Characterization of metastasis-associated antigens on RAW117 lymphosarcoma cell lines.

A syngeneic murine model system was used to study the immunobiology of metastasis. The highly malignant RAW117-H10 cell line was compared to the less malignant parental RAW117-P cell line from which it was derived, for expression of cell surface antigens. Using rabbit antisera, two major glycoprotein antigens were detected on the tumor cell surfaces. Antigen-I was uniformly distributed over the surface of these cells whereas antigen-II had a patchy, punctate distribution. Antigen-I was displayed less on RAW117-H10 cells than on RAW117-P cells, while the expression of the other serologically distinct antigen (antigen-II) was increased on RAW117-H10 cells compared to the less malignant parental (RAW117-P) cells. This differential antigen expression was assessed by immunodiffusion, a 125I-labeled protein-A binding assay, flow cytometry and rocket immunoelectrophoresis. Both these antigens had a molecular weight of 70,000 daltons. Antigen-I bound the lectin concanavalin-A whereas antigen-II did not, suggesting that antigen-I might be the viral envelope glycoprotein gp70. The identity of antigen-II is presently unknown. Syngeneic Balb/c mice injected with highly malignant and metastatic RAW117-H10 cells coated with antiserum to antigen-I were protected from early death; this effect was not seen with RAW117-H10 cells coated with antiserum to antigen-II. The opsonizing qualities of these antisera may be different due to antibody to antigen-II being shed more rapidly than antibody to antigen-I.

Enhanced antiproliferative activity by metastatic RAW117 lymphoma cells.

The highly malignant/metastatic murine large cell lymphoma cell line RAW117-H10 forms 100-200 times more liver metastatic tumors than its parental counterpart cell line RAW117-P. RAW117-H10 cells, but not the less malignant/metastatic parental cells, significantly inhibited the mitogen-induced proliferation of normal syngeneic Balb/c and allogeneic ICRC mouse spleen cells. Such an inhibition also occurred when mitomycin-C treated metastatic lymphoma cells were added 24 h after initiation of culture, indicating that no competition with mitogen binding sites on the lymphocytes was necessary for inhibition of proliferation. 'Antiproliferative' cell surface molecules were extracted non-cytolytically from the RAW117-H10 cells using butanol. The butanol extracts from the metastatic RAW117-H10 cells also inhibited the mitogen-induced proliferation and natural killer (NK) cell-mediated cytotoxicity of normal spleen cells. Our results indicate that these 'antiproliferative' cell surface molecules of metastatic murine RAW117-H10 lymphoma cells may have important role(s) in tumor-mediated host immunosuppression.

Production and preliminary characterization of monoclonal antibodies to Actinobacillus sp isolated from epididymitis lesions in a ram.

A panel of 55 monoclonal antibodies to an Actinobacillus sp isolate (As8C strain) cultured from the epididymides of an infected ram was produced. Cell lines producing 5 of these antibodies were cloned and expanded by hybridoma tumor production in Balb/c mice. An isotype profile revealed that 1 cloned antibody belonged to the immunoglobulin (Ig) M class and the 4 remaining antibodies belonged to the IgG class. Within the IgG class, 1 clone produced IgG1, 1 clone produced IgG2a, and 2 clones produced IgG2b. Ascites fluid antibody titers from the cloned hybridomas ranged from 6,400 to 51,200, as determined by enzyme-linked immunosorbent assay. Specificity of the antibodies to target As8C antigens could be demonstrated by enzyme-linked immunosorbent assay inhibition. Ascites fluid from 2 clones contained antibodies that agglutinated As8C. Two additional clones produced antibodies capable of only partial agglutination, whereas 1 clone produced antibody that did not agglutinate As8C. The indirect fluorescent antibody test revealed that target antigens for at least 4 of the 5 monoclonal antibodies were most likely located on the bacterial cell surface. Antigens were extracted from As8C, using 5 surface active chemicals. An attempt to immunoprecipitate these antigens in agarose by reacting individual extracts with each of the antibodies was unsuccessful.

Use of monoclonal antibodies to identify outer membrane antigens of Actinobacillus species.

Three monoclonal antibodies (LG17, LG30, and LG33) were used to identify outer membrane antigens of Actinobacillus sp (As8C isolate) cultured from the epididymides of an infected ram lamb. Specificity of the 3 antibodies to As8C antigens was determined by use of bacterial agglutination, the enzyme-linked immunosorbent assay, and the indirect fluorescent antibody test. Results of immunoelectron microscopy confirmed that each antibody was specific for epitopes on As8C outer membrane antigens. Evaluation by use of enzyme-linked immunoelectrotransfer blot indicated that target antigens for LG17 and LG33 antibodies had molecular weights of 10 kilodaltons and 43 kilodaltons, respectively. Multiple-band staining was observed with the LG33 antibody. The target antigen for the LG30 antibody could not be discerned by use of enzyme-linked immunoelectrotransfer blot. For each of the 3 monoclonal antibodies, enzyme-linked immunosorbent assay titers were obtained for Actinobacillus seminis, A actinomycetemcomitans, and 10 field isolates of Actinobacillus spp. Target antigens for LG17 and LG30 antibodies occurred infrequently or were absent on these bacteria. However, the target antigen for the LG33 antibody was shared by Actinobacillus seminis, A actinomycetemcomitans, and the 10 field isolates of Actinobacillus spp, indicating some diversity of outer membrane antigens between isolates.

Helper function of rat spleen cells reactive with guinea pig serum.

Treatment of rat spleen cells with normal guinea pig serum (GPS) has been shown to produce a significant loss of responsiveness to stimulation with either Con A or PHA. This phenomenon was seen even when the spleen cells were triggered with mitogens up to 96 hr after treatment with GPS, suggesting that GPS had produced a long-lasting alteration in some cells or removed a cell subpopulation. Glass-wool non-adherent spleen cells, known to produce greater responses that unfractionated spleen cells, also had their responses to Con A and PHA reduced by GPS treatment though the response was still greater than that of untreated, unfractionated cells, suggesting that the actual responder cells had been spared by GPS. Suppressor cells did not appear to be the target of GPS because such an effect would have resulted in increased responsiveness and the opposite result was obtained. That a helper cell was affected by GPS was suggested by the following observations: a) the virtually unresponsive GPS-treated spleen cells produced greater than normal responses after removal of glass-wool-adherent suppressor cells; b) the response of glass-wool-nonadherent spleen cells was significantly decreased after GPS treatment; and c) mixtures containing equal numbers of glass-wool-nonadherent and GPS-treated spleen cells also showed reduced responsiveness after GPS treatment.

Immunological properties of peripheral rat lymphocyte subpopulations reacting selectively with guinea pig complement.

Functional properties of peripheral rat T lymphocytes selectively affected by reaction with guinea pig serum (GPS) complement (C) were studied in this work. Cells sensitive to GPS cytotoxicity represented 2-7% of the nucleated cells in the spleen and 1-4% in lymph nodes. Responses of spleen and lymph node cells to Con A and PHA were markedly reduced following treatment with GPS whereas LPS-induced responses were not altered. GPS treatment also abrogated both the specific response of lymph node cells to a protein antigen and the production of leukocyte migration inhibitory factor by splenic cells. By contrast, GPS treatment significantly increased the reactivity of spleen and lymph node cells to alloantigens and enhanced the in vitro immune responsiveness of splenocytes to sheep erythrocytes. These results highlight the selective reactivity of guinea pig C with discrete subsets of immunoregulatory peripheral rat T lymphocytes.

H-Y antigen in human intersexuality.

The status of H-Y antigen was studied in 10 intersexual cases (three pure gonadal dysgenesis with XY genotype, three Klinefelter's syndrome, two true hermaphroditism with XX genotype, two male hermaphroditism) and in 18 normal adult subjects (nine males and nine females). In all these subjects, fluorescent staining and G-banding on chromosomes from cultured leukocytes confirmed their karyotype. Interestingly, H-Y antigen was found to be negative in XY females with dysgenetic gonads (PGD), while in the remaining intersexual states (viz Klinefelter's syndrome, true hermaphroditism and male hermaphroditism), it was found to be positive. These observations confirm that morphological differentiation of testis is controlled by H-Y antigen, and indicate that in the absence of the H-Y antigen, the gonad in pure gonadal dysgenetic patients (46, XY) could not be differentiated into testis, Further, it appears that H-Y antigen in no way influences the secretory function of testis.