Immunocontraceptive target repertoire defined by systematic identification of sperm membrane alloantigens in a single species.

Sperm competence in animal fertilization requires the collective activities of numerous sperm-specific proteins that are typically alloimmunogenic in females. Consequently, sperm membrane alloantigens are potential targets for contraceptives that act by blocking the proteins' functions in gamete interactions. Here we used a targeted proteomics approach to identify the major alloantigens in swine sperm membranes and lipid rafts, and thereby systematically defined the repertoire of these sperm-specific proteins in a single species. Gilts with high alloantibody reactivity to proteins in sperm membranes or lipid rafts produced fewer offspring (73% decrease) than adjuvant-only or nonimmune control animals. Alloantisera recognized more than 20 potentially unique sperm membrane proteins and five sperm lipid raft proteins resolved on two-dimensional immunoblots with or without prior enrichment by anion exchange chromatography. Dominant sperm membrane alloantigens identified by mass spectrometry included the ADAMs fertilin α, fertilin ß, and cyritestin. Less abundant alloantigens included ATP synthase F1 ß subunit, myo-inositol monophosphatase-1, and zymogen granule membrane glycoprotein-2. Immunodominant sperm lipid raft alloantigens included SAMP14, lymphocyte antigen 6K, and the epididymal sperm protein E12. Of the fifteen unique membrane alloantigens identified, eleven were known sperm-specific proteins with uncertain functions in fertilization, and four were not previously suspected to exist as sperm-specific isoforms. De novo sequences of tryptic peptides from sperm membrane alloantigen "M6" displayed no evident homology to known proteins, so is a newly discovered sperm-specific gene product in swine. We conclude that alloimmunizing gilts with sperm membranes or lipid rafts evokes formation of antibodies to a relatively small number of dominant alloantigens that include known and novel sperm-specific proteins with possible functions in fertilization and potential utility as targets for immunocontraception.

Identification of immunogenic MHC class II Tyrosinase-derived peptides using HLA-DR1 and HLA-DR4 transgenic mice.

The immunogenicity of "novel" MART-1 and Tyrosinase class-II peptides was assessed in transgenic mice. Tyrosinase(141-161) peptide was found to be immunogenic and endogenously processed in the HLA-DRbeta1*0101 and HLA-DRbeta1*0401 transgenic mice with peptide specific production of IFNgamma or IL-5 respectively. The MART-1(29-43) peptide was only found immunogenic in HLA-DRbeta1*0101 mice.

Recombinant forms of Gerbich blood group antigens: expression and purification.

Recombinant forms of normal glycophorin C (GPC), carrying the high frequency Gerbich blood group antigens, and its natural deletion mutants of Yus and Ge type (all combined with oligohistidyl tag) were expressed in CHO and COS 7 cells. The stable expression of all recombinant forms of GPC in CHO cells was obtained, but the level of expression was low and detectable only by flow cytometry. The high level of transient expression of GPC recombinant forms in COS 7 cells allowed their purification on Ni-NTA-agarose. The purified recombinant GPC and mutants of Yus and Ge type behaved in SDS-PAGE similarly to normal GPC forms from RBC membranes. The recombinant GPC.Yus and GPC.Ge mutants appeared as diffuse bands, suggesting the similar heterogeneity of glycosylation that was observed in natural GPC.Yus and GPC.Ge glycoproteins. The flow cytometry analysis of the transfected CHO and COS 7 cells showed that binding of anti-GPC monoclonal antibodies to GPC variants was accordant with the known fine specificity of these antibodies. The obtained recombinant forms of GPC carrying common Gerbich antigens may be useful in serology, and also as model molecules for structure-function studies.

Two-dimensional electrophoretic analysis of sperm antigens recognized by sperm immobilizing antibodies detected in infertile women.

In this study, high resolution two-dimensional (2-D) gel electrophoresis was used to identify human sperm antigens recognized by the sera from infertile women having sperm immobilizing (SI) antibodies. Two-D gel electrophoresis was employed to separate Percoll purified human sperm proteins using isoelectric focusing (IEF), followed by polyacrylamide gel electrophoresis (PAGE). Sperm proteins were transferred to the nitrocellulose membranes and immunoblotted with seven sera from infertile women with high titers of SI antibodies and 6 sera from those without SI antibodies. The blots were compared to the 2-D composite image of human sperm proteins [Sperm Protein Encyclopedia] and sperm surface index and the sperm surface proteins recognized by infertile sera were identified. Fifty-two human sperm surface proteins reacted with sera containing SI antibodies, while 35 of these were reactive with the SI-negative control sera. The average numbers of protein spots reacted with test and control sera were 24.6 and 15.0 respectively. A subset of sperm surface proteins which were unique to the SI antibodies were identified by the following criteria; the sperm protein spots which were highly reactive with the infertile sera containing SI antibodies but not reactive with any of the SI-negative infertile sera. The coordinates of 4 prominent immunoreactive sperm proteins were considered as possibly relevant to antibody mediated female infertility.

Molecular basis of the neutrophil glycoprotein NB1 (CD177) involved in the pathogenesis of immune neutropenias and transfusion reactions.

The human granulocyte alloantigen NB1, recently clustered as CD177, is heterogenously expressed on neutrophils of 88-97% of healthy individuals. Since its molecular nature has remained unknown, we isolated NB1 glycoprotein from granulocyte lysate by immunoaffinity chromatography. MALDI-TOF mass spectrometry identified a 50,556 Da glycoprotein which was reduced to 43,069 Da after removal of N-linked carbohydrates. Following N-terminal amino acid sequencing and NB1-specific primer construction, rapid amplification of cDNA ends PCR yielded a 1,614-bp cDNA for NB1. COS-7 cells transfected with the cDNA expressed immunoreactive NB1 glycoprotein. A 1,311-bp sequence was identified to be the entire coding region. The 5' and 3' untranslated regions consist of 27 bp and 276 bp, respectively. The open reading frame codes for 437 amino acids of which the first 21 form the signal peptide. The remaining 416 residues form a N-terminal extracellular protein with two cysteine-rich domains, three N-linked glycosylation sites and short transmembrane and cytoplasmic segments including a glycosyl-phosphatidylinositol attachment (omega) site. Database searches revealed homology to Ly-6 (uPAR) domain, suggesting that NB1 belongs to urokinase plasminogen activator receptor/CD59/Ly-6 snake toxin superfamily.

A murine monoclonal antibody against Kx protein which reacts also with beta-spectrin.

Kx is a polytopic membrane protein of human erythrocytes carrying the Kx blood group antigen, which is deficient in rare patients with McLeod syndrome. Kx is disulphide bond linked to the Kell glycoprotein, which is a bitopic type II membrane protein carrying the Kell blood group antigen. Mice immunized with a synthetic peptide predicted to be located on the second external loop of Kx produced a monoclonal antibody called 3E12 which does not recognize red cells with common Kell phenotype by agglutination and flow cytometry. 3E12 recognizes the Kx protein and the spectrin beta-chain on western blots, the affinity for these two proteins being lowered with increasing ionic strength. Linear epitopes recognized by 3E12 are E116EIEKE121 and L484AQELEKE491 on the Kx protein and spectrin beta-chain, respectively. To quantify the relative amount of Kx in Empigen BB extracts of red cell membranes, an ELISA for Kx was set up which showed conclusively that (i) there is less Kx in membranes of K0 individuals (lacking the Kell glycoprotein) than in membranes of common individuals, and (ii) that all common individuals, typed as K+k-, K-k+ and K+k+, have the same amount of Kx on their red cell membranes. When an erythrocyte membrane detergent extract from one K0 individual was chromatographed on an immobilized 3E12 column, a minute amount of authentic Kell glycoprotein was recovered in acid eluted fractions, indicating that at least the K0 individual under study may still produce some Kell protein.

Relación entre la expresión de antigenos ABH en cancer de vejiga y caracter secretor / The relationship between the expression of ABH secretor character

Existe amplia evidencia de que el cáncer está asociado con anormalidades en la regulación génetica expresada en la superficie de la membrana celular. El 80 por ciento de los individuos son capaces de secretar los antígenos ABH en saliva y otras secreciones. La presencia de estas sustancias está controlada por un gen que puede adoptar dos formas alélicas: SE dominante y SE recesiva. El objetivo de este trabajo fue investigar la relación entre la expresión antigénica ABH en célkulas de descamación urotelial y el carácter secretor en pacientes con cáncer de vejiga. Se examinaron 33 pacientes con tumores de vejiga clasificados en superficiales y profundos y una población de 40 individuos normales. Se investigó el carácter secretor en saliva y la expresión de los antígenos ABH uroteliales en sedimentos urinario. Se empleó para estos estudios la técnica de inhibición de la aglutinación. En la población normal todos expresaron los antígenos ABH en células de sedimento urinario y sólo el 80 por ciento presentó dichos antígenos en sus secreciones. En los pacientes con cáncer de vejiga el 30,31 por ciento resultó no secretor y de ellos el 70 por ciento presentó deleción antigénica ABH en sedimento urinario con mayor incidencia de tumores profundos. Nuestros resultados indicarían que los pacientes con cáncer de vejiga no secretores desarrollarían tumores con mayor grado de infiltración respecto de los pacientes secretores

Relación entre la expresión de antigenos ABH en cancer de vejiga y caracter secretor / The relationship between the expression of ABH secretor character

Existe amplia evidencia de que el cáncer está asociado con anormalidades en la regulación génetica expresada en la superficie de la membrana celular. El 80 por ciento de los individuos son capaces de secretar los antígenos ABH en saliva y otras secreciones. La presencia de estas sustancias está controlada por un gen que puede adoptar dos formas alélicas: SE dominante y SE recesiva. El objetivo de este trabajo fue investigar la relación entre la expresión antigénica ABH en célkulas de descamación urotelial y el carácter secretor en pacientes con cáncer de vejiga. Se examinaron 33 pacientes con tumores de vejiga clasificados en superficiales y profundos y una población de 40 individuos normales. Se investigó el carácter secretor en saliva y la expresión de los antígenos ABH uroteliales en sedimentos urinario. Se empleó para estos estudios la técnica de inhibición de la aglutinación. En la población normal todos expresaron los antígenos ABH en células de sedimento urinario y sólo el 80 por ciento presentó dichos antígenos en sus secreciones. En los pacientes con cáncer de vejiga el 30,31 por ciento resultó no secretor y de ellos el 70 por ciento presentó deleción antigénica ABH en sedimento urinario con mayor incidencia de tumores profundos. Nuestros resultados indicarían que los pacientes con cáncer de vejiga no secretores desarrollarían tumores con mayor grado de infiltración respecto de los pacientes secretores(AU)

Aloinmunización transplacentaria por antígenos plaquetarios específicos: prevalencia y características en una población chilena / Transplacental alloimmunization against platelet antigens: prevalence and features in a chilean population

Backgrour: Neonatal alloimmune thrombocytopenia (NAIT) is a result of fetomaternal incompatibility. Platelet destruction is caused by a maternal alntibody directed against a fetal platelet antigen inherited from the father and lacking on the mother's platelets. The incidence and features of transplacental alloimmunization depend on the frequency of expression of platelet specific antigens, which are highly variable among different populations. Aim: To determine the prevalence and characteristics of transplacental alloimmunization in a large, group of pregnant women in Chile. Material and methods: We, studied 3,041 samples obtained during the third trimester of gestation. In all samples, anti platelet antibodies were screened by ELISA with platelet membranes fixed to a microtiter plate. Positive samples were further studied for antigenic specificity with the monoclonal antibody specific immobilization of platelet antigens (MAIPA) test. Results: Anti platelet antibodies were found in 261 samples (8.5 percent). The MAIPA test identified 6 samples with antibodies directed against major platelet membrane glycoproteins, 2 anti GPIb, 2 anti GPIIb/IIIa and 2 anti GPIa/IIIa. In four cases, anti HLA antibodies coexisted. Two cases corresponded to well defined platelet antigen systems: one anti HPA-1a and one anti HPA-5b. No clinical evidence of thrombocytopenia of the newborn was detected in all these cases with anti GP antibodies. Conclusions: A prevalence of platelet specific antibodies of 0.2 por ciento with only one anti HPA-1a was detected. These findings are in contrast with those of other populations but in accordance with the low frequency of the HPA-1b/b phenotype in the Chilean population. The very low incidence of platelet specific antibodies and the lack of association with clinical thrombocytopenia in the newborn, do not support the recommendation of routine antenatal screening to all women in Chile

Contemporary non-imaging methods in diagnosis of bladder cancer: a review.

The early diagnosis of bladder cancer is central to the effective treatment of the disease. Presently, the detection of bladder tumors relies on cystoscopy and there are no methods available to easily and specifically identify the presence of bladder cancer cells. A variety of new technologies and potential tumor markers are being studied in bladder cancer and some are being translated into clinical use. It is important to realise that all available results on the diagnostic value of tumor markers do not allow firm clinical recommendations, but tests based on biomarkers will undoubtedly influence the management of bladder cancer in the near future.

A single T cell receptor recognizes structurally distinct MHC/peptide complexes with high specificity.

The 2C T cell is a CD8+, alloreactive T cell, which recognizes cells bearing Ld and Kbm3 class I major histocompatability complex molecules. Here, we characterize an allopeptide, designated dEV-8, that is a ligand in the Kbm3 molecule for the 2C TCR but is not a ligand in the Ld molecule. By biochemical and immunological properties, dEV-8 is distinct from P2Ca, the Ld allopeptide that is also recognized by the 2C TCR. Using the deduced amino acid sequence of dEV-8, we isolate a candidate endogenous source of the peptide. The endogenous protein, MLRQ, contains a peptide sequence identical to dEV-8. This degenerate recognition of two distinct peptide/MHC complexes by a single TCR has important implications for understanding allorecognition.

Isolation of a human allo-peptide presented by HLA-B51 molecules.

Recent studies have demonstrated directly that alloreactive mouse CTL recognize peptides presented by MHC class I molecules. However, there is no direct evidence that human alloreactive CTL recognize peptides presented by HLA class I molecules. We have isolated an HLA-B51 alloreactive CTL clone, 2B3, that did not kill the TAP defective cell lines T2 and .174, whereas it killed the TAP-positive cell line T1 and .174 cells transfected with TAP genes. These findings suggested that this clone recognizes a TAP-dependent allo-peptide. We attempted to isolate the human allo-peptide recognized by the 2B3 clone from HLA-B51 molecules. A naturally occurring HLA-B*5101 binding peptide isolated from T1 cells was recognized by the 2B3 clone. The peptide was also isolated from HLA-B*5101 molecules purified from C1R-B*5101 cells. In the present study, we directly demonstrated that a human alloreactive CTL clone recognizes peptide presented by HLA class I molecules.

Isolation of a kidney-specific peptide recognized by alloreactive HLA-A3-restricted human CTL.

The molecular nature of tissue-specific Ags involved in MHC-restricted CTL responses is as yet undefined. To determine the specificity of these peptides, their function, and their possible relationship to allograft rejection, we have utilized human kidney-specific CD8+ CTL clones to screen reversed-phase HPLC (RP-HPLC)-separated self peptides presented by allo-class I molecules. One of these clones is HLA-A3-restricted and the other HLA-B62-restricted, lysing human kidney cell lines but not MHC identical B lymphoblastoid cells which express the appropriate HLA molecules. We have identified a biologically active RP-HPLC fraction containing self peptides eluted from affinity-purified MHC molecules from HLA-A3+ kidney. This peptide is not expressed in HLA-A3+ spleen. Similarly, a HLA-B62-associated peptide fraction was identified in kidney but not in spleen using the HLA-B62-restricted CTL clone. Sequence analysis of the biologically active fraction from HLA-A3 kidney revealed multiple peptides. Because of the ambiguity of the peptide sequence, a mixed peptide library corresponding to this sequence was synthesized that included the HLA-A3 binding motif. The biologically active peptide library was RP-HPLC fractionated and the fraction containing HLA-A3-restricted CTL activity was sequenced. The resulting sequence of the alloreactive HLA-A3-restricted peptide epitope is GPPGVTIVK. By using this unique strategy, we describe the successful isolation and sequencing of an antigenic peptide that is recognized by a human alloreactive kidney-specific CTL clone.

An 80-kDa syncytiotrophoblast alloantigen bound to maternal alloantibody in term placenta.

PROBLEM: We have shown that most of the IgG present on term syncytiotrophoblast, membrane, microvesicles is bound to an 80 kDa protein antigen (R80K). METHODS: Microvesicles were prepared from term human placenta, and the IgG eluted at pH3. RESULTS: When IgG antibody was eluted at pH3 and reacted with acid-treated vesicles of other placentae, the alloantibody always bound to the preparation from which it was obtained, but only to about 10% of acid-treated preparations from other placentae. A similar polymorphic protein found in association with IgG antibody was found in term horse placentae. Cross-reactivity of the antibodies between species was not found. Using binding of labelled antibody, complement dependent cytotoxicity and FACS two-color analysis, the human polymorphic antigen was present on peripheral blood monocytes and B-lymphocytes. The R80k antigen on intact microvesicles was resistant to trypsin, but after acid elution of IgG, trypsin released a soluble 50 kDa fragment which reacted with the acid-eluted IgG antibody. CONCLUSION: The presence of antibodies to R80K in all term placentae studied, including first pregnancies, suggests that development of this alloantibody may be a normal requirement for successful pregnancy.

The murine BP-3 gene encodes a relative of the CD38/NAD glycohydrolase family.

The murine BP-3 antigen is a variably glycosylated glycosyl-phosphatidylinositol (GPI)-linked molecule that is selectively expressed by early B and T lineage cells and a discrete subpopulation of reticular cells in the peripheral lymphoid organs. It is also expressed on the brush border of intestinal epithelial cells, the lumenal surface of renal collecting tubules and mature myeloid cells. To further explore the nature of the BP-3 antigen, we purified the protein, obtained peptide sequences and used these to isolate cDNA clones. Two BP-3 cDNA clones were found to share the same open reading frame, but to utilize different polyadenylation sites. Expression of a full-length cDNA clone confirmed that it encodes the BP-3 antigen. Northern blot analysis with this cDNA probe revealed BP-3 transcripts of 1.3 and 2.3 kb in various tissues and cell lines representing myeloid, B and T cell lineages, while a probe containing the most 3' untranslated region of the longer cDNA clone hybridized only with the 2.3 kb RNA species. Analysis of the BP-3 cDNA sequence indicates that it represents a previously undescribed gene that shares significant homology with genes encoding nicotinamide adenine dinucleotide (NAD) glycohydrolase of Aplysia californica and the CD38 antigens in mouse and human. However, cells expressing the recombinant BP-3 protein did not exhibit NADase activity, suggesting that it may be a distant relative of NAD hydrolase with different function.

[Band patterns of human sweat proteins carrying ABO-blood-group antigens tested by SDS-polyacrylamide gel electrophoresis].

Sweats were obtained from healthy 27 male and 5 female adults (A.Se: 11, A.se:1, B.Sc:5, B.Se:5, AB.Se:2 and O.Se:8 cases) in a sauna. Human sweat proteins carrying ABO-blood-group antigens were examined by SDS-polyacrylamide gel electrophoresis followed by immunoblotting with monoclonal anti-A and anti-B antibodies. Electrophoretic bands of the sweats were generally demonstrated as follows: A band in the region of a higher molecular weight than 116,000 Da, a band in each of the regions of 96,000, 82,000, 71,000, 50,000, 43,000 and from 34,000 to 30,000 Da, and several bands (2-6 bands) in region from 28,000 to 19,000 Da. There were many protein fractions carrying ABO-blood-group antigens in the sweats, regardless of the blood groups. Individual variations of the band patterns in the number and the color depth of immunoblotting were observed. The band in the sweats from the nonsecretors did not appear clearly. A band in the region between 34,000 and 30,000 Da, and several bands in the region between 28,000 and 19,000 Da were stained with PAS. These bands are probably glycoproteins with a blood group activity in sweats. Proteins carrying ABO-blood-group antigens in human erythrocyte membranes and saliva were also tested. In erythrocyte membranes, many bands ranged widely from high to low molecular weight regions were demonstrated, and several bands with higher molecular weights than 43,000 Da were shown in saliva. The band patterns of the sweats, erythrocyte membranes and saliva differed from one another, therefore, it was considered that there are proper proteins carrying ABO-blood-group antigens in sweats, erythrocyte membranes and saliva.

A new low-frequency red cell antigen, 'SARAH'.

There are more than 100 low-frequency antigens (LFAs) which have been given International Society of Blood Transfusion (ISBT) numbers as members of systems, collections or the 700 series. In addition, there are a number of well-known (to reference laboratories) unpublished LFAs. The presence of an LFA was suspected when 2 sera were found to react with a single example of K homozygous cells. Anti-K reacting with K homozygotes was eliminated on testing with other KK cells. Testing of the reactive cell with antibodies to known LFAs and the reactive sera with cells known to carry LFAs failed to identify the specificity. A study on the family of the cell donor showed inheritance of the antigen in two generations. Further testing, which included immunoblotting and RFLPs, was carried out in Australia, the UK, Canada and the USA. By March 1993 all published LFAs had been excluded, and an application was made to the ISBT to have the antigen, SARAH, assigned a 700 series number. In April the number 700.052 was provisionally designated for this new antigen.

Polyclonal antibodies against the NB1-bearing 58- to 64-kDa glycoprotein of human neutrophils do not identify an NB2-bearing molecule.

The neutrophil-specific NB antigen system has been serologically characterized with human alloantisera. Two alleles, NB1 and NB2, have been described; however, there may be important quantitative or qualitative variation in the expression of NB1 and NB2. Human alloantibodies have been used to identify the 58- to 64-kDa glycoprotein (GP) on which NB1 antigen is located, but an NB2 antigen-bearing molecule has not yet been identified. To identify the NB2 molecule, human alloantibody to NB1 was used to isolate the 58- to 64-kDa NB1 GP, and rabbits were immunized with this GP. Two rabbit antisera were produced. Both antisera immunoblotted and immunoprecipitated the 58- to 64-kDa GP on which NB1 is located, but neither identified the molecule on which NB2 is located. The inability of two rabbit polyclonal antibodies specific for the NB1 molecule to react with the NB2-bearing molecule suggests that considerable differences may exist between these two molecules or that NB2 as currently defined is not related to NB1.

Tetrahymena gene encodes a protein that is homologous with the liver-specific F-antigen and associated with membranes of the Golgi apparatus and transport vesicles.

The F-antigen is a prominent liver protein which has been extensively used in studies on natural and induced immunological tolerance. However, its intracellular localization and biological function have remained elusive. It has generally been assumed that the F-antigen is confined phylogenetically to vertebrates. Now we have cloned and characterized a gene from the ciliated protozoan Tetrahymena thermophila encoding a protein which clearly is homologous with the rat F-antigen. The coding region of the Tetrahymena F-antigen (TF-ag) gene specifies a 46,051 M(r) protein and is interrupted by three introns. In accordance with the predicted molecular mass of the TF-ag protein, antibodies raised against a cro-lacZ'-TF-ag fusion protein specifically recognized a 45,000 M(r) protein in Western blots of total T. thermophila protein. Immunoelectron microscopy demonstrated that the TF-ag is associated with membranes of the Golgi apparatus and transport vesicles pointing to a role of TF-ag in membrane trafficking. Transcription of the TF-ag gene, as determined by run-on analyses, was only detectable in growing cells, and following transfer to starvation condition pre-existing TF-ag mRNA was rapidly degraded. The abundance of the TF-ag protein, however, declined only moderately during prolonged periods of starvation demonstrating that extensive release of the TF-ag did not take place. In combination these results suggest that the TF-ag protein is a recycled constituent of the intracellular membrane network in T. thermophila.

The identification and cloning of the murine genes encoding the liver specific F alloantigens.

The liver specific F alloantigen is a highly conserved abundant protein found in hepatic cytoplasm; smaller amounts are detected in renal tubule cells and the perikaryon cells of the central nervous system. Although the biological function of the F alloantigen is unknown, the immune response to F has been extensively studied as a murine model of tolerance and autoimmunity. Murine F exists in two allelic forms, designated F type 1 and type 2, each of approximately 43 kDa. The immune response to the allotypic form is restricted to mouse strains of I-Ak. Responding strains immunized with allotypic F break tolerance and produce precipitating antibody that reacts with both allelic forms, i.e., immunogen and self. Thus an autoantibody is produced. Using the previously isolated rat F cDNA as a probe, we report the cloning and sequencing of the two murine F allotypes. These two alleles are nearly homologous except at the extremes of the coding sequence. There are a number of regions within the F sequence that are similar to peptides that interact specifically with I-Ak. In particular, there is a sequence near the carboxy terminus, where the two allotypes differ, that has homology to the I-Ak restricted malarial antigen peptide of the ring-infected erythrocyte surface antigen (RESA).