The relationship of circulating antisperm antibodies to sperm surface antibodies in infertile men

The correlation between the amount and location of antisperm antibody binding to the sperm surface and the level measured in the serum has not been previously reported. Hence, the value and limitations of screening blood sera from men with suspected immunologic infertility are not currently known. In this study 70 paired sera and semen samples were assayed by the immunobead test (IBT). A screening protocol for blood sera was constructed to be 100% sensitive for detecting semen specimens with 20% or more of sperm binding IgG or IgA immunobeads. The specificity of this screening protocol was determined to be 79%. Serum IgA was not a good predictor of IgA on the sperm surface. The true positive predictive rate for antisperm antibodies on the sperm surface using circulating antisperm antibodies as a screening assay was estimated to be as low as 35%. There was little correlation between the site of immunobead binding following passive antibody transfer from patients' sera to donor sperm and the site of naturally occurring antibodies on the patients' sperm surface. Although direct assessment of antibodies on the sperm surface is preferred, these data suggest that serum IgG alone can be used as a sensitive screening assay for antisperm antibodies in men. A positive screen dictates that a direct assay on semen should be performed.     

Development and characterization of monoclonal antibodies to lymphocyte cell surface antigens of the rhesus monkey.

Three monoclonal antibodies directed against rhesus lymphocyte cell surface antigens are described. A pan-T mAb, T64, and a T suppressor mAb, T35, showed phenotypic and functional specificity for both human and rhesus cells. In contrast, a third mAb, N42, identifying natural killer cells in rhesus peripheral blood leukocytes, was not crossreactive with the corresponding homologous human cells. N42 reacted with the same cells identified by Leu 11a and Leu 11b in rhesus PBL and in a functional assay decreased NK activity by 80%. N42 precipitated a 50KD protein from rhesus PBL lysates and was reactive with the Fc receptor domain of the NK cell. The T-S functional activity of cells reactive with mAb T35 was demonstrated in a pokeweed-mitogen-driven system for Ig synthesis: removal of the T35 positive cells by complement-mediated lysis led to an enhanced production of Ig by rhesus PBL, and the addition of T35 positive cells to a culture of T helper and B cells resulted in a reduction of this response. T35 was determined to be an IgG2a immunoglobulin and precipitated a 34KD protein from rhesus cell lysates. An IgM immunoglobulin, mAb T64 delineated all T lymphocytes, inhibited E-rosette formation, interfered with the proliferation of cells stimulated with mitogens, and precipitated a 52KD membrane protein. The potential utilization of these mAbs in vivo for organ or tissue transplantation in the rhesus monkey is discussed.     

Discrete monoclonal antibodies define functionally important epitopes in the CD200 molecule responsible for immunosuppression function

BACKGROUND: Both murine and human CD200 fusion proteins (CD200Fc) act as immunosuppressants after engagement of cell-bound receptors (CD200R). Anti-CD200 monoclonal antibodies (mAbs) augment activity in mixed leukocyte cultures (MLCs) (increased cytotoxic T lymphocyte/cytokine production) after neutralization of endogenous CD200 activity. Previous studies documented critical regions in the N-terminal domains of both CD200 and CD200R1 for ligand:receptor binding and defined a number of synthetic CD200 and CD200R peptides that antagonize that interaction. METHODS: We used a panel of mAbs to mouse and human CD200Fc to compare the rank activities of antibodies for binding (flow cytometric analysis [FACS] or enzyme-linked immunoadsorbent assay [ELISA]) to CD200 with their abilities to augment immune reactivity in MLCs. RESULTS: Only mAbs defining epitopes in the N-terminal domain could augment MLC reactivity (or block immunosuppression by soluble CD200Fc), whereas mAbs targeting C-domain epitopes, although reactive in ELISA or FACS (targeting cell surface CD200), were inactive in MLCs. CONCLUSION: In addition to defining the importance of N-terminal epitopes for CD200 function, rank comparison of mAbs for FACS staining of CD200 expressed on various cell types indicates heterogeneity in expressed CD200.     

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.     

Use of anti-CD25 monoclonal antibody in combination with rapamycin to eliminate cyclosporine treatment during the induction phase of immunosuppression.

BACKGROUND: Cyclosporine and tacrolimus are associated with drug-induced renal dysfunction that may exacerbate recovery from ischemic injuries during the first month posttransplant. We sought to use anti-CD25 (anti-interleukin-2 receptor) monoclonal antibodies in combination with sirolimus (rapamycin) to avoid cyclosporine therapy during the early postoperative period in six renal transplant recipients deemed to be at high risk for delayed graft function. METHODS: Six consecutive patients deemed to be at high risk for delayed graft function were treated with rapamycin (2-12 mg/day), anti-CD25 monoclonal antibodies, and steroids, withholding inception of cyclosporine therapy until the serum creatinine fell below 3.0 mg/dl. RESULTS: During the first 2 months posttransplant, none of the patients displayed clinical or histopathological evidence of acute allograft rejection episodes, cytokine release syndrome, or hypersensitivity reactions. None of the patients received even empiric bolus or high-dose steroid therapy for a presumed rejection episode. All patients recovered renal graft function within 8 weeks posttransplant. To date all patients have stable renal graft function. Five patients have serum creatinine levels between 0.8 to 1.3 mg/dl at 6 months and the other patient has a serum creatinine level of 1.7 mg/dl at present follow-up of 2 months posttransplant. CONCLUSION: During the early posttransplant period anti-CD25 monoclonal antibodies combined with rapamycin and steroids offer a promising baseline therapy to avoid cyclosporine exposure and facilitate recovery from ischemic/reperfusion injuries.     

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.     

Use of theophylline to enhance sperm function.

Poor sperm motility is an important factor in male infertility. Preliminary results in our laboratory on a group of 19 men (10 suspected infertile men and 9 fertile donors) showed stimulation of sperm fertilizing ability after sperm washing with theophylline as demonstrated by zona free hamster egg penetration test. The egg penetration rate for the control spermatozoa samples from subfertile men was 16%. Incubation with theophylline (10 mM) increased the penetration rate to 46%, whereas semen incubation with theophylline (20 mM) increased the penetration rate to 51%. A similar twofold increase in egg penetration was observed in the semen of fertile men incubated with theophylline of similar concentrations. Subfertile patients with ejaculate volumes of less than or equal to 1 ml or total motile sperm count of less than or equal to 10 x 10(6)/mL or increased semen viscosity did not exhibit beneficial effects with theophylline washing as measured by hamster egg penetration test score. The increase in percentage of penetrated eggs with theophylline use in both fertile and subfertile men was significant at 10 mM concentration (p less than .001) and 20 mM (p less than .001) when compared to control (untreated) samples. No significant difference in penetration rate was seen between 10 and 20 mM theophylline concentrations. It appears that theophylline may be useful in improving the fertilizing capacity of selected human semen samples with poor motility and poor penetration ability under artificial insemination conditions.     

Use of monoclonal antibodies to quantitate T lymphocyte subpopulations in human cardiac allografts

T cell subsets have been quantitated in 40 human cardiac biopsies to characterize the surface phenotype of T lymphocytes involved in acute rejection. Seventeen biopsies were from patients receiving conventional immunosuppression, and 18 were from patients receiving cyclosporine (Cys) as the major immunosuppressive drug. Five biopsies were from nontransplanted hearts. Frozen sections were treated with monoclonal antibodies of the Leu series and an immunoperoxidase technique to determine numbers of Leu 4 (T3 or pan-T-cell), Leu 2a (T8 or suppressor/cytotoxic), and Leu 3a (T4 or helper/inducer) positive cells. Biopsies from patients on conventional immunosuppression during rejection contained 68.3 +/- 10 infiltrating leukocytes/0.048 mm2 of the biopsy, of which 47 +/- 7.4% were T cells. Most of the T cells (86%) were of the 2a/T8 phenotype. In contrast, nonrejecting hearts contained substantially fewer infiltrating leukocytes (43.3 +/- 18.8/0.048 mm2), of which only 12.6% were T cells. There was no predominance of the 2a/T8 subset in nonrejecting biopsies. It is concluded that in patients receiving conventional immunosuppression, rejection is associated with an influx of T cells, most of which are 2a/T8-positive, into the cardiac allograft. In contrast, biopsies from patients on Cys contained large numbers of infiltrating leukocytes that did not appear to correlate with the histological assessment of rejection. However, in these patients, rejection was associated with an increase in the number of T cells with no consistent pattern of subset distribution. Biopsies from nontransplanted hearts contained few infiltrating leukocytes (31.5 +/- 9.4/0.048 mm2), and none of them were T cells.     

A novel approach to production of antitumor monoclonal antibodies: antibody to a cell surface glycoprotein associated with transformation by a human oncogene

Transfection is a technique for inducing transformation of normal fibroblasts (NIH 3T3) with DNA (oncogenes) from human tumors. Our goal was to determine if these transformed cells expressed antigens associated with malignancy. NIH 3T3 cells were transfected with DNA fragments from a human acute lymphocytic leukemia (ALL 1-69), and transformed colonies were selected for growth in soft agar. Transfected cells containing human DNA sequences demonstrated by Southern blot analysis were used to immunize Balb/C mice. Monoclonal antibodies were produced and screened for binding to the parental leukemia (ALL 1-69), transfectant (17(2], and 3T3 cells in an enzyme-linked assay. A monoclonal antibody (IgM kappa) designated 17-9H3 bound to ALL 1-69 and secondary transfectant 17(2) but not to NIH 3T3 plasma membranes. Immunoperoxidase staining confirmed this binding pattern and demonstrated that the antigen was expressed on the cell surface. Expression of the antigen by transfectants directly correlated with the presence of a single 6.1 kilobase human DNA sequence. The antibody binding site of the antigen was inactivated by trypsin, glucosidase, and hyaluronidase. Binding of the 17-9H3 antibody was selective for acute lymphocytic leukemias (5/8) and osteogenic sarcomas (33/36), although other tumor types did demonstrate significant binding by immunoperoxidase staining. The majority of normal tissues did not bind 17-9H3 with the exception of some metabolically active cells (renal tubular epithelium, secretory epithelial cells, and cardiac smooth muscle), germ cells, Leydig cells of the testes, and some lymphoid cells. Monoclonal antibodies to oncogene-associated antigens may be potentially useful for cancer diagnosis and therapy and as probes for oncogene isolation.     

The specificity of nephritogenic antibodies. IV. Binding of monoclonal antithymocyte antibodies to rat kidney

Polyclonal rabbit antirat thymocyte serum (ATS) has been shown to form in situ glomerular immune aggregates following perfusion into normal rat kidney ex vivo. This may be due to the presence of T-cell-like epitopes in the rat kidney, or it may be a result of contaminating anti-connective-tissue antibodies in ATS. To exclude the latter possibility we investigated binding to the rat kidney of three different (mouse) monoclonal antirat thymocyte antibodies (anti-T-cell MoAbs), directed to Thy 1.1 antigen, as well as (control) anti-B-cell MoAbs. The MoAbs were incubated in vitro with kidney sections or perfused into the blood-free rat kidney ex vivo. It was shown using immunofluorescence (IF) and immunoelectron microscopy (IEM) (peroxidase technique) that the anti-T-cell MoAbs used, in contrast to anti-B-cell MoAbs, are able to bind with glomerular capillary walls, and with mesangial structures after incubation in vitro or perfusion ex vivo. Although staining patterns are not completely identical, the reaction product is clearly demonstrated throughout the glomerular basement membrane (GBM) and along the plasma membranes of endothelial and epithelial cells, after contact with either of the three anti-T-cell MoAbs used. It is concluded that the presence of T-cell-like epitopes in the rat kidney may lead to immune complex formation following contact with anti-T-cell MoAbs. The nephritogenicity of rabbit ATS, as well as of some batches of clinically used ATS, may also be explained by this mechanism rather than by the usually assumed presence of contaminating antibodies in these polyclonal antisera.     

Use of rituximab to decrease panel-reactive antibodies.

Patients with pre-formed antibodies may be at increased risk of rejection after organ transplantation. In this report, we describe the use of rituximab to decrease the percentage of pre-formed antibodies in a pediatric heart transplant recipient.     

Monoclonal antibody therapy. Anti-idiotypic and non-anti-idiotypic antibodies to OKT3 arising despite intense immunosuppression

The frequency, timing, and specificity of the humoral antibody response to a murine monoclonal antibody (OKT3, IgG2a) were measured in 21 consecutive renal allograft recipients. These patients received i.v. OKT3, 1-5 mg/day for 10-20 days as treatment for acute graft rejection. Maintenance immunosuppression consisted of azathioprine and corticosteroids. Using three different assays, an antibody response was detected in 75% of the 20 patients with adequate samples. The ELISA assay of the overall IgM and IgG reactivity to OKT3 revealed that IgM anti-OKT3 appeared in 65% and IgG anti-OKT3 in 50% of the patients, reaching a peak 20-33 days after the last dose of OKT3. The IgM preceeded the IgG in most cases (P less than 0.02) and in 8 cases was detected during therapy. One patient had high levels of IgM anti-OKT3 before therapy, yet responded normally to OKT3. Interference with the therapeutic effectiveness was evident in one patient who developed IgG antibodies during therapy. His serum blocked the binding of F-OKT3 to normal lymphocytes in the presence of normal BALB/c serum. The blocking assay, done by flow cytometry, measured anti-idiotypic (Id) reactivity since the sera did not affect the binding of OKT8 (another IgG2a) or anti-Leu4 (another anti-T3), and the blocking activity remained after affinity absorption with normal mouse IgG. Using this assay, 60% of the patients made an anti-Id response. One made only anti-Id, and several had anti-Id at times when other reactivities were undetectable. Antibodies to non-idiotypic, presumably isotypic, determinants represented on OKT8 occurred in only 44%, while other reactivity (OKT4; IgG2bK) was less common (12%) and weaker. While no adverse allergic reactions occurred in this group of patients, the anti-Id antibodies, which are a prominent feature of the immune response to this and probably other monoclonal antibodies, can block their therapeutic effectiveness and can arise despite intense immunosuppression. This response may require the use of different idiotypes for prolonged or repeated courses of therapy and may be the major obstacle to the use of human monoclonal antibodies.     

Identification of swine and primate cellular adhesion molecules (CAM) using mouse anti-human monoclonal antibodies

Abstract: A series of adhesion molecules of swine and Cynomolgus monkeys were identified by screening for cross-reactivity with a panel of monoclonal murine anti-human adhesion molecule antibodies obtained from the 5th International Workshop and Conference on Human Leukocyte Differentiation Antigens (Boston, MA, USA, 1993). Of 162 antibodies tested, 25 were found that cross-react significantly with swine cells, while 67 were found to react strongly with cells of Cynomolgus monkeys. Cross reactivities to swine were found with antibodies to CD 18 (β2 integrin), CD29 (β1 integrin), β7 integrin, CD49d (α4 integrin) CD49b(α2 integrin), and to a lesser extent to CD62p(P-selectin), CD62L(L-selectin), CD102(ICAM-2), CD11b, and CD49c (α3 integrin). Cross-reactivities to primate cells also included CD18, CD29, CD49d, and CD49b. In addition, reactivities to Cynomolgus monkey cells were detected with antibodies to CD11 (a, b, and c), CD31, CD44, CD49e, CD49f, CD50, CD54, CD56, CD62p, CD 102 and CD56. The tissue distribution and molecular weight of the swine antigens are similar to their human counterparts. These findings provide a spectrum of monoclonal antibodies that react with shared epitopes on homologous adhesion molecules of human, swine, and monkey cells, thus facilitating study of the role of these molecules in the immunobiology of monkeys and swine. These reagents may be useful to dissect the role of adhesion molecules in both alio- and xenoreactivity.