Regression of bovine ocular carcinoma by treatment with a mycobacterial vaccine.

Hereford cows with naturally occurring ocular squamous cell carcinoma were treated by injection of BCG cell-wall vaccine into the tumor. Regression or arrest of disease was observed in 71% of treated animals. The disease progressed in all untreated animals and animals treated with improperly compounded vaccine. At autopsy, most animals with progressive disease had lymph node metastases.

Efficacy of intratumorally administered mycobacterial cell walls in the treatment of cattle with ocular carcinoma.

Individual Hereford cows with naturally occurring ocular squamous cell carcinoma were treated by intralesional injections of mycobacterial cell walls in an oil-in-water emulsion. Six of 23 treated animals were alive (5 free of tumor and 1 with arrested disease) at a minimum of 2.5 years after treatment, as compared to 1 of 18 controls. A necropsy, lymph node metastases were found in most animals with progressive disease. The disease progressed to an advanced stage (which required death of the animals) at a faster rate for control animals than for treated animals.

Immunoprophylaxis of an ocular solid malignant tumor in cattle.

Individual Hereford cows bearing benign precursor lesions of ocular squamous cell carcinoma were treated by intralesional injection of mycobacterial cell walls in an oil-in-water emulsion in an attempt to interrupt neoplastic progression. Thirty-one months after treatment, statistical analysis of data indicated that intralesional BCG cell wall vaccine can interrupt this process and provides effective immunoprophylactic prevention of malignant disease.

A cell aggregation model for the protective effect of selenium and vitamin E on methylmercury toxicity.

Histotypic aggregation of embryonic neural retinal cells was chosen as a test model to evaluate mercury toxicity. After 24 h rotational culture with methylmercury (CH3HgCl) at 4 microM, aggregation was completely inhibited. A dose-response relationship between concentrations of methylmercury and final sizes of aggregates was found. Selenium (Na2SeO3) at concentrations of 1, 3 and 5 microM provided a protective effect for methylmercury (1 microM) toxicity. Vitamin E (DL-alpha-Tocopherol acetate) at concentrations 5, 7 and 10 microM also provided protection against the same concentration of methylmercury; however, it was less effective than selenium. Histotypic embryonal retinal cell aggregation may be a useful assay system for in vitro neurotoxic studies in morphogenesis.

Cadmium accumulation and subcellular distribution in relation to cadmium chloride induced cytotoxicity in vitro.

A bovine kidney cell culture system was used to assess what relationship cadmium (Cd) uptake and subcellular distribution had to cadmium chloride induced cytotoxicity. Twenty-four hour incubation with 0.1-10 microM Cd elicited 0-90% cytotoxicity. Fifty percent cytotoxicity was estimated to result from 0.8 microM Cd. A concentration-related Cd accumulation paralleled the cytotoxicity profile. The time-course for Cd accumulation was linear for the first 6 h of exposure and plateaued by 18 h post-exposure. When the degree of cytotoxicity was compared with the cellular Cd burden at 24 h post-treatment a least-squares linear regression analysis (r = 0.93) indicated a direct relationship. Subcellular distribution studies indicated greater than 90% Cd recovery from the soluble supernatant (105 000 g) at all levels of cytotoxicity studied. Metallothionein sequestered less than 25% of the cellular Cd. As a result of the correlation of the degree of cytotoxicity with the cellular Cd burden and the independence of subcellular distribution from cytotoxicity, a cumulative mechanism of toxicity for Cd in MDBK cells was suggested.

Detection of cell-surface antigens of bovine ocular squamous cell carcinoma.

A serologic study was conducted to identify surface antigens on cultured cells of bovine ocular squamous cell carcinoma. Sera from cattle with various stages of disease were tested for reactivity with surface antigens of cultured autologous and allogeneic cells. Radioiodine-labeled protein A assays were conducted to determine the presence of antibodies for tumor cells. It was found that all sera tested had antibodies at a high level to autologous cells, whereas the reactivity of allogeneic serum to cultured cells varied. Furthermore surface reactivity was not observed in these sera in tests for reactivity with normal epithelial cells. Reactive sera were analyzed by absorption tests with autologous and allogeneic cells. Absorbed sera showed no reactivity to surface antigens on autologous or allogeneic cells. Also, results of quantitative absorption studies indicated that absorption with precarcinoma cells eliminated the reactivity of sera from carcinoma- (cancer-) bearing animals toward cultured tumor cells. This indicates that there might be a shared antigen among the cells from precarcinoma and carcinoma lesions.

Comparison and partial characterization of the protein profiles and outer membrane antigens of Actinobacillus species isolated from ram lambs with epididymitis.

Three monoclonal antibodies (LG17, LG30, LG33) were used in the indirect fluorescent antibody test, the ELISA, and the immunoelectrotransfer blot technique to identify group-specific and strain-specific epitopes on the outer membranes of Actinobacillus seminis, A actinomycetemcomitans, and 17 field isolates of Actinobacillus spp. The field isolates had been obtained by bacteriologic culture of specimens from ram lambs with epididymitis. Only antibody LG33 consistently had specificity for an outer membrane epitope shared by most of the bacterial isolates tested. Staining of polyacrylamide gels with periodic acid-Schiff reagent, Sudan black B, and Coomassie brilliant blue R250 indicated that target antigens for antibodies LG17 and LG33 contained carbohydrate and lipoprotein components, respectively. The chemical composition of the LG30 target antigen was not determined because of its instability after exposure to sodium dodecyl sulfate. Discontinuous-gradient polyacrylamide gel electrophoresis in sodium dodecyl sulfate and spectrophotometric scans of the gels were used to analyze n-octyl-beta-D-glucopyranoside protein extracts from A seminis, A actinomycetemcomitans, and 13 representative field isolates of Actinobacillus spp. Bacterial isolates could be grouped according to their protein profiles. The first group consisted of A seminis, A actinomycetemcomitans, and 7 field isolates of Actinobacillus spp, all of which shared common protein bands with molecular masses of approximately 94 kilodaltons (kD), 64 kD, 60 kD, 52 kD, 44 kD, and 26 kD. The second group was composed of 6 field isolates, each with unique protein profiles; isolates had relatively few protein bands in common. These data suggested that members of the genus Actinobacillus cultured from ram lambs with epididymitis probably include a number of various strains.

Cryopreservation of bovine mononuclear leukocytes.

A technique of cryopreservation of bovine mononuclear leukocytes for use in lymphocyte stimulation tests and cell identification studies has been developed. Dimethyl sulfoxide was used as the cryopreservation agent. Cells were frozen to --40 C at a controlled rate of --1 C/minute and then to --80 C at a rate of --4 C/minute, and were subsequently stored in liquid nitrogen (--109 C). The cells were than recovered by rapid thawing in a 37-C water bath, diluted with tissue culture media, washed, and used for lymphocyte stimulation and cell identification tests. The magnitude of the lymphocyte blastogenic responses of frozen and thawed mononuclear leukocytes was similar to that seen with freshly collected cells. Additionally, percentages of T cells, B cells, and monocytes were similar between frozen and thawed cells and freshly collected cells.

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.

Cellular uptake and subcellular distribution of mercury in chick embryonic retinal cell aggregates.

Cellular uptake and subcellular distribution of mercury (203HgCl2) were determined in the chick embryonic retinal cell aggregation system. The accumulation of mercury was dependent upon its concentration in the medium. The uptake was rapid; a maximum deposition of mercury at 5 microM occurred within the first 30 min followed by a decline. Accumulation of mercury at 1 microM was constant between 15 min and 24 hr. The subcellular distribution of mercury was observed in the following order: nuclei and cell debris greater than mitochondria-lysosomes greater than 105,000g supernatant greater than microsomes. The activity of acid phosphatase markedly decreased in the aggregates treated with mercury at 50 to 100 microM for 24 hr. Low concentrations of mercury at 0.5 to 5 microM showed an inhibition of this enzyme activity in a cell-free system. The results indicate a relationship between the amount of mercury in the cells and the toxicity it produced on the retinal cell aggregation system.

Cytotoxicity of citrinin in cultured kidney epithelial cell systems.

Cytotoxicity of citrinin, a fungal metabolite and a common food contaminant, was evaluated in an established cell line, Madin-Darby bovine kidney (MDBK) cells and primary fetal bovine kidney (PFBK) cells. Citrinin is a known nephrotoxicant but produced a low order of cytotoxicity in cultured renal cells. A dose-dependent cytotoxic effect was observed at millimolar concentrations of the toxin. MDBK cells were more sensitive than the PFBK cells. The primary effect of this chemical was on the adherence of MDBK cells to the culture dish. Microscopic evaluation of morphologic changes indicated that cells elongated, flattened, swelled, and became rounded. The appropriateness of toxicity evaluation in culture systems in vitro is considered.

Anchorage-dependent culture of line 10 guinea-pig hepatoma cells.

A simple method for the anchorage-dependent culture of line 10 guinea-pig hepatoma cells is described.