Second International Conference on Experimental and Clinical Reproductive Immunology, 15—18 November, 2000 Amsterdam, The Netherlands

Second International Conference on Experimental and Clinical Reproductive Immunology, 15—18 November, 2000 Amsterdam, The Netherlands

Journal of Reproductive Immunology 51 (2001) 51 – 103 www.elsevier.com/locate/jreprimm Second International Conference on Experimental and Clinical R...

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Journal of Reproductive Immunology 51 (2001) 51 – 103 www.elsevier.com/locate/jreprimm

Second International Conference on Experimental and Clinical Reproductive Immunology, 15—18 November, 2000 Amsterdam, The Netherlands Abstracts of Poster Presentations Immune infertility The effect of TGFbl deficiency on female reproductive performance in mice W.V. Ingman, S.A. Robertson Department of Obstetrics and Gynaecology, Uni6ersity of Adelaide, 5005, Australia Transforming growth factor b1 (TGFb1) is a pleiotropic cytokine implicated in the regulation of proliferation and function in many cell lineages. Mice deficient in TGFb1 must be rendered deficient in the adaptive immune compartment to prevent lethal autoimmunity. TGFb1 is produced in the ovary and in vitro studies have suggested a role for this cytokine in regulation of folliculogenesis. In the implantation site, TGFb1 is synthesised by uterine epithelial cells and placental trophoblast cells. To investigate the role of TGFb1 in female reproductive function, we have studied the effect of TGFb1 deficiency on ovarian cycling, mating frequency and pregnancy outcome in scid mice. Estrous cycles were tracked in 6-week-old TGFb1−/− and control (TGFb1+ / + or + / −) littermates (n= 6 per group) for 28 days by histological analysis of vaginal smears. The average number of cycles completed over 28 days differed significantly (P B0.01) between TGFb1− / − and control mice, averaging 2.8 9 1.0 and 5.3 91.0 cycles, respectively. Mean cycle length was 9.4 9 2.3 days in TGFb1−/− mice and 5.5 91.1 days in control mice (PB 0.01). The proportion of days spent in each phase of the estrous cycle over the study period did not vary significantly between genotypes. TGFb1−/− females (8 – 10 week old) tended to mate less frequently when placed with TGFb1+/+ males (interval between co-caging and mating event 11.7 9 9.1 vs. 5.2 9 3.1 days in TGFb1−/ − and control females, respectively, P =0.06). Pregnancy was rarely maintained to parturition in TGFb1− / − females (pups were born from 3/14 vs. 8/8 mating events in TGFb1− / − and control mice, respectively). These data show that genetic TGFb1 deficiency causes considerable disruption in ovarian function and 0165-0378/01/$ - see front matter © 2001 Elsevier Science Ireland Ltd. All rights reserved. PII: S0165-0378(00)00100-5

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support of conceptus development. The mechanisms underlying estrous cycle perturbation and pregnancy failure in TGFb1 deficient mice are now being investigated.

NK and T-cell subpopulations in follicular fluid in relation to cause of infertility H.G.M. Lukassena, A. van der Meerb, E. Lindemana, A. Wetzelsa, F. Wijnands, M.J.C. van Lieropc, S. Mosselmanc, I. Joostenb, D.D.M. Braata a Department of Obstetrics and Gynecology, Uni6ersity Medical Center, Nijmegen b Department of Blood Transfusion and Transplantation Immunology, Uni6ersity Medical Center, Nijmegen. cNV Organon Oss, The Netherlands

Leukocytes and cytokines play a key role in ovarian physiology alongside gonadotrophins and traditional growth factors. Many reports describe cytokine levels in follicular fluid (FF), but only a few studies focused on the cellular content. The question arises whether distribution of immune cell populations in follicular fluid varies depending on the cause of infertility and whether this is influencing the success rate of IVF treatment. We evaluated leukocyte subpopulations present in FF of patients undergoing IVF-ET treatment for idiopathic infertility or tubal factor infertility in comparison to andrological infertility (control group). Triple colour flow cytometry was used to discriminate T-cells and NK cell subsets. So far, 121 follicles from 47 patients were analysed. Only erythrocyte contaminated FF (n=59) contained sufficient leukocytes for analyses. The data show that the immune cell composition of FF differed significantly from that of peripheral blood leukocytes from the same patients. Interestingly, FF from patients with idiopathic infertility contained a higher proportion of NK cells and a lower proportion of T cells compared to the tubal factor or andrological infertility groups. This difference was not observed in peripheral blood. Remarkably also, the composition of the NK cell subset was different, with a relatively high percentage of immunoregulatory CD16-CD56 + + NK cells. These results show striking differences in the distribution of lymphocyte subsets in FF between idiopathic infertility and other causes of infertility, possibly implicating an immunological basis for infertility. We take an interest in the functional capacity of these cells with respect to fertilization rate, oocyte maturation and embryo quality.

Detection of HLA-I and II on ejaculated human spermatozoa E. Alenany, H.D.M. Moore, I.D. Cooke Department of Obstetrics and Gynaecology, Uni6ersity of Sheffield, Sheffield, S3 7RE, UK

Detection of HLA-I and/or II molecules on ejaculated human spermatozoa has been a controversial subject for many years. We have investigated the issue using specific monoclonal antibodies in conjunction with immunolocalisation and fluorescent activated cell sorting (FACS) techniques. Semen samples were obtained from patients and potential sperm donors and were examined before and after ‘swim-up’ or Percoll selection for viable cells. Using direction immunofluorescent localisation, : 25% of sperm preparations were negative for HLA-I and II. A variable proportion of spermatozoa (5 –20%) in the remaining preparations displayed significant binding of HLA-I and/or II. There was no significant difference in the degree of HLA binding to spermatozoa from patients or donors. HLA antigen was localised to the head and midpiece of spermatozoa. A similar set of results was

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obtained by FACS analysis. Furthermore, there was no significant difference in the proportion of spermatozoa displaying HLA antigen in whole semen samples or selected motile populations. In contrast to lymphocytes, HLA-I and II expression on the sperm surface could not be induced in vitro with interferon (IFN-g, 5 ng ml − 1). However, soluble HLA-I and II (sHLA) was detected in seminal plasma from fertile and infertile individuals. Possible receptors (CD4) for these sHLA molecules were detected on spermatozoa. We conclude that a small proportion of ejaculated spermatozoa from patients and donors may display HLA on their surface, but the presence of these molecules on spermatozoa is not correlated with either sperm motility or male fertility. HLA molecules might be absorbed onto the surface of spermatozoa from seminal plasma.

Genetic analysis for Y chromosome microdeletions in Japanese infertile males K. Sakataa, S. Komoria,b, Y. Nakatab, H. Katoa, H. Sawaib, K. Koyamaa,b a Department of Obstetrics and Gynecology, Hyogo College of Medicine, Nishinomiya, Japan b Laboratory of De6elopmental Biology and Reproduction, Institute for Ad6anced Medical Sciences, Hyogo College of Medicine, Nishinomiya, Japan Introduction: The genetic basis of infertility remains unclear in a majority of infertile men. In this study, Y chromosome long arm involving DAZ (deleted in azoospermia) was screened for evaluating the incidence of microdeletion in Japanese infertile men. Methods and results: 157 infertile Japanese men with azoospermia and severe oligozoospermia were analyzed for microdeletions of the interval six of the Y chromosome by using polymerase chain reaction with sequence-tagged site markers. The eight sets of oligonucleotides primer were synthesized for polymerase chain reaction and Southern blot analysis was also performed. All analyzed infertile men were divided into five categories on the basis of sperm concentration: functional azoospermia (A) 24, azoospermia due to obstruction (AO) 20, oligozoospermia-I (OI) ( B 1× 105/ml) 33, oligozoospermia II (OII) (B 1× 106/ml) 30 and oligozoospermia III (OIII) ( B 1× 107/ml) 50. Thirty fertile men with sperm concentration \2×107/ml were also analyzed as control. Results: In 12 (7.6%) of 157 infertile men, microdeletions were identified as follows; one in category A, one in category AO, five in category OI, four in category OII and one in category OIII. On the other hand, no deletion was identified in fertile men. One common region around sY240 was identified in 11 infertile men with microdeletions. This locus might contain specific genes for spermatogenesis. The sperm concentration of nine oligozoospermic men with microdeletions was B 1×106/ml. There were no correlation between the severity of spermatogenesis and the extent of microdeletions. Conclusion: These results suggested that genes in the interval six of the Y chromosome might be important for spermatogenesis.

The role of pantophysin(h-Sp-1) in human sperm-egg interaction Shinji Komoria,b, Kazuko Sakataa, Hiroyuki Tanakaa, Ri-ichiro Kanazawaa, Yoshiyuki Tsujia, Koji Koyamaa,b a Department of Obstetrics and Gynecology, Hyogo College of Medicine, 1 -1 Mukogawacho, Nishinomiya, Hyogo, 663 -8501, Japan b Laboratory of De6elopmental Biology and Reproduction, Institute for Ad6anced Medical Sciences, Hyogo College of Medicine, Nishinomiya, Hyogo, 663 -8501, Japan

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Introduction: Previously, we isolated a human sperm antigen gene (h-Sp-1; EMBL Accession No. X68194) by screening from human testis cDNA library with human sperm specific antisera. Since the sequence analysis of the h-Sp-1 gene indicated that it had 45% homology to human synaptophysin gene, the product of the h-Sp-1 gene was termed as pantophysin. In this study, we analyzed the function of pantophysin in sperm –egg interaction. Methods and results: Northern blot analysis indicated that h-Sp-1 gene had two kinds of transcripts (2.3 and 1.1 kb). The 2.3 kb transcript expressed ubiquitously, whereas the 1.1 kb transcript expressed abundantly in human testis. In order to characterize the biological function of pantophysin in sperm, antisera were generated by immunizing rabbits with synthetic polypeptides (amino acid no. 174 –198) based on the h-Sp-1 gene. The antisera stained fixed human sperm head by indirect immunofluorescence staining. The purified antisera showed an inhibitory effect on human sperm penetration assay into zona-free hamster eggs. However, they did not show any sperm immobilizing and agglutinating activities. Since pantophysin is homologous to synaptophysin, we are now analyzing the interaction between pantophysin and sperm membrane molecules. Conclusion: These results suggested that pantophysin might play an important role in the sperm–egg interaction.

Improved digital method for detection of sperm immobilizing antibodies in infertile patients Kentaro Nakanishia, Shinji Komoria,b, Akiko Hasegawab, Yukari Hamadaa, Minoru Shigetaa, Koji Koyamaa,b a Department of Obstetrics and Gynecology, Hyogo College of Medicine, Nishinomiya, Japan b Laboratory of De6elopmental Biology and Reproduction, Institute for Ad6anced Medical Sciences, Hyogo College of Medicine, Nishinomiya, Japan

Introduction: Among various methods for detection of antisperm antibodies in infertile patients, the complement dependent sperm immobilization test (SIT) is the most useful method for the detection of antisperm antibody relevant to infertility. The SIT test has been carried out by counting the number of motile and immobile sperm under a microscope since the assay was first established by Dr Isojima in 1972. The sperm immobilization value (SIV) sometimes varied, depending on observers and laboratories. To improve this point, sperm quality analyzer (SQA: United Medical Systems, Inc.) was applied to the assessment of sperm motility. Methods and results: The mixture (20 ml of test serum, 2 ml of sperm suspension per mililitre, 4 ml of guinea-pig serum as complement) was incubated for 1 h at 34°C. As control, the inactivated complement was used. Some 10 ml of the mixture was applied to a microcapillary tube for the sperm motility assay by SQA. SIV was calculated as the sperm motility ratio of control to test sample. Quantitative sperm immobilization test was carried out by serial dilutions of test serum. The dilution required for 50% recovering of sperm motility was represented as a quantitative sperm immobilization value (SI50). SIV calculated by the digital method showed very similar values as measured by the classical method. The criteria in the classical method, that is the SIV value of \2.0 is positive, also could be applied to the new SIT method. It is thus concluded that the new digital method using SQA was highly objective and reproducible and that the values obtained by SQA method are good correlation to those by classical method. Conclusion: The new digital method using SQA was found to be a highly objective and reproductive method and a good correlation to the classical method was obtained.

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The effect of antisperm antibodies on the acrosome status of human spermatozoa M.A. Nikolaeva, E.L. Golubeva, I.V. Korotkova, A.U. Krutskikh, V.S. Repin, G.T. Sukhikh Russian Scientific Centre for Gynecology, Obstetrics and Perinatology, Moscow, Russia

Introduction: The aim of this study was to identify the impairment of acrosome status of motile spermatozoa obtained from normozoospermic subfertile men that result from ASA presence in the semen. Methods: Swim-up spermatozoa from autoimmune ejaculates (MAR% \30%, n=20) and from antibody-free semen samples (MAR% =0%, n =24) were pre-incubated for 1 h in the medium 199 supplemented by 0.5% bovine serum albumin. Acrosome reaction (AR) was examined by flow cytometry following the dual fluorescence staining of methanol fixed spermatozoa with rhodamine-labelled Arachis hypogea lectin and fluorescein-labelled Pisum sati6um lectin as spontaneous and ionophore A23187 induced. Results: The incidence of spontaneous AR in ASA-positive semen samples (28.5 9 16.0) was found to be significantly higher (PB 0.0005) than that in ASA-negative semen samples (15.095.1). The inducibility of the AR (incidence of induced minus spontaneous) in ASA-positive samples (10.7 9 23.2) was significantly lower (PB 0.003) compared to the same parameter measured in ASA-negative samples (29.6 910.7). The MAR% correlated positively with spontaneous AR (r= 0.67, PB 0.0001) and inversely with the inducibility of the AR (r= −0.37, PB0.02). A significant inverse correlation between the percentage of spontaneous AR and the AR inducibility was revealed in ASA-positive samples (r = −0.65, PB0.005). The negative values of the AR inducibility ( − 16.5917.0, range: 2 – 51%) was shown in six (30%) ASA-positive semen samples. Conclusion: The presence of ASA in human ejaculate appears to be a cause of acrosome reaction prematurity that may interfere with fertilization. The spontaneous AR of antibody-coated sperm may be blocked with ionophore A23187.

Generation of superoxide anions in conditions of infertility and the role of nitric oxide on spermatozoal cytokinematics Madhumita Purkayastha, Sukumar Chattopadhyay Department of Life Science and Biotechnology, Immunology and Tissue Culture Laboratory, Jada6pur Uni6ersity, Calcutta-700032, India

Nitric oxide is reported to be synthesized by cells located on the reproductive tract and the spermatozoa. The evidence of the localization of various isoforms of nitric oxide synthase in the organs involved in reproduction indicated the importance of nitric oxide in reproductive activity. The generation of reactive oxygen intermediates by human spermatozoa is associ’ ated with the spermatozoal function of movement. Superoxide (O2− ) ion had been shown to interact with nitric oxide (NO) and result in the generation of peroxynitrite (ONOO−) which induces cell toxicity. The possible role of reactive oxygen and nitrogen intermediates together in maintaining the spermatozoal movement is under investigation. The objective of this study was to determine the effect of nitric oxide under the conditions when the release of reactive oxygen species has been initiated. Spermatozoa population of fertile and infertile specimen was exposed to sodium nitriprusside (SNP) and subsequently incubated with cytochrome c at 37°C for 10 min. The effect of the presence of calcium on nitric oxide mediated changes of generation of reactive oxygen species was tested by exposure of spermatozoa population to

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Ca Mg A23187. The release of reactive oxygen species was measured and the effect of simultaneous exposure to nitric oxide and reactive oxygen species on percentage motile population was observed. Nitric oxide reduces the generation of reactive oxygen species. It acts as a scavenger of reactive oxygen species and enhances the percentage of motile population in fertile specimen. In cases of infertile specimen, the reduction in release of reactive oxygen species could not produce significant enhancement in cytokinematics of spermatozoa.

Genes of IL-1 system in male gonad — a quantitative analysis of transcripts Natalia Czajkowska, Dorota Fiszer, Maciej Kurpisz Institute of Human Genetics, Polish Academy of Sciences, Poznan´ , Poland

Regulation of spermatogenesis consists of two main elements: well-known pituitary hormonal control and local regulation. Identification of the local regulatory factors still remains an experimental problem. Low level secretion of regulatory peptides and their periodical release are the main causes of technical difficulties. There are several groups of para- and autocrine factors secreted by male gonad: growth factors (TGFa and b, IGF, EGF), cytokines (IL-1, IL-2, IL-6) and protooncogenes. There is a growing body of evidence that the interleukins exhibit modulatory activity on reproductive cells. In this context interestingly, a role for IL-1 appears which is produced by the testicular cells. We have analysed transcripts of IL-1 gene system (IL-1a, IL-1b, IL-1RI and IL-1RII). To determine activity of gene transcription, a quantitative PCR with isotopic detection was applied. We have detected differential expression of IL-1a and IL-1b genes in separate functional compartments of male gonad. The obtained data show the dominant expression of IL-1a gene in intratubular cells, which corresponds to our previous observations. On the contrary, IL-1b expression seems to be predominant in extratubular compartment of the gonad. It has also been found the abundant amount of IL-1RI and IL-1RA mRNA in gametogenic cells fraction in comparison to interstitial cells. Genes of IL-1 system create a complicated intragonadal environment, which seems to be very important for regulation of spermatogenesis. Some results also indicate the role of IL-1 system in apoptosis that seems to be critical for physiological differentiation of seminiferous epithelium.

An autoantigenic osteoglycin-like sperm protein discovered by 2D proteomics M.J. Wolkowicz, J. Shetty, F. Jayes, C.J. Flickinger, J.C. Herr Center for Recombinant Gamete Contracepti6e Vaccinogens, Uni6ersity of Virginia, Charlottes6ille, VA 22908, USA

Two-dimensional gel electrophoresis and Western blotting were utilized to separate and map human sperm proteins (Naaby-Hansen et al., 1997. Biol. Reprod. 56, 771 –787) and to define sperm protein auto- and iso-antigens (Shetty et al., 1999. Biol. Reprod. 61, 61 –69). Two TX-114-solubilized protein spots of 36 and 38 kDa and pI 5.1, which reacted with infertile male sera (autoantigenicity) and with ConA (indicating glycosylation), were microsequenced. Three peptide microsequences shared by the two spots matched a testicular EST, but did not match any known proteins in protein databases. The EST sequence (AA913806) was utilized to probe a lDR2 human testicular cDNA library to obtain a cDNA of 1337 bp

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possessing a 1050 bp open reading frame (ORF) encoding 350 amino acids which included the three in-frame peptides microsequenced from the original spot. The labeled cDNA clone hybridized to a single 1.6 kb testis-specific transcript on a multipletissue Northern blot and to testis and placental mRNA on a dot blot of 76 tissues. Computer analysis of the ORF demonstrated 29% identity and 49% homology over a 68 amino acid C-terminal region (amino acids 278 –343) to murine osteoglycin, a secreted extracellular matrix protein. Furthermore, a putative 20 amino acid N-terminal leader peptide as well as a consensus N-glycosylation site was predicted. Cloning of the ORF of the osteoglycin-like protein in a pET28 vector allowed expression and purification of the recombinant protein in E. coli and generation of immune sera to this human sperm autoantigen.

Alternative splicing of CBP86, a novel testis-specific CA2 + -binding protein localized in the tail of human sperm Soren Naaby-Hansen, Arabinda Manda, Michael J. Wolkowicz, Buer Sen, Jacque Retief, V. Anne Westbrook, Jagathpala Shetty, Michael Cox, Friederika Jayes, Scott A. Coonrod, Kenneth L. Klotz, Leigh Ann Bush, Charles J. Flickinger, John C. Herr Department of Cell Biology, Uni6ersity of Virginia, Charlottes6ille, VA 22908, USA

We report here a novel, polymorphic, testis-specific, calcium binding protein, CBP86, that is localized mainly in the principal piece of the human sperm tail in association with the fibrous sheath. Using 45Ca overlays on 2-D gels of human sperm extracts, the intensity of calcium binding to CBP86 was second only to calmodulin. Six alternatively spliced variants of CBP86 were cloned yielding proteins with deduced masses of 53, 51, 41, 30 and 24 kDa encoded by one [A] or two open reading frames [A and B]. Amino acids 94–493 of ORF-A bore a 25% identity with amino acids 308 –717 of human microtubule associated protein 4 however, the homologous region did not involve the microtubule binding domain of MAP4, nor were the 18-mer repeats characteristic of the microtubule binding domain of MAPs present in ORFB. A 40% similarity (25% identity) was found between aa 225-329 of ORF-B and the proline-rich extensin glycoprotein found in plant cell walls. On Western blots of reduced sperm proteins, antibodies to recombinant 53 kDa CBP86 [ORF-A] recognized prominent sperm polypeptides of 72, 43 and 31 kDa and less abundant antigenic bands at 51 and 90 –102 kDa, suggesting that CBP86 proteins are translated from the splice variants, post-translationally modified and assembled into complexes. Diagonal gels (nonreducing/reducing) indicated homo- and hetero-dimerization of the 43 and 31 kDa variants. Northern and dot blots of mRNA from 50 human tissues indicated expression of CBP86 only in the testis. Immunofluorescence of non-capacitated human sperm with rat antibodies raised to recCBP86 [ORF-A] localized CBP86 to the principal piece (intense staining) and to the midpiece and endpiece with lesser intensity. Electron microscopic immunocytochemistry localized CBP86 mainly to the fibrous sheath compartment.

Cloning and characterization of C58, a novel, hydrophobic, putative human sperm membrane antigen related to the LY-6 family of proteins J. Shetty, M.J. Wolkowicz, A. Diekman, F.L. Jayes, C.J. Flickinger, J.C. Herr Department of Cell Biology, Uni6ersity of Virginia, Charlottes6ille, VA 22908, USA

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The search for candidate sperm immunocontraceptive proteins requires the identification and cloning of molecules exposed on the sperm surface. 2-D gel electrophoretic studies of surface exposed and autoantigenic sperm proteins were carried out earlier to identify immunocontraceptive candidate molecules (Naaby-Hansen et al., 1997. Biol Reprod. 56, 771 –787; Shetty et al., 1999. Biol. Reprod. 61, 61 –69). In the present study, we have enriched and resolved on 2-D gels potential membrane proteins using surface labeling with sulfo-NHS-LC-biotin and subsequent TX-114 phase partitioning of hydrophobic proteins. A biotinylated 13 kDa (pI 4.8) protein was microsequenced by tandem mass spectrometry yielding sequence of ATSCGLEEPVSYR. Utilizing this sequence, a cDNA was cloned and sequenced containing a 372 bp ORF encoding 124 amino acids with a predicted mass of 13 kDa and a pI of 5.5, which included the embedded microsequence noted above. Notable features of C58 are: (1) a 19 aa amino terminal signal peptide; (2) a Ly-6/urokinase plasminogen activator receptor like domain (aa 22-112); (3) a potential transmembrane domain near the carboxy terminus (aa 101-124); and (4) a carboxy terminal cleavage site for transamidase (aa 97-99), suggesting C58 is possibly glycosylphosphatidylinositol (GPI) anchored. Transcripts for C58 were expressed only in testis as studied by Northern blot of eight human tissues and by dot blot analysis of 67 human tissue RNAs. Recombinant C58 was produced in E. coli using the pET 28b vector and antiserum production is underway.

The immune response of male baboons to testis-specific LDH-C4 reduces fertility but does not cause orchitis E. Goldberga, J.L. VandeBergb, M. Mahonyc, G.F. Doncelc a Northwestern Uni6ersity, E6anston, IL, USA b Southwest Foundation for Biomedical Research, San Antonio, TX, USA c Eastern Virginia Medical School, Norfolk, VA, USA Immunocontraception based on sperm antigens has been proposed for use by women, but not men, because of potential autoimmune disease. Nevertheless, immunologic infertility in males with circulating anti-sperm antibodies has been documented and there is no evidence of testicular pathology. The present study was designed to examine the effect in the male baboon, of immunization with the sperm specific antigen, LDH-C4. This vaccine suppresses fertility reversibly in female baboons. Four sexually mature male baboons were immunized with a chimeric peptide containing the B-cell epitope of LDH-C4 and a promiscuous T-cell epitope of Tetanus toxin (hC5-19:TT). The animals received booster injections, at 29, 61 and 344 days after priming. A robust immune response developed in three of the four males and was maintained for :1 year. There was no evidence of testicular histopathology in biopsies taken at days 61, 127 and 183 of the study. Spermatogenesis appeared to be normal. To assess function, sperm-zona binding was assayed. Sperm from three of the four males had a reduced hemizona index compared to non-immune controls. Therefore, a breeding trial with three males of proven fertility was undertaken. The fertility of these males was reduced by immunization with hC5-19:TT. The results suggest that immunocontraception of males with sperm antigens are not compromised by autoimmune disease and may provide an additional contraceptive choice.

Immunoinfertility inducing sperm antigen for the development of antifertility vaccine A.H. Bandivdekar, S.B. Moodbidri Institute for Research in Reproduction, J.M. Street, Parel, Mumbai 400 012, India

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A total of 80 kDa human sperm antigen (HSA) which is responsible for immunoinfertility has been identified from human sperm extract by Western blot technique using infertile woman’s serum having circulating antisperm antibodies. Purified antigen was found to be a glycoprotein of molecular size 80 kDa and cause infertility in male and female rats upon active immunization. It is conserved in human, rat and monkey spermatozoa. Immunohistochemically, it is present only in the testes and epididymis, but not in other human somatic tissues. Electronmicroscopically using immunogold technique, it is found to be localized only on the sperm cells, but not in other cells in the human testicular section. Immunofluorescent studies revealed that it is localized on the sperm head surface. Structurally, the partial N-terminal amino acid sequence of 80 kDa HAS and its peptides obtained by enzymatic digestion with endoproteinase Lys C and Glu C did not show homology with any of the known protein in database. The N-terminal and the peptide sequences obtained by endoproteinase Lys C digestion of 80 kDa HAS were synthesized. The polyclonal antibodies to these peptides were generated in rabbits. These antipeptide antibodies caused the in-vitro agglutination of human, rat and monkey spermatozoa. In addition, passive administration of the antipeptide antibodies leads to infertility in female and male rats. Thus, it is suggested that 80 kDa HAS is one of the promising candidate antigen that fulfills all the requirements for the development of contraceptive vaccine.

The use of carbohydrate epitopes in immunocontraceptive development A.B. Diekmana, E.J. Nortona, T.C. McCauleya, V.A. Westbrooka, P. Andersona, B.E. Kurtha, K.L. Klotza, R. Eastonb, A. Dellb, H. Morrisb, J.C. Herra a Department of Cell Biology, Uni6ersity of Virginia, Charlottes6ille, VA, USA b Department of Biochemistry, Imperial College of Science, Technology and Medicine, London, UK

The sperm glycocalyx represents an unexplored source of sperm-specific epitopes for immunocontraceptive development. S19, a murine IgG1 monoclonal antibody (mAb) that recognizes a sperm-surface, N-linked carbohydrate epitope, exhibits sperm-inhibitory activities in vitro including agglutination, inhibition of cervical mucus penetration and inhibition of human sperm-zona pellucida binding. The S19 mAb identified sperm agglutination antigen-1 (SAGA-1), a polymorphic, highly acidic, GPI-anchored glycopeptide that is localized over the entire human sperm surface. The SAGA-1 core peptide is identical to the sequence of CD52, a lymphocyte differentiation marker also expressed in the male reproductive tract. Comparison of anti-SAGA-1 and anti-CD52 immunoreactivities revealed that sperm CD52 exhibits N-linked glycan epitopes, including the S19 epitope, that are not detected on lymphocyte CD52. Thus, sperm and lymphocyte CD52 represent glycoforms, isoforms with the same amino acid sequence but different carbohydrate structures. Furthermore, immunochemical analysis of 28 human tissues demonstrated that the S19 carbohydrate epitope is specific to the male reproductive tract. Immunochemical and mass spectrometry analyses suggested that the S19 epitope is localized to a subset of CD52 glycoforms in the male reproductive tract. Evaluation of CD52 in multiple primate species has identified the chimpanzee as the most appropriate non-human primate model for the S19 carbohydrate epitope. Overall, localization of the S19 epitope on all sperm-surface domains, the sperm-inhibitory effects of the S19 mAb and the tissue specificity of the S19 epitope indicate the potential of the S19 carbohydrate epitope as an immunocontraceptive vaccinogen.

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Inhibition of fertility in mice immunized with recombinant mouse ZP2 I.A. Lea, E.E. Widgren, M.G. O’Rand Department of Cell Biology and Anatomy, Uni6ersity of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA

The immunological inhibition of fertility by targeting antibodies to the zona pellucida has received much interest in recent years. Most studies have focused on ZP3 as the antigen target for the antibody; ZP2 has evoked less interest. We now present studies to demonstrate the immunogenicity and fertility rates of mice immunized with near fulllength recombinant ZP2 (rZP2) and ZP2 N- and C-terminal fragments. In contrast to immunization with either N- or C-terminal fragments, which produced low antibody responses, rZP2 was highly immunogenic in female mice. In fertility trials, the pregnancy rate of mice immunized with rZP2 was reduced to 9 from 100% in adjuvant controls. However, this effect on fertility was not reversible within 32 weeks post immunization. Histological examination of the ovaries revealed normal follicular development and no apparent pathology. To understand the mechanism causing infertility, female mice were passively immunized with antibodies to rZP2. Fertility testing showed these mice to be infertile (0% pregnancy rate). Moreover, when sections of ovary were probed with antibodies to mouse immunoglobulin, antibody was detectable bound to the zona pellucida of developing oocytes. Likewise, induced ovulation in mice actively immunized with rZP2 produced normal numbers of eggs, but with antibody bound to the zona pellucida. In sperm binding assays, these eggs demonstrated a 75% reduction in the number of sperm bound compared to adjuvant control eggs. We conclude that immunization with rZP2 can bring about infertility in mice solely through the binding of antibody to the zona pellucida of the egg and without significant ovarian pathology.

Immunocontraception in macropods 2: immunisation of tammar wallabies (Macropus eugenii) and eastern grey kangaroos (Macropus giganteus) with porcine ZP and recombinant brushtail possum ZP3 A. Kitchener, L. Edds, A. Harmon, D.J. Kay Marsupial CRC, Department of Biological Sciences, The Uni6ersity of Newcastle, Callaghan, NSW 2308, Australia

As has been indicated, the main aim of the Marsupial CRC is the development of field deliverable fertility control for overabundant macropods in Australia and New Zealand. In this study, the potential for immunocontraception using the zona pellucida (ZP) as a target was examined by immunising eight female tammar wallabies with porcine zona pellucida (PZP) and eight controls with saline in Freunds adjuvant. Antibodies to PZP were assayed in serum, follicular and oviduct fluids by ELISA and in tissue by immunofluorescence. Following the establishment of a response, the wallabies were placed in a natural mating trial, followed by a further examination of fertility using artificial insemination. At the end of the trial serial sections of ovarian tissue were examined. Antibodies to PZP were detected in all fluids studied and to wallaby ZP in situ in immunised animals. Antibodies were directed at all three glycoproteins of PZP. The natural mating and AI trial demonstrated absolute infertility. Examination of ovarian tissue showed

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reduced ovarian weight and reduced numbers of large pre-antral and antral follicles. A pilot study, immunising eastern grey kangaroos with PZP, resulted in sustained levels of antibody to PZP3 (only) 14 months after a single immunisation, however there was no antibody binding to ZP in ovarian tissue of these animals. An immunisation and natural mating trial using brushtail possum recombinant ZP3 in eastern greys has commenced and results will be discussed at the presentation of this paper. However, the potential of the ZP as an immunocontraceptive target in macropods has been established.

Immunocontraception in macropods 1: immunisation of tammar wallabies (Macropus eugenii) with whole sperm D.J. Kay, A. Kitchener, A. Harman, K.L. Asquith, R.A. Knee, R.C. Jones Marsupial CRC, Discpln. Biological Sciences, Uni6ersity of Newcastle, Callaghan, NSW 2308, Australia

Overabundance of kangaroos and wallabies cause a high level of economic and environmental damage in Australia. One of the main aims of the Marsupial CRC is the development of benign management of overabundance through fertility control via a field deliverable immunocontraceptive. In this study, eight male and eight female tammar wallabies, as a macropod model, were immunised with whole epididymal sperm (and 16 saline controls) in Freunds adjuvant as a proof of concept for immunogenicity of sperm antigens and effect on fertility. Sperm antibodies were assayed in serum, bile, prostatic, epididymal, follicular and oviductal fluids using ELISA. Location of antibodies on sperm was determined by immunofluorescence and immunogold electron microscopy. Specificity of the antibodies was determined by Western blotting. antibodies to sperm were detected in serum from males and females, follicular and oviductal fluids. There was not a classical primary and secondary response but rather a steady rise to a plateau. Antibody binding to ejaculated (but not epididymal) sperm from immunised animals was mainly in the area of the midpiece and connecting piece, while serum antibodies from both males and females labelled the entire ejaculated sperm uniformly. However, follicular fluid produced staining of the head area in discrete patches. Western blotting showed specific binding to bands in the 30 and 60 kDa areas. No conclusions could be drawn from the fertility trial, however a number of immunised females produced pouch young. In a subsequent experiment, immunised and control males were mated with stimulated females followed by flushing of the reproductive tracts. More fertilised oocytes were obtained from females mated with control males compared to those with immunised males.

Mucosal antibody responses in HIV-infected individuals J. Mestecky, P.A. Goepfert, Z. Moldoveanu, L.R. Brewer, R. Kuihavy, S.J. Prince, M.J. Mulligan, S. Jackson Uni6ersity of Alabama at Birmingham, Birmingham, AL, USA

External secretions (tears, saliva, urine, nasal, seminal ejaculate, vaginal and rectal washes) and sera from 50 HIV-1-infected and 20 uninfected individuals; 60 concordant and discor-

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dant African couples; and ten HIV-1 exposed but seronegative women were examined for the levels of total and antigen-specific IgG and IgA antibodies by ELISA and chemiluminescence-enhanced Western blots. Despite the pronounced dominance of total IgA in most external secretions (e.g. saliva, nasal, and rectal washes), the levels of HIV-specific IgG antibodies were higher than those of IgA in all external secretions. Furthermore, we did not detect anti-HIV antibodies in any external secretions of seronegative individuals and we did not identify any individual who displayed the exclusive presence of IgA antibodies in secretions or sera among HIV-infected, exposed but seronegative or uninfected individuals, as reported by others. The highest levels of mucosal HIV antibodies were detected in nasal and genital tract secretions and urine; unexpectedly, intestinal secretions contained the lowest levels. The specificity of IgO HIV antibodies was the same for all external secretions sampled. IgA HIV antibodies (most frequently present in genital tract secretions and very rarely in rectal washes) were specific mostly for gp 160. Although high numbers of specific antibodysecreting cells are readily detectable in the peripheral blood of individuals infected with other viruses (e.g. influenza), a relatively low number of specific antibody-secreting cells were seen in HIV-infected individuals. Thus, in contrast to other mucosally acquired viral and bacterial infections in which antigen-specific IgA antibodies are dominant, HIV induces preferentially IgG responses in sera as well as in external secretions.

Infection and host defence Immunological memory in B cell-deficient mice conveys long lasting protection against genital tract infection with Chlamydia trachomatis by rapid accumulation of local T cells Martina Johanssona,b, Nils Lyckea a Department of Medical Microbiology and Immunology, Uni6ersity of Go¨ teborg, Sweden b Department of Obstetrics and Gynaecology, Uni6ersity of Adelaide, Australia

Using knock-out mice, we have previously shown that protective immunity against C. trachomatis is mainly conferred by CD4+ T-cells and IFN-g. Antibodies and B-cells appear to play a subordinate role. Here, we evaluated for how long protective immunity against C. trachomatis prevails and if immunological memory can be maintained in the genital tract in the absence of B-cells. Following resolution of the primary infection B-cell deficient and wild-type, C57Bl/6, mice were given a challenge inoculation. Both the B-cell deficient mice and the wild-type mice were still protected at 6 months following recovery. The immune response following challenge was evaluated. Both mMT and wild-type mice exhibited significant DTH-reactivity against specific antigen challenge. Following the challenge with live C. trachomatis, we analysed the distribution of lymphocytes in the genital tract mucosa in relation to clearance of the infection using FACS analysis. We found that CD8+ and, to a greater extent CD4+, T-cells rapidly accumulated in the genital tract mucosa of both B-cell deficient and wild-type mice. These finding were also corroborated with immunohistochemical analysis. When analysing the cell subset distribution in the genital tract, we found an unexpected dominance of a population of CD3+B220+ cells that were mostly CD4− and CD8−. IFN-g mRNA was found to be induced earlier in the genital tract after challenge than compared to following the primary infection, further corroborating the presence of specific memory cells in the genital tract. These results suggest that long-term protection against chlamydia can be induced which is of importance for vaccine development.

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Higher levels of activation and chemokine receptor expression of T-cells in the cervix than peripheral blood of normal healthy women Manyu Prakasha, Moses S. Kapembwab, Francis Gotcha, Steve Pattersona a Department of Immunology, Chelsea and Westminster Hospital, Imperial College, London, UK b Department of GU/HIV Medicine at Northwick Park Hospital, Harrow, UK

Background: Heterosexual transmission is the predominant route of HIV infection. Most research, however, has focused on the blood rather than the genital tract. The female genital tract not only acts as a site for virus initiation, but also as a source of transmission to new hosts. The relationship between cofactors and HIV transmission is complex, elucidation of cellular dynamics at the cervical mucosa may explain the role of these cofactors in HIV infection following exposure. Aim: To analyse activation marker and chemokine receptor expression on T-cells in the cervical mucosa and peripheral blood (PBMC) of normal healthy women. Furthermore, to analyse the susceptibility of unstimulated cervical leucocytes to infection by T- and M-tropic strains of virus. Methods: All women enrolled showed no inflammation of the cervix and all tested negative for sexually transmitted infections including HIV. Cervical intraepithelial cells were obtained using a cytobrush technique. Expression of activation markers (CD69, CD25, HLA-DR) and chemokine receptors (CCR5 and CXCR-4) was measured on CD4+ and CD8+ T-cells, using flow cytometry. Results: Cervical T-lymphocytes showed significantly higher levels of all activation markers. Expression of both chemokine receptors on T-cells were higher in the cervix than PBMC, CXCR-4 was the more highly expressed chemokine receptor on T-cells at both sites. The results of the in vitro infection experiments will be presented. Conclusion: The higher levels of activation marker and chemokine receptor expression on T-cells in the cervix suggest these cells are likely targets for initial HIV infection. The results of in vitro infection experiments will be used to confirm or reject the hypothesis.

Developing ovarian follicles inhibit the inflammatory reaction induced by low dose endotoxin infusion M.M. Faas, G. van der Schaaf, N. Valkhof, G.A. Schuiling Reproducti6e Immunology Medical Biology Branch, Department of Pathology and Laboratory Medicine, Uni6ersity of Groningen, The Netherlands

The low dose endotoxin-induced glomerular inflammatory reaction is more persistent in rats in the luteal phase of the ovarian cycle (pseudopregnant rats), as compared with rats in the follicular phase (cyclic rats). Therefore, we investigated the role of developing ovarian follicles in the control of the inflammatory reaction. Follicular development was induced in pseudopregnant rats (n= 10) by daily i.p. FSH injections (from day 3 onwards; FSH-rats). Control pseudopregnant rats (n= 10) received saline injections (control-rats). Also a group of cyclic rats (n=10) were used. On day 5 (cyclic rats: di-oestrus) rats were infused with 2 ml endotoxin-solution (1.0 mg/kg bw) or 2 ml saline for 1 h. Three days later, rats were sacrificed and cryostat kidney sections were immunohistologically stained for the presence of neutrophils, monocytes and expression of ICAM-1, LFA-1 and MAC-1. FSH-treated rats without developing ovarian follicles (checked by histology) were discarded from the experiment. Differences were evaluated with unpaired t-tests, with significance at PB0.05. In

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control-rats, endotoxin, as compared with saline, significantly increased glomerular number of neutrophils, monocytes and MAC-1 positive cells as well as ICAM-1 expression. FSHtreatment did not affect neutrophil number or ICAM-1 expression following endotoxin; numbers of monocytes and of MAC-1 positive cells after endotoxin, however, were significantly decreased in FSH-rats as compared with control-rats. In cyclic rats, no effect of endotoxin was observed as compared with saline. It is concluded that FSH stimulated follicles produce (a) factor(s) that prevented endotoxin-induced M¥ infiltration in PSP-rats. This (these) factor(s) may play a role in inhibiting the glomerular inflammatory reaction in rats in the follicular phase of the ovarian cycle.

Luteal phase: increasing response of the non-specific immune system A. Boumana, H. Moesb, M.J. Heinemana, G.A. Schuilinga, M.M. Faasb a Department of Obstetrics and Gynaecology, Uni6ersity Hospital and Uni6ersity of Groningen, The Netherlands b Department of Pathology and Laboratory Medicine, Uni6ersity Hospital and Uni6ersity of Groningen, The Netherlands

It has been known for many years that sex hormones (progesterone and oestradiol) can influence the non-specific immune system. The aim of this study was to investigate the monocyte cytokine response to endotoxin in the normal ovarian cycle in humans. Therefore, blood samples were taken from healthy young women (age: 27 – 34, n= 10) in both their follicular (6 –9th day of the cycle) and luteal phase (6 –9 days after positive urinary LH-detection). Whole blood was stimulated with endotoxin for 4 h at 37°C and 5% CO2. After red blood cell lysis and white blood cell permeabilisation, monocytes were stained with anti-CD14 and with antibodies against intracellular cytokines, IL-1b, IL-12 and TNF-a. Analysis was performed by flow cytometry and the results were expressed as a percentage cytokine producing cells (mean 9S.E.M.). There was no significant difference in percentage IL-12 producing monocytes. However, there was a significant increase in TNFa and IL-1b producing monocytes in luteal phase compared to follicular phase. Also, the concentration of progesterone and 17b-oestradiol were significantly increased in luteal phase. Table 1 Outcome of percentage cytokine producing monocytes in follicular and luteal phase (mean 9S.E.M.)

IL-12 IL-1b TNF-a

Follicular %

Luteal %

6.77 (1.83) 54.30 (8.18) 48.89 (6.30)

7.23 (3.68) 71.43 (8.17)* 61.01 (6.34)*

*Significant increase in luteal phase compared with follicular phase; Wilcoxon’s signed rank test, PB0.05. The fact that this significant increase in TNF-a and Il-1b producing monocytes in luteal phase coincides with a significant increase in progesterone- and 17b-oestradiol concentration,

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indeed supports the concept that the non-specific immune system can be influenced by sex hormones.

Effect of an immunomodulator in mares resistant and susceptible to endometritis E.A. Fumusoa, C. Gonza´ leza, I. Videla Dornaa, E. Inchaustia, M. Tallaricob, V. Beresteina, J. Wadec, J. Aguilarb, L. Losinnob, P. Sotoa, R. Bowdena a Facultad de Ciencias Veterinarias, U.N.C.P.B.A., Tandil (7000) Argentina b Facultad de Agronomı´a y Veterinaria., U.N.R.C., Ruta Nac 36 km.601 (5800) Rı´o Cuarto, Argentina c Vetrepharm teo, Ireland

Post-breeding endometritis is a major cause of infertility in mares. Mares that fail to clear a post-breeding inflammation from the uterus develop endometritis and are called susceptible (SM); in contrast, those that can clear it are named resistant (RM). Neutrophyles (PMN) invasion of endometrium is the main sign of acute endometritis. We evaluated the effect of cell-wall fraction of Mycobacterium phlei (Equimune IV™, Vetrepharm) applied in RM and SM upon neutrophils (PMN) found in endometrial biopsy samples. RM (n= 14) and SM (n=14) were selected using age, reproductive report, biopsy grade and clearance of Streptococcus zooepidemicus. Artificial insemination (AI) with killed semen was used as antigenic stimulus. Biopsies were obtained 24 h post-AI (M1) and 7 91 days post-ovulation (M2). In the next cycle, when mares had 35 mm-follicles, they received Equimune IV and AI. Biopsies M3 and M4 were obtained in the same interval. PMN were counted by Image-Pro Plus Analysis (Media Cybernetics™). RM and SM had high number of PMN post AI (RM: 25.093.3, SM: 27.7 92.1), in M2 it was lower (P\ 0.01) in RM (5.1 9 0.6) than in SM (21.3 90.5). M3 PMN counts were lower in both RM (18.7 9 1.4; P B0.05) and SM (15.891.2; PB0.01). There was no difference between RM (5.2 9 1.9) and SM (5.1 9 0.6) in M4. We conclude that Equimune had a significant effect in reducing acute post-breeding endometritis.

Expression of pro-inflammatory and immunoregulatory cytokines in the normal and inflammed adult rat testis O. Gerdpraserta, M.K. O’Bryana, D.J. Nikolic-Patersonb, K.L. Sebirea, K.A. Edgara, D.M. de Kretsera, M.P. Hedgera a Monash Institute of Reproduction and De6elopment, Monash Uni6ersity, Clayton, Vic 3168, Australia b Department of Nephrology, Monash Medical Centre, Clayton, Vic 3168, Australia

In spite of compelling evidence that the rodent testis is a beneficial site for grafts, it is equally certain that ‘normal’ inflammatory responses are possible within the testicular environment. In an attempt to reconcile these observations, we investigated the cytokine response within the testis following an inflammatory challenge in vivo. Adult male rats were injected (i.p.) with lipopolysaccharide (LPS, from E. coli ). Testes and liver (control) RNA samples were collected at time-points up to 72 h later. Expression of the pro-inflammatory cytokines, interleukin-1b (IL-1b), tumour necrosis factor-a (TNFa) and monocyte chemoattractant protein-1 (MCP-1) and the immunoregulatory cytokines, transforming growth

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factor-b1 TGFb1 and IL-10, was determined by Northern blot analysis. In the normal animal, expression of the pro-inflammatory cytokines and IL-10 was low to non-detectable in both liver and testis. In contrast, TGFb1 expression was at least 5-fold higher in the testis than in liver. Pro-inflammatory cytokine expression increased dramatically in the liver, 3 –6 h after LPS-treatment, but only small increases in IL-1b and TNFa were observed in the testis. Both liver and testis showed a similar very large increase in MCP-1 expression over the same time-period. IL-10 was up-regulated in parallel with the peak of pro-inflammatory cytokines in the liver (3 –6 h), but did not increase until much later in the testis (18 –72 h). TGFb1 expression was not significantly altered in either tissue after LPS-treatment. These data indicate that the production of key pro-inflammatory cytokines in response to an inflammatory stimulus is considerably attenuated in the testis. The data also suggest a role for TGFb1 in regulating immune responses within the normal testis and for IL-10 in the inflamed testis.

Autoimmunity and tolerance Update on treatment of immunologic abortion with low-dose intravenous immunoglobulin (IVIG) R.B. Strickera, A. Steinleitnerb, C.M. Bookoffa, E.E. Wingerc a California Pacific Medical Center, San Francisco, CA, USA b Astarte Medical Group, San Francisco, CA, USA c Immunodiagnostic Laboratories, San Leandro, CA, USA Background: Treatment with low-dose intravenous immunoglobulin (IVIG) appears to be beneficial for women with recurrent spontaneous abortion associated with immunologic abnormalities (Stricker et al., 2000. Fertil. Steril. 73, 536). Goal: To evaluate ongoing treatment with IVIG in women with immunologic abortion. Design: A total of 83 women were prospectively evaluated for immunologic abortion, which was defined as three or more miscarriages and the presence of specific immunologic abnormalities (see below). Prior to the next conception, women were treated with IVIG at a dose of 0.2 g/kg. Once conception was achieved, IVIG treatment was continued on a monthly basis through 26–30 weeks of pregnancy. Results: The average age of the women was 37 years (range: 28 –49 years), and the average number of miscarriages was 3.7 (range: 3 –12). Immunologic abnormalities included antiphospholipid antibodies (36%), antithyroid antibodies (53%), antinuclear antibodies (25%), antiovarian antibodies (11%), increased natural killer cells (35%), increased IgM level (22%) and increased CD4/CD8 T-cell ratio (14%). Two patients had IgA deficiency and seven patients had endometriosis. Fiftyeight of the 83 patients (70%) had more than one immunologic abnormality. Of the 83 women, 61 received initial IVIG treatment and 34 subsequently became pregnant. Of these women, 31 (91%) had a successful term pregnancy. Of the 22 women who refused IVIG therapy, 12 became pregnant and 11 (92%) miscarried. The difference in pregnancy success rate between the IVIG-treated and untreated groups was significant (P =0.001). Four women had mild allergic reactions during IVIG infusion and these reactions resolved when the IVIG brand was changed. Fetal abnormalities were not observed. Conclusion: Low-dose IVIG therapy is safe and effective for women with immunologic abortion.

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Treatment with low-dose heparin for prevention of pregnancy losses in women with antiphospholipid antibodies (APL) J. Carbonea, P. Segoviab, E. Seoanea, J.L. Ruiz-Ta, J. Bermejoa, E. Sarmientoa, A. Rodrı´guez-Hb, C. Pascualb, M. Rabada´ nc, E. Ferna´ ndez-Cruza a Immunology Department, Uni6ersity Hospital Gregorio Maran˜ o´ n, Madrid, Spain b Gynecology Department, Uni6ersity Hospital Gregorio Maran˜ o´ n, Madrid, Spain c Hematology Department, Uni6ersity Hospital Gregorio Maran˜ o´ n, Madrid, Spain Objective: To evaluate low dose heparin for the treatment of pregnant women with aPL-associated repeated abortions. Methods: The prospective study included 44 patients with aPL. Each patient had recurrent unexplained pregnancy losses. Ten patients received low dose aspirin (100 mg/daily) and low molecular weight heparin (LMW; Fragmin) 5000–10000 U subcutaneously daily. We have compared the results in this group of patients with a group of 26 pregnancies in 22 women treated with low dose aspirin alone (100 mg/daily) and with 14 pregnancies in 12 women previously treated with prednisone (20 – 30 mg/daily) and aspirin. Results: Viable infants were delivered of ten of ten (100%) pregnancies treated with heparin and aspirin and 10 of 26 (38%) pregnancies treated with aspirin alone (P=0.035). There were no significant differences between the heparin plus low-dose aspirin and the prednisone plus aspirin groups with respect to the rate of live births (100 vs. 79%, P =0.239). The only medical complication in the heparin plus aspirin group was gestational diabetes (one patient) and the frequency of preterm delivery was 10%. Positive ANA (50%), low complement C4 (30%) and C3 (40%) and increased circulating immune complexes (80%) were observed in patients treated with heparin. Women treated with prednisone and aspirin showed a higher incidence of adverse effects (21% of premature rupture of membranes, 43% of preterm delivery, 14% of fetal distress). Conclusion: Heparin plus aspirin provides a better pregnancy outcome than prednisone and that aspirin alone for the treatment of women with repeated abortion and aPL.

Antiphospholipid antibodies (APA) and recurrent pregnancy loss (RPL): treating a unique population of APA patients R.D. Franklin, W.H. Kutteh, R.W. Ke Department of Obstetrics and Gynecology, Uni6ersity of Tennessee, Memphis, TN, USA Objective: Antiphospholipid antibodies (APA) to cardiolipin (CL), lupus anticoagulant (LAC) and phosphatidylserine (PS) have been considered primary risk factors for the treatment of APA associated recurrent pregnancy loss (RPL). However, the therapeutic benefits for RPL patients that are positive for APA other than CL, LAC and PS are unknown. Thus, we assessed the efficiency of heparin and aspirin therapy in women with recurrent pregnancy loss (RPL) between APA positive populations. Study design: Women with RPL and APA were selected for comparison for antibodies against: CL and/or PS (Group 1) and phosphatidylglycerol (PG), phosphatidylinositol (PI) and/or phosphatidylethanolamine (PE) (Group 2). The APA study groups had an otherwise normal evaluation for RPL and had undergone subcutaneous heparin and aspirin therapy. Results: There were no significant differences in the patients’ age at entry, prior pregnancies, prior livebirths or miscarriages, gestational age at time of loss or percent age of losses that occurred before 12 weeks between groups. There were no differences in the 19 viable infants (76%) born to 25 women in Group 1 compared to 18 viable infants (64%) born to the 28 women in Group 2 (P=0.831). The women who miscarried showed no significant differences

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in the estimated gestational age at the time of loss, percent with a blighted ovum or percent with no fetal heart motion. Conclusion: Women with RPL and a normal evaluation except for positive PI, PG and PE, have a similar successful outcome as women with positive CL or PS antibodies when treated with heparin plus aspirin. These findings suggest that the inclusion of women with APA other than CL or PS for treatment may increase their chance of a successful pregnancy.

A biologically active antiphospholipid antibody (aPL) binds phosphatidylserine (PS) alone and phosphatidylethanolamine (PE)plus co-factors Neal S. Rote, Marjet Heizer, Kimberly Perrine Departments of Biological Sciences and Obstetrics/Gynecology, Wright State Uni6ersity, Dayton, OH 45435, USA

PS and PE normally reside on the cytoplasmic side of the plasma membrane, but are externalized under conditions of trophoblast differentiation. A monoclonal aPL, 3SB, reacts with differentiating trophoblast and prevents normal trophoblast differentiation, including intercellular fusion, hormone production, and invasion of extracellular matrix. It also promotes the coagulation cascade by removing the anticoagulant annexin V from the trophoblast surface. The specificity of this antibody was investigated using glass microspheres coated with phospholipid and tested using flow cytometry. 3SB purified of contaminating serum proteins reacted with beads coated with 50% PS with phosphatidylcholine (PC), but not with beads coated with 50% PE with PC. Reactivity of 3SB against PE coated beads was observed after the addition of 2-glycoprotein I or prothrombin and appears to react with cryptic sites in those proteins. To investigate whether PS was binding potential serum co-factors, we coated glass microspheres with 125I-PS (50% PS/PC) conjugated with a photoaffinity label. These spheres were incubated with fetal calf serum, followed by UV activation of the photoaffinity label. Annexin V served as a positive control. The beads were stripped of protein with SDS-PAGE sample buffer, the extract run on 10% PAGE, and developed to determine 125I-PS labeled proteins. Two prominent proteins were radiolabeled; molecular weights of :60 and 50 kDa. The annexin V control was 125I-PS labeled. Thus, PS specifically interacts with two serum proteins, which do not seem to function as co-factors. This monoclonal aPL recognizes an antigenic determinant that is formed by PS without co-factor requirement or by PE that complexes with co-factor proteins resulting in exposure of a cryptic epitope.

Phosphatidylserine externalization in human trophoblast differentiation and apoptosis Neal S. Rote, Bo Xu, Lin Lin Departments of Biological Sciences and Obstetrics/Gynecology, Wright State Uni6ersity, Dayton, OH 45435, USA Exposure of phosphatidylserine (PS) in the outer leaflet of the cell membrane occurs in cells undergoing apoptosis. Normal human trophoblast differentiation is also characterized by PS externalization, related to intercellular fusion. These PS-dependent sites are targets for antiphospholipid antibodies. We investigated the mechanisms of PS externalization during differentiation and apoptosis processes. BeWo, a choriocarcinoma cell line, is an in vitro model of trophoblast differentiation and apoptosis. After 24 h treatment of forskolin-in-

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duced differentiation, PS was detected on the cell surface by binding of FITC-annexin V, which increased during 24 –72 h of forskolin treatment, but was not accompanied by apoptosis monitored by TUNEL assay. Apoptosis induction by staurosporine for 1 h triggered rapid PS externalization. Two membrane-bound enzymes, floppase and scramblase, that regulate PS externalization have been described. We hypothesized that floppase is responsible for PS externalization during differentiation and scramblase during apoptosis. Inhibition of floppase with vanadate blocked differentiation-mediated but not the apoptosismediated PS externalization. The general caspase inhibitor ZVAD-fmk completely abolished staurosporine-induced apoptosis and PS externalization, but had no effect on differentiationmediated PS externalization. Thus, PS externalization is a marker for both trophoblastic differentiation and apoptosis, but differentiation-related PS externalization is regulated through activation of floppase, while activation of scramblase through the caspase system is responsible for apoptosis-mediated PS externalization.

Anticardiolipin antibodies increase apoptosis in human trophoblast of pregnancy complicated by antiphospholipid antibodies M. Sbracia, F. Scarpellini CERM, Rome, Italy

Antiphospholipid antibodies show an adverse effect on pregnancy promoting fetal wastage and spontaneous abortion. These autoantibodies seem to determine platelet aggregation and thrombosis in the blood vessels. However, the mechanisms with which antiphospholipid antibodies promote miscarriage are not well established. In this study, we evaluated the apoptosis rate in placenta of pregnancies complicated by anticardiolipin antibodies compared to normal pregnancy. Fifteen placenta of pregnancies at term complicated by anticardiolipin (treated with aspirin and heparin) and ten placenta of normal pregnancies were evaluated for apoptosis rate. Fresh placental tissue and fixed placental tissue number of the marginal regions were evaluated with TUNEL test. Furthermore, placental specimens were analysed by haematoxilin eosin staining to validate the TUNEL staining. Statistical analysis was performed by Student’s t-test and  2-test. TUNEL staining showed that placental tissues of pregnancies complicated by anticardiolipin antibodies have a higher rate of apoptosis in the sincithyotrophoblast, especially in the sincithyal knots compared to placentas of normal pregnancies (0.43 90.05 vs. 0.09 90.02%; PB 0.001). Furthermore, the vessels of decidual tissue showed an increased rate of apoptosis in pregnancies complicated by anticardiolipin antibodies with respect to normal pregnancies (1.42 9 0.21 vs. 0.33 9 0.15%; PB 0.001). These results were validated by morphological analysis of haematoxilin and eosin stained slides. Our results show that antiphospholipid antibodies promote trophoblast apoptosis and reduction of blood flow supply in placenta by promoting apoptosis of vessel wall cells in decidual arterioles.

Reproductive outcomes in mice genetically deficient in b2-glycoprotein I: a major antigen in anti-phospholipid syndrome Sarah A. Robertsona, Yonghua Shengb, Eline van Beijeringa, Steven A. Krilisb a Department of Obstetrics and Gynaecology, The Uni6ersity of Adelaide, SA 5005, Australia b Department of Immunology, Allergy and Infectious Disease, Uni6ersity of New South Wales, St. George Hospital, NSW 2217, Australia

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b2 glycoprotein-1 (b2GP1) is a phospholipid binding protein and important target antigen for anti-phospholipid antibodies, which are associated with recurrent fetal loss. Mice with a null mutation in the b2GP1 gene have been generated using conventional gene targeting techniques in embryonic stem cells. In heterozygous ( + / −) intercrosses, only 8.9% of 336 surviving offspring were homozygous for the disrupted allele ( − / −), suggesting an effect of fetal b2GP1 deficiency on fetal survival in utero (Sheng et al., accepted for publication). To investigate the role of b2GP1 as a determinant of successful pregnancy, female mice ( − / − or +/+) were mated with males of the same genotype and sacrificed at day 18 of pregnancy. There was no effect of maternal b2GP1 deficiency on the proportion of mated mice pregnant at day 18 (21/25 and 17/20 + / + and − / − females, respectively), or on litter size (mean 9 S.D.=9.091.6 and 9.0 92.7 in + / + and − / − pregnancies, respectively). Fetal and placental weights and fetal:placental weight ratios in viable sites, were comparable in −/− and +/+ mice. In a second experiment, − / − females (n =16) and +/+ females (n=17) were mated with males of the same genotype and pregnancies were allowed to proceed to term. There was no effect of b2GP1 genotype on gestational length, litter size or weight of pups at birth. Growth trajectories were altered in female − / − but not male −/− pups (mean weights of − / − pups (n =58) were 4.6, 6.2 and 9.2% greater than +/+ pups (n=67) at 3, 6 and 16 weeks of age). Together, these data suggest that while b2GP1 deficient conceptuses may be selectively disadvantaged in b2GP1 replete mothers, implantation and successful development to term is not compromised in the event of both fetal and maternal b2GP1 deficiency.

Maternally-derived ZP3 autoantibodies cause neonatal ovarian injury Yulius Y. Setiady, Kenneth S.K. Tung Department of Pathology, Uni6ersity of Virginia, Charlottes6ille, VA 22908, USA

Zona Pellucida 3 (ZP3), a glycoprotein of the zona pellucida, is the major sperm receptor. With two linear native B cell epitopes of murine ZP3, we have designed two chimeric peptides (CP), each containing a ZP3 B cell epitope (underlined) and a helper T cell epitope of bovine ribonuclease (94-104). CP2 (NCAYKTTQANK-QAQIHGPR) immunization generated a reversible anti-fertility effect without pathogenic T cell response to ZP3. CP3 (NCAYKTTQANK-FSLRLMEENW) induced antibodies to native ZP without anti-fertility effect. Herein, we report the occurrence of ovarian disease in the neonatal progeny of B6AF1 female mice immunized with CP2 or a mixture of CP2 +CP3 (in CFA). Ovarian pathology was not detected in the CP-immunized adult females, nor in the progeny of females injected with CP3 and CFA, or CFA alone. More than 70% of litters had variable degrees and incidences of neonatal ovarian disease and the ovarian disease incidences in the progeny of CP2 and CP2+CP3 groups were 33 and 26%, respectively. Neonatal ovarian disease began as monocytic and granulocytic inflammation in the ovarian interstitium and ovarian follicles. Inflammation then regressed and this was followed by either ovarian atrophy and complete depletion of primordial and growing oocytes or apparent recovery. Maternal ZP antibody titers were not predictive of ovarian disease in the progeny, thus severe ovarian disease occurred in progeny of fertile mothers with low ZP antibody titers. However, neonatal mice injected with CP2 (not CP3) antiserum developed similar ovarian pathology. Thus, (1) maternally-derived antibodies to ZP transfer to progeny severe neonatal ovarian disease; (2) the responses of neonatal and adult mice (or their ovaries) to ZP antibodies are different; (3) the process appears to be ZP3 epitope specific; and (4) unless non-pathogenic contraceptive ZP3 epitopes are identified, this complication will preclude a safe ZP3 vaccine.

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Distinction between ovarian autoimmunity and endocrine mechanisms of low ovarian responses to gonadotropin J.L. Luborskya, P. Thiruppathia, B. Rivnayb, R. Roussevc, C. Coulamc a Department of Obstetrics and Gynecology, Rush Medical College, Chicago, IL, USA b Repromedix Corporation, Woburn, MA, USA c Milleno6a Immunology Laboratory, Chicago, IL, USA

A proportion of low estradiol responses to gonadotropin is associated with ovarian autoimmunity. The objectives were to determine if: (1) ovarian antibodies in serum and follicular fluid were correlated; and (2) reproductive endocrinology of ovarian antibody positive (OVAB + ) and negative (OVAB −) low responders was similar. Serum and follicular fluid from mature follicles were obtained at oocyte retrieval during in vitro fertilization (n= 32). Low (LR) and normal responders (NR) were defined by peak estradiol/ FSH ampules administered (range: 7 –89; LR B 42). OVAB were assessed by immunoassay (Hum Reprod, 2000 15, 1046) and detected in LR (9/21) but not NR (0/11). Follicular fluid and serum OVAB were correlated (0.4, P =0.03), suggesting follicular function could be locally influenced by OVAB. Mature follicles of LR(OVAB − ) had higher progesterone (P=0.003), inhibinA (P=0.01) and VEGF (P =0.05) than NR suggesting accelerated luteinization of follicles. Likewise, follicles of LR(OVAB + ) showed evidence of accelerated luteinization. However, some LR(OVAB + ), but not LR(OVAB− ), had negligible progesterone but high inhibinA (3/9), or high progesterone and poor oocytes (4/9) ( B 10 retrieved, B4 mature, B40% fertilization). Based on serum inhibin A and B all LR(OVAB − ) (12/12), but only some LR(OVAB+ ) (5/9), had reduced ovarian reserve compared to NR. LR(OVAB+) were younger than LR(OVAB − ) (33.49 4.2 vs. 37.1 9 3.8 years; P = 0.05) and similar in age to NR (32.5 94.6 years; P= 0.5). Follicles of LR(OVAB − ) clearly exhibit accelerated luteinization, an event associated with ovarian aging. In contrast, LR(OVAB+) appear to have selective effects on steroidogenesis or oocyte function that may precede autoimmune ovarian failure.

Estrogenic hormones alter apoptosis and induce autoimmunity in normal mice S. Ansar Ahmed, D. Verthelyi, E. Karpuzogulu-Sahin Center for Molecular Medicine and Infectious Diseases, Virginia Tech, Blacksburg, VA 24061, USA

A common feature of autoimmune diseases, despite anatomical and pathological differences, is that women are more susceptible to these disorders than men. In several animal models of B-cell-mediated autoimmune diseases, It is now well established that there are marked gender differences in immunecapapbilites. Females, in general, are more immunologically reactive than males. They are also more susceptible to a wide range of autoimmune diseases, estrogens promote, while androgens abrogate these diseases. This study investigated whether estrogen can also promote autoimmunity in non-autoimmune C57BL/6J mice. Estrogen-treated mice had hypocellularity of thymus, bone marrow, spleen and lymph nodes. Estrogen dysregulated T and B cell balance to favor B cell hyperactivity. Evidence for estrogen-induced hyperactivity of B cells includes: (1) higher serum levels of immunoglobulins and autoantibodies to dsDNA and anionic phospholipids as determined by ELISA; (2) increased numbers of plasma cells in the spleen by cytology; (3) increased numbers of splenic

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Abstracts

B cells that secrete anti-dsDNA or anti-anionic phospholipids autoantibodies as determined by ELIspot; and (4) increased output of immunoglobulins and autoantibodies by individual B cells as determined by image analysis of ELIspots. B cells from estrogen-treated mice survive longer in culture. Stimulation of B cells via anti-CD40 markedly rescued cells from apoptosis and did not activate caspase-3 enzyme involved in apoptosis. Since estrogen increases IFN-g, the role of this cytokine in increased survival of B cells is currently being examined. Preliminary results suggest that blocking IFN-g increased caspase-3 levels, suggesting that IFN-g down-regulates caspase-3. In contrast to B cells, activated splenic T lymphocytes from estrogen-treated mice had increased apoptosis and caspase-3 activity. In conclusion, it is noteworthy that treatment of normal mice, solely with estrogen, can alter T and B cell regulation and overcome B cell tolerance to result in autoimmunity even in normal individuals. Immune tolerance: role of s-receptor and progesterone during pregnancy Vadivel Ganapathy Department of Biochemistry and Molecular Biology, Medical College of Georgia, Augusta, GA, USA

We recently cloned one of the subtypes of the sigma receptor (s-1 receptor) and elucidated the structure of the human and mouse genes coding for the receptor. Available evidence indicates that s ligands induce immune suppression by interfering with the production of proinflammatory cytokines and promoting the production of anti-inflammatory cytokines. To understand the role of the s-1 receptor in immune function, we investigated the expression and the ligand-binding characteristics of this receptor in Jurkat cells. The expression of the s-1 receptor in these cells was established by the presence of high-affinity binding sites for haloperidol and pentazocine in the cell membranes. The presence of s-1 receptor mRNA in these cells was shown by RT-PCR. The molecular identity of the RT-PCR product was established in a heterologous expression system. Binding studies with progesterone to Jurkat cell membranes and to the heterologously expressed RT-PCR product showed that this steroid hormone is a ligand for the s-1 receptor (dissociation constant, 90 nM). In addition, we have shown that the s-1 receptor is expressed at very high levels in the human placenta, a tissue with progesterone concentrations in the micromolar range. These findings that progesterone is a putative endogenous ligand to the s-1 receptor are important because this steroid has been shown to cause immunosuppression. We hypothesize that the interaction between progesterone and the s-1 receptor at the maternal –fetal interface during pregnancy plays a significant role in the maternal immunotolerance of the fetal allograft.

‘Open’

presentations

Amniotic fluid embolism, anaphylaxis and complement M.D. Bensona, H. Kobayashib, R.K. Silvera, H. Oib, P.A. Greenbergera, E. Haneya, T. Teraob a E6anston Northwestern Healthcare/Northwestern Uni6ersity Medical School, E6anston, IL, USA b Hamamatsu Uni6ersity Medical School, Hamamatsu, Japan

Abstracts

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Objective: We sought to evaluate the potential role of two immunologic mechanisms involving mast cell degranulation and/or complement activation in the pathophysiology of amniotic fluid embolism (AFE), a rare, unpredictable and often fatal illness of pregnant women. Methods: The study is a case series involving nine patients with AFE together with a control group of 22 women experiencing normal labor. The five main outcome measures were serum levels of complement (C3 and C4), tryptase, serum levels of a fetal antigen (sialyl Tn) and urinary histamine concentrations. Results: Serum tryptase and urinary histamine were negative in all seven patients in whom one or both measurements could be made. Seven of nine patients had elevated levels of fetal antigen. All eight patients who had serum available for testing had abnormally low levels of complement. The difference in mean complement levels between the AFE and control groups was statistically significant for both C3 and C4 (PB 0.001). Although C3 and C4 decreased slightly in the control group after delivery, the mean postpartum values were still within the normal clinical range and were also significantly higher than in the AFE cases. Conclusions: Complement activation may be important in either the etiology or mechanism of injury in amniotic fluid embolism while mast cell degranulation or anaphylaxis does not appear to have a direct role in the disease.

Molecular-genetical and biochemical methods of the fetus rhesus status B.G. Sadykov, L.R. Abdrachmanova, A.A. Zainullin Kazan Medical Uni6ersity, Medical Genetical Center, Tatarstan, Russia

We think that prenatal diagnosis has important significance in the determination of the fetus Rhesus (Rh) status, especially when the husband of the Rh-isoimmunizated pregnant has Rh heterozygotes. Part of Rh-negative pregnants has Rh antibodies in the blood from previous pregnancies and after incompatible hemotransfusion. So, fetus may be with Rh-negative factor in the next pregnancy. In this case, medical treatment and premature delivery may not be used. There are PCR techniques for the study of the fetus Rhesus status and spectrophotometry method for the determination of the bilirubin level in amniotic fluid. We carry out transabdominal amniocentesis of Rh-isoimmunized pregnants after the ultrasonography control at 20 –37th weeks of the pregnancy. We study Rh factor of the fetus deoxyribonucleic acid (DNA) of the amniocytes by the PCR methods. Rhesus factor in blood after delivery was confirmed by serological method in 100% newborns. We have received the following results: the fragments of DNA of the Rh Cc Ee and Rh of genes with the sizes: 136 bp (nucleotides sequence) − 7 exon and 186 bp 10 exon were amplificated. It is found that Rh D positive genotype is characterized by the presence of the products of the amplification with sizes of 136 and 186 bp. Rh negative genotype is characterized only by 136 bp. Besides, we began to study antibodies of ribonucleic acid (RNA) in the blood of pregnants and newborns with Rh-isoimmunization. This analysis shows that level of antibodies in RNA is high in the blood of pregnants and newborns with severe hemolytic disease. Our results confirm that the choice of the tactics in medical treatment of high risk hemolytic disease of newborn and the data of the delivery depend on the bilirubin level and on the fetus Rhesus status.

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Abstracts

Trypanosoma cruzi acute infection in mice limits fecundity and induces decidual invasion by parasites and foetal loss Carine Truyensa, Karim Mjihdia, Marie-Alexandra Lambotb, Ian Stewartc, Jean-Christophe Noe¨ lb, Yves Carliera a Laboratoire de Parasitologie, Faculte´ de Me´ decine, Uni6ersite´ Libre de Bruxelles, Brussels, Belgium b Laboratoire d’Anatomie Pathologique, Hoˆ pital Erasme, Brussels, Belgium c Human Morphology, School of Medicine, Uni6ersity of Southampton, UK

Since acute infection of mice with T. cruzi induces the production of high levels of TNF-a (Truyens et al., 1999) and IFN-g (Torrico et al., 1991), both known as abortive, we studied the effect of such acute infection on mouse gestation. Female mice infected since week 1 were mated in order to obtain gestation during the ascending phase of parasitaemia. Though the rates of mating (frequency of vaginal plugs) were similar in infected and non-infected mice, fecundity was reduced by 2/3 in infected versus control mice. In addition, whereas the rates of implantations were similar in both groups of fecundated mice, most embryos and foetuses of infected mice died during the gestation and the rare alive newborns of infected animals displayed a growth retardation (reduced birth weights) and died rapidly after birth. Such events were associated with the invasion of the decidual by parasites without foetal infection and with placental ischemic necrosis and massive leukocyte infiltration. The role of TNF-a and IFN-g in such impairment of pregnancy in T. cruzi infected mice is presently under study.

Human congenital infection induces the in utero priming of CD8( + ) T-cells Emmanuel Hermanna, Cristina Alonso-Vegab, Carine Truyensa, Patricia Rodriguezb, Faustino Torricob, Yves Carliera a Laboratoire de Parasitologie, Faculte´ de Me´ decine, Uni6ersite´ Libre de Bruxelles, Brussels, Belgium b CUMETROP/LABIMED, Faculty of Medicine, Uni6ersidad Mayor de San Simon, Cochabamba, Boli6ia

To better understand the ability of human newborns to mount an immune response, we studied the memory T-cell response in congenitally-infected newborns of mothers chronically infected with Trypanosoma cruzi. Our results show, for the first time, that human foetuses are able to expand in vivo ab CD8(+) T-cells when exposed to a live pathogen as T. cruzi. Indeed, the percentage of such cells was strongly increased in cord blood of infected newborns. In addition they expressed the CD45RO and HLA-DR molecules, but not the early activation marker CD69, indicating they had been activated in utero. Intracellular staining revealed that these CD8-T-cells were positive for perforin and were primed to produce IFN-g following stimulation of cord blood mononuclear cells (CBMC) with PMA and ionomycin. We also tested the functional responses of whole blood cells (WBC) from congenitally-infected newborns following stimulation with LPS/PHA. We observed a higher proliferative response and a higher secretion of IL-2, IFN-g, IL-4 and IL-13 as compared with uninfected neonates. All together, these data suggest that foetuses are able to generate memory CD8(+) T-cells.