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Background: In previous studies we have shown that pre-transplant hamster blood transfusion (HBT) can induce non-responsiveness in the T cell independent immunecompartment and result in tolerance towards hamster cardiac xenografts (Xgs) in T cell deficient athymic nude rats. In this study we test the combination of pre-transplant HBT with cyclosporin A (CSA) in immunocompetent Lewis rats. Methods: Before transplantation of a hamster cardiac Xg, 1 ml hamster blood was administered to nude rats or Lewis rats. CSA dissolved in olive oil was given orally at varying doses. Anti-hamster antibodies were measured by flowcytometry. Results: In nude rats HBT 3 days before transplantation resulted in 100% long-term survival >100 days (n = 9). In Lewis rats, HBT resulted in hyperacute rejection (HAR) (n = 6). Treatment of nude rats with CSA at doses varying from five to 20 mg/kg/day and treatment of Lewis rats with CSA five or 10 mg/kg/day did not effect Xg survival. However, treatment of Lewis rats with CSA 20 mg/kg/day led to long-term survival of five of nine Xgs (p

In the present study, attempts are made to improve posttransplant survival of intraportally transplanted hepatocytes in a syngeneic hepatocyte transplantation (HTX) model. Engrafted hepatocytes were detected and quantified after pretransplant labeling with 5'-bromo-deoxy-uridine. In order to enhance the survival percentage, three mechanisms that possibly influence cell survival were manipulated: (1) administration of Matrigel (extracellular-matrix components, Matrigel Basement Membrane Complex, Micronic BV, Lelystad, The Netherlands) in order to improve the environmental conditions of the transplanted hepatocytes; (2) immunosuppression of the recipients by 5 Gy total body irradiation and cyclosporin-A administration (25 mg/kg, 3 x weekly), and (3) Kupffer cell depletion by injection of dichloromethylene-diphosphonate-filled liposomes. The results were analyzed in relation to HTX without treatment. Cell survival was approximately 14% (10 days after HTX), and the transplanted hepatocytes were distributed equally throughout the various recipient liver lobes. However, no increase was attained by Matrigel coadministration, immunosuppression, or Kupffer cell depletion.

To evaluate whether further improvement in porcine islet xenotransplantation is feasible, a number of questions were addressed. Earlier we showed significant improvement in the nude mouse of the porcine islets by selection through long-term culture. Now these islets were tested in the stringent pig-to-rat model. Islets were isolated from adult pigs, cultured for 1.5–3 weeks and transplanted to rats. Possible rejection mechanisms were assessed by interference of the cellular response with cyclosporine A (CsA), blocking macrophages with gadolinium chloride (GdCl), and suppressing the humoral response with cyclophosphamide. Modifications in graft size and condition were analyzed. Untreated control recipients showed primary nonfunction (PNF). CsA treatment could fully overcome PNF and resulted in graft survival from 10 to over 134 days. Rejection was the main cause of function loss. Although rejection could not be prevented by intensifying the induction therapy, increased maintenance immunosuppression effectively blocked rejection, albeit at the expense of toxicity. Blocking the humoral response was ineffective; all grafts showed PNF. In contrast, depletion of macrophages fully prevented PNF. Combination of GdCl and CsA gave no additional effect, and grafts were rejected between 57 and 162 days. Generally, graft survivals were similar to those reported in the literature; however, long-term cultured islets required much less maintenance immunosuppression. Cessation of graft function was not always due to rejection; in some cases “islet exhaustion” was found, possibly caused by discrepancy between the graft size and the rapidly growing recipient. Neither the presence of damaged islet tissue in the graft nor the size of the graft exerted any influence on graft survival. On rejection, no real infiltration of the graft was seen; destruction gradually processed from the outside. The good functional capability of the cultured islets was illustrated by disappearance of the clinical symptoms and increase in body weight, which almost doubled in the long-term survivors.

Most studies on the immunomodulating effects of interferon-gamma (IFN-gamma) have been performed in vitro, using recombinant mouse and human IFN-gamma preparations. Recently, recombinant rat IFN-gamma (rRIFN-gamma) became available, enabling extensive studies with this new preparation in vivo. In the present study a LEW rat model was used to determine the efficacy of rRIFN-gamma on immune functions in vivo. LEW rats were treated with rRIFN-gamma by continuous intravenous infusion at a dosage of 1.5 x 10(5) U/kg.h for 2 consecutive days. Twelve hours after cessation of rRIFN-gamma administration immune functions, including NK-cell activity, phagocytosis, and mitogen-induced blastogenesis, were assessed. All experimental animals displayed a marked reduction in the number of peripheral blood and bone marrow cells when compared with controls (p less than 0.005). Assessment of immune functions revealed a significant enhancement of NK-cell activity (p less than 0.001), phagocytosis (p less than 0.05), and mitogen-induced blastogenesis (p less than 0.05). These findings indicate that rRIFN-gamma, when given in high dosages, has a stimulatory effect on various immune functions, which substantiates its important immunological role in vivo.

The effects of pre-transplant blood transfusion vary from induction of antibodies and accelerated graft rejection, to prolonged survival and even tolerance. The beneficial 'transfusion effect' in allotransplantation is believed to be merely T-cell mediated. In xenotransplantation, T-cell independent mechanisms form a major hurdle. In this study we investigated the effects of pre-transplant hamster blood transfusion on the survival of hamster cardiac xenografts in T-cell deficient athymic nude rats. Nude rats rejected xenografts after 3.8 +/- 0.5 d (n = 8), and immunocompetent Lewis rats after 4.0 +/- 0.5 d (n = 8), following a similar IgM response (P = NS). Hamster blood transfusion 3 d before transplantation in nude rats led to an IgM response and long-term xenograft survival in 17/20. Timing was of importance: blood transfusion 7 d before transplantation resulted in 45% long-term xenograft survival (n = 20). Injection of purified hamster erythrocytes, leukocytes or minced heart also led to survival of xenografts for100 d in nude rats, but not in all cases. Second xenografts transplanted to long-term survivors did not provoke an IigM response, and were accepted for100 d (n = 4). Transfer of serum from long-term survivors to untreated nude rats resulted in survival of xenografts for100 d (n = 4). In Lewis rats pre-transplant blood transfusion induced hyperacute rejection of xenografts after 158 +/- 128 min (n = 8, P0.01). We conclude that pre-transplant hamster blood transfusion can induce long-term survival of hamster cardiac xenografts in T-cell deficient athymic nude rats. This blood transfusion effect is mediated by humoral factors and can be transferred by serum. Elucidation of underlying mechanisms might provide some insight into xenotransplantation nonresponsiveness of T-cell independent immunefactors.

Antibodies play a crucial role in the rejection of xenografts. We tested the hypothesis that xenografts are protected against antibody-mediated attack early after transplantation in a concordant model. We investigated the role of xenoreactive antibodies as a stimulus for protection and the effects of a total blockade of the antibody response by the leflunomide analog malononitrilamide 279. Hamster cardiac xenografts were transplanted to Lewis rat recipients. Second transplants and retransplants of xenografts were performed to untreated rats that had a xenograft in place for 3 d. Untreated rats rejected hamster cardiac xenografts after 4.0 +/- 0.0 d. Significant levels of anti-donor IgM, as measured by flowcytometry, were present on day 3 after transplantation (11.2% +/- 2.8 vs. 1.2% +/- 0.0 on day 0, P0.001). 'Fresh' second xenografts transplanted to rats that had a first xenograft in place for 3 d and had anti-hamster antibodies, underwent hyperacute rejection. The first xenografts remained functioning. Xenografts that were removed on day 3 from untreated rats and then retransplanted remained functioning. Xenografts that were removed on d 3 from rats that had been treated with malononitrilamide 279, 15 mg/kg/d and were retransplanted underwent hyperacute rejection. IgM levels at the time of removal were 1.1% +/- 0.5 in these rats and not different from baseline (P = 0.96). We conclude that xenografts are protected against antibody-mediated damage early after transplantation. The presence of anti-donor antibodies might be an essential stimulus for the induction of protection. There seems to be a delicate balance between the injurious and protective effects of antibodies. Treatment strategies that are designed to block antibody formation completely might prevent the induction of protection.

The effect of recombinant rat interferon-gamma (rRIFN-gamma) on allograft rejection was studied in two rat heart transplantation models. Recipients were treated with rRIFN-gamma by intraperitoneal or intravenous injection, either by bolus or continuous infusion. Treatment was started after transplantation and continued during a period ranging from 4 to 10 days; dosages varied from 2.5 x 10(2) U/kg/day to 3 x 10(6) U/kg/day. Controls were infused with PBS. Treatment with rRIFN-gamma had no effect on allograft survival, irrespective of the route of administration, the dosage used or the duration of treatment. Higher dosages of rRIFN-gamma induced serious morbidity and mortality. In conclusion, systemic treatment of cardiac allograft recipients with rRIFN-gamma has no effect on graft rejection and is associated with serious toxicity and mortality when high dosages are used.

The present study was initiated to study the efficacy of donor pretreatment with interferon-gamma to induce class II antigen expression in heart tissue, and to investigate whether this pretreatment would influence heart allograft survival. BN rats were used as donors, and LEW rats as recipients. During a period of 3 consecutive days prior to transplantation, IFN-gamma was administered to BN rats via continuous intravenous infusion at dosages of 10(3), 10(4), and 5.10(4) U/hr. Control animals were infused with PBS; each group consisted of 9 animals. Analysis of IFN-gamma induced class II expression by immunoperoxidase staining revealed a significant, fourfold increase in the number of dendriticlike cells, irrespective of the IFN-gamma dose given (controls: 15 +/- 4 vs. highest dose group: 57 +/- 9 cells/mm2; P less than 0.005). Endothelial cells of arteries and venules remained class II antigen negative. Grafting of hearts from IFN-gamma perfused donors to untreated recipients (6 animals per group), did not result in a shortened or prolonged survival time in any of the experimental groups, as compared to controls. These results indicate that upgrading of class II antigen expression on dendriticlike cells is not likely to be of importance for the process of rejection.

Primary nonfunction (PNF) of hamster islets occurs when they are transplanted under the kidney capsule of diabetic Lewis rat recipients. PNF is absent if the islets are grafted into the liver. Previous results have indicated that macrophages might be involved in the phenomenon of PNF. To test this hypothesis, two procedures affecting macrophages were employed. First, recipients were pretreated with liposomes containing dichloromethylene disphosphonate (L-MDP), killing macrophages upon phagocytosis. Second, recipients were pretreated with the arginine analogue, L-NA, in order to inhibit the synthesis of nitric oxide (NO), a molecule believed to mediate beta cell dysfunction. Injection of L-MDP into the peritoneal cavity had no effect on PNF. However, co-transplantation of L-MDP with hamster islets under the kidney capsule reduced PNF significantly. Eradication of Kupffer cells with L-MDP did not prolong the survival of islets transplanted into the liver, indicating that acute xenogeneic rejection in this model is not mediated by macrophages. A single injection of L-NA, 3 h before transplantation resulted in complete annihilation of PNF, all recipients became normoglycemic within 1 day and remained so for 1.4+/-0.5 days. These results confirmed the finding that macrophages and NO play a crucial role in PNF of islet grafts.