A search for the optimum conditions for culturing allografts before transplantation.
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Biomedical subjects
Publications and source records attributed to D W Talmage.
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The blastogenic and mitogenic response of murine splenic lymphocytes to concanavalin A and the response of the resulting blasts to a lymphokine co-stimulator preparation was examined with an electronic particle counting and sizing apparatus (Coulter Counter and Channelizer). Mitogenesis, as determined by increase in cell number, correlated well with the time-integrated uptake of tritiated thymidine. It was found that co-stimulator was highly mitogenic to the lectin-induced blasts, and that the cells appearing in response to it were uniformly large (co-stimulator blasts). Furthermore, the eventual increase in cell number was proportional to the co-stimulator concentration, at least for the concentrations, cell numbers, and incubation times examined. This relatively simple assay that uses the Coulter Counter and Channelizer is therefore capable of substituting for serial measurements of tritiated thymidine uptake and yields additional information on the volume spectra of responding cells, as well as forming the basis for assaying co-stimulator activity. The co-stimulator blasts were found to be capable of killing a wide variety of allogeneic as well as syngeneic concanavalin A induced blasts. The level of killing in a 3-hr 51Cr release assay varied between 9% (for the syngeneic case) and 24% of the maximum obtained by water lysis.
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The primary and secondary cytotoxic T cell responses of C57Bl/6 (H-2b) mouse spleen cells to P-815 membrane fragments (H-2d) were examined. The primary response required the addition of a supernatant from mitogen-stimulated spleen cells, or costimulator, to the culture medium. Costimulator had little effect on the secondary response unless the spleen cells were first passed through a nylon wool column. Our data suggested that secondary cultures produced their own costimulator and that adherent cells were required for its production. The possibility that adherent cells are needed to activate helper cells is discussed.
The intraperitoneal injection of gamma-radiated, UV-radiated, or unradiated P-815 tumor cells into syngeneic or allogeneic mice suppressed the immune response to a subsequent intraperitoneal injection of sheep erythrocytes in a manner similar to the suppression caused by the injection of manner similar to the suppression caused by the injection of horse erythrocytes and termed antigenic competition. In both cases, the greatest suppression occurred when the sheep erythrocytes were injected at the same site (intraperitoneal) as the tumor or antigen several days after the tumor or antigen and in a dose of 10(8) erythrocytes or less.
The effects of different conditions of organ culture on the viability of murine pancreatic tissues and, particularly, of pancreatic islets, was assessed by morphologic and histochemical analyses. High oxygen tension (1300 mm Hg), found previously to allow successful allogeneic thyroid transplantation, was toxic to pancreatic tissue. Low temperature (22 degrees C) was associated with better islet preservation than high temperature (37 degrees C). Hydrocortisone did not reduce the survival of pancreatic islets and did appear to preserve the acinar components. Fetal and neonatal pancreas responded similarly to the various organ culture conditions. These results have potential importance in guiding future transplantation experiments.
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A marked increase in the percentage of mouse thyroids that retained function 20 days after transplantation across a major histocompatibility barrier and the percentage that lacked generalized infiltration was observed when the grafts received hyperbaric oxygen during a 4-day culture period. Perfusion of the donor animal before thyroidotomy and the addition of fetal calf serum to the culture medium did not have a significant effect on graft survival, but the percentage of grafts lacking generalized infiltration was slightly increased by the addition of hydrocortisone to the culture medium.
In vitro T cell activation requires both antigen presentation and a second stimulus provided by the lymphocyte costimulator. Neither alone is sufficient to induce specific T cell activation. The S+ phenotype of stimulating cells is dependent on the metabolic activity of these cells. This finding is consistent with the notion that production and/or release of the costimulator is a function of metabolically active cells. The costimulator acts at an early stage of the interaction between lymphocyte and antigen, and the costimulator, or a separate maintenance factor, is required throughout the culture period for the expression of full cytotoxic activity. The lymphocyte costimulator is not strain specific but is phylogenetically specific. The activation of cytotoxic T cells by S+ cells is also phylogenetically specific, and this specificity of cellular activation can be accounted for by the species specificity of the lymphocyte costimulator.
Arginine was found to be completely depleted from cell-free supernates of mixed leukocyte cultures suppressed by the addition of excess macrophages. Partial reversal of macrophage-mediated suppression was accomplished by daily addition of a cocktail containing arginine and nonamino acid nutrients. Complete reversal of the suppression was accomplished by the addition of arginine and glucose to the medium and the nonadherent cells after their separation from the adherent macrophages. A marked increase in the enzyme arginase was found in macrophages that had been cultured 24 h in vitro in Eagle's minimum essential medium plus 10% fetal calf serum, in peritoneal cells activated by prior injection of thioglycollate, and in one spleen activated by a graft vs. host reaction.
The ability of three cultured mouse tumor lines to stimulate a cytotoxic response in 5-day cultures of allogeneic lymph node cells was studied with a 51Cr release assay. Two lines of mesenchymal origin, P815 and EL-4, were found to be highly stimulatory, whereas the third cell line, CaD2, a mammary gland epithelial tumor, did not stimulate over a wide range of cell concentration. CaD2 cells were shown to contain major antigens similar to those of P815 cells by the specific lysis of both cells by lymphocytes activated to H-2d-bearing peritoneal cells.UV-irradiated P815-cells, like gamma-irradiated CaD2 cells, did not stimulate a cytotoxic response, but both cell lines were found to stimulate a full and specific response to allogeneic lymph node cells if these mixed cultures were supplemented with a supernatant harvested from concanavalin A-stimulated spleen cells.
The role of the thymocyte in its species-specific binding to macrophages has been explored. Although formalin treatment of macrophages resulted in loss of binding to thymocytes, formalin treatment of thymocytes did not have this effect. However, two differences between living and formalin-treated thymocytes were noted. Formalin-treated thymocytes bound to macrophages of any species whereas the binding of living thymocytes was species specific. Living thymocytes attained maximum binding in approximately 1 hr and then the fraction bound gradually diminished. Formalin-treated thymocytes remained bound to the macrophage and appeared to be phagocytized. Released thymocytes did not bind to fresh macrophages, but released macrophages bound to fresh thymocytes. The results suggest that the binding of thymocytes to macrophages results in maturation of thymocytes.
Monocytes were isolated by counterflow contrifugation from human peripheral mononuclear cells obtained from whole blood by the Ficoll-Hypaque technique. Examination of volume spectra of the monocytes showed a bimodal distribution with peaks at 330 and 370 micron3. The upper peak contained 61.4 +/- 3.3% of the total monocyte population, whereas the remainder were in the lower peak which overlaps the lymphocyte spectrum. The purity of the monocyte preparations from six normal adult donors, identified by peroxisomes observed by electron microscopy, was 90.4 +/- 1.6%.
The survival of thyroid allografts in mice was prolonged by either holding the grafts in vitro culture for 20 to 27 days or by cobalt-60 irradiation of the donor 2 days before transplantation with or without the intravenous injection of colloidal carbon just before removing the thyroid from the donor. In both cases the rejection process was restored by an intravenous injection of recipients with living peritoneal exudate cells (50 to 80 percent macrophages) syngeneic to the thyroid donor.
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Mouse thyroid can be maintained in organ culture for 4 weeks. Uncultured BALB/c thyroid is rejected 10-15 days after transplantation under the kidney capsule of H-2 disparate recipients (C57BL, CBA). Organ culture of thyroid tissue prior to transplantation prolongs allograft survival. This prolongation of graft survival increases with increasing time in culture and 80-90% of BALB/c thyroids maintained in culture for 26 days survive in allogeneic CBA recipients for a 60- to 70-day test period. These allografts show normal function as measured by 125I uptake, and show no histological evidence of chronic rejection. Cultured allografts can be rejected if the host's immune system is stimulated with viable leukocytes of donor origin. Host animals carrying a functioning allograft are not tolerant of donor tissues and will reject a second uncultured allograft from the same donor strain.