Prolonged Graft Survival Time Research Articles

Immunomodulation by an Adenylate Cyclase Activator, NKH477,in Vivoandin Vitro

Cyclic adenosine monophosphate (cAMP) is an intracellular second messenger which modulates T cell function. NKH477 is a direct adenylate cyclase activator derived from forskolin and now under clinical investigation as a positive inotropic agent. While the immunosuppressive effects of forskolin on lymphocytes have been reported, little is known about its effectsin vivo.In this study, we investigated whether NKH477 has immunosuppressive effects in mice, namely on cardiac allograft survival, and on the generation of cytotoxic T lymphocytes (CTL), T cell proliferation in mixed lymphocyte reaction (MLR), and production of interleukin-2 (IL-2) in MLR and in mitogen response. We assessed the effects of standard immunosuppressant cyclosporin A (CsA) on IL-2 production and on allograft survival to estimate the intensity of rejection in this acute rejection model. Saline-treated C57BL/6 (H-2b) mice rejected DBA/2 (H-2d) cardiac allografts with a median graft survival time of 10 days. In contrast, median graft survival was prolonged to 12 and 15 days in mice treated with NKH477 at 1 and 3 mg/kg/day, respectively (P< 0.01 vs control). The equivalent dose of CsA (40 mg/kg/day) to the maintenance dose after clinical cardiac transplantation prolonged median graft survival time to 15.5 days, indicating that high dose of NKH477 was as efficacious as lower dose of CsA. Addition of NKH477 to the culture medium suppressed the generation of CTL, T cell proliferation in MLR, and production of IL-2 in MLR and in mitogen response. These results suggest that NKH477 exerts a beneficial effect on murine cardiac allograft survival by modulating T cell function.

The effects of combined treatment with the novel vitamin D analogue MC 1288 and cyclosporine A on cardiac allograft survival

The efficacy of combined treatment with the novel vitamin D analogue MC 1288 (20-epi-1α,25-dihydroxycholecalciferol) and cyclosporine A (CyA) in preventing rejection following organ tranplantation was evaluated in the heterotopic cardiac allograft model. Wistar/Kyoto rats received hearts from PVG donors and were subsequently treated with MC 1288 and CyA in various dose combinations. Administration of MC 1288 0.1 μg/kg together with CyA 5 mg/kg produced significantly prolonged graft survival times as compared with single therapy with MC 1288 0.1 μg/kg ( p < 0.01) or CyA 5 mg/kg ( p < 0.001). MC 1288 and CyA were tested also in combination with the immunomodulating drug LS-2616, which, in several laboratories, is used as a model for the evaluation of new combinations of immunosuppressive drugs. LS-2616 abolishes the immunosuppressive effect of CyA and the transplanted hearts are thus rejected at the same time as grafts of untreated recipients, i.e. on day 8. The immunosuppressive effect of MC 1288 is also counteracted by LS-2616, but the effect is not absolute and the median graft survival time is 11.0 days. Combined treatment with MC 1288 and CyA in the presence of LS-2616 resulted in a median graft survival time of 14.0 days. The results obtained in our experiments indicate at least an additive effect of MC 1288 and CyA thus suggesting that in the future MC 1288, or other vitamin D analogues, might be used to control rejection, either alone, in combination with CyA or in combination with other immunosuppressive drugs with other mechanisms of action.

Effect of Deoxyspergualin on Vascular Rejection in Canine Kidney Transplantation

Deoxyspergualin (DSG), an analogue of spergualin produced by Bacillus laterosporus, has a strong immunosuppressive effect in various transplantation models. In this study, we investigated the effect of DSG on vascular rejection in canine kidney transplantation.To enhance vascular rejection, donor-specific blood transfusion (DST) was carried out on days 28, 21 and 14 preceding kidney transplantation. After DST, the donor kidney was transplanted to the recipient iliac fossa. The recipient animals were divided into five groups: namely, Group 1 (n = 7), no treatment; Group 2 (n = 6), DST only; Group 3 (n = 5), DSG only (treated with DSG intravenously at 1.2 mg./kg./day for the first 3 days after transplantation, 1.0mg./kg./day for the following 3days and 0.8mg./kg./day for the following 8 days); Group 4 (n = 6), DST and DSG treatment (same protocol as Group 3); and Group 5 (n = 5), DST and cyclosporine (CsA) (treated with CsA orally at 10mg./kg./day for 14days after transplantation).In Group 2, DST treatment significantly reduced kidney graft survival time (8.6 ± 2.2 days) compared with Group 1 (14.1 ± 5.5 days). Despite DST, DSG treatment (Group 4) significantly prolonged graft survival time (29.5 ± 2.6 days), whereas treatment with CsA (Group 5) did not prolong survival time (14.1 ± 5.5 days) (Group 4 versus 5, p < 0.01).The onset of rejection was significantly delayed in Group 4 (22.1 ± 2.7 days) compared with Groups 2 (5.7 ± 2.4 days) and 5 (13.0 ± 5.7 days) (p < 0.01). In contrast, the interval between rejection onset and animal death was significantly reduced in Groups 2 (3.0 ± 0.6 days) and 5 (2.4 ± 1.0 days) compared with Group 4 (7.3 ± 1.7 days) (p < 0.01). These findings suggest that DSG successfully prevented humoral-type (accelerated acute-type) rejections.Histologically, nonDST groups (Groups 1 and 3) showed minimum vascular rejection. In contrast, all recipients in Group 2 showed severe vascular rejection, as did 80% of CsA treated-animals (Group 5). Despite DST, however, 84% of DSG treated-animals (Group 4) showed minimal or mild vascular rejection and only 17% had severe rejection (Group 4 versus 5, p < 0.04).These data suggest that both clinically and histologically, DSG has more potent immunosuppressive effects against humoral and vascular rejection than CsA.

Lipid mediated modification of rat heart allograft survival

The effect on allograft survival of intravenous fat emulsions that differed in the ratio of functionally important n-3 and n-6 fatty acids was studied in a heterotopic cardiac transplant model in rats. Twenty percent fat emulsions were administered by continuous infusion at a dosage of 9 g fat/kg body weight per day, starting immediately after transplantation and continuing until complete rejection. The n-6 and n-3 fatty acids represent 75%, 43%, 60%, and 59% of all fatty acids in safflower oil, fish oil, soybean oil, and a 1:1 mixture of safflower and fish oil, respectively. The n-6 fatty acids predominate in safflower oil (370/1) and soybean oil (6.5/1), while the n-3 fatty acids dominate in the fish oil (7.6/1). The 1:1 mixture of safflower and fish oil has the balanced composition (n-6/n-3 = 2.1/1) recommended by Kinsella and served as oil-treated controls. Continuous infusion of safflower oil, fish oil, and soybean oil prolonged graft survival time to 13.3, 12.3, and 10.4 days, respectively, compared to 6.8 days in the oil-treated controls (P < 0.01 for all comparisons). Another control group infused with saline rejected the allografts after 7.8 days (P = NS compared to oil-treated controls; P < 0.01 for all other comparisons). The data suggest that intravenous administration of polyunsaturated fat emulsions results in an immunosuppressive effect that seems to be dependent on the n-3/n-6 fatty acid ratio of the fat emulsion.(ABSTRACT TRUNCATED AT 250 WORDS)

Lipid mediated modification of rat heart allograft survival

Abstracts The effect on allograft survival of intravenous fat emulsions that differed in the ratio of functionally important n-3 and n-6 fatty acids was studied in a heterotopic cardiac transplant model in rats. Twenty percent fat emulsions were administered by continuous infusion at a dosage of 9 g fat/kg body weight per day, starting immediately after transplantation and continuing until complete rejection. The n-6 and n-3 fatty acids represent 75 %, 43 %, 60 %, and 59 % of all fatty acids in safflower oil, fish oil, soybean oil, and a 1:l mixture of safflower and fish oil, respectively. The n-6 fatty acids predominate in safflower oil (370/1) and soybean oil (6.5/1), while the n-3 fatty acids dominate in the fish oil (7.6/1). The 1:l mixture of safflower and fish oil has the balanced composition (n-6/n-3 = 2.M) recommended by Kinsella and served as oil-treated controls. Continuous infusion of safflower oil, fish oil, and soybean oil prolonged graft survival time to 13.3,12.3, and 10.4 days, respectively, compared to 6.8 days in the oil-treated controls (P < 0.01 for all comparisons). Another control group infused with saline rejected the allografts after 7.8 days (P = NS compared to oil-treated controls; P < 0.01 for all other comparisons). The data suggest that intravenous administration of polyunsaturated fat emulsions results in an immunosuppressive effect that seems to be dependent on the n-3/n-6 fatty acid ratio of the fat emulsion. The n-6 fatty acids turned out to be just as immunosuppressive as the n-3 fatty acids if each fatty acid family was applied as the main polyunsaturated fatty acid source. Soybean oil with a n-3/n-6 fatty acid ratio, coming closer to the ratio of the oil-treated controls, was significantly less immunosuppressive than safflower oil.

Down-regulation of MHC-expression on dendritic cells in rat kidney grafts by PUVA pretreatment.

It is the aim of this investigation to elucidate the cellular mechanisms responsible for graft acceptance in rat transplantation models after PUVA pretreatment of the graft. PUVA-therapy is used successfully in the treatment of psoriasis and other dermatoses with local accumulations of dendritic cells, as well as skin-lymphomas. Following these experiences Oesterwitz et al. (1989) developed a PUVA-treatment of the graft before transplantation. In this respect PUVA means: i. v. injection of the photoactive substance 8-Methoxypsoralen into the donor and in vitro exposure of the kidney graft to long wave UVA-irradiation. This PUVA therapy significantly prolonged graft survival times in rats even across a strong MHC-barrier. It has been shown, that 40% of PUVA treated rat kidneys are accepted by the recipient for more than 100 days. MHC-class-II-positive dendritic cells present donor specific alloantigen to the recipient and induce rejection. PUVA treatment reduces the expression of MHC-class-II-molecules and may lead to tolerance of the recipient towards the graft.KeywordsDendritic CellPerivascular SpaceKidney GraftLymphatic OrganPUVA Treatment

Inhibition of rejection of hamster-to-rat heart xenografts.

Prolonged survival of concordant organ xenografts as typified by hamster-to-rat heart transplants is difficult to produce. Studies have revealed that T cells are not primarily involved in rejecting such xenografts and that the rat recipients produce high titres of lytic anti-hamster antibodies. In this study, 200 hamster-to-rat cardiac xenografts performed in 30 different experiments revealed that cyclophosphamide (CyP) and cyclosporin A (CyA) could inhibit this antibody production. CyP alone was relatively ineffective in prolonging graft survival (the median survival time was 14 days versus 3 days in untreated controls). Combining CyP and CyA virtually abolished rejection in this model. Four critically timed doses of CyP combined with continuous CyA resulted in recipients not producing anti-hamster antibodies, despite cessation of CyP therapy, and prolonged graft survival time (median survival time was more than 100 days). Cessation of CyA at 60 and 100 days resulted in the rejection of the xenografts and the appearance of the rat anti-hamster antibodies. Xenografts in recipients given only one or two doses of CyP (and continuous CyA) had a median survival time of 7 and 12 days respectively. However xenograft rejection in rats given only 1 or 2 doses of CyP could be averted by complement depletion using a 3-week course of cobra venom factor (CoF) starting on day 4 or day 7 post-transplantation respectively. Discontinuation of CoF after 3 weeks did not result in graft rejection. These results showed that immunosuppressive therapies directed at inhibiting antibody production may be of value in preventing rejection of concordant xenografts. Short-term complement depletion could rescue xenografts from rejection such that rescued grafts appear to be accommodated.

Reduced MHC class II antigen expression on rat heart and kidney cells after in vitro exposure to longwave ultraviolet irradiation

The pretreatment of both kidney and heart donor with the photosensitizer 8-methoxy-psoralen followed by in vitro longwave ultraviolet graft irradiation (PUVA therapy) significantly prolonged graft survival times in rats even across a strong major histocompatibility complex (MHC) barrier. Immunohistological studies using monoclonal anti-bodies (Mab) to rat MHC class I and II antigens showed a significant reduction of class II antigen expression after PUVA therapy in both Sprague-Dawley rat kidneys and hearts. Using MHC class I Mab there were no differences in distribution pattern in untreated as well as in PUVA treated organs. Thus, our results represent the first in vivo evidence that photochemotherapy-induced graft survival time prolongation is closely connected with its ability to reduce MHC class II antigen expression.

STUDIES OF THE INDUCTION AND MAINTENANCE OF LONG-TERM GRAFT ACCEPTANCE BY TREATMENT WITH FK506 IN HETEROTOPIC CARDIAC ALLOTRANSPLANTATION IN RATS

Immunosuppressive activities of the newly discovered FK506, isolated from Streptomyces tsukubaensis, were examined by using cardiac allotransplantation in the rat, and the mechanisms underlying induction and maintenance of FK506-induced long-term allograft survival were studied. Male rats of WKA (RT1k) and F344 (RT1lvl) strains were used as recipients and donors, respectively, and those of BN (RT1n) strain were used as third-party donors. Treatment with FK506, beginning from the day of allografting for 14, 10, or as few as 4 days, prolonged allograft survival significantly across the major histocompatibility barrier. The minimum doses for prolonging graft survival were 0.1 mg/kg/day by intramuscular treatment and 1.0 mg/kg/day by oral treatment. Treatment with FK506 at a dose of 0.32 mg/kg/day from day 4 until day 10 resulted in all the grafts surviving indefinitely and from days 5 to 10, half the grafts survived indefinitely, suggesting that the agent inhibited ongoing rejection. On the other hand, cyclosporine treatment at a dose of 20 mg/kg/day from day 2 did not prolong graft survival time statistically significantly. Induction of prolonged graft survival was not obtained by pretreatment of the prospective donor or recipient; prolonging effects were observed only when the agent was administered after allografting. Thus, the primary effect of the agent is exerted on responder lymphocytes reacting to the donor antigens in the induction phase of long-term graft acceptance. The mechanisms underlying the maintenance of long-term grafts were analyzed by testing the capacity of lymphocytes or serum of long-term graft-bearing rats to inhibit graft rejection in irradiated grafted hosts. Transfer of 2 x 10(8) lymphocytes from FK506-induced long-term F344 graft-bearing WKA rats resulted in indefinite survival of F344 heart allografts, but it did not prolong survival of third-party BN hearts. Transfer of 2.5 ml serum from long-term graft-bearing rats also prolonged graft survival of F344 hearts, but not BN hearts. These results suggest that donor strain-specific suppressor cells and humoral factor(s) are induced by treatment with FK506 in the presence of allografts, and that they play at least partial roles in the maintenance of long-term allograft acceptance.

Prolongation of rat kidney allografts by pretransplant administration of donor antigen extract or whole blood transfusion combined with a short course of cyclosporine.

The immunosuppressive effect of the combination of a three day course of cyclosporine with one i.v. injection of 3M KCL-extracted donor antigen or donor blood transfusion was tested across the strong histocompatibility barrier causing rejection within 8 days of kidney transplants from Buffalo (Buf, RT1b) to Wistar-Furth (WFu, RT1u) inbred rats. Administration of 10 mg/kg/day cyclosporine alone for three days (-1, 0, and 1) slightly prolonged graft survival time from 7 to 11 days. The combination of cyclosporine with donor Buf 3M KCl antigen or with a Buf blood transfurion administered one day prior to transplantation caused greater prolongation of graft survival--19 and 25 days, respectively. Neither third-party BN soluble antigen nor BN blood transfustions acted synergistically with cyclosporine to prolong Buf graft survival. Increasing doses of donor-soluble antigen up to an optimal dose of 5 mg proportionately prolonged graft survival; however, administration of 10 mg antigen was less effective than 5 mg. On the other hand, administration of 1 ml of donor blood achieved the maximal effect. Lymphocytes harvested ten days after transplantation from recipients that had received combined therapy with cyclosporine and donor 3M KCl antigen not only displayed specific unresponsiveness to donor stimulator cells in mixed lymphocyte culture, but also specifically suppressed the proliferative response of syngeneic, virgin WFu responder cells to allogeneic donor Buf but not to third-party BN cells. Furthermore, suppressor cell activity was suggested by diminished responses in an in vivo local adoptive mixed lymphocyte culture assay and by prolongation of Buf kidney survival following systemic adoptive transfer. These findings suggest that immunosuppression with cyclosporine permits induction of specific suppressor cells by 3M KCl donor antigen, resulting in specific unresponsiveness to allografts.

Leydig cells nonspecifically suppress lymphoproliferation in vitro: implications for the testis as an immunologically privileged site.

We have studied the influence of mouse testis cells on unstimulated lymphoproliferation; on allogeneic mixed lymphocyte reaction; and on concanavalin A-stimulated, phytohemagglutinin-stimulated, or lipopolysaccaride-stimulated responses in vitro. Co-culture with highly enriched irradiated (2000 R) Leydig cells (LC) led in each case to a considerable reduction of 3H TdR uptake. Prolonged tissue graft survival times suggest that the testis is an immunologically privileged site. This may be explained by an immune-suppressive function of LC in vivo. LC are also capable of selectively binding lymphoid and myeloid cells to their surface. This capacity, accompanied by a suppression of immune reactivity in situ, may play a role in cases of testicular relapse observed in acute leukemia.