Serum IgA antibodies to human gut luminal aspirates and human liver in alcoholic liver disease.

BACKGROUND: Elevation of serum IgA is a characteristic feature of alcoholic liver disease. It has been proposed that this occurs partly as an antigenic response to gut-derived proteins or acetaldehyde-modified liver proteins, but the principal antigens responsible remain unknown. AIMS: The goal of this study was to determine if serum IgA antibodies were present against human gut luminal antigens or liver antigens in alcoholic liver disease. PATIENTS AND METHODS: Twenty-nine patients with alcoholic liver disease, 10 with primary biliary cirrhosis, 12 with "other" liver diseases, 8 alcoholics, and 20 healthy subjects were studied. Western blotting was used to examine the reactivity of sera from these groups against human small and large bowel aspirates and liver tissue from alcoholic liver disease patients. RESULTS: Serum IgA antibodies to a 140 kDa colonic luminal protein were found in 22 (76%) patients in the alcoholic liver disease group (p < 0.0001), and 7 (24%) patients had serum IgA antibodies to a 40 kDa colonic luminal protein (p = 0.04). These responses were confined to colonic aspirates and not observed in other disease groups, alcoholics or healthy subjects. There was no significant serum IgA response to human liver proteins in alcoholic liver disease. CONCLUSIONS: Serum IgA antibodies to a human 140 kDa colonic luminal protein are frequently found in alcoholic liver disease. This novel antigen may contribute to the increased levels of circulating IgA in alcoholic liver disease.

Characterisation of a sperm coating auto-antigen reacting with antisperm antibodies of infertile males using monoclonal antibodies.

OBJECTIVE: In a previous study a number of sperm-specific antigens were identified which reacted with antisperm antibodies from both infertile and vasovasostomised males. To investigate the localisation and distribution of these antigens and their role in male fertility, monoclonal antibodies were raised against them; immunoblotting techniques were used to select only those antibodies which competed with human antisperm antibodies for these human auto-antigens. DESIGN: One antibody, NW21, reacted with an 18 kDa auto-antigen present on epididymal sperm but absent from testicular sperm. Immunohistochemical studies showed that the antigen is produced in small basal cells between the columnar epithelium of the corpus epididymis, passes up into the tubule and then coats sperm passing along the epididymis. Sperm stored in the cauda epididymis and ductus deferens stain strongly for this sperm coating glycoprotein. CONCLUSIONS: The localisation of this antigen supports the suggestion that auto-immune infertility may represent a response to epididymal rather than testicular sperm. Monoclonal antibodies raised to unique and immunologically accessible sperm coating proteins, produced in the epididymis rather than in the testis, would seem to present an excellent theoretical solution to male contraception.

Panel of anti-gp120 monoclonal antibodies reacts with same nuclear proteins in uninfected cells as those recognized by autoantibodies from patients with systemic lupus erythematosus.

Mouse monoclonal antibodies (MAbs) directed against the HIV-1 gp120 envelope protein were screened for their reactivity with intracellular antigens expressed in normal uninfected monocytes by indirect immunofluorescence and immunoblotting. Some of these MAbs reacted with the nuclei of normal uninfected cells, producing three main staining patterns by indirect immunofluorescence. Western blot analysis showed that these monoclonal antibodies recognize peptides of various molecular weights present in nuclei preparations of normal monocytes. Reactivity with peptides of similar molecular weight was also detected in sera from both HIV-infected individuals and patients with systemic lupus erythematosus. This evidence for antigenic similarities between HIV-1 gp120 and nuclear antigens represents a novel example of molecular mimicry of self-antigens by HIV envelope proteins, which supports the involvement of mechanisms of autoimmunity in HIV disease pathogenesis through recruitment of autoimmune responses to self-structures by HIV antigens.

Characterization of the circulating T-cell response after oral immunization of human volunteers with cholera toxin B subunit.

The kinetics and phenotypic characterization of the in vitro cell proliferative response to the B subunit of cholera toxin were studied using peripheral blood mononuclear cells taken from human volunteers at frequent time points after primary and booster oral immunizations. The cells induced to proliferate by oral immunization secreted IL-3, and lipopolysaccharide depletion and depletion of B cells did not affect proliferation. Flow cytometry demonstrated that activated cells were CD3- and CD4-positive. These findings indicate primed T cells proliferating specifically to the B subunit. The kinetics of the response suggested trafficking in the peripheral circulation of primed T cells from the gut, with a peak stimulation index of between 7 and 93 after first immunization, and a precursor frequency of primed cells of between 1 in 25,400 and 1 in 72,390. There was close correlation between the serum antitoxin IgA antibody levels and observed proliferation.

Circulating cellular immune response to oral immunization of humans with cholera toxin B-subunit.

Peripheral blood mononuclear cells were taken from subjects before and after oral immunization with cholera toxin B-subunit. Cells obtained from naive volunteers before immunization did not proliferate in vitro to B-subunit. Oral immunization induced a proliferative response in all volunteers with a peak stimulation index of 20, and was detected up to 1 year later. The proliferative response kinetics suggest the appearance in the blood of primed T cells from the gut coinciding with the disappearance of primed plasmablasts from the circulation, supporting the concept of a common mucosal immune system in man for T and B cells.

Cytofluorometric analysis of anti-lymphocyte antibodies in AIDS.

Anti-lymphocyte antibodies (ALA) have been detected in the plasma of 53.8% of HIV-positive patients tested (CD4/CD8 ratios: mean 0.265; range 0.01 to 0.5) using analytical continuous-flow cytofluorometry. IgG from the AIDS plasma was seen to bind to normal PBL in 53.8% of cases (14/26). In double labelling experiments CD4 + lymphocytes, CD8 + lymphocytes, and B lymphocytes were all bound by the ALA, but monocytes were not bound. Pre-adsorption of the diluted AIDS plasma onto an excess of mouse spleen cells did not remove lymphocyte binding activity. No evidence was found for preferential binding to phytohaemagglutinin-stimulated lymphocytes. ALA could not be detected in the plasma of normal subjects, patients with acute renal failure undergoing renal dialysis, or patients with high levels of circulating immune complexes.

Immunobiological studies on the alopecic (DEBR) rat.

The Dundee experimental bald rat (DEBR) has been proposed as an animal model of human alopecia areata, which is suspected of being an autoimmune disease. This study was carried out to establish whether the immunological changes observed in the lesional DEBR rat correlated with studies of human alopecia areata. The immune infiltrate was characterized using immunoperoxidase techniques on cryostat sections of vibrissa follicles. Indirect immunofluorescence was used to quantify the peripheral blood leucocytes. Some parallels were observed in the infiltration of human and DEBR rat follicles by T lymphocytes. In contrast, pre-lesional DEBR rat follicles, which are not available for investigation in human alopecia areata, were not penetrated by leucocytes and MHC class II antigens were expressed in the precortical region of the epidermal component of these follicles. Quantification of peripheral blood leucocytes showed significant increases in both T-lymphocyte subsets during lesional expression. We consider that the pre-lesional form of the rat may provide important information as a model for the pre-lesional and uninvestigated form of alopecia areata in man.

Suppress of the increase in free cytosolic calcium during the inhibition of T-cell activation by an autoantibody present in the serum of leprosy patients.

A serum factor, believed to be an IgG autoantibody, in certain patients with lepromatous leprosy inhibits the proliferation of mitogen-stimulated lymphocytes. To investigate which stage of the cell cycle was inhibited, we examined the effect of these sera on the kinetics of lymphocyte activation induced by several mitogenic agents: phytohaemagglutinin (PHA), the calcium ionophore A23187, the phorbol ester phorbol myristate acetate (PMA) and purified protein derivative of BCG (PPD). Seven out of 54 sera tested were found to inhibit PHA-stimulated proliferation. Inhibitory sera and to a lesser extent serum IgG from leprosy patients were capable of suppressing the increase in free cytosolic calcium normally observed immediately after PHA stimulation. Subsequent stages of the cell cycle, increase in cell size, the expression of the IL-2 receptor and increase in DNA were also suppressed. The inhibitory sera was not toxic and, if addition of the sera was delayed, would not inhibit lymphocytes that had already entered the cell cycle. Using mitogenic agents which act intracellularly, the normal early increase in cell size with A23187- and PMA-stimulated lymphocytes was not affected by inhibitory leprosy sera or serum IgG, but all subsequent steps in the cell cycle were suppressed; although the inhibition of proliferation in PMA-stimulated cultures was incomplete. The mechanism of action of the inhibitory sera and derived IgG, although acting through a cell surface antigen, appears to interfere with a fundamental process in activation since the effect was seen with all of the diverse stimuli examined in this study.

Changes in activation markers and cell membrane receptors on human peripheral blood T lymphocytes during cell cycle progression after PHA stimulation.

Phytohaemagglutinin (PHA)-stimulated peripheral blood lymphocytes were examined sequentially for changes in volume, the appearance of cell membrane receptors and nucleic acid synthesis. The kinetics of appearance of activation antigens were compared with the progress of the cell through the separate events of volume growth and nucleic acid syntheses, to determine points at which regulation of receptors may control further progress through the cell cycle. In all samples tested there was a consistent pattern of response in the proportion of cells progressing through the cell cycle. Most of the T cells increased in size (mean 82% at 24 hr), fewer cells entered the Gla/Glb phase with the onset of RNA synthesis (mean 68% at 48 hr) and even fewer entered DNA synthesis (mean 42% at 72 hr). The time-course of appearance and the number of cells expressing IL-2 receptors were almost identical with that of cells responding by RNA synthesis. A similar correlation was observed between expression of the transferrin receptor and DNA synthesis. Addition of anti-Tac antibody temporarily suppressed the onset of RNA synthesis and antibodies to the transferrin receptor suppressed DNA synthesis. These linkages are further evidence that IL-2 and transferrin are the specific signals for cellular RNA and DNA synthesis. With optimal concentrations of PHA, addition of IL-2 did not increase the proportion of cells bearing activation antigens or undergoing nucleic acid synthesis. Suboptimal concentrations of PHA produced a small reduction in the number of cells expressing the IL-2 receptor, but a much greater reduction in the rate of entry into RNA synthesis. There was a consistent increase in all activation parameters tested with the addition of IL-2, but the proportion of cells expressing the transferrin receptor and entering DNA synthesis was consistently lower than that of cells that expressed the IL-2 receptor or entered RNA synthesis. This suggests that regulation of the IL-2 receptor is not responsible for the reduction in the number of cells that proceed to proliferation. The CD2 antigen (T11(1] showed increasing expression in a step-wise fashion after activation, the increases coinciding with the onset of RNA and DNA syntheses.

Characterization of sperm antigens reacting with human antisperm antibodies.

Antibodies reacting with human spermatozoa have been detected by various immunological techniques in the sera of subfertile men. Different patterns of sperm agglutination are observed with different sera, either head-to-head, tail-to-tail, or tail-tip-to-tail-tip. Differences have been detected between the clinically relevant antibodies in spontaneously infertile males and the less important antibodies in males who have undergone reversal of vasectomy. It has been suggested that the variations in agglutination patterns are due either to different classes of antibody or to binding of antibody to different antigens. In the present study immunoblotting techniques were used to characterize the reactivity of solubilized sperm proteins with serum samples exhibiting different modes of sperm agglutination. This involved the electrophoretic transfer of proteins from SDS gels to nitrocellulose sheets followed by overlay with serum antibody. Using these techniques we have attempted to characterize the antigens of spermatozoa which react with sera from both spontaneously infertile and vasovasostomized men. The results showed that although antisperm antibodies bind to discrete and sperm-associated antigens, there is no substantial difference between the antigenic patterns observed with antibodies producing different types of sperm agglutination. Neither the antigens detected, nor the intensity of reaction showed significant differences although there was a tendency for head-to-head agglutinating antibodies to react more strongly with the higher molecular weight antigens. Moreover, although with sequential serum samples the patterns of agglutination may change, the antigenic pattern remains unchanged.

The clinical relevance of classes of immunoglobulins on spermatozoa from infertile and vasovasostomized males.

Following reversal of vasectomy, conceptions occur even when antisperm antibodies are present in the seminal plasma, but this is most unusual in men with similar titers of such antibodies who are spontaneously infertile. To clarify the differences between antisperm antibodies occurring in infertile men and those associated with vasectomy reversal, we have studied 23 spontaneously infertile men and 22 men who underwent vasectomy reversal, all of whom had antisperm antibodies detected in seminal plasma by the same tray agglutination test. The class of antibody on spermatozoa was defined by a double-antibody technique using diluted rabbit anti-human IgG, IgM, or IgA or secretory component, followed, after washing, by 125I-labeled donkey anti-rabbit Ig. The results have shown that similar amounts of IgG and IgM were present on the spermatozoa, but infertile men had significantly more IgA and especially more secretory component than men who underwent vasectomy reversal. This was associated with significantly greater impairment of penetration of cervical mucus in the former group. It appears that the type of antibody on the spermatozoa may vary according to the stimulus for its production.

PIP: To clarify the differences between antisperm antibodies occurring in spontaneously infertile men and in men who have undergone reversal of vasectomy, classes of immunoglobulins were compared in 23 men from the former group and 22 men from the latter group. Antisperm antibodies were detected in seminal plasma by the same tray agglutination test in all subjects. The class of antibody on spermatozoa was defined by a double-antibody technique that used diluted rabbit antihuman IgG, IgA, or secretory component, followed, after washing, by 125I-labled donkey antirabbit Ig. Although similar amounts of IgG amd IgM were present on the spermatozoa in both groups, infertile men had significantly more IgA and especially more secretory component than men who underwent vasectomy reversal. This was associated with significantly greater impairment of penetration of cervical mucus in the former group. 8 of the 20 vasovasectomized men with antisperm antibodies in their seminal plasma produced pregnancies, whereas no pregnancies have been observed in 63 spontaneously infertile untreated men with seminal plasma antibodies. Spermatozoa from 86% of vasovasectomized men penetrated the cervical mucus, while spermatozoa from only 27% of spontaneously infertile men were able to do so. It is concluded that the type of antibody on the spermatozoa may vary according to the stimulus for its production.

Characteristics of immune complexes detectable by two independent assays in gynaecological malignancies.

Immune complexes from patients with ovarian and endometrial cancer have been examined using the C1q solid phase (C1qSP) and polyethylene glycol (PEG) precipitation assays. The amount of IgG in the PEG precipitates was inversely correlated with the amount of IgM whilst the IgM values correlated positively with the C1qSP assay values. Separation studies revealed that most of the C1qSP activity was not precipitated from cancer sera by 2% PEG. The immune complexes were fractionated on Sephacryl S-300. Both the PEG precipitation and C1qSP assays detected high molecular weight immune complex like activity (greater than 19S). The C1qSP assay also detected a second peak of activity at approximately 7-8S. Most of the PEG detectable immune complexes from sera dissociated during column chromatography, whereas the C1qSP detectable complexes were relatively stable. Furthermore the IgG from fractionated PEG precipitates emerged as a monomer (7S) component. The PEG assay appeared to be detecting a high molecular weight complex containing mostly IgG rather than IgM with a low affinity for C1q, which was easily dissociated. The C1qSP assay detected a more stable high molecular weight complex containing a relatively high proportion of IgM and a low molecular weight complex, both with a high affinity for C1q.

A comparative study of complement components in polyethylene glycol precipitated immune complexes from patients with ovarian cancer and patients with rheumatoid arthritis.

Circulating immune complexes and complement components C1q, C3 and C4 were measured following polyethylene glycol precipitation of serum from patients with ovarian cancer (OC), patients with rheumatoid arthritis (RA) and control patients (CP). The C3 and C4 content of the precipitated material was significantly greater in those patients with OC in comparison to those with RA or CP, while the C1q content did not differ significantly between the two groups of patients. A statistically significant correlation between IgG, C3 and C4 levels was found in the immune complexes of both groups of patients. C1q levels correlated significantly with IgG immune complex levels in patients with RA and CP, but did not in patients with OC. These data illustrate that immune complexes from patients with OC differ from complexes precipitated from RA sera and normal sera in their complement content. It is suggested that the largely unsuccessful attempts to detect immune complexes in sera from patients with ovarian cancer, using assays dependent on the binding of C1q may be related to differences in solubility of complexes from these patients in comparison to complexes present in non-neoplastic conditions.

An assessment of sequential measurements of immune complex levels in ovarian cancer patients with respect to clinical progress.

An evaluation of immune-complex levels as an additional parameter in the clinical follow-up of ovarian cancer patients is described. These patients were treated aggressively with monthly chemotherapy following surgery. Sequential measurements of immune-complex levels have been carried out concurrently and clinical findings were recorded in a uniform manner suitable for comparison with the levels of immune complexes. The relationships between changes in tumor mass and changes in levels of immune complexes were investigated. A significant relationship between decreasing immune-complex levels and simultaneously decreasing tumor mass was found. Chemotherapy was associated with a decrease in immune-complex levels which increased again within 1 month. These data did not support an effective role for immune-complex levels in the evaluation of these patients.

Sequential studies of circulating immune complexes in gynecological malignancies.

Immune complexes have been assayed sequentially in seventeen patients with gynecological malignancies, using a polyethylene glycol precipitation assay and a Clq solid phase assay. The PEG assay demonstrated a good correlation between PEG assay levels and the course of disease in nine out of eleven patients with ovarian adenocarcinoma and all four patients with endometrial carcinoma. Three of the patients with ovarian cancer were not treated with aggressive surgery and exhibited equivocal signs of progressive disease during subsequent follow-up. There was no clear relationship between immune complex levels and the clinical condition in two of these patients. The ClqSP assay result did not correlate with disease progression and, in several instances, progressive disease was associated with a fall in ClqSP values. In a further series, twelve out of twenty patients with ovarian cancer who were initially in remission from disease but subsequently relapsed demonstrated elevated levels of PEG immune complexes between one and three months before clinical detection of recurrence whereas patients who stayed in remission maintained normal levels of PEG immune complexes. These data suggest that the PEG assay may be of clinical value in the monitoring of ovarian cancer patients with minimal residual disease following surgery.

E-rosette forming cells and cell mediated immunity in ovarian cancer.

Parameters of cell mediated immunity were examined in 50 women with ovarian cancer, and included E-Rosette formation lymphocyte number, and lymphocyte blastogenesis to PPD. Alterations in these parameters were also determined following surgery and during chemotherapy. Relapse patients demonstrated significantly decreased numbers of "active" and "total" E-Rosette forming cells, as well as a significantly decreased spontaneous incorporation of 125Iododeoxyuridine into unstimulated lymphocytes; PPD responses and total lymphocyte counts were not significantly different from the control group. Analysis of T-cell subpopulations in relapse patients suggested that these cells were suppressed in the unseparated lymphocyte state. Chemotherapy led to an expected decrease in lymphocytes but an unexpected increase in the numbers of E-Rosette forming cells initially. It was postulated that serum factors accounted for the decreased values of these cells in relapse patients rather than a true depletion.

Trophoblastic proteins in ovarian carcinoma.

Trophoblast-"specific" proteins in the plasma of 37 patients with primary epithelial carcinoma of the ovary were measured. Circulating pregnancy-specific beta1-glycoprotein (SP1) was detected in 5 subjects, human chorionic gonadotrophin (hCG) was found in 2 subjects, and human placental lactogen (hPL) was detected in 8 subjects.

Immune complexes in ovarian cancer.

Immune complexes have been assayed in ovarian cancer with a polyethylene-glycol (P.E.G.) precipitation assay and a Clq solid-phase assay. Significantly higher levels of immune complexes were detected in patients in relapse compared with those in remission. Remission values were within the range obtained for a control group. Evidence was produced that immune-complex levels rise before the clinical detection of relapse, and it is suggested that the P.E.G. assay could be a useful marker of disease progress.

Active specific immunotherapy for ovarian cancer.

Ten patients with advanced or recurrent ovarian cancer were treated with a combination of chemotherapy and active specific immunotherapy after tumour stasis had been induced. They were inoculated with irradiated allogeneic cryopreserved tumour cells and B.C.G. once monthly in addition to receiving conventional chemotherapy. The overall duration of "remission", median survival, and projected 24-month actuarial survival in the patients receiving immunotherapy were apparently better than in a retrospectively matched control group treated by chemotherapy alone.