From antibodies to sperm to antibodies in transplantation.

OBJECTIVES: a) To assess the use of "panning" procedures for the detection of antibodies to sperm. b) To develop and use human monoclonal antibodies for the analysis of human transplantation antigens. METHODS: "Panning" assays were developed. The procedures and their use in the analysis of mouse monoclonal antibodies to human sperm are described. B-lymphocytes from individuals sensitized by pregnancy, graft rejection, or blood transfusion were immortalized with Epstein-Barr virus (EBV). Selected sublines producing antibodies to human leukocyte series A (HLA) antigens were fused with mouse myeloma cells and selected clones of hybrid cells secreting monoclonal antibodies were selected. RESULTS: The results of panning assays correlated with those of agglutination assays, but not immunofluorescence or enzyme-linked immunosorbent assays (ELISA). Monospecific and cross-reactive human monoclonal antibodies to HLA antigens were produced. CONCLUSIONS: Panning assays can be used for the detection of antibodies to sperm and can be useful where target sperm are of poor motility. The assays may be more sensitive for antibodies to superficial antigens. Human monoclonal antibodies may be used for investigating the identity of, and responses to, transplantation antigens.

The RCP stroke audit package in practice.

This paper describes the use of the Royal College of Physicians 'stroke audit package' in an audit of the care of 156 patients. Recording of history and management compared favourably with that recorded on the RCP database. For a number of items, recording was significantly improved when a locally developed 'checklist', based on the RCP standards, was utilised by trainee medical staff. Benefits of the audit package, and proposed changes in practice, are briefly outlined.

Contribution of T-cell receptor-contacting and peptide-binding residues of the class II molecule HLA-DR4 Dw10 to serologic and antigen-specific T-cell recognition.

The relative contributions of putative T-cell receptor (TCR)-contacting and peptide-binding residues of a major histocompatibility complex (MHC) class II restriction element to serologic and antigen-specific T-cell recognition were investigated by site-specific mutagenesis. Amino acids 70 and 71 in the DR beta 1 domain of DR4 Dw10 are uniquely differnet from the other Dw subtypes of DR4. Residue 70 is predicted to be located at the membrane-distal surface of the class II molecule, where it may influence T-cell recognition by a direct interaction with a TCR. Residue 71 is predicted to form part of the antigen-binding groove where its influence on T-cell recognition may be mediated indirectly via an effect on peptide binding. Transfected murine L cells were produced expressing the products of DR4 Dw10B genes in which the codons for residues 70 and 71 had been mutated towards DR4 Dw14. Support for the predicted orientations of beta-chain residues 70 and 71 was lent by the observation that only residue 70 plays an important role in the formation of a serologic determinant. Mutation of this residue was sufficient to produce recovery of recognition by a human monoclonal antibody, NI, which has specificity for all the DR4 subtypes with the exception of DR4 Dw10. The human T-cell clone HA1.7, specific for influenza virus hemagglutinin (HA) peptide 307-319 and restricted by DR1 Dw1, exhibits degeneracy of MHC restriction on the DR4 Dw subtypes with the exception of DR4 Dw10.(ABSTRACT TRUNCATED AT 250 WORDS)

Production of a cytotoxic human monoclonal antibody with specificity for HLA-DR4 and -DRw10 by cells derived from a highly sensitized kidney recipient.

A human monoclonal antibody which reacts preferentially with HLA-DR4 and -DRw10 B-cell targets has been produced. A human B-cell line, secreting antibody which reacted preferentially with DR4 and DR1 targets, was derived from a highly sensitized kidney recipient who had rejected two grafts. This line was fused with the mouse myeloma P3X63Ag8.653 and a selected hybridoma cloned. The clones secrete IgM(lambda), which reacts strongly with HLA-DR4 and -DRw10 and more weakly with -DRw14 and a proportion of -DR1 B cells in cytotoxicity assays. Using B-cell lines as targets in cytotoxicity and enzyme-linked immunosorbent assays, the antibody gives a broader pattern of reaction, reacting with HLA-DR1, -DR4, -DR9, -DRw10, -DRw14, and some -DR2 targets. The antibody (NI) is currently in use as a reagent for tissue typing.

MicroELISA assays of anti-HLA activity and isotype of human monoclonal antibodies.

Both monoclonal human antibodies to HLA-DR antigens and supernatants from oligoclonal B-cell lines can be conveniently screened for activity by microELISA assays which use 1/10 of the volume of reagents used in conventional ELISA assays. Target cells are fixed to the bases of wells in Terasaki plates, 5 microliters volumes of supernatants incubated in these wells, and target bound antibody detected by peroxidase-conjugated anti-immunoglobulin followed by the substrate ABTS(2,2'-azino-di-(3-ethylbenzthiazoline sulphonate). The plates are read on a micro EIA reader. Supernatants can also be assayed for immunoglobulin content and isotype in Terasaki plates by coating the wells with isotype-specific anti-immunoglobulin, adding test supernatant and developing with appropriate peroxidase-conjugated anti-immunoglobulin sera and ABTS. When assaying for immunoglobulin content, the plates can be read either with a reader or by eye. Advantages and modifications of these procedures are discussed. There are no apparent practically important disadvantages to these procedures as compared with more conventional ELISA assays.

Production of monoclonal human antibody to HLA-DR5 (DRw11) by mouse/human heterohybridomas.

We describe here the production of a human monoclonal antibody to the HLA-DR5 antigen. A human B-cell line secreting cytotoxic antibody that reacted preferentially with DR5-positive targets was fused to the mouse myeloma P3X63Ag8.653 and the resulting heterohybridomas cloned twice. The clones secreted human IgM (lambda light chain), which showed specificity for the DR5 antigen in cytotoxicity assays and reacted with DRw11-positive but not DRw12-positive targets. These results demonstrate the potential of this approach to the production of human monoclonal antibodies to transplantation antigens.

Expression of polymorphic B-cell antigens on human kidneys.

We have examined the expression on a panel of 22 human kidneys of polymorphic B-cell determinants recognized by mouse monoclonal antibodies. Monoclonal antibodies from a mouse immunized with an antigenic preparation from a DR4 positive B-cell line reacted preferentially with kidneys from DR4 positive donors (p less than 0.005), and the pattern of reactivity with kidney tissues was similar to that of antibodies to monomorphic determinants of class II. However, these antibodies did not show clear specificity for DR4 on lymphocytes in standard serological analyses. These results provide evidence for the expression of polymorphic class II determinants on human kidneys. Reasons for the differences in the apparent specificities of the monoclonal antibodies when tested on kidney sections and lymphocytes are discussed.

Assessment of immune responses to H-Y antigen in naturally inseminated and sperm-injected mice using cell-mediated cytotoxicity assays.

An in vitro cell-mediated cytotoxicity assay has been used to assess immunity to the male-specific H-Y antigen in female mice after either insemination with male cells at syngeneic natural mating or after injection with syngeneic sperm cell suspensions. Lymphocytes showing specific cytotoxicity for the H-Y antigen could be recovered from both spleen and lymph nodes of female mice injected with sperm. However, insemination of male cells at natural mating did not apparently prime cytotoxic cells against the H-Y antigen in either the spleen or para-aortic lymph nodes draining the uterus of female mice mated once or repeatedly (3-10 X) in the absence of pregnancy. These results are discussed in relation to the factors regulating the immune responsiveness of the female to inseminated antigens.

In vitro production of anti-HLA-DR antibodies by human B-cell lines.

Human B-cell lines secreting antibodies which react preferentially with the HLA Class II antigen DR5 have been produced. Supernatants from these cell lines reacted with lymphocytes from all DR5 positive donors and a minority of DR6 positive donors but were negative on lymphocytes of other phenotypes. The cytotoxic activities of the supernatants apparently depend on IgM antibody. These results demonstrate the potential of B-cell lines for the in vitro production of antibodies to HLA-DR antigens.

Increases in the numbers of immunoglobulin-secreting cells in lymph nodes responding to sperm and other stimuli: possible relationship to immunosuppression.

It has been reported that, in early pregnancy in mice, there is an increase in the number of immunoglobulin-secreting cells in the lymph nodes which drain the uterus. This paper describes the results of further investigations provoked by interest in these early changes. Increases in the numbers of immunoglobulin-secreting cells were observed in syngeneically, but scarcely or not at all in allogeneically, mated mice. Increases were not observed in surgically sterilized female mice inseminated by normal males. However, subcutaneous injection of sperm provoked massive increases in the numbers of immunoglobulin-secreting cells in the lymph nodes draining the injection site. The changes were compared with those provoked by the injection of spleen cells and LPS. The results are discussed in relation to the nature of the interactions provoking the increases in the number of immunoglobulin-secreting cells and their possible relationship to immunosuppression, and the relative immunological unresponsiveness which the female shows to the challenge of inseminated sperm.

Detection of anti-sperm activities of monoclonal antibodies to human sperm.

The anti-sperm activities of a series of monoclonal antibodies to human sperm have been compared using agglutination, immunofluorescence, ELISA and 'panning' assays. The antibodies fell into two categories, those that could be detected by agglutination but not immunofluorescence assays and those that could be detected by immunofluorescence but not agglutination. Antibodies positive in the agglutination assays were also positive in the 'panning' assay. None of the antibodies tested was positive in the ELISA assays. These results, and others, are discussed in relation to the problems associated with the detection of anti-sperm antibodies in sub-fertile human populations.

B-cell responses to male-specific antigen(s) in mice.

It has been found that B-cell responses to male-specific antigen(s) can be clearly demonstrated by reversed plaque assays. Female mice injected with syngeneic male spleen cells showed significant increases (greater than 100 X in some strains) in the number of immunoglobulin-secreting cells in lymph nodes draining the injection site. There was a variation in B-cell responsiveness between strains and this correlated only partially with previously reported T-cell responsiveness to the H-Y antigen. C57BL (H-2b) mice were among the most responsive, while CBA (H-2k), (CBA X C57BL)F1, and BALB/c (H-2d) were all much less responsive. These results apparently open up a new approach to the investigation of B-cell responses to male-specific antigen(s).

Detection of antibody-coated sperm by 'panning' procedures.

The use of 'panning' procedures to detect immunoglobulin on the sperm surface are described. Wells on plastic plates are coated with anti-immunoglobulin molecules by either 1-step or 2-step procedures and the sperm under test are then incubated in these wells for up to 1 h and the wells washed. Antibody-coated sperm remain attached in large numbers while control sperm are washed out. These procedures have the advantages that they are cheap, simple, do not involve sperm fixation and may be used with relatively dilute cell suspensions and with sperm of low motility. The potential applications of the procedures are discussed.

Failure of spleen cell migration assays to detect cell-mediated immunity to spermatozoa after natural mating in female mice.

We have re-examined whether immunity to spermatozoa can be detected by spleen cell migration assays in inseminated female mice. Unlike previous results, however, sperm suspensions inhibited the migration of splenic leucocytes obtained from mated as well as control mice. Small differences were observed between leucocytes from untreated virgin mice and leucocytes from mice immunized with spermatozoa in adjuvant. In these experiments lymph node cells draining the injection site were added to the spleen cells.

Reactions of antisera to HCG with living human spermatozoa.

Antisera to hCG were incubated with living human spermatozoa. Agglutination and complement-dependent immobilization of the spermatozoa occurred with the anti-hCG sera but not with control sera. The antisera also reacted with human blood cells. The anti-sperm activities were largely removed by absorbing the sera with human blood cells, although these absorptions had no apparent effect on the anti-hCG activities measured by passive haemagglutination. There was, therefore, little evidence for the presence of hCG-like molecules at the surface of living human spermatozoa.

Detection of sperm surface IgG by modified haemadsorption procedures.

New haemadsorption procedures for the detection of sperm bound IgG are described. Antibody-coated sperm are fixed with formaldehyde and attached by poly-L-lysine to the bases of wells on plastic plates where they can be left for several days if necessary. Sensitised indicator red cell (for direct haemadsorption) or anti-IgG sera followed by indicator red cells (for indirect haemadsorption) are then added to these wells and the presence of antibody indicated by red cell attachment to sperm. The procedure uses simple reagents and the sperm/red cell rosettes may be fixed and stained to be kept as records.

Glucagon therapy in acute pancreatitis: prospective randomized double-blind study.

The known suppressive actions of glucagon on the secretion of pancreatic enzymes and of gastric acid, and the reported effectiveness of glucagon in treating acute pancreatitis, prompted the authors to carry out a prospective, randomized, double-blind, controlled trial of this hormone. Sixty-six patients with acute pancreatitis admitted to the surgical service of the Vancouver General Hospital were randomized into two groups of 33, receiving either glucagon or placebo in addition to their conventional therapy. The two groups were comparable with respect to the cause of the pancreatitis and the severity of the disease. Glucagon did not reduce the patients' analgesic requirements or the duration of abdominal signs, ileus, hyperamylasemia or hospital stay. Of the 66 patients, 4 died. Three of these were in the group receiving glucagon. The authors conclude that, contrary to theoretical expectations and the results of past uncontrolled trials, glucagon has no beneficial effect on the clinical course or outcome of acute pancreatitis, irrespective of the etiology or severity of the disease.