeric forms of mbl had the ability to induce c4 cleavage more efficiently than the corresponding lower oligomers (dumestre-perard et al. 2002) . how to define abnormal mbl pathway and disease associations: although, the association between mbl deficiency and risk of infection with other common diseases and death during years of follow-up has established the role of mbl in innate immune system, yet, in a large ethnically homogeneous caucasian population, there was no evidence for significant differences in infectious disease or mortality in mbl-deficient individuals versus controls and suggested that mbl deficiency is not a major risk factor for morbidity or death in the adult caucasian population (dahl et al. 2004; eisen and minchinton 2003; kilpatrick 2002; summerfield et al. 1997) . while addressing possible correlation between mbl levels and clinical conditions an issue is how to define mbl deficiency. the physiologically relevant mbl level leading to clinical manifestations is likely to differ in different diseases. in the examples given below, a number of different levels have been used as cut-off values defining mbl deficiency. judged from clinical trials it appears that at least 200 ng mbl/ml plasma is needed for reconstituting in vitro functional activity (c4b deposition) after mbl infusion in mbl deficient individuals (valdimarsson et al. 2004 ). on the other hand, in leukaemic patients a cut-off level of 500 ng/ml or more has been suggested (peterslund et al. 2001; neth et al. 2001) , and in the cases of obstetric problems even lower levels (100 ng/ml) have been indicated. a broad range of proteins binds high-mannose carbohydrates found on the surface of the envelope protein gp120 of the hiv and thus interfere with the viral life cycle, providing a new method of controlling hiv infection. while glycosylation of hiv gp120/gp41 provides a formidable barrier for development of strong antibody responses to the virus, it also provides a potential site of attack by the innate immune system through mbl/mbp. the mbl that binds to hiv depends on the high-mannose glycans present on gp120 while host cell glycans incorporated into virions do not contribute substantially to this interaction. the mbl has been shown to interact with all tested hiv strains. however, drugs that alter processing of carbohydrates enhance neutralization of hiv primary isolates by mbl. complement activation on gp120 and opsonization of hiv due to mbl binding have also been observed but these immune mechanisms have not been studied in detail. mbl has also been shown to block the interaction between hiv and dc-sign. clinical studies show that levels of mbl, an acute-phase protein, increase during hiv disease. because of apparently universal reactivity with hiv strains, mbl clearly represents an important mechanism for recognition of hiv by the immune system. however, further studies are needed to define the in vivo contribution of mbl to clearance and destruction of hiv (botos and wlodawer 2005; ji et al. 2005) . mbl that binds to high mannose glycans on hiv-1 (gp120), has been shown to neutralize the cell lineadapted strain hiv(iiib). but hiv primary isolates (pi) are generally more resistant to neutralization by antibodies. considering that pi are produced in primary cells that could alter the number of high mannose glycans on hiv relative to cell lines, ying et al. (2004) showed that both pi and cell line-adapted hiv, despite binding of mbl, are relatively resistant to neutralization by levels of mbl normally present in serum. however, binding and opsonization of hiv by mbl may alter virus trafficking and viral-antigen presentation during hiv infection (ying et al. 2004) . in search of the effect of mbl-2 polymorphisms on susceptibility and progression of hiv-1 infection in children, dzwonek et al. (2006) observed mbl deficiency more frequently in patients with severe disease. the study suggested that mbl-2 variants may be less frequent in children classified as long-term non-progressors (ltnps) and hence mbl analysis can be useful in identifying children with slow disease progression and, consequently, may not require immediate antiretroviral treatment (dzwonek et al. 2006 ). production of hiv in the presence of the mannosidase i inhibitor deoxymannojirimycin (dmm) significantly enhanced binding of hiv to mbl and increased mbl neutralization of an m-tropic hiv primary isolate. in contrast, hiv cultured in presence of alphaglucosidase i and ii inhibitors, castanospermine and deoxynojirimycin showed only slight effect on virus binding and neutralization by mbl. the study suggested that specific alterations of the n-linked carbohydrates on hiv gp120/gp41 can enhance mbl-mediated neutralization of virus by strengthening the interaction of hiv-1 with mbl (hart et al. 2003 ). respiratory syncytial virus (rsv) is the most important microbiological cause of lower respiratory tract infection (lrti) in infants. mbl deficiency is the most common immunodeficiency on the african continent. mbl deficiency has an impact on the hospitalization for lrti caused by rsv in infants from soweto, south africa. but in contrary to expectations, results suggested no association between low levels of mbl or carriage of variant alleles and lrti caused by rsv (kristensen et al. 2004 ). little is known about the innate immune response to severe acute respiratory syndrome (sars) corona virus (cov) infection. the mbl plays an important role in sars-cov infection. the distribution of mbl gene polymorphisms was significantly different between patients with sars and normal subjects, with a higher frequency of haplotypes associated with low or deficient serum levels of mbl in patients with sars. there was, however, no association between mbl genotypes, which are associated with low or deficient serum levels of mbl, and mortality related to sars. mbl could bind sars-cov in calcium-dependent and mannaninhibitable fashion in vitro, suggesting that binding is through the carbohydrate recognition domains of mbl. furthermore, deposition of complement c4 on sars-cov was enhanced by mbl. inhibition of the infectivity of sars-cov by mbl in fetal rhesus kidney cells suggested that mbl contributes to the first-line host defense against sars-cov and that mbl deficiency is a susceptibility factor for acquisition of sars (ip et al. 2005 ). susceptibility to t-cell lymphotropic virus: pontes et al. (2005) investigated the association between mbl gene polymorphism and the susceptibility to human t-cell lymphotropic virus (htlv) infection in 83 htlv-infected asymptomatic subjects. detection of mbl*a, mbl*b, and mbl*c was performed by amplifying a fragment of 349 bp (exon 1) and restriction fragment length polymorphism analysis with bani and mboii endonucleases. a strong association has been demonstrated between mbl polymorphism and htlv infection. presence of genotype bb may be associated with the susceptibility to htlv. though further studies, with a larger number of individuals, are necessary, mbl polymorphism could have a possible impact on diseases associated with htlv infection (pontes et al. 2005 ). studies demonstrating the binding of mbl to the endothelium and causing excessive complement activation and subsequent tissue damage are known (jordan et al. 2003) , where as mbl deficiency may be advantageous in some circumstances since mbl may lead to an increased cytokine secretion by macrophages (takahashi et al. 2002) . there seems to be a delicate balance as to when mbl levels may be involved in harmful or in beneficial inflammation such as in the cardiovascular and other systems. the etiology of autoimmune diseases is largely unknown. studies on associations between mbl deficiency and rheumatoid arthritis (ra) have been discussed in reviews (graudal 2004 ; barton et al. 2004) . results depend on ethnic groups, type of patients and the symptoms studied. low levels of serum mbl are associated with a higher erythrocyte sedimentation rate (esr), joint swelling score, limitation of joint motion score, and annual increase in radiographic destruction score. despite this, indications are that low mbl levels may be linked with symptoms indicating a poor prognosis as well as an earlier debut. whether variant alleles of the mbl gene causing low serum concentrations of mbl are associated with increased susceptibility to ra and erosive outcome in an inception cohort of patients with early polyarthritis was studied by jacobsen et al. (2001) . jacobsen et al. (2001) suggested that mbl variant alleles appear to be weak susceptibility markers for ra, and patients with early polyarthritis and homozygous for mbl structural variant alleles have a higher risk of developing early erosive ra. these findings, together with the positive association between mbl variant alleles and the increased serum levels of igm rf and crp, point at the mbl gene as a relevant locus in the pathophysiology of ra. graudal (2004) indicated that mbl-deficient patients have a relative risk of a severe radiographic event of 3.1 compared with the mbl competent group. the relative risk (rr) of early il-1a auto-antibodies positive patients developing serious radiographic joint destruction was significantly lower than for il-1a auto-antibodiesnegative patients. perhaps mbl and il-1a auto-antibodies are predictors of prognosis of ra and play important roles in the pathogenesis of ra (graudal 2004; lee et al. 2005) . low levels of the protein have been related to a poor prognosis in rheumatoid arthritis perhaps due to the modulatory action that mbl exerts on the secretion of tumor necrosis factor a, a central molecule in the pathogenesis of rheumatoid arthritis (graudal et al. 2000) . low mbl levels have also been associated with adult dermatomyositis and are probably related to a reduced clearance of apoptotic keratinocytes (werth et al. 2002) . genotypes related to a lower production of mbl have also been linked to the development of systemic lupus erythematosus (villarreal et al. 2001; davies et al. 1995) . genetic factors play a major role in the development of sle. more than 5% of cases are familial and the concordance rate between identical twins is 40%. genetic studies in mice suggest a complex mechanism of transmission involving interactions among several susceptibility genes and, probably, protective genes. genetic studies in humans have identified nearly 50 chromosomal areas possibly involved in lupus transmission. significant linkage has been found for at least six regions, two on chromosome 1, one near the hla region on chromosome 6, and three on chromosomes 2, 4, and 16, respectively. the genetic polymorphism of cytokines and, perhaps, of the t-cell receptor (tcr) may contribute to deregulate lymphocyte activity. the polymorphism of the fc receptors of immunoglobulins may affect immune complex clearance, thereby promoting tissue damage. further genetic studies are needed to enrich the fund of knowledge on lupus and to identify new targets for treatment (perdriger et al. 2003) . from the several investigations on mbl and sle the consensus is emerging that low levels of mbl predisposes to development of the disease. however, the connection is not like for c1q where sle develops in almost all of the rare cases of deficiency. rather, it seems that mbl deficiency aggravates the disease or the development (ohlenschlaeger et al. 2004; takahashi et al. 2005) . in sle patients, mbl deficiency increase the risk for respiratory tract infections (garred et al. 2001; takahashi et al. 2005 ) as well as the risk of developing arterial thromboses (ohlenschlaeger et al. 2004) . as with infectious disease, there is some evidence that the risk of pathology increases if there is another co-existing immune defect. for example, in a cohort of spanish patients, the odds ratio for developing sle was 2.4 for individuals with mbl deficiency, but this increased to 3.2 when there was also a co-existing partial c4 deficiency (davies et al. 1997) . studies in patients with sle have reported that mbl deficiency also influences their risk of developing certain complications, which include arterial thromboses (ohlenschlaeger et al. 2004 ) and respiratory tract infections (garred et al. 2001; takahashi et al. 2005 ). whether dysfunctional or deficient mbp variants are found with increased frequency in black patients with sle was determined (sullivan et al. 1996) . two structural polymorphisms of mbp, associated with low serum levels of mbp, were found with significantly increased frequency in the sle patient population. in contrast, a promoter haplotype associated with particularly high serum levels of mbp was negatively associated with sle. thus, it seemed that deficiencies of mbp predispose individuals to sle (sullivan et al. 1996) . the distribution of promoter variants of the mbl gene and correlations between the promoter variants and serum mbl concentrations in chinese patients with sle were investigated. significant differences in the distribution of the two pairs of promoter polymorphisms, h/l and y/x, between sle patients and controls, were observed. analysis of the correlation between promoter haplotypes and serum mbl levels revealed hy as the highest-producing, ly as the intermediate-producing, and lx as the lowest-producing haplotypes. the lx haplotype was present at a frequency of 0.259 in sle patients and 0.154 in controls and was significantly associated with sle. the lowproducing promoter polymorphism of the mbl gene is associated with sle, and a low serum mbl level is a risk factor for sle (ip et al. 1998) . whether occurrence, characteristics, and progression of sle are associated with polymorphism of the mbl gene and with serum mbl concentration, takahashi et al. (2005) reported that mbl gene polymorphism influences susceptibility to sle, but has no direct effect on disease characteristics. serum mbl levels fluctuate during the course of sle in individual patients. mbl genotyping may be useful in assessing the risk of infection during treatment of sle . mbl and fcgrii (cd32) polymorphisms have both been implicated as candidate susceptibility genes in sle. these patients carried mbl codon 54 mutant allele more frequently than controls and the haplotype hy w52 w54 w57 was significantly lower in cases compared with controls. the mbl gene codon 54 mutant allele appears to be a risk factor for sle, whilst haplotypes encoding for high levels of mbl are protective against the disease. however, differences between controls and patients were not significant when fcgriia polymorphisms were considered (villarreal et al. 2001 ). nephritis in sle, hypocomplementaemia and complement deposition have been described both in man and in experimental models. in mice, mbl is expressed in two forms, mbl-a and mbl-c. in young and old mrl-lpr and control mrl+/+, the declining levels of mbl-a and mbl-c showed a high degree of correlation. in aged mrl-lpr mice in which autoimmunity is most pronounced, high auto-ab titers and strong deposition of glomerular immune complexes were associated with deposition of c1q, c3, mbl-a and mbl-c. thus, in addition to the classical pathway and the alternative pathway, the lectin pathway of complement activation is also involved in murine lupus nephritis . sle patients were associated with a reduced functional activity of the mbl pathway of complement, in relation to expression of mbl variant alleles, increased levels of autoantibodies against cardiolipin and c1q, but not against mbl. the enhanced production of autoantibodies may be related to disturbed clearance of apoptotic material due to impaired mbl function . cohorts of mrl-lpr mice, which are known to develop age-dependent sle-like disease showed that at 2 months of age all mice already had elevated levels of anti-c1q autoantibodies, and elution of kidneys confirmed the presence of these antibodies in renal immune deposits in mrl-lpr mice and not in control mrl+/+ mice. thus, anti-c1q antibodies are already present in serum and immune deposits of the kidney early in life and therefore can play a role in nephritis during experimental sle-like disease in mice (trouw et al. 2004 ). effect of (s)-armepavine on autoimmune disease of mrl/mpj-lpr/lpr mice: (s)-armepavine (c19h23o3n; mw313) from nelumbo nucifera suppresses t cells proliferation. (s)-armepavine prevents lymphadenopathy and elongated life span of mrl/mpj-lpr/lpr mice, which have disease features similar to human sle. the action seemed to be mediated by inhibition of splenocytes proliferation, suppression of il-2, il-4, il-10, and ifn-g gene expressions, reduction of glomerular hypercellularity and immune complexes deposition, and decrease of urinary protein and anti-double stranded dna autoantibody production. it has been suggested that (s)-armepavine is an immunomodulator for the management of autoimmune diseases like sle (liu et al. 2006a ). in sle patients, there is an association between the occurrence of autoantibodies to c1q and mbl and renal involvement. the presence of autoantibodies to mbl, analogous to autoantibodies to c1q in patients with sle, contributes to the disease development. anti-mbl autoantibodies were of the igg isotype and the binding site of igg anti-mbl was located in the f(ab')2 portion. anti-mbl are present in sera from sle patients and influence the functional activity of mbl (seelen et al. 2003) . more sle patients have igg anti-mbl antibodies than normal controls. however, in sle, these antibodies are neither sensitive nor specific for this condition. they occur more frequently in (proliferative) lupus nephritis, particularly during active disease. furthermore, levels of anti-c1q rise, in many cases, prior to a relapse of lupus nephritis, suggesting a pathogenic role for the autoantibodies in immune complex-mediated renal disease. in addition, anti-c1q may interfere with the clearance of apoptotic cells, so influencing induction and expression of autoimmunity (kallenberg 2008; mok et al. 2004) . cardiovascular disease and sle: cardiovascular disease is an important complication in patients with sle. variant alleles of the mbl gene are associated with sle as well as with severe atherosclerosis. ohlenschlaeger et al. (2004) determined whether mbl variant alleles were associated with an increased risk of arterial thrombosis among danish patients with sle and suggested that among patients with sle, homozygosity for mbl variant alleles is associated with an increased risk of arterial thrombosis. the risk of venous thrombosis is not increased, indicating that mbl has a specific role in providing protection against arterial thrombosis (ohlenschlaeger et al. 2004 ). a systemic inflammatory response syndrome (sirs) is well known in patients after major surgery (bone 1992) . clinical studies of critically ill patients requiring intensive care management have shown that individuals who are mbl deficient are more likely to develop the sirs and progress to septic shock and death (garred et al. 2003b; fidler et al. 2004) , findings which may well relate to the proinflammatory cytokine response. in some cases, sirs occurs in response to infection and "sepsis" is then used to describe the symptoms. more severely a septic shock may develop with multi-organ dysfunctions (mod). the mbl levels and genotypes were investigated in 272 adults (197 with sepsis) prospectively admitted to the icu. no difference was seen between genotype frequencies in patients with sirs as compared to healthy controls. but the frequency of mbl variant genotypes was significantly higher in patients with sepsis compared with the patients without sepsis, and the risk ratios for the development of "severe sepsis" and "septic shock" ranged from 1.3 to 3.2 times higher in patients with a/o or o/o versus a/a genotype. mbl levels were inversely related to the severity of sepsis (garred et al. 2003a, b) . the snp of mbl with clinical phenotype in critically ill caucasians with sirs are associated with increased prevalence of positive bacterial cultures at admission to the icu. patients in low mbl haplotype group did not have significantly increased rates of sepsis or septic shock at admission to the icu. survival at day 28 did not differ significantly between the low mbl haplotype and high mbl haplotype groups. furthermore, mbl was not associated with a particular organism class. the prevalence of the codon 54 mutation of mbl gene in patients having repeated respiratory infections as well as the prevalence of mbl mutant genotype among patients with diffuse panbronchiolitis was further supported (gomi et al. 2004 ). in contrast, the prevalence of the mbl mutant genotype among patients with nontuberculous mycobacteria or aspergillus chronic infection was not different from that in control subjects. thus, snps in innate immunity receptors may alter recognition and clearance of bacteria without changing outcomes of critically ill adults with systemic inflammatory response syndrome (sutherland et al. 2005) . polymorphisms of both codon 54 allele and promoter variants of the mannose mbl gene in patients with primary sjogren's syndrome (ss) was suggested as one of the genetic factors that determines susceptibility to ss (wang et al. 2001) . fidler et al. (2004) analyzed the mbl levels and genotypes levels in 100 critically ill children admitted to icu. a sevenfold greater risk of developing sirs within 48 h of admission (60% of the patients) was observed for those carrying mbl variant alleles than those with wild type alleles (a/o + o/o vs. a/a). a significant relation was also found between severity of the systemic response to infection and the presence of an mbl mutation. if the severity of illness among the patients admitted with infections was divided into localized infection, sepsis, and septic shock, the median mbl levels were inversely related to severity, and the children with mbl levels below 1,000 ng/ml had a greater chance of developing sirs. a study of the frequency of sepsis in very low birth weight infants did not reveal statistical significance in clinical data between infants with and without specific mutations in a number of genes, including mbl genotypes (ahrens et al. 2004 ). following surgery of 156 patients undergoing major elective gastrointestinal surgery for malignant disease, siassi et al. (2003) reported that patients who developed sepsis or sirs showed significantly lower mean post-operative mbl levels. in colorectal cancer patients, postoperative infection is associated with poor prognosis. ytting et al. (2005) have reported significantly increased frequency of pneumonia after primary operation in colorectal carcinoma patients who were having low mbl levels. mbl deficiency appears to play an important role in susceptibility of critical ill patients to the development and progression of sepsis and septic shock, and confers a substantially increased risk of fatal outcome. there is clearly a need for improvement in defining which patient groups and which clinical data are relevant to examine. inflammatory bowel disease (ibd) is a pathological spectrum encompassing ulcerative colitis (uc), crohn's disease (cd), and indeterminate colitis. the resultant ibd phenotype is the consequence of multiple interactions between environmental factors, particularly enteric flora, and the host response to this environment, determined by immunogenetic, epithelial, and other non-immune genetic factors. mbl, as an important component of innate immunity, has engendered considerable research interest. in an early study of 340 unrelated patients with ibd genotyped for mbl2 exon 1 coding mutations, the frequency of deficient alleles was significantly lower in patients with uc than either the control group, or those with cd (rector et al. 2001 ). the study by rector et al. (2001) suggests that mbl deficiency could be protective against uc; alternatively, it could be interpreted that mbl deficiency, in individuals otherwise predisposed to ibd, may skew the phenotype away from the uc spectrum of disease towards cd. this concept is supported by another study that genotyped mbl2 in patients with cd, uc, or healthy controls (seibold et al. 2004 ). the study also assessed anti-saccharomyces cerevisiae antibody (asca) and mbl levels within the same subsets of patients, albeit slightly different numbers within each group. cd patients with mbl deficiency were significantly more likely to be positive for asca and for their lymphocytes to proliferate in response to mannan. thus, it appears that mbl deficiency could impair normal processing of mannanexpressing microbial antigens, such as those found on the cell surface of many common microorganisms. the accumulated antigens could then stimulate the immune system, and contribute to the production of asca and possibly the pathogenesis of crohn's disease. thus, mbl deficiency might act primarily to influence ibd-specific phenotype in these patients. it should be noted, however, that a follow-up study, testing a larger cohort of cd patients (n ¼ 241), failed to confirm the significant association between variant mbl genotypes and asca positivity (joossens et al. 2006) . seibold et al. (2004) suggested that the frequency of homozygous and compound heterozygous for variant exon 1 alleles differed significantly between patients suffering from cd or uc and the healthy controls. celiac disease is a multi-factorial/auto-immune disorder caused in genetically susceptible patients, by the ingestion of dietary gluten. though very little is known about the genetic factors, but there is a strong association of two hla haplotypes (dq2 or a1*05, b1*02 and dq8 or a1*0301, b1*0302) with the disease. boniotto et al. (2002) indicated an association between celiac disease and the presence of variant mbl alleles. boniotto et al. (2005) and iltanen et al. (2003) reported the frequency of homozygosity for variant mbl alleles to be higher in the patients. among 149 italian celiac patients, 116 showed the presence of dq2 or dq8. mbl2 allele and genotype frequencies varied significantly between celiac patients and healthy humans. it is likely that in those rare cases of celiac disease that are negative for dq2 and dq8, the non-hla susceptibility genotypes would exert a greater effect. the study also analyzed apoptosis within small intestinal biopsy specimens, and showed that mbl tended to aggregate to areas of apoptosis within the epithelium. mbl has been implicated in the normal clearance of apoptotic bodies (nauta et al. 2003; ogden et al. 2001) . the authors postulated that the association between mbl and celiac disease, and indeed other autoimmune conditions, could relate to impaired apoptosis, whereby mbl deficiency impairs the normal removal and clearance of apoptotic cells that may subsequently reveal previously hidden selfantigen, causing loss of self-tolerance, and spreading of autoimmunity (boniotto et al. 2005) . the association between variant mbl2 alleles and coeliac disease has also been confirmed within the finnish population (iltanen et al. 2003 ) (worthley et al. 2006) . the low mbl genotypes were strongly associated with more celiac disease symptoms as well with increased frequency of secondary autoimmune diseases. by immunohistochemistry mbl was found to be present, together with apoptotic cells, in the basal lamina under the intestinal epithelium, where they had previously found mrna for mbl. boniotto et al. suggested that impaired removal of apoptotic cells due to mbl deficiency might predispose to the development of autoimmune symptoms. mice lacking mbl have been shown to be less efficient in removal of apoptotic cells (stuart et al. 2005) . in vitro studies have also implicated mbl in removal of apoptotic cells (ogden et al. 2001; nauta et al. 2004) . alternative explanation could be that increased susceptibility to intestinal infections and diarrhea, associated with low mbl, may change the intestinal epithelia thus allowing for abnormal stimulation of anti-gliadin immune responses and triggering of the cascade leading to celiac disease. a role for mbl like in celiac disease could be easily applied to other autoimmune disorders. despite the well-established role of mbl in innate immunity, there have been relatively few studies describing the clinical effect of mbl deficiency in enteric infections. one notable exception is the association between mbl deficiency and risk of cryptosporidium parvum enteritis. this study indicated that patients with biallelic coding mutations (o/o) had a significantly greater chance of cryptosporidiosis compared to those who were either wild-type or heterozygous for mbl2 mutation (kelly et al. 2000) . the association between mbl deficiency and cryptosporidiosis was confirmed in young haitian children by kirkpatrick et al. (2006) . however, the two combined studies present compelling evidence for the role of mbl in the host defense against cryptosporidium spp. infection. however, mbl deficiency was not associated with an increased risk of escherichia coli 0157: h7 colitis nor the complication of hus (proulx et al. 2003) . h. pylori is one of the most common human bacterial infections, affecting approximately 50% of humans. several immunogenetic polymorphisms are associated with clinical outcomes in h. pylori infection, as well as with the risk of infection itself. h pylori activates mbl in vitro (c/r worthley et al. 2006) . studies demonstrated that h. pylorirelated chronic gastritis causes an increase in gastric mucosal mbl expression, but no association was found between mbl2 genotype and risk of chronic gastritis (bak-romaniszyn et al. 2006; worthley et al. 2007) . mbl has been implicated in mediating gastrointestinal ischemia/reperfusion injury in mice (hart et al. 2005) . but mbl-null mice (deficient in the murine genes encoding mbl) developed only minor gut injury after induced ischemia/reperfusion insult compared to the wild-type mice. on the contrary, mbl has been implicated as a mediator of ischemia/ reperfusion injury in both the myocardium (walsh et al. 2005) and the kidney (møllerkristensen et al. 2005 ). whereas mbl deficiency has been associated with rheumatic disorders, high mbl levels associated to disease has been reported by hansen et al. (2003) . rheumatic fever (rf) is the most common cause of acquired valvular disease in children and young adults. the pathogenic mechanisms responsible for the development of rf/rhd are associated to an abnormal host immune response (both at humoral and cellular level) to cross-reactive streptococcal antigens. the significantly elevated circulating mbl levels in patients with rhd together with the greater prevalence of mbl deficiency in controls suggest that mbl may cause undesirable complement activation contributing to the pathogenesis of rhd (schafranski et al. 2004) . probably, mbl deficiency may represent an advantage against the development of rheumatic mitral stenosis and that increased mbl levels may be related to the development of rhd. under normal conditions, mbl does not bind to the organism's own tissues, but in situations of cellular hypoxia, glycosylation of cell surfaces may occur, leading to the deposition of mbl followed by complement activation. the significantly elevated levels of mbl observed in chronic rhd suggest that mbl may represent a pathogenic factor in the complex physiopathology of the disease, whereas mbl deficient individuals might be less susceptible to develop chronic rhd. studies demonstrating the binding of mbl to the endothelium and causing excessive complement activation and subsequent tissue damage are known (jordan et al. 2003) , where as mbl deficiency may be advantageous in some circumstances since mbl may lead to an increased cytokine secretion by macrophages (takahashi et al. 2002) . under some other conditions, mbl is associated to disease severity in both infectious and autoimmune disease (garred et al. 1997 (garred et al. , 1999 . although mbl is an acute-phase protein produced by the liver, its level only shows a moderate increase and determined genetically in inflammatory diseases. the elevated mbl levels in patients with chronic rhd might corroborate the chronic inflammatory activity present in these individuals and contribute to valve injury through complement activation. in addition, mbl may act as an immunomodulatory molecule, inducing a higher secretion of cytokines by macrophages (turner and hamvas 2000) . there seems to be a delicate balance as to when mbl levels may be involved in harmful or in beneficial inflammation in the cardiovascular system. for example, kawasaki disease is a systemic vasculitis in childhood possibly caused by infections and in the developed world kawasaki disease is the most common cause of acquired heart disease in children (royle et al. 2005) . mbl as an initiator of inflammation, biezeveld et al. (2003) while studying the frequency of mbl genotypes with kawasaki disease in dutch patients found a higher frequency of mbl mutations as compared to the genotypes in controls. children younger than 1 year were at higher risk of development of cad. kawasaki disease occurs more frequent in oriental children (10 times more frequent than in caucasians) (royle et al. 2005) . in chinese, cheung et al. (2004) did not see a difference between the mbl genotypes of patients and controls. the recent observation of an association between a human coronavirus and kawasaki disease (esper et al. 2005) fits with the many indications of mbl having antiviral activity. plaque material may be removed from inside of the carotid artery (e.g., by endarterectomy) to avoid cerebral attack. rugonfalvi-kiss et al. (2005) indicated that female patients with genotypes associated with lower mbl levels had a slower rate to early restenosis, suggesting that a high level of mbl may be part of the pathophysiology of this condition. in a study of 76 patients with severe atherosclerosis, madsen et al. (1998) found that there were more patients with myocardial infarcts among norwegians with low mbl allotypes than in controls. saevarsdottir et al. (2005) found in a cohort study in iceland that the risk of developing myocardial infarction was higher in mbl deficient individuals. the relationship between markers of innate immunity and clinical outcomes in patients with heart failure (hf) after acute myocardial infarction (ami) suggested that atherogenesis and heart failure are associated with the altered control of inflammation by innate immune defenses, which include tlrs and mbl. circulating levels of mbl and stlr2 may reflect different aspects of the innate immune response and the involvement of innate immunity responses in the pathogenesis of post-mi heart failure (ueland et al. 2006) . mbl plays a dual role in modifying inflammatory responses to sterile and infectious injury. although complement is widely accepted as participating in the pathophysiology of ischemia-reperfusion injury, the specific role of the lectin pathway has been addressed by jordan et al. (2001) . since, blockade of the lectin pathway with inhibitory mabs protects the heart from ischemia-reperfusion by reducing neutrophil infiltration and attenuating pro-inflammatory gene expression, it appears that the lectin complement pathway is activated after myocardial ischemia-reperfusion and leads to tissue injury. mice devoid of mbl-dependent lectin pathway activation but fully active for alternative and classical complement pathways, are protected from cardiac reperfusion injury with resultant preservation of cardiac function. significantly, mice that lack a major component of the classical complement pathway initiation complex (c1q) but have an intact mbl complement pathway are not protected from injury. thus, the mbl-dependent pathway of complement activation is the key regulator of myocardial reperfusion ischemic injury (walsh et al. 2005) . diabetic patients at high risk for diabetic-nephropathy: whether lectin pathway of complement activation plays a role in the pathogenesis of human glomerulonephritis, is not well known. it has been proposed that mbl may bind to altered self components, which may possibly be found in diabetic patients, and mbl could thus be a potential pathogenic factor for diabetic cardiovascular complications, e.g., nephropathy saevarsdottir et al. 2005) . normo-albuminuric type 1 diabetics have been found to have higher mbl levels than non-diabetic controls, with a stepwise increase in circulating mbl levels with increasing levels of urinary albumin excretion (hansen et al. 2003) . in another study, a significantly larger proportion of patients with diabetic nephropathy presented a mbl genotype associated with higher mbl level, when compared to the group with mbl genotypes associated with low mbl levels ). the elevated serum mbl levels in type 1 diabetic patients with diabetic nephropathy were confirmed by saraheimo et al. (2005) . in a long follow up study on 270 type 1 diabetic patients, it was found that for patients with type 1 diabetes and mbl-levels below the median of 1.6 mg/ml the risk of developing micro or macro-albuminuria was 26%, while the patients with mbl-levels above the median had a risk of 41% of developing micro or macro-albuminuria (hovind et al. 2005) . these studies suggested that a high mbl geno-and phenotype is associated with an increased risk of developing diabetic kidney disease and that assessing mbl status may prove beneficial in identifying patients at risk for micro-and macro-vascular complications (c/r thiel et al. 2006) . mbl has been detected in the glomeruli of patients with lupus nephropathy, membranous nephropathy, membranoproliferative glomerulonephritis type i and anti-gbm nephritis. it was proposed that mbl binds to agalactosyl oligosaccharides of igg that terminate in n-acetylglucosamine (lhotta et al. 1999) . elevated serum mbl levels of were implicated in the pathogenesis of renal manifestations of henoch-sch€ onlein purpura (endo et al. 2000) , in iga nephropathy (endo et al. 1998) , in other forms of human glomerulonephritis (lhotta et al. 1999) , and in vascular complications of diabetes mellitus type 1 (hansen et al. 2003 ). sarcoidosis is a chronic granulomatous disease of unknown aetiology. the causative agent may be an infectious microorganism. mbl variants predisposed to sarcoidosis by increasing their susceptibility to micro-organisms among sarcoidosis patients showed that the frequencies of variants were similar regardless of severity of disease outcome. mbl gene variants did not indicate to influence susceptibility to sarcoidosis, age of disease onset, or severity of disease. the average patient ages at time of diagnosis were similar for all mbl genotypes (foley et al. 2000) . however, a 57-year-old woman patient with pulmonary sarcoidosis suggested interstitial nephritis without proteinuria and hematuria, whereas a renal biopsy showed granulomatous interstitial nephritis and mild mesangial proliferative glomerulonephritis. from this case of renal sarcoidosis, it was hypothesized that p. acnes might be involved in pathogenesis of granulomatous interstitial nephritis and that it plays a role in glomerular complement activation via the lectin pathway (hagiwara et al. 2005 ). behcet's disease (bd) is a multisystemic, recurrent inflammatory disease caused by the combination of genetic and environmental factors. the haplotypes of the mbl2 gene can influence therapeutic response in bd, thus affecting the clinical symptoms in bd patients. the promoter region, mbl2-550*c/*c (l/l) homozygote was found to have a lower frequency in bd patients than that in controls. no difference was observed in the allele frequencies of g-221 c (y/x), c + 4 t (p/q) or gly54asp (a/b) of the mbl2 gene in bd patients and in controls. the hypa haplotype contributed to bd occurrence, whereas the lypa haplotype was negatively associated with bd. bd patients with several symptoms and with an earlier disease-onset age had a higher hypa haplotype frequency. bd patients showing poor response (s) to therapy had a higher hypa frequency than those showing good response (m). it appeared that possessing hypa increases the risk of bd and that the mbl2 hypa haplotype plays a role in mbl levels and increases the susceptibility to bd (park et al. 2005 ). the mbl deficiency has been associated with poor outcome in cystic fibrosis (cf) lung disease. a mutation in masp-2 and higher serum levels of mbl than healthy controls belonging to the mbl pathway in serum have been described. thus, mbl pathway function is affected both by mbl and by masp-2 genotypes (carlsson et al. 2005) . patients undergoing abdominal aortic aneurysm (aaa) repair are exposed to an ischaemia-reperfusion injury (iri), which is in part mediated by complement activation. during iri, the patients undergoing aaa repair experience a mean decrease in plasma mbl level of 41% representing significant lectin pathway activation. this indicated that consumption of mbl occurs during aaa repair, which suggested an important role for lectin pathway in iri. hence, specific transient inhibition of lectin pathway activity can be of significant therapeutic value in patients undergoing open surgical aaa repair (norwood et al. 2006 ). in guillain-barre syndrome (gbs), complement activation plays a crucial role in the induction and extent of the post-infectious immune-mediated peripheral nerve damage. the mbl2 genotype, serum mbl level, and mbl complex activity are associated with the development and severity of gbs. the mbl2 b allele was associated with functional deficiency and relatively mild weakness. studies support the hypothesis that complement activation mediated by mbl contributes to the extent of nerve damage in gbs, which is codetermined by the mbl2 haplotype (geleijns et al. 2006 ). peritonitis is the most common and major complication in the treatment modality of peritoneal dialysis (pd) for uraemic patients. the contribution of the different complement activation pathways was studied in the host defense against experimental polymicrobial peritonitis induced by cecal ligation and puncture by using mice deficient in either c1q or factors b and c2. the c1q-deficient mice lack the classical complement activation pathway. mice with a deficiency of both factors b and c2 lack complement activation via the classical, the alternative, and the lectin pathways and exhibited the maximum mortality of 92%, indicating a significant contribution of the lectin and alternative pathways of complement activation to survival (windbichler et al. 2004) . while examining the role of serum mbl concentration and point mutations in mbl gene in pd-related peritonitis, lam et al. (2005) found that both homozygous and heterozygous patients had profoundly reduced serum level of mbl. thus, dialysis patients having lower mbl levels may increase the susceptibility of infection. mbl replacement therapy following stem cell transplantation: life-threatening complications such as graft versus host disease and infection remain major barriers to the success of allogeneic hemopoietic stem cell transplantation (sct). among various factors, mbl deficiency is a risk factor for infection in other situations where immunity is compromised. mbl2 coding mutations were associated with an increased risk of major infection following transplantation. mbl2 promoter variants were also associated with major infection. the high-producing haplotype hya was associated with a markedly reduced risk of infection. donor mbl2 coding mutations and recipient hya haplotype were independently associated with infection in multivariate analysis. thus, these results suggest that mbl2 genotype influences the risk of infection following allogeneic sct and that both donor and recipient mbl2 genotype are important (mullighan et al. 2002) . a retrospective study examining associations between polymorphisms in the gene encoding mbl, mbl2 and risk of major infection post-sct was conducted in 96 related myeloablative transplants. the study showed that "low-producing" mbl2 coding alleles, when present in the donor, were significantly associated with increased risk of major infection in the recipient following neutrophil count recovery. furthermore, a "high-producing" mbl2 haplotype, hya, when present in the recipient, was protective against infection. since mbl is under development as a therapeutic agent, findings suggest that administration of mbl may reduce the risk of infection post-transplant. further work is required to confirm these results. these results indicate a report of a genetic determinant of risk of infection post-sct, and highlight the importance of non-hla genetic factors in determining the risk of transplant complications (mullighan and bardy 2004) . genetic mbl variants are frequent and associated with low mbl serum levels. higher mbl levels may be associated with more complement-mediated damage resulting in inferior graft survival. berger et al. (2005) showed no significant difference in incidence of delayed graft function in recipients with a low mbl level (< or ¼ 400 ng/ml) compared to those with a higher mbl level (>400 ng/ml). if these data can be confirmed, pre-transplant mbl levels may provide additional information for risk stratification prior to kidney transplantation (berger et al. 2005) . complement activation is harmful for the allograft endothelium: in heart transplant recipients, fiane et al. (2005) recorded transplant-associated coronary artery disease and observed an association with mbl deficiency. they also recorded that acute rejection of the transplant was seen in 6 out of 6 with mbl deficiency as compared to 15 out of 32 with higher mbl levels. assuming that mbl may interact with the transplanted tissue and initiate complement activation, this study added to the list of studies, which suggested that complement activation is harmful for the endothelium in general, and possibly for the allograft endothelium in particular. simultaneous pancreas-kidney transplantation (spkt) is the treatment of choice for patients with type 1 diabetes and renal failure. however, this procedure is characterized by a high rate of postoperative infections, acute rejection episodes, and cardiovascular mortality. the lectin pathway of complement activation contributes to cardiovascular disease in diabetes and may play an important role in inflammatory damage after organ transplantation. mbl serum levels and mbl genotypes in patients who received an spkt from 1990 through 2000 and related graft survivals revealed that survival was significantly better in recipients with mbl gene polymorphisms associated with low mbl levels. thus, mbl is a potential risk factor for graft and patient survival in spkt. it is hypothesized that mbl contributes to the pathogenesis of inflammation-induced vascular damage both in the transplanted organs and in the recipient's native blood vessels (berger et al. 2007 ). to address further the role of mbl deficiency, verschuren et al. (2008) showed that high levels of serum mbl are associated with protection against urinary tract infections and, more specifically, against urosepsis after spkt. these results indicate an important role for the lectin pathway of complement activation in antimicrobial defense in these transplant recipients (verschuren et al. 2008 ). in paediatric oncology patients with febrile neutropenia, mbl levels are correlated to clinical and laboratory parameters. structural exon-1 mbl2 mutations and the lx promoter polymorphism were related to deficient mbl levels. the capacity to increase mbl concentrations during febrile neutropenia was associated with mbl2 genotype. infectious parameters did not differ between mbl-deficient and mbl-sufficient neutropenic children. however, most patients (61%) were severely neutropenic, compromising the opsonophagocytic effector function of mbl. mbl substitution might still be beneficial in patients with phagocytic activity (frakking et al. 2003) . five polymorphisms in the promoter and first exon of the mbl2 gene alter the expression and function of mbl in humans and are associated with inflammation-related disease susceptibility. these five polymorphisms create six well-characterized haplotypes that result in lower (i.e., lyb, lyc, hyd, and lxa) or higher (i.e., hya and lya) serum mbl concentrations. a statistically significant association was found between the x allele of the promoter y/x polymorphism (which results in a lower serum mbl concentration) and improved lung cancer survival among white patients but not among african american patients. the functional y/x polymorphism of the innate-immunity gene mbl2 and mbl2 haplotypes and diplotypes appear to be associated with lung cancer survival among white patients (pine et al. 2007) . a significantly higher incidence of mbl deficiency/insufficiency-associated genotypes was found among patients with malignant disease. findings reflecting anti-tumorigenic activity of mbl protein suggest potential therapeutic application. however, it cannot be excluded that mbl-2 mutant alleles may be in linkage disequilibrium with an unidentified tumor susceptibility gene(s) (swierzko et al. 2007 ). the genetic variations in key genes of mbl2 could influence the risk for breast cancer. a preliminary analysis of snps in mbl2 in breast cancer [166 african-american (aa) patients vs. 180 controls and 127 caucasian (cau) patients vs. 137 controls] presents evidence that common genetic variants in the 3 0 -utr of mbl2 might influence the risk for breast cancer in aa women, probably in interaction with the 5 0 secretor haplotypes that are associated with high concentrations of mbl (bernig et al. 2007 ). changes in cell surface structures during oncogenic transformation appear to promote binding of mbl to cancer cells (hakomori 2001) where the protein can mediate cytotoxic effects including mbl-dependent cell mediated cytotoxicity (ma et al. 1999; nakagawa et al. 2003) . experimental studies suggest that mbl (both wild-type and the mutant b allele) may possess anti-colorectal cancer tumor activity (ma et al. 1999) . the mbp/mbl binds specifically to oligosaccharides expressed on the surface of human colorectal carcinoma cells, sw1116. mbl binding occurs in colon adenocarcinoma cell lines (colo205, colo201 and dld-1), but not in any of the leukemic cell lines. the binding of mbl to these cell lines was sugar-specific and calciumdependent. the degree of mbl binding was correlated with the expression of lewis a and lewis b antigens on these cell lines (muto et al. 1999) . intra-tumoral administration of the recombinant vaccinia virus carrying a mbl2 gene significantly reduced tumor size as compared to controls, along with prolonged survival of mice (ma et al. 1999 ). however, these results were not reflected in clinical trials. in fact, patients with colorectal cancer have increased activation of the lectin-complement pathway and increased levels of serum mbl (ytting et al. 2004 ). in patients undergoing surgery for colorectal cancer, however, low preoperative levels of serum mbl have been linked to an increased risk of developing post-colectomy pneumonia (ytting et al. 2005) . further studies may clarify the role of the lectin-complement pathway in colorectal cancer. secondary immunodeficiencies due to disease or treatment have provided interesting patient populations within which to study the role of mbl. one such group comprises those receiving chemotherapy for malignancy. in these patients, chemotherapy induces neutropenia and increased risk of infection. studies have thus attempted to analyze the correlation between mbl deficiency and infections in such patients. it is difficult to compare these studies since these studies include patients with a variety of underlying malignancies and variety of chemotherapy, different combinations of antimicrobial agents and various other factors (klein and kilpatrick 2004) . however, there is clear evidence of the importance of mbl for protection in leukemia patients. mbl genotypes and mbl levels were correlated to the causes, frequency and duration of febrile neutropenic periods in children receiving chemotherapy (neth et al. 2001) . the majority of children were patients of acute lymphoblastic leukemia (all). children with variant mbl alleles exhibited twice as many days of febrile neutropenia as children with wild type genotypes. analysis by mbl quantification supported this as children with less than 1 mg mbl/ml had a higher number of days with febrile neutropenia. peterslund et al. (2001) described infections defined as bacteremia, pneumonia or both in hematological malignancies of 54 adults treated with chemotherapy. all patients with the infections, except one, showed mbl levels below 0.5 mg/ml. vekemans et al. (2005) conducted a prospective observational study focusing on assessment of mbl as a risk factor for infection during chemotherapy induced neutropenia in adult hematological cancer patients. they included 255 patients and determined mbl levels as well as mbl genotypes. a higher rate of severe infection was seen in mbl deficient patients. the impact was further increased when acute leukaemic patients were excluded. in a contrasting study, bergmann et al. (2003) followed 80 adults undergoing therapy for acute myeloid leukaemia (aml), which involves intense highly myelosuppressive treatment. they found no effect of mbl deficiency on frequency, severity or duration of fever and suggested that the severe immunosuppression induced by the combination of the myeloid cancer and chemotherapy may obscure the normal effector functions of mbl, though, kilpatrick et al. (2003) failed to see anything but a modest effect of mbl levels below 100 ng/ml in a retrospective study on 128 patients, most of whom were prepared for bone marrow transfer and more than half presented with aml. results cast doubt on the potential value of mbl replacement therapy in this clinical context . a growing body of evidence indicates that genetic factors are involved in an increased risk of infection. mbl gene polymorphisms that cause low levels of mbl are associated with the occurrence of major infections in patients, mainly bearing hematological malignancies, after high-dose chemotherapy (hdt) rescued by autologous peripheral blood stem cell transplantation (auto-pbsct). a retrospective examination of 113 patients treated with hdt and auto-pbsct revealed that the low-producing genotypes, b/b and b/lxa, were associated with major bacterial infection. a nation-wide study, conducted to assess the allele frequency of the mbl coding mutation in a total of 2,623 healthy individuals in japan, revealed the frequency of allele b as 0.2, almost the same in seven different areas of japan. this common occurrence suggested that mbl deficiency may play an important role in the clinical settings of immune-suppression (horiuchi et al. 2005) . studies by aittoniemi et al. (1999) in patients with chronic lymphocytic leukemia did not observe any effect of mbl on infections. a possible association between mbl genotypes and severe infections in patients with multiple myeloma receiving moderate strength induction chemotherapy has been studied. from the mbp genotypes, identified in bone marrow biopsies, the study concluded that during induction chemotherapy in patients with multiple myeloma, a general protective effect of wild-type mbl2 against chemotherapy-related infections was not apparent. however, indications were there of a reduced occurrence of septicaemia in patients with wild-type compared with variant mbl2. further studies in larger cohorts of patients are relevant (molle et al. 2006) . thus, further studies are required to describe the patients particularly at risk when being mbl deficient. the mbl knock-out mice have made possible experimental investigations of the effect of mbl deficiency. the mouse has two genes encoding different mbl molecules (mbl-a and -c) compared to one in humans. both mbls in mice are able to bind to carbohydrate surfaces and activate the complement system. a slight difference in carbohydrate specificity has been reported for the two mouse mbls. mice with only mbl-a knocked-out were first produced, but only mice with both mbl-a and -c knocked out ( no difference was seen when the bacteria were injected intra-peritoneally. however, if the mice were treated with cyclophosphamide, simulating chemotherapy-induced neutropenia, before the intra peritoneal infection, the mbl dko had more abscesses than the wild type. the mbl dko mice were also more susceptible to challenge with herpes simplex virus type 2 (gadjeva et al. 2004 ). in line with the suggested involvement of mbl in autoimmune diseases the mbl dko mice were examined for autoimmune symptoms when 18-month-old (stuart et al. 2005) . no such signs were observed. on the other hand, the ability to clear apoptotic cells was less efficient in the mbl knock-outs. it has been hypothesized that while mbl does not bind significantly to healthy tissue, changes due to abnormal conditions might reveal mbl ligands. indeed, mbl is expressed by some tumor cell lines, and gene therapy with an mbl-vaccinia construct was found protective in nude mice transplanted with a human colorectal cancer cell line (ma et al. 1999) . in vitro studies have indicated binding of mbl to cells exposed to hypoxia-reoxygenation (simulating ischemia/reperfusion) and subsequently it was shown that infusion of a blocking anti-mbl antibody would protect against myocardial destruction following ischemia/reperfusion in a rat model (jordan et al. 2001 ). using mbl dko mice møllerkristensen et al. (2005) found, in a model of kidney ischemia reperfusion (i/r) injury, that the mbl dko were partially protected as evidenced by a better kidney function in these mice after ischemia/reperfusion. increased deposition of the complement factor c3 was seen in wild type mice, and binding of mbl to sections of kidney could be inhibited with mannose. in agreement with this, devries et al. (2004) found mbl-a and -c deposited in the kidneys after ischemia/reperfusion in mbl wild type mice. the recombinant vaccinia virus carrying human mbp gene possesses a potent growth-inhibiting activity against human colorectal carcinoma cells transplanted in ksn nude mice. the treatment resulted into a prolonged life span of tumor-bearing mice. local production of mbp had a cytotoxic activity, which was proposed as mbp dependent cell-mediated cytotoxicity (mdcc). this study offers a model for the development of an effective and specific host defense factor for cancer gene therapy (ma et al. 1999; thiel et al. 2006 ). since genetically determined mbl deficiency is very common and can be associated with increased susceptibility to a variety of infections, the potential benefits of mbl reconstitution therapy need to be evaluated. in a phase i trial on 20 mbl-deficient healthy adult volunteers receiving a total of 18 mg of mbl in three 6 mg doses given (i.v.), once a week for a period of 3 weeks did not show adverse clinical changes or any sign of infusion-associated complement activation. study suggested that infusion of purified mbl as prepared by statens serum institut (ssi) is safe. however, adults have to be given at least 6 mg twice or thrice weekly for maintaining protective mbl levels assumed to be about 1,000 ng/ml (valdimarsson et al. 2004 ). considerations of mbl genotyping and association with infection opens the possibility of producing clinical grade recombinant mbl that resulted to establishing a company having this aim (jensenius et al. 2003) . the treatment of chronic disorders may possibly also be considered on the longer term. the invention led to use of at least mbl oligomer comprising at least one mbl subunit, for the manufacture of a medicament for prophylaxis and/or treatment of infection (thiel and jensenius 2007) . mutations of genes involved in the innate immune system as predictors of sepsis in very low birth weight infants opsonising immunoglobulins and mannanbinding lectin in chronic lymphocytic leukemia leishmania (viannia) braziliensis: interaction of mannose-binding lectin with surface glycoconjugates and complement activation. an antibodyindependent defence mechanism mannanbinding lectin in children with chronic gastritis polymorphisms in the mannose binding lectin (mbl) gene are not associated with radiographic erosions in rheumatoid or inflammatory polyarthritis mannose binding protein deficiency is not associated with malaria, hepatitis b carriage nor tuberculosis in africans association between mannose-binding lectin levels and graft survival in kidney transplantation low pretransplantation mannose-binding lectin levels predict superior patient and graft survival after simultaneous pancreas-kidney transplantation low levels of mannose-binding lectin do not affect occurrence of severe infections or duration of fever in acute myeloid leukaemia during remission induction therapy the mannose-binding lectin (mbl2) haplotype and breast cancer: an association study in african-american and caucasian women association of mannose-binding lectin genotype with cardiovascular abnormalities in kawasaki disease evidence for an association between mannose-binding lectin 2 (mbl2) gene polymorphisms and pre-term birth toward an epidemiology and natural history of sirs (systemic inflammatory response syndrome) variant mannose-binding lectin alleles are associated with celiac disease evidence of a correlation between mannose binding lectin and celiac disease: a model for other autoimmune diseases proteins that bind high-mannose sugars of the hiv envelope mannose binding lectin gene polymorphisms confer a major risk for severe infections after liver transplantation levels of complexity in pathogen recognition by c-type lectins deficiency of the mannan-binding lectin pathway of complement and poor outcome in cystic fibrosis: bacterial colonization may be decisive for a relationship modulating effects of mannose binding lectin genotype on arterial stiffness in children after kawasaki disease a populationbased study of morbidity and mortality in mannose-binding lectin deficiency mannose-binding protein gene polymorphism in systemic lupus erythematosus a dysfunctional allele of the mannose binding protein gene associates with systemic lupus erythematosus in a spanish population the mannosebinding lectin-pathway is involved in complement activation in the course of renal ischemia-reperfusion injury mannose-binding lectin in innate immunity: past, present and future immature dendritic cells possess a sugar-sensitive receptor for human mannan-binding lectin detection of an autologous ligand for mannan-binding lectin on human b lymphocytes evaluation and clinical interest of mannan binding lectin function in human plasma mannose-binding lectin in susceptibility and progression of hiv-1 infection in children impact of mannose-binding lectin on susceptibility to infectious diseases glomerular deposition of mannose-binding lectin (mbl) indicates a novel mechanism of complement activation in iga nephropathy complement activation through the lectin pathway in patients with henoch-sch€ onlein purpura nephritis association between a novel human coronavirus and kawasaki disease low mannose-binding lectin and increased complement activation correlate to allograft vasculopathy, ischaemia, and rejection after human heart transplantation increased incidence and severity of the systemic inflammatory response syndrome in patients deficient in mannose-binding lectin mannose-binding lectin promoter and structural gene variants in sarcoidosis the role of mannosebinding lectin (mbl) in paediatric oncology patients with febrile neutropenia mannan-binding lectin modulates the response to hsv-2 infection gene frequency and partial protein characterization of an allelic variant of mannan binding protein associated with low serum concentrations susceptibility to hiv infection and progression of aids in relation to variant alleles of mannose binding lectin mannose-binding lectin polymorphism and susceptibility to infection in systemic lupus erythematosus association of mannose-binding lectin gene variation with disease severity and infections in a population-based cohort of systemic lupus erythematosus patients mannose-binding lectin deficiency-revisited association of mannosebinding lectin polymorphisms with sepsis and fatal outcome, in patients with systemic inflammatory response syndrome mannose-binding lectin and its genetic variants mannosebinding lectin contributes to the severity of guillain-barre syndrome mannose-binding lectin gene polymorphism is a modulating factor in repeated respiratory infections the natural history and prognosis of rheumatoid arthritis: association of radiographic outcome with process variables, joint motion and immune proteins the association of variant mannose-binding lectin genotypes with radiographic outcome in rheumatoid arthritis disease associations of mannosebinding lectin & potential of replacement therapy a case of renal sarcoidosis with complement activation via the lectin pathway tumor-associated carbohydrate antigens defining tumor malignancy: basis for development of anti-cancer vaccines mannose-binding lectin (mbl) and vascular complications in diabetis elevated levels of mannan-binding lectin in patients with type 1 diabetes association between mannose-binding lectin and vascular complications in type 1 diabetes glycosylation inhibitors and neuraminidase enhance human immunodeficiency virus type 1 binding and neutralization by mannose-binding lectin gastrointestinal ischemia-reperfusion injury is lectin complement pathway dependent without involving c1q impaired secretion of rat mannose-binding protein resulting from mutations in the collagen-like domain presence of clonespecific markers at birth in children with acute lymphoblastic leukaemia association of mbl gene polymorphisms with major bacterial infection in patients treated with high-dose chemotherapy and autologous pbsct mannose-binding lectin as a predictor of microalbuminuria in type 1 diabetes: an inception cohort study the association between mannan-binding lectin gene alleles and celiac disease role of mannose-binding lectin in the innate defense against candida albicans: enhancement of complement activation, but lack of opsonic function, in phagocytosis by human dendritic cells association of systemic lupus erythematosus with promoter polymorphisms of the mannosebinding lectin gene mannose-binding lectin in severe acute respiratory syndrome coronavirus infection mannose-binding lectin: targeting the microbial world for complement attack and opsonophagocytosis the influence of mannose binding lectin polymorphisms on disease outcome in early polyarthritis recombinant mannan-binding lectin (mbl) for therapy mannose binding lectin (mbl) and hiv mannan binding lectin (mbl) gene polymorphisms are not associated with anti-saccharomyces cerevisiae (asca) in patients with crohn's disease inhibition of mannosebinding lectin reduces postischemic myocardial reperfusion injury anti-c1q autoantibodies mannose-binding lectin is a component of innate mucosal defense against cryptosporidium parvum in aids c-type lectins and phagocytosis mannan-binding lectin: clinical significance and applications no strong relationship between mannan binding lectin or plasma ficolins and chemotherapy-related infections serum mannosebinding lectin deficiency is associated with cryptosporidiosis in young haitian children is there a role for mannan/mannosebinding lectin (mbl) in defence against infection following chemotherapy for cancer? mannan-binding lectin and rsv lower respiratory tract infection leading to hospitalization in children: a case-control study from soweto, south africa mannose binding lectin level and polymorphism in patients on long-term peritoneal dialysis the mannose-binding lectin gene polymorphisms and systemic lupus erythematosus: two casecontrol studies and a meta-analysis glomerular deposition of mannose-binding lectin in human glomerulonephritis inhibition of (s)-armepavine from nelumbo nucifera on autoimmune disease of mrl/mpj-lpr/ lpr mice mannose-binding lectin and vulvovaginal candidiasis dc-sign and l-sign are high affinity binding receptors for hepatitis c virus glycoprotein e2 antitumor activity of mannan-binding protein in vivo as revealed by a virus expression system: mannan-binding protein dependent cell-mediated cytotoxicity association of mannose-binding-lectin deficiency with severe atherosclerosis association of mannose-binding lectin gene haplotype lxpa and lypb with interferon-resistant hepatitis c virus infection in japanese patients single nucleotide polymorphisms of the mannose-binding lectin are associated with susceptibility to primary biliary cirrhosis mannose-binding lectin gene polymorphisms are associated with gestational diabetes mellitus analysis of the relationship between mannose-binding lectin (mbl) genotype, mbl levels and function in an australian blood donor population antibodies to mannose binding lectin in patients with systemic lupus erythematosus chemotherapy-related infections in patients with multiple myeloma: associations with mannan-binding lectin genotypes mannanbinding lectin recognizes structures on ischemic reperfused mouse kidneys and is implicated in tissue injury mannose-binding lectin and infection following allogeneic hemopoietic stem cell transplantation mannose-binding lectin gene polymorphisms are associated with major infection following allogeneic hemopoietic stem cell transplantation human mannose-binding lectin preferentially binds to human colon adenocarcinoma cell lines expressing high amount of lewis a and lewis b antigens antitumor activity of mannan-binding protein mannose-binding lectin engagement with late apoptotic and necrotic cells opsonization with c1q and mannose-binding lectin targets apoptotic cells to dendritic cells deficiency of mannosebinding lectin and burden of infection in children with malignancy: a prospective study consumption of mannan-binding lectin during abdominal aortic aneurysm repair c1q and mannose binding lectin engagement of cell surface calreticulin and cd91 initiates macropinocytosis and uptake of apoptotic cells mannosebinding lectin variant alleles and the risk of arterial thrombosis in systemic lupus erythematosus association of hypa haplotype in the mannose-binding lectin gene-2 with behcet's disease mannose binding lectin and c3 act as recognition molecules for infectious agents in the vagina the genetic basis for systemic lupus erythematosus association between deficiency of mannose-binding lectin and severe infections after chemotherapy lung cancer survival and functional polymorphisms in mbl2, an innate-immunity gene characterization of mannose-binding lectin gene polymorphism among human t-cell lymphotropic virus 1 and 2-infected asymptomatic subjects mannan-binding lectin in children with escherichia coli o157:h7 haemmorrhagic colitis and haemolytic uraemic syndrome mannan-binding lectin (mbl) gene polymorphisms in ulcerative colitis and crohn's disease antibody-mediated activation of the classical pathway of complement may compensate for mannose-binding lectin deficiency detection of three single nucleotide polymorphisms in the gene encoding mannose-binding lectin in a single pyrosequencing reaction the diagnosis and management of kawasaki disease high rate of early restenosis after carotid eversion endarterectomy in homozygous carriers of the normal mannose-binding lectin genotype mannan binding lectin as an adjunct to risk assessment for myocardial infarction in individuals with enhanced risk mannan-binding lectin enhances susceptibility to visceral leishmaniasis on behalf of the finn diane study group, increased levels of mannan-binding lectin in type 1 diabetic patients with incipient and overt nephropathy significantly increased levels of mannose-binding lectin (mbl) in rheumatic heart disease: a beneficial role for mbl deficiency increased frequency of mannose-binding lectin insufficiency among children with acute lymphoblastic leukemia autoantibodies against mannose-binding lectin in systemic lupus erythematosus a role for mannosebinding lectin dysfunction in generation of autoantibodies in systemic lupus erythematosus genetic variants of the mannan-binding lectin are associated with immune reactivity to mannans in crohn's disease mannose-binding lectindeficient mice are susceptible to infection with staphylococcus aureus mannan-binding lectin (mbl) serum levels and post-operative infections detection of gene mutations and promoter polymorphisms in the mannan-binding lectin (mbl) gene by polymerase chain reaction with sequence specific primers rapid genotyping of mbl2 gene mutations using real-time pcr with fluorescent hybridisation probes mannose-binding lectindeficient mice display defective apoptotic cell clearance but no autoimmune phenotype mannose-binding protein genetic polymorphisms in black patients with systemic lupus erythematosus molecular basis of opsonic defect in immunodeficient children association of mutations in mannose binding protein gene with childhood infection in consecutive hospital series distinct and overlapping functions of allelic forms of human mannose binding protein polymorphisms in cd14, mannose-binding lectin, and toll-like receptor-2 are associated with increased prevalence of infection in critically ill adults mannan-binding lectin (mbl) in women with tumors of the reproductive system lack of mannose-binding lectin-a enhances survival in a mouse model of acute septic peritonitis association of mannose binding lectin (mbl) gene polymorphism and serum mbl concentration with characteristics and progression of systemic lupus erythematosus indications of mannan-binding lectin (mbl) in the treatment of immunocompromised individuals. us patent issued on clinical manifestations of mannan-binding lectin deficiency mutation of gene of mannose-binding protein associated with chronic hepatitis b viral infection anti-c1q autoantibodies in murine lupus nephritis activation of the lectin pathway in murine lupus nephritis the role of mannose-binding lectin in health and disease mannose-binding lectin: the pluripotent molecule of the innate immune system mannose-binding lectin: structure, function, genetics and disease associations mannose binding lectin and soluble toll-like receptor 2 in heart failure following acute myocardial infarction human plasmaderived mannose-binding lectin: a phase i safety and pharmacokinetic study influence of mannan binding lectin serum levels on the risk of infection during chemotherapy-induced neutropenia in adult haematological cancer patients infectious complications after simultaneous pancreas-kidney transplantation: a role for the lectin pathway of complement activation mannose binding lectin and fcgriia (cd32) polymorphism in spanish systemic lupus erythematosus patients dominant effects of mutations in the collagenous domain of mannose-binding protein decoupling of carbohydrate binding and masp-2 autoactivation in variant mannosebinding lectins associated with immunodeficiency mannose-binding lectin is a regulator of inflammation that accompanies myocardial ischemia and reperfusion injury polymorphisms of the mannose binding lectin gene in patients with sjogren's syndrome mannose binding lectin (mbl) polymorphisms associated with low mbl production in patients with dermatomyositis involvement of the lectin pathway of complement activation in antimicrobial immune defense during experimental septic peritonitis mannose-binding lectin: biology and clinical implications mannose-binding lectin and maladies of the bowel and liver mannose-binding lectin deficiency does not increase the prevalence of helicobacter pylori seropositivity interaction of mannose-binding lectin with hiv type 1 is sufficient for virus opsonization but not neutralization increased activity of the mannan-binding lectin complement activation pathway in patients with colorectal cancer preoperative mannan-binding lectin pathway and prognosis in colorectal cancer key: cord-278644-u4swxsjj authors: degn, søren e.; thiel, steffen; nielsen, ole; hansen, annette g.; steffensen, rudi; jensenius, jens c. title: map19, the alternative splice product of the masp2 gene date: 2011-10-28 journal: journal of immunological methods doi: 10.1016/j.jim.2011.08.006 sha: doc_id: 278644 cord_uid: u4swxsjj abstract the lectin pathway of complement is a central part of innate immunity, but as a powerful inducer of inflammation it needs to be tightly controlled. the masp2 gene encodes two proteins, masp-2 and map19. masp-2 is the serine protease responsible for lectin pathway activation. the smaller alternative splice product, map19, lacks a catalytic domain but retains two of three domains involved in association with the pattern-recognition molecules (prms): mannan-binding lectin (mbl), h-ficolin, l-ficolin and m-ficolin. map19 reportedly acts as a competitive inhibitor of masp-2-mediated complement activation. in light of a ten times lower affinity of map19, versus masp-2, for association with the prms, much higher serum concentrations of map19 than masp-2 would be required for map19 to exert such an inhibitory activity. just four amino acid residues distinguish map19 from masp-2, and these are conserved between man, mouse and rat. nonetheless we generated monoclonal rat anti-map19 antibodies and established a quantitative assay. we found the concentration of map19 in serum to be 217ng/ml, i.e., 11nm, comparable to the 7nm of masp-2. in serum all masp-2, but only a minor fraction of map19, was associated with prms. in contrast to previous reports we found that map19 could not compete with masp-2 for binding to mbl, nor could it inhibit masp-2-mediated complement activation. immunohistochemical analyses combined with qrt-pcr revealed that both map19 and masp-2 were mainly expressed in hepatocytes. high levels of map19 were found in urine, where masp-2 was absent. the recognition of non-self by the innate immune system is mediated by cellular and humoral pattern-recognition molecules (prms), targeting conserved pathogen-associated molecular patterns (pamps). a group of humoral prms, mannan-binding lectin (mbl), h-ficolin, l-ficolin, and mficolin, can activate the lectin pathway of complement by means of associated serine proteases, masp-1, masp-2 and masp-3 (thiel, 2007) . two additional proteins, map19 and map44, are found associated with these prms. map19, also known as smap, is one of two proteins arising by alternative splicing of the primary transcript of the masp-2 gene (stover et al., 1999b; takahashi et al., 1999) . the other product is the pro-enzyme masp-2, which is composed of a cub domain, an egf domain, a second cub domain, two ccp domains and the activation peptide, followed by the serine protease domain (thiel et al., 1997) . mature masp-2 is generated by cleavage of the activation peptide, resulting in two disulfide-linked fragments, the larger known as the a-chain and the smaller as the b-chain (schwaeble et al., 2002; thiel, 2007) . map19 consists of only the first cub domain and the egf domain, with an additional small c-terminal tail of four unique amino acid residues (eqsl) (fig. 1) . these four amino acid residues are encoded by exon 5 of the gene, which also harbors the 3′utr. like masp-2, map19 is known to be expressed in the liver and is found in plasma (stover et al., 1999a) . it forms a head-to-tail dimer, the structure of which has been solved by x-ray crystallography to a resolution of 2.5 å , and associates with mbl and the ficolins in a calcium-dependent manner. the dissociation constant for the interaction with mbl has been determined to be 13 nm, identical to the similar cub-egf fragment of masp-2, but around 16 times higher than that of full-length masp-2 (thielens et al., 2001) . map19 has been suggested to act as an inhibitor of calcium oxalate crystal growth in human urine (kang et al., 1999) . more recently, map19 has been reported to compete with masp-2 for binding to mbl, leading to attenuation of c4-cleaving activity and thus down-regulation of complement activation (iwaki et al., 2006) . map19 is relatively well conserved, the amino acid sequence for human map19 being 80% and 77% identical with mouse and rat map19, respectively, and the unique 4 c-terminal amino acids are conserved between these species (fig. 1a) . we present the generation of monoclonal antibodies specific for map19 and the construction of a solid-phase assay for quantification of map19 under dissociating conditions. using this assay we determine the mean concentration of map19 in blood and urine. we compare the mrna expression profiles of map19 and masp-2, as well as the tissue distribution of the proteins by immunohistochemical analyses. finally, we characterize the interaction of map19 with mbl and ficolins under native conditions and examine the reported inhibitory activity of map19 on activation of the complement system. fig. 1 . map19 and anti-map19 antibodies. a) alignment of the c-terminal ends of human, mouse and rat map19, demonstrating a high degree of conservation with total identity of the 4 ultimate amino acids. the peptide used for immunizations is shown. b) percent identity between full-length human, mouse and rat map19. c) western blot reactivity of anti-map19 antibodies, demonstrating specificity for map19. strips of a western blot containing purified mbl/masp complexes from human plasma were developed using hybridoma supernatants as primary antibodies. negative control is a non-reactive hybridoma (2b11) and positive control is a monoclonal antibody toward masp-2 and map19 (6g12). the parental rat serum (r166) is included, showing similar reactivity to that of the hybridoma supernatants. d) overview of the domain structure of map19 and masp-2, with indication of approximate antibody epitopes. recombinant mbl (rmbl), with complement activation capacity similar to that of mbl purified from plasma, was produced as described . plasmid encoding masp-3 has been described (dahl et al., 2001) . a pfastbac plasmid encoding human map19 cdna (thielens et al., 2001) was kindly provided by nicole thielens. the insert was excised from the pfastbac vector using bamhi and hindiii and ligated into the pcdna3.1(−) vector (invitrogen) for expression in mammalian cells. resulting colonies were screened by restriction analysis and confirmed by sequencing. recombinant masp-3 and map19 were produced by transient expression in freestyle hek293f cells in freestyle expression medium (invitrogen). the plasmids were introduced using a cationic lipid-based transfection reagent (lipofecta-mine2000, invitrogen). following a procedure we have previously described for the purification of rmap44 (degn et al., 2009) , the rmasp-3 and rmap19 were purified from the culture supernatants by affinity chromatography on rmbl-coupled sepharose 4b in a ca 2+ -containing buffer, and bound rmasp-3 or rmap19 was eluted with a buffer containing edta and high salt concentration. the purity of the products was verified by silver staining of sds-page gels. the concentrations of the proteins in the eluates were determined by od280 measurement (using molar extinction coefficients of 1.02 and 1.22 (mg/ml) -1 cm −1 for map44 and map19, respectively, calculated from the sequences using protein calculator v.3.3) and confirmed by quantitative amino acid analysis. mbl/masp complexes were purified from a pool of human plasma by affinity chromatography on mannan coupled to sepharose beads, as previously described . two monoclonal rat antibodies, 6g12 (isotype igg1), reacting with the common part of human map19 and masp-2 (fig. 1d) , and 8b5 (igg1), specific for masp-2, were previously generated (møller-kristensen et al., 2003) . likewise, a mouse monoclonal antibody, 1.3b7 (igg1κ), reacting with masp-2 and map19 was previously generated . a nonapeptide representing the c-terminus of map19 (krtcseqsl) was synthesized, thus including the four unique c-terminal amino acids of map19 and a convenient internal cysteine for coupling. the peptide was coupled to keyhole limpet hemocyanin (klh) using m-maleimidobenzoyl-n-hydroxysuccinimidyl ester (mbs) and 3 balb/c mice and 3 wistar rats were subjected to an immunization regimen with the conjugated peptide in freund's adjuvant. sera were screened as indicated below. the mice failed to respond, whereas the rats mounted very limited responses. these 3 rats and further 3 naive rats and 3 naive mice were then subjected to an immunization regiment with peptide coupled to purified protein derivative (ppd) from bacillus tuberculosis. the rats already having received freund's complete adjuvant were not primed further, whereas the naive animals were primed with 100 μl of bcg vaccine, s.c. in the neck (1.7 adult human doses of mycobacterium bovis bacillus calmette-guérin, an attenuated live vaccine, statens serum institut, copenhagen). the ppd-conjugated peptide was produced by three methods: sulfo-mbs coupling, glutaraldehyde coupling and divinylsulfone (dvs) coupling, using about 14-fold molar excess of peptide to ppd. briefly, for sulfo-mbs coupling, 300 μl of 1 mg ppd/ml was mixed with 300 μl of 1 mg sulfo-mbs/ml 25 mm phosphate buffer, ph 7.2, and incubated for 30 min end over end at room-temperature (rt). following this, the mixture was dialysed against 25 mm phosphate buffer, ph 7.2, mixed with 600 μl peptide (1 mg/ml 25 mm phosphate buffer, ph 7.2) for 3 h end over end at rt, and then dialysed extensively against pbs (137 mm nacl, 2.7 mm kcl, 1.5 mm kh 2 po 4 , 8.1 mm na 2 hpo 4 , ph 7.4). for glutaraldehyde coupling, 300 μl of 1 mg ppd/ml were added to 600 μl of 1 mg peptide/ml 25 mm phosphate buffer, ph 7.2. one ml of 0.12% glutaraldehyde in phosphate buffer was added, followed by incubation for 2 days, end over end at 4°c, and dialysis against pbs. for dvs coupling, 300 μl of 1 mg ppd/ml was dialyzed against 100 mm carbonate buffer, ph 9.0 and 2.5% (v/v) dvs added, followed by incubation at 4°c overnight (o.n.) to this mixture was added 300 μl of 1 mg peptide/ml 100 mm carbonate buffer, ph 9.0, followed by incubation o.n., end over end at 4°c. finally this was also dialyzed extensively against pbs. the three coupling reaction products were mixed in approximately equal volume and 150 μl (emulsified 6:9 in freund's incomplete adjuvant) was used for each immunization. after three rounds of immunizations the rats were still the best responders, and the highest response was generated in a rat previously subjected to the peptide-klh protocol. this rat was given recombinant map19 (rmap19, see below), i.v., before the spleen was harvested and the cells fused with x63ag8.653 murine myeloma cells, according to standard protocols (kohler and milstein, 1975; kearney et al., 1979) . resulting aminopterin resistant clones were screened as described below, and subcloned a total of 4 times to ensure monoclonality. sera from immunized animals were screened in several assays, with all reagents added at 100 μl/well: 1) on mbl complexes captured on a mannan coat and on ficolin complexes captured on a coat of acetylated bovine serum albumin (acbsa). mannan (polysaccharide from yeast and a strong ligand for mbl, purified according to (nakajima and ballou, 1974) ) or acbsa (from sigma, presenting ligands for all three ficolins (krarup et al., 2004; frederiksen et al., 2005) ) was coated in microtiter wells (fluoronunc, nunc, denmark) at 10 μg/ml carbonate buffer, ph 9.6, o.n. at 4°c. residual binding sites were blocked with 1 mg human serum albumin (hsa, statens serum institut) per ml tbs (10 mm tris-hcl, 140 mm nacl, 15 mm nan 3, ph 7.4), and the wells were washed thrice with tbs containing 5 mm cacl 2 and 0.05% (v/v) tween-20 (tbs/tw/ca 2+ ). a human serum pool diluted 20-fold in binding buffer (20 mm tris, 1 m nacl, 10 mm cacl 2 , 1 mg hsa/ml, 0.05% (v/v) triton x-100, ph 7.4) was incubated in the wells at 4°c o.n. after washing with tbs/tw/ca 2+ , animal sera were added in a 10-fold dilution series in tbs/tw/ca 2+ , from 1/100 to 1/10,000, and incubated o.n. at 4°c. wells were washed with tbs/tw/ca 2+ and developed with biotinylated rabbitanti-rat ig or biotinylated rabbit-anti-mouse ig (both antibodies from dako, z0494 and z0259, respectively, and biotinylated in-house), followed by eu 3+ -streptavidin (perkinelmer) at 0.1 μg/ml tbs/tw (tbs with 0.05% tween-20), containing 25 μm edta and incubated for 45 min before final wash and development with enhancement buffer (perkinelmer) and reading on a time-resolved fluorometer (victor3, perkinelmer). the readings are given as photon counts per second. the procedure is termed "time-resolved immunofluorometric assay", abbreviated trifma. as a control for direct reactivity with the surfaces, animal sera were tested on the same primary coats not subjected to incubation with human serum. 2) on directly coated mbl complexes or on h-ficolin complexes, purified from human plasma on mannose-tsk-75 beads and acetylated-hsa-derivatized sepharose-4b beads, respectively. plasma-derived mbl/masp or h-ficolin/masp complexes were coated in microtiter wells at 1 μg mbl or h-ficolin per ml. animal sera were added in dilution series as above, and development was performed in a similar manner. a mockcoated surface was used for background control. 3) on wells directly coated with recombinant map19, using an approach similar to the one employed for hybridoma supernatants as described below. hybridoma supernatants were screened on wells directly coated with rmap19. rmap19 was coated in microtiter wells at 0.5 μg/ml pbs o.n. at 4°c. wells were blocked with tbs/ tw, washed thrice with tbs/tw/ca 2+ , and hybridoma supernatants diluted 1:1 in tbs/tw/ca 2+ were added. after incubation and washing with tbs/tw/ca 2+ , biotinylated rabbit-anti-rat ig was added. after incubation the wells were washed with tbs/tw/ca 2+ and developed with eu 3+ -streptavidin as described above. positive supernatants were further tested in a similar assay based on coating the wells with rmasp-2 (1 μg/ml) in order to ensure lack of cross-reactivity with masp-2. supernatants tested positive in the map19 assay were tested on a western blot (wb) of plasma-derived mbl/masp complexes. forty μl pmbl/masp preparation (containing 65 μg mbl/ml) was added sample buffer containing dtt and loaded on a single-well xt-criterion 4-12% gel (bio-rad). the proteins were blotted to hybond-p pvdf membrane (amersham biosciences) and strips 0.25 cm wide were cut. strips were incubated with hybridoma supernatants diluted 1/10 in primary buffer (tbs/tw, 1 mm edta, with 1 mg hsa and 100 μg normal human igg (higg) added per ml), or with the parental rat serum 1/500. after washing 3× with tbs/tw, without azide, these strips were incubated with horseradish peroxidase (hrp)-conjugated rabbit-anti-rat ig (p0450, dako) diluted 1/1,000 in secondary buffer (tbs/tw without azide, 1 mm edta, 100 μg higg/ml). this was followed by washing and development with west dura extended dura-tion substrate (pierce). images were taken using a ccd camera (las-3000, fuji) and analyzed with the software supplied with the camera. as a control, a strip was incubated with a monoclonal rat anti-human map19/masp-2 (6g12, see below) at 1 μg/ml primary buffer, followed by hrpconjugated rabbit-anti-mouse ig (p0260, dako). recombinant map19 was coated at 0.25 μg/ml pbs, o.n. at 4°c. the wells were blocked with tbs/tw, and then washed thrice in tbs/tw. hybridoma supernatants, diluted 1:1 in tbs/tw, were added to each well and incubated for 2 h. the wells were washed thrice with tbs/tw, before the addition of 100 μl affinity-purified and biotinylated anti-rat immunoglobulin class and subclass antibodies (anti-igg1, -igg2a, -igg2b, -igg2c, -iga, and -igm, as well as a control biotinylated normal ig; zymed laboratories, rat monoab id/sp kit) in pbs/tw. two hours of incubation was followed by washing thrice with tbs/tw, the addition of eu 3+streptavidin, and development as described above. in a similar set-up, the anti-map19 antibody isotypes present in the parental rat serum was determined (serum diluted 1/10). the anti-map19 antibodies were purified on protein l agarose (sigma). approximately 1.5 ml protein l agarose was pre-washed in 0.1 m glycine, ph 2.5, then equilibrated in pbs and packed in a poly-prep chromatography column (bio-rad). ninety ml hybridoma supernatant was pre-cleared by centrifugation at 2,000 g for 10 min, added 10 ml of 10 times concentrated pbs, and loaded on the column. the effluent was collected in fractions of 10 ml. the column was washed with 15 ml (10 column volumes) pbs and elution was performed with 0.1 m glycine, ph 2.5. the eluate was collected in approximately 10 fractions of 500 μl, into 42 μl 1 m tris-hcl, ph 8.5, for immediate neutralization. the protein content of the eluates was assessed by od280 measurement, and protein-containing fractions were pooled. the anti-map19 content of the eluates, the pooled eluates and the effluent fractions was assessed by testing on directly coated recombinant map19, as described above. 2.8. test of autoreactivity of the 5 final antibodies and parental rat serum mbl/masp and ficolin/masp complexes were purified in parallel from human and rat serum. a 1:1 mixture of mannancoupled sepharose and acetylated hsa-coupled sepharose was washed twice with 25 ml tbs/tw/0.5 m nacl/10 mm edta, then equilibrated with 4 × 25 ml tbs/tw/5 mm cacl 2 . to 2.5 ml of this mixture (approximately 2 ml beads) was added either 5 ml normal human serum or 5 ml normal rat serum pre-diluted with 10 ml tbs/tw/ca 2+ . the beads were incubated with the serum samples 1 h end over end at 4°c, then spun down 5 min at 100 g and washed 3× with 50 ml tbs/tw/ca 2+ . the beads were then packed in poly-prep chromatography columns and washed with an additional 25 ml tbs/tw/ca 2+ . the column was eluted with tbs/tw/0.5 m nacl/10 mm edta and fractions of 250 μl were collected. the od280 of fractions was measured compared to the elution buffer and peak fractions were pooled. from approximately 1 ml pooled eluates of each, 500 μl was run on a reducing gel, as described above. proteins were blotted to nitrocellulose, the membranes were blocked with tbs/0.1% tween and cut into strips, which were then probed with the parental rat serum diluted 1/1,000, and the 5 final clones purified on protein l agarose diluted to 1 μg/ml, as well as a normal rat serum diluted 1/1,000, all in tbs/tw/1 mm edta/1 mg hsa per ml and 100 μg nhig per ml. a buffer only control was also included, and for the human blot, an anti mbl antibody (131-1 mouse mab) was included at 1 μg/ml along with a normal mouse serum control, 1/1,000, in order to confirm presence of mbl in the purified pool. rat antibodies were developed with hrp-rabbit-anti-rat (dako p0450) and mouse antibodies with hrp-rabbit-anti-mouse (dako p0260), both diluted 1/3,000 in tbs/tw/1 mm edta/100 μg/ml nhig. a sandwich assay based on the trifma principle was developed, involving capture with mab 6g12 and detection of bound map19 with biotinylated anti-map19 antibody (4d12) (fig. 1 ) followed by eu 3+ -labeled streptavidin. wells were coated with 4 μg 6 g12 per ml sodium acetate buffer (50 mm na-acetate, 145 mm nacl, ph 4.5) o.n. at 4°c. the wells were then blocked with hsa, 1 mg/ml tbs, washed thrice with tbs/tw, and incubated with serum samples diluted 20-fold in map19 buffer (tbs/tw, 1 m total nacl, 10 mm edta, containing 100 μg δhigg (heat-aggregated higg); beriglobin, from zlb behring gmbh, incubated 30 min at 63°c and centrifuged 10 min at 3,000 g to remove large aggregates) per ml, and 100 μg normal rat igg (lampire) per ml) o.n. at rt. the δnhig was added to adsorb any rheumatic factor present in the sample, while the normal rat igg was added to prevent cross-linking of the coating and the detecting rat antibodies by any anti-rat antibodies in the serum samples (degn et al., 2011) . after washing the wells, biotinylated 4d12 was added at 1 μg per ml tbs/tw containing 1 mg hsa/ml. following another wash, the wells were developed with eu 3+ -streptavidin as described above. a standard curve was prepared by applying a 2-fold serial dilution of a standard serum (8 dilutions) and a buffer control. along with three internal control sera (high, medium, low), this was included on every plate. all samples, standards and controls were in duplicates. sepharose beads coupled with 6g12 (anti-map19/ masp-2), 400 μl (approximately 200 μl bead volume) were washed thrice with 1 ml 0.1 m glycine, ph 2.5, then equilibrated with 10 ml incubation buffer (tbs/tw, 1 m total nacl, 10 mm edta, containing 100 μg δhigg). the equilibrated beads were incubated with 1 ml of a serum found to be low in map19 and masp-2 and pre-diluted with 3 ml of incubation buffer, for 2 h at rt end over end. the beads were spun down and the supernatant (depleted serum diluted 1/4) was saved. dilution curves were prepared using the standard serum, the low serum, the low serum depleted of map19/masp-2, the low serum depleted and reconstituted with recombinant map19, or recombinant map19 diluted in buffer only. capacity and specificity tests were carried out by measurement of dilution series of rmap19 or rmasp-2 prepared in serum diluted 1/20. serum and plasma samples were obtained from a cohort of adult danish blood donors after informed consent and according to the declaration of helsinki. the levels of mbl and masp-2 (ytting et al., 2007) and map44 and masp-3 (degn et al., 2010) were previously determined in these samples. similarly, paired blood samples and 24 h urine samples were obtained from 5 danish volunteers. samples were stored at −80°c and freezethaw cycles were kept to a minimum. urine samples were analyzed by trifma and by western blotting as described above. western blots of reduced samples were developed with 4d12, non-reduced samples with 1.3b7. map19 and masp-2 mrna expression levels were quantified in a firstchoice human total rna survey panel (applied biosystems/ambion) comprising rna from 20 human tissues, using taqman chemistry and the abi prism 7000 sequence detection system. the rna was reverse transcribed using the roche one step rt-pcr system with oligo(dt) primers. taqman gene expression assays from applied biosystems were used for masp-2 (catalog no. hs00198244_m1) and map19 (custom assay targeting exon 4-5), using β 2 -microglobulin mrna (hs99999907_m1) for normalization. the relative levels of masp-2 and map19 mrna were compared using the delta-delta cycle threshold method. immunohistochemical analysis was performed on formalin-fixed paraffin-embedded tissue multiblocks and similarly prepared multiblocks of hek293f cells transfected with map19 plasmid, masp-2 plasmid, or mock transfected. briefly, hek293f cells were transfected as described above, pelleted 72 h post-transfection, fixed o.n. in 10% neutralbuffered formalin, then subjected to standard protocols for dehydration, clearing and paraffin embedding. the 3b11 hybridoma supernatant was used to stain for map19, at a dilution of 1/300, while the purified monoclonal antibody 8b5 was used to stain for masp-2, at a dilution of 1/1,000. endogenous peroxide was blocked by pre-treatment with 1.5% hydrogen peroxide in tbs for 10 min. antigen retrieval was done by demasking with 15 minute microwave treatment in egta-containing tris buffer. primary antibodies were diluted in antibody diluent (s2022, dako) and incubated on slides for 60 min at rt. the secondary antibody was biotinylated polyclonal rabbit anti-rat ig (e0468, dako; pre-adsorbed to remove any cross-reactivity toward human or mouse immunoglobulins and fetal calf serum) diluted 100-fold in s2022. the procedure was carried out in an autostainerplus, using powervision+hrp (mono/poly) (leica biosystems) as detection system, and development was performed using the chromogen dab (k3468, dako) for 10 min. nuclear counterstain was performed using mayer's heamatoxylin for 2 min, before mounting slides for microscopy using aquatex (merck). normal human serum (nhs) was subjected to gel permeation chromatography (gpc) on a superose 6 column in either tbs with 5 mm cacl 2 or tbs with 850 mm nacl and 10 mm edta (a buffer dissociating the mbl/masp complexes) (møller-kristensen et al., 2003) . map19 was quantified in the fractions with the assay described above. fractions were also analyzed for igm, mbl and h-, l-and m-ficolin. the elution volume of purified igg and hsa was also determined. two-fold dilution series of rma19 or rmap44 ranging from 1 μg/ml to 10 ng/ml (corresponding to around 50-0.5 nm) were pre-incubated with a constant amount of rmbl (25 ng/ml) for 15 min at rt. the mixtures were then incubated with 5 ng/ml rmasp-2 for 15 min at rt, before being added to microtiter wells coated with 10 μg mannan per ml, as described above. after incubation at 4°c o.n. and washing thrice with tbs/tw/ca 2+ , the wells were incubated with 0.1 μg purified human complement component 4 (dodds, 1993) in 100 μl bbs 2+ (4 mm barbital, 145 mm nacl, 2 mm cacl 2 , 1 mm mgcl 2 , 3.8 mm nan 3 , ph 7.5) at 37°c for 90 min. the wells were then washed and a mixture of two biotin-labeled monoclonal anti-human c4 antibodies (mab 162.2 and mab 162.1, both from bioporto, 0.25 μg of each per ml tbs/tw/ca 2+ ) was incubated for 2 h at rt followed by europium-labeled streptavidin and development as described above (petersen et al., 2001) . to examine whether the competitors affected the binding of mbl to the surface, in a parallel experiment biotin-labeled monoclonal antibody against mbl (131-1) was deployed, and the wells were developed with europium-labeled streptavidin as described above. map19 is highly conserved across species and harbors only 4 c-terminal amino acids distinguishing it from masp-2 ( fig. 1a and b) . especially the c-terminal part is very well conserved, with no difference in the unique 4 residues between human, mouse and rat. nonetheless, we designed a peptide (fig. 1a) encompassing the 4 unique amino acids and conjugated it to klh or ppd (a heterogeneous protein preparation from mycobacterium tuberculosis acting as a carrier and a potent activator of the adaptive immune system), and immunized balb/c mice and wistar rats. sera were initially unsuccessfully screened in solidphase assays on mbl/masp complexes captured on a mannan surface and ficolin/masp complexes captured on an acbsa surface. however, we found that we could get signals when screening on purified mbl/masp/map complex or h-ficolin/ masp/map complex coated directly onto wells, as well as on directly coated purified recombinant map19. the best responder, a wistar rat, was boosted with rmap19 before performing the splenectomy and myeloma fusion. the resulting hybridomas were screened in solid-phase assays as before and subcloned. the final hybridoma supernatants were reactive on directly coated rmap19 and directly coated mbl/masp/map or h-ficolin/ masp/ map complexes purified from human plasma, whereas they did not give appreciable signal on recombinant masp-2 coated directly in wells. all final clones produced antibody of the igm isotype, and also the map19-specific antibodies in the serum of the rat were almost exclusively igm. the igm antibodies could be readily purified on protein l agarose and sustained biotinylation without appreciable loss of activity, but did not coat well, precluding their use as capture antibodies. the specificity of the anti-map19 antibodies was validated by performing western blotting with purified mbl/masp complex (fig. 1c) . as can be seen, the rat serum and all 6 final hybridomas were specific for map19, whereas the monoclonal antibody control (6g12) reacted with both map19 and masp-2 (as well as some degradation fragments of masp-2, which we have previously observed upon storage). there was no background staining on the control hybridoma strip. the high similarity between the peptide immunogen and endogenous rat map19 along with the absence of class-switching indicated the induction of an autoimmune response. we examined whether the monoclonal antibodies were reactive with rat map19 by western blotting. the purified antibodies and parental rat serum were tested on mbl/masp and ficolin/masp complexes purified from human serum (control, supplemental fig. 1a ) and rat serum (supplemental fig. 1b) . indeed, the purified antibodies reacted strongly with a band corresponding to the size of map19 of both human and rat origin. the levels of mrna encoding map19 and masp-2 were compared by qrt-pcr in a human tissue panel. β2-microglobulin mrna levels were used for normalization. as previously reported (stover et al., 2004; seyfarth et al., 2006) , masp-2 mrna was exclusively expressed in the liver ( fig. 2a) . the site of highest relative expression level of map19 mrna was also by far the liver, with very low expression seen in the thyroid and the kidney (fig. 2b) . in order to examine the tissue localization of map19 and masp-2, one of the final clones (3b11) and a previously generated monoclonal anti-masp-2 antibody (8b5) were applied to multi-block arrays of formalin-fixed and paraffin-embedded tissue sections (fig. 3) . the reactivity of the antibodies on formalin-fixed paraffin-embedded tissues and their specificity for map19 and masp-2, respectively, was verified by control stainings of similarly prepared blocks of cells transfected with map19 or masp-2, or mock transfected (supplemental fig. 2) . 3f11 displayed strong staining of the cytoplasm of hepatocytes at the hexagonal edges of the lobules (fig. 3a) . a grainy staining of hepatocytes surrounding the central veins was also observed. however, this was deemed to be artificial staining of lipofuscin (not shown). strong staining was also observed in alveolar macrophages, in polymorphonuclear cells, in very few cells in the thyroid, in exocrine cells of the stomach, and in prostate epithelium. in cerebellum there was weak staining of purkinje cells, while there was a diffuse staining in the kidney. the antibody specific for masp-2 (8b5) stained hepatocytes in a speckled cytoplasmic pattern (fig. 3b , see inset in liver panel for higher magnification), presumably reflecting exocytic vesicles in the cytoplasm. alveolar macrophages were strongly stained, as were exocrine cells of the pancreas and few cells in the colon. diffuse, but relatively strong staining was seen in the kidney. a solid-phase sandwich assay for map19 was constructed, in which the masp-2 and map19 specific antibody, 6g12, was used for capture of map19 from serum and bound map19 was detected with biotinylated map19-specific antibody (4d12). we produced recombinant map19 and purified this to homogeneity as assessed by sds-page and silver staining (fig. 4a inset) . in order to use the assay for quantification of map19, the concentration of this purified map19 was determined by spectrophotometry and quantitative amino acid analysis, and its titration curve was compared with that of a standard serum (fig. 4a) . as can be seen from the graph, the dilution curve of this preparation compared somewhat poorly with that of the standard serum. this was not due to a matrix effect, as diluting the recombinant material in map19/masp-2depleted serum gave a similar curve (supplemental fig. 3) . nonetheless, a credible quantitative estimation of the map19 level in serum could be obtained. furthermore, addition of 13 ng, 7 ng or 3 ng recombinant map19 to serum diluted 1/20 (containing 6 ng endogenous map19) gave recoveries of rmap19 of 84%, 102% and 103%, respectively. the total map19 measured in these samples was thus 88%, 101% and 101%, respectively. the capacity and specificity of the assay was examined by adding two-fold dilutions of recombinant map19 or masp-2 to one of the internal control sera (fig. 4b) . as seen, masp-2 was not measured in the assay, and it did not start interfering with the assay until it reached grossly non-physiological levels above 1 μg added/ml (likely by competing for binding to the capture antibody, 6g12). this corresponds to masp-2 levels of n20 μg/ml serum, which is 40 times higher than the reported mean of around 500 ng/ml (møller-kristensen et al., 2003) . conversely, a dose-response was seen with recombinant map19, until the assay began to reach saturation, above 1 μg added/ml. the interassay coefficient of variation was determined based on the inclusion in each run of three internal controls: a) mean of 139 ng/ml, cv of 13% (n =10); b) mean of 116 ng/ml, cv of 17% (n=10); c) mean of 78 ng/ml, cv of 11% (n=9). the measurement of map19 in sera from 104 danish blood donors, revealed a median map19 level of 217 ng/ml with a range from 26 to 675 ng/ml (fig. 5a ). for comparison, the mean level of masp-2 is 534 ng/ml with a standard deviation of 213 ng/ml (møller-kristensen et al., 2003) . while the levels of masp-2 are log-normally distributed, the levels of map19 were neither normally nor log-normally distributed. we compared the levels measured in serum with those measured in plasma, in a set of 70 paired serum and plasma donor samples. although slightly higher values were measured in plasma than serum (median 258 vs. 205 ng/ml) the values were highly correlated (r 2 =0.88; fig. 5b ). masp-2 levels were previously determined in the same samples (ytting et al., 2007) , allowing comparison of the map19 and masp-2 levels. based on spearman nonparametric correlation analysis, there was no correlation between the levels of the two proteins (p= 0.81; fig. 5c ). we also did not see any correlation with serum concentrations of mbl, l-ficolin, m-ficolin, masp-3 or map44, but observed a weak correlation with h-ficolin (rs = 0.23, p = 0.02; fig. 5d ). considering the reported function of map19 as an inhibitor of calcium oxalate crystal formation (kang et al., 1999) and the small size of map19, below the normal cut-off for glomerular filtration, even if dimeric, we decided to compare serum and urine levels ( fig. 6a and b) . the mean map19 level was 204 ng/ml in the sera vs. 63 ng/ml in 24 h urine of 5 male danish donors. masp-2 was measured in the same serum and urine samples yielding a median of 390 ng/ml serum, while urine levels were under the detection limit of the assay, i.e., below 1 ng/ml. there was no correlation between the serum and urine levels of the same individuals (p= 0.52, spearman). the total amount of map19 excreted in 24 h was calculated based on the total urine volume of each individual (fig. 6c) , yielding a median of 127 μg. the total excretion was not correlated with the serum levels (p= 0.45, spearman). many proteins excreted in urine are partially degraded. in order to examine whether the map19 measured in urine was indeed intact map19 and whether the measurements were reliable, we conducted western blot analysis of the urine samples, using 4d12 (reducing conditions) and 1.3b7 (anti-map19/masp-2, non-reducing conditions) (supplemental fig. 4 ). this indicated that indeed in one case, individual 4, map19 was partially degraded. as the blots developed with 4d12 and 1.3b7 were congruent, we can infer that the degradation fragment resulted from n-terminal cleavage. the presence of a degradation fragment of map19 was not dependent on storage, as the same picture was observed for samples stored frozen at −80°c and samples stored at 4°c for 10 months. generally, the band intensities observed on wb seemed to correlate well with the levels measured in trifma, i.e., individuals 1 and 4 presented the highest levels, followed by individual 5 and individuals 2 and 3. we examined the size distribution of map19 in serum by gpc under native conditions or conditions dissociating masps and maps from mbl and ficolins (fig. 7) . under native conditions, in the presence of calcium, we saw a small peak at the elution volume of mbl and ficolin complexes, and two larger peaks at elution volumes corresponding to free map19 dimer and what might be a tetramer. under dissociating conditions, i.e., high salt and edta, which dissociates masps and map19 from mbl and ficolins, the three peaks resolved into the two peaks at the size of the free dimer and putative tetramer. surprisingly, this indicates that most of the map19 is not in complex with mbl or ficolins. assuming that complexed and free dimer and tetramer map19 was measured with the same efficiency in the assay (carried out under dissociating conditions), the relative proportions of complex-bound and free map19 was estimated by integrating the area under the curve. this revealed that approximately 20% of map19 was found in complexes under native conditions, with 80% being free. we wished to examine the proposition that map19 competes with masp-2 for binding to mbl (iwaki et al., 2006) . mbl was mixed with masp-2 and various amounts of competitor, either map19 or map44, and then added to a mannan-coated surface. the wells were incubated at 37°c with c4, allowing masp-2 to cleave c4 to become deposited as c4b. increasing amounts of map44 inhibited c4 deposition, whereas addition of map19 did not result in inhibition (fig. 8) . in order to control for possible differences in mbl complex binding to the surface or a possible inhibition of this by the competitors, we in parallel developed for bound mbl and found that binding of mbl was not affected (supplemental fig. 5 ). in a move toward creating a framework for understanding the biological function of the small product of alternative splicing of the primary masp2 transcript, we have addressed a major issue, namely the difficulty of raising antibody specific for map19. this issue was resolved and we provide a number of descriptive and functional characteristics of map19, which will hopefully further investigations into its function. the generation of antibodies specific for map19 was hampered by the great similarity between map19 and masp-2, with a unique sequence of only four amino acids distinguishing map19 from the first domains of masp-2, together with the high degree of evolutionary conservation. the observed absence of isotype switching in the serum from the one responding rat might indicate a response related to the induction of autoantibodies in the animal. indeed, we found the anti-human map19 antibodies to cross-react with rat map19. protein localization may point toward function. we therefore used one of the generated antibodies, as well as a previously generated antibody toward masp-2, in immunohistochemical analyses. we also quantified mrna in various tissues to determine sites of synthesis. at the mrna level, map19 expression was found predominantly in the liver, with significant but very low expression in kidney and thyroid tissue, while masp-2 expression was completely restricted to liver. from the immunohistochemical analyses it further seemed that both map19 and masp-2 are produced by hepatocytes. very few cells were positive for map19 staining in the thyroid, and a diffuse staining was seen in the kidney. the latter may rather originate from the filtrate, especially considering the significant presence of map19 in urine. in addition to their sites of synthesis, map19 and masp-2 staining was observed in a number of tissues, perhaps indicating secondary localization, or perhaps local expression (either not included, or below detection level, in the tissue rna panels). staining of alveolar macrophages warrants caution, as these phagocytes are notorious for high lipofuscin levels, and the same consideration applies to the longlived purkinje cells of the cerebellum. in conclusion, the liver is the major site of biosynthesis of both map19 and masp-2. the median level of map19 in serum of danish blood donors was found to be 217 ng/ml. the serum level on a weight basis is thus lower than that of masp-2 (mean: 534 ng/ml (møller-kristensen et al., 2003) ), but this translates into a molar concentration of 11 nm and 7 nm for map19 (19.1 kda mature) and masp-2 (74.2 kda mature), respectively. map19 and masp-2 levels were not correlated. for comparison, the level of the other mbl/ficolin associated proteins are 1.7 μg/ml (41 nm) for map44 and 5.0 μg/ml (63 nm) for masp-3 (degn et al., 2010) . median levels of 63 ng/ml in 24 h urine and 204 ng/ml in serum were found in 5 paired urine and serum samples. somewhat surprisingly, the map19 levels in serum and urine were not correlated, and this was also not the case when looking at total urinary output. intuitively, higher serum levels should result in higher urine output, assuming that map19 gets filtered in the glomeruli as suggested by its small size. one confounding factor may be the total level of masps/maps compared to the total level of mbl/ficolins, since one could envision that mbl/ ficolins may have a carrier function for map19, despite the apparently low affinity discussed below. for comparison median masp-2 levels were 390 ng/ml serum and b1 ng/ml urine. this could be an effect both of the larger size of masp-2 and of its strong association with mbl and ficolins. map19 was originally identified as a protein co-purifying with mbl on affinity chromatography columns derivatized with mbl ligands (thiel et al., 1997) , and subsequent analyses have shown map19 to have a relatively high affinity for mbl (kd of 13 nm). it was therefore surprising to find that only about 20% of map19 is associated with mbl or ficolins under native conditions. for comparison masp-2 is found entirely in high molecular weight complexes under the same conditions (møller-kristensen et al., 2003) . a marked influence of the relatively small difference in affinity was also obvious when we found that contrary to map44, map19 showed no ability whatsoever to inhibit the lectin pathway of complement. this finding was surprising, since a previous report suggested that map19 acts as a competitive inhibitor of masp-2 (iwaki et al., 2006) . however, the relative amounts of map19, masp-2 and mbl used by iwaki and colleagues were very far from the molecular stoichiometry in vivo, as the concentration of map19 was not known at that time. thus, for their competition experiments, a constant amount of rmasp-2i (inactive mutant of masp-2) of 0.5 μg and various amounts of rsmap (rmap19), from 0 to 20 μg, were incubated with 20 μl homozygous masp-2/map19 −/− mouse serum. this corresponds to up to 1,000 μg/ml rsmap competing with 25 μg rmasp-2i for binding to 0.84 μg mbl (7 μg mbl-a and 35 μg mbl-c per ml serum in b6 mice), disregarding the presence of ficolins-a and -b and masp-1, -3, and map44. considering that map19 is less than a third of the size of masp-2, the molecular 7 . size distribution of map19 in human serum analyzed by gel permeation chromatography. serum was run either at physiological conditions (-♦-, tris-buffered saline with 5 mm cacl 2 ) or at dissociating conditions (-•-, tris buffer with 10 mm edta, 1 m nacl) and fractions were measured in the map19 assay. the void volume, total volume and elution positions of protein markers are indicated. the total protein trace is also indicated (▪, od280). fraction volume is indicated on the x-axis (ml). fig. 8 . the influence of map19 and map44 on c4 deposition. mbl was mixed with masp-2 and map19 or map44, then added to mannan coated wells. subsequently c4 was added and the wells were incubated at 37°c. the amount of deposited c4b (in counts/s) is given as a function of the concentration of competitor, either rmap44 (-■-) or rmap19 (-•-). error bars indicate standard deviation of duplicates. the experiment was performed three times with similar results, using slight differences in conditions and different batches of purified proteins. stoichiometry is even more distorted. whereas the experiments demonstrate that map19 can indeed compete with masp-2, it appears that highly non-physiological conditions are required for such observations. at our conditions we see no evidence of such competition. likewise for the c4 deposition, the lowest level shown is 100 ng rsmap per 0.5 μl serum, i.e. 200 μg per 1.0 ml, which is close to 11 μma value which is 1,000 times, 3 orders of magnitude, higher than the physiological level determined here in man. we find it highly unlikely that the physiological levels should differ this significantly in mouse from that observed in man. thus, while map19 concentrations far exceeding the physiological level may be able to compete with masp-2, in our set-up we did not observe any competition. this may be explained by the more than 10fold lower affinity of map19 for mbl, compared to that of the full-length masp-2, as well as to masp-1, masp-3 and map44 counterparts (degn et al., 2009; degn et al., 2010) . the lower affinity may in turn be explained by the absence of the cub2 domain possessed by the full-length proteases and map44 (thielens et al., 2001; cseh et al., 2002) . the determination of these dissociation constants were based on biacore analysis with curve fitting according to the langmuir 1:1 interaction model, which are hardly applicable in this case of multivalent multimeric interacting molecules. furthermore, the sensorgram for masp-2 was hardly sloping, making it unsuitable for calculations. however, using a complementary approach, chen and wallis examined the importance of the n-terminal domains of masps for binding to mbl in an inhibition assay (chen and wallis, 2001) . rat mbp-a was immobilized on the solid-phase and competition of 35 s-labeled cub-egf-cub binding was evaluated. full-length masp-1 and masp-1 cub-egf-cub yielded k i 's of 34 and 15 nm, respectively, while cub-egf yielded a k i of 1810 nm. the same domain configurations for masp-2 gave k i 's of 48, 22 and 454 nm, respectively. in both cases, this demonstrates the importance of the second cub domain, in the case of masp-1 increasing affinity 50-100-fold and for masp-2 10-20-fold. the results for masp-2 fragments can be extrapolated to map19. considering the relative levels of map19, masp-2, masp-1, masp-3 and map44, and the fact that all the other molecules associate stronger with mbl and ficolin than map19, it appears unlikely that the biological role of map19 should be to act as a physiological inhibitor of complement. in concordance with the above observations, the affinity purification procedure for rmap19 using binding to mblderivatized sepharose beads was inefficient compared to similar purification of recombinant map44, masp-3 and masp-2 using identical procedures (unpublished). our data leave open the question of the physiological role of map19. it remains a possibility that map19 has a function in calcium-homeostasis and regulation. the idea of a role in kidney function as suggested by (kang et al., 1999) is appealing. our literature review revealed a second paper pinpointing map19 in the kidney, but in relation to pathophysiology (musante et al., 2002) . musante and colleagues identified "masp-2" as one of only 6 protein factors that alter glomerular permeability in children with focal segmental glomerulosclerosis (fsgs). upon closer inspection it is clear that what these authors refer to as "masp-2" is really map19, based on the isoelectric point and size of the spot on 2d gel electrophoresis and the localization of mass spectrometry-identified peptide sequences to map19. thong-boonkerd and colleagues also identified "masp-2" in normal human urine, and again this was actually map19, based on mw and pi (thongboonkerd et al., 2002) . very recently, map19 (again referred to as masp-2) was also found to be about 3-fold upregulated in urine of individuals undergoing cardiopulmonary bypass surgery (aregger et al., 2010) . another unrelated role of map19 was suggested by a recent report that it interacts with the nucleocapsid protein of sars-cov (liu et al., 2009) . the assay presented here allows the analysis of further human populations and possibly the identification of individuals deficient for map19, potentially furthering the elucidation of the role in humans. in summary, we have produced monoclonal antibodies specific for map19, and using these, we have examined the tissue distribution of map19, we have set up a quantitative assay for map19, and we have determined the levels of map19 in serum and urine. in in vitro experiments using conditions reflecting the determined physiological concentrations of masp-2 and map19, we find that map19 is unable to compete with masp-2 for binding to mbl and hence did not impair complement activation. the physiological role of map19 remains unresolved. supplementary materials related to this article can be found online at doi:10.1016/j.jim.2011.08.006. urinary proteomics before and after extracorporeal circulation in patients with and without acute kidney injury stoichiometry of complexes between mannosebinding protein and its associated serine proteases. defining functional units for complement activation characterization of the interaction between l-ficolin/p35 and mannanbinding lectin-associated serine proteases-1 and − 2 masp-3 and its association with distinct complexes of the mannan-binding lectin complement activation pathway map44, a human protein associated with pattern recognition molecules of the complement system and regulating the lectin pathway of complement activation biological variations of masp-3 and map44, two splice products of the masp1 gene involved in regulation of the complement system assay interference caused by antibodies reacting with rat kappa light-chain in human sera small-scale preparation of complement components c3 and c4 m-ficolin, an innate immune defence molecule, binds patterns of acetyl groups and activates complement the x-ray structure of human mannanbinding lectin-associated protein 19 (map19) and its interaction site with mannan-binding lectin and l-ficolin small mannose-binding lectin-associated protein plays a regulatory role in the lectin complement pathway recombinant mannan-binding lectin (mbl) for therapy mannanbinding lectin (mbl)-associated plasma protein present in human urine inhibits calcium oxalate crystal growth a new mouse myeloma cell line that has lost immunoglobulin expression but permits the construction of antibody-secreting hybrid cell lines continuous cultures of fused cells secreting antibody of predefined specificity l-ficolin is a pattern recognition molecule specific for acetyl groups study on interaction between sars-cov n and map19 proteolytic activities of two types of mannose-binding lectin-associated serine protease levels of mannan-binding lectin-associated serine protease-2 in healthy individuals characterization of plasma factors that alter the permeability to albumin within isolated glomeruli characterization of the carbohydrate fragments obtained from saccharomyces cerevisiae mannan by alkaline degradation an assay for the mannan-binding lectin pathway of complement activation the mannan-binding lectin-associated serine proteases (masps) and map19: four components of the lectin pathway activation complex encoded by two genes extra-hepatic transcription of the human mannose-binding lectin gene (mbl2) and the mbl-associated serine protease 1-3 genes the rat and mouse homologues of masp-2 and map19, components of the lectin activation pathway of complement two constituents of the initiation complex of the mannan-binding lectin activation pathway of complement are encoded by a single structural gene organization of the masp2 locus and its expression profile in mouse and rat a truncated form of mannose-binding lectin-associated serine protease (masp)-2 expressed by alternative polyadenylation is a component of the lectin complement pathway complement activating soluble pattern recognition molecules with collagen-like regions, mannan-binding lectin, ficolins and associated proteins a second serine protease associated with mannan-binding lectin that activates complement interaction of c1q and mannanbinding lectin (mbl) with c1r, c1s, mbl-associated serine proteases 1 and 2, and the mbl-associated protein map19 interaction properties of human mannan-binding lectin (mbl)-associated serine proteases-1 and − 2, mbl-associated protein 19, and mbl proteomic analysis of normal human urinary proteins isolated by acetone precipitation or ultracentrifugation biological variation in circulating levels of mannan-binding lectin (mbl) and mbl-associated serine protease-2 and the influence of age, gender and physical exercise nicole thielens kindly provided human map19 cdna in the pfastbac vector.sed was supported by the danish graduate school of immunology and the lundbeck foundation. 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-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-023389-ilrp8vb7 authors: wefer, j.; harris, r. a.; lobell, a. title: protective dna vaccination against mog(91‐108)‐induced experimental autoimmune encephalomyelitis involves induction of ifnβ date: 2008-06-28 journal: scand j immunol doi: 10.1111/j.0300-9475.2004.01423j.x sha: doc_id: 23389 cord_uid: ilrp8vb7 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 mucin‐domain‐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 ifnγ upon recall stimulation 3 weeks after protective dna vaccination. in protected rats, we observed (1) no alterations in antigen‐specific th2 or th3 responses, (2) reduced mhc ii expression on splenocytes early after eae induction, (3) antigen‐specific upregulation of ifnβ upon recall stimulation and (4) reduced il‐12rβ2 on lymphocytes. we thus demonstrate an association of the protective effect of dna vaccination with expression of ifnβ. we are currently investigating the cellular mechanisms behind this ifnβ‐mediated protection. 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-023411-iszb5qlk authors: astrinidou‐vakaloudi, a.; xytsas, s.; diamanti, i.; . ioannidis, h; pangidis, p. title: presence of helicobacter pylori antibodies in haemodialysis patients date: 2008-06-28 journal: scand j immunol doi: 10.1111/j.0300-9475.2004.01423p.x sha: doc_id: 23411 cord_uid: iszb5qlk 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(+) 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. 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-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-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-023374-87ob1exq authors: sukhija, s.; gupta, v. k.; shah, a.; thiel, s.; sarma, p. u.; madan, t. title: levels, complement activity and polymorphisms of mannan‐binding lectin in patients of bronchial asthma with allergic rhinitis date: 2008-06-28 journal: scand j immunol doi: 10.1111/j.0300-9475.2004.01423ai.x sha: doc_id: 23374 cord_uid: 87ob1exq 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 = 0.0024, or = 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. 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-023421-1d1gf7az authors: sønder, s. u. s.; hedegaard, c. j.; bendtzen, k. title: monitoring patients treated with type 1 interferons: antiviral versus mxa induction assays date: 2008-06-28 journal: scand j immunol doi: 10.1111/j.0300-9475.2004.01423bb.x sha: doc_id: 23421 cord_uid: 1d1gf7az interferon‐α/β (ifn‐α/β) 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‐α or ‐β, 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. 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-023391-bq5w3jk9 authors: utermöhlen, o.; karow, u.; baschuk, n.; herz, j.; loegters, t. t.; krönke, m. title: delayed elimination of the lcm virus from acid sphingomyelinase‐deficient mice due to reduced expansion of virus‐specific cd8(+) t lymphocytes date: 2008-06-28 journal: scand j immunol doi: 10.1111/j.0300-9475.2004.01423l.x sha: doc_id: 23391 cord_uid: bq5w3jk9 the phagolysosomally localized acid sphingomyelinase (asmase) activated by proinflammatory cytokines such as tnf and ifn‐γ 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(+) t cells. the secretion of ifn‐γ in response to contact with target cells as well as the cytolytic activity of virus‐specific cd8(+) t cells was severely impaired. additionally, both phases of the lcm virus‐specific dth response, mediated by cd8(+) and cd4(+) t cells consecutively, were diminished in asmase(–/–) mice. however, the secondary memory response of virus‐specific ctl was not altered, and the 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(+) t cells during the acute infection of mice with the lcm virus. 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-023445-c4tqioz1 authors: lauridsen, c.; jensen, s. k. title: supplementation of vitamin c to weaner diets increases igm concentration and improves the biological activity of vitamin e in alveolar macrophages date: 2008-06-28 journal: scand j immunol doi: 10.1111/j.0300-9475.2004.01423u.x sha: doc_id: 23445 cord_uid: c4tqioz1 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‐(α‐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. 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-022631-s4n24xij authors: jonsson, m. v.; brun, j. g.; skarstein, k.; jonsson, r. title: germinal centres in primary sjögren's syndrome indicate a certain clinical immunological phenotype date: 2008-06-28 journal: scand j immunol doi: 10.1111/j.0300-9475.2004.01423h.x sha: doc_id: 22631 cord_uid: s4n24xij 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(+) patients compared to the gc(–) patients (p = 0.035). in the gc(+) group, 54.5% of the patients presented with anti‐ro/ssa compared to 43.7% in the gc(–) group. anti‐la/ssb was detected in 31.3% of the gc(+) patients compared to 25.7% of the gc(–) patients. sixty‐one percentage of gc(+) patients presented with increased levels of igg, a nonsignificant difference when compared to 39.4% in the gc(–) patients (p = 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. 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-023415-hhvmsn5b authors: karlsson, h.; larsson, p.; wold, a. e.; rudin, a. title: pattern of cytokine responses to gram‐positive and gram‐negative commensal bacteria is profoundly changed when monocytes differentiate into dendritic cells date: 2008-06-28 journal: scand j immunol doi: 10.1111/j.0300-9475.2004.01423at.x sha: doc_id: 23415 cord_uid: hhvmsn5b the normal gastrointestinal flora is crucial for the maturation of the acquired immunity via effects on antigen‐presenting 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‐γ 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. 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-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-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-023417-by18aczt authors: vilhelmsson, m.; ekman, g. j.; zargari, a.; scheynius, a. title: the malassezia sympodialis allergen mala s 11 with sequence similarity to manganese superoxide dismutase induces maturation and production of inflammatory cytokines in human dendritic cells date: 2008-06-28 journal: scand j immunol doi: 10.1111/j.0300-9475.2004.01423ae.x sha: doc_id: 23417 cord_uid: by18aczt 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. monocyte‐derived 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‐α (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. 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-023438-g0k0vvdc authors: krog, j.; jepsen, c. f.; tønnesen, e.; parner, e.; hokland, m. title: the effects of hyperbaric exposure on human peripheral blood mononuclear cells, with special emphasis on natural killer cell cytotoxicity and subsets date: 2008-06-28 journal: scand j immunol doi: 10.1111/j.0300-9475.2004.01423aa.x sha: doc_id: 23438 cord_uid: g0k0vvdc 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 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-023439-r04y1j22 authors: hedegaard, c. j.; bendtzen, k.; nielsen, c. h. title: the role of immune complexes consisting of myelin basic protein (mbp), anti‐mbp antibodies and complement in promoting cd4(+) t‐cell responses to mbp in health and multiple sclerosis date: 2008-06-28 journal: scand j immunol doi: 10.1111/j.0300-9475.2004.01423k.x sha: doc_id: 23439 cord_uid: r04y1j22 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(+) t cells, we observed the production of tnf‐α, ifn‐γ 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 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-023402-8qfmo6rq authors: reinholdt, j.; baxendale, h.; ekström, n.; kayhty, h.; poulsen, k.; kilian, m. title: pneumococcal iga1 protease activity interferes with opsonophagocytosis of streptococcus pneumoniae mediated by serotype‐specific human monoclonal iga1 antibodies date: 2008-06-28 journal: scand j immunol doi: 10.1111/j.0300-9475.2004.01423t.x sha: doc_id: 23402 cord_uid: 8qfmo6rq 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 fabα (monovalent) deprived of fcα 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 fabα. 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. 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-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-023410-eblcf902 authors: kollgaard, t. m.; reker, s.; petersen, s. l.; masmas, t. n.; vindelov, l. l.; straten, p. t. title: clonally expanded cd8(+) t cells in allogeneic bone marrow transplantation date: 2008-06-28 journal: scand j immunol doi: 10.1111/j.0300-9475.2004.01423bm.x sha: doc_id: 23410 cord_uid: eblcf902 allogeneic bone marrow transplantation (bmt) is a potentially curative therapy for patients with haematologic malignancies. several lines of evidence demonstrate that donor t cells are involved in the antitumour effects observed after bmt. thus, patients receiving t‐cell‐depleted 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(+) 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(+) 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. 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-023407-s85g7g0x authors: huang, y.‐m.; liu, x.; steffensen, k.; sanna, a.; arru, g.; sominanda, a.; sotgiu, s.; rosati, g.; gustafsson, j.‐å.; link, h. title: anti‐inflammatory liver x receptors and related molecules in multiple sclerosis patients from sardinia and sweden date: 2008-06-28 journal: scand j immunol doi: 10.1111/j.0300-9475.2004.01423d.x sha: doc_id: 23407 cord_uid: s85g7g0x 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‐α was lower (p < 0.05) in ms (mean ± sem: 3.1 ± 0.2; n = 37) compared to hc (3.6 ± 0.1; n = 37). lxr‐α was lower in ms from stockholm (2.6 ± 0.2; n = 22) compared to corresponding hc (3.4 ± 0.1; n = 22; p < 0.01) and compared to ms (3.8 ± 0.2; n = 15; p < 0.001) and hc (4 ± 0.2; n = 15; p < 0.001) from sardinia. ms patients from stockholm, but not from sassari, also expressed lower (p < 0.05) lxr‐β (−4.1 ± 0.4) compared to corresponding hc (−2.9 ± 0.3). ms from stockholm was associated with higher abca‐1 (6.1 ± 0.4 versus 5.0 ± 0.3; p < 0.05) and higher estrogen receptor‐β‐cx (2.4 ± 0.4 versus 0.8 ± 0.4; p < 0.01) compared to corresponding hc. the hc from sassari had higher androgen receptor (2.9 ± 0.2) compared to ms from sassari (1.4 ± 0.3; p < 0.01), ms (1.3 ± 0.4; p < 0.01) and hc from stockholm (1.2 ± 0.3; p < 0.01). ms from sassari had lower cyclooxygenase‐1 compared to corresponding hc (5.1 ± 0.4 versus 6.6 ± 0.3; p < 0.01) and lower prostaglandin‐e (−0.03 ± 0.5) compared to the hc (1.4 ± 0.5; p < 0.05) and ms (2.7 ± 0.4; p < 0.05) and hc from stockholm (1.9 ± 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. 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-304626-ffao7vka authors: mellors, jack; tipton, tom; longet, stephanie; carroll, miles title: viral evasion of the complement system and its importance for vaccines and therapeutics date: 2020-07-09 journal: front immunol doi: 10.3389/fimmu.2020.01450 sha: doc_id: 304626 cord_uid: ffao7vka the complement system is a key component of innate immunity which readily responds to invading microorganisms. activation of the complement system typically occurs via three main pathways and can induce various antimicrobial effects, including: neutralization of pathogens, regulation of inflammatory responses, promotion of chemotaxis, and enhancement of the adaptive immune response. these can be vital host responses to protect against acute, chronic, and recurrent viral infections. consequently, many viruses (including dengue virus, west nile virus and nipah virus) have evolved mechanisms for evasion or dysregulation of the complement system to enhance viral infectivity and even exacerbate disease symptoms. the complement system has multifaceted roles in both innate and adaptive immunity, with both intracellular and extracellular functions, that can be relevant to all stages of viral infection. a better understanding of this virus-host interplay and its contribution to pathogenesis has previously led to: the identification of genetic factors which influence viral infection and disease outcome, the development of novel antivirals, and the production of safer, more effective vaccines. this review will discuss the antiviral effects of the complement system against numerous viruses, the mechanisms employed by these viruses to then evade or manipulate this system, and how these interactions have informed vaccine/therapeutic development. where relevant, conflicting findings and current research gaps are highlighted to aid future developments in virology and immunology, with potential applications to the current covid-19 pandemic. the complement system is a heat-labile component of native plasma involving both extracellular and cell surface membrane-associated proteins which form a major constituent of the innate immune response. the whole system is comprised of over 30 proteins which have the potential to react via an enzymatic cascade in response to recognition of various stimuli, such as pathogenassociated molecular patterns (pamps) and abnormal or damaged host cells. activation of the complement system typically occurs via three distinct target recognition pathways (the classical, lectin, and alternative pathways) which converge at a single point; the cleavage of complement component 3 (c3) . each pathway has its own unique protease zymogens to recognize and respond to different antigens, but all pathways primarily work to: opsonise pathogens, lyse pathogens and infected cells, regulate the inflammatory response, and enhance the clearance of immune complexes and cell debris (1, 2) . in the context of viral infections, the complement system has been shown to exhibit numerous antiviral mechanisms via direct neutralization of both enveloped and non-enveloped viruses, and/or the promotion of other immune responses. the complement system can directly neutralize virus particles through opsonisation (3) , membrane attack complex (mac) formation on the virion (4), mac formation on virus-infected cells (5) , or targeting of intracellular viral components for proteasomal degradation (6) . other immune responses may also be modulated by the complement system to promote viral clearance, including: the regulation of inflammation/chemotaxis (7) , the induction of the antiviral state (6) , and the enhancement of adaptive immune responses specific to viral antigens (8, 9) . the effectiveness and outcome of this response can vary depending on the infectious agent and host genetic variability. the classical complement pathway is typically activated when hexameric c1q proteins bind to the fragment crystallisable (fc) ch2-domains of antigen-bound igm and/or igg immune complexes (10-12). the binding affinity of c1q to igg is dependent on the igg isotype with the greatest affinity for igg-3, then igg-1, a weak association with igg-2, and no interaction with igg-4 (13) . however, for downstream activation and complement lysis activity, the response is more efficient following igg1-c1q interactions rather than igg3-c1q interactions (14) . c1q is also a versatile pattern recognition molecule. in absence of antibodies, c1q can directly bind to apoptotic cells (15) or proteins on the cell-surface membrane of some pathogens, such as human immunodeficiency virus (hiv) (16) and dengue virus (denv) (17) . c1q can also bind other host plasma proteins such as c-reactive protein (18) , fibronectin (19, 20) , decorin (21), lactoferrin (22), pentraxin-3 (23), and serum amyloid p component (24) . the c1q molecule is an assembly of six heterotrimers, each containing three chains (c1qa, c1qb, and c1qc) with a central collagenous stem and a globular head at the c-terminus. in native plasma, the c1q molecule forms a calcium-dependent complex with two c1r and two c1s serine proteases to form the c1 complex (25). ligand recognition and binding via the c1q molecule in the c1 complex induces a conformational change and autoactivation of the c1r 2 s 2 tetramer to activate the classical complement pathway (11, 12) . activated c1s cleaves complement proteins c4 and c2 into active fragments c4b and c2a, along with an inactive fragment (c2b), and a proteaseactivated receptor (par)1/par4 ligand (c4a) which increases endothelial cell permeability (26). non-covalent binding of c4b and c2a forms the classical pathway c3 convertase, c4bc2a, responsible for cleavage of c3 into c3a (anaphylatoxin) and c3b (active component of the convertase). the newly formed c4bc2ac3b complex is a c5 convertase formed from either the classical or lectin complement pathway activation, which cleaves the c5 molecule into c5a (anaphylatoxin) and c5b. c5 proteolysis and the successive steps are then the same for each of the three complement pathways -c5b is deposited onto the activating surface and subsequent, irreversible binding of c6, c7, c8, and multiple copies of c9 to permeate the cell surface membrane and form the mac (11, 12, 27) . all three complement pathways are summarized in figure 1 . the lectin complement pathway follows the same enzymatic cascade as the classical pathway but is distinct in the antigens and proteases required for its activation. the lectin pathway is activated in response to invading pathogens via direct binding of pamps by various c1q-like lectins, complexed with mannosebinding lectin (mbl)-associated serine proteases (masps)-1/2/3. these c1q-like activators are mbl, ficolin-1 (m-ficolin), ficolin-2 (l-ficolin), ficolin-3 (h-ficolin), and collectin-11 (cl-11) (28-30). in humans, mbl is typically present as a trimer, tetramer, pentamer, or hexamer and these oligomeric structures influence its carbohydrate binding properties (31, 32). each monomeric subunit in the complex is a homotrimer with each polypeptide containing a cysteine-rich region at the n-terminus, followed by a collagen-like domain, a neck region, and a carbohydrate recognition domain capable of binding specific sugars present on pathogenic surfaces i.e., n-acetyl-d-glucosamine and dmannose (33, 34). similar to mbl, multimeric ficolin complexes are assembled through homotrimer subunits with cysteine-rich n-terminal segments which form interchain disulphide bonds, followed by collagen-like regions, but they are unique in their ability to bind distinct carbohydrates via their c-terminal fibrinogenlike domains (35-37). ficolin-1 is predominantly synthesized in monocytes and granulocytes where it can be found present on their surface or extracellularly in native human plasma. ficolin-2 is synthesized in the liver and secreted into the bloodstream where it can bind to various acetylated structures and sugars via three inner binding sites (38). ficolin-3 is synthesized in the liver and lungs and is present in native plasma at a higher concentration than ficolins-1/2, although less is known about its functional capabilities (39) . collectin-11 (cl-11) can form heterotrimeric complexes with collectin liver 1 (cl-10) in serum and can also associate with masps to activate the lectin complement pathway (40) . once one or more of these lectins have complexed with masp-2 and bound their specified target, masp-2 then cleaves c4 and c2 to form the c3 convertase (c4bc2a complex). following the proteolytic cleavage of c3, the lectin complement pathway follows the same catalytic process as the classical pathway (figure 1 ) (2) . the roles of masp-1 and masp-3 in this pathway are relatively ambiguous (29, 41). masp-1 is capable of cleaving complement component c2 and, to a much lower extent, components c3 and c4 (29, 42), whilst masp-3 may have a negative regulatory role of the lectin pathway through downregulation of masp-2 cleavage activity (43) . the alternative pathway does not require the specific proteinprotein or protein-carbohydrate interactions seen with the other two pathways. under normal physiological conditions, ∼1% of c3 per hour undergoes spontaneous hydrolysis as figure 1 | overview of the complement system following activation via antigen (classical and lectin pathways) or spontaneous hydrolysis (alternative pathway). complement activation eventuates in formation of the membrane attack complex (mac) and the cleavage products regulate inflammation (c3a and c5a) and cell-mediated immunity (c3a, c3c, c3d, c5a). the internal thioester bond is cleaved to produce c3(h 2 o). this process is augmented in the presence of various surfaces which lack complement regulatory proteins, as electrostatic and/or hydrophobic interactions adsorb c3 to the surface to induce conformational changes (44, 45) . c3(h 2 o) can then bind factor b to induce another conformational change, as factor b is then cleaved into two components by factor d: ba (which dissociates from the complex) and bb (which remains bound in the complex). the protein complex c3bbb is the alternative pathway c3 convertase, which is stabilized through the binding of properdin to produce c3bbbp and can cleave further c3 molecules through the serine protease activity of fragment bb. the alternative pathway therefore has the potential to both activate and enhance complement activity through an amplification loop; cleaved c3 components produce c3 convertases which cleave further c3 molecules (46, 47) . cleavage of c3 also yields c3a and c3b, where c3b remains bound in the complex to form the alternative pathway c5 convertase, c3bbbpc5b, and c3a acts as an anaphylatoxin. the rest of the complement cascade is then identical for all pathways (figure 1 ) (48, 49) . although complement activity typically occurs via the three pathways described, less conventional mechanisms of activation and immune modulation can occur, and have been discussed in a recent review (50) . typically, properdin is described as a positive regulator of the alternative pathway through stabilization of the c3 and c5 convertases. but its functions extend beyond this, including complement activation via direct antigen recognition of invading pathogens and apoptotic/necrotic cells (51) (52) (53) , and as a potential ligand for nkp46-mediated natural killer cell activation and subsequent secretion of x-chemokine ligand 1 (54) . similarly, c3 and its cleavage products are often described as extracellular components, yet they can have intracellular signaling roles to eliminate pathogens, alter cytokine signaling profiles and induce th1 responses (6, (55) (56) (57) . most complement proteins are primarily synthesized in the liver and secreted into the bloodstream; this process is rapidly upregulated during infection (58) . complement proteins can also be produced by epithelial cells (59) , endothelial cells (60) , and circulating immune cells such as dendritic cells, granulocytes, macrophages, and monocytes (61, 62) . local production of complement also occurs in immune privileged sites including the brain (63), eyes (64) , and testis (65) . to regulate this activity and prevent damage to healthy cells and tissues, many regulatory proteins are primarily expressed as either soluble plasma proteins or cell-surface receptors (figure 2 and table 1 ). complement regulatory proteins may be unique to a certain pathway or influence the downstream activity of all three pathways. factor h, factor i, and properdin are unique to the alternative pathway. factor h is both a soluble and cellsurface membrane regulator (124) which accelerates the decay of the c3 convertase (c3bbb) to reduce complement deposition (125) , and it functions as a factor i cofactor to cleave c3b and c4b components (111) . properdin is a positive regulator of the alternative pathway which stabilizes the c3 convertase (c3bbb) and promotes its association with further c3b molecules (129) . the c1-inhibitor (c1-inh) is a negative regulator of all three pathways. c1-inh competes with factor b to limit activation of the alternative pathway (110) , inhibits c1r and c1s to prevent classical pathway activation (41) , and inactivates masp-1 and masp-2 to prevent lectin pathway activation (41) . further proteins are required to regulate the downstream complement activity. carboxypeptidase-n/r regulates the anaphylatoxin activity of c3a and c5a via cleavage of their arginine residues (115) . c8 binding protein (114) , clusterin (123) , and vitronectin/s protein (130) are all soluble proteins which prevent the complete assembly of the mac. cd46/membrane cofactor protein (106, 107) , cd55/decay-accelerating factor (116, 117), and cd59/protectin (119) are ubiquitously expressed on the surface of host cell surface membranes to protect the cell from complement deposition. one of the key functions of the complement system is to assist in the killing and containment of invading pathogens, including bacteria (131), fungi (132) , protozoa (133) , and viruses (134) . previous reviews have discussed various evasion mechanisms adopted by viruses to dysregulate or evade this complement activity (135) (136) (137) (138) . this knowledge may then be exploited and has previously identified novel methods for vaccine and frontiers in immunology | www.frontiersin.org therapeutic intervention-an area which has not been extensively discussed in the previous reviews. the antiviral mechanisms of complement have been divided into four main sections in this discussion; physiologically however, each section is not exclusive as they work together to form a complete system. briefly, complement deposition on a virion can block interactions with host cell receptors, aggregate virus particles, signal intracellularly to induce an antiviral state, and enhance phagocytosis (3, 6, 139) . this can lead to formation of the mac and lyse lipid membranes of enveloped viruses (140) or lyse infected host cells expressing viral antigens (141) . activation of the complement system also produces pro-inflammatory anaphylatoxins (c3a, c5a, and putatively c4a) which can enhance phagocytosis and in some cases, worsen disease symptoms (142) . lastly, these processes can enhance the adaptive immune response to viral antigens, induce a th1 response (56) , modulate treg and th17 responses (143) , prolong b-cell memory and significantly increase antigen-specific antibody titres (144) . many of these functions may be evaded or manipulated by different viruses (shown in figure 3) and such examples are provided throughout. all three complement pathways can lead to virus opsonisation and complement deposition following activation. the outcome of this response largely depends on the infectious agent and could enhance viral infection, suppress viral infection, or be dysregulated by the expression of some viral proteins. the mbl protein of the lectin pathway can interact with numerous viral antigens and have varying effects on neutralization or viral enhancement. mbl can directly bind the ebola virus (ebov) glycoprotein (gp). high doses of mbl, relative to other complement proteins, can enhance ebov-gp pseudotyped virus infection into primary human macrophages and human monocyte-derived macrophage cell lines (145) . however, mbl opsonisation of the ebov-gp can neutralize ebov-gp-pseudotyped virus by preventing cell interactions via dc-sign (3). mbl has also been successfully used as a rescue therapy in 40% of mice when administered at supraphysiological levels, 24 h post-lethal challenge with a mouse adapted ebov strain (146) . so, in the context of ebov infection, the effects of mbl appear to be dependent on the cellular mbl has also been shown to bind the hiv-1 protein, gp120. this interaction was sufficient to neutralize cell-line adapted hiv infection of cd4+ h9 lymphoblasts (134) . a later study reported a similar finding, although much higher concentrations of mbl were required to achieve the same level of neutralization (50 µg/ml rather than 1 µg/ml of mbl), and these findings were not replicated when using hiv primary isolates or other cell lines for infection. in the later study, mbl was shown to be sufficient for virus opsonisation but not neutralization (147) . this highlights an important consideration for in vitro studies when investigating complement and pseudovirus interactions, as small method variations can yield significantly different results. where possible, in vivo experiments can help validate this work and address possible discrepancies. further possible implications of mbl during hiv infection have been shown in a study of single nucleotide polymorphisms (snps). snps in the mbl gene which result in low serum concentrations of mbl were associated with increased risk of hiv infection and poorer prognosis following aids diagnosis (148) . downstream from mbl binding, complement components are deposited on hiv virions which increase viral uptake and internalization into dendritic cells (dcs). both complementopsonised and complement-free hiv binding was reduced through the blockage of c-type lectins, integrins and cd4. however, the use of individual blockers showed that complement-opsonised hiv utilized β1-and β2-integrin for binding and uptake, whereas complement-free hiv utilized β2and β7-integrin (149) . a similar observation has been reported for herpes simplex virus (hsv)-2 during the infection of human dcs. complement deposition and interactions with complement receptor 3 (cr3) enhanced hsv-2 infection of immature dcs and increased the production of new virus particles, whereas complement with the use of neutralizing antibodies significantly reduced infection (150) . this highlights another important point with regards to in vitro investigations of complement and viral infection. plasma is often heat-inactivated for use in cell culture to overcome concerns of complement-mediated cytotoxicity. consequently, investigations of virus-host cell interactions may overlook important complement-mediated interactions that would normally be present during infection. the varied effects of mbl opsonisation during viral infection have also been described for severe acute respiratory syndrome coronavirus (sars-cov). multiple studies have shown the potential for mbl to bind immobilized sars-cov or the sars-cov spike protein (151, 152) . this interaction was shown to be dependent on a single n-linked glycosylation site of the spike protein and this binding could prevent spike protein interactions with dc-sign but not the angiotensinconverting enzyme 2 (ace2) receptor or cathepsin-l (152). ip et al. showed that mbl binding to immobilized sars-cov could also inhibit sars infection into fetal rhesus kidney cells and enhance deposition of c4 (151) . however, leth-larsen et al. did not observe any interactions between mbl and sars-cov spike protein in their study (153) . similar to hiv, several studies have found a significant difference of mbl snps associated with lower or deficient mbl serum levels in sars patients compared to healthy chinese population control groups (151, 154) , and a reduction of mbl protein concentrations in sars patient sera (151) . however, one other study observed no significant correlation of mbl-deficient snps in sars patients compared to healthy chinese population control groups (155) . the role of mbl in sars-cov infections appears conflicted but could be significant. as later discussed, the downstream effects of complement activation do significantly influence symptoms of coronavirus infections. other complement proteins and downstream products of its activation can opsonise virus particles. for denv and west nile virus (wnv), neutralization of the virions occurs in a c3 and c4 dependent manner following mbl binding. for wnv, neutralization was achieved independent of downstream c5 and therefore did not require formation of the mac (156). for simian virus 5 (sv5), complement-mediated neutralization is predominantly achieved through c3 deposition and the formation of virion aggregates, rather than virion lysis. for the closely related mumps virus (muv) however, the opposite effect is observed with few aggregates formed but greater susceptibility to complement lysis (157) . similarly, complement activation in the presence of influenza a virus causes virion aggregation and opsonisation of the hemagglutinin receptor. although to achieve neutralization, igm antibodies and activation of the classical pathway is required (139) . for chandipura virus (chpv), the alternative pathway and factors c3, c5, and factor b were required for complementmediated virus neutralization in absence of c8 or antibodies (158) . a different study utilized antibodies to observe classical pathway activation and reported that c1q, c3, and c4 were essential components for neutralization, but this was independent of factor b and c8 (159) . the discrepancy of the importance of factor b for chpv neutralization could depend on the presence of antibodies and the classical pathway. as mentioned previously, complement opsonisation of virions can enhance infection through interactions with complement receptors on host phagocytic cells (149, 160) . however, some complement proteins can have a protective intracellular function as well, which is independent of cell-type (6) . enveloped viruses may naturally evade the intracellular functions of complement, as the protein deposition would occur on the lipid membrane. so, for viral entry via membrane fusion or endocytosis, it is expected that the complement-opsonised viral envelope would be left on the host cell surface membrane or endosome plasma membrane. this has been demonstrated in vitro using respiratory syncytial virus (rsv), an enveloped virus which enters the cell via membrane fusion, where complement intracellular signaling was absent following infection (6) . the intracellular immune function of complement has a better-defined role for non-enveloped viruses, although the area of intracellular complement immunity is still relatively new (161) . in a c1-dependent manner and independent of downstream complement activity, c4 deposition on the capsid of non-enveloped human adenovirus 5 has been shown to contain the virus within the endosome, by blocking the fiber shedding and protein vi exposure mechanisms required for capsid disassembly (162) . the use of an adenovirus type 5 vector (adv) also showed that intracellular sensing of complement could inhibit infection and degrade the virus particle (6) . a comparison of complement-coated adv to adv only, showed that intracellular c3 signaling induced the activation of proinflammatory cytokines (ifn-β, il-6, il-1β) through nf-κb, interferon-regulatory factor (irf), and activating protein-1 (ap-1) transcription factor activation. intracellular c3 sensing was shown to be mitochondrial antiviral-signaling protein (mavs)dependent, and independent of pamps and pattern recognition receptors. sensing of complement-coated adv also targeted the virion for degradation by valosin-containing protein (vcp) and the proteasome. c3-mediated signaling could induce an antiviral state in previously uninfected cells, as the supernatant from complement-coated adv infected cells was able to protect uninfected hela cells from infection with interferonsensitive sindbis virus. lastly, some viruses have evolved evasion mechanisms to overcome the complement-mediated intracellular immune response. rhinoviruses and polioviruses were shown to inhibit the intracellular c3 complement signaling mechanism through the expression of a cytosolic 3c protease to degrade c3 (6) . discussed in more detail below, some viruses encode proteases which enhance degradation of the c3 convertase to prevent further complement deposition or mac formation. this can protect the virion from complement opsonisation and viral lysis. following complement deposition and opsonisation, the complement cascade can progress to assembly of the mac. mac formation can perturb and lyse lipid membranes of enveloped viruses or destroy infected cells expressing viral antigens to reduce viral load (4, 5, 140, 141) . again, viral proteins can be expressed to dysregulate and evade this response. zika virus (zikv) can lead to classical pathway activation via formation of antigen-antibody complexes or through direct binding of c1q. for zikv derived from insect cell lines, this interaction resulted in mac formation and a reduction of viral titres in vitro. however, zikv derived from human cell lines were more resistant to complement mediated neutralization (4) . zikv and other flaviviruses (including yellow fever virus (yfv), denv, and wnv) express and secrete the non-structural protein 1 (ns-1) to regulate complement activity. the ns1 protein has a wide variety of functions in complement regulation which include: antagonism of c4 (163), recruitment of host c4 binding protein (164) , recruitment of host factor h (165), recruitment of host vitronectin and inhibition of c9 polymerisation (166) . however, the denv ns1 protein is also capable of complement activation and the resulting soluble c5b-9 complexes have been found to correlate with disease severity in patients with dengue shock syndrome (167) . this discrepancy was addressed with the possibility that relative igm, c4 and soluble ns1 concentrations in plasma, at different sites of infection, could influence the extent of inhibition and therefore have varied effects on complement activity (163) . similarly, nipah virus (niv) exhibits factor i-like activity, either through acquisition of factor i host protein or through inherent protease activity. unlike soluble factor-i, niv exhibits no capacity for c4b cleavage and showed no significant cleavage of c3b with a cd46 cofactor, despite its integration in the niv lipid membrane. however, niv is capable of c3b cleavage into ic3b with factor i cofactors (factor h and soluble cr1) to protect against virus neutralization (168) . chikungunya virus (chikv) also exhibits factor i-like activity, likely of viral origin and dependent on host factor h concentrations, to cleave c3b into inactive ic3b and resist complement-mediated neutralization (169) . muv, sv5, and hiv-1 can all recruit host cell cd46 into the viral lipid membranes during the budding process to protect from complement deposition and neutralization (170, 171) . hiv-1 also incorporates glycosyl phosphatidylinositol-anchored cd55 and cd59 for further protection from complement mediated neutralization (170) . conversely, complement deposition has been shown to enhance hiv-1 infectivity into peripheral blood mononuclear cells through interactions with complement receptors (160, 172) . this highlights the complexity of complement and viral interactions with dualistic mechanisms, which has previously been reviewed in the context of hiv-1 infection (173) . infected host cells which present viral antigens on the cell surface membrane can activate the classical pathway, as the antigens bind igm/igg to induce complement dependent cytotoxicity (cdc). the infected cell is then lysed via the mac in an attempt to reduce viral load. for influenza a virus infection, complement-dependent lysis (cdl) monoclonal antibodies can cross-react with h1 and h2 hemagglutinin subtypes for broader protection than neutralizing monoclonal antibodies (141) . similarly, broadly neutralizing anti-hiv-1 antibodies can bind the viral envelope protein expressed on infected primary lymphocytes to initiate complement deposition. the deposition does not result in a rapid lytic effect but neutralizes viral spread to further cells (174) . for hsv-1 and hsv-2, the glycoprotein c (gc)-1 is expressed to protect virions and infected cells from complement mediated neutralization. the gc-1 protein binds c3, c3b, and c3c to prevent subsequent binding of c5 or properdin. modification of gc-1 on hsv infected cells can therefore increase their susceptibility to antibody neutralization and cdc (5, 175) . some of the cleavage products from complement activation can function as anaphylatoxins and have broader immune regulatory functions. primarily, cleavage products c3a and c5a can be generated via all three pathways and act as potent immune regulators, whilst c4a is generated via the classical and lectin pathways only (7) . the role of c4a as an anaphylatoxin is disputed as it currently has no known anaphylatoxin receptor associated with its activity (176) . however, it does function as an effector protein that is derived from complement activation, which enhances endothelial cell permeability and increases stress fiber formation via par1 and par2 (26). the roles of c3a and c5a are better described as anaphylatoxins, with the latter demonstrating higher stability and broader biological activity. c5a recruits neutrophils to the site of inflammation and both c3a and c5a can recruit: eosinophils, fibroblasts, macrophages, mast cells, and monocytes (70) (71) (72) (177) (178) (179) . these two anaphylatoxins demonstrate a large functional overlap but each have their own discrete functions. to varying degrees, both are capable of stimulating the production of pro-inflammatory mediators from monocytes and macrophages via inflammasome-caspase-1 activation (180, 181) . both can induce the degranulation of mast cells (182) (183) (184) (185) , basophils (186) (187) (188) , and eosinophils (189) . both induce respiratory bursts in eosinophils (190) and neutrophils, although only c5a shows chemotactic activity for neutrophils whereas c3a may actually prevent neutrophil mobilization from the bone marrow (191) . further, only c5a can stimulate respiratory bursts in macrophages (192) . the activity of c3a and c5a is mediated via binding to two main g-protein coupled receptors; c3ar or c5ar, respectively (193) . a secondary, non-g-protein coupled receptor (c5l2) has been shown to bind c5a and potentially regulate its biological functions in vitro, although its primary functions are not yet clear (105) . these receptors are widespread across different cell types including both myeloid cells and non-myeloid cells (e.g., astrocytes, microglia, hepatocytes, endothelial and epithelial cells) to produce various biological functions dependent on the cell type (194, 195) . c3a and c5a activity is further regulated by the enzyme carboxypeptidase-n. carboxypeptidase-n cleaves the carboxy terminal arginine amino acid of these anaphylatoxins to generate products with greatly reduced (c5a-desarg) or absent (c3a-desarg) anaphylatoxin activities (196) . the cleavage product c5a-desarg retains some chemotactic activity to recruit distant immune cells (188, 197) whilst c3a-desarg can function as a hormone for lipid metabolism (198) . beyond their roles in chemotaxis, c3a and c5a have been associated with: the induction of smooth muscle contraction (199) , regulation of vasodilation (200) , an increase in vascular permeability (201) , and the production of various cytokines including il-1β, il-8/cxcl-8, ccl5, il-6, tnfα (180, 193) . during viral infection, excessive complement activation leading to a strong pro-inflammatory response is often associated with more severe disease symptoms. the negative impact of complement activation has been associated with more severe symptoms during sars-cov and mers-cov infections. infection of c3 deficient mice with sars-cov revealed that the loss of complement activity resulted in milder disease outcomes (202) . compared to the wild-type, the c3 deficient mice showed: no significant weight loss, improved respiratory function, reduced lung pathology, and lower levels of inflammatory cytokines and chemokines (202) . proteomic analysis has shown that a product of complement activation, c3c α chain, was significantly higher in sars-patient sera compared to non-sars patient sera (203) . similarly, increased concentrations of c5a and c5b-9 were observed in sera lung tissues of hdpp4transgenic mice challenged with mers-cov. the subsequent use of a c5ar antibody to prevent c5a functional activity resulted in reduced tissue damage and a lower viral load (204) . cytotoxic effects of complement may also occur post-sars-cov infection. autoantibodies elicited 1-month after infection against epithelial and endothelial cells can mediate complementdependent cytotoxicity and enhance lysis against a549 cells and human placenta endothelial cells (205) . in patients with severe denv infection and dengue shock syndrome, overactivity of the alternative pathway has been reported with increased levels of ns1, c5a, and sc5b-9 in pleural fluids, which likely contribute to the symptoms of increased vascular permeability (167, 206) . in denv infected cells, indicators of the alternative complement pathway are upregulated, with a relatively higher concentration of factor b to factor h proteins and increased cell surface c3b deposition (206) . in mice infected with ross river virus (rrv), complement activation products have been identified in serum and inflamed tissues. similar observations have been made in the synovial fluid of rvv-infected patients. in c3 knockout mice, the signs of severe disease and tissue damage from rvv infection were diminished compared to wild-type, which suggests complement promotes rrv-induced inflammation (207) . rrv infected cells express the viral e2 protein which is glycosylated with n-linked glycans. e2 n-linked glycans are antigens for mbl and can activate complement via the lectin pathway, which results in greater inflammation and tissue damage during rvv infection (208, 209) . complement activation also plays an important role in linking the innate and adaptive immune responses. this interaction can enhance the production of antigen-specific antibody titres and shape the t-cell response to target viral pathogens more efficiently. the importance of complement in the regulation of t-cell immunity has previously been reviewed (50, 210) . cognate and co-stimulatory interactions (cd80-, and cd86-cd28, and cd40-cd40 ligand) between antigen presenting cells (apcs) and t-cells results in the local production of c3, factor b, factor d, and c5. receptors c3ar and c5ar are also upregulated on the t-cell surface whilst production of decay accelerating factor (daf) is down-regulated. the local production of complement components from immune cells enables signaling via c3ar and c5ar in an autocrine and paracrine manner. complement c3 can also be processed intracellularly, or internalized as c3 (h 2 o) from the alternative pathway, to increase pro-inflammatory cytokine expression from t-cells and recycled back to the t-cell surface (55, 57) . a major component of c3 cleavage on the t-cell surface is ic3b. tcell membrane bound ic3b binds to cr3 (and possibly cr4) on monocyte derived dcs to enhance t-cell proliferation (9) . absence of c3ar and c5ar leads to: reduced complement protein and receptor regulation, lack of co-stimulatory molecule expression, impaired cytokine production (il-1, il-23, and il-12), an induction of an itreg cell response, and suppression of t-cell proliferation (211) (212) (213) . activation of both c3ar and c5ar on dcs by their respective anaphylatoxins (c3a and c5a) can mediate the production of il-6, il-23, the il-12 receptor, and tgf-β1 to promote tcell differentiation into antiviral th1 and th17 subsets (143) . induction of the th1 response also depends on c3ar and cd46 activation on t-cells via their t-cell derived ligands (56) . in mice infected with influenza a virus, inhibition of the c5ar lead to a reduction in influenza-specific cytotoxic cd8 + t-cells (214) and c3 deficiency lead to increased viral titres and delayed viral clearance (215) . c3 is also required for the production of antigen-specific cd8 + t-cell responses during lymphocytic choriomeningitis virus infection in mice (216) . during hcv infection, the hcv core protein can interact with gc1qr on host immune cells and suppress the t-cell response. this interaction inhibits t-cell proliferation in a dosedependent manner to downregulate cd69 activation and reduce the production of ifn-γ and il-2 from t-cells (217) (218) (219) . hcv core protein interaction with gc1qr on monocyte-derived dcs inhibits il-12 production and promotes th2 cytokine production to limit differentiation into th1 cells (218) . hcv core protein interaction with gc1qr on b-cells has a differential response to the one observed on t-cells and dcs, as it increases cell surface costimulatory and chemokine receptor expression and enhances b-cell proliferation (219) . furthermore, the hcv core protein exhibits intracellular functions, as it can suppress the t-cell factor-4 transcription factor required for c9 promoter activity regulation. this reduces c9 mrna and protein levels which are required for complete mac assembly (220) . as discussed previously, complement activation can result in c3 deposition on the surface of virions. c3 and its cleavage products can interact with the b-cell receptor and b-cell coreceptor complex (cr2/cd21 ligated with cd19 and cd81) to lower the b-cell activation threshold by several orders of magnitude. this can dramatically increase antibody titres, modulate the proliferation of mature b cells, and protect the b-cells from cd95-mediated elimination (8, 144) . immune complexes coated in c3 and c3 cleavage products covalently interact with complement receptors on follicular dcs (fdcs). the c3-coated immune complexes on fdcs are then presented to b-cells in the germinal center for optimal b-cell responses, including: antibody production, somatic hypermutation, class switching, and affinity maturation (87, 221) . fdcs can then retain the c3-coated complexes within the lymphoid for extended periods of time to generate memory b-cells and promote survival (222) . alternatively, some aspects of the complement system can suppress certain responses of adaptive immunity: stimulation of cr3 on dcs can suppress the release of inflammatory cytokines (98) and c1q-differentiated dcs demonstrate an increased phagocytic capacity but reduced expression of cd80, cd83, and cd86 required for t cell activation (223) . it is apparent that the complement system has important implications for virus neutralization and development of the adaptive immune response. as our knowledge of virus and complement interactions improves, this can inform novel approaches for intervention and the development of therapeutics and vaccines. one such example is the use of rupintrivir against rhinoviral infections. rhinoviruses encode a cytosolic 3c protease which cleaves intracellular c3 to avoid the intracellular mechanisms of complement, mentioned previously. rupintrivir inhibits the viral cytosolic 3c protease to increase susceptibility of the virus to intracellular complement immunity (6) . similarly, the use of fab fragments could prevent the c4 inhibition of human adenovirus 5 vector for its use in adenoviral gene therapy to promote efficient transgene delivery (162) . due to the multifaceted and complex immune functions of the complement system, direct manipulation of complement would need to be carefully considered. inhibition of the complement system could increase susceptibility to other diseases, whilst overstimulation could result in autoimmunity and damage to host cells. a method of complement stimulation through inhibition of the cd59 regulator has been proposed for the treatment of latent hiv-1 infection in cells. the use of provirus stimulants and a cd59 inhibitor showed a dose-response effect of cell sensitization to antibody-dependent cell-mediated lysis and reduced viral load. aside from the target cells, no significant non-specific cytolytic effects were observed in vitro. cd59 protects host cells from complement activity, is ubiquitously expressed, and so its inhibition has the potential to damage host cells (224, 225) . deletion of cd59 in mice did not have a lethal outcome, however absence of the complement regulatory protein did lead to intravascular haemolysis and thrombosis (226) . treatment in the context of hiv-1 infection would be short-term however (224) and could be an exception for an otherwise incurable disease. similar approaches have been considered for other lifethreatening diseases such as cancerous conditions (227, 228) . methods of complement inhibition have also demonstrated therapeutic benefit. mentioned previously, excessive complement activation is associated with more severe outcomes of mers-cov and sars-cov infections. use of a c5ar antibody to block the pro-inflammatory effects of c5a in mers-cov infected hdpp4-transgenic mice resulted in: lower concentrations of proinflammatory cytokines, reduced viral replication in lung tissues, reduced lung and spleen tissue damage, and a reduction of viral antigen and microglia activation in the brain (204) . excessive complement activation and similar lung pathology during sars-cov infection has also been observed in h5n1 influenza cases, where the use of c3ar and c5ar antagonists reduced signs of acute lung injury and viral load in h5n1-infected mice (229) . a novel coronavirus, sars-cov-2, has recently emerged and is the causative agent of covid-19-an acute self-limiting disease which has the potential to progress to severe disease and death. symptoms of severe disease involve major alveolar damage, wide-spread lung inflammation, and progressive respiratory failure (230, 231) . the pathological features of lung, liver, and heart tissue in a severe case of covid-19 greatly resembled those seen in sars-cov and mers-cov infections which are complement-mediated (202) (203) (204) (205) 231) . mbl has been shown to activate complement via binding to sars-cov spike protein in some studies and this could translate to the sars-cov-2 spike protein, which contains n-linked glycosylation sites that are targets for mbl (151, 232) . thus, the widespread lung inflammation observed in severe cases of covid-19 could be exacerbated by excessive complement activation. furthermore, viral infections with similar lung pathology to covid-19 have demonstrated therapeutic benefit with the administration of complement inhibitors targeting c3a/c3ar or c5a/c5ar. this has been shown for h5n1 (229) , h7n9 (233) , and mers-cov (204) infections. so, it seems plausible that the lung inflammation in severe cases of covid-19 is exacerbated by excessive complement activation and this pathologic inflammation could be attenuated through use of complement inhibitors. clinical trials are currently being conducted with the use of a c5a inhibitor, the monoclonal antibody ifx-1, which has proven to be well tolerated in 300 clinical trial participants and aims to reduce inflammation whilst preserving mac formation (234) . as sars-cov-2 is an enveloped virus (235) , it is possible that mac formation could have some beneficial antiviral effects. therefore, a c5a inhibitor such as ifx-1 (inflarx) may be favorable mechanistically over a c5 inhibitor such as eculizumab (soliris), which is also considered for use in clinical trials (nct04288713) (236) . the ifx-1 monoclonal antibody targets a specific conformational epitope of the c5a molecule to block its anaphylatoxin activity, whilst c5b and downstream complement activity are preserved (237) . eculizumab is a monoclonal antibody which targets the c5 molecule to prevent cleavage into c5a and c5b, and therefore inhibits all downstream complement activity (238) . the distinction between the two antibodies is the preservation of mac activity which could be relevant against the enveloped sars-cov-2, although the effects of complement lysis and whether it occurs on sars-cov-2 is not yet known. because the efficacy and safety of eculizumab is already well characterized (239) , it is logical that this would take precedence over lesser-known options for urgent clinical trials. the use of eculizumab has already proven beneficial for treatment of severe cases of covid-19, which shows that complement is partly responsible for the symptoms in severe cases (240) . it would be interesting to compare the effects of preserving the mac during infection with the enveloped sars-cov-2, as it may offer an antiviral, as well as an anti-inflammatory, effect. but it is also possible that coronaviruses have an intrinsic evasion mechanism, perhaps similar to the ones described in this review, to avoid the lytic activity of the mac. another important consideration could be the stage of infection for implementing complement inhibitors: maintaining complement activity may have a beneficial impact early on in infection for virus neutralization and the development of adaptive immunity, and intervention may only be required to treat excessive inflammation in severe cases. the complement system has several important considerations for vaccine development, one example being its involvement in antibody dependent enhancement (ade). ade is commonly observed when non-neutralizing antibodies are present following initial priming of the immune system. non-neutralizing antibodies can still bind the viral target with the potential to cross-link with fc receptors, or activate complement and interact with complement receptors, to enhance viral infection of host cells (241) . ade is more commonly observed to be fc receptor-mediated, however complement-mediated ade has been reported for hiv-1 (242), mers-cov (243) , and ebov (244) . but complement activation can have a positive effect against viral infections in the presence of some non-neutralizing antibodies. use of the non-neutralizing influenza virus m2 extracellular vaccine in mice required functional c3 to confer protection and induce effective humoral and cell-mediated immune responses (245) . a similar effect has been reported for monoclonal antibodies against human cytomegalovirus (hcmv). following the use of gb/mf59 hcmv vaccination in humans, the immune sera had enhanced neutralization potency toward hcmv in the presence of complement. certain hcmv monoclonal antibodies rely on complement for viral neutralization, which appears distinct from cdc or virolysis, and is likely the result of blocking virus-host interactions (246) . complement activity has also been implicated for optimal protection with non-neutralizing antibody mab-13g8 against crimean-congo haemorrhagic fever virus infection in adult mice (247) . in flavivirus infections, the mechanism of ade is predominantly shown to be fc mediated (248) . complement has been shown to augment antibody-mediated neutralization of wnv in vitro (249) and the addition of c1q has been shown to lower the antibody concentrations required for wnv neutralization in vitro, which correlated with protective effects observed in vivo (250) . c1q was also shown to mediate effects of ade from flavivirus infections in a subclass specific manner, whilst mbl, factor b, or c5 depletion had no significant effect (251) . although igg subclasses are known to bind c1q with varying avidities, the mechanism to explain this effect on ade has not been identified. this could highlight the importance of selecting the right antibody subclass when considering monoclonal antibody therapies. in general, vaccines which effectively engage the complement system may gave rise to a more potent, virolytic serological response. for hiv vaccination in macaques, the presence of complement augmented virus neutralization and complement-mediated neutralizing antibody titres correlated with vaccine-mediated protection (252) . other approaches have modified vaccines to utilize aspects of the complement system for increased antigen immunogenicity, such as complement component c3d. c3d is an end-stage cleavage product from c3 activation which interacts with cr2 on b-cells, t-cells, and fdcs. when bound to an antigen, c3d can dramatically reduce the b-cell activation threshold for a stronger, more antigen-specific antibody response (8, 144, 253) . cr2 on fdcs interacts with ic3b, c3d, and c3dg to enhance antibody titres and promote long-term b-cell memory development (254) . c3d also bears t-cell epitopes so even with a lack of cr2 expression, the peptide can be internalized and presented on hla ii molecules to autoreactive t-helper cells and enhance antibody responses (255, 256) . c3d does not interact with other components of the complement system and so the associated risks are reduced, however a large enough reduction in the b-cell activation threshold could potentially lead to antibody-mediated autoimmune responses. c3d has been used as a vaccine adjuvant against several different viruses. dna vaccines encoding the envelope glycoprotein of porcine reproductive and respiratory syndrome virus were more effective at increasing antigen specific neutralizing antibody titres, ifn-γ levels, and il-4 levels when engineered with gene copies encoding the cr2 binding site of c3d in the same plasmid construct (257) . similarly, use of hepatitis e virus peptide (hev-p179) for dna vaccination in mice had enhanced anti-hev-p179 antibody titres and avidity when fused with three tandem c3d copies as genetic adjuvants (258) . c3d has also been used as genetic adjuvant for dna vaccines against newcastle disease virus and hiv-1 for increased efficacy and higher, longer-lasting antibody titres (259, 260) . fusion of c3d to target antigens is another approach for the development of safer, more immunogenic dna vaccines. coupling of c3d to the secretory form of influenza virus haemagglutinin in mice provided an effective and safer mechanism for mucosal vaccination compared to the use of other adjuvants i.e., cholera toxin b subunits and escherichia coli labile toxin (261) . so, the use of c3d as an adjuvant can help to overcome the low immunogenicity associated with dna vaccines, whilst maintaining their safety. many of the viruses discussed can activate complement, resulting in beneficial and/or detrimental effects on its survival. in the examples where viral-mediated complement activation has been more extensively studied, a viral mechanism is often identified which protects the virus from certain antiviral functions, such as the acquisition of cd46, cd55, cd59 to protect from mac formation or the expression of a regulatory protein to inhibit the complement cascade at various points. for the viruses which have been shown to activate complement but do not have a clear evasion/regulatory mechanism, such as mers-cov (204), sars-cov (202, 205) , ebov (3), and possibly sars-cov-2, it is plausible that the mechanism simply has not yet been identified. the viruses which activate complement would consequently trigger the downstream antiviral effects, both intracellularly and extracellularly. therefore, it seems plausible that these viruses would utilize a mechanism, similar to the ones described in this review, to evade this antiviral activity and promote their survival. if such a regulatory protein or process is identified, then these may present as possible antiviral targets, similar to the targeting of the rhinovirus 3c protease with rupintrivir (6). the complex interplay between viruses and the complement system can have profound implications for protection via innate immunity and the development of effective adaptive immunity. the effects of the complement system can vary between viral infections, and even during the different stages of the same viral infection, so a clear understanding of these mechanisms is important to improve efficiency of vaccine/therapeutic development whilst mitigating risk. such developments can also be applied for non-viral pathogens (including bacteria, fungi, protozoa) and to broader, more systemic functions of the complement system including: interferon signaling (262, 263) , metabolism (264), brain development (265) , and the coagulation system (266) . components of the complement system form an ancient aspect of innate immunity in vertebrates (267) and even some invertebrates (268, 269) . therefore, many animals which act as viral hosts or reservoirs for zoonoses also have an active complement system for targeting pathogens i.e., bats (270) , cows (271) , deer (272) , pigs (273) , rabbits (274) , and rats (274) , which the virus may have to overcome to avoid possible antiviral activity. further viral mechanisms of complement regulation may therefore exist which have not yet been identified and the plasticity of viral genomes could result in the emergence of novel protein regulatory functions. identifying these novel interactions could be important for the development and augmentation of vaccines and therapeutics or even the possibility of utilizing viralderived regulatory proteins as therapeutic complement inhibitors in other diseases (137) . the benefits from understanding complement mechanisms in viral diseases may have relevance for the current sars-cov-2 outbreak. previous research has demonstrated the impact of the complement system in coronavirus infections and other diseases, and this knowledge has led to the consideration of several complement inhibitors as therapeutics for severe cases of covid-19. jm wrote the manuscript and designed the tables and figures. tt, sl, and mc provided guidance and revised the manuscript. all authors contributed to the article and approved the submitted version. the complement system the complement system: history, pathways, cascade and inhibitors mannosebinding lectin binds to ebola and marburg envelope glycoproteins, resulting in blocking of virus interaction with dc-sign and complementmediated virus neutralization active human complement reduces the zika virus load via formation of the membrane-attack complex glycoprotein c of herpes simplex virus type 1 prevents complementmediated cell lysis and virus neutralization intracellular sensing of complement c3 activates cell autonomous immunity complement anaphylatoxins (c3a, c4a, c5a) and their receptors (c3ar, c5ar/cd88) as therapeutic targets in inflammation proliferation of resting b cells is modulated by cr2 and cr1 human t cell derived, cell-bound complement ic3b is integrally involved in t cell activation novel evasion mechanisms of the classical complement pathway dissection of c1q capability of interacting with igg timedependent formation of a tight and only partly reversible association human monoclonal igg isotypes differ in complement activating function at the level of c4 as well as c1q direct binding of c1q to apoptotic cells and cell blebs induces complement activation human immunodeficiency virus type 1 activates the classical pathway of complement by direct c1 binding through specific sites in the transmembrane glycoprotein gp41 c1q binding to dengue virus inhibits infection of thp-1 and cellular inflammatory responses evidence that complement protein c1q interacts with c-reactive protein through its globular head region fibronectin binds to the c1q component of complement new functional ligands for ficolin-3 among lipopolysaccharides of hafnia alvei heteromeric complexes of native collectin kidney 1 and collectin liver 1 are found in the circulation with masps and activate the complement system proteolytic activities of two types of mannose-binding lectin-associated serine protease natural substrates and inhibitors of mannan-binding lectin-associated serine protease-1 and−2: a study on recombinant catalytic fragments masp-3 and its association with distinct complexes of the mannanbinding lectin complement activation pathway c3 adsorbed to a polymer surface can form an initiating alternative pathway convertase the tick-over theory revisited: is c3 a contactactivated protein? the amplification loop of the complement pathways quantitative modeling of the alternative pathway of the complement system the central role of the alternative complement pathway in human disease membrane attack by complement: the assembly and biology of terminal complement complexes new insights into the immune functions of complement properdin can initiate complement activation by binding specific target surfaces and providing a platform for de novo convertase assembly the complement protein properdin binds apoptotic t cells and promotes complement activation and phagocytosis properdin binds to late apoptotic and necrotic cells independently of c3b and regulates alternative pathway complement activation complement factor p is a ligand for the natural killer cell-activating receptor nkp46 intracellular complement activation sustains t cell homeostasis and mediates effector differentiation human complement c3 deficiency: th1 induction requires t cell-derived complement c3a and cd46 activation a c3(h20) recycling pathway is a component of the intracellular complement system on the functional overlap between complement and anti-microbial peptides pulmonary alveolar type ii epithelial cells synthesize and secrete proteins of the classical and alternative complement pathways the endothelium is an extrahepatic site of synthesis of the seventh component of the complement system characteristics and biological variations of m-ficolin, a pattern recognition molecule, in plasma production of complement components by cells of the immune system selective expression of clusterin (sgp-2) and complement c1qb and c4 during responses to neurotoxins in vivo and in vitro chronic low level complement activation within the eye is controlled by intraocular complement regulatory proteins differential expression of complement regulatory proteins decay-accelerating factor (cd55), membrane cofactor protein (cd46) and cd59 during human spermatogenesis novel collectin/c1q receptor mediates mast cell activation and innate immunity identification of human cd93 as the phagocytic c1q receptor (c1qrp) by expression cloning murine cd93 (c1qrp) contributes to the removal of apoptotic cells in vivo but is not required for c1q-mediated enhancement of phagocytosis cd93 is rapidly shed from the surface of human myeloid cells and the soluble form is detected in human plasma activation of human neutrophils by c3a and c5a c3a and c5a are chemotaxins for human mast cells and act through distinct receptors via a pertussis toxin-sensitive signal transduction pathway c3a and c5a stimulate chemotaxis of human mast cells interleukin 3 and granulocyte/macrophage-colony-stimulating factor render human basophils responsive to low concentrations of complement component c3a the human c3a receptor is expressed on neutrophils and monocytes, but not on b or t lymphocytes expression of a functional anaphylatoxin c3a receptor by astrocytes activated human t lymphocytes express a functional c3a receptor local production and activation of complement up-regulates the allostimulatory function of dendritic cells through c3a-c3ar interaction the anaphylatoxin c3a receptor expression on human m2 macrophages is down-regulated by stimulating the histamine h4 receptor and the il-4 receptor differential expression of complement receptors on human basophils and mast cells. evidence for mast cell heterogeneity and cd88/c5ar expression on skin mast cells human t cells express the c5a receptor and are chemoattracted to c5a up-regulation of c5a receptor expression and function on human monocyte derived dendritic cells by prostaglandin e2 c5a regulates nkt and nk cell functions in sepsis the c1q and collectin binding site within c1 q receptor (cell surface calreticulin) role of surfactant proteins a, d, and c1q in the clearance of apoptotic cells in vivo and in vitro: calreticulin and cd91 as a common collectin receptor complex distribution of the α 2 -macroglobulin receptor/low density lipoprotein receptor-related protein in human tissues direct interaction between cd91 and c1q expression of complement receptors 1 and 2 on follicular dendritic cells is necessary for the generation of a strong antigen-specific igg response tumor-promoting phorbol esters stimulate c3b and c3b' receptor-mediated phagocytosis in cultured human monocytes complement receptor expression on neutrophils at an inflammatory site, the pseudomonas-infected lung in cystic fibrosis differential effects of the stimulation of complement receptors cr1 (cd35) and cr2 (cd21) on cell proliferation and intracellular ca2+ mobilization of chronic lymphocytic leukemia b cells complement receptor type 1 (cr1, cd35) expression on peripheral t lymphocytes: both cd4-and cd8-positive cells express cr1 the binding of immune complexes by the erythrocyte complement receptor 1 (cr1) complement receptor type 1 (cr1, cd35) is a receptor for c1q role of complement receptor 1 (cr1; cd35) on epithelial cells: a model for understanding complement-mediated damage in the kidney identification of the membrane receptor for the complement fragment c3d by means of a monoclonal antibody t lymphocyte expression of complement receptor 2 (cr2/cd21): a role in adhesive cell-cell interactions and dysregulation in a patient with systemic lupus erythematosus (sle) complement receptor 3 ligation of dendritic cells suppresses their stimulatory capacity cr3 is the dominant phagocytotic complement receptor on human dendritic cells crig: a macrophage complement receptor required for phagocytosis of circulating pathogens the multiligand-binding protein gc1qr, putative c1q receptor, is a mitochondrial protein c1q-mediated chemotaxis by human neutrophils: involvement of gclqr and g-protein signalling mechanisms chemotaxis of human monocyte-derived dendritic cells to complement component c1q is mediated by the receptors gc1qr and cc1qr analysis of the interaction between globular head modules of human c1q and its candidate receptor gc1qr c5l2, a nonsignaling c5a binding protein membrane cofactor protein of complement is present on human fibroblast, epithelial, and endothelial cells membrane cofactor protein (cd46) protects cells from complement-mediated attack by an intrinsic mechanism soluble forms of membrane cofactor protein (cd46, mcp) are present in plasma, tears, and seminal fluid in normal subjects the cd46-jagged1 interaction is critical for human t h 1 immunity complement 1 inhibitor is a regulator of the alternative complement pathway human factor h and c4b-binding protein serve as factor i-cofactors both encompassing inactivation of c3b and c4b regulation of complement activation by c-reactive protein: targeting of the inhibitory activity of c4b-binding protein analysis of c4 and the c4 binding protein in the mrl/lpr mouse c8 binding protein bears i antigenic determinants inactivation of c3a and c5a octapeptides by carboxypeptidase r and carboxypeptidase n identification of the complement decay-accelerating factor (daf) on epithelium and glandular cells and in body fluids decay-accelerating factor must bind both components of the complement alternative pathway c3 convertase to mediate efficient decay decay-accelerating factor regulates t-cell immunity in the context of inflammation by influencing costimulatory molecule expression on antigen-presenting cells human protectin (cd59), an 18,000-20,000 mw complement lysis restricting factor, inhibits c5b-8 catalysed insertion of c9 into lipid bilayers cd59 functions as a signal-transducing molecule for human t cell activation alternative roles for cd59 post-transcriptional cd59 gene silencing by sirnas induces enhanced human t lymphocyte response to tumor cell lysate-loaded dcs clusterin, the human apolipoprotein and complement inhibitor, binds to complement c7, c8 beta, and the b domain of c9 complement factor h: using atomic resolution structure to illuminate disease mechanisms insights into the effects of complement factor h on the assembly and decay of the alternative pathway c3 proconvertase and c3 convertase factor h as a regulator of the classical pathway activation role of human factor i and c3b receptor in the cleavage of surface-bound c3bi molecules factor i-dependent inactivation of human complement c4b of the classical pathway by c3b/c4b receptor (cr1, cd35) and membrane cofactor protein (mcp, cd46) complement and bacterial infections: from molecular mechanisms to therapeutic applications activation of the complement system by cryptococcus neoformans leads to binding of ic3b to the yeast human c1-inhibitor suppresses malaria parasite invasion and cytoadhesion via binding to parasite glycosylphosphatidylinositol and host cell receptors a human serum mannose-binding protein inhibits in vitro infection by the human immunodeficiency virus virus complement evasion strategies complement and viral pathogenesis viral-derived complement inhibitors: current status and potential role in immunomodulation complement evasion strategies of viruses: an overview. front microbiol natural antibody and complement mediate neutralization of influenza virus in the absence of prior immunity complement lysis activity in autologous plasma is associated with lower viral loads during the acute phase of hiv-1 infection complement-dependent lysis of influenza a virus-infected cells by broadly cross-reactive human monoclonal antibodies the role of anaphylatoxins c3a and c5a in regulating innate and adaptive immune responses c5a receptor-deficient dendritic cells promote induction of treg and th17 uncoupling cd21 and cd19 of the b-cell coreceptor lectin-dependent enhancement of ebola virus infection via soluble and transmembrane c-type lectin receptors high-dose mannose-binding lectin therapy for ebola virus infection interaction of mannose-binding lectin with hiv type 1 is sufficient for virus opsonization but not neutralization susceptibility to hiv infection and progression of aids in relation to variant alleles of mannose-binding lectin complement opsonization of hiv-1 enhances the uptake by dendritic cells and involves the endocytic lectin and integrin receptor families complement opsonization promotes herpes simplex virus 2 infection of human dendritic cells mannose-binding lectin in severe acute respiratory syndrome coronavirus infection a single asparagine-linked glycosylation site of the severe acute respiratory syndrome coronavirus spike glycoprotein facilitates inhibition by mannosebinding lectin through multiple mechanisms the sars coronavirus spike glycoprotein is selectively recognized by lung surfactant protein d and activates macrophages association between mannose-binding lectin gene polymorphisms and susceptibility to severe acute respiratory syndrome coronavirus infection influence of fcgammariia and mbl polymorphisms on severe acute respiratory syndrome direct complement restriction of flavivirus infection requires glycan recognition by mannose-binding lectin differential mechanisms of complement-mediated neutralization of the closely related paramyxoviruses simian virus 5 and mumps virus alternative pathway of complement activation has a beneficial role against chandipura virus infection complement-mediated neutralization of a potent neurotropic human pathogen, chandipura virus, is dependent on c1q cutting edge: productive hiv-1 infection of dendritic cells via complement receptor type 3 (cr3, cd11b/cd18) intracellular complement -the complosome -in immune cell regulation complement c4 prevents viral infection through capsid inactivation antagonism of the complement component c4 by flavivirus nonstructural protein ns1 binding of flavivirus nonstructural protein ns1 to c4b binding protein modulates complement activation west nile virus nonstructural protein ns1 inhibits complement activation by binding the regulatory protein factor h inhibition of the membrane attack complex by dengue virus ns1 through interaction with vitronectin and terminal complement proteins vascular leakage in severe dengue virus infections: a potential role for the nonstructural viral protein ns1 and complement a novel factor i activity in nipah virus inhibits human complement pathways through cleavage of c3b a factor i-like activity associated with chikungunya virus contributes to its resistance to the human complement system human immunodeficiency virus type 1 incorporates both glycosyl phosphatidylinositol-anchored cd55 and cd59 and integral membrane cd46 at levels that protect from complement-mediated destruction the paramyxoviruses simian virus 5 and mumps virus recruit host cell cd46 to evade complement-mediated neutralization complement-mediated enhancement of hiv-1 infection in peripheral blood mononuclear cells the good and evil of complement activation in hiv-1 infection anti-hiv-1 antibodies trigger non-lytic complement deposition on infected cells mechanism of complement inactivation by glycoprotein c of herpes simplex virus c4a: an anaphylatoxin in name only chemotactic responses of human peripheral blood monocytes to the complement-derived peptides c5a and c5a des arg c3a is a chemotaxin for human eosinophils but not for neutrophils. i. c3a stimulation of neutrophils is secondary to eosinophil activation the chemoattractant receptor-like protein c5l2 binds the c3a des-arg77/acylation-stimulating protein a new biologic role for c3a and c3a desarg: regulation of tnf-alpha and il-1 beta synthesis cholesterol crystals induce complement-dependent inflammasome activation and cytokine release differential effects of the complement peptides, c5a and c5a des arg on human basophil and lung mast cell histamine release the effect of human anaphylatoxins and neutrophils on histamine release from isolated human skin mast cells complement peptides c3a-and c5a-induced mediator release from dissociated human skin mast cells the c5a receptor on mast cells is critical for the autoimmune skinblistering disease bullous pemphigoid anaphylatoxin-induced histamine release with human leukocytes: studies of c3a leukocyte binding and histamine release chronic myelogenous leukemia-derived basophilic granulocytes express a functional active receptor for the anaphylatoxin c3a the degradation product of the c5a anaphylatoxin c5adesarg retains basophil-activating properties degranulation from human eosinophils stimulated with c3a and c5a c3a activates reactive oxygen radical species production and intracellular calcium transients in human eosinophils the receptor for complement component c3a mediates protection from intestinal ischemia-reperfusion injuries by inhibiting neutrophil mobilization ap-1 activation through endogenous h2o2 generation by alveolar macrophages regulation by complement c3a and c5a anaphylatoxins of cytokine production in human umbilical vein endothelial cells expression of the receptor for complement c5a (cd88) is up-regulated on reactive astrocytes, microglia, and endothelial cells in the inflamed human central nervous system the receptor for complement anaphylatoxin c3a is expressed by myeloid cells and nonmyeloid cells in inflamed human central nervous system: analysis in multiple sclerosis and bacterial meningitis human c3a and c3a desarg anaphylatoxins have conserved structures, in contrast to c5a and c5a desarg inflammatory properties of human c5a and c5a des arg/ in mast cell-depleted human skin the role of complement factor c3 in lipid metabolism expression of the complement anaphylatoxin c3a and c5a receptors on bronchial epithelial and smooth muscle cells in models of sepsis and asthma the anaphylatoxins c3a and c5a are vasodilators in the canine coronary vasculature in vitro and in vivo vascular permeability changes induced by complement-derived peptides complement activation contributes to severe acute respiratory syndrome coronavirus pathogenesis serum proteomic fingerprints of adult patients with severe acute respiratory syndrome blockade of the c5a-c5ar axis alleviates lung damage in hdpp4-transgenic mice infected with mers-cov autoantibodies against human epithelial cells and endothelial cells after severe acute respiratory syndrome (sars)-associated coronavirus infection dengue virus induces increased activity of the complement alternative pathway in infected cells complement contributes to inflammatory tissue destruction in a mouse model of ross river virus-induced disease mannose binding lectin is required for alphavirusinduced arthritis/myositis ross river virus envelope glycans contribute to disease through activation of the host complement system complement regulation of t cell immunity decayaccelerating factor modulates induction of t cell immunity locally produced complement fragments c5a and c3a provide both costimulatory and survival signals to naive cd4+ t cells absence of signaling into cd4 + cells via c3ar and c5ar enables autoinductive tgf-β1 signaling and induction of foxp3 + regulatory t cells complement c5a receptor is essential for the optimal generation of antiviral cd8+ t cell responses complement component c3 promotes t-cell priming and lung migration to control acute influenza virus infection complement component 3 is required for optimal expansion of cd8 t cells during a systemic viral infection interaction between complement receptor gc1qr and hepatitis c virus core protein inhibits t-lymphocyte proliferation hcv core protein interaction with gc1q receptor inhibits th1 differentiation of cd4+ t cells via suppression of dendritic cell il-12 production differential regulation of socs-1 signalling in b and t lymphocytes by hepatitis c virus core protein hepatitis c virus suppresses c9 complement synthesis and impairs membrane attack complex function the influence of immune complexbearing follicular dendritic cells on the igm response, ig class switching, and production of high affinity igg the generation of memory cells. i. the role of c3 in the generation of b memory cells immune modulation of human dendritic cells by complement provirus activation plus cd59 blockage triggers antibody-dependent complementmediated lysis of latently hiv-1-infected cells blockage of cd59 function restores activities of neutralizing and nonneutralizing antibodies in triggering antibody-dependent complement-mediated lysis of hiv-1 virions and provirus-activated latently infected cells targeted deletion of the cd59 gene causes spontaneous intravascular hemolysis and hemoglobinuria in vivo targeting of human neutralizing antibodies against cd55 and cd59 to lymphoma cells increases the antitumor activity of rituximab neutralization of complement regulatory proteins cd55 and cd59 augments therapeutic effect of herceptin against lung carcinoma cells inhibition of complement activation alleviates acute lung injury induced by highly pathogenic avian influenza h5n1 virus infection covid-19 infection: the perspectives on immune responses pathological findings of covid-19 associated with acute respiratory distress syndrome structure, function, and antigenicity of the sars-cov-2 spike glycoprotein treatment with anti-c5a antibody improves the outcome of h7n9 virus infection in african green monkeys inflarx starts dosing covid-19 coronavirus patients in europe the species severe acute respiratory syndrome-related coronavirus : classifying 2019-ncov and naming it sars-cov-2 eculizumab (soliris) in covid-19 infected patients -full text view -clinicaltrials.gov the inflarx technology eculizumab safety: five-year experience from the global atypical hemolytic uremic syndrome registry eculizumab treatment in patients with covid-19: preliminary results from real life asl napoli 2 nord experience antibody-dependent enhancement of viral infections. dyn immune activ viral dis mechanism for complement-mediated, antibody-dependent enhancement of human immunodeficiency virus type 1 infection in mt2 cells is enhanced entry through cd4, cd21, and cxcr4 chemokine receptors enhanced inflammation in new zealand white rabbits when mers-cov reinfection occurs in the absence of neutralizing antibody antibody-dependent enhancement of ebola virus infection complement c3 plays a key role in inducing humoral and cellular immune responses to influenza virus strain-specific hemagglutinin-based or crossprotective m2 extracellular domain-based vaccination complement enhances in vitro neutralizing potency of antibodies to human cytomegalovirus glycoprotein b (gb) and immune sera induced by gb/mf59 vaccination gp38-targeting monoclonal antibodies protect adult mice against lethal crimean-congo hemorrhagic fever virus infection structural insights into the mechanisms of antibody-mediated neutralization of flavivirus infection: implications for vaccine development complement activation is required for induction of a protective antibody response against west nile virus infection c1q reduces the stoichiometric threshold for antibody-mediated neutralization of west nile virus c1q inhibits antibody-dependent enhancement of flavivirus infection in vitro and in vivo in an igg subclass specific manner complement-mediated virus infectivity neutralisation by hla antibodies is associated with sterilising immunity to siv challenge in the macaque model for hiv/aids c3d of complement as a molecular adjuvant: bridging innate and acquired immunity complement activation and complement receptors on follicular dendritic cells are critical for the function of a targeted adjuvant novel function of complement c3d as an autologous helper t-cell target c3d adjuvant effects are mediated through the activation of c3d-specific autoreactive t cells construction and immunogenicity of dna vaccines encoding fusion protein of murine complement c3d-p28 and gp5 gene of porcine reproductive and respiratory syndrome virus fusion of c3d molecule with neutralization epitope(s) of hepatitis e virus enhances antibody avidity maturation and neutralizing activity following dna immunization enhancement of antibodies to the human immunodeficiency virus type 1 envelope by using the molecular adjuvant c3d immune effect of newcastle disease virus dna vaccine with c3d as a molecular adjuvant protection against influenza virus infection by intranasal administration of c3d-fused hemagglutinin complement, interferon and lupus complement component 3 regulates ifn-α production by plasmacytoid dendritic cells following tlr7 activation by a plant virus-like nanoparticle complement-mediated regulation of metabolism and basic cellular processes complement in the brain interaction between the coagulation and complement system evolution of the complement system: from defense of the single cell to guardian of the intravascular space sea urchin coelomocytes specifically express a homologue of the complement component c3 the role of complement in cnidarian-dinoflagellate symbiosis and immune challenge in the sea anemone aiptasia pallida specific alterations in complement protein activity of little brown myotis (myotis lucifugus) hibernating in white-nose syndrome affected sites the complement in milk and defense of the bovine mammary gland against infections complement-mediated killing of borrelia garinii-bactericidal activity of wild deer serum genetic association of the porcine c9 complement component with hemolytic complement activity inhibition of the alternative pathway of nonhuman infant complement by porin b2 contributes to virulence of neisseria meningitidis in the infant rat model key: cord-023441-q83y12sk authors: draborg, h.; roggen, e. l.; soni, n. k.; patkar, s.; friis, e. p.; lyngstrand, s. t.; christensen, l. l. h.; batori, v.; danielsen, s.; ernst, s. title: recominant expression and immunological characterization of house dust mite allergen der p 1 date: 2008-06-28 journal: scand j immunol doi: 10.1111/j.0300-9475.2004.01423ag.x sha: doc_id: 23441 cord_uid: q83y12sk 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. 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-266739-oay8gbit authors: eisen, damon p.; marshall, caroline; dean, melinda m.; sasadeusz, joe; richards, michael; buising, kirsty; cheng, allen; johnson, paul d.r.; barr, ian g.; mcbryde, emma s. title: no association between mannose-binding lectin deficiency and h1n1 2009 infection observed during the first season of this novel pandemic influenza virus date: 2011-11-30 journal: human immunology doi: 10.1016/j.humimm.2011.08.014 sha: doc_id: 266739 cord_uid: oay8gbit abstract genetic variations in host immunity may influence susceptibility to novel infections like the recently emergent pandemic influenza virus. prior studies demonstrated that mannose-binding lectin (mbl) inactivates influenza. furthermore, mbl deficiency is common and appears to predispose to respiratory virus infections. therefore, we studied whether mbl deficiency played a role in infection with the novel h1n1 2009 influenza strain in exposed health care workers. in a nested case–control study, we observed no association between phenotypic mbl deficiency, variously defined, and predisposition to h1n1 2009 influenza in 63 pairs of seropositive and seronegative participants. mbl appears to currently have little impact on innate immune responses to h1n1 2009 influenza. emerging infectious diseases may afford an opportunity to examine variations in host susceptibility caused by the absence of widespread prior immunity. the recent pandemic influenza strain h1n1 2009 emerged in mexico in april 2009 and produced a late surge in northern hemisphere influenza cases. this virus then rapidly became the dominant influenza strain in the southern hemisphere. although infections were generally mild, there was marked morbidity in pregnant and obese patients [1] . determining host genetic factors that influence the innate immune response and susceptibility to novel infections may lead to therapeutic interventions for this and future emerging infections with higher overall morbidity. mannose-binding lectin (mbl) is a pattern recognition protein of the innate immune system that recognizes pathogen-associated molecular patterns [2] . mbl binds to microbial cell surface polysaccharide residues that have characteristic chemical and spatial features that distinguish them from animal cell walls. having bound to microbial pathogens, mbl mediates killing through lectin complement pathway activation and opsonophagocytosis. mbl has been observed to bind to and neutralize the influenza virus in studies involving nonpandemic h3n2 strains [3] . in this study, the efficacy of mbl-mediated inhibition of these influenza strains that have multiple exposed glycosylation sites on the hemagglutinin head was proportional to the number of glycosylation sites available for mbl binding. mbl null mice have increased susceptibility to h3n2 influenza a viruses [4] . most recently, however, it has been demonstrated that in contrast to previous, seasonal influenza strains, mbl plays no role in murine models of influenza caused by h1n1 2009 infection [5] . mbl deficiency resulting from polymorphism in the mbl2 gene is one of the most common genetic influences on immune responses to infection. approximately 25% of the many human populations studied have lowered levels of mbl [6] and this state has been associated with predisposition to numerous infectious diseases, including those of the respiratory tract [7] . although mbl is predominantly a serum protein, significant amounts are observed in the respiratory tract in the presence of inflammation [8] . given the in vitro neutralization of influenza by mbl, we believed that mbl deficiency may predispose to infection with influenza. we tested this hypothesis in a population naive to the novel pandemic strain h1n1 2009. as part of a prospective cohort study investigating h1n1 2009 infection risks in health care workers [9] , we assessed mbl status among hospital workers who had been demonstrated to have (i) serologic evidence of infection (h1n1 2009 hemagglutination titer (hat) ն 1:40) with or without an influenza-like illness or (ii) no infection (h1n1 2009 hat ͻ 1:10) and no influenza-like illness. this study was performed at 4 melbourne tertiary referral hospitals during the influenza season of 2009 when h1n1 2009 was the predominant serotype. approval for the study was provided by the human research ethics committees at all 4 hospitals and participants gave written informed consent. mbl level was determined by mannan-binding elisa and mbl function by c4 deposition elisa as described previously [10] . we defined mbl deficiency in 3 ways, which have been used in previous infectious diseases association studies: (a) mbl level ͻ0.5 g/ml [11] , (b) c4 deposition ͻ0.2 u/l [12] , or, (c) most stringently, both levels below the preceding ranges. mbl2 genotype was not determined in this study because we have previously demonstrated that mbl level is a more sensitive and specific marker of mbl deficiency because a considerable number of individuals bear wild-type mbl2 with unmeasureable mbl [6] . to determine the sample size required for the mbl disease susceptibility study, we powered the study to detect 3-fold odds of influenza infection in mbl-deficient individuals compared with the odds of infection for mbl-sufficient participants. this odds ratio (or) was selected as being representative of respiratory infection predisposition studies wherein mbl2 polymorphisms encoding for low mbl levels were analyzed for associations with respiratory infection, including a meta-analysis in pneumococcal infection (or ϭ 2.57) [13] and respiratory infections generally (or ϭ 3.4) [14] . the required number of influenzainfected cases and uninfected controls was 65 in each group (twosided p value ϭ 0.05, 80% power), assuming a 25% frequency of mbl deficiency (as indicated by mbl level ͻ 0.5 g/ml) in the influenzaseronegative controls as is the case in healthy controls. to reach our predetermined study sample size, all seropositive and randomly selected seronegative participants from the health care worker study were invited to participate in this nested case-control predisposition study. comparison of the nonnormally distributed results for mbl level and function was performed using mann-whitney tests and frequency of mbl deficiency was compared using 2 tests. statistical calculations were performed using minitab 14 (state college, pa). a total of 231 health care workers and 215 nonclinical staff controls were enrolled in a study of the risk of infection conducted by the melbourne h1n1 clinical research team [9] . the "parent" study aimed to determine the effect of occupation and personal protective equipment on h1n1 2009 acquisition. from this cohort we recruited subjects for the mbl study reported here, based primarily on their h1n1 2009 serology results. a majority of the influenza-infected participants (63/79 h1n1 2009 hat ͼ 1:40) from the health care worker study consented to inclusion in this study and an equal number of randomly selected, uninfected controls were recruited. for the study participants, there were no symptomatic, laboratory-confirmed, influenza infections among health care workers, forcing a reliance on our serologic definition for h1n1 2009 infection. the median h1n1 2009 hat among cases was 1:80 with a range of 1:40 -1:640 and interquartile range of 1:40 -1:80. controls were matched to cases in that they worked at the same hospital only. our parent study demonstrated no association between age, sex, or number of children in the health care workers' household; on that basis, none of these factors was used in matching cases and controls. additionally, mbl levels do not change significantly with age in adult life [15] . the overall frequency of mbl deficiency as defined by mbl level ͻ0.5 g/ml was 37%, which exceeds the predicted frequency used to power our study. there were no differences in the median levels of mbl (1.04 g/ml in the seropositive group and 0.75 g/ml in the seronegative group, p ϭ 0.4) or c4 deposition (0.23 u/l in the seropositive group and 0.18 u/l in the seronegative group, p ϭ 0.4) between the groups of influenza-infected and uninfected participants (table 1) . additionally there was no significant difference in the frequency of mbl deficiency as defined by low mbl level (23/63 of seropositive participants vs 24/63 of seronegative participants, or 0.93; 95% confidence interval (ci) 0.43-2.05), low mbl function (30/63 of seropositive participants vs 34/63 of seronegative participants, or 0.78; 95% ci 0.36 -1.66), or both low mbl level and function (21/63 of seropositive participants vs 24/63 of seronegative participants, or 0.81; 95% ci 0.37-1.80). if a lower mbl level (ͻ0.1 g/ml) was used to define deficiency, there remained no difference between the frequency of mbl deficiency in seropositive (9/63) and seronegative (10/63) study participants (or 0.88; 95% ci 0.30 -2.59). there were no participants with symptomatic, proven influenza to analyze the influence of mbl deficiency on disease severity. numerous well-established host factors predispose to influenza infection or severe disease. recent studies of pandemic influenza strain h1n1 2009 disease have demonstrated that pregnancy and obesity were among important factors associated with disease severity [16] . a novel recent host factor for influenza severity appears to be igg2 subclass deficiency [17] . here we have sought information on the link between mbl deficiency and predisposition to influenza. we have demonstrated no association between mbl deficiency states and infection with the novel pandemic influenza strain h1n1 2009. numerous factors may potentially account for this lack of association. however, sample size is unlikely to have played a role. the study was powered to detect an or of 3, and the cis of the observed odds ratios are sufficiently narrow that a true or of 3 is highly implausible. whereas our sample size calculation was based on a background frequency of mbl deficiency of 25%, we actually observed a higher frequency, which would have improved the study power. similarly, there were sufficient influenza infection events based on positive h1n1 2009 serology to analyze. the absence of symptomatic influenza is not expected to have altered the result of this study but means there was no opportunity to assess mbl's influence on the severity of disease manifestations. differences in exposure to the novel h1n1 2009 influenza strain between the infected and uninfected groups are a potential source of study error. in the health care worker "parent" study [9] from which subjects for this influenza predisposition study were drawn, we observed no significant difference in the rate of influenza seroprevalence in those health care workers involved in direct patient care compared with nonclinical staff who had a background risk that approximated that of the general community. the frequency of h1n1 2009 seropositivity in health care workers involved in the "parent" study varied among the 4 study sites because the geographic distribution of symptomatic cases presenting to melbourne hospitals was notably uneven. therefore, we matched the numbers of seropositive health care worker cases and seronegative controls from each study hospital site. taken together, these points would support the argument that the overall level of exposure among the study population described here is equivalent. the obvious conclusion is that mbl is not involved in innate immune responses to influenza or other respiratory viruses. this is countered, however, by the in vitro evidence of mbl's inhibition of influenza virus [3, 18] , recent evidence of mbl's role in the development of humoral immunity to influenza [19] , and clinical association studies that demonstrate that mbl deficiency predisposes not only to respiratory tract infection in general, but also to respiratory tract infections with viral pathogens like severe acute respiratory syndrome in particular [7] . our study's choice of mbl phenotyping over genotyping in defining mbl deficiency may have affected its results. however, phenotype has a more direct biologic link with mbl activity. low mbl level is a more sensitive and specific marker of the biologic effect of mbl (mbl function as measured by c4 deposition) than mbl2 genotype [10] . large studies of healthy controls confirm that many patients with wild-type mbl2 genotype have levels of mbl in the deficient range [6] . the choice of an mbl level of ͻ0.5 g/ml to define mbl deficiency is informed by published data [6] . when we used a lower level of mbl to define deficiency, we also observed no indication that extremely low mbl levels predisposed to h1n1 2009 infection. although there were fewer individuals with mbl ͻ0.1 g/ml than with mbl levels ͻ0.5, this only slightly reduced the power to detect our proposed or of 3. unfortunately, it was not possible to recruit the greater sample size required (168 participants) to achieve power using this mbl cutoff. however, given the observed or and the ci (or 0.88; 0.30 -2.59), we can exclude with some confidence our a priori or of 3 in this population. other studies have used mbl phenotyping rather than mbl2 genotyping to determine whether mbl deficiency plays a role in disease susceptibility [20, 21] . concerns are sometimes raised as to the possibility that mbl levels may be elevated because of relatively limited acute-phase reactant changes observed in this protein [22] . however, because the patients studied here did not have clinical signs of influenza at the time of blood testing, there is no possibility of mbl levels being affected by acute-phase changes. potentially, the most potent factor that abrogates any role for mbl in protection against h1n1 2009 infection is specific features of the surface of this virus. recent data indicate that there is only 1 glycosylation site exposed on the globular head of the hemagglutinin protein of this novel virus and that mbl does not bind to this strain of influenza [23] or contribute to mouse immune responses to h1n1 2009 [5] . the absence of association between mbl deficiency and predisposition to h1n1 2009 infection we have observed is in line with and highlights the biologic significance of these in vitro data. it may be predicted that future mutation of the h1n1 2009 virus to escape the human immune response will lead to more glycosylation sites becoming exposed on the head of the hemagglutinin and that mbl may then play a role in protection against infection. it may be useful to repeat this study after future influenza seasons involving h1n1 2009 transmission. because of mbl's broad range of activity against pathogens, it has the potential for activity and even therapeutic efficacy against emerging organisms. indeed, it has recently been shown to inhibit severe acute respiratory syndrome [24] , ebola [25] , and henipaviruses (unpublished), all highly virulent pathogens demonstrated to have multiple glycosylation sites. although we have not demonstrated a clinical association between mbl deficiency and predisposition to the novel h1n1 2009 influenza strain, this may be because of the absence of exposed mbl-binding sites on this specific virus. there are no previous clinical studies on the role of mbl in innate immune defenses against influenza. the previously documented viral inhibitory activity of mbl against influenza viruses suggests that this arm of the immune system may indeed be active against influenza, including against h1n1 2009 after antigenic drift occurs. critical care services and 2009 h1n1 influenza in australia and new zealand molecular basis of opsonic defect in immunodeficient children collectin-mediated antiviral host defense of the lung: evidence from influenza virus infection of mice lack of the pattern recognition molecule mannose-binding lectin increases susceptibility to influenza a virus infection the pandemic (h1n1) 2009 influenza virus is resistant to mannose-binding lectin low serum mannose-binding lectin level increases the risk of death due to pneumococcal infection mannose-binding lectin deficiency and respiratory tract infection mannose-binding lectin is present in the infected airway: a possible pulmonary defence mechanism do frontline healthcare workers have increased risk of contracting pandemic (h1n1) 2009 influenza? a prevalence and risk factor study analysis of the relationship between mannose-binding lectin (mbl) genotype, mbl levels and function in an australian blood donor population association between deficiency of mannose-binding lectin and severe infections after chemotherapy low mannosebinding lectin complement activation function is associated with predisposition to legionnaires' disease host genetic susceptibility to pneumococcal and meningococcal disease: a systematic review and meta-analysis mannose-binding lectin concentrations, mbl2 polymorphisms, and susceptibility to respiratory tract infections in young men biological variation in circulating levels of mannan-binding lectin (mbl) and mbl-associated serine protease-2 and the influence of age, gender and physical exercise hospitalised adult patients with pandemic (h1n1) 2009 influenza in melbourne association between severe pandemic 2009 influenza a (h1n1) virus infection and immunoglobulin g(2) subclass deficiency recombinant chimeric lectins consisting of mannose-binding lectin and lficolin are potent inhibitors of influenza a virus compared with mannosebinding lectin capture of influenza by medullary dendritic cells via sign-r1 is essential for humoral immunity in draining lymph nodes low mannan-binding lectin serum levels are associated with complicated crohn's disease and reactivity to oligomannan (asca) association of mannose-binding lectin deficiency with acute invasive aspergillosis in immunocompromised patients influence of major surgery on the mannan-binding lectin pathway of innate immunity pandemic h1n1 influenza a viruses are resistant to the antiviral activities of innate immune proteins of the collectin and pentraxin superfamilies mannose-binding lectin in severe acute respiratory syndrome coronavirus infection a novel l-ficolin/mannose-binding lectin chimeric molecule with enhanced activity against ebola virus we thank mee-sook kim and philippa white for their assistance in recruiting patients for this study. this study was funded by a national health and medical research council strategic award, urgent research-h1n1 influenza number 604944. the melbourne who collaborating centre for reference and research on influenza is supported by the australian government department of health and ageing. key: cord-023425-3sjsogvq authors: røntved, c. m.; dernfalk, j.; ingvartsen, k. l. title: do high and low tumour necrosis factor‐α responders exist in dairy cows? date: 2008-06-28 journal: scand j immunol doi: 10.1111/j.0300-9475.2004.01423v.x sha: doc_id: 23425 cord_uid: 3sjsogvq 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‐α (tnf‐α) ex vivo. initially, a time‐ and dose‐dependent study was carried out to find the optimal stimulation conditions for the tnf‐α response. the tnf‐α 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‐α response. thirty‐eight danish–holstein dairy cows were investigated for their tnf‐α responsiveness ex vivo in the periparturient period. heparin‐stabilized blood samples were collected seven times over a period of 4 months (weeks −3, −1, 2, 3, 5, 9 and 13 around calving) and stimulated with 5 g/ml of e. coli lps. indeed, fluctuations in the tnf‐α responsiveness occurred over time. moreover, the mean tnf‐α 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‐α response, whereas others had high a tnf‐α response. we are currently investigating whether high and low tnf‐α responders to e. coli lps also exist in dairy cows in vivo. moreover, the importance of tnf‐α responsiveness ex vivo to dairy cows' susceptibility and clinical response to experimental e. coli infections in the udder is being investigated. 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-311913-cplhq4k9 authors: sato, satoshi; kawashima, hisashi; kashiwagi, yasuyo; fujioka, tao; takekuma, kouji; hoshika, akinori title: association of mannose‐binding lectin gene polymorphisms with kawasaki disease in the japanese date: 2009-11-23 journal: int j rheum dis doi: 10.1111/j.1756-185x.2009.01428.x sha: doc_id: 311913 cord_uid: cplhq4k9 objective: kawasaki disease (kd) is a systemic vasculitis in childhood; its etiology is unknown. the possibility that kd is an infectious disease has been discussed and investigated for decades, in light of the implication that infections are involved in the pathogenesis of kd. young children rely on their innate immune system for protection against virus and micro‐organisms. human mannose binding lectin (mbl) is a c‐type serum lectin synthesized by the liver as an acute phase protein and it plays an important role in the innate immune system. here, we investigate the relationship between the mbl gene polymorphisms and the occurrence of kd in the japanese population. method: the frequencies of the genotypes, defined as mutations in codons 52, 54 and 57, and the functional promoter variants of the mbl were determined in 45 patients with kd. results: the mbl codon‐54 polymorphism frequency of heterozygote (ggc/gac) and promoter variants were significantly higher in the kd group than that in the control group (p < 0.05). neither group showed codon 52 nor 57 polymorphisms. conclusion: it is possible that mutations of the mbl gene might be related to the trigger of the pathogenesis of kawasaki disease. kawasaki disease (kd) 1 is an acute systemic vasculitis of children, especially asian children, and presently the most common acquired heart disease in children. the diagnosis of kd is based entirely on clinical features. the clinical manifestations of kd are high fever plus: (i) bilateral non-exudative conjunctivitis; (ii) polymorphous exanthem; (iii) bilateral non-suppurative cervical lymphadenopathy; (iv) mucous membrane changes; and (v) swelling or erythema of the hands or feet. for classic kd, individuals must have a fever for 5 days and either meet at least 4/5 criteria or have evidence of coronary artery abnormality. 2 the etiology of kd is unknown. laboratory findings are nonspecific, and there are no diagnostic tests for kd. early in the course of the illness parameters of inflammation are increased: c-reactive protein (crp), erythrocyte sedimentation rate (esr), white blood cell and neutrophil counts. the disease is most common in children younger than 5 years, with an annual incidence of 90 per 100 000 population in japan 3 and 5-10 per 100 000 in europe and the usa. 4 eighty to ninety percent of cases occur in children younger than 5 years, with an average age of approximately 2 years. kd is relatively uncommon among children younger than 6 months, [5] [6] [7] and is rare in infants younger than 3 months old, which suggests the possibility that they are protected from infection by antibodies that are passively acquired from their mothers. occurrence beyond late-aged children is rare. widespread immunity to common infectious agents may explain the rarity of kd in adults. the variability in the response to infections suggests that innate immune response might be involved in an individual susceptibility to kd during the period before the production of specific antibodies. mannose binding lectin (mbl) is an important component of host innate immunity, which has a nonspecific role in complement activation and opsonization. mbl is an acute-phase reactant of hepatic origin that can bind through multiple lectin domains, repeating mannose and n-acetylglucosamine sugar motifs that are characteristically displayed at high densities on bacterial and fungal cells and on viruses. 8 the gene for mbl is located on the long arm of chromosome 10. there are three single point mutations that have been well characterized in exon 1of the mbl gene (mbl2), at codon 52 (cgt fi tgt), codon 54 (ggc fi gac) and codon 57 (gga fi gaa). the normal wild type allele is designated a, whereas the variant alleles are designated as o. any of these point mutations will lead to substantially reduced concentrations in serum of functional mbl. polymorphisms of the promoter region of the mbl2 gene have additional effects on serum mbl concentration. in particular is the one at position )221 (x/y), with the y promoter variants being responsible for high and x promoter variants for a low mbl expression activity. 9, 10 it was reported that a g to a base change at codon 54 results in a glycine (gly) to aspartic acid (asp) substitution in the collagen domain, which can disrupt the interaction between mbl and mbl-associated serine protease (masp) by causing a disruption of the mbl peptide. its function is thought to be especially important in the acute stage of primary infections, particularly in young children in the window period when maternal antibodies fall and adaptive immunity starts to develop and become mature. 11 mbl2 gene polymorphism at codon 54 has shown to be associated with systemic lupus erythematosus, 12,13 rheumatic disease, and common variable immunodeficiency. 14 in a previous study, biezeveld et al. examined the frequency of mbl2 genotypes in 90 dutch patients with kd. they described an increased risk of coronary artery lesions in patients younger than 1 year who expressed either low or medium levels of an mbl2 genotype. 9 the frequencies of kd and mbl2 genotypes are different in different populations. therefore, we think it is valuable to assess whether the mbl2 gene polymorphisms, which cause the defect in the activation of the innate immune system, are associated with susceptibility to kd in the japanese population. the subjects enrolled in this study included 45 japanese patients (29 boys and 16 girls) with kd and 34 healthy controls. the diagnosis of kd was made according to the previous criteria and echocardiographic scoring guidelines for kd. 15 dna samples were obtained from 45 patients aged 4-139 months (mean, 30.02 ae 26.4 months). six children were diagnosed as kd with coronary artery lesions. all patients were hospitalized at tokyo medical university hospital. thirty-four healthy blood donors were recruited as controls. all controls were adults who had not been diagnosed as kd. the gender distributions in the group of patients with kd and the control group were not significantly different. all participants gave written informed consent. the local ethics committee of the tokyo medical university hospital approved the study protocol. venous blood was drawn from each individual, and genomic dna was extracted from peripheral blood mononuclear cells using a qia amp blood kit (qiagen, hilden, germany). we amplified the region of the mbl gene including codon 54 using polymerase chain reaction (pcr) with a set of specific primers: 5¢-gag-gccagggatgggtcatc-3¢ (sense) and 5¢-cca aca cgt acc tgg ttc cc-3¢ (antisense). to amplify the y/x promoter fragment, the primers bar: 5¢-gat-gagcagtggggatcctaagga-3¢ and mbl kurz: 5¢-ggctaggctgctgaggtttc-3¢ were used. a direct sequence was carried out using taq dye primer cycle sequencing kit (applied biosystems, tokyo, japan). the nucleotide sequence was analyzed with an automated nucleotide analyzer, the 373a dna sequencer (applied biosystems, foster city, ca, usa). the combination of structural mutations and promoter variants in these gene results was well described in three expression groups (high, medium and low). genotype frequencies were calculated by direct counting. differences in mbl genotype frequencies among kd patients and healthy controls were determined using fisher's exact test. the odds ratio (or) and 95% confidence interval (ci) were also calculated. a probability value of < 0.05 was considered statistically significant in all analyses. the distribution of codon 54 gene polymorphisms in healthy japanese is shown in table 1 . the heterozygous type of codon 54 was seen in 23.5%, and the homozygous type was 2.9%. neither group showed codon 52 or 57 polymorphisms. we defined structural mutations in the mbl2 gene (a = exon 1 wild-type; o = exon 1 mutation.) and functional promoter variants of this gene (x/y at )221 bp [x fi c and y fi g]) by dna sequencing. table 1 shows genotypic and allelic frequencies of codon 54 in the patients with kd. a mutation in codon 54 (genotypes a/o or o/o) was reported in 22 (48.9%) of 45 patients with kd compared with nine (26.5%) of 34 healthy japanese controls (p = 0.036, or 2.657, 95% ci 1.017-6.941). table 2 shows the combination of structural mutations and promoter variants (x/y at )221 bp) in the gene (high, medium and low). the distributions of mbl genotypes in patients and controls are summarized in table 2 . the mbl genotype distributions between kd patients and controls indicated high frequencies of xa/o and xa/xa types (p = 0.028, or 2.743, 95% ci 1.069-7.035). six children were diag-nosed as having kd with coronary artery lesions. two of six patients had mbl2 gene mutations. human mannose binding lectin is one of the most important constituents of the innate immune system. many studies have reported a deficiency of mbl in different populations. the different distributions of genotypic and allelic frequencies in different populations might exist as a result of ethnic differences, in addition to migration. 16 the genotype and allele a frequencies of codon 54 did not shown significant differences compared with those of our healthy controls from other japanese, chinese and europeans. in japan, codon 52 and 57 mutations are probably absent. 17 mbl is a liver-derived serum protein that binds to sugars on the surface of pathogenic micro-organisms and triggers complement fixation. low serum levels of mbl are found in association with single nucleotide polymorphisms in the promoter region and the structural gene coding region of the mbl2 gene. deficiency of mbl due to point mutations of the mbl2 gene has been shown to predispose to infections in children. however, the roles of mbl2 genotypes in kd are still under debate. previous studies in the netherlands suggested a possible association between mbl dysfunctional alleles and kd. biezeveld et al., described that in 31 randomly selected patients with kd, median serum concentration of mbl in patients in the medium and low expression groups was significantly lower than in those in the high expression group. 9 an infectious trigger is generally believed to cause kd. several pathogens, such as hcov-nh, retroviruses, epstein-barr virus, parvovirus b19, coronavirus, and chlamydia, have been suggested by different studies as possibly important in the pathogenesis of kd. 18, 19 we recognize that the enhanced risk of the development of infection indicates the importance of the complement system in protection against diseases. many studies have shown that the mbl pathway is an independent pathway of a complement activation. mutation and deficiency of the mbl2 codon could impair the ability of the pathogen or immune complex clearance, facilitating the development of autoimmunity. therefore, we hypothesize that opsonization dysfunction of the complement system caused by mbl2 gene mutation is involved in the immune response to infection. recently, cheung described an association between premature atherosclerosis among kd patients and mbl. 10 in this study, the combination of codon-54 polymorphism and promoter variants in the mbl2 gene were significantly higher in the kd group than that in the control group (p < 0.05). however, there was no significant difference in the allele frequency of gac and serum mbl levels between patients with and without coronary artery lesions in kd cases. it is thought that mbl deficiency is a trigger for infection and kd. this study has shown that, in a population of individuals born in japan, having codon 54 variants in the mbl2 gene is significantly associated with susceptibility to kd but not with coronary arterial lesions. the disease is common among children younger than 5 years, but rare in those younger than 6 months. it is suggesting that mbl plays a role in the defense against infection at a young age, when innate immunity becomes involved in the presence of waning levels of maternal antibodies. in contrast, mbl becomes less important to counter infectious triggers at an older age, when the primary host defense against invading pathogens and subsequent clearance reactions are taken up by more mature and specific arms of the immune system, such as lymphocytes and immunoglobulins. our study suggests that mbl polymorphisms do not influence clinical manifestations of kd but influence susceptibility to kd in japanese children. this indicates that the mbl2 gene may play a role in the pathogenesis of this disease. because this study included a small number of subjects, further research in large cohorts of patients should be carried out to reach a stronger conclusion. a new infantile acute febrile mucocutaneous lymph node syndrome (mlns) prevailing in japan report of the committee on infectious diseases results of the nationwide epidemiologic survey of kawasaki disease in 1995 and 1996 in japan kawasaki syndrome hospitalizations in the united states epidemiologic features of kawasaki disease in taiwan epidemiologic pictures of kawasaki disease in japan: from the nationwide incidence survey in 1991 and 1992 kawasaki disease in infants less than one year of age mannose-binding lectin binds to a range of clinically relevant microorganisms and promotes complement deposition association of mannose-binding lectin genotype with cardiovascular abnormalities in kawasaki disease modulating effects of mannose binding lectin genotype on arterial stiffness in children after kawasaki disease acute respiratory tract infections and mannose-binding lectin insufficiency during early childhood mannose-binding lectin and susceptibility to infection in chinese patients with systemic lupus erythematosus association of mannose-binding lectin gene variation with disease severity and infections in a population-based cohort of systemic lupus erythematosus patients mannose binding lectin polymorphisms are associated with early age of disease onset and autoimmunity in common variable immunodeficiency diagnosis and therapy of kawasaki disease in children restricted polymorphism of the mannose-binding lectin gene of indigenous australians mannose-binding lectin polymorphisms in patients with hepatitis c virus infection association between a novel human coronavirus and kawasaki disease kawasaki disease: etiology, pathogenesis, and treatment none. key: cord-023392-axd0901z authors: hansen, t. k.; tarnow, l.; thiel, s.; steffensen, r.; parving, h.‐h.; flyvbjerg, a. title: association between mannose‐binding lectin and vascular complications in type 1 diabetes date: 2008-06-28 journal: scand j immunol doi: 10.1111/j.0300-9475.2004.01423i.x sha: doc_id: 23392 cord_uid: axd0901z 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 = 0.04. median serum mbl concentrations were significantly higher in patients with nephropathy than in patients with normoalbuminuria [2306 µg/l (iqr 753–4867 µg/l) versus 1491 µg/l (iqr 577–2944), p = 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 µg/l (iqr 636–5231 µg/l) versus 1741 µg/l (iqr 656–3149 µg/l), p = 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. 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-310110-haukpwtf authors: guo, jinlei; cao, yang; qin, kun; zhao, xiaopeng; wang, donghong; li, zi; xin, li; shu, yuelong; zhou, jianfang title: limited effect of recombinant human mannose-binding lectin on the infection of novel influenza a (h7n9) virus in vitro date: 2015-02-27 journal: biochemical and biophysical research communications doi: 10.1016/j.bbrc.2015.01.070 sha: doc_id: 310110 cord_uid: haukpwtf abstract mannose-binding lectin (mbl), a pattern-recognition molecule in serum, recognizes specific hexose sugars rich in mannose and n-acetylglucosamine on bacterium, yeasts, viruses as well as apoptotic cells. it has been well-identified that mbl has antiviral effects via binding to seasonal influenza h1 and h3 subtype viruses. influenza a (h7n9) virus, a novel reassortant virus to human population, possesses the surface hemagglutinin (ha) and neuraminidase (na) genes from duck and wild-bird influenza viruses and internal genes from poultry h9n2 viruses. as of dec 7th, 2014, a total of 467 human infections and 183 fatal cases have been identified. here, recombinant human (rh) mbl was tested for its binding and effects on hemagglutination inhibition (hi) and na activity inhibition (nai) of avian h7n9, h9n2 and human h3n2 viruses. we discovered that rhmbl exhibited a strong binding to h7n9 virus as human h3n2 did at high virus titers. however, it performed a significantly weaker hi activity effect on h7n9 comparing to those of h3n2 and h9n2, even at a much higher concentration (3.67 ± 0.33 vs. 0.026 ± 0.001 and 0.083 ± 0.02 μg/ml, respectively). similarly, minor nai effect of rhmbl, even at up to 10 μg/ml, was found on h7n9 virus while it displayed significant effects on both h3n2 and h9n2 at a lowest concentration of 0.0807 ± 0.009 and 0.0625 μg/ml, respectively. the hi and nai effects of rhmbl were calcium-dependent and mediated by lectin domain. our findings suggest that mbl, the host innate molecule, has differential interference effects with human and avian influenza virus and limited antiviral effect against h7n9 virus. host innate immunity plays a critical role in the early phase of infection. this first-line defense against pathogens is mediated by a variety of pattern-recognition molecules including collectins, tolllike receptors and ficolins as well as inflammatory cytokines and type i interferon or macrophages and natural killer cells. mannosebinding lectin (mbl) is one of collectins circulating in the serum and synthesized by liver. it consists of collagenous domains and carbohydrate recognition domains (crd). the crds recognize sugars including d-mannose, n-acetylmannosamine, n-acetylglucosamine and l-fucose on the surface of many pathogens in a calcium-dependent manner [1] . previous studies showed that mbl can bind to a range of clinically relevant microorganisms such as staphylococcus aureus, candida albicans [2] , hiv, sars-cov, ebola virus, hsv, influenza virus [3e6] . the binding of mbl to microorganisms is presumed to induce mbl conformational changes that allow the molecule to initiate viral neutralization or kill virus via opsonization or complement activation [7] . influenza a virus, a segmented single-stranded negative-sense rna virus, belongs to orthomyxoviridae and is subtyped according to the antigenic properties of their envelope glycoproteins, ha and na. currently, 16 ha subtypes and 9 na subtypes circulate in birds. among them, only seasonal h1n1 and h3n2 viruses circulate in human population [8] . occasionally, some subtypes of avian influenza a virus can jump into human and cause diseases with a range of clinical symptoms and outcomes, such as conjunctivitis, mild upper respiratory tract disease, as well as severe pneumonia and death [9e12] . viral ha and na assist virus binding, entry and releasing during infection cycle. their potential n-linked glycosylation sites (ngs) can be glycosylated, which might allow their binding to host mbl. it has been found that the glycan at residue 165 in h3n2 ha was of high-mannose and mbl neutralized viral infectivity via it. many lines of evidences have shown that the mbl plays an important role in fighting against seasonal flu [13e15]. however, little is known about the interactions between avian influenza virus and the innate molecules. avian influenza h7n9 virus is novel to human population [16, 17] , which contains the surface ha and na genes from duck and wild-bird influenza viruses and internal genes from poultry h9n2 viruses. unlike other h7 viruses that generally cause mild symptoms such as conjunctivitis or influenza-like illness (except one fatal case infected with h7n7 in netherlands in 2003), h7n9 virus usually results in severe pneumonia or respiratory failure in human. here, we examined the interactions of mbl with avian influenza virus h7n9, h9n2 and human virus h3n2. furthermore, we studied the molecule mechanisms for them by structure modeling. the vaccine strain a/anhui/1/2013(h7n9) (nibrg-268) was obtained from national institute for biological standards and control (uk), namely h7n9vac. the virus bears the ha and na of a/anhui/1/ 2013(h7n9) and internal genes of a/puerto rico/8/1934 (pr8, h1n1); a/brisbane/10/2007(h3n2) was named as h3n2wt in the study; h9n2 virus, a reassortant bearing the ha, na from a/hongkong/ 33982/2009(h9n2) and internal genes of pr8, was named as h9n2rg. the reassortant h7n1 ah1 haþpr8 na was with ha of a/ anhui/1/2013 and seven genes of pr8, which is rescued as previously reported [18] . h7n9vac, h3n2wt and h7n1 ah1 haþpr8 na were propagated in 9e11-day-old embryonated chicken eggs, h9n2rg was grown in madin-darby canine kidney (mdck) cells (atcc, usa) with modified eagle's medium (invitrogen, usa)containing 2 mg/ml n-tosyl-l-phenylalanine chloromethyl ketone (tpck)etreated trypsin (sigma, usa). virus stocks were purified by adsorption to and elution from turkey red blood cells (trbcs) and stored at à80 cuntil use [19] . virus titer was determined by titration in mdck cells and the tissue culture infectious dose affecting 50% of the cultures (tcid 50 ) is calculated by the reedemuench formula [20] . recombinant human mbl (rhmbl) was purchased from sino biological inc (beijing, china). ninety-six-well plates were coated with 2 â 10 5 tcid 50 influenza virus at a volume of 100 ml/well for overnight at 4 c, then were blocked for 1 h with 1% bovine serum albumin (bsa, roche, switzerland) at 37 c. different concentrations of rhmbl (0, 1, 3, 5, 7 mg/ml) were added and incubated for 1 h at 37 c. the virus-dose dependent binding assay was conducted as that wells were precoated with 2 â 10 2 , 2 â 10 3 , 2 â 10 4 and 2 â 10 5 tcid 50 influenza viruses per well. then 3 mg/ml rhmbl was added and incubated for 1 h at 37 c. the binding was detected by the biotinylated human mbl pab (0.2 mg/ml) (r&d, usa), followed by streptavidin-horseradish peroxidase (hrp) (1:200) (r&d, usa) and tetramethylbenzidine substrate solution (bd, usa), the reaction was stopped by 2 m h 2 so 4 and the optical density (od) at 450 nm was measured by elisa reader (perkin-elmer, usa). the wells coated with 10 mg/ml mannan from saccharomyces cerevisiae (sigma, usa) or coating buffer (kirkegaard & perry laboratories, usa) were used as positive control and negative control respectively. the test was performed in duplicates and in three independent experiments, absorbance from negative control was subtracted and results were normalized to positive control, data was expressed as a relative absorbance value using mean ± sem (%). hi assay was performed in v-bottom 96-well plates as previously described [20] . briefly, 25 ml influenza virus (4hau) was mixed with 25 ml rhmbl of different concentrations diluted in hank's balanced salt solution (hbss) containing 1.26 mm ca 2þ for 1 h at 37 c, then 50 ml 1% trbc was added to the mixture and incubate at room temperature for 30 min. for hi reverse assay: rhmbl was diluted in hbss containing 10 mm edta or 10 mg/ml mannan, then incubated with 4 hau of influenza virus. the results were expressed as the minimum inhibitory concentration (mic) of rhmbl that exhibited hi effect. influenza virus na activity was measured by elisa in which peanut agglutinin conjugated with hrp was used to detect b-dgalactose-n-acetylglucosamine exposed after removal of sialic acid from fetuin [21] . appropriate amounts of virus in dulbecco's 1x pbs with cacl 2 and mgcl 2 (life technologies, usa) were used to perform the nai assays. different concentrations of rhmbl were diluted in hbss containing ca 2þ and mixed with influenza virus in a total volume of 100 ml and preincubated at 37 c for 1 h, and then transferred to wells precoated with fetuin (sigma, usa) and incubated at 37 c for 4 h, after washing, 100 ml of hrp-labeled peanut lectin (3 mg/ml) was added and after 1 h at room temperature, the wells were washed and o-phenylenediamine dihydrochloride in citrate buffer was added, reaction was stopped by 2 m h 2 so 4 , and the od at 492 nm was measured. the wells only with virus were used as the positive control, the od of wells with hbss used as a negative control was subtracted. results were expressed as relative na activity (%) calculated as the od of the tested wells with virus and rhmbl divided by the od of the wells with only virus. the ha and na 3d structures were predicted by using the homology modeling method of swiss-model [22] . the modeling structures, corresponding templates and identities are shown in supplementary fig. s1 with pymol (delano scientific). the trimer structure of mbl was assembled using mbl crystal structure (pdb code: 1hup) with pisa [23] which generate the oligomeric forms of protein according to the symmetry information. to quantify the distance between rbd/na activity region and each ngs, we employed the average euclidean distance of the center (c alpha atom) of every rbd/na activity region and the center (c alpha atom) of ngs. differences between groups were tested using non-parametric kruskalewallis analysis of variance (anova) for multiple comparisons using ibm spss statistics 20.0 (ibm, usa) and p < 0.05 was considered statistically significant. the elisa results showed that rhmbl bound both seasonal and avian influenza viruses in vitro. incubation with increasing concentrations of rhmbl resulted in elevated levels of mbl bound to immobilized influenza virus, reaching a plateau at 3 mg/ml (fig. 1a) . then, 3 mg/ml rhmbl was used to determine the affinity to increasing amounts of virus, revealing the virus-dose dependent binding feature (fig. 1b) . high binding of rhmbl to the human h3n2 virus might be due to a relatively more ngs in its ha and na (table s2 ), in addition to the glycans attached at the site of 165 as previously reported [14] . beyond our expectation, h7n9 virus exhibited stronger binding than h9n2 virus, reaching a comparable level as human h3n2 at higher titers of 2 â 10 4 and 2 â 10 5 , although fewer ngs was observed in avian h7n9 than avian h9n2 (table s2 ). the differential binding of rhmbl to avian virus might result from the types of oligosaccharide attached and further investigations are needed. the finding demonstrated here implied a possible antiviral effect initiated by rhmbl at early stage of novel avian virus infection since naïve host lacked the preexisted cross-reactive or specific anti-ha or anti-na antibodies. initially, we discovered that the lowest concentration for rhmbl (mg/ml, mean ± sem) to abolish virus-mediated trbc agglutination was 1.42 ± 0.239, 0.083 ± 0.02, 0.026 ± 0.005 for h7n9vac, h9n2rg and h3n2wt (table 2) , respectively. the hi effect could be reversed in the presence of 5 mm edta or 5 mg/ml mannan, which indicates that hi was calcium-dependent and mannan-inhibitable. we then used a h7n1 reassortant obtained ha from h7n9 and other seven segments from pr8 to exclude a possible hemadsorption by n9 reported elsewhere [24] . the mic against the h7n1 was 3.67 ± 0.33 mg/ml (table 2) , which was remarkably higher than those against h3n2 or h9n2 virus, indicating a weaker hi effect of rhmbl on h7. as shown in fig. 2a , na activity of h3n2 was inhibited by rhmbl in a dose-dependent manner, and the ic 50 was 0.0807 ± 0.009 mg/ ml. similarly, the rhmbl, at a concentration of 0.0625 mg/ml, could inhibit the na activity of h9n2 (fig. 2b) . nevertheless, the relative na activity of h7n9 remained above 50% in the presence of increasing concentration of rhmbl even at up to 10 mg/ml. by contrast, the positive control, oseltamivir (roche, switzerland) at the concentration of 25 mm (equivalent to 7.109 mg/ml), could significantly inhibit the na activity of h7n9 (fig. 2c) . furthermore, the inhibition of h3n2 na activity by rhmbl could also be abolished in the presence of 5 mm edta or 5 mg/ml mannan, suggesting that the nai was calcium-dependent and mediated by lectin domain. rbd in ha, including the motifs of 190-helix, 130-and 220-loop, is one of important functional domains [25] . na activity region is composed of 8 functional residues (r118, d151, r152, r224, e276, r292, r371 and y406) and 11 framework residues (e119, r156, w178, s179, d198, i222, e227, h274, e277, n294 and e425) [18] . to elucidate a possible interference between mbl and the ngs around the functional motifs: rbd in ha or the na activity domain, we compared ngs distribution in them by structure modeling (fig. s1 ). we also obtained the trimer form of mbl using the crystal structure (pdb code: 1hup) and measured the radius of mbl crd which is at least 30 å. although there are indications that trimers of mbl are not biologically active and at least a tetramer form is needed for activation of complement [26] , 30 å can be regard as the minimum value. we subsequently calculated the average distances between the ngs located around rbd or na activity region ( table 1 ) and speculated that their distance within the size of human mbl crd, 30 å, might affect the functions of ha or na. as listed in table s2 and fig. s1 , only one conserved ngs on ha head was detected at position 240 in the avian h7n9 while different pattern was found in h3 and h9 (63, 122, 126, 133, 144 influenza a virus circulating in animal reservoirs is a continual cause for public health concern. in addition to the ever-present threat of seasonal influenza, we also face the threats from novel viruses such as h5n1 and h7n9, 2009pdm h1n1 virus in recent years. since the viruses can escape from the protection of anti-ha or anti-na antibodies due to antigenic shift, the innate immunity in naïve host is crucial for battling against newly-emerging viruses. mbl, a pattern-recognition molecule in innate immunity, is known as a b inhibitor for seasonal influenza a virus. the average concentration of mbl in healthy human plasma (aged 18e87years) is 1.72 ± 1.51 mg/ml [29] , with about 30% of individuals present mbl levels below 0.5 mg/ml [30] . the antiviral activity of mbl against seasonal influenza a virus is via its interactions with viral ha or na by blocking viral entry, fusion or releasing [15, 31] . mbl could neutralize the virus either in a complement dependent or independent manner [13, 15] , however, the effects of mbl may vary depending on specific strains. it also plays an important role in modulating inflammation and has been reported to contribute to deleterious inflammatory response to pdmh1n1 and a h9n2 avian isolate (a/quail/hong kong/g1/97) [32] . here, we found differential binding of rhmbl to human influenza h3n2 and avian h7n9, h9n2 viruses. specifically, rhmbl exhibited significant hi activity against h3n2 and h9n2 virus at a relatively low concentration (0.026 ± 0.005, 0.083 ± 0.02, respectively), while its hi activity on h7n9 virus was around 3.67 ± 0.33 mg/ml, reaching the upper limit in plasma of healthy population. in contrast, for nai on h7n9 virus, the rhmbl showed little effect even at a high concentration as 10 mg/ml. as mbl is supposed to display a steric interference with ha or na when binding to the specific hexose sugars across or adjacent to rbd in ha or activity region in na, the limited impact of mbl on h7n9 might result from its property of few ngs adjacent to functional region on the ha and na. of note, a serial of pathotypings including higher virus load, excess chemokine/cytokine response and (-) were incubated with increasing concentrations of rhmbl. na activity was measured by elisa, as described in materials and methods. c. the effects of 10 mg/ml rhmbl and 25 mm oseltamivir on na activity of h7n9vac. results were expressed as relative na activity (%) calculated as the od of the tested wells with virus and rhmbl divided by the od of the wells only with virus. the dashed line represents 50% of the original na activity. data are expressed as mean ± sem of three independent experiments. *p < 0.05. functional impairments of b and t cells have been observed in h7n9 infection cases, particularly in fatal cases [33, 34] . the aberrant inflammatory response in h7n9-infected animals could be reversed partially by anti-c5a, indicating a hyperactivated complement mediated [35] . therefore, the strong mbl-h7n9 virus interaction whereas limited effects on viral ha-receptor binding or na-mediated releasing, might amplify immune dysfunctions in vivo and confer clinical severity of h7n9 infection via activating complement pathway and further investigates are needed. isolation and characterization of a mannan-binding protein from human serum mannose-binding lectin binds to a range of clinically relevant microorganisms and promotes complement deposition interaction of mannose-binding lectin with hiv type 1 is sufficient for virus opsonization but not neutralization mannose-binding lectin in severe acute respiratory syndrome coronavirus infection mannose-binding lectin binds to ebola and marburg envelope glycoproteins, resulting in blocking of virus interaction with dc-sign and complement-mediated virus neutralization mannan-binding protein and bovine conglutinin mediate enhancement of herpes simplex virus type 2 infection in mice mannose-binding lectin and innate immunity influenza a viruses: new research developments clinical features and rapid viral diagnosis of human disease associated with avian influenza a h5n1 virus human infection with influenza h9n2 avian influenza a virus (h7n7) associated with human conjunctivitis and a fatal case of acute respiratory distress syndrome clinical and epidemiological characteristics of a fatal case of avian influenza a h10n8 virus infection: a descriptive study complement-dependent neutralization of influenza virus by a serum mannose-binding lectin human mannose-binding protein functions as an opsonin for influenza a viruses human mannan-binding lectin inhibits the infection of influenza a virus without complement human infection with a novel avian-origin influenza a (h7n9) virus biological features of novel avian influenza a (h7n9) virus mutations in polymerase genes enhanced the virulence of 2009 pandemic h1n1 influenza virus in mice adsorption of influenza hemagglutinins and virus by red blood cells world health organization. manual for the laboratory diagnosis and virological surveillance of influenza measurement of anti-influenza neuraminidase antibody using a peroxidase-linked lectin and microtitre plates coated with natural substrates swiss-model: modelling protein tertiary and quaternary structure using evolutionary information inference of macromolecular assemblies from crystalline state neuraminidase hemadsorption activity, conserved in avian influenza a viruses, does not influence viral replication in ducks structure of the haemagglutinin membrane glycoprotein of influenza virus at 3 a resolution mutations of neuraminidase implicated in neuraminidase inhibitors resistance loss of a single n-linked glycan from the hemagglutinin of influenza virus is associated with resistance to collectins and increased virulence in mice specific sites of n-linked glycosylation on the hemagglutinin of h1n1 subtype influenza a virus determine sensitivity to inhibitors of the innate immune system and virulence in mice comparison of human blood concentrations of collectin kidney 1 and mannan-binding lectin phase i safety, tolerability, and pharmacokinetic study of recombinant human mannan-binding lectin carbohydrate-binding molecules inhibit viral fusion and entry by crosslinking membrane glycoproteins mannose-binding lectin contributes to deleterious inflammatory response in pandemic h1n1 and avian h9n2 infection clinical findings in 111 cases of influenza a (h7n9) virus infection immune derangement occurs in patients with h7n9 avian influenza treatment with anti-c5a antibody improves the outcome of h7n9 virus infection in african green monkeys we thank national institute for biological standards and control (uk) and centers for disease control and prevention (usa) for providing the viruses used in our study. we greatly appreciate yu lan for instructions on influenza bioinformatics. this work was supported by national mega-projects for infectious diseases (2014zx10004002-001-004). none reported. supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.bbrc.2015.01.070. the transparency document associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.bbrc.2015. 01.070. 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-324840-ug5a9wx6 authors: de pascale, gennaro; cutuli, salvatore lucio; pennisi, mariano alberto; antonelli, massimo title: the role of mannose-binding lectin in severe sepsis and septic shock date: 2013-10-02 journal: mediators inflamm doi: 10.1155/2013/625803 sha: doc_id: 324840 cord_uid: ug5a9wx6 severe sepsis and septic shock are a primary cause of death in patients in intensive care unit (icu). investigations upon genetic susceptibility profile to systemic complications during severe infections are a field of increasing scientific interest. particularly when adaptive immune system is compromised or immature, innate immunity plays a key role in the immediate defense against invasive pathogens. mannose-binding lectin (mbl) is a serum protein that recognizes a wide range of pathogenic microorganisms and activates complement cascade via the antibody-independent pathway. more than 30% of humans harbor mutations in mbl gene (mbl2) resulting in reduced plasmatic levels and activity. increased risk of infection acquisition has been largely documented in mbl-deficient patients, but the real impact of this form of innate immunosuppression upon clinical outcome is not clear. in critically ill patients higher incidence and worse prognosis of severe sepsis/septic shock appear to be associated with low-producers haplotypes. however an excess of mbl activation might be also harmful due to the possibility of an unbalanced proinflammatory response and an additional host injury. strategies of replacement therapies in critically ill patients with severe infections are under investigation but still far to be applied in clinical practice. despite the diffusion of effective care bundles and the implementation of new technologies able to support organ function, severe sepsis and septic shock still represent a leading cause of intensive care unit (icu) admission with a case fatality rate of 30-40% [1, 2] . systemic inflammation, surrounding multiorgan failure and septic shock, results from a maladaptive unbalance between early antimicrobial immune reactions and uncontrolled local infection and inflammation. innate immune system is the primitive first-line organism's response to invasive pathogens, and it interacts with other homeostatic patterns, including inflammation and coagulation. early activation of immune response is mediated by soluble pattern recognition molecules that, in addition to complement proteins, cytokines, and coagulation factors, activate humoral and cellular effectors, identifying and neutralizing the invasive pathogen [3] . mannose-binding lectin (mbl) is a soluble pattern recognition molecule which activates the lectin pathway of the complement system and the subsequent inflammatory mechanisms [4, 5] . low mbl plasmatic levels, mainly due to genetic influences, have been largely described to be associated with susceptibility to invasive infections and poor outcome [6] . on the other hand, the excessive activation of this ancient protective system may be responsible for a detrimental unbalanced inflammatory and coagulation response, as observed in inflammatory diseases, transplant rejections, and diabetic nephropathy [7] . many authors have investigated whether mbl may influence the susceptibility to common pathogens and the development of severe infections, but, still, there is no consensus about the clinical relevance of its deficiency or the indications for replacement therapies. the purpose of this review is to summarize the results of relevant recent studies where the role of mbl in severe sepsis and septic shock has been investigated. mannose-binding lectin is a serum calcium-dependent protein, synthesized by the liver and is detectable in the sites of inflammation, particularly in epithelial-lining fluid [5] . small amounts of this protein are also produced in other organs (kidney, thymus, tonsil, small intestine, and vagina). mbl is a collectin (collagen-like lectin) and is characterized by a highordered oligomeric structure that is essential for its function and interaction with mbl-associated serine proteases (masps) [8, 9] . mbl harbors a carbohydrate recognition domain (crd) through which it binds to specific carbohydrates (i.e., mannose or n-acetylglucosamine) exposed on pathogenic agents surface, and it is therefore called a "patternrecognition molecule" [4] . subsequently masps (mainly masp-2) are able to trigger the lectin complement pathway cleaving c4 and c2 to form c3 convertase. complement system may be activated by three pathways: the classic and the alternative ones are antibody dependent and belong to the adaptive immune response; the lectin one is antibody independent and, as part of the innate immune system, comes into play within the first 12 hours from microorganisms' contact [10] (figure 1 ). mbl plays a central role as a firstline defense against invading pathogens by triggering complement system, directly mediating opsonophagocytosis, and possibly functioning as a toll-like receptor coreceptor [11] . in humans there are two genes that might code for mbl, but only mbl2 gene is functional, located on the long arm of chromosome 10 [12] . mbl deficiency may be due to the presence of single nucleotide polymorphisms (snp) either in the gene-coding or in the promoter regions. the wildtype gene is called "a" (homozygous haplotype, a/a); instead the variants alleles are widely classified as "0" (0/a and 0/0). three points mutations involve the exon 1, identifying allele b (codon 54), c (codon 57), and d (codon 52). additionally, many snps may affect the promoter region and determine low mbl protein serum levels and activity (variants h/l, y/x, and p/q). even though all these mutations could be combined with exon 1 alleles, seven haplotypes are typically found in humans genome [13, 14] . these polymorphisms determine the production of unstable proteins, with shorter half-life, mainly due to the absence of the high-ordered oligomeric structure. these variants are not able to efficiently activate the complement pathway [15] . healthy individuals (genotype a/a) generally present mbl levels above 1000 ng/ml. in the newborn this protein is detectable at concentrations of two-thirds of their mothers. normal levels are reached within a month [16] . mbl levels are not influenced by age, circadian cycle, and physical exercise and, during inflammation, do not increase over 3-4 folds than baseline level [17] . mbl deficiency is generally defined by plasmatic protein levels below 500 ng/ml or by an mbl function lower than 0.2 u/ l c4 deposition [18] . the detection of pulmonary mbl concentration is quite difficult. some authors have reported bronchoalveolar lavage (bal) levels ranging between 20 and 80 ng/ml, but these results were not corrected for dilution factors (i.e., urea or other lung proteins with known lung concentration) that could explain the large distance from the minimum concentration needed to activate complement proteins (300-400 ng/ml) [19] . plasmatic levels ranging between 500 and 1000 ng/ml are generally detected in heterozygous patients (a/o genotype); instead homozygous variant mbl2 alleles usually present very low concentrations (<50 ng/ml). similarly the haplotypes that include mutations in promoter regions are associated with significant reduction of mbl protein production and activity. however, even though the degree of mbl deficiency is strictly dependent upon patients' genotypes, in some cases low mbl plasmatic levels have been also associated with wild-type genes [13] . this gene was already present in early invertebrates more than fifty million years ago and has been highly conserved throughout animal and human evolution. this would suggest that the correct function of mbl protein is crucial for the survival of living animal species. it is of interest to note that there is geographic distribution of different alleles: the b variant is predominant in eurasian populations, the c variant mainly among asians and america indians, and the d haplotype seems to be frequently expressed in caucasian region. this particular distribution might be linked to the initial human migrations table 1 : mbl deficiency and susceptibility to diseases (human and animal studies). (vi) hsv out of africa and induced by some specific advantages due to mbl deficiency. for example, high mbl production has been observed to be associated with higher incidence of preterm births; instead moderately low levels could protect the organism from mycobacteria systemic infection and from the complement induced inflammatory-mediated damage of some diseases (i.e., meningococcemia and rheumatoid arthritis) [20, 21] . additionally sporadic reports have not found a clear association between mbl deficiency and increased rate of infectious episodes [22] [23] [24] . the wide range of clinical effects linked to mbl haplotypes has also been attributed to the role of associated mutations in other genes encoding proteins with similar functions (i.e., l-ficolin, masp2, and surfactant proteins) [25, 26] . however, to date, there are few data upon the clinical role of these combined deficiencies. the prevalence of mutations in one or both mbl2 gene alleles is relevant, ranging between 30% and 40% in analyzed populations [13, 27] . during the last twenty years an increasing body of evidence has indicated that mbl deficit, due to specific haplotypes, generally increases frequency and severity of infectious episodes [28, 29] (table 1) . however the structure of our immune system is redundant, and this may explain why in many cases polymorphisms of mbl2 gene were not observed to influence susceptibility to infections [24] . the role of this lectin is particularly relevant when adaptive immune system is immature or compromised [30] . in a case-control study upon 47 infants, lower mbl cord blood concentrations were associated with a higher incidence of gram-negative sepsis ( = 0.036) [31] , and an observational cohort study upon 100 pediatric icu patients identified mbl2 gene exon 1 polymorphisms as a main determinant of progression from sepsis to septic shock [32] . additionally, the incidence and outcome of severe infections appear to be influenced by the levels and activity of mannosebinding lectin. in a cohort of leukemic patients undergoing chemotherapy, severe infections (bacteremia, pneumonia or both) occurred more frequently in those individuals with lower mbl concentrations ( < 0.001) [33] . in an ethnically homogeneous english population, homozygotes for mbl codon variant alleles showed a significantly higher risk of invasive infections due to streptococcus pneumoniae, "the captain of men of death" [34] . similarly allelic variants of this gene seem to be associated with increased susceptibility to meningococcal disease [35] . among respiratory tract infections, independently from the causal pathogen, mbl insufficiency has been observed to predispose to higher severity and poor outcome [36] . even though legionella spp. act as an intracellular pathogen, mbl function was lower in infected cases during an australian legionnaires' disease outbreak [37] . increased susceptibility and worse outcome in 212 caucasian patients with acute respiratory distress syndrome (ards) were also observed in presence of mbl2 gene polymorphisms [38] . regarding viruses, in chinese population, the presence of mbl2 gene b variant was associated with increased risk of coronavirus infection [39, 40] ; instead normal mbl function seems to worsen pandemic h1n1 and avian h9n2 infections by potentially upregulating inflammatory response [41] . furthermore, in a recent large retrospective study involving 102 donor-recipient orthotopic liver transplantation pairs, patients who received mbl-deficient livers showed a threefold increased risk of clinically significant infections including cytomegalovirus-related diseases [42] . few authors have studied the role of mbl in severe fungal infections. polymorphisms of this gene were observed in seven of ten white patients with chronic necrotizing aspergillosis compared with 25% of controls [43] . in addition variations of mbl plasmatic levels seem to correlate with the occurrence of invasive candidiasis [44] . mbl genetic, plasmatic, and functional profiles were investigated in numerous clinical settings obtaining different results. the critically ill patient, affected by severe infections with severe sepsis and septic shock, might be a field of particular interest for a better knowledge of their clinical relevance and the possible development of novel therapeutic strategies. mannose-binding lectin is not only part of the innate recognition system of invasive pathogens but effectively modulates the cytokines' production by macrophages during phagocytosis. this effect, upon interleukin (il)-6, il-1 , and tumor necrosis factor-, was clearly shown in an "ex vivo" model of immediate immunity response to neisseria meningitidis infection [45] . mbl deficiency may be associated with unbalanced proinflammatory responses to infective and noninfective triggers. in a cohort of critically ill pediatric patients, fidler and coworkers observed that mbl levels less than 1000 ng/ml, consistent with mbl-2 gene exon 1 polymorphisms, significantly increased the risk of developing systemic inflammatory response syndrome (sirs) and progression to severe sepsis/septic shock [32] . additionally in patients with sirs, mbl insufficiency degree was observed to correlate with severity of systemic infection, according to the genetic profile [46] . the association between the deficiency of this protein and worse outcome during severe systemic infections (i.e., evolution to refractory septic shock) may be also related to the significant interaction between complement activation, inflammatory cytokines' "storm", and coagulation cascade. the influence of complement activation upon septic shock development was largely investigated. many studies have shown how the classical and alternative pathways are activated during septic shock and are involved in mechanisms aimed to clear endotoxin. this role has been more recently studied also for lectin complement activation due to mbl [47, 48] . disseminated intravascular coagulation (dic) may worsen the course of septic shock but the occurrence of this severe complication is unpredictable. however recent data suggest that mbl deficit may be a significant risk factor for the early development of dic and organ failure during severe infections [9] . conversely, excessive mbl expression might be harmful, since this molecule may contribute to the pathogenesis of inflammatory induced vascular damage and organ failure, as observed in patients undergoing solid organ transplantation [49] . hence mbl, due to its pivotal role in the crosstalking among complement activation, coagulation, and systemic inflammation, may represent a key point for the understanding of the development of systemic severe infections, as interestingly investigated in animal models and clinical studies involving patients with severe sepsis/septic shock. even though many differences between animal models and humans limit the "translationalability" of preclinical data, several mouse experiments support the role of mbl deficiency in severe infections, especially after bacteria inoculation. two functional mbl genes exist in the mouse, and the generation of double knockout gene-deficient mice has increased the investigations in this field. after inoculation of 5 × 10 7 cfu staphylococcus aureus, mbl-null mice showed at 48 hours 100% mortality compared with wild-type (wt) mice which survived in a percentage of 55%. additionally, pretreatment of mbl-null mice with rhmbl increased their survival rate of about 50% [50] . in another model of mbl and/or masp 1/3 deficient mice takahashi and coworkers observed that this deficiency was associated with early occurrence of dic and liver injury after s. aureus inoculation, suggesting the role of this protein in the development of organ failure and systemic coagulation activation during severe infections [9] . another study demonstrated that mbl is able to strongly bind to o-antigen region of lps, contributing to mice platelets activation and rapid occurrence of septic shock [48] . susceptibility of mbl null mice to pseudomonas aeruginosa postburn infection was also investigated [51] . all mbl-null mice, after burn and bacterial inoculation, early developed septic shock and died; instead the majority of wt animals (two-thirds) survived. these observations underline the relevance of innate immunity and mannose-binding lectin in the susceptibility and outcome of severe bacterial infections occurring in this population. regarding fungal diseases, the protective role of this lectin was also observed in murine models of invasive pulmonary aspergillosis after ex vivo mbl administration [52] . however lectin pathway activation does not only depend on mbl function. some authors have observed how deficient mice models, without the capability to activate mblindependent lectin cascade (i.e., ficolins and other collectins), are more susceptible to develop severe systemic pneumococcal infections [53] . although most of literature evidence obtained by animal studies supports the importance of mbl in the acquisition and outcome of severe infections, these observations, due to unresolved several limits of animal studies, may not be considered conclusive and strongly need clinical human studies to definitely identify its clinical relevance. studies. the mbl key role as part of innate immunity is the reason why haplotypes associated with its deficiency mainly influence infectious episodes involving neonates and children or immunosuppressed adults. in a population-based prospective study performed in greenland upon almost 300 eskimo children, both heterozygous and homozygous subjects, aged 6 to 17 months, for variant alleles presented a twofold increased risk of acute respiratory infections, including pneumonia [30] . additionally, capoluongo and colleagues, analyzing 75 preterm newborns, identified two mbl2 gene variants as independent risk factors associated with unfavorable outcome, including higher bronchopulmonary dysplasia prevalence [54] . turkish authors have investigated the possible relationship between cord blood mbl levels and neonatal sepsis. the results indicated that lower mbl levels during fetal inflammatory response syndrome (firs) were associated with higher risk of sepsis development independently from gestational age and birth weight [55] . another prospective study conducted on 62 neonates (27 of them were preterm) showed how lowest mbl levels were detected in infants with septic shock, especially in case of fatal outcome ( < 0.05). relevant sensitivity, specificity, positive, and negative predictive values for detecting sepsis episodes were also documented [56] . in a recent swiss investigation, mbl levels were detected in cord blood of 141 newborns. forty-seven developed sepsis (28% within the first 72 hours of life) and 13% required catecholamines because of septic shock. after excluding those infants who underwent surgery, low mbl concentrations resulted independently being associated with increased risk of early-onset gram-negative sepsis [31] . in pediatric oncological patients, mbl deficiency was associated with susceptibility, poor outcome, and duration of febrile neutropenic episodes [57] . in a prospective study mbl deficit was observed to increase the severity of disease during pediatric icu admission after febrile neutropenia [58] . additionally also mbl-related proteins deficit was mediators of inflammation 5 investigated in this setting. in a cohort of 94 children treated with chemotherapy for cancer, masp-2 deficit (<200 ng/ml) significantly increased the risk of febrile neutropenia and bacteraemia development and prolonged cumulative duration of hospitalization and antimicrobial treatment [59] . the importance of mbl function during the first months of life, when the efficacy of innate immunity is crucial, has induced some authors to propose its dosing as part of a biomarkers panel for the early detection of severe neonatal infections in low-resource settings [60] . impaired innate immune mechanisms may also increase the risk of nosocomial infections in critically ill patients as observed by sutherland and colleagues. in a genetic association study, the authors identified the relationship between snp in cd 14, mbl and toll-like receptor-2 with increased prevalence of positive cultures and sepsis [61] . in a cohort of 195 adult septic patients, mbl deficiency resulted also independently being associated with higher sequential organ failure assessment (sofa) score at day 3, suggesting its role as a risk factor for the development of severe sepsis and septic shock [62] . additionally in a multicenter prospective study involving eight adults icus in u. k., the association between mbl-2 exon and promoter polymorphisms with the outcome of 174 patients affected by severe sepsis and septic shock was studied [63] . compared with healthy subjects, mbl deficient patients were at increased risk of sepsis, with a significant higher mortality rate in presence of levels below 1000 ng/ml (47.2% versus 22.2%, = 0.05). during severe sepsis and septic shock, the increase of mbl plasmatic levels, as acute phase response molecule, may be different. in a report of 128 adult critically ill patients, dean and colleagues observed that regardless of mbl-2 genotype those patients who were mbl deficient at study entry were not able to reach normal plasmatic levels during severe sepsis and septic shock [17] . furthermore, a well-conducted prospective study, performed in denmark, investigated the mbl genetic and plasmatic profile in a population of 272 critically ill icu patients with documented sirs [46] . among enrolled patients 172 met the criteria for severe sepsis and 70 for septic shock. compared with noninfectious sirs, these patients shared the carriage of mbl variants alleles and low serum levels according to the severity of disease ( = 0.03). another recent korean study in icu patients investigated whether mbl2 gene polymorphisms and serum levels might influence severity and prognosis of sepsis [64] . the authors compared 26 septic patients with 398 healthy controls, analyzing three snp and dosing mbl serum levels on day one. among sepsis group, homozygosis for the polymorphism at codon 54 (a/a) resulted in a significant risk factor for severe sepsis development ( = 0.001). mbl serum levels ≥ 1.3 mcg/ml were associated with a lower 28day mortality rate in the septic shock group ( = 0.02). the role of mbl deficiency in critically ill patients with severe pneumonia, a still leading cause of death due to an infectious disease, has been investigated by many authors. in a large case-control study, 848 patients affected by community-acquired pneumonia (cap) were compared with 1447 healthy control subjects and 519 patients without relevant infectious diseases. mbl2 and masp2 haplotypes were equally distributed among those subjects. in the multivariate analysis, mbl deficiency was associated with poor outcome measures (i.e., severe sepsis, acute respiratory failure, multiorgan dysfunction syndrome, and death) [36] . eisen and colleagues have reanalyzed data from six studies involving 675 patients affected by severe infections [18] . first, the authors defined a mbl cutoff value of 0.5 mcg/ml as a reliable predictor of low producing status (negative predictive value 98%). they confirmed that mbl deficiency significantly increased the risk of death due to severe infection, also in icu setting, especially when streptococcus pneumoniae was the invasive causative agent (odds ratio 5.6, 95% confidence interval, 1. 27-24.3) . the association between mbl deficiency and s. pneumoniae invasive infection outcome has been recently investigated in a spanish prospective cohort study [65] . during the study period 117 patients with invasive pneumococcal infection were enrolled: the rate of allelic variants was 32%. snp mbl2 (ao/oo) and septic shock were the factors independently associated with in-hospital mortality. otherwise early adequate antibiotic dose ≤ 4 hours resulted in a significant protective determinant. mbl deficiency role was also studied in some other systemic infections due to specific organisms. resman and colleagues recently described the case of a necrotizing myositis and septic shock due to haemophilus influenzae in a patient where igg3 and mbl deficiency were diagnosed [66] . in a well-conducted prospective study, the correlation between mbl2 gene polymorphisms and the outcome of escherichia coli pyelonephritis was investigated [67] . although no association was found with the incidence of e. coli infections and the presence of bacteremia, those patients who shared lowexpression mbl2 genotypes showed a significant higher risk of septic shock development (odd ratio: 9.1, 95% confidence interval: 1.23-65.9; = 0.03). finally, in nonbacterial severe systemic infections, invasive candidiasis (ic), especially candidemia, still remains a leading cause of death due to infections in critically ill patients. serum mbl levels were measured in 68 patients with proven ic, 82 hospitalized not infected patients, and 70 healthy subjects [44] . even though mbl concentration was significantly higher in ic patients than controls, the authors identified a marked decrease in its plasmatic levels during the first days of infection in association with mannans increase. these observations, although limited, suggest a crucial role of mbl also in the early phase of candidiasis. therapy. mbl substitution therapy in patients with recognized lectin deficit has been proposed. apart from genetic analyses, antigenic measurement is widely diffused as diagnostic test. even though mbl serum levels < 500 ng/ml or mbl activity < 200 u/ml may be considered a significant deficiency, there are not standard guidelines aimed to define which patient categories need to be tested (i.e., in presence of severe recurrent respiratory infections or acquired immunesuppression). recombinant human mbl use, to supplement mbl deficiency status, has been investigated in animal and phase i/ii human studies [68, 69] . although its clinical efficacy has not been clearly established, still now no adverse effects were observed. sixty-five mbl infusions were given to 12 mbl deficient chemotherapyinduced neutropenic children. the observed postadministration level was 1.06 mcg/ml (range: 0.66-2.05) which may be considered protective [70] . a similar pharmacokinetic profile was observed in 20 healthy mbl-deficient volunteers and two patients with staphylococcus aureus septicemia [71] . however, beyond these preliminary observations, mbl replacement needs to be further investigated in deficient patients affected by acute severe infections, especially in presence of multiple-level immune system impairment. an increasing body of data support the role of mbl as central player of innate immunity. several gene polymorphisms have been identified in association with decreased serum levels and activity. many authors have showed the association of this molecule deficit with recurrent severe infections, particularly involving the respiratory tract and encapsulated bacteria. additionally growing evidence suggests its importance during systemic severe infections as severe sepsis and septic shock. this correlation might derive from the crosstalking among complement system, coagulation patterns, and proinflammatory cytokines. even though many patients with systemic infections, who present mbl serum levels below the functional threshold, are at higher risk to develop severe complications and poor outcomes (i.e., septic shock, multiple organ failure), in some cases low levels have appeared to be protective, probably reducing the inflammatory cytokines' storm. moreover not all published studies have identified a clear association between deficiency and increased risk of infections. replacement therapy with recombinant human protein during severe sepsis and septic shock affecting deficient patients has been proposed but it still remains an experimental treatment. hence, until new promising and robust data will be available, the strict adherence to current standard recommendations still remains the mainstay of severe sepsis/septic shock management. surviving sepsis campaign: international guidelines for management of severe sepsis and septic shock: 2012 fluid therapy in septic shock the immune system evolved to discriminate infectious nonself from noninfectious self the mannose-binding lectin: a prototypic pattern recognition molecule mannan-binding lectin and its role in innate immunity impact of mannose-binding lectin on susceptibility to infectious diseases mannose-binding lectin: clinical implications for infection, transplantation, and autoimmunity the lectin-complement pathway-its role in innate immunity and evolution mannosebinding lectin and its associated proteases (masps) mediate 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levels in neonatal sepsis and septic shock deficiency of mannose-binding lectin and burden of infection in children with malignancy: a prospective study mannosebinding lectin (mbl) as prognostic factor in paediatric oncology patients deficiency of mannose-binding lectin-associated serine protease-2 associated with increased risk of fever and neutropenia in pediatric cancer patients emerging biomarkers for the diagnosis of severe neonatal infections applicable to low resource settings polymorphisms in cd14, mannose-binding lectin, and toll-like receptor-2 are associated with increased prevalence of infection in critically ill adults low mannosebinding lectin function is associated with sepsis in adult patients mannose-binding lectin polymorphisms in severe sepsis: relationship to levels, incidence, and outcome association of mannose-binding lectin-2 genotype and serum levels with prognosis of sepsis genetic variants of the mbl2 gene are associated with mortality in pneumococcal sepsis necrotizing myositis and septic shock caused by haemophilus influenzae type f in a previously healthy man diagnosed with an igg3 and a mannosebinding lectin deficiency association between mannose-binding lectin deficiency and septic shock following acute pyelonephritis due to escherichia coli human plasma-derived mannose-binding lectin: a phase i safety and pharmacokinetic study infusion of plasma-derived mannan-binding lectin (mbl) into mbl-deficient humans safety and pharmacokinetics of plasma-derived mannosebinding lectin (mbl) substitution in children with chemotherapy-induced neutropaenia the pharmacokinetic profile of plasma-derived mannan-binding lectin in healthy adult volunteers and patients with staphylococcus aureus septicaemia the authors declare that there is no conflict of interests. key: cord-268902-npug5c8p authors: liu, yang; liu, jianying; pang, xiaojing; liu, tao; ning, zhijie; cheng, gong title: the roles of direct recognition by animal lectins in antiviral immunity and viral pathogenesis date: 2015-01-29 journal: molecules doi: 10.3390/molecules20022272 sha: doc_id: 268902 cord_uid: npug5c8p lectins are a group of proteins with carbohydrate recognition activity. lectins are categorized into many families based on their different cellular locations as well as their specificities for a variety of carbohydrate structures due to the features of their carbohydrate recognition domain (crd) modules. many studies have indicated that the direct recognition of particular oligosaccharides on viral components by lectins is important for interactions between hosts and viruses. herein, we aim to globally review the roles of this recognition by animal lectins in antiviral immune responses and viral pathogenesis. the different classes of mammalian lectins can either recognize carbohydrates to activate host immunity for viral elimination or can exploit those carbohydrates as susceptibility factors to facilitate viral entry, replication or assembly. additionally, some arthropod c-type lectins were recently identified as key susceptibility factors that directly interact with multiple viruses and then facilitate infection. summarization of the pleiotropic roles of direct viral recognition by animal lectins will benefit our understanding of host-virus interactions and could provide insight into the role of lectins in antiviral drug and vaccine development. animal lectins will benefit our understanding of host-virus interactions and could provide insight into the role of lectins in antiviral drug and vaccine development. keywords: lectin; virus; direct interaction; antiviral immunity; viral pathogenesis lectins, a highly diverse group of proteins that recognize carbohydrates, have been demonstrated to play a vital role in numerous life processes and to be critical for several viral infections and pathogeneses in a variety of organisms [1, 2] . based on their conserved structure of sequence motifs for sugar binding and carbohydrate specificities, lectins have been categorized into many families conventionally designated as calnexin, c-type, l-type, p-type/mannose-6-phosphate receptors (mprs), i-type/siglecs, m-type, f-type (absent in mammals), r-type, f-box, chitinase-like lectins, galectins and intelectins (table 1 ) [3] . the features of carbohydrate recognition domains (crds), such as structure peculiarity, carbohydrate binding selectivity and geometrical arrangement of multiple crds, determine the different properties of lectins [2] [3] [4] . furthermore, the diversity of locations and functions indicates the importance of lectins in the basic life processes of organisms. lectins are exploited as susceptibility factors to facilitate viral entry, replication or assembly ( figure 1 ). insight into direct lectin-based viral recognition will provide a deep understanding of host-virus interactions. mammalian lectins have been categorized into multiple classes according to the features of their crds, as well as their sugar recognition specificity. some lectins generally play a role outside the cell, whereas others are predominantly intracellular and located on cytoplasmic organelles. extracellular lectins, including c-type, r-type, i-type/siglecs lectins and galectins, are secreted into the extracellular milieu or are localized to the plasma membrane and are capable of mediating cell adhesion, immune signaling and pattern recognition activities for host-pathogen interactions. however, intracellular lectins, such as the calnexin family, m-type, l-type and p-type lectins, are located in luminal compartments of the secretory pathway and function in the trafficking, sorting and maturation of glycoproteins [3] [4] [5] . as lectins play diverse roles in mammalian physiological processes, we only focus herein on a portion of lectins that directly interact with viral components and describe their functions in viral propagation and pathogen-host immune responses. c-type lectins (ctls) are a large group of proteins in metazoans that were originally named according to their property of ca 2+ (c-type)-dependent carbohydrate binding. sequence and structural comparisons of c-type lectins have suggested that their carbohydrate-binding activity is mediated by a specific crd that is conserved in a variety of organisms. although some c-type lectins do not possess carbohydrate-binding activity, all of them show distinct sequence similarity and are believed to descend from a common ancestor during evolution [6] [7] [8] . to date, c-type lectins have been divided into 14 subgroups according to their domain architecture and the phylogenetic relationship between their crd sequences [9] . in general, c-type lectins can be separated into two groups, mannose-binding and galactose-binding c-type lectins, based on the specificity of their carbohydrate-binding activity. the binding specificity of these two groups is mediated by diverse residues flanking the conserved cis-proline in the long loop region, in which the sequence of the core motif is e-p-n for mannose-binding and q-p-d for galactose-binding specificity [10, 11] . previous studies have demonstrated that interchange of the e-p-n and q-p-d sequences is sufficient to switch the mannose-and galactose-binding specificity ( figure 2 ) [10] . however, several lectins are exceptions to this rule. for example, surfactant protein a, possessing an e-p-k but not an e-p-n motif, binds to mannose sugars [12, 13] . although human tetranectin contains a galactose-type q-p-d motif, it is not responsible to the lectin-carbohydrate interaction [14] . therefore, other determinants, including modifications around the binding sites and stereochemical factors, should be taken into consideration when examining binding specificity [15] [16] [17] . robust investigations have shown that multiple mannose-/galactose-binding c-type lectins play important roles in viral infections in mammals. mbl, one of the most intensively studied lectins, is a member of the collectin family, a subgroup of c-type lectins ( figure 3a ). the mbl molecule contains four domains that are standard for collectin family proteins: a cysteine-rich region, a collagen-like domain, a neck region and a carbohydrate recognition domain. the native functional form of mbl is a hexamer; however, although mbl can form several oligomeric forms, the dimers and trimers do not have biological activity, and at least a tetramer form is needed to activate the complement cascade [18] . mbl functions as a soluble pattern recognition receptor in the host complement system and is involved in resistance to many viral infections [19] . the crds of mbl multimers recognize carbohydrate patterns on the virus surface, and consequently, the binding of mbl and viral particles results in activation of the lectin pathway of the complement system. masps (mannose-binding lectin-associated serine proteases), which are protease zymogens (an inactive form of an enzyme) similar to the c1r and c1s molecules of the classical complement pathway, are activated to cleave complement components c4 and c2 into c4a/c4b and c2a/c2b, respectively. interactions between c4b and c2b produce the c3 convertase, which continuously activates c3 further downstream in the cascade to eliminate viruses ( figure 1a ) [20] . current investigations have reported a resistance role of mbl in infections of multiple human viruses, including human immunodeficiency virus (hiv) [21] , hepatitis b virus (hbv) [22, 23] , hepatitis c virus (hcv) [24] , west nile virus (wnv) [25] and dengue virus (denv) [25] . specific recognition of these viral particles by mbl is a central event for activation of the lectin-based complement cascade. the mechanism of viral elimination by the mbl-based complement cascade is still unclear. unlike bacterial elimination by the complement system, mbl-based complement components do not appear to form a membrane-attacking complex on the viral surface; therefore, viral eradication may not be mediated by known complement mechanisms. three possible mechanisms have been proposed for mbl-mediated viral elimination. (1) mbl-mediated complement c3/c4 deposition onto the viral surface. viral neutralization can be processed in cell-free serum and is completely dependent on c3 and c4 activation, but not on c1q and c5, suggesting that neither opsonization nor the classical/alternative complement pathway is sufficient for viral neutralization [25] . (2) mbl can directly neutralize viruses. pre-incubation of serial concentrations of recombinant mbl with hiv cell-derived living particles was found to dramatically neutralize hiv infection [21, 26] . however, other independent investigations have suggested that some primary hiv isolates resist direct neutralization by mbl [27] , indicating that the possible neutralizing activity depends highly on the different carbohydrate structures on the surfaces of various viral strains. (3) mbl can block the recognition of viruses and receptors. dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin (dc-sign), which is present on the surface of dendritic cells, functions as a key attachment factor used for the recognition and uptake of multiple viruses [28] [29] [30] [31] [32] . a study reported that mbl can prevent interaction between hiv and dc-sign, thereby inhibiting the hiv infection of t cells, which is mediated by dc-sign [33] . furthermore, mbl interacts with the viral envelope glycoproteins of ebola and marburg viruses (marv), resulting in the impairment of viral internalization by blocking virus-dc-sign interaction ( figure 1b ) [34] . two soluble collectins, designated sp-a and sp-d ( figure 3b ,c), have been found to be involved in the recognition of viral particles for limiting infection in humans. sp-a is produced within the respiratory tract, gastrointestinal tract, and possibly other sites; conversely, sp-d is primarily synthesized in the respiratory tract [35] . these factors are constitutively secreted into the lungs by alveolar type ii cells, unciliated bronchial epithelial cells and other mucosal tissue cells [36, 37] . the specific location of surfactant proteins suggests a defensive role against viral invasion of the respiratory system. both sp-a and sp-d interact with different strains of influenza a virus (iav) via glycosylated hemagglutinin (ha) and neuraminidase (na) on the viral surfaces. the binding of iav to sp-a leads to agglutination of the virions, inhibition of iav infectivity and dissemination and also facilitates clearance by macrophages and neutrophils ( figure 1a ,b) [38] . sp-d binds to iavs and thereby inhibits virus attachment and entry by viral aggregation ( figure 1b ) [39] [40] [41] [42] [43] and also controls iav infection in human by activating neutrophil chemoattraction ( figure 1a ) [44, 45] . respiratory syncytial virus (rsv) infects humans via the respiratory tract. sp-a has been reported to bind fusion (f) and adherence (g) glycoproteins on the surfaces of rsv virions, resulting in opsonization to reduce infection by enhancing viral uptake by peripheral blood mononuclear cells (pbmcs) and alveolar macrophages ( figure 1a ) [46, 47] . sp-d also directly interacts with rsv surface g protein to modulate host immune responses to control rsv infection [48] . dc-sign (cd209) and its homolog l-sign (also called dc-signr, cd209l) are one of the most investigated c-type lectins involved in viral infection ( figure 3f ). unlike the main role of collectins in host defense, dc-sign and l-sign have been widely reported to be susceptibility factors that facilitate viral entry into host immune cells [28, 31, 32, 49] . both dc-sign and l-sign are trans-membrane proteins that are composed of a short cytoplasmic tail, which is responsible for signaling and internalization, a transmembrane region, a neck domain, which consists of eight repeat regions of 23 amino acids, and a carbohydrate recognition domain [50] . the roles of dc-sign/l-sign in viral infections have been summarized and reviewed previously [50, 51] . studies have shown that dc-sign/l-sign are capable of binding to the surface proteins of hiv [28] , cytomegalovirus (cmv) [30] , denv [32] , wnv [52, 53] , severe acute respiratory syndrome coronavirus (sars-cov) [54] [55] [56] , hcv [57, 58] , ebola virus [29] and marv [54] and consequently facilitating viral entry ( figure 1c ). differential glycosylation patterns of viral surface proteins strongly influence the efficiency of viral recognition by dc-sign/l-sign [59, 60] . for example, only mannosylated envelope (e) glycoproteins on denv, but not e proteins with complex glycosylation, have been shown to interact with dc-sign-expressing cells [60] . the mannose receptor (mr, cd206) is another c-type lectin that functions as a viral recognition receptor on the cell membrane ( figure 3g ). mr is mainly expressed in multiple immune cells, including macrophages and dendritic cells, and is a key susceptibility factor for denv infection of human macrophages. binding of mr to denv e glycoproteins enhances viral attachment, thus facilitating denv internalization into macrophages, and deglycosylation of the denv e glycoprotein enables the abrogation of this binding, and denv infection of primary human macrophages can be blocked by anti-mr antibodies [61] . moreover, the interaction between mr and hbv surface antigen (hbsag) enhances viral uptake by dendritic cells (dcs), resulting in the impairment of dc function and the ineffective antiviral response of chronic hbv [62] . the recognition of viral surface glycoproteins by mr is also beneficial to influenza virus [63, 64] and hiv [65] invasion into host cells ( figure 1c ). overall, dc-sign/l-sign and mr function as receptors/attachment factors for viral entry into particular cell types. clec5a/mdl-1 (myeloid dap12-associating lectin) is a c-type lectin associated with dap12 (12-kda dnax-activating protein) on myeloid cells such as monocytes, macrophages and neutrophils ( figure 3h ) [66] . a recent study has found that clec5a binds to dengue glycoproteins. however, in contrast to other c-type lectin receptors, the association between clec5a and denv does not result in viral entry, but rather induces dap12-mediated immune signaling to stimulate the release of pro-inflammatory cytokines that potentially contribute to the pathogenesis of dengue hemorrhagic fever [67, 68] . clec5a also directly interacts with japanese encephalitis virus (jev) to induce dap12 phosphorylation in macrophages and therefore plays a role in jev-induced neuro-inflammation and lethality ( figure 1c ) [69] . the blocking of clec5a in mice can significantly reduce the infiltration of jev-harboring leukocytes into the central nervous system, thus attenuating neuro-inflammation and protecting the animals from jev-induced lethality [69] . the discovery of a role of clec5a in flaviviral pathogenesis suggests that the extracellular crd modules are generally responsible for the recognition of viral glycoproteins; nonetheless, the intracellular modules determine the role of c-type lectins in viral infection. langerin (also known as cd207), containing a single ca 2+ -dependent crd domain, is a type ii transmembrane c-type lectin that is specifically expressed on langerhans cells ( figure 3i ). the physiological function of langerin is to trigger the cellular membrane superimposition and zippering that benefit birbeck granule (bg) formation [70] . langerin is capable of directly binding to hiv-1 envelope protein gp120 and thus serves as a potential receptor for hiv-1 infection in langerhans cells ( figure 1c ) [71, 72] . however, a recent study reported that langerin is a natural barrier for hiv-1 transmission among langerhans cells. langerin is capable of directly capturing hiv-1 and sequentially degrading it in bgs to promote t cell elimination of hiv-1 infection [73] , suggesting that langerin plays a pleiotropic role in hiv infection. furthermore, langerin functions as an attachment factor to facilitate measles virus (mv) infection in langerhans cells [74] . a large number of host proteins are abundantly glycosylated. therefore, microbial recognition by c-type lectins relies on the mechanism for distinguishing carbohydrate structures between self and non-self, and the c-type lectin structure largely influences binding avidity and selectivity in the recognition of self and non-self carbohydrate structures [75, 76] . based on the x-ray crystal structures of mannose-binding lectin (mbl), the mbl crd sites in the trimer form are too far apart to spatially interact efficiently with common mammalian high-mannose oligosaccharides. however, the dense and repeated arrays of carbohydrates present on the microbial surface can span the distance between the binding sites in mbl, resulting in highly avid multivalent interaction [75, 77] . moreover, the number of crds is another determinant for the avidity and strength of differential binding by c-type lectins. the eight different c-type crds of mr contribute to its high binding affinity for single sugars, even though each individual crd motif only displays weak affinity. the crd domain organization also confers mr with the ability to recognize the wide range of different carbohydrates found on the pathogen surface and to distinguish between self and foreign glycoproteins [75] . galectins are a group of secreted proteins that associate with specific cell surface glycans containing beta-galactosides ( figure 3d ,e) [78] . although mammalian galectins lack conventional signal sequences, they reach the cell surface via a particular mechanism. galectins accumulate directly beneath the plasma membrane and are subsequently involved in the establishment of membrane-bound vesicles that pinch off before release outside the cell; galectins then bind to glycoconjugates on the plasma membrane or remain in the extracellular matrix [79] [80] [81] . fifteen galectins have been identified in mammals and are categorized into three structural forms: dimeric, tandem or chimeric. dimeric galectins, also called prototypical galectins, are homodimers and include galectin-1, -2, -5, -7, -10, -11, -13, -14 and -15. tandem galectins contain at least two distinct crds within one polypeptide and include galectin subtype-4, -5, -8, -9 and -12. galectin-3 is specific to mammals, has one crd and a long non-lectin domain, and exists in either a monomeric form or a multivalent complex associated via the non-lectin domains of monomers [82] ; this property allows galectin-3 to effectively bridge different ligands to form adhesive networks. current investigations have indicated that direct recognition between galectin-1/-3 and viral-surface glycoproteins is important for host-virus interaction. [83] . because of its particular binding specificity for galactosides, galectin-1 recognizes the surface envelope proteins of many human viruses and therefore is involved in viral infection. galectin-1 binds to niv-f, a viral envelope glycoprotein of nipah virus (niv), to reduce the niv-f-mediated fusion of endothelial cells and thereby inhibit niv-induced syncytium formation [84] . galectin-1 directly interacts with the envelope glycoproteins of influenza a/wsn/33 virus and inhibits its hemagglutination activity, resulting in the reduction of influenza virus infectivity ( figure 1b) [85] . however, galectin-1 has also been reported to be a susceptibility factor for viral entry. hiv-1 exploits galectin-1 to enhance gp120-cd4 interaction, leading to faster viral entry and more robust viral replication ( figure 1c ) [86] [87] [88] [89] . in addition to galectin-1, the role of galectin-3 in viral infection has been elucidated by several studies. galectin-3 has been shown to interact with herpes simplex virus-1 (hsv-1). rnai-mediated knockdown of galectin-3 in human corneal keratinocytes significantly impaired hsv-1 infection, suggesting that hsv-1 exploits galectin-3 to enhance its attachment to host cells [90] . recently, proteomic-based studies have identified galectin-3 as a host-binding partner of parvovirus minute virus of mice (mvm). the authors proposed that galectin-3 binding facilitates the access of mvm to its receptor(s) at the plasma membrane and thus promotes mvm endocytosis ( figure 1c ) [91] . the above-mentioned evidence indicates a pleiotropic role of galectins during viral infections. the endoplasmic reticulum (er) of mammalian cells contains molecular chaperones and foldases, which are required for forming the active structures of newly synthesized peptides and thus serve as components of the er quality control system. the er-resident chaperones include bip, calnexin ( figure 3l ) and calreticulin (a calnexin-like soluble form without the transmembrane region) [92, 93] . both calnexin and calreticulin are lectin-like, membrane-bound molecular chaperones that associate with newly synthesized proteins in the er. in addition, several studies have indicated that calnexin and calreticulin preferentially interact with glycoproteins that carry monoglucosylated n-linked oligosaccharides [94] [95] [96] [97] . the maturation of virus-encoded proteins occurs in the er, and calnexin family proteins have been shown to transiently interact with multiple viral proteins that consequently undergo rapid maturation ( figures 1d and 4a) . both calnexin and calreticulin can transiently interface with envelope glycoproteins f and hn of sendai virus (sev) [98] and glycoproteins g1/g2 of uukuniemi virus (uukv) (bunyaviridae family) [99] to facilitate the rapid maturation of these proteins. during sars-cov infection, maturation of the viral s protein due to its interaction with calnexin is essential for the formation of infective virions [100] . calnexin/calreticulin also plays a role in the assembly and secretion of hbv middle (m) envelope protein [101] , hiv-1 envelope protein gp160 [102] and gp120 [103] . furthermore, during the rotavirus life cycle, calnexin binds to the er-associated viral transmembrane protein nsp4, a nonstructural glycoprotein that acts as a toxin capable of inducing diarrhea in animals [104] [105] [106] . calnexin/calreticulin is also associated with the glycosylation of hantaan virus (htnv, also known as hantavirus) envelope proteins gn and gc and plays a crucial role in the folding of htnv glycoproteins with a high content of high-mannose oligosaccharides [107] . accumulated evidence suggests that the lectin-like calnexin proteins interact with viral components to largely facilitate viral assembly and protein maturation. the p-type lectin/mannose 6-phosphate receptors (mprs) are transmembrane glycoproteins that target lysosomal enzymes located in either intracellular organelles or the plasma membrane ( figure 3j ,k). mprs can bind newly synthesized lysosomal hydrolases in the trans-golgi network (tgn) and deliver them to pre-lysosomal compartments. the mpr crd was originally identified in two types of proteins, cation-independent and cation-dependent mannose 6-phosphate receptors (ci-mpr and cd-mpr, respectively), both of which recognize mannose-6-phosphate (m-6-p) to identify and route lysosomal enzymes to the lysosomal compartment [108] . a previous study demonstrated that human herpes simplex virus (hsv) glycoprotein d (gd) binds to both ci-mpr and cd-mpr. these mprs sort glycoproteins modified with m-6-p to lysosomes in the trans-golgi compartment and divert them to the endosomal pathway ( figures 1e and 4b) [109, 110] . mprs were also found on the surfaces of mammalian cells as serving as putative cellular receptors for hsv entry and cell-cell viral spread; furthermore, chemo-or immuno-blocking mprs was shown to inhibit hsv entry and the production of hsv plaques in monkey cells ( figure 1c ). mouse cells lacking both ci-mpr and cd-mpr remain sensitive to hsv infection [110] , suggesting that the expression of mprs is not essential for hsv invasion. varicella zoster virus (vzv) is known as a highly infectious human pathogen, and multiple vzv envelope glycoproteins are modified by m-6-p; therefore, ci-mprs appear to be important for vzv infection. intracellular ci-mpr contributes to the transport of enveloped vzv to late endosomes, and the plasmalemmal form is necessary for cellular entry through cell-free vzv particles [111] . l-type lectins are widely distributed in plants and animals. animal l-type lectins are intracellular luminal proteins that are involved in protein sorting in the luminal er-golgi compartments of animal cells. there are four l-type lectins in mammals: ergic-53 ( figure 3m) , ergl, vip36, and vipl [112, 113] . a recent study has shown that intracellular cargo receptor ergic-53 interacts with the glycoproteins of arenavirus, hantavirus, coronavirus, orthomyxovirus, and filovirus particles. ergic-53 is also essential for the propagation of arenavirus, coronavirus, and filovirus; in the absence of ergic-53, viral particles can be formed but are noninfectious (figures 1e and 4c ) [114] . in addition to the above-mentioned mammalian lectins, there are some other lectin classes in animals, e.g., m-type, r-type, i-type, chitinase-like, f-box lectins and intelectins. as we have not found reports on direct interactions between these lectins and viral components, we cannot determine the role of these lectins in viral infection based on the current knowledge. lectin crds are conserved throughout evolution, and many lectin homologues have been identified and reported in invertebrates. a homolog of galectin plays a role in opsonization for bacterial clearance in marsupenaeus japonicus [115] . similarly, a galectin-like factor is expressed on the surface of oyster hemocytes and plays a role in oyster physiology through the recognition of oligosaccharides [116, 117] . several proteins identified in crassostrea hongkongensis [118] , venerupis philippinarum [119] and trypanosoma cruzi [120] have been categorized as homologs of mammalian i-type lectins/siglecs with high sialic acid-binding activity. in arthropods, multiple lectins identified in shrimp, such as l-type, p-type/mprs, m-type, and calnexin family factors, have been proposed to be important in shrimp innate immunity [121] . many c-type lectin homologues in aedes and anopheles mosquitoes have been found to be involved in insect immune responses and pathogenesis [122] [123] [124] . the current investigations of the immune roles of arthropod lectins mainly focus on their anti-bacterial or anti-parasite functions, including microorganism-induced lectin up-regulation, lectin-mediated microorganism recognition and opsonization [125, 126] . however, little is known about the molecular details of lectins in arthropod immunity and pathogenesis, especially with regard to the function in arthropod-virus interactions. a recent study on c-type lectins in aedes aegypti initially assessed lectin functions in viral infections of arthropods. tens of c-type lectins were identified in aedes [122, 123] and anopheles [124] mosquitoes, and most are soluble forms [127] . previous studies have shown that an aedes aegypti c-type lectin, mosquito galactose-specific c-type lectin-1 (mosgctl-1), interacts with wnv in a calcium-dependent manner to form a mosgctl-virus complex. this complex consequently interacts with mosquito protein tyrosine phosphatase-1 (mosptp-1), a mosquito homolog of human cd45 in a. aegypti, to enable viral attachment to the plasma membrane and enhance viral entry. in vivo experiments showed that mosgctl-1 and mosptp-1 function as part of the same pathway and are critical for wnv infection of mosquitoes [127] . further investigations identified that another 9 mosgctl paralogs facilitate dengue infection of mosquitoes. these mosgctls interact with denv-2 surface e protein and virions, functioning as susceptibility factors for dengue viral entry into mosquito cells. however, mosptp-1 did not influence dengue infection in mosquitoes, suggesting that other membrane receptors may recruit the denv-mosgctl complex onto the cell membrane for viral entry [122] . in agreement with the findings in mosquitoes, a recent study has identified a c-type lectin in the shrimp marsupenaeus japonicus that interacts with an envelope protein of white spot syndrome virus (wssv) and consequently associates with a cell-surface calreticulin, which serves as a membrane receptor that facilitates viral entry in a cholesterol-dependent manner [128] . the study therefore suggested that c-type lectins might play a broad role in expediting many viral infections of arthropods. the role might not be limited to wnv/denv in mosquito and wssv in shrimp but might extend to other virus infections in arthropods. lectins are potential targets for the development of antiviral drugs and vaccines. such lectin-based antiviral strategies are divided into two parts: (1) lectin-based immune activation and (2) blockade of lectin receptors against viral entry [129] . many envelope viruses are protected by their dense carbohydrate shield against efficient recognition and persistent neutralization by the host immune system. various natural and synthetic carbohydrate-binding agents have been screened to refine candidates that can reinforce the recognition of specific pathogens, enhance the cascade amplification of the innate immune response and interrupt virus attachment to receptors. in fact, lectins have been considered as drug targets for many years. several heterologous lectins derived from various organisms have been already selected and introduced into pre-clinic trials for hiv therapy, including svn (scytovirin), a 9.7-kd lectin isolated from aqueous extracts of the cyanobacterium scytonema varium [130] , and uda (stinging nettle lectin), a 8.5-kd plant lectin isolated from urtica dioica [131, 132] . furthermore, the combined usage of uda with hha (amaryllis lectin, from a hippeastrum hybrid) and gna (snowdrop lectin from galanthus nivalis), another two carbohydrate-binding agents, showed broad anti-viral activity against four serotypes of denv in monocyte-derived dendritic cells by preventing virus attachment [133] . additionally, an interesting monoclonal antibody, 2g12, which interacts with specific, highly conserved glycosylation sites on hiv envelop protein gp120 shows a broad anti-hiv neutralizing activity. the mechanism of this antibody specifically targeting n-linked glycans is very similar to that of lectins [134] [135] [136] . with regard to arthropod-borne viruses, vector ligands that interact with pathogens are ideal targets for interfering with the successful acquisition of the virus from the vertebrate host. due to the importance of c-type lectins in dengue infection of mosquitoes, these lectin factors may be proposed as targets for the development of vaccines or antiviral drugs. studies show that treatment with mosgctls antisera dramatically interrupted denv-2 infection of mosquitoes through blood feeding. therefore, the humoral response against mosgctls in mammals could feasibly impair dengue infection of mosquitoes. the approach to blocking mosquito c-type lectins may direct a future avenue for the development of a transmission-blocking vaccine that interrupts the mosquito-borne viral life cycle and reduces disease burden [122] . lectins comprise highly diverse proteins with different carbohydrate recognition activities and play pleiotropic roles in the immune responses and pathogenesis of many viral infections (summarized in table 2 ). the interaction between lectins and viral glycoproteins may lead to the three following consequences: (1) lectins, such as mbl and sps, function as pattern recognition molecules that bind a repertoire of viruses and activate antiviral immune responses; (2) lectins are employed as attachment factors that recruit viral particles to the cell membrane to enhance viral entry, e.g., some mammalian lectins (dc-sign, l-sign, mr and mprs) or their homologs in arthropods (mosgctls); and (3) some intracellular lectins, such as calnexin and ergic-53, function as susceptibility factors associated with virus-encoded proteins to facilitate viral replication or assembly (please refer to figures 1 and 4) . interestingly, the same lectin may show opposing roles in different virus infections. for example, galectin-1 binds to niv to inhibit syncytium formation and recognizes iav to reduce its infectivity [84, 85] ; however, galectin-1 was also reported to be a susceptibility factor that enhanced gp120-cd4 interactions, thus facilitating hiv entry [86] [87] [88] [89] . the current accumulated knowledge indicates that lectins are crucial host factors with complex and profound roles in the process of viral infection. lectins for investigation of proteins and carbohydrates molecular structure animal lectins animal lectins: a historical introduction and overview demonstration of carbohydrate recognition activity in diverse proteins which share a common primary structure motif two distinct classes of carbohydrate-recognition domains in animal lectins mannose-binding proteins isolated from rat liver contain carbohydrate-recognition domains linked to collagenous tails. complete primary structures and homology with pulmonary surfactant apoprotein the c-type lectin-like domain superfamily engineering galactose-binding activity into a c-type mannose-binding protein binding of sugar ligands to ca(2+)-dependent animal lectins. ii. generation of high-affinity galactose binding by site-directed mutagenesis studies on the carbohydrate-binding characteristics of human pulmonary surfactant-associated protein a and comparison with two other collectins: mannan-binding protein and conglutinin the major lung surfactant protein, sp 28-36, is a calciumdependent, carbohydrate-binding protein interaction of tetranectin with sulphated polysaccharides and trypan blue. scand lectin-carbohydrate interactions: different folds, common recognition principles mechanism of n-acetylgalactosamine binding to a c-type animal lectin carbohydrate-recognition domain mechanism of ph-dependent n-acetylgalactosamine binding by a functional mimic of the hepatocyte asialoglycoprotein receptor human mannose-binding protein carbohydrate recognition domain trimerizes through a triple alpha-helical coiled-coil collections and ficolins: humoral lectins of the innate immune defense two mechanisms for mannose-binding protein modulation of the activity of its associated serine proteases interaction of mannose-binding lectin with hiv-1 is sufficient for virus opsonization but not neutralization high frequency of variant alleles of the mannose-binding lectin 2 (mbl2) gene are associated with patients infected by hepatitis b virus mannose-binding lectin in chronic hepatitis b virus infection mannose-binding lectin mbl2 gene polymorphisms and outcome of hepatitis c virus-infected patients direct complement restriction of flavivirus infection requires glycan recognition by mannose-binding lectin a human serum mannose-binding protein inhibits in vitro infection by the human immunodeficiency virus glycosylation inhibitors and neuraminidase enhance human immunodeficiency virus type 1 binding and neutralization by mannose-binding lectin dc-sign, a dendritic cell-specific hiv-1-binding protein that enhances transinfection of t cells c-type lectins dc-sign and l-sign mediate cellular entry by ebola virus in cis and in trans human cytomegalovirus binding to dc-sign is required for dendritic cell infection and target cell trans-infection dc-sign and l-sign can act as attachment receptors for alphaviruses and distinguish between mosquito cell-and mammalian cell-derived viruses dc-sign (cd209) mediates dengue virus infection of human dendritic cells inhibition of dc-sign-mediated trans infection of t cells by mannose-binding lectin mannose-binding lectin binds to ebola and marburg envelope glycoproteins, resulting in blocking of virus interaction with dc-sign and complement-mediated virus neutralization localization of lung surfactant protein d on mucosal surfaces in human tissues immunocytochemical localization of surfactant protein d (sp-d) in type ii cells, clara cells, and alveolar macrophages of rat lung expression sites of the collectin sp-d suggest its importance in first line host defence: power of combining in situ hybridisation, rt-pcr and immunohistochemistry mechanisms of anti-influenza activity of surfactant proteins a and d: comparison with serum collectins pulmonary surfactant protein d in first-line innate defence against influenza a virusinfections site-directed mutagenesis of cys-15 and cys-20 of pulmonary surfactant protein d. expression of a trimeric protein with altered anti-viral properties mechanism of binding of surfactant protein d to influenza a viruses: importance of binding to haemagglutinin to antiviral activity assessment of the antiviral properties of recombinant porcine sp-d against various influenza a viruses in vitro inhibition of influenza viral neuraminidase activity by collectins evidence for a protective role of pulmonary surfactant protein d (sp-d) against influenza a viruses recombinant sp-d carbohydrate recognition domain is a chemoattractant for human neutrophils surfactant protein a binds to the fusion glycoprotein of respiratory syncytial virus and neutralizes virion infectivity surfactant protein a enhances uptake of respiratory syncytial virus by monocytes and u937 macrophages respiratory syncytial virus and pulmonary surfactant c-type lectin receptors on dendritic cells and langerhans cells dc-sign: escape mechanism for pathogens the c type lectins dc-sign and l-sign: receptors for viral glycoproteins nile virus discriminates between dc-sign and dc-signr for cellular attachment and infection dc-sign enhances infection of cells with glycosylated west nile virus in vitro and virus replication in human dendritic cells induces production of ifn-alpha and tnf-alpha dc-sign and dc-signr interact with the glycoprotein of marburg virus and the s protein of severe acute respiratory syndrome coronavirus ph-dependent entry of severe acute respiratory syndrome coronavirus is mediated by the spike glycoprotein and enhanced by dendritic cell transfer through dc-sign -sign) is a receptor for severe acute respiratory syndrome coronavirus l-sign (cd 209l) is a liver-specific capture receptor for hepatitis c virus hepatitis c virus glycoproteins interact with dc-sign and dc-signr dendritic-cell-specific icam3-grabbing non-integrin is essential for the productive infection of human dendritic cells by mosquito-cell-derived dengue viruses dendritic cell-specific intercellular adhesion molecule 3-grabbing non-integrin (dc-sign)-mediated enhancement of dengue virus infection is independent of dc-sign internalization signals the mannose receptor mediates dengue virus infection of macrophages the mannose receptor acts as hepatitis b virus surface antigen receptor mediating interaction with intrahepaticdendritic cells involvement of themannose receptor in infection of macrophages by influenza virus reading pc macrophage receptors for influenza a virus: role of the macrophage galactose-type lectin and mannose receptorin viral entry oligomerization of the macrophage mannose receptor enhances gp120-mediated binding of hiv-1 immunoreceptor dap12 bearing a tyrosine-based activation motif is involved in activating nk cells clec5a is critical for dengue-virus-induced lethal disease clec5a is critical for dengue virus-induced inflammasome activation in human macrophages clec5a regulates japanese encephalitis virus-induced neuroinflammation and lethality langerin, a novel c-type lectin specific to langerhans cells, is an endocytic receptor that induces the formation of birbeck granules the role of dendritic cell c-type lectin receptors in hiv pathogenesis diversity of receptors binding hiv on dendritic cell subsets langerin is a natural barrier to hiv-1 transmission by langerhans cells human langerhans cells capture measles virus through langerin and present viral antigens to cd4+ t cells but are incapable of cross-presentation the c-type lectin superfamily in the immune system self-and nonself-recognition by c-type lectins on dendritic cells trimeric structure of a c-type mannose-binding protein galectins: a family of animal beta-galactosidebinding lectins secretion of the galectin family of mammalian carbohydrate-binding proteins secretion of the baby hamster kidney 30-kda galactose-binding lectin from polarized and nonpolarized cells: a pathway independent of the endoplasmic reticulum-golgi complex plasma membrane targetting, vesicular budding and release of galectin 3 from the cytoplasm of mammalian cells during secretion galectins: regulators of acute and chronic inflammation galectins: structure, function and therapeutic potential endothelial galectin-1 binds to specific glycans on nipah virus fusion protein and inhibits maturation, mobility, and function to block syncytia formation galectin-1 binds to influenza virus and ameliorates influenza virus pathogenesis galectin-1 promotes hiv-1 infectivity in macrophages through stabilization of viral adsorption galectin-1 acts as a soluble host factor that promotes hiv-1 infectivity through stabilization of virus attachment to host cells galectin-1 and hiv-1 infection host-soluble galectin-1 promotes hiv-1 replication through a direct interaction with glycans of viral gp120 andhost cd4 binding of transmembrane mucins to galectin-3 limits herpesvirus 1 infection of human corneal keratinocytes proteomic analysis identifies a novel function for galectin-3 in the cell entry of parvovirus role of n-oligosaccharides endoplasmic reticulum processing reactions in glycoprotein folding and degradation lectins as chaperones in glycoprotein folding association of folding intermediates of glycoproteins with calnexin during protein maturation role of n-linked oligosaccharide recognition, glucose trimming, and calnexin in glycoprotein folding and quality control transient, lectin-like association of calreticulin with folding intermediates of cellular and viral glycoproteins glycan-dependent and -independent association of vesicular stomatitis virus g protein with calnexin kinetics of interactions of sendai virus envelope glycoproteins, f and hn, with endoplasmic reticulum-residentmolecular chaperones, bip, calnexin, and calreticulin transient association of calnexin and calreticulin with newly synthesized g1 and g2 glycoproteins of uukuniemivirus (family bunyaviridae) monitoring of s protein maturation in the endoplasmic reticulum by calnexin is important for the infectivity of severe acute respiratory syndrome coronavirus role for calnexin and n-linked glycosylation in the assembly and secretion of hepatitis b virus middle envelopeprotein particles calreticulin interacts with newly synthesized human immunodeficiency virus type 1 envelope glycoprotein, suggesting a chaperone function similar to that of calnexin effects of inefficient cleavage of the signal sequence of hiv-1 gp 120 on its association with calnexin, folding, and intracellular transport the rotavirus nonstructural glycoprotein nsp4 possesses membrane destabilization activity rotavirus nonstructural glycoprotein nsp4 alters plasma membrane permeability in mammalian cells the molecular chaperone calnexin interacts with the nsp4 enterotoxin of rotavirus in vivo and in vitro analysis of n-linked glycosylation of hantaan virus glycoproteins and the role of oligosaccharide side chains in protein folding and intracellular trafficking lysosomal enzyme binding to mouse p388d1 macrophage membranes lacking the 215-kda mannose 6-phosphate receptor: evidence for the existence of a second mannose 6-phosphate receptor herpes simplex virus glycoprotein d acquires mannose 6-phosphate residues and binds to mannose 6-phosphate receptors role of mannose-6-phosphate receptors in herpes simplex virus entry into cells and cell-to-cell transmission mannose 6-phosphate receptor dependence of varicella zoster virus infection in vitro and in the epidermis during varicella and zoster a putative novel class of animal lectins in the secretory pathway homologous to leguminous lectins structures of the carbohydrate recognition domain of ca 2+ -independent cargo receptors emp46p and emp47p the intracellular cargo receptor ergic-53 is required for the production of infectious arenavirus, coronavirus, and filovirus particles a galectin from the kuruma shrimp (marsupenaeus japonicus) functions as an opsonin and promotes bacterial clearance from hemolymph hemocytes and plasma of the eastern oyster (crassostrea virginica) display a diverse repertoire of sulfated and blood group a-modified n-glycans the galectin cvgal1 from the eastern oyster (crassostrea virginica) binds to blood group a oligosaccharides on the hemocyte surface a novel sialic acid binding lectin with anti-bacterial activity from the hong kong oyster (crassostrea hongkongensis) cloning and characterization of a sialic acid binding lectins (sabl) from manila clam molecular interaction of siglecs (sialic acid-binding ig-like lectins) with sialylated ligands on trypanosoma cruzi diversity and multiple functions of lectins in shrimp immunity transmission-blocking antibodies against mosquito c-type lectins for dengue prevention cloning and characterization of a mannose binding c-type lectin gene from salivary gland of aedes albopictus the genome sequence of the malaria mosquito anopheles gambiae purification, characterization and cdna cloning of a novel lipopolysaccharide-binding lectin from the shrimp penaeus monodon a novel c-type lectin (fclec4) facilitates the clearance of vibrio anguillarumin in vivo in chinese white shrimp a c-type lectin collaborates with a cd45 phosphatase homolog to facilitate west nile virus infection of mosquitoes collaboration between a soluble c-type lectin and calreticulin facilitates white spot syndrome virus infection in shrimp targeting the c-type lectins-mediated host-pathogen interactions with dextran overexpression and purification of scytovirin, a potent, novel anti-hiv protein from the cultured cyanobacterium scytonema varium the mannose-specific plant lectins from cymbidium hybrid and epipactis helleborineand the (n-acetylglucosamine)n-specific plant lectin from urtica dioicaare potent and selective inhibitors of human immunodeficiency virus and cytomegalovirus replication in vitro carbohydrate-binding agents cause deletions of highly conserved glycosylation sites in hiv gp120 a new therapeutic concept to hit the achilles heel of hiv broad antiviral activity of carbohydrate-binding agents against the four serotypes of dengue virus in monocyte-derived dendritic cells the broadly neutralizing anti-human immunodeficiency virus type 1 antibody 2g12 recognizes a cluster of alpha 1â��2 mannose residues on the outer face of gp120 antibody domain exchange is an immunological solution to carbohydrate cluster recognition dissection of the carbohydrate specificity of the broadly neutralizing anti-hiv-1 antibody 2g12 key: cord-342176-tewfm8it authors: kjærup, rikke m.; dalgaard, tina s.; norup, liselotte r.; bergman, ingrid-maria; sørensen, poul; juul-madsen, helle r. title: adjuvant effects of mannose-binding lectin ligands on the immune response to infectious bronchitis vaccine in chickens with high or low serum mannose-binding lectin concentrations date: 2013-11-08 journal: immunobiology doi: 10.1016/j.imbio.2013.10.013 sha: doc_id: 342176 cord_uid: tewfm8it mannose-binding lectin (mbl) plays a major role in the immune response as a soluble pattern-recognition receptor. mbl deficiency and susceptibility to different types of infections have been subject to extensive studies over the last decades. in humans and chickens, several studies have shown that mbl participates in the protection of hosts against virus infections. infectious bronchitis (ib) is a highly contagious disease of economic importance in the poultry industry caused by the coronavirus infectious bronchitis virus (ibv). mbl has earlier been described to play a potential role in the pathogenesis of ibv infection and the production of ibv-specific antibodies, which may be exploited in optimising ibv vaccine strategies. the present study shows that mbl has the capability to bind to ibv in vitro. chickens from two inbred lines (l10h and l10l) selected for high or low mbl serum concentrations, respectively, were vaccinated against ibv with or without the addition of the mbl ligands mannan, chitosan and fructooligosaccharide (fos). the addition of mbl ligands to the ibv vaccine, especially fos, enhanced the production of ibv-specific igg antibody production in l10h chickens, but not l10l chickens after the second vaccination. the addition of fos to the vaccine also increased the number of circulating cd4+ cells in l10h chickens compared to l10l chickens. the l10h chickens as well as the l10l chickens also showed an increased number of cd4−cd8α−γδ t-cells when an mbl ligand was added to the vaccine, most pronouncedly after the first vaccination. as mbl ligands co-administered with ibv vaccine induced differences between the two chicken lines, these results indirectly suggest that mbl is involved in the immune response to ibv vaccination. furthermore, the higher antibody response in l10h chickens receiving vaccine and fos makes fos a potential adjuvant candidate in an ibv vaccine. the first line of host defence against pathogens involves the innate immune system. pathogens have specific microorganismassociated molecular patterns (mamps) that are recognised by pattern-recognition receptors (prrs). two kinds of prrs exist: surface prrs and soluble prrs. prrs trigger intracellular signalling cascades upon mamp recognition culminating in activation of antigen presenting cells and production of co-stimulatory molecules as well as pro-inflammatory cytokines. the production of costimulatory molecules and pro-inflammatory cytokines initiates the early host response to infection and also partakes in the activation and shaping of the adaptive immune response (crozat et al. 2009; de visser and coussens 2005; hoffmann et al. 1999) . several soluble prrs have been described, and an example of such is mannose-binding lectin (mbl). mbl has various functions in, for example, complement activation, promotion of complementindependent opsonophagocytosis, modulation of inflammation, recognition of altered self-structures and apoptotic cell clearance (dommett et al. 2006) . mbl is a collectin consisting of multiple identical polypeptide chains oligomerised into different sizes. the chains are made up of four distinct domains. these are a cysteinerich n-terminal domain, a collagenous domain, a neck domain, and a calcium-dependent carbohydrate-recognition domain (crd) at the c-terminal (takahashi 2011) . it is the crd that permits mbl to bind in a ca 2+ -dependent manner to mamps such as polysaccharides. in fact, terminal sugars, such as d-mannose, l-fucose and n-acetyl-d-glucosamine, found on the surface of many microorganisms, contain equatorial 3-and 4-hydroxyl groups to which mbl binds. mbl does not bind to d-galactose and sialic acid found on the surface of many animal cells. beside sugars, mbl has also been found to bind to phospholipids, nucleic acids, and non-glycosylated proteins (ip et al. 2009 ). the mbl genes in human (madsen et al. 1994 (madsen et al. , 1995 steffensen et al. 2000; sumiya et al. 1991) , porcine (juul-madsen et al. 2011a; lillie et al. 2007) , bovine (liu et al. 2011) and chickens (kjaerup et al. 2013) have been found to have polymorphisms resulting in wide variations in mbl serum levels in the organisms. over the last decades mbl deficiency and the influence on the susceptibility to different types of infections have been subject to extensive studies as reviewed (heitzeneder et al. 2012; mayilyan 2012; takahashi 2011) . some results indicate that mbl deficiency may actually be beneficial with regard to disease, for example visceral leishmaniasis (santos et al. 2001 ). however, most results suggest that mbl deficiency leads to a weaker immune response. in humans, several studies have shown that mbl participates in the protection of hosts against virus infections, such as infections with influenza a virus (chang et al. 2010 ), hepatitis c virus (brown et al. 2010) , ebola virus (michelow et al. 2011) , and severe acute respiratory syndrome (sars) coronavirus (ip et al. 2005; zhou et al. 2010) . thus, mbl in chickens may also play a role in the pathogenesis of chicken virus infections and the production of antibodies as suggested by juulmadsen et al. (2007) . selective breeding of chickens for low or high serum mbl concentrations has been performed for several generations at our department as published by juulmadsen et al. (2007) . this has resulted in two distinct chicken lines designated high (l10h) or low (l10l) with mean serum mbl concentrations of 33.4 g/ml serum (l10h) and 7.6 g/ml serum (l10l) (f14 generation, unpublished) . studies using these chicken sublines as well as outbred chickens have shown an inverse relationship between the mbl concentrations and the pathogen-specific antibody response (juul-madsen et al. 2007; schou et al. 2008 ). studies in mice have shown that mbl deficiency may result in a higher igg antibody response after infections (carter et al. 2007 ) and vaccinations (guttormsen et al. 2009 ). from these results it can be hypothesised that basal mbl plasma levels may influence specific humoral immune responses. this explanation for this may be that either: (1) mbl pushes the immune response into a more cellular response (th1 vs. th2); (2) mbl efficiently neutralises the pathogen via the complement membrane-attack complex and no adaptive immune response is needed; or (3) mbl influences the pro-inflammatory cytokine production via interaction with surface receptors, such as toll-like receptors (ip et al. 2008) . infectious bronchitis (ib) is a highly contagious disease of economic importance in the poultry industry with symptoms such as sneezing, tracheal rales, and coughing. furthermore, ib may cause a decline in egg quality and production in layers (raj and jones 1997) . ib is caused by the coronavirus infectious bronchitis virus (ibv) which is highly able to genetically mutate and recombine. as a result, there is a continuous development of new strains throughout the world. different strains can co-circulate within a region, and the severity of the disease varies from strain to strain and from flock to flock (capua et al. 1999; cavanagh 2007; cook et al. 2012) . consequently, applied vaccines sometimes provide insufficient protection, as vaccination with one strain of ibv may not be protective against other strains. vaccine efficacy may be improved by the use of adjuvants. good candidates for vaccine adjuvants are carbohydrates since they are mostly of low toxicity and high biocompatibility and furthermore play major roles within the immune system (petrovsky and cooper 2011) . carbohydrates such as mannan (liu et al. 2012) , chitosan (rauw et al. 2010) , and fructooligosaccharide (fos) (benyacoub et al. 2008 ) have previously been used in vaccines or diets as modulators of the immune response. these three carbohydrates are potential mbl ligands owing to their content of sugar units. the hypothesis of this study was that immunity after ibv vaccination may be improved after temporarily inhibition of the mbl function. this was achieved by adding an mbl ligand (mannan, chitosan, or fos) to the vaccine given to chickens and thereby creating an artificial mbl deficiency during vaccination. innate as well as adaptive immunological parameters were measured throughout the experimental period. all chemicals were obtained from sigma-aldrich, ballerup, denmark, except when noted. dreamtaq tm master mix was obtained from qiagen, and oligonucleotide primers and probes were obtained from eurofins mwg operon, ebersberg, germany. purified chicken mbl was bought from the department of cancer and inflammation research, university of southern denmark. it was purified from chicken serum as previously described (laursen et al. 1998a ). the binding capacity of mbl to ibv was measured using elisa. dilutions of purified mbl and serum samples from l10l or l10h chickens were made with or without addition of saccharides. biowhittaker ® veronal buffer (lonza, walkersille, md, usa; cat. no. 12-624e) adjusted to a final concentration of 5 mm mgcl 2 and 10 mm cacl 2 was used for diluting the samples. for the titration of mbl binding, concentrations of 0, 1, 2, 4, 8, 16, 24 and 32 g/ml purified mbl were used, and 5 l serum samples were diluted 1:50. the concentrations of the saccharides added were as follows: mannan 100 mg/ml; fos 100 mg/ml; chitosan 10 mg/ml; galactose 9 mg/ml; and edta 20 mm. one-hundred microlitres of the dilutions and saccharides were mixed in a nunc 96-well polypropylen microwell plate (thermo fisher scientific, slangerup, denmark; cat. no. 442587) and incubated for 5 min before the samples were transferred to a 96-well microtitre plate coated with ibv antigen (from the proflok ® ibv antibody test kit from synbiotics corporation, san diego, ca, usa; cat. no. 96-6506). wells receiving only buffer were used as negative controls. all dilutions were added in triplicates. the plate was then incubated at room temperature for 1 h. after a washing step with gibco ® dpbs supplemented with calcium and magnesium (life technologies europe bv, naerum, denmark; cat. no. 14080-048) and ph adjusted to 7.4 followed by supplementation with 0.1% bsa (hereafter called pbs+), the wells were incubated for 45 min at room temperature with 1 g/ml of biotinylated monoclonal mouse anti-cmbl (bioporto diagnostics a/s, gentofte, denmark; cat. no. hyb 182-01) in pbs+. after another washing step streptavidin horseradish peroxidase (sav-hrp) (bd bioscienses, albertslund, denmark; cat. no. 554066) diluted 20,000-fold in pbs+ was added. after 30 min of incubation and washing with pbs+, the presence of sav-hrp was detected by adding 100 ml of substrate solution (<0.05%, wt/wt; 3,3 ,5,5 tetramethylbenzidin). colour development was stopped with a 1 m h 2 so 4 solution. the colour development was determined by reading the absorbance at 405 nm with absorbance at 650 nm as reference. generation 13 from the two au inbred sub-lines l10l and l10h (juulmadsen et al. 2007 ) was used in this study (n = 72). chickens from l10 consist of 67.5% um-b19 and 33.5% white cornish (laursen et al. 1998b ). the offspring were reared together in a biosecured ibv-free environment until they were 3 weeks of age and then allocated into 4 different treatments groups with 9 birds from each subline in each group. the first treatment group was treated with 100 l deionized water containing one dose of live attenuated nobilis ® ib ma5 (msd animal health, ballerup, denmark; danish product license no. 8674) per animal. the second treatment group was treated with 100 l deionized water containing one dose of live attenuated nobilis ib ma5 vet and 200 mg/ml purified mannan from saccharomyses cerevisiae per animal aiming at 100 g mannan per gram body weight (juulmadsen et al. 2011b ). the third treatment group was treated with 100 l deionized water containing one dose of live attenuated nobilis ib ma5 vet and 50 mg/ml deactylated chitosan from shrimp shells per animal. the fourth treatment group was treated with 100 l deionized water containing one dose of live attenuated nobilis ib ma5 vet and 200 mg/ml fos (orafti ® p95 from alsiano a/s, birkerød, denmark) per animal aiming at 100 g fos per gram body weight. all solutions were shaken before nasally applied to the chickens. after three weeks, the four treatment groups were vaccinated again with the same amount of vaccine and saccharides as for the first vaccination. the chickens were fed diets that met or exceeded nrc requirements. food and water were provided ad libitum. the experimental procedures were conducted under the protocols approved by the danish animal experiments inspectorate and complied with the danish ministry of justice law no. 382 (10 june 1987) and acts 739 (6 december 1988) and 333 (19 may 1990) concerning animal experimentation and care of experimental animals. serum was collected from blood samples (0.5-0.7 ml) taken from the jugular vein or the wing vein from the experimental chickens on days 0, 1, 2, 3, 4, 5, 7, 14, 21, 22, 23, 24, 25, 26, 28, 35, 42, 49 and 56 post vaccination 1 (pv1). heparin-stabilised blood for immunophenotyping was collected once a week on days 0, 7, 14, 21, 28, 35, 42, 49 and 56 pv1 and the collected blood (0.5-0.7 ml) was divided into one serum tube and one heparin tube. oropharyngeal airway (opa) swab samples were collected on days 0, 2, 3, 4, 5, 7, and 23 pv1. swab samples were kept in 0.5 ml virus media (20.4% gibco ® penicillin-streptomycin (life technologies europe bv, naerum, denmark; cat. no. 15140-122); 74% biowhittaker ® pbs (lonza, verviers, belgium; cat. no. be17-517q); 5% biowittaker ® foetal bovine serum (lonza, verviers, belgium; cat. no. de14-801f); and 0.01‰ phenol red (merck, darmstadt, germany, cat. no. 1072410005) at −20 • c until testing by real-time quantitative reverse transcription pcr (qrt-pcr) after the termination of the experiment. mbl haplotypes were determined by means of the taqman ® snp genotyping technique (applied biosystems, foster city, ca, usa). two assays were designed, using the custom taqman ® assay design tool according to the instructions on the website (https://www5.invitrogen.com/custom-genomic-products/tools/ genotyping/). these assays distinguish between the cg and ta alleles of snp1 and the gggg and agga alleles of snp2 (kjaerup et al. 2013) . the assays were validated and run on 384-well microtitre plates in an abi prism 7900ht sequence detection system instrument, using version 2.2 of the sds software. a total reaction volume of 10 l was applied, each sample containing 5 l taqman ® universal pcr mastermix (applied biosystems, life technologies europe bv, naerum, denmark; cat. no.4364338), 0.25 l assay mix, 3.75 l h 2 o, and 1 l dna at a concentration of 5 ng/l. to ensure optimal clustering, 24 positive controls, representing animals of known genotypes, were included in each run, together with four non-template (negative) controls. amplification was obtained through an initial incubation period of 10 min at 95 • c followed by 40 cycles of denaturation for 15 s at 92 • c and annealing/extension for 1 min at 60 • c. after amplification, end-point reads were carried out, and analysis was performed with 2-cluster calling enabled in the sds software version 2.2. forty-seven microlitres of three swab samples from each subline, treatment group and day were pooled; thus, 3 pools per subline were analysed for each time point. rna purification was done using the qiaamp ® viral rna mini kit (qiagen, copenhagen, denmark; cat. no. 52904) according to the manufacturer's instructions. the reverse transcription pcr was carried out using the high-capacity cdna archive kit (applied biosystems, life technologies europe bv, naerum, denmark; cat. no. 436881) according to the manufacturer's instructions with 25 l of rna. the real time quantitative reverse transcription pcr (qrt-pcr) reaction was performed with the forward primer ibv5 gu391 (5 -gcttttgagcctagcgtt-3 ), the reverse primer ibv5 gl533 (5 -gccatgttgtcactgtctattg-3 ) and the dual-labelled probe ibv5 g probe (5 -fam-caccaccagaacctgtcacctc-bhq1-3 ) previously described to amplify at least 15 different strains of ibv (callison et al. 2006 ). the total reaction volume was 20 l containing 11 l taqman ® universal pcr master mix (applied biosystems, life technologies europe bv, naerum, denmark; cat. no. 4304437), 0.9 m primers, 0.125 m probe and 0.9 l cdna as template. the reaction was performed in an abi prism 7900ht sequence detection system at 50 • c for 2 min, 95 • c for 10 min with optic off; 40 cycles of 95 • c for 15 s followed by 60 • c for 1 min with optics on. standard curves were included in each qrt-pcr run and were generated from dilutions of cdna originating from purified rna from the live attenuated nobilis ib ma5 vet. the standard curves were made as 5-fold dilutions starting at one dose, corresponding to minimum 10 3 eid 50 according to the manufacturer's instructions. results are expressed as viral load according to the standard curve. however, the viral load is only a measure of viral rna, not true viral replicates. each qrt-pcr experiment contained triplicate no-template controls, test samples and dilution series of nobilis ib ma5 vet cdna. the igg-specific antibody titres against ibv in serum were measured as previously described (juul-madsen et al. 2011b ) using the proflok ® ibv antibody test kit from synbiotics corporation (san diego, ca, usa; cat. no. 96-6506) according to the manufacturer's instructions. the absolute numbers of different t-cell subsets in peripheral blood were measured once a week using a no-lyse no-wash flow cytometric method. fifty microlitres of 25 times diluted heparinstabilised blood was mixed with 50 l antibody solution containing and 0.25 l anti-cd8␣-cy5 (clone 3-298) in facs buffer (0.2% bsa, 0.2% sodium azide and 0.05% normal horse serum in pbs). all monoclonal antibodies were obtained from southern biotech (birmingham, al, usa). the samples were incubated at room temperature for 20 min in a 4 ml tube in darkness, and immediately before acquisition 400 l facs-buffer and 25 l flow-count tm fluorospheres (beckman coulter ireland, mervue, galway, ireland; cat. no. 7547053) were added. b-cell numbers were only measured at week 9 pv1. the protocol was as described above but using 1 l anti-bu-1-rpe (clone av20) in facs buffer. bu-1 positive macrophage and monocyte subsets were avoided by fsc/ssc gating on small lymphocytes. all flow cytometric analyses were performed on a bd facscanto tm (bd biosciences, san jose, ca, usa) equipped with a 488 nm blue laser and a 633 nm red laser. using the facsdiva software each sample was acquired and recorded for 1 min at medium flow rate. single-stained compensation controls as well as negative fluorescence minus one (fmo) controls were included and titration of all antibodies was performed prior to the experiment in order to determine the optimal staining concentrations. the count of cells in the samples was calculated according to the manufacturer's instructions as: cells/l = (total number of cells counted × dilution factor × l fluorospheres added × assayed concentration of fluorospheres)/(total number of fluorospheres counted × total volume). license to conduct the animal experiment was obtained from the danish ministry of justice, animal experimentation inspectorate by helle r. juul-madsen. the experiment was conducted according to the ethical guidelines. all data, except for the viral load measured by qrt-pcr, were found to be normally distributed. to analyse the data from the mbl-ibv binding assay the values from no addition of mbl ligand were compared with the values from each of the specific ligands added using the analysis of variance principle. in cases of significant effect (p < 0.05) the given mbl ligand was considered to affect the mbl-ibv binding. the viral loads were, due to pooled samples (only 3 pools per group), only tested for subline differences. the viral loads were far from being normally distributed and so an 2 -test was used to test if the sublines had an effect on the viral load. the test was performed on data from days 1 to 7 pv1. for test of the subline effect the observation was distributed into three viral load categories: viral load = 0, 0 < viral load < 0.12 × 10 −5 , and viral load > 0.12 × 10 −5 . the 2 -test tested the null hypothesis: that the observations were distributed equally over the sublines for all categories. the model used for the ibv-specific igg antibody titre measured by elisa was: y ijk = + t i + w j + l k + twl ikj + e ijk , where = overall means, t i = fixed effect of treatment i, w j = fixed effect of week j pv1, l k = fixed effect of line k. the various interaction effects and y ijk and e ijk were expected to be normally distributed. to avoid weeks with little or no antibody titre, the antibody titre was only statistically analysed from week 2 to 9. the model used for the absolute counts of tcell subsets measured by flow cytometry was: y ijk = + t i + w j + l k + lw jk + tl ik + twl ikj + e ijk , where = overall means, t i = fixed effect of treatment i, w j = fixed effect of week j pv1, and l k = fixed effect of line k. the various interaction effects and y ijk and e ijk were expected to be normally distributed. the model used for the absolute counts of b-cells measured by flow cytometry was: y ij = + t i + l j + tl ij + e ij , where = overall means, t i = fixed effect of treatment i, l j = fixed effect of line j. the various interaction effects and y ij and e ij were expected to be normally distributed. the analysis of the variance was performed by the glm procedure of the sas software (sas institute inc. 2009). an elisa protocol was used for measuring the binding of mbl to ibv. the results (fig. 1) showed that purified mbl binds to ibvcoated plates in a dose-dependent manner (fig. 1a) . mbl in serum samples was also shown to bind to ibv ( fig. 1b and c) . this binding was inhibited by adding mbl ligands. thus, the addition of mannan and fos reduced the mbl-ibv binding significantly (p < 0.01) for l10h serum samples. addition of chitosan only showed a tendency for inhibition of mbl-ibv binding (p = 0.0513). for the mbl in l10l serum the binding was significantly inhibited by mannan and fos (p < 0.01), but not by chitosan. addition of edta to the serum samples inhibited the mbl-ibv binding significantly (p < 0.01) in both sublines, indicating that the binding is calcium dependent. on the other hand the negative control, galactose, had no influence on the mbl-ibv binding. chickens were tested for their mbl haplotype as described by kjaerup et al. (2013) and were determined through the taqman ® snp genotyping technique (data not shown). all l10l chickens were homozygous for the a1 haplotype. all l10h chickens were homozygous for the a3 haplotype, except for five chickens which were heterozygous a1/a3 and were found in groups 1 (n = 2), 3 (n = 2) and 4 (n = 1). ibv-specific qrt-pcr was used for monitoring the presence of ibv genomes in the opa swabs sampled from days 0 to 21 pv1 (fig. 2) . three swab samples from each subline were pooled based on treatment group and day. the results were statistically analysed regardless of treatments (table 1) , since three measurements per subline, treatment group and day were considered insufficient for statistics. in general, the l10l chickens had a higher viral load than the l10h chickens. the viral load in l10h peaked at day 2 pv1, whereas in l10l it peaked at days 2-3 pv1. the viral load in both (table 1) . l10h had a significantly larger number of chickens with low viral load (<0.12 × 10 −5 ) than l10l. contrary to this, l10l had a significantly larger number of chickens with high viral load (>0.12 × 10 −5 ) than l10h (p < 0.01). hence, the viral loads indicated that l10h chickens were less severely affected by the infection than l10l chickens. the ibv-specific igg antibody titres were measured using elisa (fig. 3) . a few chickens were non-responders and remained seronegative. these chickens were two l10h from treatment groups 1 (n = 1) and 4 (n = 1), and four l10l from treatment groups 1 (n = 1), 3 (n = 1) and 4 (n = 2). they were excluded from the statistical analysis of the ibv-specific igg titre. these chickens responded as the other chickens in all other parameters. therefore, the measurements for these chickens were maintained in the statistical analysis of all other parameters. when comparing the sublines, group 4 was the only group where l10h and l10l differed significantly from each other in the ibv-specific igg antibody titre (p < 0.01). in this group the antibody titres in l10h chickens were higher than the antibody titres in l10l chickens after the second vaccination (week 3) and for the rest of the experimental period. the antibody titres showed no difference between the l10l chickens in the four treatment groups. on the other hand, the antibody titre in l10h showed differences between the treatment groups at week 4 (group 1 / = groups 3 and 4; and group 4 / = groups 2 and 3), week 5 (group 4 / = groups 1, 2 and 3), week 6 (group 4 / = groups 1, 2 and 3) week 7 (group 4 / = group 1), week 8 (group 4 / = group 1) and week 9 pv1 (group 4 / = group 1), where p < 0.04. in summary, the ibv-specific igg antibody titres were the same for l10l chickens between different treatments, whereas the addition of an mbl ligand to the vaccine, especially fos, seemed to enhance the production of ibv-specific igg antibody in l10h chickens, but not in l10l chickens. an absolute count flow cytometric protocol using a no-lyse no-wash method, as described by seliger et al. (2012) was used for quantifying t-cell subsets in whole blood. the t-cell subsets were identified by fsc/ssc gating on small lymphocytes, followed by identification of cd3+ cells (t-cells) and finally by subdividing the cd3+ cells into total cd4+ cells (i.e. cd4+cd8␣+ and cd4+cd8␣−), cd4−cd8␣+ cells, and cd4−cd8␣− cells (fig. 4a and b). cd4+cd8␣+ and cd4+cd8␣− cells were combined as total cd4+ cells, due to individual differences in the counts of cd4+cd8␣+ cells between animals which have an heritable origin (hala et al. 1992) . the results are presented in figs. 5 and 6. comparing the counts between the two sublines (l10h and l10l) within each treatment group (fig. 5) , the numbers of total cd4+ cells showed no difference between l10h and l10l in groups 1 and 3. in group 2 the counts of total cd4+ cells in l10h were significantly higher than in l10l at week 6 pv1 (p = 0.03). a more pronounced difference between l10h and l10l was seen in group 4, where the counts of total cd4+ cells were significantly higher for l10h than l10l at weeks 2, 5, 6, 7, 8 and 9 pv1 (p ≤ 0.03). counts of cd4−cd8␣− cells for l10h were higher than l10l in groups 3 and 4 at week 1 pv1 (p < 0.05), whereas counts of cd4−cd8␣− cells were higher for l10h than l10l in group 2 at weeks 0, 1, and 2 pv1 (p ≤ 0.02). cd4−cd8␣+ cells only differed between l10h and l10l within groups in group 1 at week 5 (p = 0.02). briefly, the addition of fos to the vaccine increased the number of total cd4+ cells in l10h chickens compared to l10l chickens, and the l10h chickens showed a significant increased number of cd4−cd8␣− cells after the first vaccination when an mbl ligand was added to the vaccine compared to l10l chickens. the comparison of counts between treatment groups within each subline (l10h and l10l) is presented in fig. 6 . the counts of total cd4+ cells showed no difference between treatment groups neither for the l10h nor l10l sublines, except for the l10l subline at week 6 pv1 (group 3 / = group 4) where the l10l chickens in treatment group 3 showed a significantly higher count of total cd4+ cells than l10l chickens in treatment group 4 (p = 0.03). the numbers of cd4−cd8␣+ cells for the l10h sublines differed significantly between treatment groups at weeks 6-9 pv1 (group 1 / = group 3), where the l10h chickens in treatment group 3 had significantly lower numbers than the l10h chickens in treatment group 1 (p < 0.05). besides this, the numbers of cd4−cd8␣+ cells differed significantly for l10h between treatment groups at week 6 pv1 (group 2 / = group 4), where p = 0.01. the numbers of cd4−cd8␣+ cells for l10l only differed significantly between treatment groups at week 3 pv1 (group 1 / = groups 2 and 4), where p = 0.04. the number of cd4−cd8␣− cells differed significantly for the l10h sublines between the treatment groups at week 1 (group 1 / = groups 2 and 4), week 2 (groups 1 / = 2 and 5), week 3 (group 1 / = 2), week 5 (group 1 / = 3), and week 6 pv1 (group 1 / = 3), where p ≤ 0.04. the number of cd4−cd8␣− cells for l10h in treatment group 1 was lower than the other treatment groups at weeks 1, 2, 3, 5 and 6 pv1. besides this, the number of cd4−cd8␣− cells differed significantly for l10l between the treatment groups at week 1 (group 1 / = 3), week 2 (group 1 / = 4), and week 5 pv1 (group 1 / = 3) where p ≤ 0.05. the l10l chickens in treatment group 1 were lower in the number of cd4−cd8␣− cells than the other treatment groups at weeks 1, 2, and 5 pv1. briefly, the addition of an mbl ligand had no influence of total cd4+ cells within a l10h or l10l subline. however, the addition of chitosan to the vaccine decreased the number of cd4−cd8␣− cells in the l10h chickens at week 6 to 9 pv1. also, the l10h chickens as well as the l10l chickens also showed an increased number of cd4−cd8␣−␥␦ t-cells when an mbl ligand was added to the vaccine, most pronouncedly after the first vaccination. the b-cells were identified by fsc/ssc gating on small lymphocytes, followed by identification of bu-1 positive cells ( fig. 4a and c) . results are shown in table 2 . a significant difference between fig. 3 . ibv-specific igg antibody titres as measured by elisa. results are shown as mean values ± sem. the asterisks indicate statistically significant differences (p < 0.05) between groups or sublines. the double asterisks indicate statistically significant differences (p < 0.01) between groups or sublines. h or l indicates the sublines with high or low mbl serum concentration, respectively; 1, 2, 3 or 4 indicates the treatment groups "vaccine" (1), "vaccine + mannan" (2), "vaccine + chitosan" (3) or "vaccine + fos" (4). l10h and l10l chickens were observed in treatment group 1 where the absolute counts of b-cells were significantly lower for l10h chickens than for l10l chickens (p = 0.001). no significant differences were observed between l10h and l10l chickens in the other treatment groups. the main purpose of the present study was to investigate innate and adaptive immune responses following ibv vaccination when temporarily inhibiting mbl function by co-administering mbl ligands. the binding of pathogens by prrs, such as mbl, is an important step to initiate the early host response to infection. if mbl efficiently neutralises the pathogen via the complement membrane-attack complex, no adaptive immune response is needed. thus, poor adaptive memory is obtained and the purpose of the vaccine is unachieved. to our knowledge the current study is the first to show that mbl binds to ibv. binding was shown to occur through the crd of mbl since binding was inhibited by edta as well as mannan. previous studies have shown that human mbl binds to several viruses, such as hiv (saifuddin et al. 2000) and sars (ip et al. 2005) . these bindings have also been shown to occur through the crd of mbl. further, the current study shows that mbl binds to ibv in a dosedependent manner. besides this, it also provides evidence that fos is an mbl ligand with the potential to inhibit mbl-ibv binding. molecules (epstein et al. 1996; takahashi et al. 2011 ). in the serum samples from l10h chickens chitosan showed a tendency to inhibit the mbl-ibv binding (p < 0.0513) indicating that chitosan may also be an mbl ligand. chitosan is a highly basic polysaccharide obtained by deacetylation of chitin which is a linear polymer of n-acetyld-glucosamine (ravi kumar 1999) , which is known to be an mbl ligand (epstein et al. 1996) . chitosan is insoluble in water, which is why there is an uncertainty concerning the precise amount of chitosan added in the binding assay and to the vaccine given to the chickens. in a previous study both chitosan and its derivative n,n,ntrimethylated chitosan (tmc) were used as adjuvants in vaccines in raccoons (fry et al. 2012) . this study showed a higher number of responders to the vaccine when tmc was added instead of chitosan. tmc is water-soluble and have the same immunogenic and adhesive properties as chitosan (kotze et al. 1997) , which is why, with hindsight, that tmc instead of chitosan probably would have been a better choice as a possible mbl ligand added to the ibv vaccine to avoid the uncertainty about the concentrations used. following the first ibv vaccination the presence of viral genomes was observed in the opa of all the birds. however, the viral loads were lower for l10h chickens than l10l chickens (fig. 2) , indicating that l10h chickens were less severely affected by the infection than l10l chickens. previously, juulmadsen et al. (2011b) did not find any statistical difference in virus load between l10l and l10h chickens after vaccination. some of the incongruence between the current study and the vaccination part of the previous study could be explained by the larger animal groups in the current study, giving a more accurate outcome. besides, the estimation of viral load using qrt-pcr is more accurate than estimating the viral load by gel. the higher viral load in the l10l does, however, support the previous suggestions that mbl is associated with susceptibility to ibv infection (juul-madsen et al. 2007; juul-madsen et al. 2011b) . the ibv-specific igg antibody titres after the first vaccination did not differ between sublines (l10h and l10l) or treatment. after the second vaccination (week 3) of chickens receiving vaccine alone no boosting effect of the vaccine was found in the two sublines. on the other hand, the ibv-specific igg antibody titres after the second vaccination for l10h chickens receiving an mbl ligand together with the vaccine increased more than the antibody titre for l10h receiving vaccine alone (fig. 3) . this was most pronounced for the l10h chickens receiving vaccine and fos. this is also in contrast to the challenge study by juulmadsen et al. (2011b) , where the titre was lower for l10h chickens receiving mannan together with the vaccine compared with chickens receiving vaccine alone -we have no explanation for that. the antibody titres were the same for the l10l chickens between treatment groups. this, in combination with the results of the mbl-ibv binding assay, where the mbl-ibv binding was clearly inhibited by the addition of ligands in serum from l10h but not l10l, implies that the combination of high mbl serum concentrations and the addition of an mbl ligand have an impact on the development of ibv-specific igg antibodies. an increased antibody titre has earlier been reported in chickens vaccinated against eimeria (janardhana et al. 2009 ) and in mice vaccinated and infected with salmonella (benyacoub et al. 2008 ) when feeding a diet containing fos. the dramatic increase in the antibody titre after vaccination with fos may be beneficial in breeding, since the transfer of maternal antibodies to offspring is of importance in protecting the newly hatched chicks. this protection would be increased by an increase in the amount of antibodies in the dams, since there is a direct relationship between ibv-specific igg levels in the dam and those in her offspring (hamal et al. 2006) . flow cytometry-based methods for counting absolute numbers of peripheral blood cells have previously been described (burgess and davison 1999; seliger et al. 2012 ). in the current study flow cytometry was used to identify phenotypic differences between the chicken sublines vaccinated with or without the addition of mbl-ligand. no general differences were observed in the amount of total cd4+ cells for both sublines and between treatment groups. nor was any difference between the two sublines within each treatment group observed in groups 1, 2 and 3. however, the addition of fos to the vaccine gave a higher number of total cd4+ cells in the l10h chickens than in the l10l chickens (fig. 5) . despite of this, no difference in the amount of b-cells at week 9 pv1 (table 2) was observed in this group, indicating that the increase may have been caused by th1-cells instead of th2-cells. this is supported by a study by guo et al. (2008) which indicates that th1-cells are activated immediately after ibv infection. the response of cd4−cd8␣+ cells, mostly cytotoxic t-lymphocytes (ctl), is crucial for the elimination of the virus from local infection sites (collisson et al. 2000; guo et al. 2008; juul-madsen et al. 2011b ). an increased number of cd4−cd8␣+ cells were indeed observed in the current study both pv1 and pv2 which may support the suggestions by collisson et al. (2000) that ctls are important in the protection against ibv. however, this increase was repressed in l10h chickens treated with fig. 4a and b. the absolute counts of total cd4+ cells are shown in the left column, the absolute counts of cd4−cd8␣+ cells are shown in the middle column, and the absolute counts of cd4−cd8␣− cells are shown in the right column. the upper panels show the comparison of sublines in group 1, the second upper panels the comparison of sublines in group 2, the second lower panels the comparison of sublines in group 3, and the lower panels the comparison of sublines in group 4. results are shown as mean values ± sem. the asterisks indicate statistically significant differences (p < 0.05) between groups. the double asterisks indicate statistically significant differences (p < 0.01) between groups. h or l indicates the sublines with high or low mbl serum concentration, respectively; 1, 2, 3 or 4 indicates the treatment groups "vaccine" (1), "vaccine + mannan" (2), "vaccine + chitosan" (3) or "vaccine + fos" (4). vaccine and an mbl ligand at weeks 6-9 pv1 (fig. 6) . as this study does not contain a mock vaccine group, it cannot be ruled out that the increase is age-dependent. involvement of other receptors specific for the used mbl ligands may have influenced some of the differences observed in the current study. however, mbl has an influence as the difference was more pronounced for the l10h chickens than l10l chickens for both the ibv-specific igg antibody titres and the numbers of cd4−cd8␣+ and cd4−cd8␣− cells. the chicken cd3+cd4−cd8␣− cells in circulation are mostly ␥␦ t-cells which may contribute with up to 60% of the circulating tcells in a healthy adult chicken (dalgaard et al. 2010 ) indicating that they may play an important role in the chicken immune system. for the three groups receiving vaccine and an mbl ligand, the ␥␦ t-cell response was more pronounced in the l10h chickens than in the l10l chickens pv1 (fig. 5) . there was also a tendency (p = 0.0517) for this in the group receiving vaccine alone. this increase was also observed in the study by juulmadsen et al. (2011b) . this study showed a higher increase in the percentage of ␥␦ t-cells after ibv vaccination in the group receiving vaccine and mannan. in the current study no difference was observed in the ␥␦ t-cell response pv2 between l10h and l10l chickens within treatment groups (fig. 5) . these inconsistencies may be explained by the use of live virus in the previous study versus the use of attenuated live virus in the current study as already suggested. however, at least a tendency for a higher number of ␥␦ t-cells was observed in the current study until week 6 pv1 for l10h chickens receiving vaccine and mbl ligand compared to l10h chickens receiving vaccine alone (fig. 6) . the same was observed for l10l chickens receiving vaccine and chitosan or fos until week 3 pv1 and at week 5 the asterisks indicate statistically significant differences (p < 0.05) between groups. the double asterisks indicate statistically significant differences (p < 0.01) between groups. h or l indicates the sublines with high or low mbl serum concentration, respectively; 1, 2, 3 or 4 indicates the treatment groups "vaccine" (1), "vaccine + mannan" (2), "vaccine + chitosan" (3) or "vaccine + fos" (4). pv1 for l10l treatment groups receiving vaccine and mbl ligand (fig. 6) . the function of chicken ␥␦ t-cells is still under debate, but studies have indicated that ␥␦ t-cells respond to pathogens and provide a protective immune response after immunisation with both live attenuated and non-attenuated salmonella strains (berndt et al. 2006) . the results of the current study may support these indications since our results showed that cd4−cd8␣−␥␦ t-cells are increased after ibv vaccination when mbl is inhibited. a difference in the amount of b-cells at week 9 pv1 (table 2) was observed between the l10h and l10l chickens receiving only vaccine, but no difference was observed for ibv-specific antibody titres in this group. in the treatment group receiving vaccine and fos a major difference between l10h and l10l chickens was observed for the ibv-specific antibody titres, even though only a tendency (p = 0.078) for a difference in the amount of b-cells at week 9 pv1 was observed between these chickens. these findings support the suggestions made by guttormsen et al. (2009) that an increased igg response in mbl-deficient mice is not only attributed to an increase in the number of b-cells. the increase in igg may also have been caused by ig class switching, as previously observed in mice for dietary fos by nakamura et al. (2004) . previous challenge studies have shown an up regulation of igg in species with low amount of mbl in both mice (guttormsen et al. 2009 ) and chickens (juul-madsen et al. 2007 ). likewise, ruseva et al. (2009) argue that genetic environment influences the modifying effect of mbl. we cannot conclude whether the different amounts of bcells at week 9 pv1 observed in chickens receiving vaccine alone in the current study was caused by vaccine or age. if the difference was caused by the vaccine, the mbl ligands eliminate this difference. in conclusion, the ibv-specific igg antibody titres were the same for l10l chickens between different treatments, whereas the addition of an mbl ligand to the vaccine, especially fos, seemed to enhance the production of ibv-specific igg antibody in l10h chickens, but not in l10l chickens. the addition of fos to the vaccine also increased the number of total cd4+ cells in l10h chickens, in combination with an unchanged amount of b-cells at week 9 pv1 compared to l10l chickens. the l10h chickens as well as the l10l chickens also showed an increased number of cd4−cd8␣−␥␦ t-cells when an mbl ligand was added to the vaccine, most pronouncedly after the first vaccination, suggesting that cd4−cd8␣−␥␦ t-cells may also play a role in the immune response against ibv. these results indicate, as previously suggested by juulmadsen et al. (2011b) that mbl is involved in the adaptive immune response to ibv vaccination. mbl inhibition may therefore be beneficial to achieve high antibody response during vaccinations. further studies are needed to elucidate whether the addition of mbl ligands to ibv vaccine gives a better immune protection against ibv infection. (fp7, 2007 (fp7, -2013 , research infrastructures action, under the grant agreement no. fp7-228394 (nadir) feeding a diet containing a fructooligosaccharide mix can enhance salmonella vaccine efficacy in mice circulating gamma delta t cells in response to salmonella enterica serovar enteritidis exposure in chickens specific interaction of hepatitis c virus glycoproteins with mannan binding lectin inhibits virus entry counting absolute numbers of specific leukocyte subpopulations in avian whole blood using a single-step flow cytometric technique: comparison of two inbred lines of chickens development and evaluation of a real-time taqman rt-pcr assay for the detection of infectious bronchitis virus from infected chickens co-circulation of four types of infectious bronchitis virus (793/b, 624/i, b1648 and massachusetts) mannose-binding lectin a-deficient mice have abrogated antigen-specific igm responses and increased susceptibility to a nematode infection coronavirus avian infectious bronchitis virus lack of the pattern recognition molecule mannose-binding lectin increases susceptibility to influenza a virus infection cytotoxic t lymphocytes are critical in the control of infectious bronchitis virus in poultry the long view: 40 years of infectious bronchitis research crosstalk between components of the innate immune system: promoting anti-microbial defenses and avoiding immunopathologies flow cytometric assessment of chicken t cell-mediated immune responses after newcastle disease virus vaccination and challenge the interplay between innate and adaptive immunity regulates cancer development mannose-binding lectin in innate immunity: past, present and future the collectins in innate immunity mucosal adjuvants to improve wildlife rabies vaccination molecular mechanisms of primary and secondary mucosal immunity using avian infectious bronchitis virus as a model system deficiency of mannose-binding lectin greatly increases antibody response in a mouse model of 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control basal serum level of mannan-binding protein complement genetics, deficiencies, and disease associations high-dose mannose-binding lectin therapy for ebola virus infection dietary fructooligosaccharides up-regulate immunoglobulin a response and polymeric immunoglobulin receptor expression in intestines of infant mice carbohydrate-based immune adjuvants infectious bronchitis virus: immunopathogenesis of infection in the chicken the positive adjuvant effect of chitosan on antigen-specific cell-mediated immunity after chickens vaccination with live newcastle disease vaccine chitin and chitosan fibres: a review mannan-binding lectin deficiency modulates the humoral immune response dependent on the genetic environment dietary fructooligosaccharides and potential benefits on health interaction of mannose-binding lectin with primary isolates of human immunodeficiency virus type 1 mannanbinding lectin enhances susceptibility to visceral leishmaniasis sas proprietary software mannan-binding lectin (mbl) in two chicken breeds and the correlation with experimental pasteurella multocida infection a rapid high-precision flow cytometry based technique for total white blood cell counting in chickens detection of structural gene mutations and promoter polymorphisms in the mannan-binding lectin (mbl) gene by polymerase chain reaction with sequence-specific primers molecular basis of opsonic defect in immunodeficient children mannose-binding lectin and the balance between immune protection and complication dietary sugars inhibit biologic functions of the pattern recognition molecule, mannose-binding lectin a single asparagine-linked glycosylation site of the severe acute respiratory syndrome coronavirus spike glycoprotein facilitates inhibition by mannose-binding lectin through multiple mechanisms the authors wish to thank l.r. dal and h. svenstrup for technical assistance and k.v. østergaard for critical reading of the manuscript. this work was supported by the project "development of genetic selection technology for polyvalent resistance to infectious diseases" (poly-reid) (grant number 10-093534) granted by danish council for strategic research, danish poultry council, the hatchery hellevad, and cobb-vantress, and grants from the graduate school of science and technology at aarhus university and european community's seventh framework programme the authors declare that they have no conflicts of interest. key: cord-306111-wn1gxhk9 authors: dommett, r. m.; klein, n; turner, m. w. title: mannose‐binding lectin in innate immunity: past, present and future date: 2006-09-01 journal: tissue antigens doi: 10.1111/j.1399-0039.2006.00649.x sha: doc_id: 306111 cord_uid: wn1gxhk9 the human collectin, mannose‐binding lectin (mbl), is an important protein of the humoral innate immune system. with multiple carbohydrate‐recognition domains, it is able to bind to sugar groups displayed on the surfaces of a wide range of microorganisms and thereby provide first‐line defence. importantly, it also activates the complement system through a distinctive third pathway, independent of both antibody and the c1 complex. three single point mutations in exon 1 of the expressed human mbl‐2 gene appear to impair the generation of functional oligomers. such deficiencies of functional protein are common in certain populations, e.g. in sub‐saharan africa, but virtually absent in others, e.g. indigenous australians. mbl disease association studies have been a fruitful area of research and implicate a role for mbl in infective, inflammatory and autoimmune disease processes. overall, there appears to be a genetic balance in which individuals generally benefit from high levels of the protein. however, in certain situations, reduced levels of circulating mbl may be beneficial to the host and this may explain the persistence of the deleterious gene polymorphisms in many population groups. it is now 60 years since the australian nobel prize winner sir frank macfarlane burnet, together with john mccrea, identified three inhibitors in serum (called a, b and g), which were able to inactivate influenza virus (1) . we now know that the b inhibitor was, in fact, a protein called mannosebinding lectin (mbl), a component of the innate immune system (2) . during the past 30 years, our understanding of this protein has steadily increased as a result of extensive research activity in three main areas: (a) bio/immunochemistry (including molecular genetics), (b) microbiology and (c) immunodeficiency. work in these areas initially proceeded independently as evidence for both an inexplicable biological function and a clinical deficiency state emerged. the isolation and characterization of the protein were necessary in order to illuminate the observations of the so-called rarf bactericidal activity (3, 4) in the microbiology area and the opsonic deficiency reported in many paediatric populations. some of the main developments are summarized in table 1 . this review briefly addresses issues relating to the early history of mbl, its structure, function, genetics and disease associations. finally, future developments including the potential use of both plasma-derived and recombinant mbl are discussed. the existence of mammalian serum lectins was first predicted in 1975 by robinson et al. (5) , and the protein was first isolated in 1978 from cytosolic fractions of rabbit liver by kawasaki et al. (6) . subsequently, wild et al. (7) were able to isolate mbl from both human and rat liver. more recently, extrahepatic transcription of mbl has been reported and this may have implications regarding its role in localized host defence (8) . mbl belongs to a family of proteins called the collectins, which possess both collagenous regions and lectin domains. the other major human collectins, surfactant protein a and surfactant protein d, possess structural characteristics similar to those of mbl and are found predominantly in the lung and other mucosal sites (9) . plasma-associated phagocytic defect (28) 1975 existence of mammalian serum ôlectin-like proteins specific for mannoseõ predicted (5) 1976 association of opsonic defect with frequent infections in infancy, but deficiency also present in 5% of the general population (29) 1978 mbl isolated from rabbit liver (6) 1980 opsonic deficiency in infants with chronic diarrhoea (30) 1981 association of yeast opsonization defect with suboptimal c3b deposition (31) 1982 description of mouse rarf: a complement-activating bactericidal protein (3) 1983 human mbl isolated from liver (7); human serum mbl isolated (121) prospective study of opsonic deficiency in infancy (122) 1984 rarf activity present in vertebrate classes (4) 1985 bovine serum mbl described (123) opsonic defect linked to absence of an unidentified co-factor of the complement system (124) 1987 mbl activation of classical complement pathway (18) 1988 rat serum mbl a and c described (125) ; description of c-type crd (126) 1989 gene for human mbl cloned (33, 34) ; human mbl has bactericidal activity (127) ; opsonic nature of mbl demonstrated (35) mbl inhibits in vitro infection by hiv (85) correlation of opsonic defect with low serum mbl levels (32) 1990 bovine and mouse serum b inhibitors of influenza a virus identified as mbl (2) correlation of mbl levels with classical complement pathway activation at low serum concentrations (128) 1991 mouse mbl a and c described (129) opsonic deficiency and low mbl levels linked to single point mutation in codon 54 (variant b) (50) 1992 human mbl levels in acute-phase responses (59) ; crystallography of mbl crd (130) ; novel protease (masp-1) and complement activation by mbl (19) human rarf identical to mbl-masp (131) low mbl levels in africans linked to codon 57 (variant c) mutation in the mbl gene (51) 1994 third mbl mutation in codon 52 (variant d) described (52) 1995 polymorphisms found in promoter region of mbl gene (55) 1997 second masp found to activate complement (20) mbl mutations are an important risk factor for infections in children (132) 1998 reconstitution of opsonizing activity by infusion of purified mbl into mbl-deficient humans (112) 1999 truncated form of masp-2 -map19 (21) 2000 complement-activating complex of ficolins and masp (133) mbl shown to bind to clinically relevant organisms (15) structural aspects of mbl the protein structure of mbl has been studied extensively, and aspects are presented in figures 1 and 2 . the protein consists of multimers of an identical polypeptide chain of 32 kda. each chain comprises four distinct regions encoded by different exons of the mbl-2 gene, as will be discussed in more detail later. each chain has a c-terminal, calcium-dependent carbohydrate-recognition domain (crd); a short, a-helical, hydrophobic neck region (in the so-called coiled-coil configuration); a collagenous region containing 19 gly-xaa-xaa triplets and a cysteine-rich n-terminal region. three polypeptide chains form a triple helix within the collagenous region, stabilized by hydrophobic interactions and interchain disulphide bonds within the n-terminal cysteine-rich region. this is the basic building block of all circulating molecular forms of mbl. in serum, mbl consists of oligomers ranging from dimers to hexamers, and x-ray crystallographic studies/electron micrographs have revealed that these oligomers have a sertiform or a bouquetlike structure due to an interruption in the collagenous region, giving rise to a kink/hinge. the ability of the protein to bind effectively to microorganisms and activate complement appears to depend on the presence of higher order oligomers (tetramers and above). work by drickamer and colleagues (10, 11) and also by ezekowitz and colleagues (12) has provided an insight into the structure of the crd. each crd binds a calcium ion, enabling it to form co-ordination bonds with the 3-and 4-hydroxyl groups of specific sugars including mannose, n-acetyl-d-glucosamine, n-acetyl-mannosamine, fucose and glucose. the three crds in each structural subunit are separated by a constant 45-å distance (12) . clustering of the structural subunits provides a flat platform, permitting binding of mbl to the arrays of repeating sugar groups on microbial surfaces. although the binding affinity of each individual crd-sugar interaction is relatively low at 10 23 m (13), the formation of higher order oligomers provides multiple crds, which are able to bind simultaneously with high avidity. mbl is a major pattern-recognition molecule of the innate immune system. it primarily recognizes specific sugar groups (as above) on the surface of microorganisms, enabling it to distinguish self from non-self. it can also bind to phospholipids, nucleic acids (14) and non-glycosylated proteins. mbl has been shown to bind promiscuously to a wide range of bacteria, viruses, fungi and protozoa and some selected examples are listed in table 2 . neth et al. used flow cytometry to demonstrate mbl binding to clinically relevant bacterial isolates from immunocompromised children and noted differences in binding within some species such that one isolate might show strong binding, whereas another was much weaker (15) . the role of specific structural features of microorganisms (e.g. the capsule), which permit or prevent binding to mbl, has been explored in several studies. the earliest work was probably by kawakami et al. on the socalled rarf complex (which was later identified as mbl) and its interaction with salmonella enterica serovar typhimurium (3). this suggested that the structure and composition of lipopolysaccharide play a crucial role in mbl binding and function. other mechanisms that enable microorganisms to avoid recognition and killing by mbl include lipooligosaccharide sialyation (16, 17) . despite much progress in this area, many puzzles remain to be addressed, mostly related to the exact disposition of sugars on microbial surfaces. our understanding of mbl function has grown rapidly over the past three decades. it is now recognized to have a role in processes as diverse as complement activation, promotion of complement-independent opsonophagocytosis, modulation of inflammation, recognition of altered self-structures and apoptotic cell clearance. a role for mbl in host defence was first proposed in 1987 when ikeda et al. observed that the protein was able to activate the classical pathway of complement (18) . however, it is now clear that mbl activates a novel third pathway of complement, often termed the mbl pathway, in an antibody-and c1-independent fashion as illustrated in figure 3 . this functional activity reflects the fact that mbl circulates in association with a group of mbl-associated serine proteases (the so-called masps). in 1992, matsushita and fujita demonstrated the presence of a novel complement enzyme in serum, which was thought to generate the c3 convertase (c4bc2a), associated with classical pathway activation (19) . however, this activity was later found to be mediated by masp-2 (20) , and the original enzyme is now known as masp-1 and may activate c3 directly. subsequently, a small separately synthesized fragment of masp-2 termed smap or map19 was identified (21, 22) and a third masp (masp-3) with no known function was also described (23) . current understanding suggests that on binding to microorganisms, autoactivation of masp-2 occurs, permitting cleavage of c4 and c2 to form a c3 convertase, which is indistinguishable in specificity from the convertases found in the other two activation pathways of complement (24) . it should be noted that the so-called mbl pathway is also activated by another family of proteins called ficolins. the ficolins are structurally similar to collectins, with collagenous domains linked to fibrinogen-like domains having sugar-binding properties. l-and h-ficolins are humoral factors synthesized by hepatocytes, although h-ficolin has also been observed in bronchial/alveolar fluid and in bile (25) . in contrast, m-ficolin is found on peripheral blood mononuclear cells, polymorphonuclear cells and type ii lung epithelial cells (26) . ficolins are also found in complexes with the masps and are considered to have different binding specificities compared with mbl (27) . in 1968, miller et al. reported a plasma-associated defect of phagocytosis in a child with severe recurrent infections, failure to thrive and diarrhoea (28) . in vitro work revealed a failure of the childõs plasma to opsonize heat-killed bakers yeast (saccharomyces cerevisiae). this defect was later detected in the sera of children with recurrent unexplained infections (29) and chronic diarrhoea of infancy (30), but, interestingly, studies in the general population also revealed a relatively high frequency of the defect (5%). in 1981, studies linked this opsonic deficiency to the complement figure 3 complement activation pathway. the lectin pathway of complement is activated by mbl and ficolins. on binding to appropriate targets, mbl-masp-2 complexes cleave c4 and c2 to form c3 convertase (c4bc2a). mbl-masp-1 complexes may activate c3 directly. ficolins also work in combination with the masps. the classical and alternative pathways also generate c3 convertase enzymes, which cleave c3. the lytic pathway (c5-c9) is common to all three routes of c3 cleavage. mbl, mannose-binding lectin; masp, mbl-associated serine proteases; masp-1, mbl-associated serine protease-2; masp-2, mblassociated serine protease-2. system by demonstrating that sera with the deficiency deposited less c3b on yeast surfaces (31) . however, it was not until 1989 that the common opsonic defect was found to be associated with low levels of the mannose-binding protein, which we now refer to as mbl (32) . in that same year, the gene for mbl was cloned (33, 34) (genetics of human mbl). in a study of mbl-coated salmonella montevideo, kuhlman et al. reported that mbl was able to interact directly with cell surface receptors and promote opsonophagocytosis (35) . subsequently, a number of putative mbl-binding proteins/receptors have been proposed including cc1qr/ calreticulin (36), c1qrp (37) and cr1 (38, 39) . however, it is unclear whether mbl is acting as a direct opsonin or is merely enhancing other complement pathways and/or antibody-mediated phagocytosis. the role of mbl as a modulator of inflammation appears to be complex and, accordingly, its mechanism of action remains unexplained. one possible explanation is that mbl is able to trigger proinflammatory cytokine release from monocytes (40, 41) . this concept was addressed in studies by jack et al. using neisseria meningitidis incubated with increasing concentrations of mbl before being added to mbl-deficient whole blood. release of tumour necrosis factor a, interleukin (il)-1b and il-6 from monocytes was enhanced at mbl concentrations below 4 mg/ml but suppressed at higher concentrations (42) . clinical studies in this area are discussed later. the role of mbl in the recognition of altered self and apoptosis a role for mbl in the clearance of apoptotic cells was first proposed by ogden et al. in 2001 (43) . mbl was found to bind directly to apoptotic cells that expose terminal sugars of cytoskeletal proteins, thereby permitting their recognition and directly facilitating their phagocytosis by macrophages. defects in the clearance of apoptotic cells have been implicated in the pathogenesis of certain autoimmune conditions, although the precise role of mbl, if any, remains elusive. for example, in 2005, stuart et al. reported that although mbl-deficient mice displayed defective apoptotic cell clearance, they did not develop autoimmune diseases (44) . in animal studies, mbl has been implicated in the pathophysiology of ischaemia reperfusion injury due to its ability to recognize altered self-structures. stahl and colleagues have proposed the lectin pathway as a mediator of this process in certain organs, and the absence of mbl/masp pathway activation appears to afford protection in these disease models (45, 46) . however, the relevance of these findings to human health needs to be established. changes in cell surface structures during oncogenic transformation appear to promote binding of mbl to cancer cells (47) where the protein can mediate cytotoxic effects including mbl-dependent cell mediated cytotoxicity (48, 49) . the relative importance of such mechanisms in tumour immunology is, at present, unknown. there are two human mbl genes, but mbl-1 is a pseudogene and only mbl-2 encodes a protein product. the functional mbl-2 gene is located on chromosome 10 (q11.2-q21) and comprises four exons as illustrated in figure 1 . exon 1 encodes the signal peptide, a cysteine-rich region and part of the glycine-rich collagenous region. exon 2 encodes the remainder of the collagenous region and exon 3 encodes an a-helical coiled-coil structure, which is known as the ôneckõ region. exon 4 encodes the crd, which adopts a globular configuration. the promoter region of the mbl gene contains a number of regulatory elements, which affect transcription of the protein. in 1991, the complete nucleotide sequence of all four exons of the human mbl-2 gene was determined by sumiya et al. in two british children with recurrent infections and low mbl levels (50) . in both individuals, a point mutation was observed in codon 54, changing the codon sequence from ggc to gac and substituting aspartic acid for glycine in the translated protein. familial studies confirmed that the defect was inherited in an autosomal dominant fashion. in 1992, lipscombe et al. identified a second exon 1 mutation in codon 57 (gly / glu), when studying a sub-saharan african population (51) , and in 1994, madsen et al. reported a mutation in codon 52 (arg / cys) (52) . these point mutations are now commonly referred to as variants b, c and d respectively, with variant a indicating the wild type. the b variant mutation occurs at a gene frequency of approximately 25% in eurasian populations. in contrast, the c variant is rare in eurasians but is commonly seen in sub-saharan african populations, with frequencies of 50%-60%. population studies suggest that the b variant mutation may have arisen between 50,000 and 20,000 years ago (53) since no structural gene mutations have been identified in studies of indigenous australian populations who arrived on the continent approximately 50,000 years ago, whereas the b variant mutation was probably introduced into both north and south america at the time of the last glaciation approximately 20,000 years ago. the effect of these exon 1 mutations on the protein product continues to be the focus of study. they are believed to impair oligomerization and lead to a functional deficiency. the b and c mutations result in the replacement of critical axial glycines in the triple helix by dicarboxylic acids, resulting in distortion of this important part of the protein (50) . in contrast, the d mutation results in the replacement of arginine with cysteine. this extra cysteine has been proposed to cause formation of adventitious disulphide bonds that hinder higher oligomer formation (54) . several polymorphisms have also been reported in the promoter region of the gene. studies by madsen et al. investigating the large interindividual variation in serum mbl levels revealed three polymorphisms, h/l, x/y and p/q at positions 2550, 2221 and 14 of the mbl gene (55, 56) . subsequently, four common haplotypes were identified, namely lxp, lyp, lyq and hyp. of these, hyp, which is associated with medium to high levels of mbl and lxp, which is associated with low levels of the protein, appear to be most important. these promoter haplotypes are in strong linkage disequilibrium with the exon 1 mutations, resulting in seven common extended haplotypes, namely hypa, lypa, lyqa, lxpa, hypd, lypb and lyqc. other rare haplotypes have also been described (57) . figure 4 illustrates the frequency of these various haplotypes in selected populations and highlights the degree of ethnic variation. the combination of structural gene and promoter polymorphisms results in a dramatic variation in mbl concentration in apparently healthy individuals of up to 1000-fold (caucasian: range <20-10,000 ng/ml). in addition, ezekowitz and colleagues presented evidence in 1988 that mbl was an acute-phase reactant (58) . in these investigations, rna was isolated from a ônormalõ liver taken as part of a staging biopsy for hodgkins disease and was compared with rna isolated from a fresh post-mortem liver of a victim with severe trauma. the authors found that mbl messenger rna transcripts were barely detectable in normal liver but that induction was seen in liver exposed to acute stress. subsequent studies have shown that mbl levels can increase between 1.5 and threefold during the acute phase, but this response is variable between individuals (59) . it should also be noted that even during an acutephase response, individuals heterozygous or homozygous for mbl mutations appear unable to achieve the protein levels of those possessing a wild-type genotype. approximately one-third of the caucasian population possess genotypes conferring low levels of mbl, with approximately 5% having very low levels. no absolute level of mbl deficiency has been defined. genotype and phenotype show a relatively strong correlation and studies often use just one measure to infer deficiency. however, there is ôadded valueõ in performing both measures and we would strongly advocate this approach whenever possible. mbl occurs in two distinct forms in rodents and rhesus monkeys (60), but only one form is found in humans and chickens. as discussed previously, there are two human mbl genes, which are most likely due to a gene duplication event (61) . however, mbl-1 is a pseudogene and the potential mechanisms responsible for silencing the mbl-1 (63). such substitutions were also found in other higher primates including chimpanzees and gorillas but not in more distant primates such as the rhesus monkey. the authors concluded that both the mbl-1 and the mbl-2 genes have been selectively silenced by the same molecular mechanisms, but skewed in time resulting in overall downregulation of mbl levels in the present human population. the high frequency of variant alleles observed in certain populations was initially puzzling since it suggests that functional mbl deficiency may well be advantageous. similarities have been proposed between the mbl genetic system and the role of the sickle cell gene in protection against malaria as occurs in carriers of the sickle cell haemoglobin allele (64) . the argument runs as follows: certain intracellular parasites use c3 opsonization and c3 receptors on monocytes/macrophages to enter their host. therefore, any reduction in complement-activating function of the host may reduce the probability of parasitization. in support of this notion is a study on patients with visceral leishmaniasis, which revealed that such patients are more likely to have high mbl levels than uninfected controls (65) . a small study of ethiopian patients with lepromatous or borderline lepromatous leprosy also found that their mbl levels were significantly higher than those of healthy blood donors (66) . an alternative explanation of the unexpectedly high frequency of low mbl phenotype individuals found in many tropical regions is that excessive complement activation can result in immunopathologically mediated host damage; therefore, any mechanism that reduces complement activation may be beneficial (51) . the identification of mbl deficiency as the cause of the so-called common opsonic defect has been followed by a plethora of disease association studies aimed at defining the precise role of this protein. a number of the early studies concentrated on paediatric populations and mbl was suggested to provide substitute ôantibodyõ-like activity during the ôwindow of vulnerabilityõ (approximately 6-24 months), when maternal immunoglobulin g (igg) antibody levels have waned but the infantõs own adaptive immune response is still immature (32) . nevertheless, studies in adults suggested that there might be a role for mbl throughout life (67) . notwithstanding these reports, the majority of individuals possessing a variant mbl allele apparently suffer no ill effects and remain essentially healthy. in a study that apparently confirms this, dahl et al. monitored 9245 adults in a danish caucasian population and found no evidence for significant differences in infectious disease or mortality in mbl-deficient individuals compared with controls (68) . similar findings were reported by tacx et al. in unselected adults admitted to hospital with infections (69) . nevertheless, these studies should not be regarded as proof that mbl levels have no clinical relevance. many groups have undertaken case-control studies, which do indeed suggest that mbl is an important immunological modulator. in some cases, there is evidence that the significance of mbl deficiency is more readily appreciated when there is another co-existing defect (70), as we first proposed in 1991 (71). space does not permit a comprehensive review of all the mbl clinical studies that have been undertaken to date, and the topics covered below have been selected in order to illustrate examples of possible roles for mbl in a variety of clinical situations. most studies have explored the role of mbl in relation to the acquisition of an infectious organism (susceptibility) and the nature of the associated clinical course (severity). in clinical practice, this distinction can be difficult. however, for the purposes of this review, we will highlight examples of infections in which mbl appears to have an influence on one or other of these two aspects of infectious diseases. hamvas et al. have recently shown a role for mbl in mycoplasma infection (72) . they studied cases of infection in patients with primary antibody deficiencies (pad) that are known to be particularly susceptible to such organisms and compared them with a control population. more than two-thirds of pad patients with mycoplasma infections were mbl deficient (in possession of an exon 1 variant allele) compared with one-third of the control group. in the same study, they were able to demonstrate binding of mbl to three strains of mycoplasma using flow cytometry and proposed a role for mbl in prevention of invasive disease. in 2003, severe acute respiratory syndrome (sars) emerged as a highly infectious disease caused by a novel coronavirus (sars-cov). it provided a new challenge to previously unexposed individuals predominantly in asia. specific antibodies to sars-cov could be detected 10 days after the onset of symptoms, making sufferers reliant on innate immune mechanisms during the early phase of infection. since the structure of the virus was rapidly established (73, 74) , it also became clear that this novel infectious agent was rich in the sugars known to be targeted by mbl and it was hypothesized that this lectin might well be involved in first-line defence against this infection. subsequent studies found significant differences in the distribution of mbl-deficient genotypes in patients with sars compared with those in controls (75, 76) . these studies suggested that mbl plays a role in susceptibility to the infection but does not influence subsequent severity. in their investigations, ip et al. were also able to demonstrate binding of mbl to the virus and its ability to inhibit infection (75) . (77). the b variant allele was found more commonly in patients with symptomatic hepatitis b cirrhosis and in those with spontaneous bacterial peritonitis. it was also noted that mbl levels were lower in this patient cohort with chronic infection. screening for mbl mutations in such patients was suggested in order to enable identification of those at increased risk of complications who may benefit from prophylactic antibiotic treatment. in 2005, chong et al. also reported that mbl genotypes correlating with low protein levels were associated with the occurrence of cirrhosis and also hepatocellular carcinoma in hepatitis b carriers (78) . they also demonstrated that mbl is able to bind hepatitis b surface antigen. in the same year, thio et al. published the results of a nested case-control study of 527 patients who had either naturally recovered from hepatitis b (n ¼ 338) or had persistent infection (n ¼ 189). they found that mbl genotypes correlating with high serum levels were associated with recovery from infection, whereas those correlating with lower levels were associated with persistence of the virus (79) . it should be noted that approximately half of the subjects were also infected with human immunodeficiency virus (hiv), but the authors concluded that this did not influence the results obtained. matsushita et al. investigated the influence of mbl mutations in hepatitis c infection and found that sufferers who were homozygous for b variant alleles were less likely to respond to interferon treatment (80) . further work would be warranted in order to define the role of mbl in the pathogenesis of hepatitis infection. secondary immunodeficiencies due to disease or treatment have provided interesting patient populations within which to study the role of mbl. one such group comprises those receiving chemotherapy for malignancy. these patients are rendered neutropenic by their treatment (or underlying disease process) and are subsequently at increased risk of infectious complications. in 2001, two studies were published reporting an effect of mbl deficiency in such patients. neth et al. studied 100 children and measured mbl levels and genotype. children in possession of mbl variant alleles spent twice as many days in hospital with febrile neutropenia during the first 6 months of their treatment compared with wild-type individuals (81) . in the other study, peterslund et al. followed 54 adults undergoing chemotherapy for various haematological malignancies and found that those who developed ôsignificantõ infections (bacteraemia, pneumonia or both) in the 3-week periods post-treatment had significantly lower levels of mbl compared with those without significant infections (82) . subsequent studies have shown differing results, but drawing comparisons between them is inherently difficult. these patients are a highly heterogeneous population, with different underlying disease processes, undergoing treatment regimens of differing intensity, resulting in various degrees of immunosuppression. in one contrasting study, bergmann et al. followed 80 adults undergoing therapy for acute myeloid leukaemia, which involves intense highly myelosuppressive treatment. they found no effect of mbl deficiency on frequency, severity or duration of fever and suggested that the nature of the treatment overwhelmed any potential influence of mbl (83) . further clinical studies in such patients are required in order to delineate the exact role of mbl. an mbl double-knockout mouse model has been used to explore the above clinical conundrum. in 2004, shi et al. demonstrated that mbl null mice were highly susceptible to intravenous inoculation with staphylococcus aureus, all dying within 48 h, compared with 55% survival of mbl wild-type mice. however, when the mice were inoculated via the intraperitoneal route and rendered neutropenic (using cyclophosphamide), neutropenic mbl null mice were found to have higher accumulations of bacteria in the blood and organs compared with neutropenic wild-type mice. by day 8 post-infection, the neutropenic wild-type mice had cleared their blood, but the neutropenic mbl null mice had persistent bacteraemia. the authors were able to reverse the phenotype by treating the mbl null mice with recombinant mbl (84) . to date, nearly 40 million humans have been infected with hiv. the clinical consequences of viral exposure are variable. some individuals can be repeatedly exposed to the virus but remain free from infection. others can be infected but remain free from clinical disease. while numerous viral and host factors will determine the fate of an individual exposed to hiv, there are data to indicate that mbl can influence both susceptibility and severity of hiv infection. the likely target for hiv binding is the heavily glycosylated glycoprotein, gp120. while mbl can be readily demonstrated to bind to purified gp120 (85) , the capacity of mbl to neutralize primary hiv isolates is less convincing. recent data indicate the mbl can opsonize hiv but does not induce neutralization at the levels at which it is normally present in serum. however, binding and opsonization of hiv by mbl may alter virus trafficking and viral antigen presentation during hiv infection. mbl may influence uptake by dendritic cells (dc), which express a cell surface lectin called ôdc-specific intracellular adhesion molecule 3-grabbing non-integrinõ (dc-sign). dc-sign has been shown to mediate a type of infection called ôtransõ-infection, where dc bind hiv and efficiently transfer the virus to t cells. preincubation of hiv strains with mbl prevents dc-sign-mediated trans-infection of t cells and indicates that at least in vitro, mbl may inhibit dc-sign-mediated uptake and spread of hiv (86) . whatever the mechanism of mbl interactions with hiv, a number of clinical studies have suggested that deficiency of mbl is a risk factor for acquiring hiv infection. mbl deficiency appears to increase the acquisition of hiv infection by between three-and eightfold (87) (88) (89) (90) . there is also an increased risk of vertical transmission from infected mothers to their offspring (91) . however, these findings have not been replicated in all populations, with some studies failing to demonstrate a role for mbl in hiv infection (92) (93) (94) . there is even less clarity with regard to the role of mbl in hiv disease progression. garred et al. (87) demonstrated that men with mbl variant alleles had a shorter survival time following the onset of acquired immune deficiency syndrome (aids) than did patients with wild-type mbl alleles. however, in a well-characterized cohort of homosexual men, variant mbl alleles had an insignificant effect on survival following the diagnosis of aids (95) . in this latter study, there appeared to be a protective effect of mbl variant alleles, with a delay in the development of aids from the time of hiv seroconversion. patients with mbl variant alleles had lower cd4 counts at the time of developing aids, indicating that mbl deficiency may influence the onset of aids for any given cd4 count. furthermore, mbl mutations appeared to protect against the development of kaposi sarcoma, a finding that was difficult to explain (95) . in another study, prohaszka et al. (90) found that mbl levels were lower in asymptomatic hiv-positive individuals compared with hiv-negative controls. however, the protective effect of mbl was lost in patients with an aids diagnosis; patients with high mbl levels had significantly lower numbers of cd4 cells. a possible explanation is that enhanced proinflammatory cytokine production in advanced hiv disease acts to increase mbl synthesis (96) , elevating levels in patients with late-stage disease. indeed, a recent study has shown in vitro that mbl can enhance proinflammatory cytokine production and viral replication (97) . in the light of studies indicating a role for mbl in inflammatory modulation, it is tempting to suggest that under some circumstances, mbl may act to promote inflammatory cell activation, thereby accelerating the rate of cd41 t-cell depletion. few studies have assessed the impact of mbl in the context of effective antiviral therapy. however, one study has attempted to relate mbl status and hiv-infected longterm non-progressors (ltnps) (98) . mbl levels were consistent with a wild-type genotype in the six ltnps studied. amoroso and colleagues had also suggested such an effect in a study showing that children with rapidly progressing disease were more likely to have mbl variant alleles (codon 54) than slower progressors (99) . cystic fibrosis provides an example of a clinical condition where mbl appears to be exerting its role as an infection susceptibility gene and inflammatory modulator. garred et al. were the first group to report that patients with mbl variant alleles have significantly impaired lung function and decreased life expectancy in comparison with wild-type individuals (100) . the effect of mbl deficiency on the severity of lung disease was most apparent in patients with chronic pseudomonas aeruginosa infection and it was also found that burkholderia cepacia infection was more common in patients with mbl deficiency. in 2004, davies et al. reported that an effect of mbl was only seen in adults homozygous for mbl mutations. these patients had significantly reduced lung function, more frequent hospital admissions and raised systemic inflammatory markers. however, there was no evidence of increased susceptibility to burkholderia cepacia and pseudomonas aeruginosa (101) . whether mbl has an effect on early colonization with burkholderia cepacia and pseudomonas aeruginosa or subsequent secondary viral infections or whether there is an (anti)inflammatory effect on subsequent lung damage remains unclear. clinical studies of critically ill patients requiring intensive care management have shown that individuals who are mbl deficient are more likely to develop the systemic inflammatory response syndrome (sirs) ( figure 5 ) and progress to septic shock and death (102, 103) , findings which may well relate to the proinflammatory cytokine response. it should also be noted that chronic inflammation is now increasingly accepted to be a risk factor for myocardial infarction (mi), and a recent study by saevarsdottir et al. has found that patients with high mbl levels have a decreased likelihood of suffering a mi -again suggesting a potential role for mbl in modulating the inflammatory response (104). as a component of the complement system with similarities to c1q, but also as a player in infectious and inflammatory processes, the structure and function of mbl have prompted studies exploring a possible role in autoimmune conditions. systemic lupus erythematosus (sle) has been the focus of a number of mbl genotyping studies, but the results have been somewhat inconsistent. nevertheless, a recent meta-analysis has reviewed studies in this area and found that mbl variant alleles are indeed sle risk factors (105) . as with infectious disease, there is some evidence that the risk of pathology increases if there is another co-existing immune defect. for example, in a cohort of spanish patients, the odds ratio for developing sle was 2.4 for individuals with mbl deficiency, but this increased to 3.2 when there was also a co-existing partial c4 deficiency (106) . studies in patients with sle have reported that mbl deficiency also influences their risk of developing certain complications, which include arterial thromboses (107) and respiratory tract infections (108, 109) . a role for mbl in the pathogenesis of rheumatoid arthritis has also been suggested. malhotra et al. reported that changes in igg glycosylation secondary to the underlying disease results in mbl-associated complement activation (110) . such complement activation then contributes to chronic inflammation of the synovial membrane. however, graudal et al. found that patients with lower mbl levels experienced earlier, more severe, symptoms and had more rapid joint destruction as visualized radiologically (111) . several recent research publications suggest the directions in which future work on this collectin and its associated molecules may proceed. these include therapeutic interventions, functional assays and the evaluation of the importance of mbl in disease. these are considered briefly below. therapeutic potential of mbl mbl replacement was first attempted (without any knowledge of the deficiency) when fresh frozen plasma was given to patients and found to correct the opsonic defect (28, 29) . since then, affinity-purified, plasma-derived mbl has been safely given to many patients, resulting in normalization of enzyme-linked immunosorbent assay detectable mbl and complement-mediated opsonic activity (112) . a phase 1 study showed the half-life of the protein to range between 18 and 115 h (113) . the development of recombinant mbl is also at the phase 1 trial stage and such developments provide exciting prospects for the future exploration of the therapeutic potential of mbl. exactly who would benefit from replacement therapy is under debate and the importance of targeting well-defined patient groups will be vital to its success. the discovery of other components of the lectin pathway including the ficolins and the masps indicates that this limb of the immune system is complex and extends beyond mbl and masp-2 alone. this knowledge enables us to question the impact of these molecules either in isolation or in combination. functional assessment of the lectin pathway may be a far more accurate and clinically relevant measurement than mbl level and/or genotype alone. a number of different assays have been reported, which assess activity at different stages of the functional pathway; therefore, the results must be interpreted accordingly (114, 115) . the impact of deficiencies of the various adjunctive components is also the subject of much current research. in 2003, stengaard-pedersen et al. reported the first identified case of masp-2 deficiency (116) . functional analysis of the ability of mbl to activate the lectin pathway, estimating c4b deposition on a mannan surface, was performed on a group of patients with suspected immunodeficiency. one patient was found to have deficient pathway activity despite having sufficient mbl. no masp-2 or map19 was found in the plasma, and genetic analysis indicated that the patient was homozygous for a point mutation in exon 3 of the gene (d105g). clinically, the patient suffered from recurrent infections and autoimmune symptoms. subsequently, the frequency of this mutation has been assessed in a small number of populations and values range from 1.3% to 6.3% (117) . as discussed previously, the contribution of masp-1 and masp-3 in the pathway remains unexplained. the role of ficolins is now beginning to be addressed in clinical studies. like mbl, no absolute levels of deficiency have yet been defined. atkinson et al. studied more than 300 children with recurrent respiratory tract infections and measured l-ficolin levels (118) . an association with mbl deficiency in the same patient cohort had already been reported (119) . in this study, low levels of l-ficolin were more common in patients than in controls and most common in patients with co-existing atopic disorders, suggesting a role for l-ficolin in protection from microorganisms complicating allergic disease. polymorphisms in the ficolins have been identified, although their clinical significance is as yet unknown. mbl is an ancient molecule, which has probably been subject to a large number of evolutionary pressures. the last 50,000 years of human evolution have been associated with major changes as hominids moved from an essentially nomadic lifestyle to increasingly crowded living arrangements in large settled communities. associated with these changes, the spectrum of common infectious diseases would also have changed. more recently, the introduction of antibiotics, the emergence of novel infections and increasing use of immunosuppressive therapies have provided new challenges to our innate host defence system. despite all these changing evolutionary pressures, mbl gene polymorphisms persist at high frequencies, suggesting that they offer potential advantages to the host. thus, there exists a balance in which certain individuals benefit from the expression of high levels of the protein, whereas others (living in differing environments, eg. the tropics) may benefit from reduced levels of circulating mbl ( figure 6 ). mbl status may also be either advantageous or disadvantageous when considered from the viewpoint of the severity of a particular illness. thus, it is known that those with higher levels of mbl are better able to modulate inflammation, probably through an effect on cytokine responses. in contrast, those deficient in mbl appear to be at risk of sepsis and sirs. for these reasons, we believe that analyses of the relevance of mbl (120) should be extended beyond its role in infectious disease and include clinical areas such as autoimmunity and inflammatory disorders. inhibitory and inactivating action of normal ferret sera against an influenza virus strain bovine and mouse serum beta inhibitors of influenza a viruses are mannose-binding lectins properties of a new complement-dependent bactericidal factor specific for ra chemotype salmonella in sera of conventional and germ-free mice a group of bactericidal factors conserved by vertebrates for more than 300 million years affinity chromatography of human liver alpha-d-mannosidase isolation and characterization of a mannan-binding protein from rabbit liver isolation of mannosebinding proteins from human and rat liver extra-hepatic transcription of the human mannose-binding lectin gene (mbl2) and the mbl-associated serine protease 1-3 genes collections and ficolins: humoral lectins of the innate immune defense structure of the calcium-dependent lectin domain from a rat mannose-binding protein determined by mad phasing trimeric structure of a c-type mannose-binding protein human mannose-binding protein carbohydrate recognition domain trimerizes through a triple alpha-helical coiled-coil binding of sugar ligands to ca(21)-dependent animal lectins analysis of mannose binding by site-directed mutagenesis and nmr nucleic acid is a novel ligand for innate, immune pattern recognition collectins surfactant proteins a and d and mannose-binding lectin mannose-binding lectin binds to a range of clinically relevant microorganisms and promotes complement deposition activation of complement by mannose-binding lectin on isogenic mutants of neisseria meningitidis serogroup b the lipopolysaccharide structures of salmonella enterica serovar typhimurium and neisseria gonorrhoeae determine the attachment of human mannose-binding lectin to intact organisms serum lectin with known structure activates complement through the classical pathway activation of the classical complement pathway by mannose-binding protein in association with a novel c1s-like serine protease a second serine protease associated with mannan-binding lectin that activates complement a truncated form of mannose-binding lectin-associated serine protease (masp)-2 expressed by alternative polyadenylation is a component of the lectin complement pathway two constituents of the initiation complex of the mannan-binding lectin activation pathway of complement are encoded by a single structural gene masp-3 and its association with distinct complexes of the mannan-binding lectin complement activation pathway crystal structure of the cub1-egf-cub2 region of mannose-binding protein associated serine protease-2 hakata antigen, a new member of the ficolin/opsonin p35 family, is a novel human lectin secreted into bronchus/alveolus and bile human m-ficolin is a secretory protein that activates the lectin complement pathway l-ficolin specifically binds to lipoteichoic acid, a cell wall constituent of gram-positive bacteria, and activates the lectin pathway of complement a familial plasma-associated defect of phagocytosis defective opsonization. a common immunity deficiency yeast opsonisation in children with chronic diarrhoeal states a study of c3b deposition on yeast surfaces by sera of known opsonic potential association of low levels of mannan-binding protein with a common defect of opsonisation exon structure reveals its evolutionary relationship to a human pulmonary surfactant gene and localization to chromosome 10 structure and evolutionary origin of the gene encoding a human serum mannose-binding protein the human mannose-binding protein functions as an opsonin human leukocyte c1q receptor binds other soluble proteins with collagen domains mannose binding protein (mbp) enhances mononuclear phagocyte function via a receptor that contains the 126,000 m(r) component of the c1q receptor complement receptor 1/cd35 is a receptor for mannan-binding lectin complement receptor type 1 (cr1, cd35) is a receptor for c1q activation of human monocytes by streptococcal rhamnose glucose polymers is mediated by cd14 antigen, and mannan binding protein inhibits tnf-alpha release induction of tnf-alpha in human peripheral blood mononuclear cells by the mannoprotein of cryptococcus neoformans involves human mannose binding protein mannose-binding lectin regulates the inflammatory response of human professional phagocytes to neisseria meningitidis serogroup b c1q and mannose binding lectin engagement of cell surface calreticulin and cd91 initiates macropinocytosis and uptake of apoptotic cells mannose-binding lectin-deficient mice display defective apoptotic cell clearance but no autoimmune phenotype gastrointestinal ischemia-reperfusion injury is lectin complement pathway dependent without involving c1q mannose-binding lectin is a regulator of inflammation that accompanies myocardial ischemia and reperfusion injury tumor-associated carbohydrate antigens defining tumor malignancy: basis for development of anti-cancer vaccines antitumor activity of mannan-binding protein in vivo as revealed by a virus expression system: mannan-binding proteindependent cell-mediated cytotoxicity antitumor activity of mannan-binding protein molecular basis of opsonic defect in immunodeficient children high frequencies in african and non-african populations of independent mutations in the mannose binding protein gene a new frequent allele is the missing link in the structural polymorphism of the human mannan-binding protein restricted polymorphism of the mannose-binding lectin gene of indigenous australians molecular determinants of oligomer formation and complement fixation in mannose-binding proteins interplay between promoter and structural gene variants control basal serum level of mannan-binding protein different molecular events result in low protein levels of mannan-binding lectin in populations from southeast africa and south america a new strategy for mannosebinding lectin gene haplotyping a human mannose-binding protein is an acute-phase reactant that shares sequence homology with other vertebrate lectins the concentration of the c-type lectin, mannan-binding protein, in human plasma increases during an acute phase response characterization of two mannose-binding protein cdnas from rhesus monkey (macaca mulatta): structure and evolutionary implications characterization of murine mannose-binding protein genes mbl1 and mbl2 reveals features common to other collectin genes the human ortholog of rhesus mannose-binding protein-a gene is an expressed pseudogene that localizes to chromosome 10 the ôinvolutionõ of mannose-binding lectin protection afforded by sickle-cell trait against subtertian malareal infection mannan-binding lectin enhances susceptibility to visceral leishmaniasis dual role of mannan-binding protein in infections: another case of heterosis? mannose binding protein gene mutations associated with unusual and severe infections in adults a population-based study of morbidity and mortality in mannose-binding lectin deficiency mannan binding lectin in febrile adults: no correlation with microbial infection and complement activation mannan binding lectin deficiency and concomitant immunodefects the molecular basis of a common defect of opsonization role for mannose binding lectin in the prevention of mycoplasma infection characterization of a novel coronavirus associated with severe acute respiratory syndrome the genome sequence of the sars-associated coronavirus mannose-binding lectin in severe acute respiratory syndrome coronavirus infection association between mannose-binding lectin gene polymorphisms and susceptibility to severe acute respiratory syndrome coronavirus infection mannose binding lectin gene mutations are associated with progression of liver disease in chronic hepatitis b infection mannose-binding lectin in chronic hepatitis b virus infection mannose binding lectin genotypes influence recovery from hepatitis b virus infection hepatitis c virus infection and mutations of mannose-binding lectin gene mbl deficiency of mannose-binding lectin and burden of infection in children with malignancy: a prospective study association between deficiency of mannose-binding lectin and severe infections after chemotherapy low levels of mannose-binding lectin do not affect occurrence of severe infections or duration of fever in acute myeloid leukaemia during remission induction therapy mannose-binding lectin-deficient mice are susceptible to infection with staphylococcus aureus a human serum mannose-binding protein inhibits in vitro infection by the human immunodeficiency virus interaction of mannose-binding lectin with hiv type 1 is sufficient for virus opsonization but not neutralization susceptibility to hiv infection and progression of aids in relation to variant alleles of mannose-binding lectin mannan-binding lectin in the sub-saharan hiv and tuberculosis epidemics the level of the serum opsonin, mannan-binding protein in hiv-1 antibody-positive patients mannan-binding lectin serum concentrations in hiv-infected patients are influenced by the stage of disease polymorphisms in the mbl2 promoter correlated with risk of hiv-1 vertical transmission and aids progression absence of association between mannose-binding lectin gene polymorphisms and hiv-1 infection in a colombian population mannose-binding protein in hiv-seropositive patients does not contribute to disease progression or bacterial infections circulating levels of mannose binding protein in human immunodeficiency virus infection presence of the variant mannose-binding lectin alleles associated with slower progression to aids human mannose-binding protein gene is regulated by interleukins, dexamethasone and heat shock modulatory effect of mannose-binding lectin on cytokine responses: possible roles in hiv infection low mannose-binding lectin serum concentrations in hiv long-term nonprogressors? polymorphism at codon 54 of mannose-binding protein gene influences aids progression but not hiv infection in exposed children association of mannose-binding lectin gene heterogeneity with severity of lung disease and survival in cystic fibrosis impaired pulmonary status in cystic fibrosis adults with two mutated mbl-2 alleles association of mannose-binding lectin polymorphisms with sepsis and fatal outcome, in patients with systemic inflammatory response syndrome increased incidence and severity of the systemic inflammatory response syndrome in patients deficient in mannose-binding lectin mannan binding lectin as an adjunct to risk assessment for myocardial infarction in individuals with enhanced risk the mannose-binding lectin gene polymorphisms and systemic lupus erythematosus: two case-control studies and a meta-analysis a dysfunctional allele of the mannose binding protein gene associates with systemic lupus erythematosus in a spanish population mannose-binding lectin variant alleles and the risk of arterial thrombosis in systemic lupus erythematosus association of mannose-binding lectin gene variation with disease severity and infections in a population-based cohort of systemic lupus erythematosus patients association of mannose binding lectin (mbl) gene polymorphism and serum mbl concentration with characteristics and progression of systemic lupus erythematosus glycosylation changes of igg associated with rheumatoid arthritis can activate complement via the mannose-binding protein mannan binding lectin in rheumatoid arthritis. a longitudinal study reconstitution of opsonizing activity by infusion of mannan-binding lectin (mbl) to mbl-deficient humans human plasma-derived mannose-binding lectin: a phase i safety and pharmacokinetic study an assay for the mannan-binding lectin pathway of complement activation functional analysis of the classical, alternative, and mbl pathways of the complement system: standardization and validation of a simple elisa inherited deficiency of mannan-binding lectin-associated serine protease 2 deficiency of the mannan-binding lectin pathway of complement and poor outcome in cystic fibrosis: bacterial colonization may be decisive for a relationship l-ficolin in children with recurrent respiratory infections mannan-binding lectin insufficiency in children with recurrent infections of the respiratory system human mannose-binding lectin in immunity: friend, foe, or both? isolation and characterization of a mannan-binding protein from human serum a common congenital immunodeficiency predisposing to infection and atopy in infancy mannan-binding protein and conglutinin in bovine serum suboptimal c3b/c3bi deposition and defective yeast opsonization. i. evidence for the absence of essential co-factor activity isolation and characterization of two distinct mannan-binding proteins from rat serum two distinct classes of carbohydrate-recognition domains in animal lectins a serum lectin (mannan-binding protein) has complement-dependent bactericidal activity the level of mannan-binding protein regulates the binding of complement-derived opsonins to mannan and zymosan at low serum concentrations molecular characterization of the mouse mannose-binding proteins. the mannose-binding protein a but not c is an acute phase reactant structure of a c-type mannose-binding protein complexed with an oligosaccharide human mannose-binding protein is identical to a component of ra-reactive factor association of mutations in mannose binding protein gene with childhood infection in consecutive hospital series cutting edge: complementactivating complex of ficolin and mannose-binding lectin-associated serine protease mannose-binding lectin accelerates complement activation and increases serum killing of neisseria meningitidis serogroup c activation of the lectin complement pathway by h-ficolin (hakata antigen) differential recognition of obligate anaerobic bacteria by human mannose-binding lectin differential binding of mannose-binding lectin to respiratory pathogens in cystic fibrosis human mannose-binding protein inhibits infection of hela cells by chlamydia trachomatis binding of mannan-binding protein to various bacterial pathogens of meningitis interaction of human mannose-binding protein with mycobacterium avium interaction of mannose-binding lectin with primary isolates of human immunodeficiency virus type 1 high mannose glycans and sialic acid on gp120 regulate binding of mannose-binding lectin (mbl) to hiv type 1 mannose binding lectin (mbl) and hiv mannan-binding protein and bovine conglutinin mediate enhancement of herpes simplex virus type 2 infection in mice mannan-binding lectin modulates the response to hsv-2 infection mannose binding protein is involved in first-line host defence: evidence from transgenic mice binding of host collectins to the pathogenic yeast cryptococcus neoformans: human surfactant protein d acts as an agglutinin for acapsular yeast cells mannose-binding lectin is a component of innate mucosal defense against cryptosporidium parvum in aids recognition of plasmodium falciparum proteins by mannan-binding lectin, a component of the human innate immune system the major surface glycoprotein of trypanosoma cruzi amastigotes are ligands of the human serum mannose-binding protein novel masp2 variants detected among north african and sub-saharan individuals analysis of mannose-binding lectin 2 (mbl2) genotype and the serum protein levels in the korean population association of mannose-binding lectin gene haplotype lxpa and lypb with interferon-resistant hepatitis c virus infection in japanese patients 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-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-023429-x52gbklw authors: ruseva, m.; gajdeva, m.; takahashi, k.; ezekowitz, a.; thiel, s.; jensenius, j. c. title: mannan‐binding lectin inhibits humoural responses date: 2008-06-28 journal: scand j immunol doi: 10.1111/j.0300-9475.2004.01423an.x sha: doc_id: 23429 cord_uid: x52gbklw chronic hepatitis b virus (hbv) infection affects about 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 ca‐dependent 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 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-023390-5hcgdlmt authors: bhuvanath, s.; nilkaeo, a. title: inflammatory cytokine modulation of cancer cell proliferation date: 2008-06-28 journal: scand j immunol doi: 10.1111/j.0300-9475.2004.01423bi.x sha: doc_id: 23390 cord_uid: 5hcgdlmt 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. 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-322933-5xnxjqm5 authors: murugaiah, valarmathy; tsolaki, anthony g.; kishore, uday title: collectins: innate immune pattern recognition molecules date: 2020-03-10 journal: lectin in host defense against microbial infections doi: 10.1007/978-981-15-1580-4_4 sha: doc_id: 322933 cord_uid: 5xnxjqm5 collectins are collagen-containing c-type (calcium-dependent) lectins which are important pathogen pattern recognising innate immune molecules. their primary structure is characterised by an n-terminal, triple-helical collagenous region made up of gly-x-y repeats, an a-helical coiled-coil trimerising neck region, and a c-terminal c-type lectin or carbohydrate recognition domain (crd). further oligomerisation of this primary structure can give rise to more complex and multimeric structures that can be seen under electron microscope. collectins can be found in serum as well as in a range of tissues at the mucosal surfaces. mannanbinding lectin can activate the complement system while other members of the collectin family are extremely versatile in recognising a diverse range of pathogens via their crds and bring about effector functions designed at the clearance of invading pathogens. these mechanisms include opsonisation, enhancement of phagocytosis, triggering superoxidative burst and nitric oxide production. collectins can also potentiate the adaptive immune response via antigen presenting cells such as macrophages and dendritic cells through modulation of cytokines and chemokines, thus they can act as a link between innate and adaptive immunity. this chapter describes the structure-function relationships of collectins, their diverse functions, and their interaction with viruses, bacteria, fungi and parasites. used by malhotra et al. (1992) . they are known to mediate pathogen recognition through calcium-dependent carbohydrate recognition domains (crds). the following nine collectins have been identified to date: mannan-binding lectin (mbl), three bovine serum collectins, conglutinin, cl-43 and cl-46, lung surfactant proteins sp-a and sp-d, and more recently discovered collectins including, collectin kidney 1 (cl-k1, also called cl-11), collectin liver 1 (cl-l1, also called cl-10) and collectin placenta 1 (cl-p1 also called cl-12). the overall functions of collectins include microbial aggregation and neutralisation, opsonisation, complement activation, and modulation of inflammatory responses. collectins are oligomers of trimeric subunits, for most collectins, the subunits are homotrimers (made up of three identical polypeptides) but heterotrimers can be found for sp-a, which is made up of highly homologous spa-1 and spa-2 polypeptides. hetrotrimers can also form in the case of cl-10 and cl-11 ( fig. 4.1 ). the subunit of each collectin is composed of (i) a short n-terminal (7-28 amino acid residues) cysteine-rich domain, involved in multimerisation (by disulphide bridging); (ii) a collagen-like domain composed of gly-x-y triplets repeats, where x and y represent any amino acids; (iii) a short segment which can form coiled-coil helices, and (iv) , and mbl (c). representations of the trimeric "head" of collectins. these structures represent the 'neck', and the crds of three polypeptides which make up the trimeric subunit. the helix interacts with a neighbouring carbohydrate recognition domains (kishore et al. 2006; skjoedt et al 2012) the c-terminal globular c-type lectin domain, also called the crd (carbohydrate recognition domain) (uemura et al. 2006) (fig. 4.1) . the triple-helical collagen region provides significant rigidity and stability to the molecule (colley and baenziger 1987) . another structural feature of the collagenlike domain of collectins is that it can be o-glycosylated (colley and baenziger 1987) . both mbl and sp-a show an interruption of the gly-x-y triplet repeats, which introduces a bend in the otherwise straight triple helix. this enables the fully assembled multi-subunit structure to angle away from the central core, producing a structure resembling a bouquet of flowers (fig. 4 .2) (voss et al. 1991) . several distinct functions of the collagen domain of collectins have been reported. the collagen domains of sp-a and mbl are involved in receptor-mediated properties. a gekgep specific motif found within the collagen domain of mbl is suggested to bind c1q receptor (arora et al. 2001) , and mediates the enhancement of phagocytosis through c1qr (arora et al. 2001) . a similar motif is within the collagen domain of sp-a (white et al. 1985) , which is also involved in the interaction with c1q receptor (malhotra et al. 1992; malhotra et al. 1990) , and mediates phagocytosis of staphylococcus aureus by monocytes (geertsma et al. 1994) . furthermore, the collagen domain of mbl is shown to bind mbl-associated serum proteases, masp1, 2 and 3, which mediate complement activation via the lectin pathway (thiel et al. 1997; tan et al. 1996) . additionally, the positively charged collagen region found in the membrane bound cl-p1 is involved in the uptake of oxidised ldl particles (ohtani et al. 2001) . the cysteine residues found within the n-terminal domain (7-28 amino acids) form disulphide bonds between monomers, thereby, stabilising trimeric subunits as well as a larger multimers. it was believed that at least two cysteine residues are required at the n-terminal domain for the formation of multimers of trimeric subunits (brown-augsburger et al. 1996; mccormack et al. 1999; mccormack et al. 1997a, b) . however, in the case of cl-43, it is secreted as a single trimeric subunit, despite having two cysteine residues (rothmann et al. 1997; lim et al. 1994a, b) . therefore, other factors contribute to oligomerisation of trimeric subunits, in addition to the of n-terminal cysteine residues. the c-terminal region contains a coiled-coil trimerizing neck region (residues 112-130 in human mbl) ( fig. 4.1) , and the crd (residues 134-245 in human mbl) which folds up into an independent globular carbohydrate-binding structure for each polypeptide chain. each subunit is held together covalently through disulphide bonds, or non-covalently structured into oligomers of up to six subunits. c-type crds are connected to the collagen-like domain through the 'neck' region (24-28 amino acid residues) . furthermore, the neck region is involved in aligning the collagen chains. a broad carbohydrate specificity is required by collectins in order to recognise and bind a large repertoire of (pathogen-associated molecular patterns) pamps. such broad specificity is achieved by an open and flexible trough -like binding pocket found within the crds. the selection of ligands by this site depends on the positioning of vicinal hydroxyl groups of sugars, which form coordination bonds with a ligated calcium ion, hydrogen bonds and a polar van der waals contact (ng et al. 1996) . ligand specificity of collectins is divided into two main sub-classes (mannosebinding or galactose-binding type), which is based on a three amino acid residue motif found in the ca ++ ion binding site. the sequence 185-glu-pro-asn is associated with binding of mannose-like sugars, while the sequence 185-gln-pro-asp is associated with binding galactose-like sugars. the molecular differences based on which crds discriminate between mannose and galactose-type ligands depend on the orientation of c3 and c4 vicinal hydroxyl groups presented on monosaccharides. mannose-specific crds bind ligands in which hydroxyl groups at the c3 and c4 positions are in an equatorial orientation (mannose, glucose, glucosamine), while in galactose these vicinal hydroxyls are in an axial orientation (drickamer and taylor 2015) . inhibition studies using monosaccharides have shown that most likely, all the above described collectins, except cl-p1, prefer mannose ligands over galactose (ohtani et al. 2001; holmskov et al. 1994) . however, a wider range of binding specificity has been reported for mbl and lung surfactant proteins sp-a and sp-d, as these collectins are also capable of binding to nucleic acids (nadesalingam et al. 2003) , phospholipids (sano et al. 1999) , as well as non-glucosylated proteins. fucose, a hexose deoxy sugar is bound by mannose-specific crds in a different manner as it has equatorial hydroxyl groups placed on its c2 and c3 position of the sugar ring, not the c3 and c4 (weis et al. 1991a, b; ng et al. 1996; iobst and drickamer 1994) . computational docking studies have demonstrated that αd-glucose docks into the crd of sp-d via vicinal equatorial hydroxyl groups on the 2-and 3-position of its sugar ring (allen et al. 2001a, b) . although mbl affinity is reported to be very low for monosaccharide galactose, mbl crystallographic studies demonstrate that galactose is ligated in the mbl binding region via coordination bonds with hydroxyl groups placed at c1 and c2 position of the sugar ring (ng et al. 1996) . in addition to galactose and mannose, binding of collectins to a range of sugars has also been studied ; they exhibit preferences for certain sugar residues over others. for instance, despite sp-d being structurally similar to conglutinin, it displays a greater affinity for maltose, a glucose disaccharide, which is a weak ligand for conglutinin. sp-d is suggested to have a lower affinity for glcnac, which is the best ligand for conglutinin. moreover, binding of cl-43 to sugars is closely related to mbl, although the structure of cl-43 is closer to sp-d and conglutinin (lu et al. 2002) . the sugar-binding specificity of cl-11/cl-k1 has been investigated (venkatraman girija et al. 2015) . it has a larger recognition interface than mbl, and recognises predominantly mannose-rich structures, interacting with two sugar residues at a glycan terminal, rather than a single sugar. human mbl is synthesised by hepatocytes and secreted into the blood stream (sastry et al. 1991; ezekowitz et al. 1988; hansen et al. 2000) . initially, mbl was isolated from the liver of the rabbit, rat and chicken, where expression levels were detected in the soluble cytosol, rather than on the cell surface. two forms of mbl (mbl-a and mbl-c) were detected in rodents (hansen et al. 2000; drickamer et al. 1986 ), rabbits (kawasaki et al. 1978 kozutsumi et al. 1980 ) and rhesus monkeys (mogues et al. 1996) . however, only one form of mbl is present in humans and chimpanzees (mogues et al. 1996) . although the liver is the main production site of mbl-a and mbl-c in mice, mrna expression of mbl was also detected in various tissues (table 4 .1) (shushimita et al. 2015) . substantial expression levels of mbl-a and mbl-c were reported in kidney and intestine (table 4 .1). detection of mbl proteins in the small intestine suggests that mbl may have similar roles to secretory iga (reichhardt et al. 2012 ). the collectins sp-a and sp-d are primarily detected in the alveolar space of the lungs, and synthesised by alveolar type-ii cells (table 4 .1) (voorhout et al. 1992 , nayak et al. 2012 , and nonciliated bronchial epithelial cells, also known as clara cells (voorhout et al. 1992; crouch et al. 1992) . although the lung is the main site of sp-a and sp-d synthesis, presence of sp-d has also been reported at extrapulmonary sites. sp-d expression has been shown immunohistochemically in human trachea, brain, heart, kidneys, testis, salivary gland, placenta, prostate, small intestine, and pancreas (table 4 .1). a low expression level has been detected in spleen, uterus, adrenal gland and mammary glands (fisher and mason 1995; madsen et al. 2000; herías et al. 2007) . furthermore, immunoreactivity of sp-d has also been shown in the epithelial cells of both small and large ducts of the parotid gland, lacrimal and sweat glands, epithelial cells of intra-hepatic bile ducts and gall bladder, as well as esophagus, exocrine pancreatic ducts, and in the urinary tract (madsen et al. 2000; bräuer et al. 2007 ). in the case of sp-a, low levels are detected in small intestines from human and rat (table 4 .1) (lin et al. 2001 , van iwaarden et al. 1990 ). in addition to its presence in the murine uterus, very low sp-a expression is found in human prostate, amniotic fluid, thymus and salivary gland . sp-a and sp-d have also been localised in the fetal membranes, and choriodecidual layer of the late pregnancy uterus (miyamura et al. 1994) . as a result of pulmonary microbial infection, the protein levels of both sp-a and sp-d have been reported to increase in the alveolar compartment (atochina et al. 2001) . thus, the level of sp-d increases in response to allergen-induced eosinophilia (kasper et al. 2002) , suggesting that both sp-a and sp-d may function as acute phase reactants within the lungs. furthermore, hypoxia results in an increased concentration of both sp-a and sp-d in the alveolar compartment (white et al. 2001) . conglutinin, cl-46 and cl-43 are serum collectins identified in bovidae and synthesised in the liver . these collectins provide a first line of defense against microbial pathogens. cl-l1 mrna was detected in the liver, and studies using northern blot analysis have suggested that low levels occur in the placenta. although most collectins are secreted, cl-l1 was found in the cytosol of hepatocytes, which may suggest its interaction with intracellular ligands . the presence of cl-p1 was reported in vascular endothelial cells (table 4 .1); cl-p1 is suggested to be membrane bound, and it contains an intracellular domain (ohtani et al. 2001) . expression of mbl, sp-a and sp-d at the mucosal surfaces suggest the innate immune roles of these collectins against invading pathogens. during helicobacter pylori infection, an increased level of sp-d has been detected, suggesting the possible role of sp-d in the mucosal defense outside the lungs (murray et al. 2002) , eg. gastrointestinal tract. collectins are important soluble pattern-recognition receptors (prrs) of the humoral arm of the innate immune response. collectins are able to recognise and bind to a wide variety of microbes and are involved in their clearance and forming a central link to adaptive immunity against microbial infections. in this section, we will discuss the well-known collectins: mbl, sp-a and sp-d, as well as newly discovered collectins: liver collectin (cl-l1), kidney collectin (cl-k1), and placenta collectin (cl-p1). we will also briefly discuss bovine collectins, conglutinin, cl-43 and cl-46. microbes can be cleared by collectins via a number of mechanisms such as aggregation, opsonisation, phagocytosis, microbial growth inhibition, complement activation, as well as modulation of adaptive immunity. pulmonary surfactant is composed of 90% phospholipids and 10% proteins (made up of surfactant proteins, sp-a, sp-b, sp-c and sp-d. whilst, sp-b and sp-c are hydrophobic and essential for the physiology of the alveolar surfaces, sp-a and sp-d are hydrophilic and contribute to lung immunity. an early study showed that pulmonary surfactant enhanced the killing of staphylococcus aureus by alveolar macrophages (am), in vitro (laforce et al. 1973) . both gram-negative and grampositive bacteria are recognised by sp-a and sp-d, enhancing their phagocytosis by ams (fig. 4 .3) (pikaar et al. 1995) . for gram-negative bacteria, sp-a and sp-d both bind to lipopolysaccharide (lps) but differ in preferential targets on the molecule. sp-a binds to the lipid a moiety of rough lps (which lacks the o-antigen and shortened oligosaccharides) (van iwaarden et al. 1994) , and enhances phagocytosis of bacteria by am (kalina et al. 1995) , but not to smooth lps (which contains the o-antigen) (van iwaarden et al. 1994) . in contrast, sp-d binds strongly to smooth lps from escherichia coli and salmonella species but does not recognise the lipid a moiety or oligosaccharide deficient lps (kuan et al. 1992) . this indicates that sp-d preferentially targets the core terminal saccharides in the bacterial ligand, whilst sp-a prefers lipid a. sp-d has also able been shown to bind to rough lps via its trimeric carbohydrate recognition domain (crd), (targeting shortened oligosaccharides) and agglutinating e. coli (kuan et al. 1992) , and rough lps from klebsiella (lim et al. 1994a, b; kishore et al. 1996) . in addition to lps, sp-a is able to bind to capsular polysaccharides of klebsiella species, enhancing their phagocytosis by am (kabha et al. 1997) . however, bacterial peptidoglycan is not a ligand for sp-a (murakami et al. 2002) . sp-a and sp-d directly inhibit the growth of several gram-negative bacteria by increasing the membrane permeability of the bacterial cell wall (fig. 4 .3) . sp-a and sp-d also inhibit biosynthetic functions in strains of e. coli, k. pneumoniae and enterobacter aerogenes . similarly, sp-a inhibits the growth of p. aeruginosa by increasing membrane permeability (van iwaarden et al. 1994 ), but the bacterium can resist through quorum-sensing and the secretion of a flagellum-mediated exoprotease that degrades sp-a (kuang et al. 2011a). furthermore, sp-a downregulates tnf-α secretion via toll-like receptor 2/nf-κb mediated pathway, indicating its role in modulating inflammatory responses against bacterial ligands (murakami et al. 2002) . sp-a can bind to the outer membrane protein (omp) of haemophilus influenzae type a and to a lesser extent, type b (mcneely and coonrod, 1994) . sp-a can also aggregate and opsonise h. influenzae type a, facilitating killing by am (mcneely and coonrod 1994) . similarly, sp-a binds to the capsular polysaccharide of some strains of k. pneumoniae, agglutinating the bacteria and increase phagocytosis by macrophages (kabha et al. 1997) , and treatment with sp-a plus sp-b n (n-terminal saponin domain of sp-b) significantly reduced bacterial infection and enhanced neutrophil recruitment (coya et al. 2015) . sp-a has a bacteriostatic effect on mycoplasma pneumoniae via binding to di-saturated phosphatidylglycerols on the bacterial membrane (piboonpocanun et al. 2005) . sp-a can interact with mycobacterium tuberculosis putative adhesin apa glycoprotein on its surface (ragas et al. 2007 ). sp-d can also bind to gram-positive bacterial ligands such as lipoteichoic acid and peptidoglycan via its crd (van de wetering et al. 2001) and to lipoarabinomannan (lam) from m. tuberculosis and mycobacterium avium (ferguson et al. 1999; kudo et al. 2004) . sp-d is also able to interact with cell membrane lipids of m. pneumoniae (chiba et al. 2002) . it is intriguing that although both sp-a and sp-d bind and agglutinate m. tuberculosis, they have opposing effects on phagocytosis by macrophages. sp-a enhances phagocytosis via increased expression of mannose receptor on the host cell surface (beharka et al. 2002) , whilst sp-d inhibits phagocytosis by blocking the interaction of lam with macrophage mannose receptor, and not as a result of bacterial agglutination by sp-d (ferguson et al. 1999 . in a mouse model of tuberculosis infection, sp-a −/− , sp-d −/− , and sp-a/d −/− knockout mice still had the ability to phagocytose and clear m. tuberculosis when given a low-dose aerosol challenge of the pathogen, suggesting that both sp-a and sp-d could be redundant in this animal model (lemos et al. 2011) . similarly, both sp-a and sp-d can also bind to legionella pneumophila, but seem to inhibit intracellular bacterial growth in the macrophage (sawada et al. 2010) . sp-a and sp-d can also directly facilitate phagocytosis without the need for microbial binding, by up-regulating the expression of cell surface phagocytic receptors in macrophages, such as mannose receptor (beharka et al. 2002; kudo et al. 2004) . in sp-a −/− knockout mice, expression of mannose receptor is down-regulated, showing that sp-a is important in regulating the expression of this receptor (beharka et al. 2002) . similarly, sp-a is able to enhance phagocytosis of streptococcus pneumoniae by am, independent of its binding to the bacterium, via the increased expression of scavenger receptor a (sr-a) . interestingly, the vast majority of clinical strains of the opportunist pseudomonas aeruginosa secrete an elastase that degrades sp-a and facilitates evasion of opsonisation by the collectin during phagocytosis (kuang et al. 2011b) . sp-a and sp-d can play important roles in modulating the intracellular environment after phagocytosis by stimulating reactive oxygen and nitrogen intermediates facilitating the killing of intracellular pathogens. this is of particular note in mycobacteria, which are specialist intracellular bacteria. sp-a enhances the killing of intracellular mycobacterium bovis bcg by increasing nitric oxide (no) production, in addition to enhancing the release of inflammatory mediators such as tnf-α (weikert et al. 2000) . in contrast, in ifn-γ primed am, sp-a decreases no production in response to intracellular infection with m. tuberculosis and m. avium by inhibiting tnf-α secretion and nuclear factor-kappa b (nf-κb) activation (pasula et al. 1999; hussain et al. 2003) . sp-a can also enhance the intracellular killing of mycoplasma pulmonis via a no dependent mechanism (hickmandavis et al. 1998) . bacteria-derived cell-wall molecules such as lps and peptidoglycan are potent stimulators of inflammation and can also interact with pattern-recognition receptors (prrs) such as cd14 or toll-like receptors (tlr), via pathogen-associated molecular patterns (pamps), and activate downstream intracellular signalling. sp-a and sp-d can also directly bind to prrs (e.g. tlr and cd14) and thus can modulate the inflammatory response. sp-a and sp-d can alter lps interactions with cd14 via different mechanisms (sp-a via neck domain; sp-d via crd) (sano et al. 2000) . furthermore, via direct interaction with cd14, sp-a inhibits production of tnfα induced by smooth lps, but not rough lps in u937 macrophages (sano et al. 1999 ). in sp-a −/− knockout mice, tnf-α induced by smooth lps, significantly increased, compared to wild-type mice (borron et al. 2000) , whilst sp-a has also been shown to inhibit tnf-α induction by peptidoglycan via direct binding to tlr-2 (murakami et al. 2002) . thus, sp-a significantly decreases peptidoglycan or smooth lps-induced pro-inflammatory responses (via nf-κb activation). sp-a has no effect or increases the inflammatory response induced by rough lps. in tuberculosis, sp-a has pleiotropic effects being able to promote inflammation in the presence of infection and suppresses inflammation in uninfected macrophages, probably protecting uninfected lung areas from the deleterious effects of inflammation (gold et al. 2004) . in humans, sp-a exists in two isoforms, sp-a1 and sp-a2, which are encoded by distinct genes. fully assembled sp-a protein contains both gene products. a number of studies have described polymorphisms in these genes and the sp-d gene which may have a role in susceptibility to microbial infection, particularly tuberculosis. polymorphisms within and flanking the sp-a1 and sp-a2 genes have been described which indicate protection or susceptibility toward pulmonary tb in the populations studied in mexico, ethiopia, india and china (floros et al. 2000; madan et al. 2002; malik et al. 2006; vaid et al. 2006; yang et al. 2014) . two sp-a1 alleles (sftpa1 307a, sftpa1 776t) and two sp-a2 alleles (sftpa2 355c and sftpa2 751c) were associated with tuberculosis susceptibility in ethiopia (malik et al. 2006 ). the sftpa2 751a/c polymorphism and the haplotype 1a 3 in sp-a2, which both affect the amino acids in crd region of sp-a, may alter binding to m. tuberculosis and thus were found to be strongly linked with tuberculosis susceptibility (malik et al. 2006) . another study also found two polymorphisms (sp-a2 g1649c and sp-a2 a1660g) in the introns of sp-a1 that were associated with tuberculosis in an indian population, but none in the sp-a1 gene (madan et al. 2002) . in a chinese population, the polymorphism 1649g in the sp-a2 gene was strongly associated with tuberculosis, mirroring the findings in the ethiopian and indian populations (yang et al. 2014 ). the sp-a2 1649g leads to a transversion (proline to alanine), affecting the triple helical structure of sp-a (yang et al. 2014) . in sp-d, the polymorphism, g459a, is significantly associated with tuberculosis susceptibility in an indian population, but the molecular basis for susceptibility is not understood (vaid et al. 2006) . these observations illustrate the complexities of host-pathogen interactions in bacterial infection mediated by these collectins. mbl is the recognition subcomponent of the lectin pathway of the complement system and is present mostly in the serum. the structure of mbl is similar to that of sp-a, and in the presence of ca 2+ , it has been observed to target terminal sugars (e.g. d-mannose, l-fucose, and n-acetyl-d-glucosamine), on the surface of a number of gram-positive and gram-negative bacterial species (ip et al. 2009; lugo-villarino et al. 2011) . the binding of mbl to microbial surfaces can activate complement through mbl-associated serine proteases (masps), resulting in enhanced microbial clearance via opsonisation (c3 and c4 deposition) and complement-mediated lysis. however, mbl also has complement-independent activity such as inhibition of bacterial adhesion (jack et al. 2005 ) and opsonisation to enhance bacterial uptake (kuhlman et al. 1989; polotsky et al. 1997; jack et al. 2005) . strong in vitro binding of mbl to s. aureus, streptococcus pyogenes, listeria monocytogenes and nonencapsulated neisseria meningitidis has been described (levitz et al. 1993; van emmerik et al. 1994; neth et al. 2000) . moderate levels of mbl binding were observed in e. coli, haemophilus influenzae and klebsiella species, whilst no binding has been observed for p. aeruginosa, enterococcus species and streptococcus pneumoniae (levitz et al. 1993; van emmerik et al. 1994; neth et al. 2000) . bacterial pathogens have involved strategies to interfere with mbl binding and functions for survival, via the synthesis of a polysaccharide capsule and sialylation of lps ligands on the bacterial surface which reduces the binding of mbl (jack et al. 2005; krarup et al. 2005 ). this effectively masks or alters the bacterial ligands for mbl interaction. a number of studies have characterised the bacterial ligands for mbl. mbl is able to bind to peptidoglycan and lipoteichoic acid from s. aureus (polotsky et al. 1996; nadesalingam et al. 2005a, b) , lam from m. avium (polotsky et al. 1997 (jack et al. 1998; devyatyarova-johnson et al. 2000; jack et al. 2001; gulati et al. 2002) . m. meningitidis can also interfere with mbl binding through encapsulation (van emmerik et al. 1994) , whilst m. gonorrhoeae is not able to form capsules. encapsulation seems to be less robust at decreasing mbl binding than sialyation of los (jack et al. 1998) . bound mbl can activate complement and the ability of neisseria species to cascade complement all the way to c9 (membrane attack complex (mac)) is crucial for protection against infection, since they are otherwise poorly phagocytosed by neutrophils and macrophages when opsonised by c3 (ross and densen 1984) . mbl bound to the surface of neisseria is able to increase bacterial killing via increased complement activation (jack et al. 1998 (jack et al. , 2001 gulati et al. 2002) , and similar observations of bactericidal activity have been reported for e. coli and salmonella species (kawasaki et al. 1989; ihara et al. 1991) . for most other bacteria, complement activation to the c3 deposition stage is enough for protection via increased phagocytosis through opsonisation by complement products on the bacterial cell surface. mbl can increase c3b deposition on s. aureus (neth et al. 2002) , but this does not appear to result in increased complement activation (cunnion et al. 2001) . mbl targets wall teichoic acid in s. aureus and this interaction is particularly important in infants that have not developed adaptive immunity, leading to bacterial clearance via mbl-mediated complement activation (kurokawa et al. 2016 ). in addition to its complement-mediated activities, mbl is also has various intrinsic effects, being able to act as an opsonin independently and other direct effects. mbl enhances uptake and intracellular killing of salmonella by neutrophils and monocytes (kuhlman et al. 1989) , but this may also involve interaction with fibronectin (ghiran et al. 2000) . recently, mbl has also been shown to have a direct inhibitory effect on flagellar activity in pathogenic salmonella bacteria, impairing their motility (xu et al. 2016) . mbl can also increase uptake of mycobacteria by macrophages (polotsky et al. 1997 ) and n. meningitidis by neutrophils, monocytes and macrophages (jack et al. 2001) , but this uptake by neutrophils may not result in intracellular killing (drogari-apiranthitou et al. 1997) . mbl also appears to improve the efficiency of internalisation of bacteria bound to the macrophage plasma membrane (neth et al. 2002) . mbl co-interacts with tlr2 in sensing s. aureus and thus influencing the subsequent inflammatory response (nauta et al. 2003; ip et al. 2008) . mbl deficiency increases susceptibility to microbial infection even though the majority of mbl-deficient individuals are usually healthy (eisen and minchinton 2003) . the concentration of mbl in the plasma varies considerably in humans (0-10, 000 ng/ml) due to polymorphisms in the mbl gene (steffensen et al. 2000) . mbl deficiency is commonly observed in around 25% of caucasians (having low levels (<500 ng/ml)), which renders them susceptible to infection (valdimarsson et al. 2004 ). mbl-deficient mice are susceptible to s. aureus infection , whilst mbl deficiency increases susceptibility to postburn infection with p. aeruginosa (moller-kristensen et al. 2006) . a large cohort study has also found a strong association between mbl deficiency and meningococcal infection, and pneumococcal pneumonia, in patients undergoing chemotherapy (gaynor et al. 1995; kronborg et al. 2002; roy et al. 2002) . in contrast, normal or increased levels of mbl are linked to frequent infection with m. tuberculosis and m. leprae (garred et al. 1994 (garred et al. , 1997b , probably through complement-mediated phagocytosis of the pathogen. up to 30% of healthy individuals have polymorphisms linked to mbl deficiency and these, together with serum levels, have been associated with susceptibility to tuberculosis and other inflammatory diseases in some ethnic populations (takahashi and ezekowitz 2005; thiel et al. 2006; goyal et al. 2016 ). of the three more recently discovered collectins (cl-l1, cl-k1, cl-p1), cl-l1 and cl-p1 have been shown to have bacterial interactions. cl-k binds to e. coli, k. pneumoniae, p. aeruginosa and m. tuberculosis (keshi et al. 2006; hansen et al. 2010; troegeler et al. 2015) , whilst cl-p1 can bind to e. coli and s. aureus jang et al. 2009 ). both cl-l1 and cl-k1 can activate the complement lectin pathway as can the heteromeric form cl-lk, which interacts with the masps (henriksen et al. 2013) . cl-p1 can activate the alternative and classical pathways via its interaction with c-reactive protein (crp) (roy et al. 2016 ). there is limited data on the activity of cl-lk in vivo and in vitro, but due to average serum concentrations being below that of mbl (0.3 μg/ml vs. 1.5 μg/ml), pathogen recognition and clearance through complement activation is likely to have a minor role to play for these collectins. it is not clear whether these collectins can act directly as opsonins in a complement-independent manner. cl-l1 can bind d-mannose, nacetylglucosamine, d-galactose, l-fucose and d-fructose in a ca 2+ dependent manner axelgaard et al. 2013) . similarly, cl-k1 can also bind l-fucose, d-mannose and n-acetylmannosamine hansen et al. 2010 ). furthermore, cl-lk was recently demonstrated to be a prr for m. tuberculosis, being able to primarily target mannose-capped lipoarabinomannan (manlam), in a ca 2+ dependent manner, on the surface of the mycobacterium, but not to m. smegmatis due to the lack of mannose caps on its lam (troegeler et al. 2015) . mice deficient in cl-k1 did not show altered susceptibility to m. tuberculosis infection and cl-lk opsonised m. tuberculosis did not result in altered phagocytosis or intracellular survival of the pathogen in human macrophages (troegeler et al. 2015) . interestingly, the levels of cl-lk in serum of tuberculosis patients is reduced, compared to controls, correlating inversely to the immune response to m. tuberculosis and suggesting that it may be useful as a biomarker for the disease (troegeler et al. 2015) . conglutinin was the first mammalian collectin to be discovered and is found in bovidae, together with other lesser known collectins (cl-43 and cl-46) . conglutinin is similar in overall structure to sp-d and is able to bind to microbial surfaces in the presence of ca 2+ . conglutinin is secreted by the liver and found predominantly in bovine serum at an average concentration of 12 μg/ml . conglutinin has been shown to have anti-microbial properties. low serum levels of conglutinin have been associated with acute infections (e.g. pneumonia and metritis) and predisposition to respiratory infection (ingram and mitchell 1971; holmskov et al. 1998) . conglutinin is able to bind many microbes, including gram-negative bacteria such as e. coli and salmonella typhimurium (friis-christiansen et al. 1990; friis et al. 1991) , lps and peptidoglycan (wang et al. 1995) and gram-positive bacteria such as mycobacteria (dec et al. 2012; mehmood et al. 2019) . conglutinin is uniquely able to bind to ic3b, via the mannose sugars on the α-chain of ic3b (laursen et al. 1994 ). conglutinin is able to bind and agglutinate ic3b-coated erythrocytes (lachmann and muller-eberhard 1968; laursen et al. 1994) , and as well as e. coli, increasing the respiratory burst of phagocytes (friis et al. 1991 ). conglutinin has also been shown to be protective against bacterial infection in vivo, being able to increase the survival of mice experimentally infected with highly virulent strains of s. typhimurium (friis-christiansen et al. 1990) . a recombinant truncated form of conglutinin, composed of the α-helical neck region and the crd of conglutinin (wang et al. 1995) , was recently shown to bind to able to bind to the vaccine strain mycobacterium bovis bcg (probably via lam), and act as an anti-opsonin both in the presence and absence of complement deposition. thus, conglutinin can interfere with the uptake of the bacterium by thp-1 macrophages and alter their inflammatory response (mehmood et al. 2019 ). this suggests that conglutinin interfers with uptake of mycobacteria by macrophages via two important mechanisms: (1) blocking interaction of mycobacterial lam with mannose receptor, and (2) blocking ic3b interaction with complement receptors cr3 and cr4 (mehmood et al. 2019) . these data potentially have important implications for bovine tuberculosis. cl-43 and cl-46 are also bovine-unique collectins, but their role in the physiology and innate immunity against bacteria has not been fully studied. there is one report of cl-43 binding to e. coli strain k12, enhancing attachment to phagocytes . there are numerous studies that describe direct interaction of sp-a and sp-d with a range of viruses, enhancing their phagocytosis, as well as neutralising viral infection of host cells (fig. 4.3) . experiments on sp-a −/− and sp-d −/− knockout mice infected with influenza a virus (iav) suggest that both collectins are protective, but this is dependent on viral strain-specific factors, such as the nature of glycosylation in ha and na (levine et al. 2001 (levine et al. , 2002 hawgood et al. 2004) . also, mice lacking both sp-a and sp-d, have an iav infection phenotype almost identical to sp-d −/− mice (hawgood et al. 2004 ). moreover, sp-d, but not sp-a, enhanced the clearance of iav infection in the mouse lung (levine et al. 2001) . thus, these studies suggest that sp-d plays a more significant role than sp-a in the host innate immune response to infection with iav. sp-a binds to iav, neutralises the virus and inhibits the release of viral particles from infected cells, by targeting mannose residues of viral surface haemagglutinin (ha) or neuraminidase (na) benne et al. 1995) . sp-d strongly inhibits hemagglutination activity of iav, resulting in viral aggregation and neutralisation (hartshorn et al. 1994) . sp-d is also able to inhibit na activity, with inhibition being stopped in the presence of d-mannose (reading et al. 1997) . sp-d has a stronger inhibitory effect on na compared to sp-a (tecle et al. 2007 ). sp-d binds to mannosylated, n-linked sugars on viral ha and na via its crd, resulting in potent anti-iav infectivity (hartshorn et al. 1994 (hartshorn et al. , 2000 . sp-d was able to inhibit virus-induced ha activity, block the enzymatic activity of viral na, and neutralise the ability of seasonal h1n1 strains of iav to infect human respiratory epithelial cells (job et al. 2010) . however, in the same study, some pandemic h1n1 were found to be resistant to sp-d inhibition that correlated with the degree of n-glycosylation in the globular head of ha (job et al. 2010) . it has been shown that porcine sp-d has an increased ability to inhibit, not just seasonal iav strains, but also a number of pandemic and avian strains (van eijk et al. 2003; hillaire et al. 2012 ). this is important as pigs are a source of iav pandemic strains (h1n1) that can be transmitted to humans, so studying porcine sp-d could provide further insights into this host reservoir. a recombinant truncated form of sp-a (rfhsp-a) made up of α-helical neck and crd, promotes iav infection, replication, upregulation of viral factors (m1) in lung epithelial a549 cells and enhances the pro-inflammatory response (al-qahtani et al. 2019 ). this contrasts with full-length sp-a which inhibits iav infection and dampens the pro-inflammatory response, demonstrating that the full-length sp-a molecule is required for iav protection (al-qahtani et al. 2019). however, in a similar study, a recombinant truncated form of sp-d (rfhsp-d) was shown to inhibit iav entry, down-regulate viral factors (m1) and down-regulate the pro-inflammatory response (al-ahdal et al. 2018) . these opposing effects of rfhsp-a and rfhsp-d provide further insight into iav pathogenesis and the possible utility of rfhsp-d as a therapeutic molecule. in bronchoalveolar lavage (bal), sp-d enhances iav uptake and virus-induced respiratory burst by neutrophils , but other collectins (sp-a), mucins and gp-340 dampen sp-d's effect, and thus, significantly reduce the ability of sp-d to promote neutrophil oxidative response . therefore, the net effect of bal is to increase neutrophil uptake of iav while reducing the respiratory burst response to virus . sp-a is also able to bind to herpes simplex virus type 1 (hsv-1) via viral n-linked sugars and enhance phagocytosis of the virus by macrophages (van iwaarden et al. 1991; van iwaarden et al. 1992a, b) . the mechanism of binding of sp-a to hsv-1 is similar to binding to iav, involving interaction with the sialylated carbohydrate on the collectin's crd. sp-a also has an opsonin activity, increasing uptake of hsv-1 by am (van iwaarden et al. 1991) . similarly, sp-a binds to cytomegalovirus and enhances viral entry into rat lung cells (weyer et al. 2000) . it is unknown whether sp-d has any activity against other herpesviridae. sp-a is able to bind to respiratory syncytial virus (rsv) targeting the f2 subunit of the viral f antigen and is able neutralise the virus (ghildyal et al. 1999; sano et al. 1999 sano et al. , 2000 . children with severe rsv infection have reduced levels of sp-a and sp-d in bal samples compared to healthy controls (kerr and paton 1999). in sp-a −/− knockout mice, rsv infection was more severe than in sp-a +/+ mice and the addition of exogenous sp-a to sp-a −/− mice reduced viral load and inflammation, and enhanced rsv clearance (levine et al. 1999) . sp-d can bind to rsv protein g and is able to neutralise rsv infectivity in vitro (hickling et al. 1999) . interestingly, rsv itself can alter sp-a expression in human pulmonary epithelial cells, upon infection by interfering with protein translation (bruce et al. 2009 ). sp-a binds to human immunodeficiency virus 1 (hiv-1) via the viral envelope gp120 glycoprotein and inhibits direct infection of cd4 + t cells (gaiha et al. 2008 ). yet, in dendritic cells (dc), sp-a increases hiv uptake, through enhanced binding to gp120 and facilitates transfer of hiv from dc to cd4 + t cells (gaiha et al. 2008) . sp-d is also able to bind to hiv gp120 and inhibit viral infectivity (meschi et al. 2005) , whilst rfhsp-d was also able to bind to gp120 and prevent infection of jurkat t cells, u937 monocytic cells and pbmc, and significantly suppress the hiv-1 induced cytokine storm in these cells (pandit et al. 2014 ). interestingly, a direct protein-protein interaction between rfhsp-d and dc-sign (dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin) modulates the capture of hiv-1 and its transfer to cd4 + t cells, revealing a novel and distinct anti-viral mechanism against hiv-1 by sp-d (dodagatta-marri et al. 2017) . this same rfhsp-d has also been recently shown to restrict the transfer of hiv across the vaginal epithelial barrier, by altering the gene expression signature of the epithelium (pandit et al. 2019) . these recent studies demonstrate the therapeutic potential of rfhsp-d against hiv infection. elevated levels of serum sp-d have been reported in severe acute respiratory syndrome (sars) coronavirus infected patients (wu et al. 2009 ). sp-d is able to bind to the glycosylated spike protein (s-protein) on the sars coronavirus (leth-larsen et al. 2007 ). both sp-a and sp-d bind to coronavirus strain hcov-229e, and inhibit viral infection of human bronchial epithelial (16hbe) cells. whilst sp-a only modestly reduced infection in am, whereas sp-d had no effect (funk et al. 2012) . human and porcine sp-d can interact with ebola virus glycoprotein and enhance viral infection in pulmonary cells, suggesting that sp-d may enhance viral spread (favier et al. 2018) . sp-a has been shown to enhance clearance of pulmonary adenovirus infection and inhibit lung inflammation (harrod et al. 1999) . bovine sp-d is also able to bind to bovine rotaviruses via the vp7 glycoprotein and neutralise infectivity (reading et al. 1998 ). sp-d binds to the a27 protein of vaccinia virus. sp-d −/− knockout mice challenged with vaccinia virus resulted in increased mortality, compared to sp-d +/+ mice, suggesting that sp-d has a protective role against vaccinia infection (perino et al. 2013 ). mbl is able to interact with a number of viral pathogens and its effect is generally protective, although there are examples of negative as well as positive outcomes for infection as a result of mbl-mediated binding (fig. 4.4) . several studies have shown that mbl is a potent inhibitor of iav infection (hartley et al. 1992; hartshorn et al. 1993b; reading et al. 1995 reading et al. , 1997 . moreover, mbl also has the added ability to deposit complement on iav-infected cells (reading et al. 1995) . there are also elevated levels of mbl in the lung during iav infection, suggesting that it may be important for protection against iav pathogenesis (reading et al. 1997; fidler et al. 2009 ). mbl can inhibit viral hemagglutination and directly neutralise iav in either a complement-dependent or independent manner (hartshorn et al. 1993b; anders et al. 1994; kase et al. 1999) . mbl binds to iav ha and na, and without involving complement, neutralises the virus ). however, some iav strains are resistant to the effects of mbl which is dependent on the degree of glycosylation on the viral ha globular domain (reading et al. 1997; job et al. 2010; tokunaga et al. 2011) . furthermore, mbl −/− mice were more susceptible to infection from highly glycosylated viral strains of iav than wild-type mice (chang et al. 2010 ). however, pandemic strain h1n1 and avian influenza a h9n2 produced more severe disease (enhanced production of pro-inflammatory response) in wild-type mice compared to mbl −/− mice, suggesting that mbl may have a deleterious effect in some iav infections (ling et al. 2012) . mbl is able to neutralise hiv-1 in vitro by binding to the n-linked mannose glycans of viral gp120, and binding to hiv-1 infected cd4 + t cells and monocytes and inhibiting reverse transcriptase activity (ezekowitz et al. 1989; teodorof et al. 2014) . another study has also shown mbl can also bind to viral gp41 as well as gp120 (saifuddin et al. 2000) , whilst mbl also activates complement on gp120 binding (haurum et al. 1993) . studies have shown a tentative link between low levels of mbl and increased risk of hiv-1 transmission or progression to aids, but this remains contentious (garred et al. 1997a; takahashi and ezekowitz 2005; ballegaard et al. 2014 ). there has also been a report of a positive correlation between the rate of aids progression and mbl plasma concentration (mangano et al. 2008) . however, other studies have found no correlation between mbl levels and aids disease progression (nielsen et al. 1995; mcbride et al. 1998) . in general, sp-d is better able to inhibit hiv-1 than mbl, but as is the case for mbl, sp-d's inhibitory activity against hiv-1 is lower than what has been observed for iav (meschi et al. 2005 ). mbl has also been shown to contribute to hiv-1 pathogenesis, where mbl mediates enhancement of hiv-1 dissemination to the brain by soluble gp120, which is taken up by the cxcr4 receptor on neurones, and then intracellularly trafficked by mbl, thus resulting in the apoptosis of neuronal cells (bachis et al. 2006; teodorof et al. 2014) . epidemiological studies have revealed association of mbl with hepatitis b virus (hbv) and hepatitis c virus (hcv) infection and disease severity, based on genetic polymorphisms (thomas et al. 1996; matsushita et al. 1998; yuen et al. 1999; sasaki et al. 2000; hakozaki et al. 2002) . however, one study found no link between mbl polymorphisms and hbv infection (hohler et al. 1998) . mbl is able to bind to hcv envelope glycoproteins, e1 and e2, and is able to activate complement (via masp-2), resulting in the neutralisation of hcv particles (brown et al. 2010) . mbl probably binds to n-linked glycosylated forms or hbv surface antigen (hbsag) (brown et al. 2007 ), but it is unknown whether this interaction neutralises the infectivity of the virus. mbl is also able to bind to ebola virus via its envelope glycoprotein (gp), which contains high mannose glycan sites, and is able to inhibit the binding of ebola (and marburg) viruses to dc-sign, blocking attachment to host cells and also neutralise the virus through complement activation (ji et al. 2005) . furthermore, soluble gp is a key component of ebola viral pathogenesis and mbl was found to be able to negate gp activity and the virally induced cytokine storm (escudero-perez et al. 2014) , and thus mbl could be involved in protection against increased vascular permeability which is a characteristic of ebola haemorrhagic disease. nevertheless, high dose mbl therapy in a mouse model, where mice we given recombinant human mbl at levels greater than seven times above average human levels, survived otherwise fatal ebola viral infection and became resistant to reinfection (michelow et al. 2011 ). there is limited or circumstantial data on the interaction of mbl with a number of other viral pathogens. in mice, mbl appears to modulate the immune responses to hsv-2 ( gadjeva et al. 2004) , mbl deficiency seems to be linked with recurrent infections (gadjeva et al. 2004; seppanen et al. 2009 ). mbl also binds to flaviviruses such as dengue and west nile virus and is able to neutralise infection through complement-dependent and independent mechanisms (avirutnan et al. 2011 ; fuchs et al. 2011) . genetic polymorphism affecting mbl serum levels may also contribute to the pathogenesis and disease severity of dengue fever (avirutnan et al. 2011). for collectins cl-l1, cl-k1, cl-p1, there is limited data on their interaction with viruses. only cl-k1 has been shown to bind iav and partially decrease the infectivity of the virus (hansen et al. 2010; selman and hansen 2012) . the binding of cl-l1 and cl-p1 to viruses has not been reported. like sp-d, conglutinin binds to iav resulting in the inhibition of hemagglutination and infectivity of the virus (hartshorn et al. 1993a ). conglutinin binds via its crd to the high mannose sites on the viral ha. iav treated with conglutinin also boosted neutrophil respiratory burst (hartshorn et al. 1993a ). conglutinin, cl-43 and bovine sp-d have been reported to bind the bovine rotavirus nebraska calf diarrhoea virus, targeting its vp7 glycoprotein (reading et al. 1998) . binding resulted in hemagglutination and neuralisation of rotavirus, with cl-43 showing the highest activity against the virus; it is the first report of collectin activity against a nonenveloped virus (reading et al. 1998 ). however, conglutinin has a higher inhibitory activity against iav (strain hkx31) than bovine sp-d or cl-43 (reading et al. 1998) . conglutinin binds to hsv-2 and enhances infection in mice (fischer et al. 1994) . it is also able to bind to hiv-1 gp160 and inhibit interaction of the virus with the cd4 receptor (andersen et al. 1991) . interestingly, a collectin-like protein analogous, to bovine conglutinin, was purified from human serum (called conglutinin-like protein) and this was demonstrated to bind to hiv-1 gp120 via its crd and inhibit viral infectivity (ushijima et al. 1992 ). collectins are able to recognise and bind to a number of fungi, both primary and opportunistic fungal pathogens, at various stages in their life cycle. collectins can exhibit direct growth inhibition and enhance phagocytosis of fungi; in some cases, they can contribute to the fungal pathogenesis. both sp-a and sp-d can bind to the conidia of aspergillus fumigatus, via its β-(1-6)glucan carbohydrate structures on the fungal cell surface in a ca 2+ dependent manner (fig. 4. 3) (madan et al. 1997a; allen et al. 2001a, b) . sp-a and sp-d can cause inhibition of conidia infectivity via agglutination, enhancement of phagocytosis and intracellular killing of a. fumigatus conidia by neutrophils and am (madan et al. 1997a ). the fungal ligands of sp-a are 2 n-linked glycosylated glycoproteins (gp45 and gp55) isolated from culture filtrate and are also used for elisa diagnosis of allergic aspergillosis (madan et al. 1997b) . fungal melanin was recently determined to be the primary ligand for sp-d on the a. fumigatus conidia cell surface, and is able to facilitate fungal phagocytosis and modulate the anti-fungal immune response (wong et al. 2018) . utilising a mouse model of invasive pulmonary aspergillosis (ipa), sp-d, but not sp-a, was found to be protective against a normally fatal challenge of a. fumigatus conidia (madan et al. 2001a, b) . in this study, ipa mice-treated intranasally with purified human sp-d or rfhsp-d showed 60 and 80% survival respectively (madan et al. 2001a, b) . the basis of this therapeutic protection by sp-d and rfhsp-d was observed to be enhanced phagocytosis of conidia by macrophages and neutrophils, fungistatic effects on the growth of conidia and a dampening of pathogenic th2 cytokines (il-4 and il-5), whilst enhancing protective th1 cytokines (tnf-α and ifn-γ) (singh et al. 2009 ). sp-d −/− knockout mice are more susceptible to ipa (madan et al. 2010 ). however, sp-a −/− knockout mice demonstrate resistance to ipa, suggesting that sp-a may be involved in the pathogenesis of ipa (madan et al. 2010 ). both sp-a and sp-d also have a direct effect on histoplasma capsulatum, inhibiting its growth by increasing the permeability of the fungal membrane ). however, no aggregation of h. capsulatum was observed by sp-a or sp-d, and neither collectin altered the phagocytosis of the fungus or inhibited the growth of macrophage-infected h. capsulatum . sp-a is also able to bind to cryptococcus neoformans, both in its encapsulated and non-encapsulated yeast form, but this does not result in increased phagocytosis of the acapsular form (walenkamp et al. 1999 ). sp-a binding was ca 2+ -dependent and was inhibited by glucose and mannose, but not galactose (walenkamp et al. 1999 ). intranasal infection with c. neoformans gave the same survival outcome in sp-a −/− knockout mice and wild-type mice, suggesting that the fungus is resistant to sp-a mediated host defence mechanisms (giles et al. 2007) . a subsequent study found that sp-d increases the phagocytosis of hypocapsular c. neoformans by murine macrophages and that this facilitated fungal survival (geunes-boyer et al. 2009 ). other studies have also shown that sp-d can agglutinate c. neoformans and a. fumigatus (schelenz et al. 1995; madan et al. 1997a ). furthermore, sp-d can bind to both encapsulated and acapsular c. neoformans and can aggregate acapsular c. neoformans in particular (van de wetering et al. 2004a ). the cryptococcal capsular components glucuronoxylomannan (gxm) and mannoprotein 1 (mp1) are the ligands for sp-d (van de wetering et al. 2004a ). sp-d is able to facilitate c. neoformans infection further by protecting the fungus against oxidative stress allowing for disease progression in the mouse model of infection (geunes-boyer et al. 2012) . sp-d is also able to bind blastomyces dermatitidis, via β-glucan on its surface, and subsequently block interactions with β-glucan-receptors on am (lekkala et al. 2006 ). this study also showed a reduction in tnf-α, dampening the host inflammatory response and thus may facilitate disease progression (lekkala et al. 2006 ). sp-d and sp-a can also bind to coccidioides posadasii via its surface antigens. in a mouse model of infection, c. posadasii infection is able to suppress the expression of pulmonary sp-a and sp-d, possibly facilitating fungal disease progression and dissemination (awasthi et al. 2004 ). sp-d can also bind to candida albicans via its surface antigens and agglutinate the pathogen and directly inhibiting its growth without the requirement of macrophage dependent phagocytosis (van rozendaal et al. 2000) . similarly, sp-a is able to bind to c. albicans and interfere with attachment to am, inhibiting phagocytosis (rosseau et al. 1997) . sp-a is also able to dampen the pro-inflammatory response elicited by c. albicans by human am and monocytes, which may be important in regulating excessive inflammation in the lung during candida infection (rosseau et al. 1999) . in saccharomyces cerevisiae, sp-d is observed to bind to its surface, but not sp-a, whilst the fungal ligand for sp-d is yeast β-(1-6)-glucan (allen et al. 2001a, b) . the opportunistic fungus, pneumocystis is able to infect a number of mammals with each species of the fungus displaying strict host specificity. for example, p. carinii and p. wakefieldiae infect rats, p. murina infects mice, p. oryctolagi infects rabbits, and p. jirovecii infects humans. sp-a and sp-d are able to recognise and bind pneumocystis species via the major surface glycoprotein (msg; also known as gpa) of the fungus mccormack et al. 1997a, b) . msg contains an n-linked carbohydrate chain made up of glucose, mannose, and n-acetylglucosamine and is involved in attachment of the fungus to alveolar epithelium in pneumocystis pneumonia (zimmerman et al. 1992; vuk-pavlovic et al. 2001) . cruciform dodecamers and other large oligomers of sp-d have a higher affinity of binding to p. carinii than do smaller oligomeric versions of sp-d (vuk-pavlovic et al. 2001) . sp-d is also able to recognise pneumocystis cysts via surface β-glucans (vuk-pavlovic et al. 2001 ). however, sp-d binding does not appear to increase the phagocytosis of the fungus (mccormack et al. 1997a, b; vuk-pavlovic et al. 2001) . despite this, sp-d does aggregate p. carinii in large complexes that may restrict phagocytosis by macrophages and may allow for persistence of the fungus within the host lungs (vuk-pavlovic et al. 2001) . pneumocystis pneumonia does alter the expression of sp-a in the lungs (atochina et al. 2001) , with a threefold increase in the levels of sp-a and sp-d (phelps et al. 1996; aliouat et al. 1998; qu et al. 2001 ), but decreases total phospholipid content (atochina et al. 2001) . human sp-a enhances attachment of p. carinii to rat am in vitro (williams et al. 1996) . sp-a also reduces phagocytosis of p. carinii in human am in vitro (koziel et al. 1998) . these data suggest that increased levels of sp-a during pneumocystis pneumonia (phelps et al. 1996 ) may contribute to the pathogenesis through binding to the fungus and interfering with its am recognition (koziel et al. 1998 ). immunosuppressed sp-a −/− mice also have increased susceptibility to p. carinii infection (linke et al. 2001) , whilst removal of immunosuppression resulted in efficient clearance of the infection , showing that sp-a does not enhance p. carinii clearance, but does modulate the host immune response during the resolution of infection. sp-d modulates interaction of p. carinii with am and also aggregates p. carinii, impairing phagocytosis by am (yong et al. 2003) . in sp-d −/− mice, there was delayed clearance of p. carinii infection, increased inflammation and altered nitric oxide response (atochina et al. 2004) . similarly, in immunosuppressed mice, sp-d was found to enhance p. carinii infection (vuk-pavlovic et al. 2006 ). mbl has been reported to interact with various primary and opportunistic fungal pathogens. low serum levels of mbl have been linked to increased likelihood of fungal disease (mullighan et al. 2002; granell et al. 2006) . mbl is able to bind to a. fumigatus (neth et al. 2000) , b. dermatitidis (koneti et al. 2008 ), c. albicans (kitz et al. 1992 neth et al. 2000; ip and lau et al. 2004; van asbeck et al. 2008) , candida parapsilosis (van asbeck et al. 2008) , and c. neoformans (chaka et al. 1997; van asbeck et al. 2008 ). the ligands for mbl binding to c. albicans and c. neoformans are mannan and mannoprotein, respectively (chaka et al. 1997; ip and lau, 2004) , whilst 1,3-β-glucan and mannose are the mbl ligands on b. dermatitidis and a. fumigatus, respectively (neth et al. 2000; koneti et al. 2008) . mbl is able to bind a. fumigatus conidia showing aggregation, enhancing phagocytosis, and complement deposition ). however, mbl binding of conidia did not always result in the killing of a. fumigatus by phagocytes (madan et al. 2005a, b; kaur et al. 2007 ). moreover, mbl may be less important in this context, since it is mainly a serum protein and may not be in significant levels in the lung. nevertheless, genetic polymorphisms in the mbl gene have been shown to be associated with severe aspergillosis (crosdale et al. 2001; vaid et al. 2007) . similarly, mbl deficiency is a risk factor for aspergillosis in immunocompromised patients, cancer patients and transplant recipients. in the mouse model of infection, mbl deficiency does not necessarily affect the survival of mice infected with a. fumigatus conidia, due to redundancy since mice having two copies of the mbl gene (mbl1 and mbl2), encoding mbl-a and mbl-c proteins in mouse serum (hogaboam et al. 2004) . however, treatment with recombinant mbl does enhance survival in ipa mice ). thus, the role of mbl in a. fumigatus infection may also depend on the route of infection and the level of immunosuppression of the host. mbl interaction with b. dermatitidis has only been studied in the mouse system. both mbl mouse proteins (mbl-a and mbl-c) bind to b. dermatitidis yeast cells (koneti et al. 2008) . inhibition of macrophage response to b. dermatitidis is also mediated by mbl, binding to 1,3-β-glucan ligand on b. dermatitidis, and thus inhibiting 1,3-β-glucan interaction with dectin-1 receptor on macrophages and also decreasing tnf-α production kimberg and brown 2008) . moreover, macrophage production of g-csf, ifn-γ, mcp-1, and rantes were significantly inhibited by mbl in response to b. dermatitidis, but not il-6 (brummer et al. 2007) . mbl can bind to c. albicans yeast and pseudohyphae and to c. parapsilosis yeast cells (denton and disalvo 1964; sugar and picard 1988; brummer et al. 2005; van asbeck et al. 2008) . mbl is able to aggregate c. albicans resulting in its growth inhibition and complement deposition of c4b and c3b on its surface via masps (ip and lau 2004; van asbeck et al. 2008) . similar levels of mbl-mediated complement deposition were also observed for c. parapsilosis (van asbeck et al. 2008 ). however, the binding of mbl to c. albicans may inhibit its phagocytosis by macrophages or dendritic cells (zimmerman et al. 1992; schelenz et al. 1995; chaka et al. 1997; vuk-pavlovic et al. 2001; ip and lau 2004; van de wetering et al. 2004a) . mbl seems to inhibit candida-induced macrophage responses in thp-1 cells through tlr-2 and tlr-4, suggesting that c. albicans modifies tlr signalling pathways in the macrophage (wang et al. 2013) . however, in the case of neutrophils, mbl enhances the phagocytosis of both c. albicans and c. parapsilosis yeast cells (van asbeck et al. 2008) . mbl greatly facilitates complement-mediated uptake of c. albicans via cr1 receptor in neutrophils and this results in the stimulation of reactive oxygen species by intracellular dectin-1, which recognises the phagocytosed fungal β-1,3 glucan (li et al. 2012) . the binding of mbl with c. albicans yeast also increases tnf-α production by monocytes in vitro (ghezzi et al. 1998 ) and in vivo (lillegard et al. 2006) . double knockout (mbl-a and mbl-c) mice were found to be more susceptible to systemic infection with c. albicans compared to wild-type mice (held et al. 2008) . vaginal candidiasis is an important mycosis in women. mbl protein is present in vaginal secretions (pellis et al. 2005) ; mbl levels seem to increase in vulvovaginal candidiasis. however, mbl levels were found to be lower in women with recurrent vulvovaginal candidiasis, because of polymorphisms in their mbl gene (babula et al. 2003; liu et al. 2006; giraldo et al. 2007; donders et al. 2008; milanese et al. 2008 ). the precise role of mbl in candidiasis remains to be fully explored. mbl can bind to acapsular c. neoformans yeast cells (chaka et al. 1997) , but this does not cause aggregation (eisen et al. 2008) . however, mbl binding to acapsular c. neoformans did facilitate complement deposition and enhancement of fungal phagocytosis by neutrophils (van asbeck et al. 2008) . furthermore, tnf-α production was induced in peripheral blood mononuclear cells by c. neoformans mannoprotein and this effect was enhanced by mbl (chaka et al. 1997) . it is unknown whether mbl binds to h. capsulatum or p. carinii. it is unlikely that mbl binds to h. capsulatum, since the cell wall contains 1,3-α-glucan (rappleye et al. 2007 ); however, in p. carinii, the cell surface of cyst forms does contain β-1,3-glucan (williams et al. 1996) , which may bind mbl. in coccidioides species, it is also unknown whether mbl interaction occurs, but patients with active coccidioidomycosis have been shown to have low serum mbl levels, compared to healthy individuals previously infected with coccidioides, and that low levels of mbl were associated with polymorphisms in their mbl gene (corredor et al. 1999 ). very few studies have investigated the interaction of these minor collectins with fungal species. cl-k1 can bind c. albicans (selman and hansen 2012) and cellular extracts (mannan) of s. cerevisiae (keshi et al. 2006; selman and hansen 2012) . cl-p1 has also been reported to bind to s. cerevisiae and mediate phagocytosis of yeast-derived zymosan, suggesting that cl-p1 mediates phagocytosis for fungi in the vascular endothelium jang et al. 2009 ). interestingly, cp-p1 also partially binds to a. fumigatus, via its crd, and in association with properdin, can activate the complement alternative pathway, resulting in c3b deposition and formation of the membrane attack complex (ma et al. 2015) . this shows a novel mechanism of triggering the alternative pathway of complement (ma et al. 2015) . there are no reports of cl-l1 interaction with fungi. there are also limited reports of the binding of bovine-unique collectins to fungi. cl-43 is able to bind to acapsular c. neoformans in vitro in a ca 2+ -dependent manner (schelenz et al. 1995) , and immobilised yeast mannan . conglutinin is able to bind to glycoproteins and polysaccharides derived from s. cerevisiae (n-acetylglucosamine, mannose, mannan) (loveless et al. 1989; lim and holmskov 1996) . an area of collectin that is yet to be fully explored is the interaction of collectins with protozoal and helminth pathogens, which are responsible for some of the most important global infections. there are limited studies and these are mostly based on genetic polymorphisms in collectin genes that are associated with predisposition or severity of these diseases. there is a limited number of functional studies on the role of collectins in protozoal and helminth infections. increases in levels of sp-d were observed in serum, renal and cerebral tissues in mice experimentally infected with plasmodium berghei, compared to control mice (cahayani et al. 2016) . low mbl serum levels and genetic polymorphisms in the mbl gene have been associated with more severe malaria, particularly in children (luty et al. 1998; holmberg et al. 2008) . mbl can bind to p. falciparum protein extracts, but it does not appear to inhibit the parasite directly (klabunde et al. 2002) . mbl does not opsonise p. falciparum, but it can bind to p. falciparum-infected erythrocytes, recognising the 78-kda glucose-regulated stress glycoprotein of the parasite . mbl binding to p. falciparum merozoite adhesins have also been reported, having the ability to activate complement (korir et al. 2014) . the complement lectin pathway can be activated by trypanosoma and leishmania (cestari et al. 2013) . mbl binds to glycosylated antigens on trypanosoma cruzi, on the surface of metacyclic trypomastigotes, resulting in complement activation (cestari idos et al. 2009 ). in a mouse model of t. cruzi infection, mbl influences host resistance and pathology (rothfuchs et al. 2012) . in some strains of t. cruzi, mbl mediates resistance to complement lysis of the parasite and enhances invasion of host cells (evans-osses et al. 2014) . mbl also binds to major cell surface glycoconjugates (lipophosphoglycans) on leishmania parasites, triggering lectin pathway activation and promastigote lysis (green et al. 1994; ambrosio and de messias-reason 2005) . certain genotypes of the mbl2 gene were also predictive for the risk for developing visceral leishmaniasis and other clinical complications in infections with leishmania chagasi (alonso et al. 2007) . similarly, there was a strong correlation found between serum levels of mbl and the probability of developing visceral leishmaniasis (santos et al. 2001 ). monocytes challenged with mbl-opsonised l. chagasi promastigotes secreted higher levels of tnf-α and il-6 than controls, suggesting that mbl may play an important role in pathogenesis (santos et al. 2001) . in helminth infections, mbl binds to the surface glycoproteins of schistosoma mansoni cercariae and adult worms and is able activate the lectin pathway (klabunde et al. 2000) . curiously, no differences in serum mbl levels were observed between patients infected with schistosoma and in healthy controls (klabunde et al. 2000) . another study has shown that high mbl serum levels are associated with protection in schistosomiasis (antony et al. 2013) . interestingly, high levels of mbl and cl-k1 were inversely correlated with urogenital infections with s. haematobium (antony et al. 2015b) . although cl-k1 has not been shown to bind directly to the parasitic worm, it was observed to be a risk factor for urinary schistosomiasis (antony et al. 2015a). furthermore, concomitantly elevated il-6 levels were also observed in urinary schistosomiasis cases compared to controls that correlated with mbl levels (antony et al. 2015b). similar findings linking il-6 and mbl have also been described in patients with visceral leishmaniasis (santos et al. 2001; antony et al. 2015b) . sp-d has also been shown to bind to fucosylated glycoconjugates (α-1-3 linked fucose) on the surface of s. mansoni larval stages, although the significance of this interaction remains unclear (van de wetering et al. 2004b, c) . however, a recent study has suggested that sp-d is essential for protection against helminth infection, using the experimental model nematode nippostrongylus brasiliensis (thawer et al. 2016) . n. brasiliensis infection of sp-d −/− knockout mice resulted in severe susceptibility to parasitic disease, whilst treatment with rfhsp-d enhanced parasite clearance and anti-parasitic immune responses (thawer et al. 2016) . sp-d was determined to bind to n. brasiliensis larvae via its crd, and to enhance their killing by am (thawer et al. 2016 ). a considerable number of in vitro and in vivo studies have focused on the immunomodulatory functions of collectins and their contribution to the host defense system. through activation of complement, and production of pro-inflammatory cytokines, mbl makes a major impact on the generation and regulation of the immune-mediated inflammatory response. allergen-mediated activation of the complement lectin pathway has been demonstrated (varga et al. 2003) . allergen extracts (parietaria (pa) and house dust (hd) mite) were shown to bind purified mbl, and trigger the lectin complement pathway. differences in plasma mbl levels may affect the degree of complement activation in different individuals, thus, susceptibility to allergic diseases. significantly elevated serum mbl levels were observed in pediatric mild-asthma patients, suggesting the possible role of mbl in the pathogenesis of asthma by contributing to airway inflammation, or increasing the risk of asthma development (uguz et al. 2005) . enhanced levels of serum mbl also correlate with an increased peripheral blood eosinophils in these individuals. it is also suggested that oxidative stress increases the mbl synthesis, and triggers complement activity. mbl can initiate complement activation following oxidative stress in asthma (collard et al. 2000; nadeem et al. 2003; uguz et al. 2005) , and aggravate inflammation. significantly increased mbl levels and mbl pathway was also detected in patients with bronchial asthma, rhinitis and allergic bronchopulmonary aspergillosis (abpa) (kaur et al. 2005) . a higher level of plasma mbl is likely to contribute to activation of lectin pathway, and an increased severity, including enhanced blood eosinophil counts. in addition, production of mbl in the liver is suggested to increase by up to three fold in response to environmental stimuli. therefore, higher levels of plasma mbl in allergic patients, compared to the non-allergic patients, may result from elevated hepatic synthesis caused by allergen exposure. furthermore, the circulating level of mouse mbl-a was also measured in aspergillus fumigatus allergen-sensitised and non-sensitised mice. increased level of mmbl-a was observed following allergic sensitisation, suggesting that challenging these mice with allergen may contribute to a higher level of mbl in sensitised mice as well as allergic patients (kaur et al. 2005) . earlier in vivo studies using mouse mbls have reported the likely role of mbl-a as a mediator of inflammation (santos et al. 2001; takahashi et al. 2002) . moreover, a substantial decline in the airway hyperresposiveness to a. fumigatus conidia was seen in mbl-a-deficient mice (mbl-a −/− ) when compared to mbl-a +/+ control mice, which suggest the possible role of mbl-a and its ability to trigger progression of airway hyper-responsiveness (hogaboam et al. 2004) . since levels of plasma mbl are genetically determined, it is of interest to study the genetic polymorphisms in mbl in relation to allergic susceptibility. in order to address the correlation between polymorphisms in the mbl gene and the progression of atopic diseases, nagy et al. found a contribution of variant mbl alleles to the susceptibility to acute or chronic chlamydia pneumoniae infection in asthmatic children . another study that focused on the genetic association of mbl related single nucleotide polymorphisms (snps) with allergic patients , reported the identification of g1011a, an intronic snp found in the mbl gene, and presence of 1011a allele of snp g1011a to be associated with an enhanced level of plasma mbl., snp g1011a has also been suggested to play a role in regulating mbl expression. additional polymorphisms were found at positions 550 (h/l variants) and 221 (x/y variants) in the promoter region of the mbl gene, which associated with high mbl levels in the plasma. 1011a allele was also associated with bronchial asthmatic patients with allergic rhinitis and abpa, which positively correlated with allergic markers, including high peripheral blood eosinophil counts, and reduced levels of forced expiratory volume at timed interval of 1 s (fev1) in these patients. however, no structural snps have been observed within the mbl gene in these allergic patients. as carbohydrate recognition immune molecules, both sp-a and sp-d have been shown to interact with gp55 and gp45 of a. fumigatus in a calcium and carbohydrate specific dependent manner (madan et al. 1997b) . both these collagenous molecules inhibit specific ige binding to these glycoproteins, and block allergen triggered histamine release from human basophils isolated from derp-and a. fumigatus-sensitised patients (madan et al. 1997a, b) . dodecameric forms of human sp-d mediate binding, aggregation, and phagocytosis of starch granules, containing grass pollen allergens from dactylis glomerata and phleum pratense via alveolar macrophages (erpenbeck et al. 2005) . sp-d can suppress proliferation of pbmcs isolated from children with derp-sensitive asthma (wang et al. 1998) , and suppress secretions of il-2 by pbmcs (borron et al. 1998) . suppressive effects of sp-a on the production and release of il-8 by eosinophils were also reported, which is stimulated by ionomycin in a concentration-dependent manner (cheng et al. 1998 ). since ige cross-linking, release of histamine and pmbcs proliferation are crucial immunological factors contributing to the development of asthmatic symptoms, both sp-a and sp-d are crucial immune modulators in resisting allergenic challenge, as well as suppressing substantial hypersensitivity reactions in the lungs (kishore et al. 2002) . intranasal administration of sp-d or rfhsp-d caused reduced levels of peripheral and pulmonary eosinophilia, and the effect persisted up to 16 days in the abpa mice. these observations therefore indicate the potential of sp-d as a therapeutic agent (kishore et al. 2002; madan et al.2001a; 2005a, b) . in addition, protective role of rhfsp-d has also reported in murine model of derp allergens-induced pulmonary hypersensitivity (singh et al. 2003) . shifting of th2 to a th1 following sp-d treatments appeared to be crucial to the protective mechanism, since, ifn-γ gamma is suggested to inhibit differentiation of th2 in response to il-4 (elser et al. 2002) . additionally, production of nitric oxide was significantly inhibited when derp mice derived alveolar macrophages are pre-incubated with rfhsp-d, and resulted in low levels of tnf-α in the rfhsp-d treated derp mice (liu et al. 2005) . culturing alveolar macrophages with allergen and sp-d has induced an increased production of il-10, il-12, and ifn-γ, indicating a positive correlation between macrophages and sp-d triggered inhibition of airway inflammation and airway hyper-responsiveness (ahr) (takeda et al. 2003) . a study by madan et al. has focused on the susceptibility of sp-a −/− and sp-d −/− mice to challenge with a. fumigatus allergen compared to wild-type mice (madan et al. 2005a, b) . intrinsic hypereosinophilia and seven fold increase in il-5 and il-13 levels were seen in both sp-a −/− and sp-d −/− mice. however, lower levels of ifn-γ to il-4 ratio in the lungs were observed, suggesting the possible th2 basis of immune response. treating these mice with exogenous intranasal sp-a and sp-d resulted in reversal of th2 polarisation. sp-d −/− mice was reported to be more susceptible to a. fumigatus allergen-induced pulmonary hypersensitivity when compared to wildtype mice. however, resistant to sensitisation was seen with sp-a −/− mice. intranasal administration of sp-d or rfhsp-d led to rescue of the sensitised sp-d −/− mice, while sp-a −/− mice demonstrated an enhanced levels of il-5 and il-13, causing greater pulmonary eosinophilia. genetic polymorphisms in the collagen region of sp-a2 (sp-a2 g1649c and sp-a2 a1660g) may also increase susceptibility to allergic bronchopulmonary aspergillosis (abpa) (saxena et al. 2003 ). the collectins are structurally related to the complement protein c1q (having a collagenous region and similar overall tertiary structure. a common receptor for sp-a, mbl and c1q was described in 1990 (malhotra et al. 1990) (fig. 4.5) , as collagen region binding cc1qr. this was subsequently identified as calreticulin (~56kda). two other candidate receptors were subsequently proposed: fig. 4 .5 collectin receptors on immune cells. collectins have been shown to bind a number of receptors and putative receptors, which lead to immunomodulatory responses. binding of collectins to toll-like receptor 2 (tlr-2), tlr-4, sp-a receptor 210 (sp-r210), cd91-calreticulin, and signal inhibitory regulatory protein-α (sirp-α) alters production of pro-inflammatory mediators. for example, sp-a and sp-d binds to sirp-α via their collagenous tails, and stimulates pro-inflammatory chemokine production via calreticulin/cd91 interaction. furthermore, bacterium bound collectins induces the conformational changes of calreticulin/cd91 interaction, which then activates p38-mitogen-activated protein kinase (mapk) signalling pathway, leading to transcriptional activation of nf-κb (nuclear factor kappa-light-chain-enhancer of activated b cells), and expression of pro-inflammatory cykines, including tumour necrosis factor alpha (tnf-α). sirpα is abundantly expressed in macrophages, and ligation of sirp-α by lung collectins are crucial in preventing damage to the airways caused by the production of pro-inflammatory responses. thereby, phosphorylated cytoplasmic region of sirp-α recruits shp-1 (src homology region 2 domain-containing phosphatase-1), which in turn dephosphorylates protein substrates involved in mediating physiological effects. thus, the interaction between sirp-α and shp-1 negatively regulates p38-map kinase signalling, and stimulates nf-κb activity, and cells become resistant to tnf-mediated effects, such as apoptosis (1) c1qrp (cd93) (c1q receptor associated with phagocytosis stimulated by c1q, mbl or sp-a): but this has subsequently been shown not to bind any of these ligands. it may be an adhesion receptor (mcgreal et al. 2002; norsworthy et al. 2004 ). (2) cr1, the complement c3b receptor, does interact with c1q and mbl, but functional aspects are not yet widely explored (jacquet et al. 2018 ). (3) calreticulin remains the main candidate as a receptor/adapter involved in phagocytosis mediated by c1q and collectins (ogden et al. 2001; vandivier et al. 2002) . calreticulin bound to the cell surface cd91 mediates uptake of apoptotic cells to which c1q, mbl, sp-a and sp-d are bound. it also mediates uptake of microorganisms targeted by the collectins. sp-a and sp-d can interact with phagocytic receptors and are able to influence receptor-mediated uptake of bacteria (lawson and reid 2000) . sp-a enhances the phagocytosis of s. aureus by monocytes but does not induce intracellular killing or the production of reactive oxygen intermediates (roi) (geertsma et al. 1994) . sp-a is also able to enhance the uptake of m. tuberculosis and m. avium by macrophages via the increased expression of mannose receptor (gaynor et al. 1995; kudo et al. 2004) , whilst sp-a enhances the scavenger receptor a (sr-a)-mediated uptake of streptococcus pneumoniae by am . sp-a is also reported to bind to a 210-kda sp-a receptor (spr210) in u937 macrophages and rat am and mediate uptake of mycobacterium bovis bacillus calmette-guérin (bcg) via this receptor (chroneos et al. 1996; weikert et al. 1997) ; in rat macrophages, this led to enhanced mycobacterial killing and an increase in the production of inflammatory mediators, tnf-α and nitric oxide (weikert et al. 2000) . sp-a and sp-d can also bind to the major lps receptor, cd14 that is present on alveolar macrophages. sp-a binds to the peptide portion of cd14, whilst sp-d interacts with the glycan of the receptor (sano et al. 2000) . sp-a modulates lps-induced cellular responses by direct interaction with cd14 (sano et al. 1999) , serving as an important mechanism for the recognition and clearance of this endotoxin. as noted above, sp-a and sp-d can interact with the versatile protein calreticulin (malhotra et al. 1990 ). when sp-a and sp-d bind to surface target ligands such as lps or apoptotic cells, multiple collagen regions are presented on the surface, which interact with the calreticulin -cd91 receptor complex (gardai et al. 2003) . this can then lead to the promotion of phagocytosis and initiation of cell signalling pathways leading to the production of pro-inflammatory cytokines and priming of adaptive immunity (ogden et al. 2001; vandivier et al. 2002; gardai et al. 2003) . in contrast, sp-a and sp-d can also suppress inflammatory responses by binding to signal regulating protein α (sirp-α) on macrophages and epithelia via their crd region, and this leads to a signalling pathway that blocks pro-inflammatory mediator production ( fig. 4 .5) (table 4 .2) (gardai et al. 2003) . therefore, the orientation of binding of sp-a and sp-d (collagen or lectin (crd)) domains to host receptors calreticulin-cd91 or sirp-α, respectively, have opposing effects and illustrate a dual function of these collectins, which could be, (1) protection of the naïve lung via maintenance of immune homeostasis and (2) protection via the triggering of inflammation to clear pathogens, allergens, necrotic and apoptotic cells. another receptor, gp-340 has also been found to bind to sp-d and sp-a (holmskov et al. 1997a; tino and wright 1999) , but any microbial interaction has been described for influenza virus (hartshorn et al. 2006a, b) and not bacteria, whilst binding of sp-a to gp-340 stimulates macrophage chemotaxis (tino and wright 1999) . calreticulin (cc1qr) is a collectin binding protein of 56 kda, and is known to bind c1q, mbl, sp-a, conglutinin and cl-43 (fig. 4 .5) (table 4 .2) (malhotra et al. 1993 ). this interaction is independent of calcium ions, and ionic in nature. it is mediated by the collagen domain 'c' of the collectins (cc1qr). the c1q binding site has regulation of phospholipid secretion and cytokine production, phagocytosis, and inhibition of t-cell proliferation been mapped to the s-domain of calreticulin (stuart et al. 1996) . conglutinin and cl-43 also bind c1qr and calreticulin (dec and wernicki 2006) . sp-a receptor 210 (sp-r210) is a 210 kda oligomeric molecule that earlier has been purified from the macrophage cell line u937 by affinity chromatography (fig. 4 .5 (table 4 .2) (chroneos et al. 1996) . sp-r210 is another candidate receptor for sp-a, and also found in type ii cells and alveolar macrophages. the direct interaction between sp-r210 and sp-a occurs through the collagen region of sp-a. antibodies raised against sp-r210 inhibit sp-a binding to alveolar type ii cells and alveolar macrophages, thus, prevent sp-a-mediated uptake of mycobacterium bovis, and block sp-a-bacillus calmette-guerin complexes to phagocytes. furthermore, sp-r210 inhibited sp-a-mediated phospholipid secretion by alveolar type ii cells (weikert et al. 1997 ). additionally, a study by yang et al. reported sp-r210 as a cell surface myosin 18a (yang et al. 2005) , and expressed in multiple isoforms. gp340 is a 340 kda sp-d binding glycoprotein purified from lung bronchoalvelar lavage of alveolar proteinosis patients , and belongs to the scavenger receptor superfamily, consisting of multiple scavenger receptor type b domains (holmskov et al. 1999) . furthermore, gp340 has been shown to be identical to salivary agglutinin, and its binding interaction with streptococcus mutans and helicobacter pylori has been reported (ligtenberg et al. 2001) . the direct binding of sp-d to gp340 occurs in a calcium dependent manner, and is inhibited by edta. the interaction is not affected by the presence of maltose, suggesting that the binding is a protein-protein interaction via the crd region of sp-d rather than a lectin-carbohydrate interaction. similar binding pattern was observed between gp340 and rfhsp-d, composed of trimeric neck region and crd . gp340 exists in a soluble form, and acts as an adaptor for sp-a and sp-d, but how gp340 is anchored in the membrane and the mechanism of binding to cell surface has not been elucidated fully yet (table 4 .2). sp-d can bind to human neutrophil defensins (hnps) via its neck and crd region (hartshorn et al. 2006a, b) . the interaction between sp-d and hnps can trigger anti-viral activity in the bal fluid. hd6, human β-defensins, and human neutrophil peptide (hnp)-4 bind sp-d with weaker affinity, while hnp-1-3 bind sp-d with high affinity and trigger inhibition of sp-d mediated anti-viral activity (doss et al. 2009 ). additionally, sp-d binds human decorin from amniotic fluid in a calcium dependent manner via sulphated n-acetyl galactosamine moiety of decorin (nadesalingam et al. 2003) . rfhsp-d interacts with dermatan sulphate moiety of decorin, and core protein of decorin interacts with sp-d via the collagen-like region. the interaction between lung collectins (sp-a and sp-d) and native as well as recombinant cd14 has been reported (table 4 .2) (sano et al. 1999) . cd14 is a soluble receptor for lps, and the neck domain of sp-d has been shown to bind the leucine-rich peptide portion of cd14, whilst the carbohydrate moiety of cd14 interacts with the lectin domain of sp-d (sano et al. 1999) . thus, both these surfactant proteins appear to modulate the cellular response to smooth and rough lps by interaction with cd14. (sano et al. 1999) . furthermore, the association of sp-a and sp-d with toll-like receptors (tlr), and or the tlr-associated molecule, could be one of the mechanisms by which they function as modulators of inflammation an inflammatory mediators (sano et al. 1999) . studies have reported direct involvement of sp-a in tlr2 signalling, and inhibition of downstream gene activation (murakami et al. 2002) . sp-a interacts directly with tlr4 and myeloid differentiation factor 2 (md-2), which are known critical signalling receptors for lps (billod et al. 2016) . thus, binding of sp-a with extracellular domain of tlr4 and md-2 was revealed in a calcium-dependent manner, involving the crd region. additionally, sp-a has attenuated cell surface binding of smooth lps, and induced nf-κb activation in cells expressing tlr4 and md-2 (murakami et al. 2002) . sp-d does not have significant effect on tlr4 expression, but it down-regulates the tlr4-mediated signalling against lps (henning et al. 2008 ). thus, sp-a's ability to dampen tlr2 and tlr4 signalling is associated with decrease in the phosphorylation of ikappabalpha, a key regulator of nf-κb activity, and nuclear translocation of p65, resulting in reduced tnf-α secretion in response to tlr ligands. furthermore, the same study also reported diminished phosphorylation of akt, an essential regulator of nf-κb and potentially mapks. therefore, there is a critical role for sp-a in modulating lung inflammatory reactions by regulating macrophage-mediated tlr4 activity. as noted above, calreticulin was identified as a common receptor for c1q, mbl and sp-a (malhotra et al. 1990) . calreticulin is mainly an intracellular protein but it is present on cell surfaces bound to cd91 and, thus, acts as an adaptor or co-receptor while binding to collagenous region of these collectins (fig. 4 .5) ( table 4 .2) (ogden et al. 2001; vandivier et al. 2002) . uptake of apoptotic cells by phagocytes mediated by c1q or mbl binding to calreticulin-cd91 complex was revealed (vandivier et al. 2002) . hla class i heavy chain (arosa et al. 1999 ), or cd59 have been shown to act as a calreticulin-binding proteins on cells which do not express cd91, allowing c1q or mbl coated particles to adhere to the cells. although a number of collectin receptors have been identified, there is still a need to fully elucidate how collectins stimulate phagocytes, and mediate phagocytosis, as well as other signalling transduction pathways. more studies on the structural aspects of receptors are needed, especially how these receptors are anchored in the membrane of immune and non-immune cells and, which co-receptors and signalling pathways are involved. in conclusion, collectins appear to play important roles in controlling lung allergy, inflammation and hypersensitivity, in addition to dealing with a wide variety of pathogens at pulmonary and extra-pulmonary sites. they act against pulmonary allergens through their ability to resist allergen challenge by interfering with allergen triggered ige interaction, degranulation of mast/basophils, cellular infiltration, and polarisation of helper th response. their roles have been also implicated in altering profiles of pro-inflammatory cytokines and chemokines as a result of infection, and allergen challenge. further research is needed to characterise specific collectin receptors that are crucial for collectin functions other than phagocytosis. drickamer k, dordal ms, reynolds l (1986) mannose-binding proteins isolated from rat liver contain carbohydrate-recognition domains linked to collagenous tails. complete primary structures and homology with pulmonary surfactant apoprotein. j biol chem 261 (15) entry inhibition and modulation of pro-inflammatory immune response against influenza a virus by a recombinant truncated surfactant protein d surfactant changes during experimental pneumocystosis are related to pneumocystis development polysaccharide recognition by surfactant protein d: novel interactions of a c-type lectin with nonterminal glucosyl residues interactions of surfactant proteins a and d with saccharomyces cerevisiae and aspergillus fumigatus pulmonary surfactant protein a up-regulates activity of the mannose receptor, a pattern recognition receptor expressed on human macrophages interactions of surfactant protein a with influenza a viruses: binding and neutralization computational approaches to toll-like receptor 4 modulation recombinant rat surfactant-associated protein d inhibits human t lymphocyte proliferation and il-2 production surfactantassociated protein a inhibits lps-induced cytokine and nitric oxide production in vivo detection of surfactant proteins a and d in human tear fluid and the human lacrimal system dectin-1 is a major beta-glucan receptor on macrophages mannan binding lectin and viral hepatitis specific interaction of hepatitis c virus glycoproteins with mannan binding lectin inhibits virus entry site-directed mutagenesis of cys-15 and cys-20 of pulmonary surfactant protein d. expression of a trimeric protein with altered anti-viral properties respiratory syncytial virus infection alters surfactant protein a expression in human pulmonary epithelial cells by reducing translation efficiency lectindependent enhancement of ebola virus infection via soluble and transmembrane c-type lectin receptors immunological basis for susceptibility and resistance to pulmonary blastomycosis in mouse strains production of il-6, in contrast to other cytokines and chemokines, in macrophage innate immune responses: effect of serum and fungal (blastomyces) challenge increased cd11b and hypoxia-inducible factors-1alpha expressions in the lung tissue and surfactant protein-d levels in serum are related with acute lung injury in severe malaria of c57bl/6 mice role of early lectin pathway activation in the complement-mediated killing of trypanosoma cruzi mechanisms of complement lectin pathway activation and resistance by trypanosomatid parasites induction of tnf-alpha in human peripheral blood mononuclear cells by the mannoprotein of cryptococcus neoformans involves human mannose binding protein lack of the pattern recognition molecule mannose-binding lectin increases susceptibility to influenza a virus infection suppressive effects of sp-a on ionomycin-induced il-8 production and release by eosinophils human surfactant protein d (sp-d) binds mycoplasma pneumoniae by high affinity interactions with lipids purification of a cell-surface receptor for surfactant protein a complement activation after oxidative stress: role of the lectin complement pathway identification of the post-translational modifications of the core-specific lectin. the core-specific lectin contains hydroxyproline, hydroxylysine, and glucosylgalactosylhydroxylysine residues isolation of paracoccidioides brasiliensis from the nine-banded armadillo dasypus novemcinctus, in an endemic area for paracoccidioidomycosis in colombia natural anti-infective pulmonary proteins: in vivo cooperative action of surfactant protein sp-a and the lung antimicrobial peptide sp-bn mannose-binding lectin gene polymorphisms as a susceptibility factor for chronic necrotizing pulmonary aspergillosis surfactant protein d: subcellular localization in nonciliated bronchiolar epithelial cells capsule production and growth phase influence binding of complement to staphylococcus aureus conglutinin, cl-43 and cl-46-three bovine collectins effect of conglutinin on phagocytic activity of bovine granulocytes isolation of blastomyces dermatitidis from natural sites at augusta, georgia the lipopolysaccharide structures of salmonella enterica serovar typhimurium and neisseria gonorrhoeae determine the attachment of human mannose-binding lectin to intact organisms protein-protein interaction between surfactant protein d and dc-sign via c-type lectin domain can suppress hiv-1 transfer mannose-binding lectin gene polymorphism and resistance to therapy in women with recurrent vulvovaginal candidiasis interactions of alpha-, beta-, and theta-defensins with influenza a virus and surfactant protein d recent insights into structures and functions of c-type lectins in the immune system the lectin pathway of complement activation contributes to protection from west nile virus infection infection of human alveolar macrophages by human coronavirus strain 229e mannan-binding lectin modulates the response to hsv-2 infection surfactant protein a binds to hiv and inhibits direct infection of cd4 + cells, but enhances dendritic cell-mediated viral transfer by binding sirpalpha or calreticulin/cd91, lung collectins act as dual function surveillance molecules to suppress or enhance inflammation dual role of mannan-binding protein in infections: another case of heterosis? susceptibility to hiv infection and progression of aids in relation to variant alleles of mannose-binding lectin mannanbinding lectin in the sub-saharan hiv and tuberculosis epidemics mannose-binding lectin is a disease modifier in clinical malaria and may function as opsonin for plasmodium falciparum-infected erythrocytes pulmonary surfactant protein a mediates enhanced phagocytosis of mycobacterium tuberculosis by a direct interaction with human macrophages binding of surfactant protein a to c1q receptors mediates phagocytosis of staphylococcus aureus by monocytes surfactant protein d increases phagocytosis of hypocapsular cryptococcus neoformans by murine macrophages and enhances fungal survival surfactant protein d facilitates cryptococcus neoformans infection serum-mediated enhancement of tnf-alpha release by human monocytes stimulated with the yeast form of candida albicans surfactant protein a binds to the fusion glycoprotein of respiratory syncytial virus and neutralizes virion infectivity complement receptor 1/cd35 is a receptor for mannan-binding lectin cryptococcus neoformans is resistant to surfactant protein a mediated host defense mechanisms mannose-binding lectin gene polymorphism, vulvovaginal candidiasis, and bacterial vaginosis surfactant protein a modulates the inflammatory response in macrophages during tuberculosis c-type lectin receptors in tuberculosis: what we know mannan-binding lectin pathway deficiencies and invasive fungal infections following allogeneic stem cell transplantation recognition of the major cell surface glycoconjugates of leishmania parasites by the human serum mannan-binding protein regulation of the mannan-binding lectin pathway of complement on neisseria gonorrhoeae by c1-inhibitor and alpha 2-macroglobulin mannose-binding lectin and the prognosis of fulminant hepatic failure caused by hbv infection lung surfactant protein d (sp-d) and the molecular diverted descendants: conglutinin, cl-43 and cl-46 purification and characterization of two mannan-binding lectins from mouse serum cl-46, a novel collectin highly expressed in bovine thymus and liver collectin 11 (cl-11, cl-k1) is a masp-1/3-associated plasma collectin with microbial-binding activity sp-a enhances viral clearance and inhibits inflammation after pulmonary adenoviral infection two distinct serum mannose-binding lectins function as beta inhibitors of influenza virus: identification of bovine serum beta inhibitor as conglutinin conglutinin acts as an opsonin for influenza a viruses human mannose-binding protein functions as an opsonin for influenza a viruses evidence for a protective role of pulmonary surfactant protein d (sp-d) against influenza a viruses mechanism of binding of surfactant protein d to influenza a viruses: importance of binding to haemagglutinin to antiviral activity innate defense against influenza a virus: activity of human neutrophil defensins and interactions of defensins with surfactant protein d salivary agglutinin and lung scavenger receptor cysteine-rich glycoprotein 340 have broad anti-influenza activities and interactions with surfactant protein d that vary according to donor source and sialylation complement activation upon binding of mannan-binding protein to hiv envelope glycoproteins pulmonary collectins modulate strain-specific influenza a virus infection and host responses increased susceptibility of complement factor b/c2 double knockout mice and mannan-binding lectin knockout mice to systemic infection with candida albicans pulmonary surfactant protein a regulates tlr expression and activity in human macrophages heteromeric complexes of native collectin kidney 1 and collectin liver 1 are found in the circulation with masps and activate the complement system expression sites of the collectin sp-d suggest its importance in first line host defence: power of combining in situ hybridisation, rt-pcr and immunohistochemistry a recombinant trimeric surfactant protein d carbohydrate recognition domain inhibits respiratory syncytial virus infection in vitro and in vivo surfactant protein a mediates mycoplasmacidal activity of alveolar macrophages the number and position of n-linked glycosylation sites in the hemagglutinin determine differential recognition of seasonal and 2009 pandemic h1n1 influenza virus by porcine surfactant protein d mannose-binding lectin deficiency alters the development of fungal asthma: effects on airway response, inflammation, and cytokine profile no association between mannose-binding lectin alleles and susceptibility to chronic hepatitis b virus infection in german patients mannose-binding lectin variant associated with severe malaria in young african children collectins: collagenous c-type lectins of the innate immune defense system affinity and kinetic analysis of the bovine plasma c-type lectin collectin-43 (cl-43) interacting with mannan isolation and characterization of a new member of the scavenger receptor superfamily, glycoprotein-340 (gp-340), as a lung surfactant protein-d binding molecule the plasma levels of conglutinin are heritable in cattle and low levels predispose to infection cloning of gp-340, a putative opsonin receptor for lung surfactant protein d collectins-soluble proteins containing collagenous regions and lectin domains-and their roles in innate immunity surfactant protein a decreases nitric oxide production by macrophages in a tumor necrosis factor-alpha-dependent mechanism major component of ra-reactive factor, a complement-activating bactericidal protein, in mouse serum conglutinin level in dairy cattle: changes associated with disease binding of sugar ligands to ca 2+ -dependent animal lectins. ii. generation of high-affinity galactose binding by site-directed mutagenesis role of mannose-binding lectin in the innate defense against candida albicans: enhancement of complement activation, but lack of opsonic function, in phagocytosis by human dendritic cells mannose-binding lectin enhances toll-like receptors 2 and 6 signaling from the phagosome mannose-binding lectin and innate immunity activation of complement by mannose-binding lectin on isogenic mutants of neisseria meningitidis serogroup b mannose-binding lectin accelerates complement activation and increases serum killing of neisseria meningitidis serogroup c mannose-binding lectin enhances phagocytosis and killing of neisseria meningitidis by human macrophages c1q and mannosebinding lectin interact with cr1 in the same region on ccp24-25 modules scavenger receptor collectin placenta 1 (cl-p1) predominantly mediates zymosan phagocytosis by human vascular endothelial cells mannose-binding lectin binds to ebola and marburg envelope glycoproteins, resulting in blocking of virus interaction with dc-sign and complement-mediated virus neutralization pandemic h1n1 influenza a viruses are resistant to the antiviral activities of innate immune proteins of the collectin and pentraxin superfamilies sp-a enhances phagocytosis of klebsiella by interaction with capsular polysaccharides and alveolar macrophages interaction of surfactant protein a with bacterial lipopolysaccharide may affect some biological functions human mannan-binding lectin inhibits the infection of influenza a virus without complement increased surfactant protein d in rat airway goblet and clara cells during ovalbumin-induced allergic airway inflammation plasma mannan-binding lectin levels and activity are increased in allergic patients elevated levels of mannan-binding lectin (mbl) and eosinophilia in patients of bronchial asthma with allergic rhinitis and allergic bronchopulmonary aspergillosis associate with a novel intronic polymorphism in mbl protective role of mannan-binding lectin in a murine model of invasive pulmonary aspergillosis pulmonary collectins enhance phagocytosis of mycobacterium avium through increased activity of mannose receptor the human mannose-binding protein functions as an opsonin the staphylococcal surface-glycopolymer wall teichoic acid (wta) is crucial for complement activation and immunological defense against staphylococcus aureus infection pulmonary surfactant protein a augments the phagocytosis of streptococcus pneumoniae by alveolar macrophages through a casein kinase 2-dependent increase of cell surface localization of scavenger receptor a the demonstration in human serum of "conglutinogenactivating factor" and its effect on the third component of complement inactivation of staphylococci by alveolar macrophages with preliminary observations on the importance of alveolar lining material bovine conglutinin binds to an oligosaccharide determinant presented by ic3b, but not by c3, c3b or c3c the roles of surfactant proteins a and d in innate immunity effect of lung surfactant collectins on bronchoalveolar macrophage interaction with blastomyces dermatitidis: inhibition of tumor necrosis factor alpha production by surfactant protein d dispensability of surfactant proteins a and d in immune control of mycobacterium tuberculosis infection following aerosol challenge of mice the sars coronavirus spike glycoprotein is selectively recognized by lung surfactant protein d and activates macrophages surfactant protein-a enhances respiratory syncytial virus clearance in vivo surfactant protein d enhances clearance of influenza a virus from the lung in vivo absence of sp-a modulates innate and adaptive defense responses to pulmonary influenza infection effect of mannose-binding protein on binding of cryptococcus neoformans to human phagocytes mbl-mediated opsonophagocytosis of candida albicans by human neutrophils is coupled with intracellular dectin-1-triggered ros production human salivary agglutinin binds to lung surfactant protein-d and is identical with scavenger receptor protein gp-340 recognition of candida albicans by mannan-binding lectin in vitro and in vivo expression of the carbohydrate recognition domain of bovine conglutinin and demonstration of its binding to ic3b and yeast mannan primary structure of bovine collectin-43 (cl-43). comparison with conglutinin and lung surfactant protein-d expression of the carbohydrate recognition domain of lung surfactant protein d and demonstration of its binding to lipopolysaccharides of gram-negative bacteria surfactant protein-d modulates interaction of pneumocystis carinii with alveolar macrophages both human sp-a1 and sp-a2 genes are expressed in small and large intestine mannose-binding lectin contributes to deleterious inflammatory response in pandemic h1n1 and avian h9n2 infection immunosuppressed surfactant protein a-deficient mice have increased susceptibility to pneumocystis carinii infection efficient resolution of pneumocystis murina infection in surfactant protein a-deficient mice following withdrawal of corticosteroidinduced immunosuppression therapeutic effect of surfactant protein d in allergic inflammation of mite-sensitized mice mannose-binding lectin and vulvovaginal candidiasis bovine serum conglutinin is a lectin which binds non-reducing terminal n-acetylglucosamine, mannose and fucose residues collectins and ficolins: sugar pattern recognition molecules of the mammalian innate immune system c-type lectins with a sweet spot for mycobacterium tuberculosis mannose-binding lectin plasma levels and gene polymorphisms in plasmodium falciparum malaria soluble collectin-12 (cl-12) is a pattern recognition molecule initiating complement activation via the alternative pathway binding of pulmonary surfactant proteins a and d to aspergillus fumigatus conidia enhances phagocytosis and killing by human neutrophils and alveolar macrophages lung surfactant proteins a and d can inhibit specific ige binding to the allergens of aspergillus fumigatus and block allergen-induced histamine release from human basophils surfactant proteins a and d protect mice against pulmonary hypersensitivity induced by aspergillus fumigatus antigens and allergens protective role of lung surfactant protein d in a murine model of invasive pulmonary aspergillosis association of polymorphisms in the collagen region of human sp-a1 and sp-a2 genes with pulmonary tuberculosis in indian population susceptibility of mice genetically deficient in the surfactant protein (sp)-a or sp-d gene to pulmonary hypersensitivity induced by antigens and allergens of aspergillus fumigatus susceptibility of mice genetically deficient in the surfactant protein (sp)-a or sp-d gene to pulmonary hypersensitivity induced by antigens and allergens of aspergillus fumigatus susceptibility of mice genetically deficient in sp-a or sp-d gene to invasive pulmonary aspergillosis localization of lung surfactant protein d on mucosal surfaces in human tissues expression and localization of lung surfactant protein a in human tissues human leukocyte c1q receptor binds other soluble proteins with collagen domains interaction of c1q receptor with lung surfactant protein a structure and homology of human c1q receptor (collectin receptor) binding of human collectins (sp-a and mbp) to influenza virus variants of the sftpa1 and sftpa2 genes and susceptibility to tuberculosis in ethiopia detrimental effects of mannose-binding lectin (mbl2) promoter genotype xa/xa on hiv-1 vertical transmission and aids progression association of mannose-binding lectin gene haplotype lxpa and lypb with interferon-resistant hepatitis c virus infection in japanese patients mannose-binding protein in hiv-seropositive patients does not contribute to disease progression or bacterial infections the cys6 intermolecular disulfide bond and the collagen-like region of rat sp-a play critical roles in interactions with alveolar type ii cells and surfactant lipids the carbohydrate recognition domain of surfactant protein a mediates binding to the major surface glycoprotein of pneumocystis carinii deletion mapping of n-terminal domains of surfactant protein a. the n-terminal segment is required for phospholipid aggregation and specific inhibition of surfactant secretion macrophageindependent fungicidal action of the pulmonary collectins human c1qrp is identical with cd93 and the mni-11 antigen but does not bind c1q aggregation and opsonization of type a but not type b hemophilus influenzae by surfactant protein a complement dependent and independent interaction between bovine conglutinin and mycobacterium bovis bcg: implications in bovine tuberculosis surfactant protein d binds to human immunodeficiency virus (hiv) envelope protein gp120 and inhibits hiv replication high-dose mannosebinding lectin therapy for ebola virus infection mbl2 genetic screening in patients with recurrent vaginal infections surfactant proteins a (sp-a) and d (sp-d): levels in human amniotic fluid and localization in the fetal membranes characterization of two mannose-binding protein cdnas from rhesus monkey (macaca mulatta): structure and evolutionary implications deficiency of mannose-binding lectin greatly increases susceptibility to postburn infection with pseudomonas aeruginosa mannose-binding lectin gene polymorphisms are associated with major infection following allogeneic hemopoietic stem cell transplantation surfactant protein a inhibits peptidoglycan-induced tumor necrosis factor-alpha secretion in u937 cells and alveolar macrophages by direct interaction with toll-like receptor 2 expression of surfactant protein d in the human gastric mucosa and during helicobacter pylori infection increased oxidative stress and altered levels of antioxidants in asthma identification and characterization of a novel interaction between pulmonary surfactant protein d and decorin collectin surfactant protein d binds antibodies and interlinks innate and adaptive immune systems mannose-binding lectin recognizes peptidoglycan via the n-acetyl glucosamine moiety, and inhibits ligand-induced pro-inflammatory effect and promotes chemokine production by macrophages the development of asthma in children infected with chlamydia pneumoniae is dependent on the modifying effect of mannosebinding lectin mannose-binding lectin engagement with late apoptotic and necrotic cells an insight into the diverse roles of surfactant proteins, sp-a and sp-d in innate and adaptive immunity mannose-binding lectin binds to a range of clinically relevant microorganisms and promotes complement deposition enhancement of complement activation and opsonophagocytosis by complexes of mannose-binding lectin with mannose-binding lectinassociated serine protease after binding to staphylococcus aureus structural analysis of monosaccharide recognition by rat liver mannose-binding protein ca 2+ -dependent structural changes in c-type mannosebinding proteins the level of the serum opsonin, mannan-binding protein in hiv-1 antibody-positive patients murine cd93 (c1qrp) contributes to the removal of apoptotic cells in vivo but is not required for c1q-mediated enhancement of phagocytosis c1q and mannose binding lectin engagement of cell surface calreticulin and cd91 initiates macropinocytosis and uptake of apoptotic cells molecular cloning of a novel human collectin from liver (cl-l1) the membrane-type collectin cl-p1 is a scavenger receptor on vascular endothelial cells surfactant protein d interacts with pneumocystis carinii and mediates organism adherence to alveolar macrophages surfactant protein d inhibits hiv-1 infection of target cells via interference with gp120-cd4 interaction and modulates proinflammatory cytokine production surfactant protein d reverses the gene signature of transepithelial hiv-1 passage and restricts the viral transfer across the vaginal barrier surfactant protein a suppresses reactive nitrogen intermediates by alveolar macrophages in response to mycobacterium tuberculosis mannose binding lectin and c3 act as recognition molecules for infectious agents in the vagina protective effect of surfactant protein d in pulmonary vaccinia virus infection: implication of a27 viral protein surfactant protein-a levels increase during pneumocystis carinii pneumonia in the rat surfactant protein a binds mycoplasma pneumoniae with high affinity and attenuates its growth by recognition of disaturated phosphatidylglycerols opsonic activities of surfactant proteins a and d in phagocytosis of gram-negative bacteria by alveolar macrophages interactions of human mannose-binding protein with lipoteichoic acids interaction of human mannose-binding protein with mycobacterium avium alteration of surfactant proteins a and d in bronchoalveolar lavage fluid of pneumocystis carinii pneumonia the mycobacterium tuberculosis cell-surface glycoprotein apa as a potential adhesin to colonize target cells via the innate immune system pulmonary c-type lectin surfactant protein a histoplasma capsulatum alpha-(1,3)-glucan blocks innate immune recognition by the beta-glucan receptor a serum mannose-binding lectin mediates complement-dependent lysis of influenza virus-infected cells collectin-mediated antiviral host defense of the lung: evidence from influenza virus infection of mice antiviral activity of bovine collectins against rotaviruses the salivary scavenger and agglutinin binds mbl and regulates the lectin pathway of complement in solution and on surfaces complement deficiency states and infection: epidemiology, pathogenesis and consequences of neisserial and other infections in an immune deficiency phagocytosis of viable candida albicans by alveolar macrophages: lack of opsonin function of surfactant protein a surfactant protein a down-regulates proinflammatory cytokine production evoked by candida albicans in human alveolar macrophages and monocytes mannose-binding lectin regulates host resistance and pathology during experimental infection with trypanosoma cruzi structural characterization of bovine collectin-43 mbl genotype and risk of invasive pneumococcal disease: a casecontrol study collectin cl-p1 utilizes c-reactive protein for complement activation interaction of mannose-binding lectin with primary isolates of human immunodeficiency virus type 1 pulmonary surfactant protein a modulates the cellular response to smooth and rough lipopolysaccharides by interaction with cd14 surfactant proteins a and d bind cd14 by different mechanisms mannan-binding lectin enhances susceptibility to visceral leishmaniasis mannose-binding lectin polymorphisms in patients with hepatitis c virus infection molecular characterization of the mouse mannose-binding proteins. the mannose-binding protein a but not c is an acute phase reactant pulmonary collectins protect macrophages against pore-forming activity of legionella pneumophila and suppress its intracellular growth association of polymorphisms in the collagen region of sp-a2 with increased levels of total ige antibodies and eosinophilia in patients with allergic bronchopulmonary aspergillosis binding of host collectins to the pathogenic yeast cryptococcus neoformans: human surfactant protein d acts as an agglutinin for acapsular yeast cells structure and function of collectin liver 1 (cl-l1) and collectin 11 (cl-11, cl-k1) mannose-binding lectin 2 gene polymorphism in recurrent herpes simplex virus 2 infection mannose-binding lectin-deficient mice are susceptible to infection with staphylococcus aureus mannanbinding lectin is involved in the protection against renal ischemia/reperfusion injury by dietary restriction protective effects of a recombinant fragment of human surfactant protein d in a murine model of pulmonary hypersensitivity induced by dust mite allergens therapeutic effects of recombinant forms of full-length and truncated human surfactant protein d in a murine model of invasive pulmonary aspergillosis crystal structure and functional characterization of the complement regulator mannosebinding lectin (mbl)/ficolin-associated protein-1 (map-1) detection of structural gene mutations and promoter polymorphisms in the mannan-binding lectin (mbl) gene by polymerase chain reaction with sequence-specific primers localisation of the c1q binding site within c1q receptor/calreticulin experimental blastomycosis pneumonia in mice by infection with conidia mycobacterial antigen 85 complex (ag85) as a target for ficolins and mannose-binding lectin the role of the mannose-binding lectin in innate immunity lack of mannose-binding lectin-a enhances survival in a mouse model of acute septic peritonitis surfactant protein d regulates airway function and allergic inflammation through modulation of macrophage function improvements on the purification of mannan-binding lectin and demonstration of its ca( 2+ )-independent association with a c1s-like serine protease inhibition of influenza viral neuraminidase activity by collectins intracellular mannose binding lectin mediates subcellular trafficking of hiv-1 gp120 in neurons surfactant protein-d is essential for immunity to helminth infection a second serine protease associated with mannan-binding lectin that activates complement clinical manifestations of mannan-binding lectin deficiency mutation of gene of mannose-binding protein associated with chronic hepatitis b viral infection glycoprotein-340 binds surfactant protein-a (sp-a) and stimulates alveolar macrophage migration in an sp-a-independent manner the pandemic (h1n1) 2009 influenza virus is resistant to mannose-binding lectin collectin cl-lk is a novel soluble pattern recognition receptor for mycobacterium tuberculosis surfactant protein a without the interruption of gly-x-y repeats loses a kink of oligomeric structure and exhibits impaired phospholipid liposome aggregation ability mannose-binding lectin levels in children with asthma inhibition of human immunodeficiency virus-1 infection by human conglutinin-like protein: in vitro studies association of sp-d, mnl and i-nos genetic variants with pulmonary tuberculosis distinct alleles of mannose-binding lectin (mbl) and surfactant proteins a (sp-a) in patients with chronic cavitary pulmonary aspergillosis and allergic bronchopulmonary aspergillosis human plasma-derived mannose-binding lectin: a phase i safety and pharmacokinetic study mannose binding lectin plays a crucial role in innate immunity against yeast by enhanced complement activation and enhanced uptake of polymorphonuclear cells characteristics of surfactant protein a and d binding to lipoteichoic acid and peptidoglycan, 2 major cell wall components of gram-positive bacteria collectins: players of the innate immune system aggregation of cryptococcus neoformans by surfactant protein d is inhibited by its capsular component glucuronoxylomannan surfactant protein d binding to terminal alpha1-3-linked fucose residues and to schistosoma mansoni porcine pulmonary collectins show distinct interactions with influenza a viruses: role of the n-linked oligosaccharides in the carbohydrate recognition domain binding of mannan-binding protein to various bacterial pathogens of meningitis pulmonary surfactant protein a enhances the host-defense mechanism of rat alveolar macrophages surfactant protein a is opsonin in phagocytosis of herpes simplex virus type 1 by rat alveolar macrophages rat surfactant protein d enhances the production of oxygen radicals by rat alveolar macrophages binding of surfactant protein a (sp-a) to herpes simplex virus type 1-infected cells is mediated by the carbohydrate moiety of sp-a binding of surfactant protein a to the lipid a moiety of bacterial lipopolysaccharides role of pulmonary surfactant protein d in innate defense against candida albicans role of surfactant proteins a, d, and c1q in the clearance of apoptotic cells in vivo and in vitro: calreticulin and cd91 as a common collectin receptor complex studies on the mechanisms of allergen-induced activation of the classical and lectin pathways of complement molecular basis of sugar recognition by collectin-k1 and the effects of mutations associated with 3mc syndrome immunocytochemical localization of surfactant protein d (sp-d) in type ii cells, clara cells, and alveolar macrophages of rat lung structural comparison of recombinant pulmonary surfactant protein sp-a derived from two human coding sequences: implications for the chain composition of natural human sp-a carbohydrate recognition domain of surfactant protein d mediates interactions with pneumocystis carinii glycoprotein a surfactant protein d enhances pneumocystis infection in immune-suppressed mice pulmonary surfactant protein a binds to cryptococcus neoformans without promoting phagocytosis a recombinant polypeptide, composed of the alpha-helical neck region and the carbohydrate recognition domain of conglutinin, self-associates to give a functionally intact homotrimer inhibitory effect of pulmonary surfactant proteins a and d on allergen-induced lymphocyte proliferation and histamine release in children with asthma mannan-binding lectin inhibits candida albicans-induced cellular responses in pma-activated thp-1 cells through toll-like receptor 2 and toll-like receptor 4 sp-a enhances uptake of bacillus calmette-guerin by macrophages through a specific sp-a receptor surfactant protein a enhances mycobacterial killing by rat macrophages through a nitric oxide-dependent pathway drickamer k (1991a) physical characterization and crystallization of the carbohydrate-recognition domain of a mannose-binding protein from rat structure of the calciumdependent lectin domain from a rat mannose-binding protein determined by mad phasing surfactant protein a binding to cytomegalovirus proteins enhances virus entry into rat lung cells isolation and characterization of the human pulmonary surfactant apoprotein gene elevated expression of surfactant proteins in newborn rats during adaptation to hyperoxia respiratory innate immune proteins differentially modulate the neutrophil respiratory burst response to influenza a virus human surfactant protein a enhances attachment of pneumocystis carinii to rat alveolar macrophages fungal melanin stimulates surfactant protein d-mediated opsonization of and host immune response to aspergillus fumigatus spores surfactant proteins a and d inhibit the growth of gram-negative bacteria by increasing membrane permeability elevated plasma surfactant protein d (sp-d) levels and a direct correlation with anti-severe acute respiratory syndrome coronavirus-specific igg antibody in sars patients mannose-binding lectin inhibits the motility of pathogenic salmonella by affecting the driving forces of motility and the chemotactic response identification of the surfactant protein a receptor 210 as the unconventional myosin 18a correlation analysis between single nucleotide polymorphisms of pulmonary surfactant protein a gene and pulmonary tuberculosis in the han population in china surfactant protein dmediated aggregation of pneumocystis carinii impairs phagocytosis by alveolar macrophages ) h progression of liver disease in chronic hepatitis b infection 120-kd surface glycoprotein of pneumocystis carinii is a ligand for surfactant protein a