Sperm antigens recognized by antisperm antibodies present in sera of infertile adults and prepubertal boys with testicular failure

Immunoblotting of a repertoire of sperm antigens reacting with antisperm antibodies present in sera of infertile adults and prepubertal boys with testicular failure was performed. In the subgroups selected for this study, 55% of examined infertile women, 65% of infertile men and 64% of prepubertal boys with gonadal failure gave positive results by Western blotting with extracted sperm antigens. Sperm antigens with molecular weights of 57, 58, 62, 63 and 66 kDa were the most immunodominant entities recognized by antisperm antibodies from prepubertal boys. No positive reactions were detected by Western blotting in a control population of fertile adults, whereas in a group of prepubertal healthy boys only one sample revealed reactivity against sperm antigens of 58 and 70 kDa.     

Flow cytometric determination of binding of monoclonal antibodies to human spermatozoa

Summary. Although several monoclonal antibodies to sperm antigens are available, only few systematical reports on the appearance of distinct antibody binding structures in spermatozoa of infertile patients are available. We studied the binding frequency of six monoclonal antibodies (TÜS-1, -2, -10, -17, -19, -20) by means of a flow cytometer in different semen samples and their possible relationship to other sperm parameters, in particular to the motility parameters as determined by computer-assisted semen analysis. The percentage of vital spermatozoa binding the different antibodies was 21.5% (TÜS-2) to 52.1% (TÜS-20). The percentage binding was higher in samples with normozoospermia than in those with oligo-asthenoteratozoospermia. A significant correlation occurred between sperm concentration and the percentage of spermatozoa stained by TÜS-2 and TÜS-17; between motile spermatozoa and those binding TÜS-17 and TÜS-20; the velocity average path and those binding TÜS-17; an inverse correlation occurred between the morphologically-abnormal spermatozoa and those binding TÜS-20; an inverse correlation between TÜS-17 and acrosin. We conclude from our results that although the antigens of the antibodies concerned are not yet characterized, the determination of antibody binding will give additional information on the functional status during capacitation and acrosome reaction of spermatozoa.     

Evaluation of the expression of sperm proteins in normozoospermic and asthenozoospermic men using monoclonal antibodies

Recent studies have shown that infertility affects estimated 15% of all couples. Male infertility is the primary or contributory cause in 60% of these cases. Consequently, the application of assisted reproduction is increasing. These methods could benefit from an extended evaluation of sperm quality. For this reason, we analyzed sperm proteins from 30 men with normal spermiograms and 30 men with asthenozoospermia. Ejaculates of both groups were tested by flow cytometry (FCM) and fluorescence with a set of well-characterized anti-human sperm Hs-monoclonal antibodies (MoAbs), which were generated in our laboratory. No statistically significant differences were found between normospermics and asthenospermics in the expression of the sperm surface protein clusterin, evaluated with Hs-3 MoAb, and semenogelin, evaluated with Hs-9 MoAb. However, FCM revealed quantitative differences in the acrosomal proteins between normozoospermic and asthenozoospermic men, namely, in glyceraldehyde-3-phosphate dehydrogenase, evaluated with Hs-8 MoAb, valosin-containing protein, evaluated with Hs-14 MoAb, and ATP synthase (cAMP-dependent protein kinase II, PRKAR2A), evaluated with MoAb Hs-36. Asthenozoospermic men displayed a highly reduced expression of intra-acrosomal proteins, with a likely decrease in sperm quality, and thus a negative impact on successful reproduction. Asthenozoospermia seems to be a complex disorder involving intra-acrosomal proteins.     

Proteomic identification of sperm antigens using serum samples from individuals with and without antisperm antibodies

The aim of the study was to identify human sperm antigens reacting with polyclonal antisperm antibodies. Protein sperm extracts were subjected to electrofocusing, and next immune reactions (immunoblotting) were carried out with positive for antisperm antibodies and control (not containing antisperm antibodies) serum samples. Proteomic analysis of human sperm proteins resulted in identification of 80 sperm antigens that could be divided into three groups: antigens specific for patients with antisperm antibodies (32), antigens recognised by both infertile patients and control sera (35) and antigens detected by control serum samples only (13). Among antigens specific for infertile patients, there were 12 sperm entities known to be involved in fertilisation process. We have also characterised three protein entities identified only by sera of infertile women. Altogether, the proteomic analysis resulted in identification of 27 sperm entities not reported previously in human sperm proteome. Identified proteins are sperm antigens that could be potentially responsible for immunological infertility. The study also sheds new light on the sperm antigens in aspect of gender specificity. The investigation of human sperm proteome by the use of antisperm antibodies‐containing sera of infertile individuals not only may indicate new proteins but also can draft their immunological nature.     

Localization of binding sites of naturally occurring antisperm antibodies on human spermatozoa by immunofluorescence

Antisperm antibodies (ASA) may affect sperm motility, acrosome reaction, sperm penetration of cervical mucus, binding to the zona pellucida, and sperm-egg fusion. We investigated the localization of ASA of infertile men or men after vasectomy bound on the sperm surface using an immunofluorescence method. Binding occurred in the acrosomal region, midpiece, and tail. Most of the ASA in both groups of patients bound to the midpiece alone or in combination with other regions of spermatozoa. Only few ASA samples showed binding to all the three sperm regions. A combination of binding to the acrosomal region and to the midpiece was never observed. In infertile patients with ASA, the binding site was compared with sperm parameters. ASA binding to the sperm head influenced the acrosome reaction. Binding of ASA on tail and/or midpiece was not associated with a significant alteration of viability and motility. Immunofluorescence appears to be a valuable tool in the diagnosis of immune infertility, in particular when impairment of the acrosome activity is suggested.     

Production of human monoclonal antibodies against Galalpha(1-3)Gal

The hyperacute rejection observed in models of pig-to-human xenotransplantation is mainly because of the presence of natural antibodies in human blood with specificity for the Galα(1–3)Gal (Gal) carbohydrate moiety present on the surface of porcine endothelial cells. Human monoclonal anti-Gal antibodies could be of use both in the study of the basic mechanisms of hyperacute rejection as well as in its clinical prevention. In the present study we prepared 42 heterohybridomas (human–mouse) secreting antibodies with specificity for the Gal epitope. All of the antibodies produced were of the IgM isotype, according to a dot-blot assay. Twenty-seven antibodies were further characterized, and shown to be specific for Gal by different methods, including an enzyme-linked immunosorbent assay, in which the plates were sensitized with mouse laminin as a source of Gal. Specificity was also confirmed using purified Gal carbohydrate in a hemagglutination inhibition assay. The antibodies were shown to mediate lysis of Gal-expressing rabbit erythrocytes in the presence of complement. However, the heterohybridomas themselves were shown to express Gal, a result of the mouse P3x63Ag8.653 hybridoma cells used during hybridoma generation. The presence of this epitope on the surface of anti-Gal-producing cells, and on the antibody itself, represents a limitation to the production of high affinity anti-Gal antibodies.     

Male antisperm antibodies: association with a modified sperm stress test and lipid peroxidation

We previously reported a modified sperm stress test (MOST), low scores (< 0.39) in which were associated with sperm-related abnormal in vitro fertilization. Preliminary observations suggested that the presence of male sperm antibodies (ASA) could give low MOST scores. It was therefore decided to undertake a study to verify this possible association and also to ascertain if such a relationship was causal in nature. Six hundred and fifty semen samples from patients consulting for infertility were assessed for basic seminal characteristics, motion parameters (CASA), ASA and MOST. Thirty-nine samples (6%) were ASA-positive. Samples with and without ASA showed similar characteristics, except for percentage of normal forms and MOST scores (0.35 +/- 0.03 vs. 0.67 +/- 0.01, P < 0.001, for ASA-positive and -negative, respectively). There was a strong statistical association between presence of ASA and low MOST scores (P < 0.0001). One-hundred per cent of ASA-positive samples displayed low MOST scores. To verify the nature of this relationship, we incubated ASA-free spermatozoa with ASA-positive and -negative (control) sera. Despite an increase in the percentage of ASA-bearing spermatozoa in those aliquots incubated with ASA-positive serum, their original (pre-incubation) MOST scores remained unchanged. Furthermore, the rate of lipid peroxidation, indirectly reflected in MOST scores, was not different in the aliquots incubated with ASA. In conclusion, there seems to be a strong association between presence of ASA and low MOST values in semen samples of infertile patients; however, the relationship does not appear to be causal.     

Assessment of antisperm antibodies in a sample of Egyptian patients with hepatitis C virus infection

Association of hepatitis C virus (HCV) with autoimmune phenomena and impaired semen parameters has been previously reported. The aim of this study was to investigate the influence of HCV infection on the development of antisperm antibodies (ASAs) in HCV‐positive males. The study was conducted on 30 HCV‐infected individuals and 30 healthy control subjects. In both patients and control groups, liver enzymes and reproductive hormones were measured; computer‐assisted semen analysis (CASA) was performed; HCV‐RNA in serum was measured and IgG and IgA ASAs in semen were determined. Free testosterone, sperm concentration, progressive and total motility were significantly lower in HCV patients than in the control group, whereas ASAs of the IgG and IgA classes were significantly higher in HCV patients. However, correlations between viral load and the examined semen parameters and ASAs were nonsignificant. In conclusion, HCV may be responsible for the increased ASAs detected in HCV patients in the present study, possibly providing another plausible explanation for the decreased sperm motility reported in HCV patients. These findings could be of value in fertility management of HCV patients.     

Monoclonal antibodies in neuro-oncology

Many studies have suggested the possible existence of tumor-associated antigens in brain gliomas. Strong evidence for the existence of such cell determinants was provided by recent investigations using hybridoma technology. The possibility of obtaining monoclonal antibodies (MAbs) against glioma-associated antigens should help to allow their identification, purification, and characterization.Utilizing MAbs as reagents of predefined specificity, a number of central and peripheral nervous system antigens could be detected. The molecules recognized by MAbs in glioma cells can be subdivided into four categories: [1] biochemical defined proteins, [2] specificities shared by nervous system-lymphoid cells, [3] oncoembryonic-oncofetal determinants, and [4] tumor-restricted antigens. Of greater significance is the heterogeneity of antigen expression among various individual glioma cells observed in frozen sections of tumor biopsies. Using a panel of MAbs, the phenotypic heterogeneity, i.e., the variation in antigen expression can be documented within and among malignant gliomas and cell lines derived from them. In spite of this the characteristic pattern of antibody binding to brain tumors makes MAbs the potentially best reagents for immuno-histochemical application in surgical neuropathology. Moreover, immuno-cytological screening of tumor cells in the cerebrospinal fluid has also proved to be valuable.The localization of radio-labelled MAbs in experimental and human gliomas growing subcutaneously and intracranially in athymic nude mice were explored by radioscintigraphy and autoradiography. Imaging experiments with¹³¹I-labelled MAbs recognizing epitopes on the glioma cell surface showed high levels of specific activity in xenografts. Preliminary data indicate that administration of¹³¹I-MAbs as well as drug conjugates (daunomycin-MAbs) causes a depression of glioma cell proliferation in vitro as well as delayed tumor growth and thus prolonged survival time of tumor-bearing mice. The mechanisms of antibody delivery and transport of immunotoxins from the vascular compartment to intracerebral tumor tissue are presently a subject of discussion. The complexity of this area necessitates comprehensive experimental work in order to define the factors involved in the delivery of MAbs to brain tumor tissue and thus optimize the rate of blood-to-tumor transport. Current investigations have shown that it is possible to image malignant human gliomas using radio-labelled antibodies. The next step will be to attain target immunotherapy. The use of MAbs as carrier molecules for clinical applications might soon be possible.     

Identification and characterization of monoclonal antibodies that partially block human natural antibody binding to pig endothelial cell xenoantigens

Abstract: The shortage of human donors for clinical transplantation has led to a serious consideration of the use of non-human species as organ donors. The major barrier to the clinical use of xenografts from species such as the pig in human transplantation has been the aggressive nature of the immune-mediated rejection of the graft. We have recently identified the molecular weights of several endothelial cell surface proteins that may be targets of human antibody-mediated responses to pig aortic endothelial cells (PAEC). In this series of experiments, we produced a panel of rat monoclonal antibodies (Mabs) to PAEC in an effort to identify Mabs that detect pig xenoantigens. Mabs were selected based on flow cytometric binding to PAEC, pig platelets, and various pig cell lines, including a pig kidney cell line (LLC-PK1) reported to react with human natural antibodies (HNA). Eleven of the eighty-three antibodies produced were cytotoxic for PAEC. Six of the cytotoxic clones recognized a 44 kDa protein and two of the clones recognized a 115 kDa protein expressed on the surface of PAEC. Since PAEC target antigens recognized by human natural antibodies include both 115 and 44 kDa antigens, these Mab clones were selected for further study. Several distinct patterns of tissue reactivity were demonstrated within this group of antibodies by immunohistochemical analysis; however all monoclonal antibodies were highly reactive with endothelial cells in all tissues examined. Two monoclonal antibodies recognizing antigens that are highly expressed on pig endothelial cells (92–98%) and pig platelets (74–92%), but moderately expressed on pig splenocytes (33–38%), were capable of reproducibly blocking 48–53% of human IgM binding to pig endothelial cells when analyzed with flow cytometry. This data suggests that these Mabs may recognize epitopes of potential significance in the human-to-pig xenograft reaction.     

Intralesional administration of I-131 labelled monoclonal antibodies in the treatment of malignant gliomas

Summary The authors report their preliminary experience with the use of radiolabelled monoclonal antibodies (MAb) as an adjuvant treatment for 33 malignant gliomas. MAbs employed in this study are raised against Tenascin (TN) which is an antigen of the extracellular matrix of the tumour. It has also been found in neoplastic cells but never in normal brain tissue. This therapy is aimed to give a local high dose radiation (boost) while sparing healthy brain structures.This treatment has always been well tolerated and no adverse reactions at the level of CNS or major extraneural organs has been observed. Significant improvement of median survival has been obtained but this result should be cautiously evaluate since the study is non-randomized. Comparison with other current adjuvant technique is briefly discussed.     

Analysis of human sperm membrane antigens reacting with sera from antisperm antibody positive and negative patients by Western blotting

Summary. Immunological infertility is thought to be caused by the binding of antibodies to 'fertility-related' antigen(s) on the sperm membrane. We compared antibody profiles in sera from 20 ASA (+) and ASA (-) men, using a sperm membrane extract as an antigen. Antigens were separated by SDS-PAGE under reducing conditions. The patients were classed as ASA (+) by the MAR (> 50%), d-IBT (> 20%) and TAT (> 1:64). The results showed that immunoreactive bands in both the ASA (+) and ASA (-) groups were heterogenous and included bands covering the whole molecular weight range. Statistical analysis showed significantly more patients in the ASA (+) group having immunoreactive bands at molecular weights of 32 Kd ( P > = 0.006) and 79 Kd ( P > = 0.02) when compared to the ASA (-) group. In the ASA (-) group significantly more patients had reactive bands at 81 Kd ( P > = 0.01) when compared to the ASA (+) group. The 32 Kd antigen reacted only with sera from ASA (+) patients. We conclude that differences exist between the ASA (+) and ASA (-) groups when this extraction method is used and that the isolation and purification of the 32 Kd protein may justify further investigation.     

Purification of estramustine-binding protein and production of monoclonal antibodies to its different components

Estramustine-binding protein (EMBP) is a heterodimeric 46-kDa glycoprotein that is secreted from the prostate. Upon reductive cleavage it decomposes into two closely related components, C1 and C2, and the shared glycosylated peptide C3. EMBP binds estramustine and estromustine, the active metabolites of estramustine phosphate (Estracyt®), which is a drug with antimitotic properties used in the treatment of prostatic carcinoma. In the present study, a two-step procedure (i.e., anion-exchange and Con A-Sepharose chromatography) is described for the isolation of EMBP in high yield from rat prostate tissue. Mouse monoclonal antibodies (mAbs) were produced using the major DEAE-Sepharose fraction of EMBP as an immunogen. Eleven mAbs were selected by screening in a solid-phase ELISA. One displayed high-affinity binding with soluble EMBP (K_a ≈ 3 × 10¹⁰ M^?1) and crossreacted with a human prostate tumor extract in a radioimmunoassay. The epitopes defined by these mAbs were analyzed by Western immunoblotting. All constituents of EMBP, except component C1, were identified by at least one antibody. Nine visualized either one or both of the two EMBP subunits under denaturing conditions, two of which retained their reactivity after reduction of disulfide bridges. One epitope was exposed to its mAb only when the antigen was in its reduced state. The immunoreactivity was eliminated by protease treatment, whereas deglycosylation with glycopeptidase F had a minimal effect. EMBP has been detected in tissues other than the prostate as well as in prostate neoplastic specimens and in several other human malignancies. Hence, these mAbs will be a useful tool in the characterization of EMBP in different tissues and in evaluating existing and in defining new indications for Estracyt therapy. © 1995 Wiley-Liss, Inc.     

Role of CD18-dependent and CD18-independent mechanisms in the increased leukocyte adhesiveness and in the variations of circulating white blood cell populations induced by anti-CD3 monoclonal antibodies

Anti-CD3 antibodies induce a quick and profound depletion of peripheral blood mononuclear cells (PBMCs) that is not well understood. We studied the effect of OKT3, a mouse monoclonal antibody againts the human CD3 complex, on the in vitro adhesion of human PBMCs to monolayers of fresh and fixed human umbilical vein endothelial cells (HUVECs). OKT3 induced an increased adhesiveness of PBMCs. This phenomenon was blocked with anti-CD18 antibodies, indicating the participation of 2 integrins. As this increased adhesiveness could explain the lymphopenia by adhesion of PBMCs to endothelial cells and their sequestration in some peripheral vascular beds, we studied the effect of anti-CD18 antibodies in vivo on mice injected with 145/2C11, a hamster monoclonal antibody against murine CD3. Mice treated with 145/2C11 presented with a transient granulocytopenia and a sustained reduction in PBMCs. A monoclonal anti-CD18 antibody prevented the granulocytopenia but had no effect the drop in PBMCs. Consequently, the in vivo depletion of PBMCs after administration of an anti-CD3 monoclonal antibody involves CD18-independent mechanisms, while the transient drop in polymorphonuclear cells appears to be CD18-dependent.     

Human glioma associated and related antigens,identified by monoclonal antibodies

Summary Tumour specific and various tumour-associated antigens expressed on human glioma cells were reported from many laboratories. These have been identified by xeno-, allo-, or auto-sera and more recently by utilizing monoclonal antibodies produced by hybridoma technique.In this article, the results of reports are summarized comparing the studies with conventional antisera and monoclonal antibodies. The present and future clinical applications of these monoclonal antibodies are described.     

Identification of the sperm antigen interacting with antibodies in serum from an infertile woman

Serum (IS) obtained from an infertile woman induced head-to-head agglutination of human sperm. The immunoglobulin G (IgG) fraction of the IS was prepared by ammonium sulfate fractionation and DEAE cellulose chromatography. The IgG localized to the post-acrosomal region of the sperm, determined by indirect immunofluorescence and interacted with a human sperm protein with an estimated Mr of 80 kDa, determined by immunoblotting. The identity of the interacting sperm antigen was verified by isolating the 80 kDa sperm protein by affinity chromatography. The present results suggest that the anti-80 kDa antibodies may be responsible for the infertility.     

Monoclonal antibodies against antigens expressed by human sperm: Monoklonale Antikörper mit Spezifität für humane Spermatozoenantigene

Summary.  The production and characterization of 21 mouse monoclonal antibodies (TüS1 - TÜS21) with specificity predominantly for human spermatozoa antigens is described. Reactivity of cells from human ejaculates, peripheral blood and several organs was determined using the alkaline phosphatase -anti-alkaline phosphatase (APAAP)-technique as well as the indirect immunofluorescence test. 15 of the monoclonal antibodies reacted with various regions of human sperm and often also with their precursor cells in the testis. Cross-reactivity with animal spermatozoa was frequently observed.
Zusammenfassung.  Die Produktion und Charakterisierung von 21 monoklonalen Anti-körpern (TüS1 - TÜS21) der Maus mit Spezifität vorwiegend für humane Spermatozoenantigene wird beschrieben. Die Reaktivität mit Zellen in humanen Ejakulaten, im peripheren Blut sowie Organen wurde mit der alkalischen Phosphatase-anti-alkalischen Phosphatase (APAAP)-Technik sowie dem Immunfluoreszenztest bestimmt. 15 der monoklonalen Antikörper reagierten mit Spermatozoen und überwiegend auch mit deren Vorläuferzellen. Kreuzreaktivität mit tierischen Spermien wurde häufig beobachtet.     

Characterization of monoclonal antibodies raised against the prostatic cancer cell line PC-82

For production of monoclonal antibodies (McAbs), hybrid cells were prepared by fusion of spleen cells of BALB/c mice immunized with the human prostatic cancer cell line PC-82 and the P3-X6(3)Ag8.653 murine myeloma cell line. Supernatants of approximately 500 hybrid clones were screened for prostate specific antibodies using an ELISA on PC-82 cells. A selection of antibodies was further tested for their specificity on a large series of different tissues. A broad cross reactivity pattern was obtained. Most cross reactivity was with pancreatic tissue, kidney, and bowel. One antibody turned out to react with prostate stromal cells. Two McAbs (ER-Pr 1 and ER-Pr 2) reacted solely with prostatic epithelium. Monoclonal antibody affinity chromatography combined with SDS-PAGE showed that both antibodies were directed against a 35-kD protein. Immunoblotting revealed that this protein is identical to prostatic antigen (PA). The epitope detected by ER-Pr 1 and ER-Pr 2 was largely preserved after formalin-fixation of prostatic tissues which renders these antibodies very suitable for routine examination of tissue sections.     

重组人睾丸精子结合蛋白对人精子运动参数的影响

目的:探讨重组人睾丸精子结合蛋白(TSBP)对体外培养人精子运动参数的影响。方法:将22例生育男性的精液经Percoll密度梯度离心后,分别与0.01mg/ml及0.1mg/ml的重组His6-TSBP在体外共同孵育1h或3h,同时设立对照组,Western印迹检测重组His6-TSBP与精子膜的结合情况,计算机辅助精液分析(CASA)系统测定重组His6-TSBP对精子运动参数的影响。将12例弱精子症患者的精液按同样方法处理,检测重组His6-TSBP对弱精子症患者精子运动参数的影响。结果:0.1mg/ml重组His6-TSBP与生育男性精子作用1h可以提高体外培养精子的前向运动百分率(a+b级精子百分率),培养3h后前向运动百分率和活率均有所提高,差异具有显著性(P<0.05);0.01mg/ml重组His6-TSBP对检测各指标均无显著性影响。0.1mg/ml重组His6-TSBP与弱精子症患者精子作用3h可以提高精子前向运动百分率(P<0.05),但对活率无显著影响。结论:0.1mg/ml的重组His6-TSBP在体外可以提高生育男性精子的前向运动百分率和活率及弱精子症患者精子的前向运动百分率。