Pathophysiological effects of human TNF-alpha-producing tumor xenografts in immunosuppressed mice.

Groups of CBA mice immunosuppressed with anti-thymocyte serum (ATS) treatment were xeno-transplanted with either HeLa human cervical carcinoma cells or genetically modified cells expressing the human tumor necrosis factor-alpha (TNF) gene (All cells). Both cell lines were highly resistant to the cytotoxic effects of TNF. If 3 x 10(6) tumor cells were inoculated s.c. into female mice, HeLa cells grew progressively into large tumors and killed 74% of the recipients, while TNF-expressing All cells caused fatal tumor growth only in 22% of the mice. 3 x 10(6) or 1.5 x 10(7). All cells produced progressive tumor growth and lethality in all male recipients. In sera of all the A11-cell-transplanted mice, biologically active TNF was detected shortly (4.5 h) after tumor inoculation (6 39 U/ml), decreasing to below detection level in the circulation by day 3. In recipients of 15 million A11 cells, circulating TNF reappeared and reached high levels (12-1000 U/ml) 3 to 7 weeks later, when the animals bore large tumors (14-23 mm). Generally, such mice became cachectic, severely anemic, hypothermic, and soon died. On account of calcium mobilization from bones, their serum Ca levels were high. Electron microscopy revealed severe liver damage, but there were no signs of chronic arthritis. These results suggest that ATS-treated mice xenotransplanted with TNF-gene-transfected A11 human tumor cells provide a new model for studying the pathophysiological and anti-tumor effects of TNF.

Impaired T cell functions preceding lymphoproliferative disorders in mice neonatally tolerized to transplantation antigens.

In A/J (H-2a) (A) mice, the neonatal i.v. injection of (B10 x A)F1 spleen cells (SC) induces partial transplantation tolerance (TT) to C57BL/10ScSn (H-2b) (B10) skin allografts, chronic host-versus-graft disease (HVGD) and lethal lymphoproliferative disorders (LPD). They produce anti-T-cell autoantibodies (ATA), and the proliferative responses of their SC to the T cell mitogen Con A are decreased. We found that, similar to ATA, the hyporeactivity of T cells developed earlier (at 1-2 weeks of age) than splenomegaly. The proportions of both CD4+ and CD8+ T cells were not reduced in the spleens of tolerized mice without manifest LPD. The supernatants (SN) of Con A-stimulated tolerized SC contained no, or only small amounts of interleukin-2 (IL-2). Thus, in the tolerized mice, ATA and T cell deficiency preceded the development of LPD. ATA and the decreased amount of the T cell growth factor IL-2 might play a role in the defective T cell activation.

Host-versus-graft disease in mice after the induction of neonatal transplantation tolerance by two different methods.

Newborn A mice were injected either with a single i.v. dose (Group A) or with repeated doses of (B10 x A)F1 semiallogeneic spleen cells (SSC) (Group B). A similar degree of partial transplantation tolerance (TT) to B10 skin allografts was revealed in both groups. No signs of acute, rapidly fatal host-versus-graft disease (HVGD) (anemia, leukocytosis, severe thrombocytopenia, hepatic infarcts, gastrointestinal bleedings) were found, rather a chronic HVGD developed [moderate thrombocytopenia, autoimmune antithymocyte antibodies (ATA)] in both groups. The mortality due to lymphoproliferative disorders (LPD) was significantly higher in Group A. Thus, repeated perinatal injections of (B10 x A)F1 SSC into A mice did not increase the tolerogenic and the LPD-inducing effect either, and they did not elicit acute HVGD in contrast to observations in other F1 donor-recipient combinations [1]. Consequently, the development of acute HVGD depends on immunogenetic factors and not on the repeated administration of SSC.

Spleen cells from antithymocyte serum pretreated mice do not induce GVHD but exert increased repopulating activity in irradiated semiallogeneic recipients.

Graft-versus-host disease (GVHD) due to allogeneic bone marrow transplantation can be prevented by depleting the T cells from the marrow graft in vitro. However, the elimination of the donor T cells results in a higher frequency of graft failure, secondary infections and, in case of leukaemia, relapse. We found, that, in contrast to normal spleen cells, spleen cells from A or B10 donor mice pretreated with xenogeneic antithymocyte serum (ATS) in vivo did not induce GVHD in non-irradiated (B10 x A)F1 hybrids. Spleen cells of ATS-pretreated A donors did not cause GVHD in allogeneic CBA mice made neonatally tolerant to the A donor strain either. Furthermore, spleen cells from ATS-treated donors did not cause GVHD in irradiated F1 hybrid recipients, moreover, they decreased the lethal effect of irradiation. The in vivo ATS pretreatment improved the repopulating capacity of spleen cells in irradiated syngeneic recipients, too. The effect of the ATS treatment does not rely solely upon the elimination of T cells, since flow cytofluorometric analysis revealed only a partial depletion of both the CD4+ and CD8+ T cells of the ATS-pretreated animals. These observations may also have clinical relevance.

gamma-Hydroxybutyrate mediated protection of liver function after long-term hypothermic storage.

Liver function was measured after 20 hr of hypothermic preservation in University of Wisconsin (UW) solution and in modified UW (MUW) solution containing gamma-hydroxybutyrate (GHB). Rat livers were rapidly cooled by in situ portal flushing with chilled UW or MUW solution, then removed and stored at 4 degrees C. After 20 hr of storage, liver hemodynamics and function were studied during 90 min of reperfusion in an isolated perfused liver system. Three groups were investigated: livers flushed with and stored in a commercial UW solution for 20 hr (UW group) or in a modified UW solution with 500 mg/L of GHB added (MUW group), and livers flushed with UW solution and reperfused immediately thereafter (control group). Addition of GHB to the cold storage solution significantly improved liver function after 20 hr of cold storage. Livers in the MUW group produced bile at a much higher rate then those in UW group (3.47 +/- 0.34 vs. 0.87 +/- 0.29 ml/100 g liver weight/min at 60 min of reperfusion), while the control livers produced 4.60 +/- 0.40 ml bile/100 g liver weight/min. At the same time, liver blood flow at a perfusion pressure of 11 cm H2O was significantly higher in the MUW group than in the UW group (391 +/- 32 ml/min/100 g liver vs. 177 +/- 33 ml/min/100 g liver) and only slightly lower than in the control group (494 +/- 49 ml/min/100 g liver). Aspartate amino-transferase (AST) and alanine aminotransferase (ALT) levels in perfusate samples taken from the venous effluent were raised during reperfusion in all groups. However, AST and ALT values were significantly lower (503 +/- 88 IU/L/100 g AST, 184 +/- 33 IU/L/100 g ALT) at 90 min of reperfusion in the MUW group than in the UW group (1567 +/- 330 IU/L/100 g for AST and 644 +/- 227 IU/L/100 g for ALT). This study clearly demonstrates that GHB greatly improves liver function and integrity after hypothermic preservation and has the potential to substantially increase the acceptable storage time of donor livers before transplantation.

Autoimmunity, hyporeactivity to T cell mitogens and lymphoproliferative disorders following neonatal induction of transplantation tolerance in mice.

We have reported that, in A/J (A) (H-2a) mice, a partial tolerance to C57BL/10ScSn (B10) (H-2b) skin allografts and a high incidence of lethal lymphoproliferative disorders (LPD) can be induced by the neonatal i.v. injection of 2 x 10(7) semiallogeneic (B10 x A)F1 spleen cells (SC) (Végh, P., Baranyi, L. and Jánossy, T., Cell. Immunol. 1990, 129: 56). In this study, we show that the incidence and mortality of LPD were continuously growing from 1 month of age until the end of the experiment at 1 year (64% and 36%, respectively). Based on histology, 27% of the diseased mice suffered from lymphoid malignancies. In the remaining cases (73%), reactive histopathological changes were seen in the spleen, lymph nodes (LN), liver and kidneys. The proportion of CD4+ T cells in the spleen and LN as well as that of splenic B cells decreased, while the percentages of mature and immature myeloid cells doubled. The total cell number of each (sub)population, however, was elevated in both lymphoid organs. The cells taking part in the lymphoproliferation were of host (A) and not of donor (F1) origin. Preceding the development of apparent LPD, the SC, LN cell and thymus cell suspensions of 1-month-old tolerized mice showed reduced in vitro proliferative responses to T cell and T cell-dependent B cell mitogens (Con A or PWM), while their reactivity to a T cell-independent B cell mitogen (lipopolysaccharide) was essentially unimpaired. This hyporeactivity seems to be functional, because neither histology nor immunophenotyping by flow cytometry revealed significant alterations in the spleen and thymus of such animals, apart from a slight reduction in the ratio of CD4+/CD8+ T cell subpopulations in the spleen. The in vivo T cell-mediated immune response of the tolerized mice was practically normal to third party CBA/Ca (H-2k) allografts. Antithymocyte autoantibodies (ATA) were detected in the sera of 76% of the tolerized mice at 1 month of age (i.e., even before the mass appearance of LPD). ATA as well as antinuclear Ab were present in 65% of the adult tolerized mice, independently of the presence of LPD. Taken together, in A mice neonatally injected with (B10 x A)F1 SC, a partial, specific allograft tolerance and a chronic host-vs.-graft disease-like syndrome developed. The latter is manifested in hyporeactivity to T cell mitogens, development of autoantibodies and, subsequently, in progressive LPD and lymphoid malignancies.

MHC-specific graft-protective and delayed-type hypersensitivity (DTH) suppressive activity of a CD4+ CD8+, alpha beta T cell receptor (TCR) positive lymphoma isolated from a tolerant mouse.

Two lymphomas were found in, and isolated from A (H-2a) mice in which permanent transplantation tolerance was induced to CBA (H-2k) histocompatibility antigens by the neonatal injection of (CBAxA)F1 spleen cells. They proved to be of recipient origin and were transferable to syngeneic A mice, growing as disseminated lymphomas (L33 and L46) and killing the recipients rapidly. Analysis of the cell surface antigens disclosed that both lymphomas had an immature T cell phenotype [Thy-1+, CD5+, CD3low, TCR alpha beta low, CD4low, CD8high, heat-stable antigen (HSA) positive, and CD44-, MHC class II-, CD45R-, sIg-, Gr-1-, CD11b-]. Intraperitoneal (i.p.) injection of syngeneic A mice with viable L33 lymphoma cells resulted in a dose-dependent, significant prolongation of the mean survival times of "specific" CBA and MHC-identical B10.BR skin allografts as compared to the survival of appropriate grafts in non-lymphoma-bearing controls. The survival times of third party MHC-incompatible B10 (H-2b) and B10.D2 (H-2d) allografts were only slightly prolonged in A mice inoculated with L33 cells. The graft-protective effect was not abrogated if the proliferative capacity of the L33 cells was blocked by in vitro mitomycin C (MMC) pretreatment. Furthermore, the inoculation of L33 lymphoma into A mice significantly inhibited their DTH response to the sensitizing CBA histocompatibility antigens. In contrast, the L46 lymphoma had no effect on the survival of CBA allografts and the DTH reactivity. These data suggest that the CD4+CD8+TCR alpha beta + L33 T cell lymphoma originating from a neonatally tolerant mouse has a specific immunosuppressive effect on the in vivo reactivity of syngeneic mice to the tolerance-inducing (MHC class I) alloantigens.

Lethal systemic graft-vs-host disease in neonatally tolerant, but not in F1 hybrid mice.

The possibility, conditions, and quantitative aspects of eliciting GVHD in CBA (H-2k) mice made neonatally tolerant to the alloantigens of the donor A (H-2a) strain were studied. The intravenous injection of different doses (10(7)-2 x 10(8)) of A spleen cells caused a severe, often fatal, systemic GVHD in 12-month-old tolerant mice. The GVHD was found to be specific: spleen cells of a third party strain (B10) did not induce any disease. The intensity and the mortality of the GVHD depended on the cell dose and on the age of the recipients. In contrast, unirradiated (CBA x A)F1 recipients proved to be resistant to the lethal disease. In spite of their different susceptibility to the systemic GVHD, the tolerant and F1 hybrid recipients showed equally strong local GVH reactivity in the popliteal lymph node enlargement assay. Neonatally tolerant mice offer a new, sensitive model for the induction of lethal GVHD without the need of immunosuppression or irradiation.

Abrogation of neonatally induced permanent transplantation tolerance in mice: quantitative aspects.

Neonatal transplantation tolerance was induced in CBA (H-2k) mice by the intravenous injection of 20 million (CBAxA)F1 spleen cells to the transplantation antigens of the A mouse strain. Those mice which carried an A (H-2a) skin allograft without any sign of rejection for at least 120 days, were considered to be permanently tolerant and were selected for further experiments. Abrogation of permanent transplantation tolerance was achieved by injecting the tolerant mice with different doses (50, 100 and 200 millions, respectively) of normal syngeneic (CBA) lymphoid (spleen) cells. Dynamics of the rejection of the test skin allografts tolerated so far revealed well reproducible dose-response curves. Further groups of tolerant CBA mice were given 10, 50, 100, or 200 million "sensitized" (G + 16) CBA spleen cells: "sensitization" by A-skin allografting was performed 16 days before. The sensitized spleen cells abolished the state of tolerance more vigorously and effectively than the normal CBA spleen cells did. In a third group of experiments, the abrogating capacity of 50 million sensitized CBA spleen cells 16, 120, 240, or 360 days after sensitization was compared. The efficacy of the sensitized cells in abolishing the state of tolerance decreased continuously, but, even 360 days after sensitization a remarkably strong immunologic memory was demonstrable. The excellent quantitative correlations found between the number of the injected lymphoid cells and the dynamics of the abrogation of tolerance offer a highly promising new possibility for studying the immunological activity, the immunologic memory, etc., of the different lymphoid cell (sub)populations in performing the transplantation immune reactions.

Induction of transplantation tolerance and development of lymphomas in mice: lack of interdependence.

Neonatal transplantation tolerance was induced in five different H-2-incompatible donor----recipient mouse strain combinations by the iv injection of semiallogeneic (F1 hybrid) spleen cells. The highest degree of tolerance (expressed by the survival time of the specific test skin allografts) was observed in the (CBA X A)F1----CBA combination (approximately 95% permanent tolerance) but some degree of tolerance was achieved in all strain combinations. An increased incidence of malignant lymphoproliferative disorders was observed in all groups of mice which underwent neonatal tolerance induction. The highest incidence of lymphoproliferative malignancies was observed in the (B10 X A)F1----A tolerance induction system, in which approximately 50% of the recipient mice died within 1 year. In further experiments, spleen cells of mice which proved to be permanently tolerant after the neonatal tolerance induction were transferred into syngeneic, normal, adult, ATS-pretreated, allografted recipients; by this method, in approximately 50% of the recipients permanent "adult" tolerance was achieved. The spleen cells of the "adult" tolerant mice were able to transfer the tolerance to other adult, syngeneic, ATS-pretreated recipients. Even the fourth serial transfer resulted in essentially the same degree of tolerance in the new recipients. We consider the serial transfer a classical instance of "infectious tolerance" based on suppressor mechanisms. However, an increasing number (and malignancy) of lymphomas occurred in the course of the serial transfers and prevented the "indefinite" transfer of tolerance after the fourth occasion. We conclude that both the degree of transplantation tolerance and the high frequency of lymphomas are determined by (immuno)genetic factors but the two phenomena are not interrelated. Thus, successful transplantations do not seem to be necessarily accompanied by an increased incidence of malignancies.

Anesthesia approach to hepatic transplantation.

Anesthesia support for patients undergoing orthotopic liver transplantation can be complicated because of multiple medical problems in such patients and rapid hemodynamic, metabolic, and coagulation changes intraoperatively. Preoperative assessment should include careful review of the cardiovascular, respiratory, and hematologic systems. Use of isoflurane as the main anesthetic agent will minimize toxicity to the liver. During liver transplantation, hemodynamic monitoring and immediate laboratory studies should be available. In our experience during the first 100 liver transplantations performed at our institution, use of a rapid infusion pump and venovenous bypass has helped normalize hemodynamic and renal function.

Hemodynamic and metabolic changes in hepatic transplantation.

In this study, we retrospectively analyzed the intraoperative hemodynamic, laboratory, and coagulation data on the first 83 patients who underwent an initial liver transplantation procedure at our institution. The major hemodynamic changes at the time of reperfusion of the donor liver were significant decreases in arterial blood pressure, systemic vascular resistance, and pulmonary artery temperature and significant increases in cardiac output and pulmonary capillary wedge pressure. The alterations in laboratory values reflected intraoperative therapeutic manipulations. Citrate toxicity is a concern, and the amount of calcium chloride administered reflected the volume of blood transfused. On reperfusion, the fibrinogen concentration decreased and both the prothrombin time and the activated partial thromboplastin time increased. This coagulopathy was also evident in the thromboelastographic values. Aggressive monitoring and prompt intervention are necessary to maintain hemodynamic and metabolic homeostasis in these patients.

Plasma glucose concentrations during liver transplantation.

We reviewed the intraoperative plasma glucose concentrations in 100 consecutive patients who underwent orthotopic liver transplantation. The plasma glucose concentration increased significantly (P less than 0.05) from 110 +/- 46 mg/dl (mean +/- SD) to 204 +/- 60 mg/dl during the preanhepatic phase of transplantation (phase I). No significant change in plasma glucose concentrations occurred during the anhepatic phase (phase II). During the reperfusion phase (phase III), the mean plasma glucose concentration increased significantly (P less than 0.05) from 201 +/- 56 mg/dl to 384 +/- 72 mg/dl. The only glucose administered was that contained in the blood products. No correlation was found between the amount of glucose administered with the blood products and the changes in plasma glucose concentrations in these patients. None of the patients became hypoglycemic during any phase of the transplant procedure. All patients demonstrated a tendency toward hyperglycemia.

Mechanism of the A/Ph.MC.S1 tumor graft rejection in syngeneic mice.

A considerable number of normal mice injected with a low dose of A/Ph.MC.S1 syngeneic methylcholanthrene-induced tumor cells showed regression of the tumor after a transient period of growth. The regressor mice eliminated a challenge inoculum in an accelerated fashion. Splenic lymphocytes from such regressor mice inhibited the growth of the same tumor in the local adoptive transfer assay. This capacity required the presence of thymus-derived cells. The regressor animals developed a delayed-type hypersensitivity response against the tumor In vivo and their lymph-node cells produced macrophage migration inhibitory factor in the presence of tumor cells in vitro. The growth of this tumor was facilitated by treating the primary recipients with carrageenan, and the strong tumor-inhibitory capacity of regressors was also depressed by this agent. Early macrophage infiltration of the tumor in regressor mice was demonstrable histochemically, preceding the inhibition of tumor growth. A lymphokine-producing T cell-mediated, macrophage-dependent delayed-type hypersensitivity-like mechanism is proposed to be the dominant mechanism of the resistance in this tumor-host model.

Manifestation of K+ transport alterations in cultured tumour cells of mice.

Sarcoma was induced by injection of human adenovirus type 12 into newborn, isogeneic CBA mice and maintained in adult female CBA mice by serial passages. Cells obtained from the tumours were cultivated by 3-4 passages in vitro. Normal fibroblastic cell cultures were gained by the same manner from isogeneic CBA mouse embryos. Characteristics of potassium transport in cultures of malignant cells and of normal fibroblastic cells were analysed. As a chemical tracer of K+ movements, 86Rb+ was applied. No significant difference could be detected either in the potassium concentration, or in the 86Rb+ uptake of the two types of cultured cells. However, when the cells were exposed to ouabain, the malignant cells showed a significantly reduced response, thus, the malignant cells accomplished a much less decrease in either cellular potassium concentration or in that of 86Rb+ uptake rate than the normal cells. These findings well fit the hypothesis advanced by several authors that malignant cells have a reduced density of ouabain receptor on the membrane.

Studies on the mechanism of chronic graft rejection in mice.

Acute and chronic types of rejection after transplantation are distinguished. The exact mechanism, however, by which the different types of rejections have been brought about are not clarified. In this study, experimental data are presented on some characteristics of the chronic type of graft rejection. Allografting was performed between isogeneic mouse strains differing at their strong histocompatibility antigens. Recipients were treated with antithymocyte serum (ATS). Those recipients which showed a chronic type of graft rejection were selected and regrafted with a second-set or a third party allograft. It was found that, in most cases, the chronic rejection of first-set allografts did not results in a sensitization (= accelerated rejection), but rather in a partial specific tolerance (= prolonged graft survival) to a second-set allograft. Furthermore, transfer of spleen cells from mice of chronic rejection into normal, adult, ATS-treated, allografted mice did not result in any significant change of the mean graft survival as compared to mice not receiving spleen cell transfer. On the other hand, transfer of spleen cells from mice with acutely rejected grafts resulted in a shortened graft survival. It is supposed that the partial specific tolerance observed after chronic graft rejection may be maintained rather by a specific suppressor mechanism than clonal deletion.