Canine Transmissible Venereal Sarcoma Research Articles

Review Of Canine Transmissible Venereal Tumour (TVT) In Dogs

Transmissible Venereal tumour (TVT) also known as stickers sarcoma (Sticker, 1904) transmissible venereal tumour, venereal granuloma, canine condyloma or infectious sarcoma (Madewell and Theilen, 1987; Nielsen and Kennedy, 1990 and Roger, 1997). Canine transmissible venereal Sarcoma (TVS) (Yang, 1988) is the most notable among the three recognized transmissible tumours in domestic animals. It is a naturally occurring contagious round – cell neoplasia of dogs. It is a well characterized sexually transmitted neoplasm commonly affecting the external genitalia in dogs (Brown et al., 1980; Richardson, 1981 and Cohen, 1985). TVT is primarily located in the mucous membrane of the external genitalia of either sex. This tumour is unique in oncology, because it was the first tumour to be transmitted experimentally. This was achieved by the Russian Veterinarian, Nowinsky in 1876 according to Epstein and Bennett (1974) and Yang, et al (1991). Canine transmissible venereal tumour (CTVT) is virtually the only tumour transmitted by cell transplantation under natural conditions (Yang, 1988). Viable CTVT cells can be transmitted to susceptible animals and transfer, in a given dog, to other mucous membranes (e.g. the oral cavity) spontaneously through injured skin, or mucosa during coitus, licking, fighting/ biting, sniffing or scratching (Stubbs and Furth, 1934). In addition to the genital system (Higgins, 1966), CTVT can occur any where on the body including the skin, oral and nasal cavities, conjunctiva and even the anal mucosa (Neilsen and Kennedy, 1990, Calvert, 1984; Harmir, 1986; Amber and Adeyanju, 1986; Adeyanju et al., 1987 and Betamuzi and Bittegeko, 1991) gluteal region, maxilla gingival, dorsal skin of thorax (Gurel et al., 2002). TVT can be experimentally transmitted to wolves, Jackals, Coyotes and red foxes (Madewell and Theilen, 1987; Neilsen and Kennedy, 1990 and Roger, 1996).Recent research in Australia which revealed the existence of a newly emerged tumour in Tasmanian Devils (Devils are an endangered marsupial species) that appears to be caused by transmissible cancer cells, in this case by biting (Claudio et al., 2006). The tumour is recognized in all dog breeds in various part of the world, especially in tropical and sub tropical zones (Gurel, et al., 2002). Due to the nature of transmission by sexual contact, free roaming, sexually active dogs in region with poorly enforced leash laws are at greatest risk for contrasting this disease. Although, CTVT does not often metastasized, it has been reported in the regional lymph nodes, liver, pancreas, spleen, lung, kidney, ocular cavity and brain (Mcleod and Lewis, 1972; Oduye et al., 1973; Adams and Slaughter, 1970; Prier and Brodey, 1963, Yang, 1987, Perez et al., 1998, Ferreira et al., 2000). CTVT transplantability in animal model showed that tumour cell differentiation and, in turn, spontaneous regression could be induced. (Yang, 1988 and Angell, 2000). Nigerian Veterinary Journal Vol. 28 (1) 2007 pp. 54-70

Review of canine transmissible venereal sarcoma.

Canine transmissible venereal sarcoma (CTVS) is usually a sexually transmitted neoplasm of the external genitalia of dogs. This tumour is unique in oncology because it was the ¢rst tumour to be transmitted experimentally, this being achieved by the Russian veterinarian Nowinsky in 1876. This stimulated a lively interest among scientists and became a new starting point for the study of oncology. CTVS has been studied and reported extensively since the early days of nineteenth century, but a recent, widely circulated, review of this condition has not been published. This review is designed to unify the epidemiological, pathological, immunological and therapeutic concepts related to CTVS.

Augmentative effects of phytohemagglutinin-P on proliferation and cytotoxicity of interleukin-2-activated canine peripheral blood lymphocytes.

Canine peripheral blood lymphocytes (PBL) were simulated with recombinant human interleukin-2 (rhIL-2) alone, or with phytohemagglutinin-P (PHA) and subsequent rhIL-2 in order to obtain large numbers of lymphokine-activated killer (LAK) cells. Incubation of PBL with rhIL-2 alone allowed proliferation of large granular lymphocyte (LGL)-like lymphocytes, and the cytotoxic activity of the lymphocytes against canine transmissible venereal sarcoma cells was detected 5-7 days after the culture onset. However, the lymphocytes died within 2 weeks of culture, resulting in limited generation of functional LAK cells. Thus, PBL pretreated with PHA are subjected to rhIL-2-dependent culture. Small- or middle-sized lymphocytes predominantly proliferated in response to rhIL-2, and proliferation of the lymphocytes was sustained for longer than 30 days by repeated stimulations with PHA and subsequent rhIL-2. Cytotoxicity reached significant levels from 2 weeks after the culture onset and thereafter remained almost constant for at least 2 weeks, leading to large-scale production of the LAK cells. Pretreatment of PBL and PHA seems to enhance the LAK cell functions through modification of the precursors of the effector LAK cells.

Changes in Lymphokine-activated Killer Activity in Peripheral Blood Lymphocytes from Canine Transmissible Venereal Sarcoma Models.

Time course changes in anti-tumor activity induced in peripheral blood lymphocytes (PBL) with recombinant human interleukin-2 (rhIL-2) and phytohemagglutinin-P (PHA) were studied in dogs implanted with canine transmissible venereal sarcoma (CTVS) as a tumor-bearing model. The rhIL-2-dependent and PHA-dependent cultures allowed selective proliferation of lymphocytes expressing Thy-1 antigens. The lymphocytes acquired a prolonged anti-tumor activity against the CTVS cells, starting from 2 weeks after the culture, indicating generation of lymphokine-activated killer (LAK) cells. The LAK cells showed serial growth in rhIL-2-containing culture medium for at least a further 2 weeks without loss of the anti-tumor activity.

Role of lymphocytes in spontaneous regression of experimentally transplanted canine transmissible venereal sarcoma.

Sensitized peripheral blood lymphocytes (PBL) obtained from canine transmissible venereal sarcoma (CTVS)-regressed dogs were more cytotoxic against CTVS cells than non-sensitized PBL from untreated dogs. Cytotoxicity shown by sensitized PBL was inhibited significantly by the addition of anti-major histocompatibility complex (MHC) class II mouse monoclonal antibody as well as that of anti-dog thymocyte rabbit serum. The degree of cytotoxic activity shown by lymphokine activated killer (LAK) cells induced from non-sensitized or sensitized PBL was similar to that of the activity shown by sensitized PBL. These LAK activities were also prohibited by the addition of anti-dog thymocyte rabbit serum. Immunohistochemical examination demonstrated that MHC class II antigens were expressed on the surface membrane of CTVS cells and thymocyte antigens were detected on the surface of the tumor infiltrating lymphocytes. From the results mentioned above, lymphocytes which play a central role in tumor regression are considered to be T cells. These cells might recognize MHC class II antigens on the surface membrane of CTVS cells in tumor regression.

Characterization of dog interleukin-2 activity.

Proliferative activity of murine interleukin-2 (IL-2) dependent T cells (CTLL-2) was detected in the culture supernatant of canine peripheral blood lymphocytes (PBL) stimulated with phytohemagglutinin-P (PHA-P), and was defined as dog IL-2. The highest production of IL-2 was obtained under the conditions in which a PBL population of 2 x 10(6) cells/ml was stimulated with PHA-P at a concentration of 10 micrograms/ml at 38 degrees C for 48 hr. Dog IL-2 activity was significantly inhibited by heating at 65 degrees C, acidification under pH 4, alkalification over pH 10, and trypsin exposure. A peak of dog IL-2 activity was detected in the fraction with a molecular weight of approximately 31,000 by gel filtration. Long-term culture of canine lymphocytes was successful over 10 passages by using dog IL-2 with PHA-P-stimulation every 3 passages. The cultured cells mostly consisted of small- and medium-sized lymphocytes. These cells reacted to anti-dog thymocyte rabbit serum and anti-dog Thy-1 mouse monoclonal antibody. These cells were therefore considered to originate in T-lineage lymphocytes. Cytostasis of PBL from intact dogs reacting to canine transmissible venereal sarcoma cells was increased significantly when PBL was cultured for more than 30 days with homologous IL-2.

An inhibitor of tumor cell growth from normal horse serum

During our studies of cytostatic cytokines in the mixed leukocyte culture, we found that horse serum in the medium control contained a tumor cell growth-inhibitory factor. The fraction isolated by molecular sieving and ion exchange chromatography inhibited the growth and DNA synthesis of the primary culture and passaged cell line of the canine transmissible venereal sarcoma, murine T (L5178Y) and B (P3-X63-Ag8.653) lymphoid tumor cells, murine mammary tumor cells (RIII), bovine lymphoid tumor cells (BL3), and the nontransformed cell line of baby hamster kidney cells. Nontransformed cell lines such as African green monkey kidney (Vero) and Madin-Darby bovine kidney cells and normal mammary cells of the dog and goat were inhibited only at high concentrations. The active component is a protein with an Rf value of 0.09 upon electrophoresis in native 7.5% polyacrylamide gels. The eluate from the native gel could be further separated into three polypeptides with molecular weights of approximately 67, 65, and 55 kDa under reduced conditions in sodium dodecyl sulfate-polyacrylamide gels.

Tumor size, leukocyte adherence inhibition and serum levels of tumor antigen in dogs with the canine transmissible venereal sarcoma.

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.

Role of humoral immunity in progressive and regressive and metastatic growth of the canine transmissible venereal sarcoma.

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.

Growth stage dependent expression of MHC antigens on the canine transmissible venereal sarcoma.

Canine transmissible venereal sarcoma (CTVS) is a naturally occurring contagious neoplasm which can be transplanted with intact viable cells across major histocompatibility (MHC) barriers within the species and even to other members of the canine family, such as foxes, coyotes, and wolves. After 2 to 4 months of progressive growth the tumour regresses spontaneously in adults but metastasizes in immunosuppressed hosts and neonates. The mechanisms of how the tumour cells manage to overcome histocompatibility barriers so successfully for such a long period and yet succumb later are not known. In the present study we found that CTVS cells were not stimulatory to the lymphocytes of normal or tumour-bearing animals in mixed lymphocyte-tumour reaction (MLTR), although the lymphocytes from tumour-bearing animals in mixed lymphocyte-tumour reaction (MLTR), although the lymphocytes from tumour-bearing hosts responded well to either phytohaemagglutinin (PHA) or third-party allogeneic lymphocytes. Immunofluorescent antibody (IFA) assay of MHC antigens by monoclonal antibodies (MoAb) to monomorphic Class I and Class II MHC antigens showed that progressor tumour cells lacked the expression of the antigens whereas 30 to 40% of regressor tumour cells expressed them.

Immunoelectron Microscopic Localization of a Tumor-Associated Antigen (TAA) in the Canine Transmissible Venereal Sarcoma by Anti-TAA-Antiferritin Hybrid Antibodies<xref ref-type="fn" rid="FN1">2</xref><xref ref-type="fn" rid="FN2">3</xref>

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.

Regulation of leukocyte glass adherence and tube leukocyte adherence inhibition (LAI) reactivity by serum factors in dogs with progressing or spontaneously regressing canine transmissible venereal sarcoma (CTVS).

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.

Detection of immune complexes in sera of dogs with canine transmissible venereal sarcoma (CTVS) by a conglutinin-binding assay

The canine transmissible venereal sarcoma (CTVS) is capable of extended growth in an allogeneic host. Since immune complexes can enhance allograft survival in other animal models, we used an enzyme-linked conglutinin-binding assay to determine the presence and amount of circulating immune complexes in dogs with CTVS. With the conglutinin-binding assay, 23 of 64 dogs (36 per cent) bearing CTVS had concentration of immune complexes 3 standard deviations greater than those detected in normal canine serum. When dogs with circulating immune complexes were separated into 3 groups based on whether the size of CTVS had increased (progressor), decreased (regressor) or remained the same (steady-state) during the week before collection of serum, no significant difference was found in the amounts of immune complexes in sera from progressor and regressor dogs. In 3 regressor dogs that received a second transplant of CTVS, the mean concentration of circulating immune complex was significantly greater than the mean for progressor dogs. In progressor dogs, amounts of immune complexes decreased with increasing tumour volume. Following sucrose density gradient ultracentrifugation analysis of sera from a normal and progressor dog, 27S complexes containing both IgG and IgM were detected in serum from a progressor dog. Thus, it appears that the conglutinin-binding assay is a useful and sensitive method for detecting immune complexes in canine serum.

Canine Transmissible Venereal Sarcoma: Quantitation of T-Lymphocyte Subpopulations During Progressive Growth and Spontaneous Tumor Regression<xref ref-type="fn" rid="FN2">2</xref>

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.

Amplification of lymph node cell tube leukocyte adherence inhibition (LAI) reactivity by leukocyte adherence inhibition factor (LAIF)

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.

Canine transmissible venereal sarcoma: tumor cell and infiltrating leukocyte ultrastructure at different growth stages.

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: distribution of T and B lymphocytes in blood, draining lymph nodes and tumours at different stages of growth.

The levels of T, B and null lymphocytes in the peripheral blood, draining lymph nodes, and tumour masses at different growth stages in dogs transplanted with canine transmissible venereal sarcoma (CTVS) were determined by immunofluorescence techniques. The tumours were classified at excision into "progressor", "steady state", and "regressor" stages of growth. The percentage of B cells in the lymphocytes infiltrating into the progressively growing tumours (n = 10, 37.3 +/- 7.4%) was significantly higher (P less than 0.025) than that in regressing tumours (n = 21, 26.1 +/- 1.9%). In contrast, the percentage of T cells in the lymphocytes infiltrating into the regressing tumours (n = 21, 61.2 +/- 2.6%) was significantly higher (P less than 0.005) than that in the progressively growing tumours (n = 10, 34.0 +/- 5.1%). The tumours at the steady-state growth stage (n = 9) had 50.8 +/- 5.7% infiltrating T-cells, which was significantly higher (P less than 0.005) than the progressors and lower (P less than 0.005) than the regressors. The percentage of null cells of progressors (n = 10, 26.0 +/- 6.9%) was significantly (P less than 0.025) higher than in regressors (n = 21, 13.5 +/- 2.9%). The draining lymph nodes of progressor dogs (n = 5) had significantly fewer (P less than 0.025) B cells (8.2 +/- 2.3%) than in normal (n = 5, 16.1 +/- 3.1%), regressors (n = 12, 19.1 +/- 1.7%) and steady-state dogs (n = 5, 15.8 +/- 2.6%). Although there was slight lymphopenia and fluctuation of null cells, no significant differences in T- and B-lymphocyte levels were noted in the peripheral blood of the tumour dogs (n = 44) studied.

Sequential changes in peripheral blood leukocyte adherence inhibition (LAI) reactivity during progressive growth and spontaneous regression of canine transmissible venereal sarcoma.

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 suggest that LAI reflects a function 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.

Identiflcatlon and Tumor-Associated Venereal Sarcoma Physicochemical Characterization of a Antigen From Canine Transmissible<xref ref-type="fn" rid="fn2">2</xref><xref ref-type="fn" rid="fn3">3</xref><xref ref-type="fn" rid="fn4">4</xref>

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.

Canine transmissible venereal sarcoma: leukocyte adherence inhibition (LAI) reactivity of various lymphoid tissues of dogs with tumors at different stages of growth.

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.