key: cord-0042109-reu3ixop authors: nan title: XXVIth Meeting of the Seandinavian Society for Immunology and the Xlth Summer School Goteborg, Sweden, 25–28 May 1995 date: 2006-06-29 journal: Scand J Immunol DOI: 10.1111/j.1365-3083.1995.tb03616.x sha: 238a7184d9d9876b23b92b957489b91c98365c39 doc_id: 42109 cord_uid: reu3ixop nan pathogenesis of CIA, we have estabbshed T cell hybridomas from DBA/t and C3H.Q (H-2i) mice immunized with rat CII. All (he hybrid clones selected recognized the immtinodominant epitope included in the bromide-cleaved fragmenl CB 11 fragment of rat CII bul not of mouse CII. The analysis by DNA sequencing of the TCR of II clonally distinct T cell hybridomas revealed a high diversity of the gene segment usage Furthennore the Cl>R3-like regions responsible for antigen recognition were also very diverse in length and amino acids composition. We suggest that the variable glycosylation of the epitope increase.s the diversity of the TCR. Immunoglobulins as modulators of protein reactivities K. Andersson, U.-B. Hansson, U. Alkner, and J. Bjorklund. Dept. of Biochemistry, Liind University, Sweden. Fax:-l-46 46 104534. We have examined the modulating eifects of human immunoglobulin on the reactions of specific antibodies with their antigen. The effects of non-immune polyclonal human IgG and/or rheumatoid factors from rheumatoid sera were estimated immunologically in two conventional antigen-antibody systems with polyclonal antibodies (HSA-rabbit anti-HSA and tetanus toxoid (TT)-human anti-TT). Using enzymes (glucose-6-phosphate dehydrogenase and human placenta! alkaline phosphatase) as antigens for polyclonal rabbit antibodies, efFects on biological activities could be followed. Non-specific immunoglobulins were found to inhibit the binding of antigen by specific antibodies of both himian and rabbit origin. Furthermore, human immunoglobulins were also able to modify the composition of preformed antigen-antibody complexes. The observed changes could not be ascribed only to the activities of specific antibodies in the immunoglobulin preparations. In addition, we have also observed some odd interactions of immunoglobulins with affinity ligands which further indicate that immunoglobulins may interact with each other and with other proteins not only as antibodies against antigens, but also through interactions which are distinct from antigen-binding. A network of such "non-immunological" interjictions would be of great importance in providing suitable conditions for physiological protein activities. It is also easy to conceive a regulatory function of immunoglobulins simitar to the allosteric regulation of, for instance, enzymatic activities throiigh this kind of interactions. The adhesion of leukocytes to vascular endothelium and their migration into tissues i,s mediated hy adhesion molecules on the endothelial cells and leukocytes. Vascular adhesion protein-1 (VAP-l) is a 180/90 kDa endothelial molecule expressed most prominently in high endothelial eells in peripheral lymph node (PLN) type lymphatic tissues. The expression of VAP-1 is upregulaled in inllammation at other siies as well. VAP-1 mediates lymphocyte binding to peripheral lymph nodes, tonsil and synovium. In the present work we examined the expre.ssion. structure and function of VAP-1 in normal and inllamcd skin. In psoriasis, lichen ruhcr planus, pemphigoid and allergic lesions. VAP-1 was significantly upregulated. The expression of VAP-1 was also clearly increased in the macroscopically healthy skin ol' the skin disease patients. The VAP-1 molecule induced in .skin diseases is identical in size to that found in tonsil HEV and is also modified with abundant sialic acid residues. VAP-1 in inflamed skin is functional since inhibition with an anti-VAP-l mAh IB2 causes a 70% reduction in lymphocyte adhesion to vascular endothelium. In conclusion, VAP-1 is likely to contribute to lymphocyte homing to inflamed skin. Aionsson B., Smedman. L, & Troye-Blomberg, M. Dept. of Immunology, Stockholm University, S-106 91 Stockholm, Sweden. Fax +46 8 157356 Measles remains to be one of the most important child health problem in developing countries. Studies of protective immune responses and identification of relevant B-and T-cell epitopes are required for a better understanding of immunity to measles. We have established techniques to measure antigen specific T-cell responses in whole-blood cultures from capillary specimens. These techniques were used to study proliferation and TH1/TH2 heterogeneity in response to various measles antigens when stimulating lymphocytes from immune adult donor's or from children before and after vaccination. Separated lymphocytes from adult immune donors proliferated in response to crude measles antigen, i.e. Complement Fixation Antigen (CFA), but not to control antigen. However such response was not detected when stimulating whole-blood cell cultures obtained from vaccinated children. T-cell activation can be manifested by cytokine secretion rather than proliferation, we therefore measured IFN-7 in supernatants from cell-cultures stimulated with CFA. Increased, dose dependent, IFN-y production was detected by ELISA in cultures with separated lymphocytes from adult immune donors, as compared to control antigen and medium alone. Whole-blood cultures from vaccinated children are presently being examined in the same way. We are currently refining the technique for detection of low amounts of IL-4. Fetal Antigen 1 (FAl) was first described by Fay et aL 1988 Eur.J.Obstet.Gynecol.Repro.Biol. 29, 73-85 , and has recently been characterized with respect to primary structure and glycosylation by Jen. sen etal, 1994 Eur.J.BioI.Chem. 225. 85-92. In the context of diabetes there are striking similarities between human FAl and the 38 kDa insulin secretory granule protein isolated from the rat (Roep etal, 1990 Nature 345, 632-634) with respect lo Mr and the tissue expression. The 38 kDa has been described as a major autoantigen in IDDM. We have established a murine animal model for the study of the biological significansof FAl including: (i) purification of mFAl, (ii) production of antibodies, (iii) immunohistochemical location studies, (iv) establishment of ELISA technique. In brief the preliminary data are: • murine amniotic fluid contains 2500 times more mFA! than normal mouse serum. • fetal scrum day (16) (17) (18) (19) (20) (21) contains mFAI at about the same level as amniotic fluid. • the content of mFAl in sera from pregnant mice increases at least 30 times before delivery and decreases in less than 6 hours after delivery to normal range of mFA 1 in serum at adult mice. • newbom mice have a mFAl serum level 2500 times that of for adult mice decreasing to normal range at sexual maturity. Sinasi Bayrak**, Rudolf DoUing*, Roland LausterM atthias Hesse^ Vivianne Malmstrom*, Rikard Holmdahl* & Avrion Mitchison §Deutsches Rheuma-Forschungszentrum, Berlin, Germany; Fax 49-3I>-454;090; *Biotechnologiezenlruin Berlin-Buch GmbH, Berlin, Germany; #Lund University, Depanment of Medical Innammation Research, Lund. Sweden; Fas 46.46-I03110 Recognition of one or more epitopes on the mouse type-II-collagen (CII) by autoreactive T-cells is decisive in developing collageninduced arthritis (ClA) in mice. The main aim of this project \s localization of T-cell epicopes on the mouse CII. a maner of imporuinct not only for understanding the nature of CII autoimmunity in the mouse, but atso of importance in connection with clinical trials. We have two tools for the epitope analysis: starting from mouse type-II-collagen cDNA. kindly given us by Professor Vuorio (Turku), we cloned and expressed segments of the mouse CII gene. Some 80*0 of the mouse Cn protein sequence is covered by engineered pepUdes expressed as fusit)n proteins with the maltose-binding protein. These allow one to identify cryptic epitope;:. i.e. epitopes not revealed by immunization with intact protein. In addition »e obtained a series of synthetic peptides. covering the same 80^ on the protein, for epitope mapping. We localized I-Aq restricted autologous epitopes on the mouse type-D-collagen which have not been earlier identified. One epitope. giving the highest response, has been characterized further: T-cellis) responding to it appear to be endogenously primed and upregulated during inflammation, suggesting a role for this epitope in the pathogenesis of CIA. Furthermore, epitopes identified in this study share a common binding motive to the I-Aq molecule. The capacity of adjuvants to stifnulate antigen-presenting cells (APC) to produce cytokines is likely to be important during the initiation of the immune response. Iscoms and its matrix have been reported to induce the production of cellbound and secreted IL-1. Purified components of Quitlaja extracts have been used to develop novel adjuvant fomiulations based on iscoms. We studied the capacity of four new matfix formulations to stimulate production of IL-1 ifi an in wYro system. Murine peritoneal cells were cultured with various concentrations of the matrixes and the supernatants were tested for IL-1a by immunoassay. The most efficient fofmulation was 7.0.3. The influence of the physical conformation of the adjuvant on the capacity to stimulate APC was evaluated by the production of IL-1 mediated by 7.0.3 either in iscom form (containing influenza antigens), or in matrix form or as free component. Iscom was the most efficient form. The matrix and free components also stimulated IL-1 secretion but at lower level and highef concentrations were required. The in vitro results were confirmed by in wVo experiments. A significant pfopoftion of IL-1a+ splenocytes were detected by FACS in mice following treatment with iscoms. CD30 is a 120 kDa surface antigen expressed by Reed Stemberg cells of Hodgkin s disease, as well as by activated B and T lymphocytes. The immunologic function of CD30 is not yet determined It is known, however, that 85 % of the CD30+ T cells co-express the T-iielper (Th) marker CD4 To investigate whether CD30 defines a subtype of T helper cells, seven Pifyrosporum orbiculare specific CD4+ T cell clones from a patient with atopic dermatitis were assessed for CD30 surface and gene expression. The clones were activated with 0KT3 (anti-CD3 antibodies) and defined as Thl {n=2), ThO (n=2), and Th2 (n=3), according to their cytokine mRNA production detected by RT-PCR 0KT3 activation induced CD30 expression on more than 85% of the cells in all clones within one day as measured with tlow cytometry using fluorescein-conjugated anti-CD30 (Ber-H2, Dakopatts, Glostrup, Denmark) . A difference between the clones was noted in that the Th2 clones remained highly positive in CD30 reactivity whereas the other clones started to decline from day 3 Fourteen days after stimulation the Th2 clones expressed CD30 on 69 % of the cells, Ihe ThO clones on 15 % and the Thl clones on 6 % of the cells. Still, 24 day5 after stimulation, we determined approximately 40 % CD30+ cells among the Th2 clones but only 5% or less in the ThO and Thl clones The gene expression for CD30 as detected by RT-PCR correlated well to the cell surface expression of CD30. These data indicate that CD30 identifies activated CD4+ T cells and is sustained expressed in Th2 cells-CA^* MOBIUZATION tN PHYSIOLOGICALLY STIMULATED SINGLE T CELI5 CORRELATES WITH T CELL RECEPTOR SURFACE EXPRESSION. E. Blichfeldt, L.A. Munthe. J.S. R0tnes, B. Bogen. University of Oslo, Norway. Fax: +47 2285 1209. We have investigated the relationship between TcR expression and * signaling. Intracellular free Ca^* concentration li) was monitored, by use of an imaging system, in single Fura-2 loaded CD4* T cells stimulated with their physiological ligand, i.e. antigenic peptide bound to MHC molecules on antigen-presenting cells. 6 Thl clones derived from mice transgenic for the a^ TcR {Vol. Jal9; Vp8.2, D9I, J0L2) of the CD4* BALB/c T cell clone 4B2A1 were used. The specificity of the transgenic receptor is towards the 91-101 fragment of the MOPC315 Xl L-chain. presented on the MHC class III-E** molecule. The different Thl clones express varying levels of the transgenic TcR as detected by staining with the clonotype-specific mAbGBIB. Such nonhomogenousexpression of tbe transgenic TcR among the clones is due to endogenously rearranged TcR genes; i.e., most of the clones express two distinct TcR's. The relative intensity of staining of the Tbl clones varied from 56 % (3G11, a clone also expressing TcR V^S) to 100 %. compared 10 4B2A1 (the donator clone of the transgenes, expressing only one receptor). Both the maximum and mean [Ca^*]i increase evoked in the various cloned T cells by 91-101(>.2"')pulsed, I-E'* expressing L cell fibroblasts correlated linearly with the relative intensity of GB113 staining. This indicates that quantitative differences in TcR levels may be an important factor in T cell activation. After preparing and characterizing dextran-cholera toxin B subunit (CTB) conjugates, we studied their immunogenicity in mice following systemic or mucosal immunizations Dextrans of different molecular weights were conjugated via adipic acid hydrazide and N-succinimidyl-3-(2-pyritiyldithio) propionate (SPDP) to CTB Intranasal immunizations with conjugates evoked local IgA antibody responses against dextran in the lung and perora! immunizations did the same in the small intestine. Intranasal immunizations also elicited serum antibody titers that were significantly higher or equal to those afler subcutaneous immunizations. The antibody responses were dose-dependent. We have also studied the effect of preexisting antibodies to CTB on the magnitude of the anti-dextran response The results show that it is possible to evoke a local as well as systemic antibody response against a polysaccharide by conjugating it to CTB and using an appropriate route of immunization. Ilie effect of MHC-I ligation in luimaii T-cells was investigated using immobilized aiiti-M!IC-l niAb as a niodelsystem. Our data shows tliat MHC-I ligation induces proliferation in PBMC winch is co-stimulated by CD28 ligation. MHC-I induced proliferation ill T-ceils is dependent cellular interactions with non-T-cell. Exposure of PBMC for 24 hours to anti-MHC-1 niAb and subsequent cuUuie without stimulation induces proliferation and development of cytotoxicity in human T-cells. Tuiictional maturity is accompanied hy aiUi-MllC-l mAb induced upregiilatioii of tiie 1 CR and CD28 molecules 011 T-ceQs. Tlie upregulation of tlie CD28 molecule is greatly enhanced by simultaiiious ligation of the 1 CR. Tliese results clearly demonstrate that MIIC-I complexes may be impoiiaiit inducers of positive signaling in human T-cells. liiis is the first lepoit on a fiiiictionnl relationship between llic MHC-I complex and the CD28 receptor. Ihe lesirlts also suggest that when T-cells act as APC tliey may become activated upon co:itaci witli an antigen specific T-cell. T lymphocyte participation in infectious arthritis induced hy superantigen-producing staphylococci Bremen T. and A. Tarkowski, Depts of Clinical linmunolo^^y dt Rheumatology, University of Goleborg, S-413 45 Goleborg, Sweden. Fax:+46 31 826791. In order to study factors contributing to septic arthritis we have analyzed the influence of bacteria! superantigen production as well as T lymphocyte participation in the course of iS. aureus arthritis. Methods: Healthy Sprague-Dawley rats were inoculated i.v. with 5 superantigen producing 5. aureus strains and one nonsuperantigen producing 5. aureus control strain. The animals were assessed with regard to clinical course, and serum levels of lL-6, immunoglobulins and rheumatoid factors (Exp I). Rats inoculated with S. aureus AB-1 producing staphylococcal enterotoxin A were further analyzed by the use of histopathology and immunohistochemistry 1 year). The expression of IFNy, ILIO and IL12 was increased in SFMC as compared to RA peripheral blood mononuclear cells (PBMC). In addition, the expression was higher in RA SFMC than in PBMC from healthy control individuals. Although high levels of TGFp mRNA was found in SFMC, a significantly decreased TGFp / p2tTiicroglobulin (p2m) ratio was found as confipared to PBMC from patients and control individuals. IL4 mRNA could not be detected, either in SFMC or in PBMC. Cytokine expression in RA PBMC did not differ from control PBMC, with the exception of a decreased TGFp / p2m ratio in RA patients with early disease. Our findings of IFNy and IL12 mRNA expression but undetectable levels of IL4 mRNA, suggest that the synovitis is characterized by a type 1 immune response. The presence of TGF^ and ILIO mRNA indicates that immunosuppressive cytokines may also operate in the inflamed joint, although their level of expression rnay not be sufficient for down-modulation of the immune activation. Broinander/i.K.'. Ekinan,l..'.Kahler. G." Kopf, M." and NLycke*. 'DcpLol Medical Microbiology and immunology, Univ. of Cidleborg, S-41346 GOtcborg. Sweden. "Max-Ptanck-lnstitul Oil lmmunbiologie, Sttlbcweg 51 D-79108 freiburg. Cicrmany. Apart from being an important intlammatoiy cytokine pnxluccd hy many cetls in Ihe gut epithelium IL-6 has also been ascribed a regulatory role in IgA B ditTerentiation. In support of this, 11,-6 deficcnl mice (0,-6-/-) were found lo exhibit poor mueosal IgA responses to vaccinia vims infection, suggesting a defect in ihe mueosal immune system of these mice. We used IL-*-/-mice to investigate whether mucosal inuniine responses were impaired alter oral immunizalion with soluble protein antigens given together with cholera toxin (CT) as tlie adjuvant. Interestingly, we found similar capacity to respond with anti-KLH Sh'C in lamina propria and spieen after oral immunizalion in IL-6-/-and wilJ type mice Although, we have previously found thai CT up-regulates 0,-6 production by anligenpresenting-as well as gut epilhelial cells Ihe present study clearly demonstrated Ihat, as in normal mice, CT exerted strong adjuvant effects in IL-6-/-mice, indicating thai IL-6 is not required for the adjuvant mechanism of CT. Locul tgA responses appeared not lo be impaired in IL-6-/-mice because specific IgA liters and total IgA levels in gut lavage were of similar magnitude as in wild type mice. Furthermore, we found unaltered frequencies of membrane IgA cells and total IgA SFC numbers in PP as compared lo wild type mice. Also, level of I'NA-slaining of germinal centers in PP which co-localized mIgA cells was comparable between IL-6 -/-and wild type mice. Also, immiuiohistoehemical analysis of lamina propria demonstrated similar numbers of IgA cells in IL-6-/-and wild type mice. Our experiments using soluble protein antigens do not supptai the notion that lL-6 is critical for mucosal IgA responses. Protein phosphatases PP1/PP2A play a critical role in interieukin-2 induced, LFA-1 dependent, homotypic adhesion in human CD4 T cell lines. Brockdorff, J,, Nielsen, M., ''Svejgaard, A,, Oduin, N. from The Ceil Cybernetics Laboratory, University of Copenhagen Fax:+45 35327851 and §Tissue Typing Laboratory, State University Hospital, Copenhagen, Denmark. Besides its function as a growth factor for T lymphocytes, interleukin-2 {IL-2) induces LFA-1 mediated adhesion, migration, and extravasation of T lymphocytes, it is, however, largely unknown, how IL-2 receptors {1L-2R) are coupled to the LFA-1 adhesion pathway. Here, we show that calyculin A, an inhibitor of protein phosphatases PP1/2A, strongly inhibited IL-2 induced proliferation and homotypic aggregation in CD4 T cells In contrast, ligand induced down regulation of IL-2 receptors was not inhibited In in vitro assays, calyculin A inhibited PP1/2A-mediated dephosphorylation of phosphoryiase A, a physiological substrate for PFI/PP2A phosphatases Likewise, in vivo pretreatment with calyculm A mhibited PP1/PP2A phosphatase activity by aproximately 70 %. Inhibitors of other protein phosphatases, such as cyclosporm A, had no effect on IL-2 induced homotypic adhesion or proliferating In contrast, cyclosporm A almost compleately blocked CD3 mediated proliferation m parallel experiments In conclusion, we provide evidence that protein phosphatases PP2I/PP2A play a critical role in IL-2 induced, LFA-1 dependent adhesion The amino acid structure of RF produced in healthy immunized donors, patients with Rheumatoid Arthritis (RA) and patients with lympho-proliferative diseases were compared. Of 13 RF all using the DP-10 VH and Kv325 VL germline gene the usually extremely diverse CDRH3 regions show a remarkable restriction in length and sequence. Two pairs of RF have a particularly high homology in this region. The CDRH3S of RF-MR1 from a healthy immunized donor and Bor, a mixed cryoglobulinemia RF, have only two amino acid differences. Further, the CDRn3s of two sets of clonally related RF, one found in a patient with RA and one found in a healthy immunized donor have only three amino acid differences from each other. Even more restriction was seen in the CDRH3 in three RFs, one from each of these three conditions, using another heavy chain germline gene, DP-54. The homologies seen in the V regions of these RF in both health and disease, suggests that they have been selected against a very similar, if not identical epitope at some stage in B cell development. In the present study, the V8 and VY segment usage of-yB T cells in PB and ainong intraepithelial lymphocytes (IEL) of non-inflamed and inflamed intestine of patients with inflammatory bowel disease (IBD) was analyzed. Cell surface expression of the protein and gene transcription was assayed by using flow cytometry using a panel of V gene subtype specific mAb and PCR analysis using V gene specific primers. The T6 T cell receptor (TCR) expressing cells were increased in PB of patients with Crohn's disease (CrD), but not in patients with ulcerative colitis (UlC). In patients with IJIC, higher frequency of ^5 T cells was found in the non-inflamed than in the inflamed intestine. In IBD patients, the V51 subset was clearly dominant among i^ T cells in the intestine both as analyzed by flow cytometry and by PCR analysis. A major fraction of ^6 T cells expressed VY8, but a significant fraction was stained also with mAb directed against Vyl, VY3 and Vy^ products. In normal PB the V78 subset was a minor subset only expressed on approximately 5 % of the 75 T cells, whereas in PB of IBD patients the VY8 subset was highly increased and constituted around 25 % of all 76 T cells. Furthermore, the V51 subset was highly increased in PB of IBD patients. These results indicate that there is an expansion of i6T cells in PB of IBD patients which resemble the dominant "gut-like" phenotype. Interestingly, we found some patients with acute IBD of short disease duration, who had an extremely large proportion of ^5 T cells in PB expressing two distinct Vy proteins on the cell surface. In vitro secretion of Interleukin-4 and Interferon-7 in response to specific allergens in peripheral mononuclear cells frotn atopic and non-atopic individuals. CD4+ T cells can be divided into two major subsets, T helper (Th)l and Th2 cells. Interleukin-4 (IL-4) is produced by Th2 cells and Induces switching to Immunoglobulin E which induces histamine release from mast cells in allergy. Interferon-y (IFN-y) produced by Thl cells counteracts the effects of iL-4. In this study we wanted to investigate whether the elevated specific IgE-Ievels in allergic patients were reflected by an increased number of IL-4 producing cells in response to allergens. The number of IL-4 and IFN-y producing cells (ELISPOT) in response to specific allergens (birch polleji and hair from cat) in peripheral mononuclear cells from atopic and healthy individuals were compared. In the atopic donors there was an increase in the number of IL-4 producing cells in response to specific antigen as compared to healthy donors (p<0.(101). When the allergen was combined with a suboptimal dose of PHA, there was a synergized increase in the number of IL-4 producing cells in the atopic donors, which was not seen with the number of IFN-y producing cells. In conclusion, these data indicate that the elevated IgE levels in atopies are reflected by an increased number of allergen specific IL-4 producing cells. The aim of the present study was to investigate the immune response in rats orally fed ovalbumin (OVA) compared to control rats fed a normal diet after immunization with OVA and an orally challenge with a bacterium producing OVA. Rats were made partially tolerant by feeding an OVA-containing diet from weaning for one to four consecutive weeks. Controls were weaned onto a standard diet without OVA. One week after ending OVA feeding, the animals were immunized with a mixture of OVA and human serum albumin (HSA) in Freiind 's complete adjuvant (FCA) ai one site of the back. Blood was collected two weeks later and the delayed type hypersensitivity (DTH) response was evaluated by challenging the rats with OVA and HSA in the ears. Five days thereafter the animals were ihen orally challenged by colonizing wiih an Escherichia coli producing OVA. All rats fed OVA showed a significantly suppressed DTH response to OVA compared to the rats fed standard diet. Funhermore only lhe rats fed OVA. showed bystander tolerance i.e. had a significantly lower DTH response to HSA than the controls fed a normal diet. Immunohistochemistry of sections from the intestine showed a significantly higher IL-2 receptor expression, mainly in goblet cells and greater MHC class II expression both in the villous core and epithelium in the controls than in lhe rats fed OVA. Results from the present study showed that rats partially tolerant to one antigen can show a suppressed T-ccU response to an unrelated antigen given simultaneously. This also leads to a reduced local inflammatory response when colonized with a bacterium producing OVA. Efficient production of protective anti malaria antibodies requires an intact and functional T-cells system. In mice, regulatory CD4+ T-cells can be divided into two functionally distinct subpopulations denoted THI and TH2 cells based on the cytokine profiles they produce. In human, recent studies also suggested functional lieteiogeneity of CD4+T-cells. THI cells produce IL-2 and IFN-Y and TH2 cells produce IL 4. lL-5 and IL-13. THI cells mediate DTH and aie involved in inflammatory reactions, while TH2 cells regulate specific antibody production. We have reported earlier that, in individual donors. iL-4 secretion was not associated with either proliferation or IFN-y production, but was well correlated with elevated concentrations of serum anti-malaria antibodies (IgG) to the same peptides u.sed for T-cell activation. Since IL-4 is an important factor involved in immunoglobulin class switching from IgM/IgG to IgE. it was of interest to study the role of IL-4 in the regulation of anti-malaria IgE production. For this purpose, two groups of donors were selected. One with high and the other with low serum anti-malaria IgE antibody levels. Cells from the donor group with high serum anti-malaria IgE antibodies produced significantly higher IL-4 in response to a recombinant P155/RESA and a crude P.falciparum extract. Similarly individuals from this group had a higher potential to produce IL 4. The levels of serum anti-malaria IgE antibodies were well conelatcd with the capacity of cells to produce IL-4. These findings suggest a regulatory role of IL-4 in the production of anti-malalia IgE antibodies. The role and biological significance of these antibodies in malaria are presently under investigation. The enzyme-linked immunospot (ELISPOT) assay was used to enumerate the number of IFN-y and IL-4 producing cells after in vitro stimulation with the malaria antigen Pfl55/RESA or synthetic peptides corresponding to its major T-cell epitopes. Two groups of naturally primed individuals living in rural areas of Burkina Faso were studied. The donors comprised one group of healthy (non-parasitemic) mainly adult people and one parasitemic mainly younger people. IL-4 producing cells were detected in response to PHA but no such cells were detected in response to the malarial antigens. The most frequent IFN-y responses were seen with Pfl55/RESA. Thus, after stimulation with this antigen 52% of the donors responded positively in the ELISPOT assay, while only 27% responded to the synthetic peptides, suggesting that the r-Pfl55/RESA contained T-cell epitopes not covered by the peptides used in this study.The number of IFN-Y producing cells in response to the malarial antigens did not differ between the two groups. However, IFN-y levels found in sera from the parasitemic individuals were significantly higher than in those from healthy donors. This latter finding and the lack of differences seen in the number of IFN-7 producing spots in the two groups indicate that IFN-Y producing cells may have sequestered to other organs in the parasitemic group. Collagen-induced arthritis{CIA) is an experimental model for Rheumatoid Arthritis. In the DA rat it is induced with rat collagen type II(CII) which gives a chronic disease dependent on both B-and T-cells.The aim of this study was to investigate the antibody reactivities and the cellular picture in the synovial flutd(SF) and compare them with RA data. CIA was induced in DA rats by intradermal injections of Cll. At the peak of disease, SF was withdrawn and synovial tissue(ST) dissected. ST sections and SF cell smears were stained, and SF was tested in ELISA for antibodies to Cll and to heat shock protein(Hsp). In addition, we investigated the presence of Rheumatoid factors(RF) and gamma interferon(g-INF). The major cell population in SF was granulocytes, but plasma cells and lymphocytes could be detected. High anti-CII-and RF-ievels were observed both in the SF and systemically. Notably. RF levels were also increased in normal DA rats. A moderate increase in anti-Hsp antibody levels were recorded in arthritis both in SF and systemically. No detectable levels of g-INF were seen in SF. In conclusion, the appearance of cells in the SF is similar in CIA in rats and in human RA. The antibody production data indicate an unusually high production of RF in the arthritis-prone DA rat, but gives no support for a strong production in SF of anti-Hsp or anti-Cll antibodies. No detectable levels of g-INF were recorded in SF. The murine dendritic cell line D2SC/!, immortalized with relrovirus, was stimulated by the viruses Herpes simplex virus (HSV), Sendai virus (SV) and the bacteria E. coli and S. aureus Cowan I (SAC). All induced production of IFN-a and -3 in the D 2SC/1 cells, determined at 24 h by immunoassays. IFN-p levels were usually higher than IFN-a level.s. The HSV was clearly the most efficient inducer. Precultivation of D2SC/1 cells with mGM-CSF (24 h) and mlFN-jJ (2 h) markedly enhanced IFN-a/p production, except for SV. The latter inducer therefore uses another mechanism than e.g. HSV. Cells containing IFN-a and -|i mRNA at 6 h were identified by in situ hybridization with 35Slabelled cRNA probes. Most cells showed a relatively low and uniform IFN-a/p mRNA expression, but infrequent cells ( 100-fold reduced binding affinity for MHC class II compared to native SEA. The fusion protein was successfully used to direct T cells to B-CLL cells coated with different B lineage specific (CDI9. 20) or associated (CD37. 40) mAbs. The PA-SEAm protein was lO-IOO-fold more potent against mAb coated compared to uncoated HLA class Il-t-B-CLL cells. No correlation was seen between the amount of mAb bound to the cell surface and sensitivity to lysis. Preactivation of B-CLL ceils by phorbol ester increased their sensitivity, and lysis was dependent on ICAM-1 molecules. However, no preactivation of the target cells was needed when a cocktail of 2 or 4 mAbs was used. Circulating leukemia and spleen cells were equally well killed. We conclude that tbe natural target specificity of SEA -MHC class IIcan be reduced by mutagenesis and novel binding specificity can be introduced by linkage to tumor reactive mAbs. Our findings encourage the construction of recombinant SEA mutant fusion proteins for specific T cell therapy of hematopoetic tumors such as B-CLL. CRP is the prototypic aciJte-phase serum protein in man. Biological effects of CRP include its interactions with wide ligands spectrum including phosphorilcholine containing molecules, some sera proteins, nuclear material and so on. We have found that IgG immobilized on the solid phase binds soluble hiiman CKV. The binding of CRP by IgG was relatively sensitive to ionic concentration, being maximal at physiological NaCl concentrations and low pH and was calcium-independent. Dissociation constant of this interaction was about 5,910"^ M. The interaction depended slightly on IgG subclasses and IgG species. The immune complexes ofrabbit IgG antibodies to numan albumin with this antigen immobilized on the solid phase also bound CRP. It is possible tbat all or most of the CRP-binding capacity was localized on Fc-fragment of IgG. That is confirmed by the following facts: 1) (Fab')2 fragments bound CRP very slightly; and 2) interaction of IgG antibodies to albumin with this antigen was not inhibited by CRP. From the other hatij soluble aggregated IgG inhibited binding CRP with solid phase immobilized IgG. Also CRP changed complement activation ability of aggregated IgG. It possible that CRP may be involved into immune complexes formation via IgG and change the immune complex methabolism. Grdic. P.. Hernqiiist. E . and Lyckc N . Dcpl of Medical Microbiology and Immunology. University of GOtcborg. S 413 46 Gateborg, Sweden. Oral tolerance has been associated wiih systemic suppression excricd by CDs' cells as well as by CD4' cells of lhe Th2-phcnol>-pe. We investigated whether the complete absence of CDS T cells or IL-4. responsible for Th2-difTerentiation, would affect lhe ability lo respond wiih oral tolerance (OT) to fed proicin antigens. Therefore wildlype and gene targeted CD8-/-or IL-4-/-mice were orally fed KLH and subsequcnily challenged s>stemically with antigen plus BJBl adjuvant. Interestingly, ali llircc types of mice normal, CD8-/-and Th2-{II-4-/-) deficient mice exhibited similar levels of s>stemic tolerance after antigen feeding as compared to orally PBS-trcated eontrol mice: Both splenic anti-KLH SFC and T cell IFN-y production were significantly reduced in orally fed mice of all strains irrespective of the complete lack of CDS'-or Th2 (IL-4 -/-) ceils. By conlrasl. oral feeding of KLH in Ihe presence of cholera toxin (CT) abrogated oral tolerance in normal, CDS-/-as well as in IL-4 -/-mice. This finding clearly demonstrates thai CT can break oral lolcrance in lhe complete absence of CDS T cells, indicaling ihat the imnnmomodulaling effect of CT may not require CDS T cells. Moreover, the OT-breaking ability of CT in IL-4-/-mice was achieved even though these mice have been found impaired in their ability lo respond to spccifie mucos;il imniuni/alion using CT as lhe adjuvant . The mechanism responsible for OT and CT"s effect in these models are currently being characterized. Oral administration of DSS to mice induces a disease with clinical and histological features reminiscent of human ulcerative colitis. We have investigated the local production of cytokines during the development of DSS colitis in mouse strains representing different forms of immune dysfunction, such as athymic (nu/nu) mice and mice with severe combined immunodeficiency (SCID). DSS was administered in the drinking water to specific pathogen free CD-1(BR), CD-1 (BR) nu/nu and CB-17 SCID mice. The mice were killed after 7 days and their intestines were cut out. Colon length and fecal water content were recorded and the colon was cut into small pieces and placed in 2% saponin buffer for extraction of cytokines. The cytokine content in the extract was measured using ELISA methods specific for IFN-y, IL-6 and TNF-a. Colitis in euthymic and athymic animals, presented as shorteriing of the colon and diarrhea, was associated with elevated levels of IL-6 and to some extent IFN-y and TNF-a in colon tissue. DSS induced severe colitis also in SCtD mice (n=5) but no cytokinos were deteoted in colon extracts. Cytokines in colon tissue (pg/mg tissue, ±SD) CD-1 (n=5) CD-1 nu/nu ( In BlOA mice, the response to pigeon cytochrome c (PCC) is largely restricted to T cells expressing vaU/v33 TCR. Less than 1% of the vall/vP3 cells possess canonical sequences associated with respotise to PCC in vivo. We quantified the occurrence of val l/v|33 T cells within splenic compartments by double immunohistology, following PCC immunisation The background level of vall/vP3 cells in follicles is insignificant, but comprise approximately 0.5% of the cells in the T zones. T cells expressing va]l/vP3 start to accumulate 4 days after primary immunisation within both the germinal centre and mantle of secondary follicles. This specific foliicular response continues for at least a further 10 days. Up to 10% of foUicular T cells take up 5-Bromo-2'-Deoxyuridine following 2 hour pulse labelling, indicating that these cells are in active proliferation within the follicle No changes in vall/vP3 T cell numbers occurred in follicles in response to chicken y-globulin. In the T zones the number of val I/vp3 T cells rises transiently between days 4 and 7 after immunisation, then falls rapidly to levels not significantly different from those in non-immunised mice. At the peak of the T zone response the number of val l/vp3 cells is double that of background The data indicate that the clonal expansion of antigen-specific T cells in the follicles is at least as important as that in T cell tich areas. Surface display compared to periplasmic expression of a malarial antigen in Salmonella typhimurium and its implications on the immunogenicity D. Haddad, S. Liljeqvist', S.Kuniai-. S. Stihl', P. We have used two different expression systems for the production of an 80 aminoacid sequence from the C-terminus of the Flasmidium falciparum blood stage antigen Ffl55/RESA in an attenuated Salmonella typhimurium Aro A vaccine .strain. Upon expression, the malarial sequence is targeted either to the periplasm as a soluble fusion protein containing two igG-binding domains from the staphylococcal protein A (ZZ) or to the bacterial surface, inserted into the outer membratic protein A (OMPA) derived from Escherichia coli and Shigella dysenteriae. Both ZZM3 and 0MPAM3 were stably expressed in the periplasm or on the surface of Salmonella respectively, but the ZZ expression system yielded 100 times more malarial immunogen than the OMPA system. Live recombinant Salmonella expressing ZZM3 or 0MPAM3 were used to immunize mice. The ZZM3 hybrid induced antibodies to M3, ZZ and to Pfl55/RESA whereas, 0MPAM3 induced similar levels of antibodies reactive to M3 but not to Pfl55; neveitheless, both recombinants induced a memory response to M3 and lo Pfl55/RESA. The high levels of M3 produced by the ZZ system make it suitable for the expression of heterologous antigens in Salmonella. However, in spite of the quantitative difference in M3 expression, both the ZZ and OMPA carriers provided comparable immunogenicity to M3. indicating that the display of M3 on the surface of Salmonella is equally efficient in priming the immune system as is periplasmic targeting. IgE antibodies are able to enhance the specific antibody response in mice via the low affinity receptor for IgE (CD23). B cells and foliicular dendritc cells (FDC) are the only murine cell types expressing CD23, and one of the suggested modes of action of IgE/CD23 is to increase the ability of B cells to present antigen to T cells. Another possibility is that FDC capture the IgE/antigen-complcxes and present these directly to B cells. One approach trying to differentiate between these two cell types is to make use of the MHC restriction of the IgE-mediated enhancement. Mouse strains with the H-2b haplotype are nonresponders in this system, wliereas H-2''.M-k.s.p are responders. Nonresponder C57BL/6J mice are irradiated and reconstituted with spleen or bone marrow cells from (C57BL/6J x DBA/2)F1 mice which are responders in this system. The chimeric mice are then immunized with IgE and antigen, and the production of antigen specific antibodies is tested in ELISA. Only the B cells will express CD23 since FDC do not appear to be of bone marrow origin, and FDC are very fragile and will be destroyed when spleen cell suspensions are prepared. A positive result in the chimeric mice would suggest that B cells are required for Igl; mediated enhaticement. Preliminary results indicate that mice reconstituted with spleen cells are nonresponders. wherea.s mice reconstituted with bone marrow cells are responders. The phenotype of intraepithelial lymphocytes (IEL) in human jejunum, ileum and colon was studied in situ as well as after isolation. y6 T cells constituted on average 30% of IEL at all levels of the intestine. The majority of these 78 IEL were CD4" CD8" and preferentially expressed V8IV78. In contrast, ap IEL showed a large variation in frequency and phenotype between different gut levels. CD8+ ap IEL dominated in jejunum while cells with the unusual T cell phenotype, CD4-CD8'TCRap+, constituted a major population of colonic lEL. CD4"*" aP IEL were equally represented as a minor population, at all three levels of the gut. These data suggest that y8 IEL are involved in surveillance of the epithelial cells whereas ap IEL may participate in immune responses to luminal antigens. A small proportion of IEL with thymocyte-like phenotypes (CDl + TCR/CD3-. CD2+ TCR/CD3-as well as CDI+ TCR-^+ and CDl + TCRap+ IEL) were detected in jejunum. Furthennore. expression of recombinase activating gene-1 (RAG-1) mRNA was detected in jejunal but not in colonic IEL as determined by RT-PCR. RAG-1 expression was confined to jejunal IEL with immature phenotypes (CD2"*'TCR/CD3' and CD3'*"/rCR" IEL). This strongly suggests that human small intestinal epithelium is a site for extrathymic T cell maturation. Recent reports have suggested interleukin-4 (IL-4) as a selective inducer of VCAM-! on human umbilical vein endothelial cells (HUVEC). VCAM-1 is an endothelial ligand for VLA-4 (CD29CD49d) expressed on lymphocytes, monocytes and eosinophils but not on neutrophils. IL-4 could thus serve to recruit selectively VLA-4-expressing leukocytes into areas of inflammation without attracting neutrophils. In a new culture system for human intestinal microvascular endothelial cells (HIMEC, in press. Gut) we arrive at a different conclusion. Protein expression was examined by cell-ELISA and flow cytometry. mRNA was examined by reverse transcription-PCR. Adhesion of leukocytes was measured in an fluorescence based multiwell assay (Calcein-AM). VCAM-1 was upregulated in a dosedependent fashion on HIMEC after stimulation with LPS (0-1 f4g/m\). IL-lp or TNF-a (both 0-1000 U/ml) and displayed characteristic time kinetics similar to HUVEC. IL-4 (0-1000 U/ml) on the other hand appeared lo exert only a negligible stimulatory effect on HIMEC compared with HUVEC. However, IL-4 induced raised mRNA levels compared with unstimulated controls. Adhesion studies were performed to evaluate the functional implications of these findings. Our data show that IL-4 is not able to induce VCAM-1 selectively in HIMEC. Preferential recruitment of mononuclear cells to the gut may thus be mediated by other hitherto unknown mechanisms. The role of infected monocytes in measles pathogenesis i. *; still poorly understood. We studied measles virus (MV) infection in cells of the human myelold lineage with different matunilion stages, from human bone marrow granulocyte/macrophage progenitors to macrophages. MV was able to infect myeloid progenitors CFU-GM (granulocyte-macrophage colony forming units), monocytes and monocyte-derived macrophngcs. MV infection in granulocyte-macrophage colonies of progenitor ccll.s was productive, unlike in monocytes and macrophages. which supported virus RNA and protein .synthesis without releasing infectious virus. Stimulation of infected monocytes by inlerleukin-3 and/or granulocyte-macrophage colony stimulating factor was not sufficient to change a restricted infection to a productive one. Maturation of infected monocytes to macrophages did not enhance measles virus production either. In contrast to the results obtained with monocytic cells, human promyelocytic and promonocytic cell lines HL-60, U-937 and THP-1. each of which represents a different stage of maturation, supported virus replication, and TPA (12-0tetradecanoyl-phorbol-13-acetate) -induced maturation of the cells to the macrophage-Uke did not markedly alter virus replication. Mjclin basic protein (N/lliP) is a candiditic auluanligen in muKipk sclerosis (MS) Analysis ol" a (ctrarepacl seqiicncc 1 Kb 5' of itie MBP gene in Finnish .MS palicnls has indicated both linkage and association with certain alleles ol a 1.2 Kb PCR ainplificalion fragmcnl. However, ihcse findings have nol been tonlinncd in subsequent analyses ol' North American and British patients. Likewise, analysis of HLA genes in MS has shown linkage in Finland and dose segregation in a few Swedish families in contrast wilh negative results in rutxnt investigations elsewhere. This could indiciitc ethnic differences widi btlween Nordic .MS and Western I-,urnpcan MS, i.e. a geographic genetic heterogeneity. Thus it is of interest to study the MBP gene in a MS jHipuhition as simitar a^ [ussible to th Finnish. If) Swedish nuclear families with 2, 3 or 4 patients with MS were itivcsligatcd for an 8 allele ainplirieation fragment (length 220-248) of the lulrarepeat 5' ol" the MBP gene, as well as 100 patients wilh relapsing remitting MS and 28 patients with primarily chronic progressive .MS iind 'J4 healthy controls. In this analysis, I'C.R primers were labeled wilh '^S and products visualized by autoradiography rwo-lociis linkage analysis laikd to reveal any linkage in the investigated Taniilies, regardless of differeni iixxlcls of inheritance, gene frequencies and liability classes. Best fit revealed a iii:)'uiive I.OD-score. Neither were there any differences in the distribution of alleles between familial or sporadic cases, nor between elinical subgroups, and uHilrols. Our conclusion is ihac the MBP gene does not appear to inlluence the susceptibility to MS in Swedish p.-itients This in contrast with Finnish [Mtieiils but in good agreement with other ethnic groups of tiuropean origin Unlike type 1 diabetes, where the gene coding for the ciindidale autoiuitigen msiilin has been firmly linked to susceptibility, the MBP gene, the primary candidate autoantigen in .MS. docs not seem to have this role. MALARIA. n. Helmby, H. Perlmann, M. Troye-Blomberg. and P. Perlmann. Department of Imiiuinology, Stockholtn University, S 106 91 Stockholm, Sweden. Fax;+46 8 157356. Humans repeatedly infected with the malaria parasite PlasnuuHiim falciparum have elevated serum levels of IgE. In order to investigate the mechanisms of IgE elevation In malaiia we studied mice itnmtinized with the rodent malaria Plasmodium chabaudi chabauili for total IgE and IgE antimalarial antibodies. Hyperimmune mice had elevated levels of total IgE in their sera as compared to non-infected cotitrol mice. The levels of total IgE were comparable to those of mice repeatedly infected with the helminth Schistosoma niansohi, known to give rise to strongly increased IgE production. Sera taken from mice 3 weeks after one infection with P.chabuudi showed almost no IgE-elevation. indicating that prolonged or repeated exposure to the parasite is necessaiy for the itiduction of an igR response. When tested in immunoblotting and ELISA, the .sera were found to contain IgE antibodies specific for a vatieiy of P.chabaucH antigens. The results indicate that malaria infection is directly involved in the induction of enhanced IgE levels. IgE antibodies specifically enhance the antibody response to BSA-TNP in vivo via CD23, the low affinity receptor for IgE. The enhancement of the BSA-response is MHC-restricted, since mouse strains of H-2''-*l-P4-s haplotype are responders, whereas H-2b mice, such as C57BL/6, are not. This nonresponsive phenotype is recessive because it is lost when the animal is crossed with a responder strain. From studies of recombinant inbred strains we have found that nonresponsiveness is linked to the A locus of MHC class II. However, the region between the crossover points is large enough to harbour other genes in addition to those encoding the A molecule. We have crossed C57BL/6 mice which are transgenic for either the Aa or the Ap gene of H-2k haplotype. The offspring was tested by Southern blot and expression of A^ was analyzed by flow cytometry. Double transgenic, double negative as well as {C57BL/6 x CBA)F1 mice (BCFl, H-2b/k) were immunized with IgE and BSA-TNP. After 1 and 3 weeks, animals were bled and sera assayed in ELISA. Neither the double transgenic mice nor the control littemiates could produce BSA-specific antibodies, whereas a 150-fold enhancement was detected in BCFl animals already after 1 week. Thus, tack of IgE/CD23mediated enhancement of the BSA-specific antibody response in H-2b mice may not be due to the A molecules themselves, but to other gene products encoded nearby. The purpose of this study was to establish whether a pneumococcal type 4 polysaccharide protein-conjugate vaccine {PS4TT) gives a better antibody response and protection than a conventional 23-valent polysaccharide vaccine (Pneumovax^N) Two inbred strains of mice {BALB/cABoni and C57BL/6JBom) and one outbred strain (Hsd NIHS) were immunized with PS4TT or Pneumovax, and antibody levels to type 4 polysaccharide were analysed Survival and bacteremia were determined after i.p. challenge with 100 LDjo of the highly virulent pneumococcus type 4. 7/10 NIHS mice and 3/10 C57BL/6J mice showed an IgG response to the conjugate, whereas no BALB/c responded In contrast, all BALB/c mice developed an IgM response Protection with the conjugate was 100%, 89% and 73% in NIHS, C57BL/6J and BALB/c mice, and protection with Pneumovax was 80%, 80% and 33% respectively. Conclusions. The mouse model allows distinction between different levels of protection. Generally, PS4TT induced a better imtnune response and protection than Pneumovax. There was a discrepancy in individual mice between antibody response and protection. The restored proliferative response in mercury lowresponder mice in vitro H Hu and G. Molier, Department of Immunology, Arrhenius Laboratories for Natural Sciences, Stockholm University, S-106 91 Stockholm, Sweden Fax:+46 8 154163 Abstract In vivo mercury causes autoimmune disease in the susceptible mice and rats. However, it is still not known how mercury ions primarily activate the immune system Previous studies tTom our lab have shown that in vitro mercuric chloride induce a high proliferative immune response in mercurysusceptible mice, i e A.SW and Balb/c mice, in which both CD4' and CDS' T cells were activated, while in resistant mice as DBA/2 mice, a low proliferative response was induced and only CD8* T cell were activated. We expanded the studies and found mercuric chloride induced a high proliferative response in other mercury-susceptible mice like SJL mice (high responder). and always a very low response in resistant mice like A/J mice or CBA mice (low-responder). After culturing spleen cells from low-responder mice with mercury for certain period of time, we washed the mercury away and the recovered cells were incubated for one day or two days more. We found that a high proiiferative response was restored in these low-responder mice, i e. DBA/2, A/J and CBA mice The restored proliferative response was higher in DBA/2 than CBA and A/J mice The phenotype analysis showed that in the restored response both CD4' and CD8^ T cells from low-responder mice were activated to proliferate. Our results suggest that in vitro spleen lymphocytes from both in vivo mercury-susceptible and resistant mice have the potential to respond to the mercury primarily. Acute P. falciparum is associated with a pronounced reduction in the ability of peripheral T cells to respond to antigenic stimulation in vitro, while the mechanism behind this T-cell dysfunction is not fully understood. One possible cause is in vitro production of the immunosuppressive cytokine IL-10, previously implicated in peripheral T-cell non-responsiveness in acute visceral leishmaniasis. In the present study we have examined the role of IL-10 in malaria induced T-cell non-respon^ivetiess. Our preliminary results indicate that: L In vitro proliferation can nol be restored by addition of a blocking monoclonal antibody to IL-10. 2. No inverse relation between IL-10 production and proliferation in vitro can be detected. 3. Significant reduction of in vitro lymphoproliferation of healthy T cells requires tnuch higher levels of IL-10 than those measured in patient T-cell supernatants. These results indicate that the well-established in vitro nonresponsiveness of peripheral T cells from acute P. falciparum malaria patients is unlikely to be due to IL-10 produced in vitro. Results of these and further experiments oti the mechanisms in malaria-induced T-cell noti-responsiveness will be discussed. Psoriasis vulgaris has HLA associations. We have previously defined certain HLA risk haplotypes for psoriasis. The aim was now to see if the risk haplotypes correlate with the clinical disease. The series consisted of 64 patients and the clinical parameters were age of onset, family history of psoriasis, arthritis and clinical treatment periods at hospital. The HLA risk haplotypes including Cw6, DR7 and DQAl*0201 have heen foimd in 30% ofthe patients. The presence of these risk haplotypes correlated with earlier age of onset (P=0.027) and family history of psoriasis (P=0.022) but not with arthritis or the number of clinical treatment periods. Conclusion: HLA haplotypes with Cw6, DR7 and DQAl*0201 increase the penetrance of psoriasis genes in the family. The risk haplotypes precipitate the onset of psoriasis at an earlier age. Y. Jiang and G, Mbller, Department of Immunology, Stockholm University, S-106 91 Stockholm, Sweden, Fax: +46 8 154163, Mercury-induced autoimmunity has been described in susceptible strains of rats and mice It is still unclear how mercury modulates the immune system under a stringent genetic control, leading to autoimmune development in predisposed individuals (responders) Our previous in vitro studies have demonstrated that HgCb was able to activate murine lymphocytes from both responder and nonresponder strains We have also shown that HgCb induced both proliferation and transformation of CD4' and CD8' T cells from responder BALB/c mice, in contrast, only CDS' T cells from nonresponder DBA/2 mice were transformed In the present report, we have studied the expression of CD69, the very early activation antigen, as an early activation marker on in vitro HgCljstimulated lymphocytes We found that the expression of C[>69 was upregulated as early as 6 hrs after exposure to mercury on both CD4' and CD8' T celts from either BALB/c and DBA/2 mice However, the upregulation on BALB/c CD4' cells was long-lasting and disappeared by day 4 In contrast, the upregulation on DBA/2 CD4" cells was veiy transient, which could not be observed at 24hrs of culture A similar dynamic expression of CD69 was observed on CD8' celts from both straias, i e,, after an early transient upregulation, the expression of CD69 decreased to the level as on mercury-unstimulated cells, but was upregulated again in the late period of culture However, the second wave of upr^ylation was earlier (from day 4) and stronger on DBA/2 CDS* cells, TTius, consistent with our previous findings, CD4 T cells were preferably activated in responder BALB/c strain, suggesting that helper T cells play a crucial role for the immunoloipcal effects caused by HgCb and determine the ability of differait strains to respond to HgCt:, Weak response of immunosuppressive cytokines (IL-10, IL-4 and TGF-P), cause severe protracted and relapsing experimental autoimmune encephalomyelitis (SPR-EAE) in DA rats, lssazadeh S, Mustafa M,!,, Lorentzen J.C, Hdjeberg B,, Olsson T, Institute for Molecular Medicine, Karolinska Hospital, Karotinska Institute, Sweden. Fax: +46 8 7744822 DA rats immunized by svngcnic spinal cord emulsified in FIA develop a severe protracted and relapsing t>pe of EAE, a model for MS, Here we used an in silu hybridizalion to siudy the dynamies of cellular mRNA e,'Lpression of cjiokines in the central nervous system (CNS) and lymphoid organs of SPR-EAE in DA rats and acute monophasie EAE in Lewis rats, A number of proinflammalon' cylokines: T helper (Th)-l (interferon /IFN-y, tumor necrosis factor/ TNF-[i) and inlcrleukin /IL-12, TNF-a, IL-l[i and cytolysin and putative immunosuppressive cytokines; Tli-2 (IL-4, IL-UI) and transforming growth factor /TGF-p was investigated, A high number of cells e,\pressing mRNA for IL-12, IFN-7, TNF-a, IL-lp, TNF-p and cjtolysin was detected before clinical signs of SPR-EAE and then dramatically increased at acute phase of SPR-EAE, All these cytokines were also seen augmented at relapses, furthermore lL-12 and TNF-p were found elevated even at remission. Interestingly all proinflammatory cytokines were also detected in high numbers in acute monophasie EAE at height of the disease While TGF-P and IL-10 raised during recovery' phase of acute monophasie EAE, In contrast these two c>iokines as well as IL-4 were almost absent in SPR-EAE in remission. Thus we conclude that (i) ThI cytokines. lL-12. TNF-a, IL-lp and cylolysin are related to initiation and efTector phase of atitoaggressive ncuroinfiammation in both monophasie and SPR-EAE, (ii) TGF-P and IL-10 are associated with disease recovery in acute tnonophasic EAE in Lewis rats There is a great interest in developing vaccines against sexually transmitted diseases (STD) eg, infections caused by Chlamydia and HIV This requires knowledge about how to stimulate and how to measure immunity in the genital mucosa We chose cholera toxin (CT) and its B subunit (CTB) as model antigens since especially CT is known to elicit a strong immune response at different mucosal sites CT/CTB is also interesting to use as a carrier/adjuvant to antigens chemically coupled to CTB To fmd out how to immunise to get an optimal local IgA response we immunised mice by several different routes The repeated local vaginal immunisations, alone or in conjunction with rectal immunisation, and the intranasat route seems to be the best way for inducing an IgA response in the genital mucosa The responses were loralised to the vagina and cervix, whereas responses in the uterus and the fallopian tubes were low or absent. Thus it seems that to achieve a local IgA antibody response in the genital tract repeated vaginal or intranasal immunisations have to be made. Based on this knowledge we have also coupled peptide antigens from Chlamydia and been able to induce significant speciftc IgA responses in the genital tract mucosa APO-1/Faj RECtnOR EXPRESSION IN HUMAN AND EXPERIMENTAL SJOGREN'S SYNDROME. R. Jonsson and K. Skarstein. Brocgelmann Res Lab, Univ of Bergen, N-502] Bergen. Norway. Fax; -H1755974689. Defective /^ as-mediated apoptosis of the immune system may contribute to the pathogenesis and development of autoimmune diseases such as SjOgren's syndrome (SS). With ihis background we have analyzed the distribution of the Fas protein in salivary glands of human and murine SS with anti-fax mAb and immuno-histologicai technique (ABC-immunoperoxidase). Inflamed salivary gland tissue was obtained from patients with primary and secondary SS and from MRL/lpr mice. The human SStissue presented a very diversified Fas staining pattern. The distribution of fas-positive cells among the infiltrating mononuciear cells was also disparate ranging from very few but mostly -30 % of the MNC. In the human SS tissue staining was mostly confined to acinar and ductal epithelium, but also very intense in the intcrstitium indicating presence of soluble Fas. Despite mutation of the Fas gene in the MRL/lpr strain, there was an expression of lhe apoptosis related Fas protein increasing with age in salivary glands of these mice (~1-3% of the MNC). Furthermore, specific antibody therapy against oligoclonally expanded TCR Vp families did not significantly influence the Fas expression. Overall, the Fas expression was much lower in the autoimmune mice than in humans. The pattern of fas expression in glandular tissue in SS suggests an involvement of this apoptosis-signalling receptor molecule in the pathogenesis and/or lymphoid cell accumulation. We have developed an unbiased single-sided specific PCR technique to study the expressed human kappa immunoglobuiin liglit chain genes iu mononuclear cells from peripheral blood (MNC). Approximately 50 gene products from a MNC pool from 11 individuals and 50 from a single individual were cloned from cDNA, sequenced, and assigned to the germline gene with the closest over-all homology. Some V^ families and genes were found to be used more often and some less often than expected from their occurence in the germ-line. Thus, family Vglll was found to be the most frequently expressed followed by V^l. The repertoire expressed in the pool was more versatile than in the single individual, but in both cases three germline genes {A27 (Hum k\'32S), L6 (T'^) and A3/A19) were dominating. Overall, approximately 80 % of the sequences could be assigned to eight germline genes. The Jg proximal Vg genes was preferentially used. In the pool }^3 was expressed rarely. The sequences were very mutated with an average homology to the closest germline gene of only 93 %. N-additions were found in about half of the sequences. Unexpected lengths of CDRl were found in a few of the sequences assigned to germline genes belonging to family VKIII. Thus a sequence assigned to the L6 gene had five additional codoiis between codon 27 and 28. The sympathetic nervous system has been shown to influence inflammation and immune responsiveness. The aim of this study was to investigate if lymphoid cells are able to produce catecholamines and if catecholamines, once secreted, influence the immune reactivity. Mclhods: The dopamine (DA) and norepinephrine (NE) content in mouse spleen cells, peritoneal macrophages and T and B cell hybridomas v^as examined by capillary electrophoresis. Tbe impact of L-DOPA. DA and NE on proliferation, measured as thymidine uptake, and differentiation, measured as cytokine and immunogiobiilin production, was studied in vitro in mitogen driven cell cultures. Spleen cells were cultured with L-DOPA and caiecholamines and induction of apoptosis was studied by propidium iodide labeling and FACS detection of hypodiploid cell nuclei. Results: DA was detected in spleen cells, peritoneal macrophages, B cell hybridomas and 2 of 3 Tcell hybridomas in concentrations varying between 7x10'^''and 7x10''^ mole dopamine/cell. Also NE was found in a T cell clone and splenocytes. L-DOPA, dopamine and norepinephrine dose-dependently inhibited Con A and LPS stimulated proHferaiion, immunoglobulin production and formation of lL-2. INF-y and IL-6 by spleen cells. L-DOPA, DA and NE dose-dependently induced apoptosis of splenocyles. Conclusion: Since lymphocytes a) produce DA and NE, b) are suppressed by catecholamines and c) express catecholamine receptors there are biological prerequisites of an autocrine regulation of the activity of lymphocytes by catecholamines. A. Jorgensen*, N. 0dum', C. Ropke' •Department of Medical Anatomy A, University of Copenhagen. Tlie Panum Institute. 2200-N Copenhagen, Denmark. Fax: +45 35 32 72 69. IDepartment of Medical Microbiology and Immimology, University of Copenhagen, The Panum Institute. The role of cultured human thymic epithelial cells (TEC) as aiitigenp resenting cells was investigated with the use of superantigens (SAg): SEA and SEB. Coculture of TEC and mature CD4+ T cells showed that TEC are able to present both alloantigens and superantigens to the CD4+ T ceils, as measured by increased proliferation of the T cells. The presentation is dependent on expression of MHC class II and possibly other costimuiatory adhesion molecules on TEC. TEC express ICAM-1, LFA-3 and CD40, TEC themselves are not stimulated to increased proliferation, but production of cytokines (IL-1 and IL-6) from TEC enhances the CD41 T ceU respons. TEC do not express B7-1 but show a low expression of B7-.2 and the SAg presentation can be further increased by the addition of monoclonal antibody against CD28. TEC also present SAg to subpopulations of human thymocytes: CD4+8+. CD4+ and DN CD3 positive thymocytes. Thus TEC act as antigenpresenting cells but less eftective than professional antigenpresenting cells, due to the absent or weak expression of B7. This may support, that TEC are more important in positive than in negative selection. Oral tolerance was induced in Wisiar rats by feeding ovalbumin (OvA)-cont3ining pellets during 2 weeks. Two weeks after the end of feeding the rats were imrnunised with a mixture of OvA and human serum albumin (HSA) in Freund's complete adjuvant. Every fourth day between day 7 and day 26. the popliteal lymph nodes (draining the immunisation site) was removed. The enlargemenr of tbe lymph nodes, the number of CD4+ and CD8+ T-cells and tbe expression of TGF-p and IFN-y in the lymph node was monitored. In a parallcll set of rats, lhe specific antibody production was nionilored for 18 weeks afler immunisation. In lhe OvA-fed rats there was no size-increase of the draining lymph nodes afler immunisation, while tbe nodes of the control rats were heavily enlarged from day 13 to day 24 after immunisation. Furthennore, from day 7 lo day 15 scattered TGF-p expressing cells were present in the T-cell area of the lymph nodes of OvA-fed rats only. Tbe anti-OvA levels in sera from the rats revealed an initial B-cell priming of the OvA-fed group, with levels higher than [be control group during the Tirst to weeks. Tbereafter. suppression governed lhe respon.se, and from week 4 to 18 lhe anti-OvA levels were considerably lower ihan the controls. The suppression also affected, lhe response to the by.stander antigen HSA. as even the anti-HSA levels were lower in the OvA-fed group than in the control group. The application of parallel distributed processing to immune networks has first bpiMi introduced by Vertosick and Kelly [1]. By making use of Oiiidiii Cazeneva enigma, tlie sharing of idiotypic determinants by antibodies directed tn non-cross reacting epitopes [2], they further led to the concept of content-addrrstiable immune meinoiies [3] . This concept is especially interesting, since it ex* plains the structure behind the ability of pattern completion abilities of the immune system. In this study, we develop further the model introduced by Seiden and Celada [4] in order to study the pattern storage capacities of tlie immun*? systetn. This model is based on a non-standard rpllular automata. The results obtained shows clearly that the Ouidin-Cazenave enigma enables the pattern completion ability of the immune system. In other words, it is possible that a secondary response towards a partly changed antigen (f.ex. a mutated pathogen) is not altered, if these changps are under a certain limit. [1] Vertosick, F., Kelly, R. Immunology, li)H!t, 66,1-7. [2] Oudin, J., Cazenave, P. Proc. Nttti Amd. Sci. USA, 1971 USA, , 68,2016 USA, -2620 [3] Vertosick, F., Kelly, R. ./. theor. Biol, 1991. 150,225-237. [4] Seiden, P., Celada, F. The mucosal Ig response (numbers of IgA-, IgM-and IgGproducing immunocytes per defined mucosal length unit) as well as the local IgA subclass distribution were studied in by-passed jejunal segments with bacterial overgrowth and compared with normally functioning segments in adults. Sterile ilea! urinary conduits were likewise examined by immunohistochemistry in children; the mucosa showed metaplasia and glandular atrophy, but the median numbers of IgA, IgM and IgG immunocytes per mucosal length unit only tended to be decreased, Tbe jejunal segments with bacterial overgrowth showed minor histological changes and contained fairly normal numbers of IgA and IgG immunocytes, but there was a significant reduction (p<0.05) of IgM immunocytes (12 cells/length unit) compared with control mucosa (24 cells/length unit). The subclass distribution of IgA ininiunocytes showed for lgA2 tbat the median number per mucosal length unit was significantly decreased (p<0.01) in ileal conduits (7 cells or 30% of total IgA) but increased {p<0.05) in jejunal segments with bacterial overgrowth (42 cells or 43% of total IgA) compared with Ihat in normal ileum (15 cells or 40% of total IgA) and jejunum (24 cells or 23% of total IgA), respectively. These data suggested an association between intestinal bacterial load and topical IgA subclass production. Inierferon gamma and IL-4 have been found to play major regulatory roles in Thl and Th2 CD4 T cell differcniiation and therefore also crilically niiay affect mucosal immune responses. Since IFN-y production is abundant in mueosal tissues and lias recently been aseribed A critical role in epithelial cell functions, including llic expression of SC and irancylosis of secretoiy igA, we addressed whether lack of IFN--)' functions would afTect IgA responses following oral immuni/ations. Wild lype and IFN-y receplor (IFN-yR ) gene knockout mice were immunized pcrorally with KLH and cholera to.\in (CT) as lhe mucosal adjuvanl. Surprisingly, we found no evidence of an impaired response lo oral immunization in IFN-yR deHcienl mice. Similar numbers of anti-KLH IgA SFC were recorded in wild type and IFN-yR deficient mice. However, at the systemic level signiricantly reduced specific lgG2a and IgG3 serum aniibody levels were observed in IFN-yR deficient mice. Also, in vitro stiniiitalions of perorally primed T cells with recall antigen gave 2-3-fold stronger proUferative responses ;ind liiglier IFNy production as compared to wild t>pc T cells. IgA B cell differentiation and epithelial trancytosis of secretory IgA appeared normal in IFN-yR deClCTcnl mice with comparable levels of lotal IgA and anu-CT IgA in lavage in immunized mice. Indeed, the local gut mucosal immune response was functional because antitoxic protection as analyzed by the ligiited loop test did not differ between wild lype and IFN-yR deficient mice Finally, we tested whether oral tolerance may be stimulated in the absence of IFN^. Consistently, we found strong suppression of systemic anti-KLH antibody responses if IFN-yR deficient mice fed KLH prior to the parenteral immunization We conclude that mucosal IgA imnuiiiity and oral tolerance appear to be unaffected by the lack of lFN-y function, suggesting that other factors such as IL-4 may compensate in vivo, Experiments in IFN-yR/lL-4 double knockout mice are underway. The levels of IgM and IgG antibodies to Re glycolipid of Salmonella minnesota strain Re 595 was determined in the blood sera of 56 children aged 4-12 years with chronic pyelonephritis by enzyme immunoassay 80% purity) 0/N then recultured witfi Con A. Reduction in T cell proliferation with Con A stimulation can be seen in purified T cell cultures (day I to 2 but txjt on d^ 3). The presence of remaining APCs mi^t cause interference at tfie presentation level i.e. SEB would be presented to T cdls leading to proliferation instead of anergy. The success of these methods would allow an extensive study of f)en[^eral tolerance in \ilro for tfie firet time Cellular immunity in cynomolgus monkeys rectally exposed to a low dose of SIVSM Barbro Makitalo, K Karlen, E Rudl. G Biberfeld. P Putkonen, R Thorstensson Swedish Institute for Infectious Disease Cotitrol, Stockholm, Sweden, 1 Health Canada, Ottawa, Canada Objective: To characterize the virus specific immunity, especially the cell-mediated immunity in monkeys which received a subinfecbous dose of a macaque cell grown stock of SIVSM (SMM-3). Methods: Cynomolgus monkeys were inoculated with a SIVSM stock propagated in vitro on cynomolgus monkeys PBMCs. Tenfold serial dilutions of stock virus ranging from 10"' to 10"3 was autraniatic applicated intrarectally (iR) in 6 monkeys (2 animals for each dilution). Infection was monitored by virus isolation and PCR{gag, pol, env, LTR). Virus specifc immunity was investigated by ELISA, Western blot, T-cell proliferation(HIV-2 viral lysate, SIV env synthetic peptides) and CTL{gag/pol, nef). Results: Infection was demonstrated by positive virus isolation and PCR and by se reconversion in two of Iwo monkeys given the 10"' dilution and 1/2 became infected given the 10"2 dilution. The remaining three IR inoculated animals did not become infected as shown by negative virus isolation, serology and PCR but showed SIV specific gag/pol and nef CTL and T-cell proliferation against SIV env synthetic peptides. Conclusions: Monkeys exposed to low concentrations of SIV developed SIV specific CTL and T-cell proliferation but no demonstrable viral antibodies. These animals have recently been rechallenged with a higher dose to determine if the cellular immunity can confer protection against challenge. In previous studies in this laboratory preformed IC were employed in flowcytometrical analyses of the distribution of immune complexes (IC) on blood cells and the buffering capacity of erythrocytes (E). In an effort to approach more closely physiological conditions, we have chosen to examine the binding to whole blood celts of IC formed in situ from FITCconjugated tetanus loxoid (TT) and natural anti-TT antibodies contained in the donor serum. To this end, the anti-TT levels of a range donors were assessed by ELISA, and selected donors were employed in the investigations. The binding of IC formed in situ and preformed IC, incubated for 1 hr. at 37°C (both at a ratio corresponding to the equivalence point) to whole blood cells was compared. It was found that uptake by leukocytes was considerably delayed for the IC formed in situ. In contrast, maximal binding to E occurred at 3 min. for both types of IC. Shifting the ratio towards antigen-as well as antibody-excess retarded the time point of maximal binding to E of in situ formed IC. The mechanisms underlying these observations as well as capacity of E to restrict the uptake of in situ formed IC are currently under investigation. AnrvMFev.CdlBioi^. 397-425,1989) . Thenon-stationary model also explainsgrowth signal firing, and vwthdraw from the ceD cycle, in terms erf the alternating interaction (L Matsson. HiysHeu. E48. 2217-31, 1993) . Wthan assessed Kj^.=La+0.5nM these data do not aanply with the mass-action based respcaise (Fig. B) . Apart from assumed absence of membrane induced receptor ccrrelations. and assumed linear dependence on occupancy, the mass-action based model also lacks an altemadng dynamics due to the assumptiai of a staticnary state. Antibody responses against putative virulence factors ofH. pylori vjcTz determined in sera and gastric aspirates from patients with duodenal ulcers (n=19), in asymptomatic H. pytori carriers (n=18) and in non-infected age-matched controls (n=20). This was done to evaluate if antibody specificities are found in asymptomatic carriers that are lacking in ulcer patients and if immune responses may be induced locally in the stomach but not in serum Antibody levels against H. pylori lipopolysaccharide, flagellin, urease and two surface antigens, i e a 26 and a 30 kDa protein, and for control purposes against whole membrane proteins (MF), were tested in gastric aspirates and sera by means of different ELISA methods A majority ofthe infected subjects had significantly higher levels of specific antibodies against the flagellin, the urease and the MP in sera and in gastric aspirates than the healthy controls In no instance were antibody responses against any particular H. pylori antigen seen in the asymptomatic but not in the symptomatic H. pylori carriers The antibody responses were generally more pronounced and more abundant in sera than in gastric aspirates- Oral vaccination with cholera vaccine is known to induce antibody secreting cells (ASC) against the B-subunit of the cholera toxin (CTB) in duodenal mucosa. in this study we wanted to evaluate if the vaccine also is capable of inducing antibodies against the bacterial component ofthe vaccine and if vaccine-specific ASC:s can be induced in the stomach mucosa Eight healthy non-infected and 6 H. pylori-infected adult Swedes were given two doses of CTB-whole cell cholera vaccine at two week intervals From each subject several (12-14) punch biopsies were taken from the antrum and from the upper part of the duodenum immediately before and 7 days after completed vaccination. The lymphocytes were isolated from the biopsies and the numbers of total and vaccine-specific immunoglobulin secreting cells determined by enzyme-linked immunospot assay (ELISPOT) A majority of the infected as well as the non-infected subjects responded to the vaccination with increased numbers of ASC:s against CTB and the bacterial component in the duodenum. Most of the //, p>7on-infected but only one of the non-infected subjects also responded to the vaccine in the stomach. Munthc', Anders Sollicn", Zlatko Dembic*, Peter Hofgaard*, Hildc Omholl*, and Bjamc Bogen* •Institute of Immunology and Rheumatolog>, Univ of Oslo, Norway, Fax 47 22 207287 #bep of Biology, PRPT. HofTmann-La Roche Lid,, Basel, Switzerland CD4' T cells usually recogni^x: peptide bound to MHC class II molecules while CD8' cells recognize peptide on class I molecules Such a correlation between coreceptor expression and MHC-reslriclion is thought to be a consequence of positive selection in thcth\inus. Here we describe an exception to this nilc, CDS' T cells are surprisingly frequent in mice transgenic for an ap TCR which recognize an lg light chain (X,2"') idiotj-pic peptide, presented on the I-E'' class II molecule. These CD8* cells are now demonstrated lo be X.2'''-speciric and I-E" class Il-restricted, CD8 expression seems to be of no functional importance for the e!ass-II restricted specificity. Most ofthe CDS"ĉ ells express endogenous TCRa chains. Such endogenous a-chains appear lo enhance positive selection because the CD8* population is dramatically reduced in recombination deficient TCR-transgcnic SCID mice. The repertoire of endogenous TCRa chains in CD8' cells is skewed, indicating that only certain Va domains may augmeni positive selection Expression of endogenous TCRa chains becomes more frequent as ihymocUes make Ihc transitions from CD4'CD8' -• CD4'"''CD8* -* CD8', A mechanism for positive selection of T cells with two TCR, OS well as a mismatch between coreceptor and MHCrcstriction, is discussed. The CD8' ciass Il-restricted T cells Will LPS blasts from J.2''Mransgenic mice, suggesting that such T ceils could function as idiot>'pc-specifie suppressors of B cells. Despite that the MHC class II molecules Aq and AP only display a 4 amino acid difference within the B-chain, A^l mice are susceptible to collagen-induced arthritis (CIA), whereas AP mice are resistant. We have investigated the ability of AP-vs Aq-expressing APCs to activate Cll-reactive T-cell hybridomas from Aq mice. The T-cell hybridomas recognize the immunodominant peptide 011(256-270). and are either reactive with the naked peptide or demands the presence of hydroxylysine-linked carbohydrates within this determinant. Whereas the glycopeptide-reactive T-cell hybridomas were strictly dependent on Aq (or presentation, T-cell hybridomas recognizing the "naked peptide" accepted AP APCs, provided that the synthetic peptide or carbohydrate-depleted CII were used as antigens. No response could be detected among the latter cells, however, if glycosylated CII was used as antigen. We propose that the carbohydrates within the 011(256-270) determinant have dual roles: either as epitopes (for the glycopeptide-reactive T-cells) or as a steric hindrance, allowing the qlvcopeptide to bind Aq but not Ap. Development of an inflammatory self-limiting polyarthritis can be provoked in DA rats by Freunds incomplete adjuvant (FIA), Rats from the same strain develop a more severe and chronic arthritis when rat type II collagen (RCII) is administred with FIA. If an irrelevant antigen is added to these arthritogens. in this case ovalbumin (Ova), the development of arthritis is completely inhibited. This inhibition is disease-specific and long-lasting. To investigate mechanisms responsible for induction, as well as inhibition of arthritis, a kinetic study of local cytokine production in lymph nodes has been performed in rats immunized with the above mentioned agents. By using in situ hybridization techniques, the mRNA expression of TNFa, IL2, IFNy and IL4 was determined. A rapid and pronounced expression of TNFa mRNA was recorded in RCII/FIA and FIA immunized rats, already 2 hours after immunization. This pronounced expression was not observed in Ova/FIA injected animals, which instead were the only animals where the IL4 gene was expressed. The expression of IPN7 mRNA was limited in RCIl/FlA-and FIAimmunized rats, whereas IL2 mRNA expression was only detected after RCII/FIA injection. Lymph node cells from RClI-immunized animals generated a high amount of TNFa after restimulation with RCII. whereas restimulation with the mitogen Con A generated a cytokine mRNA response dominated by IL2 and IFNy. These and other results indicate that a strong local expression of TNFa, induced by arthritogenic stimuli, may be important for the induction of athritis. Moreover, the elicitation of an immune reaction to an irrelevant antigen, may inhibit arthritis development by modulating the arthritis associated cytokine response. The inflammatory nature of multiple sclerosis (MS) implicates the involvement of immunoregulator>' c>'tokines. Evidence is accumulating that IFN-Y. TNF-a and TNF-P have disease promoting properties in MS, while TGF-p, IL-10 and, probably, IL-4 may e,rt some protective effects. In the present study we used in situ hybridization with cDNA oligonucleotidc probes to delect and enumerate mononuclear cells (MNC) expressing IFN-y, TNF-a. TNF-p, TGF-P. IL^ and IL-10 mRNA in blood and cerebrospinal fluid (CSF) from 77 patients with MS, 23 with optic neuritis (ON) and controls The MS patients had elevated numbers of blood MNC expressing all 6 c>tokines both when enumerated without previous culture and after culture with myelin basic protein (MBP), IFN-y, TNF-a, TGF-P and IL-4 were further enriched in the CSF of MS patients. Numbers of TNF-a niRNA expressing blood MNC correlated positively with the severity and progression of MS, In contrast, TGF-P and lL-4 were significantly higher in patients with shorter duration and minor disability, suggesting beneficial downregulating effect of IL-4 and TGF-p in MS, Patients with ON. in many instances representing early MS. had similarly augmented numbers of TNF-a, TNF-p and IL-10 positive blood MNC as clinically definite MS, ON patients examined within one month after onset had lower numbers of MBP induced IL-10 mRNA expressing cells compared to remission, indicating a possible protective role of IL-10, The results suggest that IFN-y. TNF-a, TNF-p. TGF-P, IL-4 and IL-10 are in\olved in MS, TNF-a could be usetiil as a disease activity marker in MS, and regimens promoting TGF-P and IL-10 could influence MS beneficially. EM. Nilsenl.G, Haraldsenl.T, Scholz^andP. Brandtzaeg'' LIIPAT, Insts. of Pathol.'. and Surg. Res.;2 and Surg. Dept. B^. Univ. of Oslo, Rikshosp., N-(X)27 Oslo. Norway. Fax;+47-22ii226i Increasing evidence suggests that leukocyte emigtution to areas of inflammation depends on endothelial ceil activation, which may also result in production of impwrtant cytokines by such cells. These cytokines may further enhance the inflammatory process, but organ-specific differences must be taken into account. Methods: To evaluate cytokine profiles of putative importance for the development of inflammatory bowel disease, we examined human intestinal microvascular endothelial cell (HIMEC) mRNA expression for 12 cytokines with reverse Iranscriptase-PCR (RT-PCR). The cytokine profiles were compared with those obtained in human umbilical vein endothelial cells (HUVEC). Cells were cultured to confiuence in I3-weIl plates and then stimulated for 4 h with rh IL-1 p (100 U/ml) or rh IL-* (100 U/ml). Different cytckine mRNAs were delected by cDNA synthesis and PCR amplification of polyA RNA isolated from cultured cells. Results: Unstimulated HIMEC expressed low levels of mRNA for IL-ip, IL-6 and TNFa. However. IL-lp, and to a lesser degree IL-4, increased mRNA for these three cytokines. We were unable to detect mRNA for ILla. IL-2. IL-3. IL-4. IL-5. IL-10 or IFN-y in HIMEC. By comparison, in HUVEC IL-lp stimulated mRNA for IL-1 a,IL-lp and IL-6, but not for the other cytokines. Message for TGF-P and IL-8 was present in both cell types and was not subjected to regulation by IL-1 p and IL-4. Conclusion: Our results suggest that TNF-aexpression is preferential for HIMEC and that both HIMEC andHUVECproducelL-lp, IL-6. IL-8 and TGF-p. Some of these cytokines are known to be powerful inducers of endothelial cell adhesion molecules, and TNF-a may contnbute to tissue damage in various gut diseases. Erythrocytes (E) express low numbers of complement receptor, type 1 (CRl, CD35). via which they bind opsonised immune complexes (IC) in competition with leukocytes expressing high numbers of CRI as well as other complementand Fc-receptors. We examined the cellular distribution on whole blood cells or isolated leukocytes of preformed tetanus toxoid (TT)/ human anti-TT immune complexes, opsonised in situ in 80% serum. Reflecting the kinetics of C3-fragment incorporation, E took up IC rapidly, accounting for the major proportion of the total early cellular IC-binding (median: 83% after 30 sec. and 92% after 3 min.). The presence of E reduced the leukocyte uptake of IC after 30 sec. by >60%. Subsequently, the IC-binding to E and the restrictive effect exerted by H decreased. After 15 min.. E inhibited the IC-uptake by phagocytes by <20% and promoted a progressive uptake by B cells, which could be abolished by blockade of E-CRl with the MoAb, 3D9. CRl-blockade inhibited the initial IC-uptake by phagocytes by >85%. and <45% after 15 min. Our findings support the hypothesis that E act as a buffer, restricting the IC-uptake by circulating phagocytes within the first minutes of incubation during which CRl is the dominant receptor, and suggest that the presence of E enhances the late IC-uptake by B cells. To characterize the antibody (ab) response in cynomolgus macaques immunized with native HIV-2 envelope glycoprotein gpl25 coupled to immune stimulating complexes (iscoms) or Ribi adjuvant. METHODS: Nine monkeys were immunized at 0. 2. 4, 6. and 9 months with HIV-2/SBL6669 gpl25, purified by affinity chromatography using Galanthus nivalis agglutinin. Five monkeys received 30 |J.g of gpl25 in iscoms and four monkeys received 50 |ig gp 125 in Ribi adjuvant. Serum samples collected fourteen days after each immunization were analysed for ab against HIV-2/SBL6669, gpl25 and the V3 region of gpl25. Ab titers were determined using ELISA technique. RESULTS AND CONCLUSION: Immunization with gpl25 in iscoms resulted in maximum ab levels already after two immunizations and did not increase on boosting. In the gpl25-Ribi immunized monkeys low ab titers were initially observed. On completion ofthe immunizations ab titers had reached levels comparable to those in the gpl25-iscom immunized monkeys. Fourteen days after the final booster the monkeys were challenged with 30 MID50 of homologous HIV-2/SBL6669. No differences with respect to induction of protection was observed since two of five gpl25-iscom immunized monkeys and two of four gpl25-Ribi immunized monkeys were protected. Immunology and Rheumatology, University of Goteborg. Sweden. Fax: +46-31-826791 S. aureus arthritis is a highly erosive disease where both host and bacterial determinants are of importance in its induction and progression. The aim of our study was to assess the role of sar gene as a virulence determinant in the pathogenesis of septic arthritis. Materials and methods. 5. aureus strain, isogenic for sar locus, was inoculated i.v. into Swiss mice. Clinical, bacteriological and histopathological progresion of the disease was then studied. Results. The peak frequency of arthritis within I week after inoculation of bacteria was 79% in the group of mice inoculated with wild, sar+, strain whereas corresponding frequency in sar" miiiant was 21 % (p<0,01). Also, infection with sar"*" staphylococci led to significantly higher weight loss. To assess the relationship between clinical signs and spread of bacteria we analyzed the early homing pattern and persistence of.?, aureus in joints, kidneys, spleen, liver, and peripheral blood. Organs from sar"^ inoculated subjects contained at later stages a considerably higher amounts of iive bacteria. Conclusion. Our results suggest that the sitr system of S. aureus is an important virulence determinant in the induction and progression of septic arthritis. Nordahl, M , Nielsen, M,, 'Svejgaard, A., 0dum. N from The Cell Cybernetics Laboratory, University of Copenhagen Fax:+45 35327851 and §Tissue Typing Laboratory, State University Hospital, Copenhagen, Denmark. Interleukin-2 ts a critical regulator of T lymphocyte proliferatton The exact mechanism by which IL-2 receptors (IL-2R) regulate transcription of different genes is partly unknown It was recently shown (1) that IL-2 tnduced tyrosme phosphorylation and nuclear translocation of STAT3, a newly identified member of the signal transducers and activators of transcription (STAT) family of protems Here we show, that IL-2 induces tyrosine phosphorylation of STAT3 and a STAT5-like protein in allospecific CD4 T cell lines In contrast, STAT 1 and 2 proteins were not tyrostne phosphorylated Interestingly, the STAT5-like protein had an apparent molecular mass of 100 kDa. STAT5 was originally identified as a 92 kDa protein in mammary epithelial cells (2) suggestmg that the IL-2 responsive. 100 kDa protein may be a new, STAT5-like, member of the STAT family of proteins, Cytokine induced tyrosine phosphorylation of the STAT 3 and STAT5-like proteins followed similar kinetics In conclusion, we provide new evidence that STAT proteins are involved in IL-2R signaling in antigen specific, human T NK cells take part in innate immune responses by cytolysis of certain virally infected and tumor cells. They have also been ascribed a role m controlling myelopoesis and lymphocyte development. We set out to investigate if NK cells have a role in controlling the intensity of in vivo inflammatory responses. Methods: In outbred SWISS mice, NK celts vre depleted by repeated intrapcritonca! injections with an antibody to the NKl.l. antigen, derived from the mouse hybridoma PK136. Controls received an antibody to Herpes virus. Granulocyte mediated inflammation was induced by injection of 30 microliters of olive oil intradcrmally in the hind ftxjtpad. 24 hours later, swelling of the footpad was registered. In other experiments, mice were immunized and boostered with oxa/.olonc to asess delayed type hypersensitivity (DTH). Results: Granulocyte mediated inflammation was not significantly changed 24 hours after NK cell depletion. Alter 8 days, a significant (p < 0,05) increase of swelling was registered. Concomitantly. there was a tendency towards increased numbers of peripheral blood lymphocytes and granulocytes. In the DTH model, a tendency for increased reaction was seen in NK cell depleted mice. Conclusion: Our results ptimt to an important role lor NK cells in regulating the intensity of inflammatory response. However, we believe that the NK cells down-regulate the development of effector cells, neutrophils and possibly also monocytcs/macrophagcs. rather than excerting suppression during the inflammatory response itself. A new hypothesis based on experimental work with various non-specific immunological stimulators is presented for better characterization and deeper understanding of the underlying processes in immunologiac response. Nocicepsis is the description of the disturbance of cell surface homeoslasis by intimate physicochemical force leading to nerve cell stimulus. Nocicepsis can also be applied to the ignition pbe nomeon in the lympho-reticulo-endothclial defence mechanisms. The triggering force in the contact of intruding microtie.pollen or other non-self moiety with the responding L-RES cell is exe cuted by the electrochemical differnce in the intimate cell surface milieu. This nonspecific electrochemical shock brought by the intruding agent leads then to revealing and aclivalion of more specific signal mechanisms. This primary triggering is a well conserved unicellular phanomenon in contrast lo the sequelae, where the functioning is dependenl sophisticated cell lo cell contacts and interactions. Such evolutionally younger interaction mechanisms include various adhesion and immunological rcseptors as well the array of local and more general modifying mechanisms served by cyto kines.inflmmalory,allergic and neural mediators and various hormones. Their eletncntary function is to inhibit the spreading of tlie harmful element by needed amplification or suppression of various required cell poplations in a regulated manner leading in the end to extermination of the intruder. M. tyl;)gri;ijipgi^/M. leprae specific T cell iines and clones from healthy donors vaccinated with heat killed M± leprae or BCG were screened for proliferative reactivity against the recombinantly expressed heat shock proteins (HSPs) HSP18, HSP65, and HSP70. Overlapping synthetic peptides covering the respective HSP sequences were then used to map epitopes of the HSP reactive T cell lines or clones identified whereas a combination of blocking and panel studies were applied to identify the HLA molecules presenting the individual epitopes. Two epitopes of the HSP18 molecule presented by HLA-DR4,Dw4 and DRl, respectively, was identified. T cells recognized the mycobacleriai HSP65 antigen in the context of HLA-DR 1, DR2, DR5, DR7, and interestingly DR4 (Dw4 and Dwl4). All of these epitopes (except DR5 and DR7 recognition) were defined at the sequence level. By combining a DNA subclone and peptide approach, we identified T celi epitope sequences from HSP70 presented by HLA-DR2, DR3, and DR7. HLA-DRl, DR5, as well as HLA-DRw53 were shown to present epitopes not yet defined at the peptide level. The results will be discussed in relation to vaccine design. selection of scFv antibodies binding to tissue expressed antigens. J. Olsson. T, Brodin and T. Kailand Pharmacia AB Oncology-Immunology, Box 724 S-220 07 Lund. Sweden. Fax: -i-46 46 191134. Recently we developed a method based on phage display technology for selection of scFv antibodies directed against novel antigens on the surface of viable cells. Now we present the development of a procedure for phage selection using tissue expressed antigens, i.e. the use of tissue sections to expose intracellular, membrane bound and extracellular tissue epitopes to phage antibody libraries from which binders of novel specificities can be selected. This would allow the discovery of new markers characterizing phenotypes expressed exclusively in vivo, such as pathological processes, cellstromal interactions, organ-architecture dependent cellular phenotypes. markers of embryonic development or other markers of interest to histological in situ analysis. In model experiments phage reacting to an epithelial tissue antigen, C215. could be enriched 20-700 times over non-specific phage while pre-blocking with C215 Mab erased specific phage enrichment. Linear correlation of the enrichment factor versus the relative proportion of antigen positive surface area could be demonstrated. Finally, an immune library was subjected to positive selection to find scFv antibodies that could be demonstrated to bind to frozen sections of human tumor tissue. The effects of serum concentration and factor 1 depletion on the deposition of C3 fragments on human B cells following spontaneous in vitro complement activation. In our attempt to optimize the deposition of C.^ fragments on the surface of human B cells, we investigated the effect of increased serum concentration and depletion of factor I (FI) from normal human .serum (NHS) on ihi.s depo.siii()n. We incubated peripheral blood leucocytes in 25-75% NHS iVoin healthy AB-positive donors at M°C. This was done in the pit^.senee of Mg-EGTA to block the ela.s.sieal complement pathway while allowing activation via the aUernalive pathway to continue. We then mea.sured the depo.sition of C3 fragments on B lymphocytes, using FITCeonjuguted polyclonal ant!-C3c and C3d antibodies. The deposition was mea.sured by llowcyiometry. specirieaiiy seleeting lor B eelLs with PE-conjugated monoclonal anti-CDiy antibody. We found that increased serum concentration enhanced the deposition of C3 fragments on human B cells. Surprisingly (he depletion of FI from NHS led to decreased depo.sition in spite of the fact that C3 conversion lo C3 fragments in the lluid phase was enhaneed. We conclude from this work that i) increasing the availability of complement enhances C3 fragment deposition on human B cells and that ii) this deposition is a result of loea! (.surfaeemembrane), raiher than general (lluld phase) activation. The T cell surface molecules CD6 and CD28 have been shown to be receptors for accessory signals in T cell activation. We here demonstrate that in the absence of any other activating stimulus, simultaneous ligation of CD6 and CD28 by mAb induces T cell proliferation. An essential requirement for the induction of T cell proliferation via the ligation of both molecules is the immobilisation of the anti-CDG and anti-CD28 mAb by using rabbit anti-mouse immunoglobulins (RAM Igj. The T cell proliferation induced by crosslinking of CDO and CD28 is IL-2 dependent, demonstrated by the cell surface expression of the IL-2R and the inhibition of the proliferative response by anti-lL-2 MAbs . In addition the proliferating T cells activated via CDG and CD28 expressed lL-2 mRNA. The CD6/TPA-induced T cell proliferation was completely inhibited by cyclosporin A, however the proliferative response after crossiinking of CDG and CD28 was partially inhibited by the immunosuppressive drug. Our data provide evidence for a pathway of antigen-independent T cell activation via CDG and CD28 , which is lL-2 dependent. Storage mites are a well recognized source for allergic diseases such as astlitna and rhinitis Lepidoglyphus desinictor (Ld) is the most common storage niitc species in iiortheni and caitrat Europe An Ld population was obtained from Allergon AB. Angeiholni, Swodcn Poly-A selected niRNA was punfied according to statidard methods Degenerate primers were designed from tlie previously partiaiiy determined amino acid sequtaicc of the major allergen of Ld, Lep c/I Reverse transcnptase polymerase chain reaction (RT-PCR) and PCR was performed using 3'RACE and 5'RACE protocols and tlie resultant cDNA fragments were cloned into TA plasmids (hivitrogen) Tlie nucleotide scquaices were computer analyzed using DNASIS software program Tlic probable leader sequence was 16 ammo acid residues long Tlie encoding reminder ofthe sequence showed 125 amino acids residues for Lep dl. Tlie full cDNA IS approximately 550-600 nucleotides long Companson of several independent PCR products indicate polytnorphism, the sequences show some altered amino acid composition m tlie aicoding part and multiple mutations to tlie extend of block deletion in the non-coding pait of tlic 5' and the 3' end of the mRNA. Tlie complete cDNA will be used m an expressionsystem to obuin tlie recombinant altergai Oncology Immunology, Lund. Sweden, -^Department of Immniunology, University of Lund, Sweden. Characterization of T cell signalling pathways is of fundanicma] importance to understand many aspects of ihe biology of T cells. We have earlier shown that stimuUiion of human CD4''' T cells wiih SEA presented on CHODR transfeclanw coexpressing eilhcr B7 oi LFA-3 resuUed in distinct cylokinc profiles. We now denionsirate that B7, but nol LFA-3, strongly costimuiated Lranstripiion and lL-2 mRNA expression in CD4^ cells. A 40-fQld increase in IL-2 transcriplion wa* ra-ordcd wiih CHO-DR/B7/tJA-3, suggesting a cooperative effeci of B7 and LFA-J ai Ihc iranscriplional level. Gel shifl analysis demonstrated that slimulalion of COA* T cells wiih CHODR and SEA was suffiLiem to induce signincuni amounts of NF-kB binding prolciiis, whuicas induclion of AP-1 binding proteins required cosliinulaiion. LFA-3 induced moderate levels of AP-1, bul did nol influence the levels of NF-kB. whili-B7 cosiimulalion strongly induced boUi AP-1 and subsianiially enhanced NF-kB binding proteins. The CHO-DR/B7/LFA-3 triple iriinsfeciani induced a furihet increase in AP-I and NF-kB binding proteins c:ompaicd to lhe double uansfectants. The level of Oci-1 binding protcini remained similar in all samples. Super-shift analys leveaied ihai the NF-tB complex of costimulaied CD4''' T cells contained large amounts of p50, subslantial amounts of p65 and marginal levels of c-Rel proteins. The API binding proteins contained c-jun, jun-D and Fra-1 but marginal amounl.s of jun-B and c-Fos. Our results indicate disiinci effect.^ of B7 and LFA-3 costimulalion on lhe activity of AP-1 and NF-kB. Tliese may partly account for the differeniial effecis of B7 and LFA-3 coslimulation on IL-2 expression- Immunoassay Department, Mechnikov Research Inst. for Vaccines & Sera, Moscow, Russia, Fax:+7 095 9160320 The common immunochemical identification and classification of Streptococcus pneumoniae is based on the properties of capsular polysaccnarides. This approach does not cover noncaDsuIar strains, which makes if difficult to study the role of these forms of bacteria, as well as fonns with a small capsule, in human pathology-\ye analyzed variability of pneumococcal strains from me international collection by the Isolation of immunochemically active pneumococcal proteins. The protein preparations of 46 § neumococcal strains of known serotypes were subiected to DS get electrophoresis with subsequent immuno^blptting. Immunochemically active components were detected with the use of blood sera from patients with bacieriologically identified pneumococcal infection, as well as blood sera from clinically healthy adults. The presence of immunochemically active proteins with mol. wt. between 50 and 95 kD, as well as protein with a mol. wt. of ca-80 kD, common for all strains under study, was established. Some of these proteins were common for strains of different serological tyDes (strain 77/64 of type 22A and strain 39/57 oF type T?), while some of them exhibited an identical pattern for one serperoup strains of one serogroup (strains 55/51 and 52/57, serogroup 35). The possibility of using this active protein antigens as markers of pneiimococcal infection is considered. We could ihus distinctly differentiate S.pnenmoniae strains by some features other than the size of their capsules and their type. It would be interesting to find out the existence of epidemiqlogical correlation Detween protein types and the morbidity rate and severity of pneumococcal infection. Malaiia infections mean long lasting antigenic exposure of the human host. Sucli persistant stimulation affects the balance between CD4+ cells of Tli 1 and Th2 type with consequences for the course and final outcome ofthe infection. Th2 cell produced lL-4 Is responsible for the switch of the immunoglobulin isotype production of B cells from IgM/IgG to IgE. Besides being the major immunoglobulin causing allergic reactions, IgE elevation is also a hallmark of helminthic infections. However high IgE levels have recently also been encountered in protozoal diseases, for example in Leishmania infections. More recently, we and others have also fotind significantly elevated levels of total as well as IgE anti-malarial antibodies in F.falciparum malaria. That IgE is indticed by the malaiia parasites is supported by the finding of elevated IgE in mice experimentally infected with F.chabaudl where the final clearance of the infection has been shown to be dependent on a Th2 response. In contrast we saw no elevation of IgE in mice infected with F.vinckei vinckei where immunity is dependent on a Thl response. The influence of host genetic factors is suggested by the remarkable stability of the IgE antibody profiles over time and irrespective of season in an area with high and perennial Uansmission . Significantly higher IgE in Gambian children with cerebral F.falciparum disease than in uncomplicated cases might suggest that IgE is pathogenic in malaria-However IgE mediated reactions involving cells equipped with Fcg receptors have been shown to efficiently kill helminthic pai'asites in vitro. Further studies will hopefully give insight in the question of IgE's role for protection and/or pathogenesis in malaria. Complement i.s a .system oi more than 30 proteins found both in plasma and on cell membranes. The complement system has se\'eral important functions in the immune response raniiins from lniiiation ol' inflammation, neutralization and eliminalton of pathogens, and regulation of antibody responses, to clearance of immune complexes and disruption of cell membranes. Under certain conditions complement can, however, aci us a mediator of Jeletenous inliammatory reactions and the complement activation has heen implicated in the pathogenesis of some autoimmune disorders, bioincompatibility reactions and decompression sickne.ss. Our tibjectivc is to disrupt in vivo mouse genes coding for ihe third complement component iC3) and factor B rospeciiveiy. The C3-deficient mice will serve as a model of complement deficiency where the complement activation by any of the activation pathways is prevented at an early stage. These mice will be used to study the role of the complement system in Ihe pathogenesi.s oi some immune complex diseases, the rei;ulation oi antibody response, in the pathogenesis of decompression sickness and some bioincompatibility reactions. Factor B deficiency is expected to lead to .selective absence of the altemanve pathway '.'ii complement activation and will be used to evaluate the role of this pathway of complement activation in the above described phenomena. The progress of the work will be presented. We characterised DQ alleles of 31 Finnish coeliac disease (CD) families and of 32 random controls We also studied a gene dosage effect of risk alleles. All patients with either full or latent CD, but only 28 % of the random controls (p<10^) and 48 % of the healthy sibs {p<10') had DQAI'0501 and DQB1*O2O1 allele, A clear gene dosage effect was seen: 45% of the CD patients had more than two risk alleles, whereas this was observed in only 29 % of the silent CD patients, 9 % of the random controls (p<10"') and 3% of the healthy sibs (p<10" ') 72 % of the random controls (p<10"'') and 52 % of the healthy sibs (p<10*) had no or only one susceptibility allele. Nine of 13 (69 %) CD index cases with more than two susceptibility allele had a second pair of DQA1*O5OI and DQBl*0201 alleles, three (23 %) of them had a second DQBl*020l allele and one (8 %) had a second DOAI»050I allele. No major difference between the two alteles was seen The gene dosage effect was also analysed in relation to the sex of the patients and the onset of CD, but no evident associations could be observed. Allo selective NK cells are resistant to Cyclosporin A Ii Pelcrsson', M Dohlsten'-Î ) The Wallenberg Laboratory. Lund University, S-220 07 Lund. Sweden I~iix: +46 46 104201 and 2) Pharmacia Oncology Inununoiogy, LuriJ. Sweden Transplantation ol' tissue over allogencic barriers results in ihe activation of T lymphocyies. which have hcen shown lo play a pivtxal rote as effectors in rcjcciion iinmunily. T lymphncyics with an allo sclck;iivc cyloliixii.-effcci were induced when Wistar Kurtb (WF) rats were imtnunii^cd wilh alliigcncie Bri)wn Norway (BN) eells. When BN rats were immunized wiili WF cells such a re.spc»n.se wa,s noi achieved bul instead allo selective naiural killer (NK) eells were induced (Erics.son ei al., J. Immunof 149: 1504. 1992). These allo seleclivc NK eells were of the CD3-CD8+NKR-PI inierriiediale phenolype and were highly effieient in killing the appropriate target eclIs when not exhibiting self MHC cla.ss I molceuics. In ihis study we show that inimuni/aiiim of BN and WF rats wilh allogcneic cells in comhinaiion with CyclDsporin A (CsA) had only marginal effects on the induction ol' allo selective NK cells, while ihe same dose totally inhibited ihe induction of allo selective CTL. CsA suppressed the induetion of altoreactivc CTLiotoxicity with little or no NK activity. In order to evaluate whether the TIL lines had reaciivity against p53 epitopes, we incubated ihe H'-A-A2.1+ cell bne T2 with a range of p53 peplides. p53 peptide pulsed T2 cells were specifically killed by some, but not all, of our expanded TIL lines. We have analyzed TCR a-gene rean-angemenis during the early T-cell ontogeny by hybridoma production and PCR cloning. Our results of fetal thymocyte hybridomas demonstrate that the first TCR a-gene rearrangements are detectable on developmental day 16. Interestingly, in all fetal day 16 thymocyte hybridomas the most proximal Ja-gene {Ja50) was rearranged. This rearrangement was always found on one chromosome only with a concomitant 8 rearrangement on the homologous chromosome. Four of the rearranged Ja50 genes were cloned and sequenced. In one hybridoma the V61D52element, which is thought to be specific for the 5-region, was rearranged to Ja50-gene. It was not only found in hybridomas but it was detected in fetal thymus DNA by nested PCR as early as day 15.5 of development. Because the Ja50-gene is a pseudogene. which can not encode a functional TCR receptor on the cell surface, we wanted to determine whether thymocytes with this aberrant reauangement could further develop. We used modified inverted-PCR technique to detect the replacement of the V51D82Ja50 rearrangement by secondary Ja -rearrangement. Thymus DNA from day 17.5 of development was amplified and the excision products which contained both the first Ja-region rearrangement (V61D52Ja50) and the reciprocal joint of the secondary Jaregion rearrangement (Va3Ja49) were sequenced. We suggest that the V51D52Ja50-rearraiigement belongs to the normal thymocyte development. Therefor our results also indicate that a/p and T/S lymphocytes have a common precursor. Murine NK cells arc known to mediale Fi-hybrid anliparental graft rejection responses. This phenomenon has been linked to ihc MHC, and in particular, to lhe al/a2 domains of the MHC class 1 molecules. Here, we have addressed the role of MHC class I bound peptides in NK cell-mediated Fi-hybrid anii-parentai rejection by studying the resistance of F)-hybrids between B6 and differcnl bm muiant strains to B6-dcrived RBL-5 lymphoma cell line. Tumor development occurred at a similar frequency in all combinations of (B6Xbm)Fi mice and control B6 mice. These results suggest ihat absence of a specific MHC class I presented peptide repertoire on grafted cells is not sufficient lo induce NK cell-mediated Fl-hybrid anti-parental rejection responses. Contributed equally to this study. Class I molecules of the major histocompatibility complex (MHC) affect target cell sensitivity lo natural killer (NK) cell lysis, and influence the development of the NK cell repertoire. In the mouse, absence of MHC class I molecules on target cells is associated with an increased susceptibility to NK cell mediated lysis, while syngeneic class 1 molecules confer protection. In contrast to the protective role of syngeneic class I molecules, less is known about the interaction between murine NK cells and allogeneic class 1 MHC molecules. In theory, such could either be triggering, inert or inhibitory. To directly address the role of allogeneic class I in Interaction with NK cells of the mouse, a panel of polyclonal allogeneic murine NK cells were exposed to H-iP class I positive or negative target cells, and susceptibility to lysis was assessed. For all effectors studied, regardless of H-2 haplotype or genetic background, a preferential killing of class I deficient targets was observed. This pattern was observed JH vitro with tumor as well as lymphobiast targets, and in vivo in rapid elimination studies of radiolabeled tumor cells. The results demonstrate that protection from murine NK celt mediated lysis can be conferred by the expression of allogeneic class I molecules. No evidence for a triggering effect caused by the expression of allogeneic class I molecules was observed. The data are discussed in relation to current models on NK cell/MHC class 1 interactions, alloreactivity mediated by NK cells, and the role of NK cells in allogeneic graft rejection responses. STAPHYLOCOCCUS AUREVS ARTHRITIS IN MICE I-Sakinieae. T Hremcll. A, Tarkowski E)epanmcnts of Clinical Immunology and Rheumatology. University of Gbteborg, S413 46 Goteborg, Sweden J-ax +46-31-826791 Bacterial arthniis is a serious rapidly progressing disease wiih high morbidity and mortality despite antimicrohiaj therapy The erosive arthnUs is mediated by bacterial products as well as inflammatory cells. We evaluated the effect of corticosteroids in the treatment of septic Staphylococcus aureus arthritis. Materials and methods. Swiss mice were injected i.v. with S. aur^uj LS-1 Three days after inoculation of bactena i p treatment with cloxacillin anl demethasone was started. The mice were followed individually Arthritis was evaluated clinically and histopathologically. Serum samples and bacteriological isolates were obtained Results No The selection of allogeneic T cells after injection into neonatal SCID (H-2^} mice was analyzed. In the spleen of young recipients (<. 21 days old) injected with CBA/J (H-2'') T cells, CD4+ donor cells had increased two limes and CD8+ donor lymphocytes had increased six times in cell number, with a six-fold enhancement in the frequency of CDK^ Vpl4+ T cells. This was paralleled by clinical signs of GVH disease. Donor cells from these mice did not proliferate in vitro when stimulated w ith spleen cells from nude mice of H-21'. H-2'', or H-2'' haplolype. In eight weeks old recipients howe\ er, spleens were of a size similar to that of untreated SCID, containing 25% donor lymphocytes with a normalized CD4/CD8 cell ratio and TCR Vp repertoire, and the proliferdlive inhibition was confined to H-2'' reactive donor cells. Thus, H-2^'+ cells from these mice also proliferated against syngeneic non-T-cells, suggesting reversion of anergy of autoreaclive populations when parked in SCID mice. When CBA/J donor cells that had resided in SCID mice for two months were transferred into syngeneic CBA/Ca nu/nu recipients a significant reversion from tolerance to H-2^ e.\pressing spleen cells was obserd. When injecting C57BI/6 T cells, the SCID recipients developed a more severe GVHD resulting in death in the niajonty of the mice within three weeks. Here we also detected differences in the distribution of the donor cells and in the cytokine pattern in the sera compared to SCID mice injected with CBA/J. The significance of immunoregulatory ceils in GVHD will be discussed. M. Salmi, and S Jalkanen MediCity Research Laboratory, Turku University. Tykistokatu 6, 20520 Turku, Finland Fax.+358-21-6337000 The regulated interactions of leukocytes with vaseular endortielial ceils are crucial in controlling leukocyte trafficking between blood and tissues Vascular adhesion protein-l (VAP-l) is a novel, human endotheiial cell moiecuie that mediates tissue-selective lymphocyte binding Two species of VAP-l exist in lymphoid tissues Glycosidase digestions revealed that the mature 170 kD form of VAP-l expressed on the lumenal surfaces of vessels is a heavily siaiyiated glycoprotein containing a mucin-like domain The sialic acids were indispensable for the function of VAP-l, since the desialyiated form of VAP-i no longer mediated lymphocyte adherence We aiso show that L-seiectin is not required for iymphocyte binding to VAP-l under conditions of shear stress These data indicate that L-seiectin negative lymphocytes can bind to peripheral iymph node type venules via VAP-I mediated pathway thus circumventing the need for the MECA-79 defmed peripheral lymph node addressin -L-selectin interaction Moreover, our findings extend the role of carbohydrate mediated binding in iymphocyte-endothelial interactions beyond the selectins. In coneiusion. VAP-i naturally exists as a 170 kD siaiomucin-like protein that uses sialic acid residues to interact with non-L-selectin ligand(s) of lymphoeytes under non-static conditions. Psoriasis is a chronic infiammatory disease charaterized by hyperproliferation of keratinocytes and accumulation of activated T-cells in epidermis. Psoriasis has been associated with infections by Streptococcus pyogenes and cross-reactions between streptococcal M-proteins and human keratins have been demonstrated. The aim of this study was to investigate the possibie roie of cross-reactive T-cells the pathogenesis of psoriasis. Peripheral T-ceii responses to keratin, M-protein and four M-peptides with shared sequences were analysed in untreated psoriatic patients and healthy controls by proliferation and production of IFNy and lL-4 using ELISPOT There was a dominating IFNy response to M-proteins and keratin, characteristic for Thi-cells, with little IL-4 production and proliferation. Compared to controls psoriatic patients showed markedly stronger IFNy-response to all the peptides, but less difference was observed in response to whole proteins. These results are consistent with the hypothesis that streptocoecai infections may activate keratin reactive Thi-ceiis in susceptible individuals, contributing to the pathogenesis of psoriasis. Psoriasis is a chronic inflammatory disease charaterized by hyperproliferation of keratinocytes and accumulation of activated T-cells in epidermis Psoriasis has been associated with infections by Streptococcus pyogenes and cross-reactions between streptococcal M-proteins and human keratins have been demonstrated. The aim of this study was to investigate the possible role of cross-reactive T-cells in the pathogenesis ot psoriasis. Peripheral T-cell responses to keratin, M-protein and four M-peptides with shared sequences were analysed in untreated psoriatic patients and healthy controls by proliferation and production of IFNy and IL-4, using ELISPOT. There was a dominating IFNy response to M-protein and keratin, characteristic for Thl-celts, with little IL-4 production and proliferation. Compared to controls psoriatic patients showed markedly stronger IFNy-response to all the peptides, but less difference was observed in response to whole proteins. These results are consistent with the hypothesis that streptococcal infections may activate keratin reactive Thl-cells in susceptible individuals, contributing to the pathogenesis of psoriasis. Cross-linking of MHC Class I molecules (MHC-l) induces signals in human T-cells. We have studied the biochemical signal-pathway that leads to IL2 production and IL2 receptor (CD25) upregulation. Our results suggest that MHC-I signaling iniiially is dependent on tyrosine kinase activity whicli induces tyrosine phosphorylation and activation of PLC-yl, leading to a rise in the intracellular free Ca^* concentration. MHC-I signal transduction is dependent on surface expression of the TCR/CD3 and CD45 molecules. We are presently studying whether any specific tyrosine kinases are activated following MHC-I cross-linking. We have also investigated the later distal signal-pathway. Preliminary results suggest that an hereto unknown protein is specifically translocated to the cell nucleus following MHC-I cross-linking. Tlie nature of this protein is currently under investigation S. O. Linna* and A. Tiilikainen, Depts. of Medical Microbiology and Pediatrics*, Univ. of Oulu, 90220 Oulu, Skin testing was compared with in vitro production of sulfidoleukotrienes (sLT) in the diagnosis of immediate hypersensitivity, because a reliable in vitro test offers obvious clinical and financial advantages over an in vivo test. -Patients. Eight children with asthmatic symptoms and one adult with atopic eczema were tested. Allergens were the most common ones in the living environment of the patients: dog, cat, horse, timothy grass, birch, fish, egg and milk. Skin tests were performed with routine prick testing. sLT were released from isolated leucocjrtes in the presence of allergen and measured by a novel test (Buhlmann Laboratories, Basel, Switzerland), the Cellular Allergen Stimulation Test (CAST-ELISA) hased on the detection of LTC4, LTD4 and LTF4 by a monoclonal antibody. Correlation between skin tests and sLT production: sLT production Infection of inbred C57BL/6 mice by asexual blood-stages of P. berghei malaria parasites invariably results in a fata! disease course. However, there are two distinct clinical syndromes, related to the size of the infective inoculum.'Either death occurs during the second week of infection with marked neurological symptoms (known as experimental cerebral malaria or ECM), or several weeks later as the result of gross anaemia and hyperparasitaemia without any apparent neurological involvement. We have investigated in detail how the course of infection in this system depends on the size of the infective inoculum. Mice were infected intraperitoneally with 1x10^ to 1x10^ blood-stage P. berghei Kt73 parasites, and the course of Infection was followed daily by measuring parasitaemia, reticulocyte count, and rectal temperature. Low infection doses resulted in ECM in all mice, while no mice developed ECM after high dose infection. Intermediate dose infection caused some but not al! mice to develop ECM. Detailed results of these experiments will be presented, and the possible background for the dependency of the clinical outcome on the size of the infection inoculum will be discussed. Coronavirus transmissible gastroenteritis virus (TGEV) induces an actJte and fatal diarrhea in newborn piglets, as well as an intense and early production of interferon u (IFN-«). We have previously shown that in vitro Indudble porcine IFN-u secreting cells {IFN-ix SC) were present in pig fetal hematopoietic organs at early stages of gestation (Splichal et al.,Immunol. Letters, 1994, 43. 203-208) . In the present study, miniature pig fetuses were treated by TGEV via umbilical cord vein injection after laparotomy and uterotomy of anaesthetized pregnant sows. Control fetuses were subjected to the same surgery. All fetuses were delivered 20 hrs after surgery. Cell suspensions from blood, liver. bone-martow and spleen were incubated fcr 10 hrs in tissue culture microplates or nitrocellulose-bottomed microplates coated with anti-pig IFN-it mAb. IFN-u titers in supernatants were determined by a specific ELISA. whereas IFN-a SC frequency was obtained by an ELISPOT assay. Flow cytometry was performed to characterize CD 45, CD 4. SLA class il and 74-22-15 G/M positive cells. IFN-.x SC and IFN-u production were detected after in utero TGEV injection of fetuses at the second half period of gestation. IFN-a SC were more abundant in nonadfierent cell preparations than in total cell suspensions. These results show the presence of IFN-u SC in porcine fetal lymphohematopoietic organs following in utero exposure to TGEV. Comparability of measurement results is essential for research studies. Validation of analytical methods by a method evaluation study is therefore required before methods are used for research purposes. In this study a method evaluation of an Enzyme-Linked Immunosorbent Assay (ELISA) for determination of IgA in human serum was performed by use of the programme AMIQAS. The ELISA was set up as a sandwich ELISA using antibody against IgA for coating microtiterptates. Method evaluation samples were prepared from a serum sample with a high concentration of IgA (6.5 g/L) and an IgA-deficient serum sample in the ratio 1+5, 2+4, 3+3. 4+2, 5+1, 6+0, The serum samples were run in a dilution of 1/40,000. The detection range established by this method evaluation was shown to be 0.3 -6.5 g/L. The method evaluation analysis showed that the method was in analytical and statistical control. No significant systematical errors could be demonstrated. IgA may be used as a marker of exposure to microorganisms or to stress. The method is currendy being used for investigations of IgA levels in serum from waste collectors and from workers employed in the textil industry. Waste collectors are exposed to a variety of different microorganisms during collection of waste. These microorganisms are mainly fungi and actinomycetes, and to a lesser extent bacteria. Investigations were performed with waste collectors collecting compostable waste in order to determine the microbial exposure and their specific antibody response against microorganisms. Waste collectors were equipped with filters for collection of airborne microorganisms. The microorganisms found on filters wen; cultivated on selective media. The predominant microorganisms were found to be fungi of the Penicillium and Aspergillus species, and actinomycetes. Serum from the waste collectors was investigated for antibody response (IgG, IgE, IgA) against the cultivated microorganisms in SDS-PAGE and immunobloiting. Various antiboby responses (IgG) were found against Aspergillus fumigatus. which is a fungus that can give respiratory symptoms. Other antibody responses were found against different microorganisms cultivated from exposed filters. These are being further investigated. This study is a part of the research programme "Waste Colleeiion and Recycling" (CORE). We report that aIpha-2-macrogIobulin (a;M) can form complexes with a high molecular weight porcine mannan-binding protein (pMBP-28). The 0(jM/pMBP-28 complexes were isolated by PEG precipitation and affinity chromatography on mannan-Sepharose, protein A-Sepharose and anti-IgM Sepharose. The occurrence of o(2M/pMBP-28 complexes was further indicated by use of an anti-a^M affinity column and chelating Sepharose loaded with Zn'*. The eluates from these affinity columns showed a^M and pMBP-28 in SDS-PAGE and Westem blotting. Furthermore, the OjM/ pMBP-28 complexes were demonstrated by electron microscopy. Fractionation of pMBP-containing D-mannose eluate from mannan-Sepharose on Superose 6 showed two protein peaks which reacted with anti-Cls antibodies in ELISA, one of about 750 kDa which in addition contained pMBP-28 and anti-ajM reactive material, the other with an M, of 100-150 kDa. The latter peak revealed rhomboid molecules (7 x 15 nm) in the electron microscope and a 67 kDa band in SDS-PAGE under reducing conditions. This band was also seen in eluates from the anti-a^M and chelating columns. Based on these obset^ations we propose a model for the interaction of pMBP-28 with OjM. Serum amyloid P component (SAP) and C-reactive protein (CRP) are members of the pentraxin protein family. SAP is the precursor protein to amyloid P component present in all forms of amyloidosis. The prevailing notion is that SAP in circulation has the form of a double pentameric molecule (decamer) whereas CRP is a single pentameric molecule. We have investigated by gel permeation chromatography the M, of SAP in freshly collected human serum and of SAP purified by carbohydrate affinity chromatography and anion exchange chromatography. SAP was monitored by quantitative immunoelectrophoresis and ELISA, and SAP peak fractions were analyzed by use of SDS-PAGE, Western blotting, and electron microscopy. The results indicate that native SAP circulates as a single pentamer, part of which forms complexes with C4b-binding protein. The properties of SAP changed during purification as indicated by immunoelectrophoresis and electron microscopy. Thus, electron micrographs of purified SAP showed a predominance of decamers. However, the SAP decamers reversed to single pentamers when purified SAP was incorporated into SAP-depleted serum. KM. Thompson. M. Bgrretzen. I. Randen, 0, Fgrre, J.B. Natviq. Institute of Immunology and Rheumatology, The National Hospital, Fr.Qvams gt.1, 0172 Oslo, Nonway We have compared the structure of IgM rheumatoid factors {RF) produced as monoclonal antibdodies from healthy individuals, following immunization, to RF derived from RA patients. RF from the synovial tissues of RA patients preferentially use VH 3 gene segments together with a diverse array of V,,, This contrasts with RF derived from the peripheral blood of healthy donors, v^here the great majority utilize light chain segments of the kappa 3 family, and VH1 heavy chain segments are frequently used. RF from both groups were frequently found to be extensively mutated in the VH segment However, inspection of the R:S ratios in the CDR1+2 of these antibodies revealed that there was a strong selective pressure acting against mutations leading to amino acid replacements in the RF from healthy individuals. This was not seen in RF from RA patients. Affinity measurements indicate that RF in healthy individuals do not undergo affinity maturation, and that RF in RA may develop high affinity for IgG which may be of significance to the pathology of the disease. Preliminary studies suggest the existence of several different serotypes of//, pylori In this study, the capacity of//, pylori to induce type-specific Immune responses was studied. Sera and gastric aspirates from 11 //. pylori infected Swedish adults were tested for specific antibodies against membrane preparations (MPs) and lipopolysaccharides (LPS) prepared from the study subjects' own strains and with corresponding antigens from two reference//, pylori strains using different ELISA methods It was found that sera from 6 ofthe 11 subjects had significantly higher IgA antibody titers and 4 sera had higher IgG titers against MPs from the homologous strains than from the reference strains Five sera had higher IgA titers and 5 sera had higher IgG titers against LPS prepared from the subject s own strain than against LPS preparations from the two reference strains. Similarly, gastric aspirates from six subjects had higher IgA titers against the homologous than the heterologous MPs and five aspirates reacted in higher IgA titer with the homologous LPS. In immunoblotting analyses, sera showed stronger reactivities with homologous than with heterologous LPS preparations. The results support the existence of different serotypes of H. pylori expressing strain-specific antigenic determinants that are immunogenic in vivo. Mice of various inbred strains of conventional euthymic mice were fed by gliadin in attempts to induce enteropathy. Gliadin was administered intragastrically for two months starting in 10 days old mice, bovine serum albumin (BSA) fed mice served as controls.As conventional mice were not sensitive to gliadin feeding, immunodeficient strains of mice: athymic nu/nu Baib/c mice, mice with targeted disruption of IL-2 gene (IL-2 K.O.) and mouse strain with severe combined immunodeficiency (SCID), were used to determine the possible effect of long term gliadin feeding on their intestinal mucosa. Profound changes of intestinal mucosa were found in specimens of jejuna of gliadin fed athymic nude mice. Computer image analysis revealed markedly lower values of villi lengths and areas in gliadin fed nude mice as compared with BSA fed controls. IL-2 K.O. and SCID mice exert only minimal changes. The differences in response to gliadin feeding among various experimental models suggest that impairment of immunoregulatory mechanism is responsible for induction of intestinal lesions. T lymphocytes accumulate in the rheumatoid synovium. TheT cell derived cytokines IFNy and IL4 are produced following activation and affect the activity of other cells in the compartment. However, there are contlicting results concerning the production capacity of cytokine production in the synovium. In this study We compared the in vitro activation ofT-lymphocytes from synovial tissue (ST) and fx:ripheral blood (PB) in patients with RA by measuring IFNy and IL4 levels after in vitro stimulation of mononuclear cell suspensions. Methods: ST and PB samples from 18 patients with RA wei-e obtained. Mononuclear cell suspensions were cultured for 72 hours in medium containing ConA. Cell-free supernatants were analysed for IFNg-and IL4 contents. Results: No spontaneous production of IFNv wa.s detected in any of the PB or ST cultures. In contrast, after stimulation with ConA was detected inl2/14 PB {3400+4200U/ml) and 13/14 ST (5300+6300U/ml) cultures A spontaneous IL4 production was detected in 8/17 PB (600±300pg/ml) and 8/17 ST (10(>+400pg/ml) cultures. A mitogen-induced IL4-production was detected in 12/16 PB(3200+4100pg/ml) and 13/16 ST (900+1900 pg/ml) cultures. In 10 of these 16 patients the synovial IL4 prtxiuction was lower than in matched PB samples. Conclusion: No significant differences in the activation between PB and ST T-lymphocytes are found with respect to the mitogen induced synthesis of IFNg and IL4. In addition, there was no apparent dominance of Thl or Th2 population in any of the two compartments analysed in patients with RA. Tlie Chinese population in Hong Kong has extremely low incidence of invasive H. influenme type b (Hib) infecUon as well as of cwriage of the microorganism Although there is no vaccinaiion against Hib in ttong Kong significant amounl of IgG antibody against Hib capsular polysaccharide (CPl was found in healthy children and adults with ELISA (Lau ei al.,1994) . The origin of these anUbodies may be cross-reactive antigens like CP of E.coti K 100 which is very similar to Hib CP. The aim was to detennine the isotype and idiotype distribution of natural antibody against Hih CP in healthy Hong Kong residenu. ELISA of 20 sera using biotinylaied Hib CP showed antibodies against Hib CP in all individtials. The antibodies were mainly IgG2. btit in most samples also IgA and IgCi4. Three intlividuaLs had IgGl and two IgM antibodies. The only child had the highest IgGl anUbody level. Isoelectric focusing/immunoblotdng (IRF) pattems of lgG2 antibody cloties against CP of Hib and E.coti K 100 were very similar in almosi ail cases. Three individuals had additional clones for K 100 CP as compared to Hib CP. which disappeared after absorpUon with K 100 CP in two cases. Spectrotypic analysis of IgG antibodies belonging to the Hib idiotype 1 (Id-1) a-ucas et.al.,1991) revealed much stronger EF patterns as compared to the Hib CP specific IgG2 antibody wiUi bands in different locations. The high reactivity or serum IgG IgA and IgM against monoclonal antibody to Hib Id-I (a-Id-l) was also found in ELISA. This reacUvily was not abolished after absorpUon of the sera either with Hib CP or K 100 CP. Tbe data indicate the higb prevalence of ld-1 in the Hong Kong populaUon. However, the share of Id-1 among Hib CP specific antibodies was significant only in one individual. Others bad much lower activity of Id-1 anti-Hib CP antibodies as compared to the total IgG Id-I. T^at may indicate thai the Hong Kong subjects have Id-1 positive anUbodies in their serum that are not specific for Hib CP. This is consistent wilh the nature of Id-1 which is a marker of the A2Vi^ region nsage rather than a marker of Hib CP paratopes. We suggest that natural antibodies to Hib CP found at high levels in the sera of healthy Hong Kong Chinese arc the product of exposure to crossreactive andBens different figm E.coli K 100 CP. Colonization of the embryonic thymus by hematopoietic progenitors depends on the adhesion of these cells to the thymic vascular surface, extravasation, and transepithelial migration. Using the chicken, which offeis excellent access to the embryo, we searched for T cell progenitor-specific antibodies that could interfere with thymus homing. Monoclonal antibody (mAb) 2-264 recognized thymocytes from embryonic day 12 onwards, but the expression level of the antigen decreased upon thymocyte differentiation. In the adult, only low expression was found on 5-10% of blood and spleen cells. The expression of this protein is highest on CD4'8' thymocytes. intermediate on CD4+8+ thymocytes. and low on CD4-8* thymocytes. CD4+8" thymocytes lack the 2-264 antigen. A subpopulation of embryonic bone marrow cells and vascular endothelial cells are 2-264+. From thymocytes and a transformed T cell line. mAb 2-264 precipitated a molecule of 110 kD both under reducing and nonreducing conditions. An embryonic chicken thymocyte cDNA library in the vector pCDNA3 was saeened in a COS cell transient transfection system, and a 1.8 kb cDNA clone was isolated. The protein predicted from the nucleotide sequence is an adhesion molecule belonging to the immunoglobulin superfamily. It consists of five extracellular Ig domains, a transmembrane region and a shon cytopiasmic tail. As 2-264+ cells in 13-day embryonic bone marrow in a high frequency give rise to T cells after intrathymic reconstitution in the congenic chickens, we suggest that this protein is involved in T cell precursor migration into the thymus. Staphylococcus aureus is the most prevalent infectious agent in hematogenously spread kidney infections in man.The aim of our study was to develop a model of this disease and to characterize in situ inflammatory reactions. Materials and methods: Sixty Swiss mice were inoculated i.v. with 0,8 xlO''S.aureus of LS-1 strain. At days 1,4.7.14,21 and 32 after inoculation the mice were sacrificed. Bacj^dal cultures from blood, iirine and kidneys were obtained.In adition . histopathological and immunohisiochcmical evaluation of kidneys was performed. Results; Within the first week after inoculation peak values of tissue resident S.aureus were observed,Histopathologically both focal and diffuse inflammatory infiltrates were seen in kidney cortex and medulla. Immunohistochemical evaluation revealed high numbers of Mac-1"*" phagocytic cells as well as CD 4"'' and CD 8"'' lymphocytes. Many of T cells stained for Vbeta 4,7, lOb and 11 TCR families. We describe a first model of hematogenousiy acquired S.aureus nephritis.Immunohistochemical data suggest that T lymphocytes vividly participate in the course of this disease. We have found that IL-4-targcted mice (IL-4-/-) are grossly impaired in (heir mucosal immune response to conventional protein antigens. In the present study we sought to understand the mechanisms responsible for the defecl in these miee. Wild type and IL-4 -/-mice were orally immunized with OVA together with cholera toxin (CT) adjuvant. Irrespective of the dose of antigen we failed to detect but poor anti-OVA local and s>slemic responses while anti-CT responses were roughly 50% cf those round in wild type mice Even when lL-4 -/-mice had already responded with high anti-OVA serum antibody liters afler i.v. immunization we could not elicit local anti-OVA IgA responses through oral immunizations. Further attempts to immunize perorally with OVA entrapped in microparticles failed. By contrast, if OVA was covalcntly coupled to CT strong responses were detected both systemically and in particular in gut lamina propria. Whereas only poor T cell priming in gut associated tissues was achieved with OVA given admixed with CT the conjugated form of OVA fully restored specific T cell priming as determined by proliferation and IFNy production Contrary to what has been proposed earlier CT acts in the absence of IL-4, and probably Th2-ce]]s, and uses an lL-4 ind^ndent pathway to stimulate gut mucosal immune responses. Intercellular adhesion molecule 1 (ICAM-1) is a member of the immunoglobulin superfamily interacting with the integrins. This interaction is critical for leukocyte extravasation into inflamed tissue. The aim of this study was to assess the role of ICAM-1 in the pathogenesis of Staphylococcus aureus septicemia and arhtritis. Method: Mutant mice, lacking the ICAM-1 gene, and normal wild-type congenes were intravenously injected with S. aureus strain LS-1. The animals were foltowed-up clinically during six days, regarding development of arthritis and mortality. Results: Six days after inoculation 50% of the animals in the ICAM-1 mutant group have died but none in the wild-type group. The mutants developed less severe arhritis than the wild-type animals. Histopathological examination of the lCAM-1 deficient mice displayed decreased erosive arthritis than in the controls. The sera from the mutants contained lower levels of IFN-ycompared to the congenes. This is in agreement with the m vitro data, where spleen cells from naive ICAM-1 deficient mice stimulated with TSST-1 and cell walls, produced lower levels of IFN-Y than spleen cells from healthy controls. Naive mutant mice had more circulating neutrophils and lymphocytes but lower number of cells capable of phagocytosis than the wild congenes. Conclusion: Mice lacking the ICAM-I gene exhibit higher mortality in S.aureiis arthritis than normal controls. Lower levels of IFN-Y production a"^ lower number of phagocytizing ceils compared to the wild-type mice may contribute to this. However, decreased extravasation of leukocytes due to the lCAM-1 deficiency, leads to a less destructive course of septic arthritis in comparison with the wild littermates. MHC molecules and maybe most notably classll molecules are, depending on allcle. correlated with susceptibility or resistance to particular autoimmune diseases. In a model for Rheumatoid Arthritis that we study. Collagen Induced Arthritis, the susceptibility is strongly correlated with the Aq molecule. We have previously shown that an Ab'' transgene expressed on an H-T background makes resistant mice arthritic. We have noy/ extended this study andexpres.sed the transgene on BIO congenic strains of the H-T, H-2'. H-2" and H-2'' haplotypes. Somewhat surprisingly the transgene is able to fonn a cell surlace expressed dimer with several of the endogenous alpha chains. All but Aa" pair with Ap'' q to form cell surface dimers. We thus have more tools for studying the role of Ap** in restricting T eell response to self antigens. IgE antibodies specific for TNP are able to upregulate the antibody response to TNP-coupled carrierproteins via the low afftnity receptor tor IgE, CD23. In previous studies, the serum level of BSA-specific antibodies in mice given IgE + BSA-TNP was l(X)-fold higher, when measured in ELISA. as compared to tbat in control mice given antigen alone. In order to find out whether this difference was exclusively duetto affinity maturation or if there was an actual increase in the number of antibody producing cells, we used an ELISPOT assay which made it possible to detect the number of BSA specific cells in different lymphoid tissues. IgE antibodies induced significantly higher amounts of IgM-and IgG producing BSA-speeific cells in the spleen and the peak of the response was seen as early as five to six days after immunization. Furthermore, experimental parameters for lgE-mediated enhancement such as administration route, age and sex of the mice and hapteti density were investigated. IgE was found to enhance the response when antigen was given without adjuvants intravenously, intraperitoneally as well as subcutanously. The stimulation was independent of age and sex of the animals and required a low or medium coupling degree of bapten molecules to the carrier. Our findings show that IgE-induced responses are seen under several experimental conditions and that the B-cell response in spleen is upregulated very early after immunization. Patients with extrinsic allergic alveditis (EAA) have accumulations of T lymphocytes in their lungs, and CD8+ lung T cells have been implicated in the pathogenesis of EAA. In this study we analyzed the T cell receptor (TCR) Va and Vp gene usage of CD4+ and CD8+ lung and peripheral blood (PBL) T lymphocytes. Materials and methods. 10 patienLs with clinical signs of EAA were studied (7 were exposed to mould; 3 to birds). From seven of these patients, samples were also available after elinical reeovery. Lung cells, obtained by bronchoalveoloar lavage (BAL), and paired PBL samples were analyzed by flow eytometry using a panel of anti-TCR V speeifie monoclonal antibodies. Results. 5/7 patients had expansions, restricted to the lung, of CD8+ T cells using a particular Va or Vp. with a normalized V gene usage on follow-up. In four patients minor expansions using the same Vp in both CD4+ and CD8+ subsets were identified, suggesting a possible role for superantigens in this disease. Conclusion. In EAA we found most expansions, correlating with clinical activity, of T eells utilizing a partieular TCR Va or vp gene segment, to oceur in the lung CD8+ T cells. This further implicates these cells in the pathogenesis of EAA. We intend to procceed studying the functional role of CD8+ T cells in EAA by stimulating T cells with the relevant antigen in vitro. Vitamin A deficiency is associated with a 3-4 times higher incidence of infectious diseases and increased mortality in children in developing countries. Accordingly we have seen that vitamin A deficient rats, inoculated iv. with Staphylococcus aureus. develop septic arthritis with a 2 times higher frequency than ihe control rats. Since phagocytes play an important role in the defence against bacierial infections, we wanted to study if the function of those cells is influenced by vitamin A deficiency. Peripheral blood from vitamin A deficient and control rats was used to determine the percentage of phagocytic cells as well as Ihe phagocytosis activity per cell. This was done by measuring the number of fluureceinisothiocyanate liibelled bacteria ingested by granulocytes and monocytes. To determine Ihe capacity of intracellular killing of bacteria phagocytes from the peritoneal cavity of vitamin A deficient and conlrol rats were recovered and incubated with staphylococcus aureus for 24 hours. Thereafter Ihe cells were lysed and the incorporated bacteria cultured on horse blood agar plates. The percentage of phagocytic cells in the peripheral blotid of vitumin A deficienl rats was about 40 % higher (p = 0.001) and the number of bacteria ingested per cell 30 % higher in viiamin A deficient than in control rats. However, the intraceilutar killing capacity of the phagocyies from vitamin A deficient rats was significantly impaired (p = 0.02), since a higher number of bucteria remained alive in the phogocytes after 24 hour incubation. The results from our study show that the relative number of blood phagocytes is increased during vitamin A deficiency. The ingestion of bacteria by these cells is nol affected but Ihe ability to kill bacteria is impaired. This might be an important mechanism explaining the increased susceptibility to infections, respectively to septic arthritis during vitamin A deficiency. TRANSFER OF MODIFIED HUMAN IL-IB INTO TUMOR CELLS Anette Gjorloff Wingren', Olle Bjorkdahl', Tord Labuda', Gunnar Hedlund'-^ , Hans-Otov Sjogren', Terje Kalland'-^, Bengi Widegren', and Mikael Dohlsten' -^. 'The Wallenberg Laboratory, Department of Tumor Immunology, University of Lund, Lund, Sweden. Fax.no; +46-46-104201. ^Pharmacia Oncology Immunology, Lund, Sweden. Two different human IL-113 cDNA constructs coding for the mature protein were prepared in order to develop a model for studying the biological effects of lL-lB release. To obtain a released form of human IL-1B. a signal sequence from the related IL-1 receptor antagonist was fused to the lL-lB encoding gene construct. A panel of murine cell lines was transduced with retroviral technique and analyzed for Ihe expression of IL-1B, with (ssIL-IBl or without (lL-lB) a signal sequence. Cell lines transduced with IL-IB contained large amounts of the IL-I6 protein i.e. whereas cells expressing sslL-lB .secreted most of the IL-16 protein. Treatment of ssIL-lB transduced cells with Brefeldin A,an inhibitor of protein transport to the endoplasmatic reticulum, induced accumulation of the protein i.e. Release of the mature form of IL-IB from monocytes has been connected with cell death. In mouse fibroblast, melanoma, lymphoma and fibrosarcoma cell lines transduced to secrete IL-IB, no accompanying cell death could be detected. Preliminary in vivo results using IL-IB and ssIL-lB transduced tumor cells demonstrated distinct regulation on tumor growth. I.M. Wold. I.N. Farstad, P Krajci, F.E Johansen and P.Brandtzaeg, Laboratory for Immunohistochemistry and Immunopathology (LIIPAT), Institute of Pathology, University of Oslo, Rikshospitalet, N-0027 Oslo, Norway. Fax: +47 22112261 The J chain is a 15-kDa polypeptide present in dimeric IgA and pentameric IgM (collectively called pig). This peptide is cnjciai for the affinity of pig to the epithelial secretory component (SC or pig receptor) and therefore plays a key role in secretory immunity.To define at which stage of B-cell differentiation J-chain expression starts we have characterized J-chain expression in several human B-cell lines representing different stages of differentiation. Northem blot analyses were used to screen for J-chain expression and quantitative PCR was established to quantify the copies of mRNA per cell. An intemal standard for the PCR reaction was constructed that utilizes the same primer pairs as the J-chain mRNA.The cell lines were phenotyped by How cytometry for surface markers including CD38 and the integrin 37 subunit, J chain was expressed in all cell lines except the pre-B-cell lines. This is in contrast to murine B cells where J chain is expressed only after activation. J-chain expression at the mRNA level was also detected in plasmacytoid B-cell lines that secrete IgG or IgE. The level of J-chain mRNA quantified by PCR ranged from 19000 to 350000 copies per cell. Protein and mRNA expression were well correlated. A new technique, liquid-liquid partition chromatography in an aqueous polyethylene glycol/dextran two-phase system, was used to analyse differences in surface properties of antibodies with different antigen-binding sites. Employing welt-characterized monoclonal IgG antibodies, intact as well as Fab/Fc fragments thereof, and chimeric IgG antibodies, we found a remarkable relationship between the structure of the antibody combining sites and the chromatographic behaviour of the antibodies. The surface properties of IgG antibodies are dominated by those of its antigen-binding regions. In addition, the constant parts of IgGl, 2 and 4 seemed to fotm similar scaffoldings, on to which CDRs of variable shapes and sizes are interspaced. The surface properties of antibodies of IgG, IgM, IgA and IgE type were also compared as were the events, in terms of conformational changes, occurring upon binding of hapten by these antibodies. The importance of the observed differences in surface properties/conformational changes are discussed. Reactive arthritis develops after certain gastrointestinal or urogenital infections in susceptible HLA-B27 positive individuals. Abnormal interaction between the causative microbes and the host is suggested to play a role in the pathogenesis of this disease. We fed peripheral blood monocytes of healthy HLA-B27 positive persons with Yersinia enterocolitica 0:3 and followed the expression of HLA-B27 on the cell surfaces using monoclonal antibodies against different epitopes of HLA-B27 molecule. We found that phagocytosis of Y. enterocolitica 0:3 induced changes in the expression of different epitopes of HLA-B27. This suggests that Y. enterocolitica O:3 altered the three-dimensional structure of HLA-B27 molecules. The modifications were in close proximity to the antigen binding cleft. The antigen presentation by modified HLA-B27 molecules may have a role in the pathogenesis of seronegative spondylarthropathies via impaired elimination of arthritis-triggering microbes or via an autoimmune mechanism. A.Yarilin and N.Sharova. Laboratory of Lymphocyte Differentiation, Institute of Immunology, Moscow, Russia Fax:+7 095 1171027 Epithelial cell line HTSC from human thymus was produced. HTSC cells are large polymorphic cells with numerous cytoplasmic inclusions; they express cytoceratins and products of MHC classes I and II but lack or lytnphocyte and macrophague markers. Proliferation and secretory activity are absent in monocultres of HTSC. Addition of viable human thymocytes induces epithelial cell proliferation and secretion of al-thymosin and high-molecular factors with T-cell co-stimulatory activity. The secretion can be modulated by some hormones, cytokines and physical factors. Thymocytes adhere to HTSC cells and die by the mechanism of apoptosis during first day of co-culturing and then are eliminated. Human blood T cells and tnouse thymocytes are considerably less effective in inducing HTSC cell activation and resistant to apoptosis induction. The presence of idiotype-reactive T cell subsets and their relation to the tumor load were analyzed in 9 patients with monoclonal gammopathy of undetermined significance (MGUS), 12 patients with multiple myeloma (MM) clinical stage I and 9 patients with MM stage II/III. An enzyme-linked immunospot assay was used to identify interferon (IFN)-Y-, interleukin (IL)-2-or IL-4-secreting T cells after stimulation by F(ab')2 fragments of monoclonal IgG. The response to autologous IgG was significantly higher than that induced by isotypic monoclonal IgG. Comparable results were obtained in a proliferation assay (^H-thymidine incorporation), Eight of 9 patient.s with MGUS, 7 of 12 patients with MM .stage I and 3 of 9 with MM stage II/III had T cells secreting lFN-y and/or IL-2 (T helper [Th] type-1 cells), whereas ceils secreting both Thl and Th2 or ThO types of cytokines were more frequent in patients with MM, particularly in those with MM stage Il/m. The number and frequency of Thl-type cells were significantly higher in MGUS patients as compared to MM stage II/III. The T cell response was inhibited by an anti-DR antibody. The results indicate that idiotype-reactive T cells of the Thl and Th2 or ThO subsets were present in monoclonal gammopathies, and might provide indirect evidence that idiotype-reactive Thl-type cells may have a regulatory impact on the human tumor B cells. Experimental allergic neuritis (EAN) is an inflammatory demyeiinating disorder of the peripheral nervous system. It is a T cell mediated and cytokines including interferon-y (IFN-y) and transforming growth factor (TGF-B) are involved in the pathogenesis By adopting in situ hybridization, we studied the mRNA expression of immune response up-and down-regulating cytokines in EAN. IFN-y, interleukin-4 (IL-4) and the TGF-(J were examineded in the target organ, i.e. sciatic nerve, and in lymph nodes and spleen over the course of EAN, The dynamics of IFN-y mRNA expression in the sciatic nerve followed approximately the clinical course of EAN with peak values around day 14 post immunization (p.i), whereas IFN-y was transcribed earlier in spleen and lymph nodes with maximum on day 7 pi. On the contrary, transcription of IL-4 and TGF-fl was only slightly enhanced in EAN, with minor fluctuations in sciatic nerves peaking on days 11 and 28 pi. The highest numbers of TGF-fl mRNA positive cells in lymph nodes were observed during clinical improvement of EAN, The data argue for a major proinflammatory role for IFN-7 and a hypothetical disease downregulating (unctions for TGF-6 at the target site in EAN. We have previously demonstrated that staphylococcal enterotoxins contribute to the anhritis and mortality during staphylococcal infection. To further explore the relationship between staphylococcal superantigens and T cell responsiveness in the pathogenesis of hematogenic SEA producing S aureus infection we used TcR Vp3 transgenic (TGVp3) mice. TGVp3 mice and non-TG littermates were inoculated intravenously with S.attreus AB-1 strain producing a large amount of SEA, which specifically reacts with TcR Vp3. TGVp3 mice were highly susceptible to the acute bacterial infection. Within 9 days after inoculation, 85% of TGVp3 mice died compared with 31% of non-TG littermates (p