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.

Inhibition of lymphocyte blastogenesis by serum leachates of fly ash.

Samples of 8 ashes were leached with canine serum for 24 h to remove metal ions from the particle surfaces. The particles were removed by filtration, and the concentrations of 11 metal ions in the serum leachates were determined by atomic absorption spectrophotometry. The leachate samples were evaluated using the canine whole-blood lymphocyte stimulation test (WB/LST). The serum extracts of oil-related ashes were highly inhibitory, while lower biological activity was observed for the extracts of coal ashes. The observed inhibition in the WB/LST was correlated with the concentration of each metal ion using Kendall's rank correlation test. The highest correlations were observed for Mn and V. The results are compared with previous WB/LST studies on pure metal salts.

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.

Chronic and acute trypanosomiasis in mice: a study by in vitro cloning of lymphohaematopoietic progenitors.

A semi-solid culture system was used to study the effects of trypanosome infection in two species of mice on the propagation of progenitor cells from the bone marrow and spleen. The deer mouse (Peromyscus maniculatus) survived infection with Trypanosoma (T.) equiperdum for more than 15 days. During the first 10 days there was inhibition of development of granulocyte-monocyte colonies from progenitor cells in the bone marrow. B-lymphocyte progenitors in the spleen showed increased activity, producing colonies 140-300% above normal control groups during the same period. - Conversely, all Balb/c mice infected with the trypanosomes died within 10 days with fulminating parasitemia and massive spleen enlargement. There was a general activation of progenitor cells; B-lymphocytes from the spleen and bone marrow and granulocyte-monocyte colonies from bone marrow, although this was not sustained more than 4 days after infection. - In chronically infected deer mice the pattern of response of the bone marrow and spleen progenitor cells was significantly different over successive weekly intervals. Periodicity of response in these organs was displayed by recurring waves of activation and depression of the progenitor cells. - Thus, there were significant differences in response patterns of deer mice and Balb/c mice to T. equiperdum infection which could be established by the behavior of host lymphohaematopoietic progenitor cells in culture. We therefore suggest that such in vitro cultures may be useful in assessment of the immune response to trypanosomiasis by the host and also for the study of the pathology of both chronic and acute trypanosomiasis.

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.

Assessment of the immune responsiveness of mice exposed to a 1.5-Tesla stationary magnetic field.

Humoral and cell-mediated immune responses were assayed following a 6-day exposure of LAF1/J mice to a 1.50 Tesla (1 T = 10(4) Gauss) stationary magnetic field. In tests of the immune response to sheep erythrocytes, the number of Jerne plaques formed by spleen lymphocytes and the level of serum IgM were not significantly different for the exposed mice in comparison with control animals. Tests for mitogen-induced lymphocyte proliferation also demonstrated no significant differences in the response of spleen lymphocytes from exposed and control groups of mice.

Radiation-induced inhibition of human lymphocyte blastogenesis: the effect of superoxide dismutase and catalase.

Mitogen-induced lymphocyte blastogenesis was measured following X-irradiation (0-4 Gy) in the presence or absence of superoxide dismutase (SOD), under aerobic and anaerobic conditions. There were no significant differences between radiation survival curves under these different conditions, nor did SOD have any radioprotective effect. This demonstrates the lack of oxygen dependence of radiation-induced inhibition of lymphocyte blastogenesis. Following X-irradiation at 2 Gy, neither SOD nor catalase, alone or together, added before or after irradiation, were radioprotective. In comparison to controls, both enzymes depressed lymphocyte proliferation when added at levels as low as 25 microgram catalase or 100 microgram SOD/ml media. When SOD and catalase were added together, the greatest depression of blastogenesis was obtained with increasing levels of SOD relative to increasing levels of catalase, indicating that SOD was largely responsible for this depression. The suppressive effect of administration of SOD (p less than 0.05), catalase (p less than 0.001) and SOD + catalase (p less than 0.001) on lymphocyte division was significantly greater when given prior to X-irradiation. The lack of an oxygen effect and the inability of SOD and catalase to protect human lymphocytes from X-irradiation suggest that 2- and /or H2O2 are not involved in radiation-induced inhibition of lymphocyte blastogenesis.

Growth of macrophage colonies from normal canine peripheral blood: morphological, cytochemical and functional parameters.

Canine macrophage colonies were grown at high colony-forming efficiencies (average of 4.5 macrophage colonies/10(4) mononuclear cells plated) from gradient-separated peripheral blood mononuclear cells. The colonies were first observed on day 5 of the culture period, reached maximum numbers between days 10-14, and differed kinetically from CFU-GM colonies. Colony cells had typical macrophage morphology at the cellular and ultrastructural levels, were nonspecific-esterase positive, specific-esterase negative, and were actively phagocytic. Colony growth in semisolid cultures and 3H-TdR incorporation in liquid cultures occurred following stimulation with rabbit anti-canine immunoglobulin antisera (anti-Ig), anti-bovine serum albumin and normal rabbit serum. Addition of 2-mercaptoethanol (2-Me) to stimulated cultures significantly enhanced the proliferative response. A maximal response was obtained using anti-IgM and 2-ME. Preincubation of anti-Ig with goat anti-rabbit IgG or purified canine immunoglobulin in the presence or absence of 2-ME significantly reduced the proliferative response, suggesting the presence of both specific and nonspecific components of stimulation. The growth of canine macrophage colonies from peripheral blood provides a method for non-invasive, sequential and kinetic studies of macrophage progenitor cells in large animals.

Growth of human T lymphocyte colonies from whole blood: culture requirements and applications.

Growth of human lymphocyte colonies from whole blood following stimulation with PHA, Con A, or PPD is described. Individual colony cells were identified as T lymphocytes on the basis of surface marker and enzyme cytochemical characterizations. Colony formation increased as a power function over a wide range of cell concentrations above a critical minimal concentration. The whole blood culture system eliminates possible selective effects of lymphocyte colony techniques utilizing gradient-enriched lymphocyte fractions and more closely approximates the in vivo milieu. The whole blood colony method is more sensitive for the detection of low-level radiation effects on lymphocytes than widely used tests that measure 3H-thymidine incorporation. In preliminary studies, we used the whole blood method to determine the relative radiosensitivity of lymphocytes from humans with various hematopoietic disorders, and observed abnormalities in mitogen responsiveness and colony formation in some of the patient groups. This method has wide application for studies in cellular and clinical immunology.

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.

Increased radiosensitivity of a subpopulation ot T-lymphocyte progenitors from patients with Fanconi's anemia.

In vitro radiation survival of peripheral blood T lymphocytes was studied in 15 clinically normal adults and 4 patients with Fanconi's anemia. Tritiated thymidine incorporation in a whole blood lymphocyte stimulation test (LST) and a newly developed whole blood T-lymphocyte colony assay were used to measure lymphocyte blastogenesis and colony formation in response to phytohemagglutinin (PHA) or concanavalin-A (Con-A) stimulation. Lymphocyte colony formation was found to be consistently more sensitive than the LST for detection of low-level radiation effects using both normal cells and lymphocytes from Fanconi's anemia patients. Lymphocytes from patients with Fanconi's anemia were significantly more sensitive to in vitro x-irradiation than lymphocytes from clinically normal individuals as measured by their ability to divide when stimulated by PHA in the LST (patients, D37 = 198 R; normals, D37 = 309 R, p = 0.057) and colony formation assay (patients, D37 = 53 R; normals, D37 = 109 R, p = 0.016). No significant difference in the radiosensitivity of the Con-A response was observed between the two groups. The PHA-responsive T-lymphocyte subpopulation in Fanconi's anemia patients appears to be intrinsically defective. The nature of this defect, significance in the disease process, and relevancy of these findings to the establishment of radiation protection standards are discussed.

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.

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.

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.

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.