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Tumor antigen (TA) associated with the canine transmissible venereal sarcoma (CTVS) was detected in the sera of dogs bearing the tumor. Rabbit antisera specific for tumor antigen and 3 M KCl extracts of CTVS cells were used in both a competitive enzyme-linked immunosorbent assay (ELISA) and antigen-capture ELISA to quantify levels of circulating TA. In a study of 29 dogs bearing the transplanted CTVS, levels of circulating TA correlated positively with tumor volume. In a longitudinal study of four dogs receiving a transplant of 10(8) viable CTVS cells, circulating CTVS antigen was detected transiently 2 days after transplantation, while persistent levels of TA associated with increasing tumor volume were demonstrable 19-34 days after transplantation. In three of four tumor-bearing dogs, levels of serum TA correlated inversely with values obtained with peripheral blood leukocytes in the leukocyte adherence inhibition (LAI) assay; elevated levels of circulating TA found in dogs with large (greater than 7 cm3) tumors were associated with decreased LAI reactivity of peripheral blood leukocytes. TA could not be detected in sera 48-72 h after surgical removal of CTVS whereas LAI reactivity of peripheral blood leukocytes to CTVS antigen rebounded 1-3 weeks following tumor excision. Results of this study support the use of the competitive ELISA and LAI techniques in assessing levels of circulating tumor antigen, tumor burden and tumor-specific immunity.

1. 1. The total amount of DNA present in L5178Y cells treated with antiserum in the absence of complement was decreased whereas the rate of incorporation of [3H]thymidine into DNA during a 1 hr pulse was increased as compared with those of controls. 2. 2. This increase in specific activity of [3H]thymidine in the DNA of antiserum-treated cells appeared to be due, at least in part, to an increase in thymidine kinase activity.

The structure of canine transmissible venereal sarcoma (CTVS) has been examined from 14 to 71 days after implantation. During early growth, the tumour appears to be composed primarily of loosely arranged, round cells and a few fibroblast-like cells. As the tumour mass increases, the round cells become tightly packed with highly interdigitating plasma membranes. The number of irregularly shaped round cells and fibroblast-like cells increases with increasing tumour mass. Collagen and reticular fibres can be found in early tumours, frequently in association with the round cells, and in regions devoid of fibroblast-like cells. During tumour regression, cellular degradation is evident in fibroblast-like and irregularly shaped cells as well as round cells. The data suggest that transformation may occur in the course of tumour growth, causing morphological change from round to fibroblast-like cells, and that CTVS is an undifferentiated round-cell sarcoma capable of differentiation in a fibroblastic direction. Also present, primarily in tumour cells from newborn dogs, are cytoplasmic lamellar arrays and crystalline virus-like structures, both previously described in other forms of tumor cells. Images Figs. 1-3 Fig. 4 Figs. 5-8 Fig. 9 Figs. 10-12 Figs. 13-14

Leukocyte adherence inhibition (LAI) reactivity of various lymphoid tissues of dogs with canine transmissible venereal sarcoma (CTVS) at different stages of growth was determined by the tube LAI test. Tumors were classified at the time of excision into progressor, steady state, and regressor stages of growth. The LAI reactivity to CTVS antigen extract of spleen, draining and non-draining lymph-node cells, and peripheral blood leukocytes of regressors (non-adherence index--NAI of 172.8 +/- 46.8, 148.1 +/- 64.7, 138.7 +/- 47.3, and 172.2 +/- 60.7, respectively) was significantly greater than that of progressors (46.1 +/- 20.0, 38.5 +/- 21.5, 50.2 +/- 30.0, 24,6 +/- 37.2, respectively, p less than 0.001) and normal dogs (47.5 +/- 22.8, 54.6 +/- 24.6, 26.7 +/- 14.0, 50.9 +/- 22.4, respectively, p less than 0.001). In contrast, LAI reactivity of progressor lymphoid tissues to CTVS antigen extract did not differ significantly from that of normal dogs. LAI reactivity of lymphoid tissues from steady state tumor bearers (97.9 +/- 39.2, 80.7 +/- 47.3, 87.1 +/- 40.0, 85.1 +/- 53,9, respectively) was intermediate between and significantly different from LAI reactivities of regressor (p less than 0.05) and progressor (p less than 0.01) lymphoid tissues. Significant LAI reactivity observed in regressors suggests the presence of a functional effector cell mechanism associated with spontaneous regression of CTVS. The three distinct patterns of LAI reactivity observed in tumor-bearing dogs appear to correlate with the clinical course of tumor growth.

Erythrocyte antibody (EA) rosette techniques employing sheep red blood cells sensitized with canine (homologous) and rabbit (heterologous) IgM and IgG antibodies were used to determine the number of cells with Fc receptors for IgM (Tμ) and IgG (Tγ) among T lymphocytes isolated from peripheral blood and lymph nodes of dogs. The percentages of Tμ and Tγ lymphocytes detected were found to be independent of the species origin of sensitizing antibody. Among peripheral blood T lymphocytes there were 53.0 ± 2.7% Tμ cells and 18.4 ± 3.6% Tγ cells. T lymphocytes obtained from lymph nodes were 62.1 ± 5.4% Tγ and 15.7 ± 2.6% Tγ. The number of Tμ cells detected increased from 20.0% when freshly isolated to 49.1 ± 4.1% after in vitro culture for 2–16 hr. The expression of the Fc-μ receptor in culture was inhibited by cycloheximide, demonstrating a requirement for active protein synthesis. In contrast, the number of Tγ lymphocytes detected did not vary between freshly isolated cells and those which had been cultured for 16 hr. Expression of the Fc-γ receptor during this time period was not inhibited by cycloheximide.

Sequential changes in canine transmissible venereal sarcoma (CTVS) extract-induced leukocyte adherence inhibition (LAI) reactivity of peripheral blood leukocytes (PBL) were monitored in dogs from the time of tumor inoculation through progressive growth and spontaneous regression or surgical excision of CTVS. The time-course profiles of LAI reactivity of tumor dog PBL correlated with the clinical stage of tumor growth, substantiating our previous findings in a cross-sectional study. The kinetics of the time-course appearance of LAI reactivity observed and the demonstration of a rapid anamnestic response following tumor challenge indicate further that LAI is a reliable in vitro measure of in vivo tumor immunity. A significant increase in CTVS antigen extract-induced LAI reactivity observed in the PBL during tumor regression suggests that LAI reflects a functional effector cell mechanism associated with spontaneous regression of CTVS. By contrast, absence of significant LAI reactivity of PBL during progressive tumor growth suggests the presence of serum blocking factors. In addition, a substantial rebound in LAI reactivity observed in the PBL of dogs 3 to 7 days following surgical excision of progressively growing CTVS provides further evidence that tumor-cell components are associated with progressive tumor growth and diminished LAI reactivity.

A tumor antigen (TA) associated with murine leukemia-lymphoma L5178Y cells has been identified by the enzyme-linked immunosorbent assay (ELISA) and indirect immunofluorescence (IIF) techniques. The antigen was present in both non-solubilized and 0.5% NP-40 solubilized membrane extracts. Rabbit anti-L5178Y lymphoma serum (RALS), extensively absorbed with normal mouse tissues, identified TA in extracts of L5178Y lymphoma and L5178Y leukemia cells grown in horse serum (L5178Y/HS), but not in extracts of L5178Y cells grown in fetal calf serum (L5178Y/FCS). Similarly, absorbed rabbit anti-L5178Y/HS serum specifically reacted with extracts of lymphoma and L5178Y/HS but not with L5178Y/FCS cells. Membrane IIF showed positive reactivity in 88% of lymphoma and 73% of L5178Y/HS cells, whereas splenic lymphocytes and L5178Y/FCS cells were negative. Goat anti-AKR virus serum reacted with soluble extracts of lymphoma, L5178Y/HS, and L5178Y/FCS as well as with normal DBA/2 tissues in the ELISA. However, goat anti-AKR virus serum did not block the reactivity of RALS to lymphoma in the blocking ELISA (BELISA). Expression of TA, but not murine leukemia viral antigen(s), was correlated with the in-vivo tumorigenicity of the L5178Y cells. The antigenic activity of lymphoma extract was reduced by incubation for 1 h at 56 and 65°C, by trypsin digestion, and by exposure to pH 2.8 or 11.0 for 1 h. The antigen, sequentially purified by gel filtration and Lentil-lectin affinity chromatography, was a glycoprotein, with a molecular weight of approx. 64,000 daltons, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis.

A technique is described for finding the pineal body of the dog. The posterior half of the skull is cut a little behind the parietofrontal suture, through the occipital condyles. The cerebral hemispheres and cerebellum are carefully sliced, disclosing the pineal at the frontal edge of the colliculi. Two types of cells are present, those with completely round nuclei, and others with vesicular and variably shaped nuclei. In the histological pattern, ependymal cells were observed on the edges, pinealocytes and glial cells within the gland.

Electron microscopic study of the canine transmissible venereal sarcoma (N = 26) at different stages of growth showed that tumors in a progressive phase of growth (N = 12) were comprised mostly of large, round cells with prominent nuclei and nucleoli, a few spindle-shaped cells and collagen fibers, focal areas of necrosis, and lymphocyte infiltrates. Regressing tumors (N = 9) had the fewest viable round cells, greater numbers of infiltrating leukocytes, and were comprised almost entirely of connective tissue (collagen bundles) in the latest stages of regression. The infiltrating leukocytes consisted primarily of lymphocytes, some eosinophils, and neutrophils, but very few macrophages. Round-cell nuclei contained numerous perichromatin and interchromatin granules. Their cytoplasm often had annulate lamellae complexes and large deposits of glycogen. Loosely packed tumor cells had extensive proliferation and elongation of cytoplasmic extensions (filopodia). Evidence for cell-mediated tumor cell lysis was demonstrated by degenerative changes in tumor cells which were closely apposed to lymphocytes. Spindle-shaped cells in regressing tumors had intravacuolar collagen fragments, suggesting a possible degradative capacity by these cells. Canine transmissible venereal sarcoma, also called transmissible venereal tumor of dog and Sticker's sar- coma, is a naturally occumng round cell neoplasm of the genital tracts of male and female dogs. Although normally transmitted through coitus, tumors may be induced experimentally by inoculation of viable tumor cells into normal, non-preconditioned dogs. In either neonatal or immunosuppressed dogs, this sarcoma usu- ally grows vigorously and meta~tasizes,~.~~ but the his

We examined the regulation of leukocyte glass adherence and tube leukocyte adherence inhibition (LAI) reactivity by serum factors in dogs with regressing or progressing canine transmissible venereal sarcomas (CTVS). Both regressor and progressor peripheral blood leukocytes (PBL), draining and non-draining lymph node cells (LNC), and splenic leukocytes were significantly responsive to CTVS antigen extract in tube LAI. In contrast, a significant decrease in basal glass adherence of progressor PBL, draining and non-draining LNC, and splenic leukocytes was observed. Normal glass adherence was restored to progressor leukocytes by extensive washing with warm serum-free media, while significant tube LAI responsiveness to CTVS antigen extract was maintained. Preincubation of regressor PBL and LNC with progressor sera in two-stage tube LAI decreased the basal glass adherence of treated leukocytes. This effect of progressor sera was heat labile, a characteristic of CTVS antigen. Collectively, these findings suggest that progressor leukocytes and progressor sera treated regressor leukocytes were activated by interaction with serum CTVS antigen and thus behaved in tube LAI as stimulated cells, even in the absence of CTVS antigen. Regressor but not progressor sera were shown to contain anti-CTVS IgG with specific arming activity for normal dog PBL, but not LNC in two-stage tube LAI. The nonadherent response of peripheral blood neutrophils in two-stage tube LAI was proportional to the concentration of arming IgG, whereas no change was observed in glass adherence of PBL. The results of this study define the role of progressor and regressor serum factors in the mechanism of tube LAI and demonstrate a relationship between leukocyte glass adherence and the clinical course of CTVS. These findings show that tube LAI is a simple and reproducible measure of active factors in the immune response to a tumor.

Virus-like particles measuring approximately 110 nm in diameter were found, along with parvoviruses, in a fecal specimen of a parvovirus-infected dog. Under the electron microscope, the outer component of the particles was found to be composed of what appeared to be numerous fine spikes or a solid coat perforated by many regular small slits. The inner ‘core’ of the particle had an irregular border, measured approximately 60 nm in diameter and was occasionally eccentrically located.

A tumor antigen associated wtih canine transmissible venereal sarcoma (CTVS) has been identified and partially characterized. The antigen was demonstrated in 3-M KCI and saline extracts of washed CTVS cells. Rabbit anti-CTVS antisera absorbed wih glutaraldehyde cross-linked normal dog serum and pooled homogenates of canine spleen, thymus, lymph node, and liver were used to identify the antigen. The specificity of the rabbit anti-CTVS antiserum for the CTVS antigen was established by use of immunodiffusion, two-dimensional electrophoresis, and the enzyme-linked immunosorbent assay (ELISA). Extracted CTVS antigen was fractionated by Sephadex G-200 chromatography, and the antigen factions were identified with the use of absorbed rabbit anti-CTVS antiserum and the ELISA technique. Antigen activity was detected in samples with estimated molecular weights greater than 70,000 daltons. Antigen fractions reacted with absorbed anit-CTVS antiserum to form a single precipitation band in immunodiffusion studies. Extraction of CTVS antigen with 3 M KCl and saline in the presence of a protease inhibitor did not significantly alter the antigen activity or molecular weight of the CVTS antigen. Cytoplasmic and nucleolar fluorescence were observed in an indirect immunofluorescence test with the use of acetone-fixed CTVS cells and absorbed anti-CTVS antiserum. The CTVs antigen activity was greatly reduced by trypsin digestion, by incubation for 1 hour at 65 degrees C, and by exposure to pH 2.8, 4.0, and 11.0 for 1 hour each.

Heat-inactivated rabbit antiserum, in the absence of complement, induced a 1.5–2-fold increase in cyclic AMP levels in target cells L5178Y leukemia lymphoblasts within 10–20 min after the experiment. This change preceded the previously reported delayed inhibitory effects of antiserum on cell growth such as inhibition of RNA, DNA, and protein synthesis and cell proliferation, suggesting that cyclic AMP may be one of the mediators of the antigen-antibody reactions which occur at the cell surface. Furthermore, the addition of cyclic GMP or excess calcium to either antiserum or cyclic AMP-treated cultures alleviated the growth inhibitory effects of either antiserum or cyclic AMP, substantiating further the hypothesis proposed.

Intraarticular inoculation of Mycoplasma bovis into the joints of six-week-old calves caused severe arthritis in five inoculates and mild arthritis in a sixth. Intraarticular inoculation of killed M. bovis did not cause arthritis.Arthritic calves had fever, joint swelling, lameness, neutrophilia, and intercurrent pneumonia from which M. bovis could not be recovered.Gross lesions were massive fibrinosuppurative synovitis and tenosynovitis, erosion of cartilage, and its replacement by polypoid granulation tissue. Histologic lesions were extensive ulceration of synovial membranes, leukocytic infiltration of the suhsynovium, congestion, hyperemia, and thrombosis of the subsynovial vessels. Cartilage erosion was accompanied by chronic osteomyelitis and formation of pannus tissue.The presence of thrombi and platelet aggregates suggests that the inflammatory process in the synovium may arise from the interaction of M. bovis with the vasculature and the coagulation process of the host.

Canine transmissible venereal sarcoma (CTVS) is a contagious neoplasm which regresses spontaneously in adult dogs but metastasizes and kills puppies transplanted with the neoplasm at a very young age. Immunofluorescence studies showed that 30 +/- 14% of cells from steady-state and 22 +/- 7% of cells from regressing tumors had membrane-bound antibodies which could be eluted out with warm washes at 24 degrees C, whereas the cells from progressor tumors had very little such antibody (6 +/- 6%). Time-course kinetics of anti-CTVS antibodies in the serum of tumor-bearing dogs did not correlate well with tumor volume, however, the presence of such antibodies in adult dogs (47 +/- 13%) but absence (0%) in the puppies with tumor metastasis suggested the importance of antibodies in resistance to metastasis.

The levels of T-lymphocyte subpopulations expressing IgMFc and IgGFc receptors (i.e., T mu lymphocytes and T gamma lymphocytes, respectively) were quantitated for T-lymphocytes obtained from peripheral blood, draining and nondraining lymph nodes, and the tumor mass during progressive growth and spontaneous regression of the canine transmissible venereal sarcoma. Analysis of the T-lymphocyte subpopulations in these lymphoid compartments demonstrated that distinct profiles of T mu and T gamma lymphocytes correlated respectively with the growth and regression statuses of the tumor. The percent of T gamma lymphocytes in the peripheral blood of dogs with progressing, steady-state, and late-regressing tumors was significantly increased over that of control dogs (P less than .05, P less than .001, and P less than .001, respectively) and over that of dogs with early-regressing tumors (P less than .05, P less than .01, and P less than .02, respectively). The percent of T gamma lymphocytes in the lymph nodes draining the tumor was observed to have a significant increase in dogs with progressing (P less than .01), steady-state (P less than .01), and late-regressing (P less than .001) tumors compared with that in control dogs. The percentage of T gamma lymphocytes was observed to have a significant increase in the nondraining lymph nodes of dogs with steady-state and late-regressing tumors compared with that of control dogs (P less than .01 and P less than .002, respectively) and that of dogs with progressing tumors (P less than .001 and P less than .0005, respectively). The percent of tumor-infiltrating T gamma lymphocytes was lowest in tumors that were growing progressively. A significant increase in T gamma lymphocytes was observed in steady-state (P less than .05), early-regressing (P less than .001), and late-regressing (P less than .05) tumors. Early-regressing tumors contained significantly (P less than .005) greater levels of T gamma lymphocytes than did late-regressing tumors.

Studies were performed to determine whether decreases in transport of calcium and glucose might be among the earliest changes triggered by the antigen-antibody reactions occurring on the cell surface of murine leukemia L5178Y cells after treatment with rabbit antisera. After treatment with antisera, in the absence of complement, these cells exhibited a decreased uptake of 45Ca, 2-deoxy[3H]glucose, and 3-0-methyl[3H]glucose. These changes occurred rapidly, within 2 minutes after the addition of antiserum, in contrast to the previously reported inhibitory effects of antiserum on DNA, RNA, and protein synthesis, which became demonstrable only after 4 to 8 hours. The kinetics of uptake of the radioactive substrates was biphasic, with a very rapid initial uptake followed by less rapid linear uptake. The precise mechanism of cell growth inhibition remains to be elucidated, but one of the initial effects of antiserum treatment may be a perturbation at the cell membrane such that transport of specific nutrients is decreased, resulting in the observed effects on macromolecular synthesis.

This investigation examines the immunologic basis for specific antigen-induced tube leukocyte adherence inhibition (LAI) reactivity of draining lymph node cells (LNC) from dogs with canine transmissible venereal sarcoma (CTVS). CTVS regresser LNC, macrophage-depleted LNC, and enriched T lymphocyte fractions, but not enriched B lymphocyte fractions, were specifically reactive to CTVS antigen extract in direct tube LAI. In addition, regressor LNC amplified tube LAI responses by generating supernatants with leukocyte adherence inhibition factor (LAIF) activity for normal dog indicator LNC and enriched peripheral blood mononuclear cells (PBMC) in an indirect tube LAI assay. However, macrophage-depleted LNC and enriched T lymphocyte fractions failed to generate supernatants with LAIF activity, suggesting that macrophage accessory cells play a central role in the amplification of tube LAI. Interestingly, CTVS regressor peripheral blood leukocytes (PBL) and PBMC, which were specifically reactive in direct tube LAI, also failed to generate supernatants with LAIF activity. These findings demonstrate a distinction between LAIF-mediated amplification and direct tube LAI reactivity, and suggest that leukocyte populations with differing cellular proportions and from different immunologic compartments may participate in tube LAI via different mechanisms.

Immunoferritin and immunoperoxidase reagents were used to localize a tumor-associated antigen (TAA) of the canine transmissible venereal sarcoma (CTVS). Round tumor cells from the CTVS at different stages of growth, i.e., progressive, steady state, and regressing, had TAA diffusely distributed throughout the cytoplasm. In general, TAA was not found on the plasma membrane, within the nucleus, between inner and outer membranes, in cytoplasmic vacuoles, or specifically with any part of the cytocavitary system. Transitional tumor cells, which are intermediate cell types between round cells and spindle-shaped cells and which appear in the tumors at steady state and regressing stages, contained less TAA in their cytoplasm than did the round cell type. The microvilli of tumor cells also contained TAA, suggesting that, in addition to whole cell lysis, shedding of all or parts of these processes may be a mechanism of TAA release as evidenced by the presence of antigenic activity in the extracellular material.