Regression of feline sarcoma virus-induced sarcomas in dogs. II. Immunologic investigations.

The serial development of cell-mediated immunity (CMI), cytotoxic antibody activity, serum blocking activity, and virus-neutralizing antibody levels were monitored in vitro for beagle and mongrel puppies inoculated with feline sarcoma virus (FeSV) and were compared to in vivo histologic markers of regression of induced sarcomas. CMI developed rapidly and maintained a high level of in vitro activity throughout the tumor life-span. Cytotoxic antibody levels similarly rose rapidly to peak just before clinically detectable regression and then declined during most of the regression sequence. This suggested antibody fixation at the tumor site, correlating with the histologic finding of focal necrosis and neutrophilic infiltrates. Inactivation of antibody by circulating antigen with subsequent immune complex formation was a possibility. Levels of virus-neutralizing antibody in sera paralleled those of cytotoxic antibody; their relationship in the circulation was not clear, but each related to virus-determined antigenic specificities. Serum blocking activity rose rapidly, leveled off during most of the tumor life-span, and rose slightly during the last stages of regression. This partly explained the lack of in vivo tumor lymphoid infiltrates to correlate with the striking in vitro CMI. Blocking activity was also present, however, when lymphoid infiltrates were seen histologically. Thus in vitro-in vivo correlation was best for cytotoxic antibody, which suggested that antigen-antibody reactions involving neutrophil-mediated regression sequences were important in effecting tumor-cell destruction.

Regression of feline sarcoma virus-induced sarcomas in dogs. I. Morphologic investigations.

In a study of morphologic changes in the development and regression of feline sarcoma virus (FeSV)-induced tumors in dogs, 27 weaned and newborn beagle and mongrel puppies were inoculated with FeSV in doses from 1.0 to 3.0 gEq; 2 beagle and 2 mongrel puppies were used as uninoculated contact controls. All animals were examined daily, and crude tumor volume was calculated from length, width, and depth measurements of the neoplasms. Biopsies were done at various stages of tumor development and regression. When tumors were no longer palpable, all puppies were necropsied. Two of 8 (25%) weaned beagle puppies, 10 of 12 (83%) newborn beagles, and 1 of 7 (14%) newborn mongrels developed tumors, all histologically confirmed fibrosarcomas. No metastatic tumor foci were detected. The tumor life-span was divided into approximately equal periods of growth and regression. The initial regression period was characterized by focal necrosis and accompanying neutrophil infiltration. The later stages of regression were characterized by lymphocytic or mixed mononuclear infiltrates. Thus the regression histopathology was not uniform and suggested that different immunologic mediation systems effect regression. Nonneoplastic morphologic changes consisted largely of lymphoid depletion and necrosis in lymph nodes and thymus after inoculation.

A "whole-blood" technique for the quantitation of canine "T-lymphocyte" progenitors using a semisolid culture system.

A whole blood technique is described for the growth of concanavalin A (Con A) stimulated canine lymphocyte colonies in semisolid medium. By eliminating the routine Ficoll-Paque (F-P) gradient lymphocyte isolation, this method avoids potential problems of growth modulation due to elimination of non-lymphoid accessory cells and the influences on colony formation associated with the selective effects of F-P on lymphocyte subpopulations. Thus, the technique more closely approximates the in vivo milieu. The whole-blood method also produces higher cloning efficiencies than methods using gradient isolation of lymphocytes. Studies over a wide range of blood concentration produced a linear response of in vitro colony formation although extrapolation of the cell-dose colony-response curve did not intersect zero. Mitogen titration data indicates that a relatively large dose of Con A is required for whole blood colony formation compared to the standard F-P method. The colonies ultrastructurally were composed of lymphoblastic and lymphocytic elements which were negative for non-specific esterase activity. Characterization of cells retrieved from the colonies using rosetting techniques indicates a high percentage of the colony cells relative to canine peripheral blood cells form rosettes with human erythrocytes.

Seasonal variation in cell mediated immunity of clinically normal dogs.

A whole blood lectin-induced lymphocyte stimulation test, using Con A and PHA, was used to assess the cell mediated immune status of 52 beagle dogs over a period of 16 months. The data indicated the presence of a seasonal variation in immune response with the peak estimated to be in July and an estimated trough in January. The relative amplitudes around the mean, for the two mitogens, were about 50%, so that the responses on lymphocyte division ranged from 50 to 150% of the mean during the course of the year. The possible implications of this finding for human health are discussed.

The selective growth of human T lymphocyte colonies from whole blood in a semisolid culture system.

Human T lymphocyte colonies may be selectively grown from whole blood in a single phase semisolid culture system following stimulation with PHA-P, Con-A, or PPD. This technique eliminates the requirement for gradient-enriched lymphocyte fractions, and provides a sensitive system for the study of T lymphocyte progenitors that more closely approximates the in vivo milieu. Whole blood colonies were composed of lymphoblasts and mature lymphocytes. Individual colony cells, identified as T lymphocytes, lacked lipase and specific esterase activity, formed E rosettes, did not phagocytize latex beads, and were largely ANAE positive. Whole blood was plated at a final concentration of 3%. Optimal mitogen/antigen concentrations were 125 microgram Con-A, 80 microgram PHA-P and 50 microgram PPD/ml culture media. Peak colony growth occurred between days 7 and 8. Colony formation increased as a power function over a wide range of cell concentrations (5 x 10(3)-5 x 10(4) lymphocytes plated). Maximal whole blood colony formation occurred when 5 x 10(4)-10(5) lymphocytes were plated. There was a significant increase in the cloning efficiency using whole blood as compared to gradient-separated cells. This method has wide application for the study of radiation effects, lymphocyte alterations in various disease states, antigen recognition, and the induction and amplification of T cell function.

The formation of human lymphocyte colonies in semisolid cultures in response to allogeneic mixed-lymphocyte stimulation.

A technique is described for the growth of human lymphocyte colonies in semisolid culture systems in response to allogeneic lymphocyte stimulation. Colonies did not form to any major extent using autologous lymphocyte stimulation. Both one-way and two-way mixed-lymphocyte reactions were investigated. Ultrastructurally, such colonies are composed of cells with lymphoblastic and lymphocytic morphology. The majority of the lymphoid elements composing the colonies were T-cells based on their ability to rosette with sheep red blood cells. Our studies suggest that the colonies are clonogenic in origin and therefore the technique offers the potential for isolation of specific clones, or subpopulations of lymphocytes involved in allogeneic reactions and characterization of their function. Studies directly comparing the stimulation indices achieved with standard mixed lymphocyte cultures utilizing 3HTdr-incorporation to the colony-forming assay indicate that the cloning technique produces higher stimulation indices for allogeneic/autologous reactions and produces less autologous (background) response than the 3HTdr incorporation technique. In addition to lymphocyte colonies, we also observed colonies of surface-adherent populations of macrophages, including multinucleated giant cells. Thus, the technique appears to provide a new and potentially more sensitive method for the study of transplantation immunology and cell-mediated immunity in humans.

Graft-versus-host disease in two immunocompetent dogs.

Graft-versus-host disease developed in two dogs injected with lymphocytes from BCG immunized donors. The disease was characterized by bone marrow depression, ulcerative enteritis, necrotizing cholangiohepatitis, thymic atrophy, pancreatitis, lymphadenopathy, inflammation of mucous membranes and weight loss. In one of the two dogs repopulation of bone marrow and lymphoid tissue by donor cells was demonstrated by cytogenetics. The development of GVHD was considered unusual because both animals received on immunosuppressive treatment and both responded well to PHA in lymphocyte transformation assays indicating they were immunocompetent. It was hypothesized that stimulation of donor lymphocytes by BCG enhanced their ability to induce a graft-versus-host reaction.

Production of mesenchymal tumors in nude mice by Ph1 negative fibroblasts obtained from a Ph 1 positive CML patient: a preliminary report.

An experimental model system is presented for the investigation in humans of the role of hematopoietic stromal elements in the regulation of hematopoiesis as well as in the pathogenesis of myelofibrosis in myeloproliferative disorders. The model is based on the simultaneous application of three experimental techniques: (1) growth of bone-marrow derived fibroblastic colonies in vitro, (2) cytogenetic demonstration of marker chromosomes associated with hematopoietic malignancies, and (3) the transplantation of isolated stromal elements into athymic (nude) mice. Using this model, we describe the induction of mesenchymal tumors in nude mice by Ph1 negative fibroblasts obtained from the bone marrow of a patient with a Ph1 positive chronic myelogenous leukemia. Mesenchymal tumors also were induced in nude mice with bone marrow-derived fibroblasts from a patient with aplastic anemia, who was successfully treated with bone marrow transplantation, and from a normal human volunteer. Morphologic, cytogenetic and electron microscopic studies of bone marrow mesenchymal elements in culture and of tumors induced in nude mice from the CML patient indicate the cells composing the tumor are of human origin and are negative for the Ph1 chromosome. The results provide the first in vivo morphological and cytogenetic support using human materials, of the hypothesized relationship of progenitors of in vitro fibroblastic colonies to marrow stromal elements.

Growth of canine T-lymphocyte colonies in vitro.

Canine lymphocytes from peripheral blood, lymph nodes, thymus and bone marrow were stimulated with phytohemagglutinin-P (PHA) or concanavalin-A (CON-A) to form colonies in methylcellulose. Lymphocytes exposed to mitogens in liquid phase formed clumps the size of colonies. Lymphocyte clumping was eliminated by plating cells directly into methylcellulose, but high concentrations of mitogens (CON-A or PHA is greater than 10 mg/10(6) lymphocytes) were required in order to get subsequent colony formation. Thus, in contrast to published reports, exposure of lymphocytes to mitogen prior to plating was not required for cloning of canine peripheral blood lymphocytes. Colonies from thymus, lymph node, or peripheral blood consisted predominantly of T lymphocytes, whereas cultures from bone marrow also produced colonies with macrophage morphology and surface-adherent colonies with mesenchymal morphology.

Increased clonogenic (CFU-C, PFU-C) populations from bone marrow and spleen of nude mice.

Clonogenic populations from bone marrow and spleen of nude mice and their normal littermates were enumerated in vitro using a methylcellulose supported culture system. This technique allows for the simultaneous quantitation of progenitors of granulocyte-monocyte pathways (colony forming units in culture, CFU-C) and for progenitors of "mesenchymal" elements (plaque-forming units in culture or PFU-C). These populations were distinguished in culture by their growth, characteristics and morphology. CFU-C gave rise to suspended colonies of granulocyte-monocyte composition while PFU-C formed surface-adherent colonies of mesenchymal morphological features (fibroblastic and reticuloendothelial morphology). Significant elevations in the relative and absolute numbers of CFU-C and PFU-C were observed in the bone marrow and spleen of 6 wk old nu/nu mice relative to heterozygous littermates. The results are discussed in terms of non-T cells components involved in cell-medited immunity against neoplastic development.

Spontaneous cell-mediated cytolysis by peripheral blood cells obtained from whole-body chronically irradiated beagle dogs.

The level of natural killer (NK) activity of continuously gamma-irradiated (whole body) beagle dogs and their nonirradiated controls was studied. For analytical purposes, irradiated dogs were segregated into groups according to their clinical status: clinically normal, hypocellular, or with acute non-lymphocytic leukemia. Since unirradiated control animals exhibited a wide range of NK responses, the data from each irradiated animal were compared to its own age-matched or litter-matched unirradiated control. Of the eight clinically normal irradiated dogs (median = 146% activity of control) only one animal had a NK activity lower than that of its control. The hypocellular group (n = 5, median = 21.8% of control) and the leukemic group (n = 4, median = 52.5% of control) each contained one responder with higher activity than its control. The difference between the percentage of control of the clinically normal and clinically abnormal dogs was found to be significant (P less than 0.05). There is a negative correlation between the NK results obtained and the total accumulated dose of radiation at the time of sampling (correlation coefficient = -0.739, P less than 0.01), suggesting a radiation effect upon natural killer activity, which is evidence by enhancement at lower doses and depression at higher doses of irradiation.

Chemiluminescence of canine peripheral blood lymphocytes stimulated by mitogens.

Luminol-dependent chemiluminescence of peripheral blood lymphocytes from dogs stimulated with concanavalin A (Con A) or phytohemagglutinin P (PHA) was measured with a Pico-Lite luminometer. 10 microliter of luminol gave optimal quantum yield from 1 X 10(6) lymphocytes sensitized with either 80 micrograms Con A or 160 micrograms PHA. Addition of superoxide dismutase did not influence the course of chemiluminescence. Whereas catalase produced 41% increase in quantum yield, mannitol caused a 51% inhibition of chemiluminescence. Lymphocytes exposed to varying doses of short term x-irradiation or lymphocytes isolated from dogs kept under continuous exposure through a gamma irradiation source showed dose-related depression of chemiluminescence. Membrane factors may be involved in lymphocyte stimulation to chemiluminescence as pulse experiments with Con A and PHA revealed. It is proposed that chemiluminescence measurements may be useful in monitoring early events in lymphocyte stimulation by antigens and mitogens.

Effect of trace elements found in coal fly ash, on lymphocyte blastogenesis.

To evaluate the potential health effects of coal fly ash, a byproduct of coal combustion for electrical energy production, on the immune system, we studied the effects of trace elements found in fly ash on lymphocyte blastogenesis. Of the sixteen trace elements studied, seven inhibited lectin-induced lymphocyte division, six showed no inhibition and three produced inconsistent effects. The ranking of the toxicity of the elements is Mn, V, As (III), Cu, Cd, Se, and Be. Our data indicate that whole blood lectin-induced lymphocyte blastogenesis is a sensitive and reproducible test for in vitro screening of trace elements affecting the immune system.

Time and dose-response study of the effects of vanadate on rats: morphological and biochemical changes in organs.

Vanadate at a dosage level of 0.9 mg V/kg per day produced acute toxic signs in rats when injected subcutaneously for 16 days. These signs were weakness, loss of appetite, dehydration, significant reduction in body weight, nose bleeding, and death. The pathological and biochemical changes were most severe in kidney tissue. The kidney lesions were bilateral and multifocal. At two days, degenerative and necrotic changes of the tubular and glomerular epithelium, thickening of glomerular membrane, vascular congestion, and edema were observed. At five days, proliferation of tubular epithelial and interstitial cells was observed. At 12 days, the cellular proliferation in both cortex and medulla was significantly greater. Fibrosis was observed at glomerular tuft, preglomeruli, pretubules, and interstitium (cortex and medulla). At 25 days, the collagen deposition reached the highest level in all regions, cellular proliferation decreased, and thickening of the arteriolar wall became prominent. The renal lesions were coupled with changes in the levels of protein, RNA, DNA, and hydroxyproline. At 40 days, the kidney showed signs of recovery. Blood urea nitrogen levels were significantly elevated at 2-25 days post-treatment. Stained tissue sections from liver, lung, heart, spleen, thymus, lymph nodes, testes, and adrenal glands of the treated rats were examined microscopically and appeared normal. Biochemically, significant changes (p less than .05) in protein, RNA, DNA, and hydroxyproline were also observed in these organs. At lower dosage (0.6 mg V/kg per day for 16 days), similar but less severe pathological and biochemical changes in kidneys and other organs were observed. At 0.3 mg V/kg per day for 16 days, the changes in the tissues were detected only at the biochemical level. These results indicate that the toxic effects of vanadium are cumulative and that vanadium-produced fibrosis in tissues is dose-dependent.

Canine osteosarcoma karyotypes from an original tumor, its metastasis, and tumor cells in tissue culture.

Radiation-induced osteosarcoma, its metastasis, and cells grown in tissue culture were karyotyped. Both hypodiploid and hyperdiploid stem lines were observed. The hypodiploid line contained 45-55 chromosomes with 10-15 abnormal metacentric and submetacentric chromosomes and one subtelocentric marker. The hyperdiploid line contained 90-105 chromosomes with 20-30 abnormal metacentric and submetacentric chromosomes with two subtelocentric markers. Karyotypic analysis can be used to monitor osteosarcomas maintained in tissue culture.

Double electroimmunodiffusion: a rapid diagnostic test for canine coccidioidomycosis.

Double electroimmunodiffusion (EID) was adapted for detection of antibodies to Coccidioides immitis. In a limited experiment with canine serums, the test was found to be qualitatively as sensitive as the complement-fixation (CF) test. Advantages of EID over CF are that EID takes only 30 minutes to perform and requires only 10 mul each of antigen and antibody.

Lymphocyte transformation in the dog: response of lymphocytes from normal and immune dogs to phytohemagglutinin, coccidioidin, and purified-protein derivative.

Antigen- and mitogen-induced in vitro transformation of dog lymphocytes was quantitated by pulse labeling with 3H-thymidine (3H-TdR). Dosages of antigen and duration of incubation were varied to determine the dose and incubation time that would allow a clear distinction between sensitized and nonsensitized dogs. Each of the 2 antigens tested, coccidioidin and purified protein derivative of tuberculin, induced a higher rate of 3H-TdR incorporation in lymphocyte cultures from dogs immunized to the homologous antigen than in lymphocyte cultures from dogs immunized to either the heterologous antigen or no antigen. Lymphocyte cultures from all dogs showed a high rate of 3H-TdR incorporation in response to phytohemagglutinin stimulation. Sequential lymphocyte transformation tests were done on 4 dogs immunized with either Calmette-Guérin bacillus alone or Calmette-Guérin bacillus in Freund's complete adjuvant. A positive response to purified protein derivative became evident in all 4 dogs 11 days after immunization and peaked at 18 days after immunization.

Delayed cutaneous hypersensitivity in the dog: reaction to tuberculin purified protein derivative and coccidioidin.

A quantitative skin test for delayed-type hypersensitivity was developed in the dog. The test procedure involved testing animals in the pinna of the ear and quantitating the reaction by measuring the change in ear thickness. Skin test reactions to tuberculin-purified protein derivative and coccidioidin were found to be specific and to correlate with the immunization histories of the 27 dogs tested. Kinetic studies on the tuberculin reaction indicated that ear thickness increased slowly following antigen injection, reaching a peak at about 48 hours. Cellular infiltrates at reaction sites were primarily responsible for the increase in ear thickness. They consisted predominantly of mononuclear cells, although a marked number of neutrophils were also present. Multiple skin tests with tuberculin-purified protein derivative and coccidioidin on 8 nonimmunized (normal) dogs indicated that a skin test was capable of actively sensitizing a portion of the animals tested.

Correlation of in vitro and in vivo tests for cell-mediated immunity in the dog.

Using tuberculin (purified protein derivative) as the test antigen, 29 dogs with different vaccination histories were tested with the lymphocyte transformation (LT) assay, the indirect agarose leukocyte migration inhibition (LMI) assay, and the skin test for delayed type hypersensitivity. All three tests were done simultaneously on each dog. The LT assay results were found to correlate well (r = 0.88) with the skin test results, whereas LMI results were found to correlate poorly (r = 0.55) with the skin test results. It was concluded that the LT assay is a more reliable measure of cell mediated immunity in the dog than is the LMI assay.

Comparison of whole blood and purified canine lymphocytes in a lymphocyte-stimulation microassay.

The optimum mitogen concentration and time required for using whole blood from dogs in a microassay were determined, and this test then was compared with a standard lymphocyte-stimulation microtest, using gradient-isolated lymphocytes, 2 different mitogens (phytohemagglutinin and concanavalin A), and 2 different culture media. Statistical analysis of the data from 10 dogs showed that whole blood was significantly more reactive than were gradient-isolated lymphocytes (P less than 0.05). Waymouth's medium was significantly better than RPMI 1640 (P less than 0.001), and concanavalin A was significantly more mitogenic than phytohemagglutinin (P less than 0.001). The interaction between lymphocyte source and mitogens was the only one of the various interactions that was significant at P less than 0.05.