Factors determining prolongation of rat heart allograft survival by perioperative injection of donor spleen cells

Previously, we have shown that a perioperative injection of donor mononuclear cells in combination with cyclosporine treatment on day 2 after transplantation prolongs heart allograft survival in rats. In this study we determined whether the efficacy of this treatment was influenced by the same factors that have been shown to affect the efficacy of preoperative administration of donor cells. The effect of the following factors were investigated: dosage and repetition of the donor cell injection, viability of the donor cells, immunosuppressive drugs other than cyclosporine, and the rat strain combination. We found that there was an optimal dosage of donor cells; dosages of 4 x 10(7) or 1 x 10(8) cells gave the best heart graft survival. Repetition of the donor cell injection was not useful. Reducing viability of the cells by irradiation did not abrogate the prolonged graft survival, whereas killing of the cells did. Methylprednisolone, azathioprine, or cyclophosphamide in combination with the perioperative donor cell injection did not prolong heart graft survival in comparison with treatment with the drug only. The efficacy of this treatment was also influenced by the rat strain combination. In some combinations, this treatment prolonged graft survival, whereas in others an effect was absent or undetectable. Importantly, this treatment never adversely affected graft survival. We conclude that the efficacy of this treatment is influenced by similar factors as found for preoperative treatment with donor cells. A major advantage of this treatment over preoperative blood transfusions is that it avoids sensitization.     

Graft survival and immune regulation of pancreas allograft recipients induced with thymoglobulin, sirolimus, and cyclosporine

BACKGROUND: Our objective was to determine the impact of thymoglobulin-sirolimus-cyclosporine immunosuppression on the alloimmune response of pancreas-kidney transplant recipients. METHODS: Thirty-six pancreas transplant recipients received an induction protocol of thymoglobulin, sirolimus, reduced-dose cyclosporine, and corticosteroids. A subset of 10 recipients were also enrolled in a study to measure immune responsiveness. Flow PRA-determined HLA antibody, donor-specific flow cytometry crossmatching (FCXM), T-cell subset, and suppressor cell assays were performed at various timepoints during the first posttransplant year. RESULTS: One-year patient, kidney, and pancreas survivals were 97%, 94%, and 92%, respectively. There was 1 death due to sepsis, and 1 kidney and 2 pancreas graft losses. There were no acute rejection episodes. Recipients in the immune-monitoring study displayed depression of CD3, CD4, and CD8 counts (<80%) until 3 months posttransplant. At transplantation, 9 of 10 patients displayed <10% class I HLA antibody. By 3 months, 7 of 10 showed a transient elevation in class I HLA antibodies, with 2 patients expressing >80% flow PRA. At transplant 1 patient was FCXM-positive, whereas, by 3 months posttransplant, 2 of 10 patients demonstrated a positive FCXM. There were no clinical consequences to either the presence of HLA antibody or the positive FCXMs. By 6 months, 7 of 9 patients expressed immunoregulatory suppressor cell activity. CONCLUSIONS: The absence of acute rejection events was likely due to inhibition of donor-specific immunity. Seventy percent of patients demonstrated an early non-donor-directed HLA antibody response that had no adverse effect on graft function and 78% of the patients displayed immunoregulatory suppressor cell function, probably contributing to the successful clinical outcome.     

Synergistic interaction of 3 M KCl-extracted donor antigens (e-HAg) with cyclosporine or cyclosporine/sirolimus for prolongation of rat heart allograft survival

Extracted donor histocompatibility antigens (e-HAg) may potentiate the effects of drugs to protect organ allografts from rejection. We examined the capacity of e-HAg when combined with cyclosporine (CsA) alone, sirolimus (rapamycin, RAPA) alone, or CsA/RAPA combinations to prolong heart allograft survival in rats. Wistar-Furth (WF: RT1^u) rats that received CsA (10 mg/kg/day) by oral gavage for 3 (days 0, 1 and 2) or 7 (days 0, 1, 2, 3, 4, 5 and 6) consecutive days displayed modest prolongation of Brown Norway (BN; RT1ⁿ) heart allograft survival from a mean survival time of 7.2 ± 0.8 days in untreated controls to 12.2 ± 1.1 days and 18.6 ± 2.7 days, respectively (p < 0.01). Although administration on the day of transplantation (day 0) of a single intravenous (i.v.) dose of BN e-HAg (5 mg/kg) failed to affect allograft survival, both three (days 0, 1 and 2) and five (days 0, 1, 2, 3 and 4) injections significantly potentiated the effect of a 3-day course of oral CsA (18.6 ± 1.3 days (p < 0.01) and 20.0 ± 1.4 days (p < 0.01), respectively) and of a 7-day course of oral CsA (25.3 ± 4.4 days (p < 0.05) and 33.5 ± 9.3 days (p < 0.01), respectively). Median-effect analysis confirmed a synergistic interaction between CsA (0.5 mg/kg × 7 days, i.v.) and e-HAg with combination index (CI) values less than 0.7 (CI = 1 shows additive interactions, CI < 1 synergistic, and CI> 1 antagonistic, interactions). In contrast, e-HAg failed to affect the immunosuppressive effect of RAPA. However, e-HAg (5.0 mg/kg × 3 days) significantly potentiated the effects of a 7-day or 14-day course of RAPA (0.01 mg/kg)/CsA (0.5 mg/kg) combination therapy, namely from 26.0 ± 4.8 days with a 7-day treatment of CsA/RAPA alone to 32.6 ± 3.6 days (p < 0.01) and from 28.2 ± 2.7 days with a 14-day course of CsA/RAPA alone to 42.0 ± 4.9 days (p < 0.05), respectively (CI = 0.2–0.5). Thus, e-HAg potentiates the immunosuppressive effects of CsA alone and of the CsA/RAPA combination, but not of sirolimus alone.     

Prolongation of allograft survival by repeated cycles of donor antigen and cyclosporine in rat kidney transplantation

Combination therapy with a short course of cyclosporine (CsA) on the day prior to, to day of, and the day after transplantation and one dose of 5 mg 3M-KCl-extracted donor-soluble antigen (Ag) prolongs the survival of Buffalo (Buf, RT1b) kidney allografts in Wistar-Furth (WFu, RTu) inbred rats because of the induction of specific suppressor cells. Four systems were utilized to demonstrate suppressor cell activity in vivo. First, pooled lymphocytes from CsA-Ag-treated hosts suppressed the capacity of admixed, syngeneic WFu cells to display an in vivo mixed lymphocyte culture reaction toward donor Buf, but not third-party Brown-Norway (BN, RT1n), hosts. Second, systemic adoptive transfer two days prior to, or on the day of, transplantation of 5 x 10(8) putative suppressor cells harvested ten days after combined Ag-CsA treatment prolonged graft survival slightly but significantly from 7 to 9 days in virgin, secondary hosts. Third, admixture of 5 x 10(8) cells from Ag-CsA-treated hosts vitiated the capacity of 5 x 10(8) virgin WFu spleen cells to restore the capacity of recipients sublethally irradiated with 500 rads to reject. Buf allografts at 7.9 days rather than 16.7 days. Fourth, i.p. administration of low-dose cyclosphophamide (CY) 7 days after transplantation, a regimen known to inhibit suppressor cells, reduced the capacity of the Ag-CsA regimen to prolong graft survival. Two additional cycles of CsA therapy at 10-day intervals administered in an attempt to maintain T suppressor dominance over T helper cells prolonged median graft survival to 65 days. Similar prolongation was not achieved using donor blood transfusion as the immunogen, or using cycles of CsA alone. These findings suggest that 3M KCl donor antigen amplifies the induction of specific suppressor cells, and that CsA by virtue of helper T cell inhibition facilitates the establishment of suppressor cell dominance, eventually leading to host unresponsiveness.     

PUVA augments cyclosporine A-mediated rat cardiac allograft survival.

PUVA, the administration of the photosensitizer 8-methoxypsoralen (8-MOP) followed by exposure of the skin to longwave ultraviolet radiation (UVA, 320-400 nm), is employed clinically for the treatment of skin diseases. PUVA is immunosuppressive and we have shown previously that it can significantly prolong skin allograft survival. This enhanced survival is associated with reduced spleen cell cytotoxic activity against donor cell targets with preserved ability of treated animals to be immunized to third-party alloantigens 5 days after exposure to a course of PUVA. To examine whether PUVA may potentiate the effect of cyclosporine A (CYA) in inhibiting cardiac allograft rejection, we employed a rat cardiac transplant model. Lewis rats (RT1(1) received cardiac allografts at a heterotopic site from Lewis Brown Norwegian (RT1(1/n)) hybrid donors. Seventy animals were equally divided into 10 groups. Starting on the day of surgery, three groups received a suboptimal doses of CYA (1.5, 4.5, or 9.0 mg/kg im), three groups received the same doses of CYA and 1.0 mg/kg of 8-MOP injected ip followed by 6.35 J/cm2 of UVA radiation to their shaved dorsums (PUVA), one group received PUVA alone, one group received UVA radiation alone, one group received 8-MOP alone, and the final group received no treatment. Therapy was carried out daily for 7 days and survival of the allograft was assessed by daily palpation of the transplanted heart.(ABSTRACT TRUNCATED AT 250 WORDS)     

Circadian stage-dependent prolongation by cyclosporine of segmental pancreatic allograft function in the rat

While the toxicity of many drugs has been reduced by their administration at certain circadian rhythm stages (if not at certain times of day), as shown herein, direct therapeutic benefit from the improvement of the desired effect can be obtained by the circadian timing of intraperitoneal cyclosporine (Cs) for Lewis rats bearing an ACI segmental pancreas allograft. Under conditions of light (L) and darkness (D) alternating at 12-hour intervals, staggered by 8 h in 3 rooms kept at 24 degrees C, the effect of Cs in delaying graft rejection was improved by timing. When the mean time to rejection during the L span is equated to 100%, graft function is prolonged by 40% at the right time (injection daily during the D span) as compared to the wrong time (injection daily during the L span).     

Piceatannol in combination with low doses of cyclosporine A prolongs kidney allograft survival in a stringent rat transplantation model

BACKGROUND: The discovery of new immunosuppressive agents has enhanced short-term graft survival. However, current immunosuppressants often induce toxicities that limit their clinical use. Thus, there is a need for new immunosuppressants for use in clinical transplantation. Piceatannol blocks Syk and ZAP-70, tyrosine kinases involved in immune cell activation. We examined whether piceatannol prolongs kidney allograft survival in the stringent ACI-to-Lewis rat model. METHODS: Kidney recipients were divided into four groups. Group 1 (n=8) received piceatannol 30 mg/kg per day intravenously and cyclosporine A (CsA) 2 mg/kg per day intramuscularly from day -3 to day 7 after transplantation. At day 8, piceatannol was reduced to 10 mg/kg per day and the combined treatment continued until day 60. Group 2 (n=9) received 2 mg/kg per day CsA alone from day -3 to day 60. Group 3 (n=4) received piceatannol alone as in group 1. Group 4 (n=2) received only the vehicle dimethyl sulfoxide from day -3 to day 60. Graft rejection was defined as either a serum creatinine level more than 2 mg/dL or animal death. RESULTS: Group 1 animals survived for at least 115 days (n=8, P<0.05), with several animals maintaining their grafts for more than 200 days. In contrast, 8 of 9 animals in group 2 rejected their grafts within 10 days of transplantation; one animal survived for 71 days. Excellent graft function was maintained in group 1 animals despite withdrawal of immunosuppression. CONCLUSIONS: These results are the first to show that piceatannol, when combined with subtherapeutic dosages of CsA, prevents graft rejection, suggesting that targeting Syk and Zap could be useful for preventing graft rejection.     

Allogeneic hepatocyte transplantation in the rat spleen under cyclosporine immunosuppression

An innovative approach for stimulating the rapid growth of allogeneic hepatocytes implanted into splenic tissue with maintenance of the structural integrity is described. Single cell suspensions of hepatocytes from normal male ACI-strain rats (RTIa) were injected (2 X 10(6) cells) into the spleen of allogeneic male Fischer (RTI1) recipient rats. A 70% partial hepatectomy (PH) was performed at the same time as hepatocyte transplantation. Animals were treated for 4 days prior to, and 1 day after, transplantation with a feeding regimen containing 0.05% 2-acetylaminofluorene (AAF) to inhibit regeneration of the residual host liver. Animals received cyclosporine (CsA) 3 mg/kg/day s.c. posttransplantation. Histological examination of a standard longitudinal section of the recipient spleen two days posttransplant revealed an approximately 0.54-mm2 area replaced by hepatocytes. By 7 days this had increased to 0.97 +/- .15 mm2. Without CsA administration, hepatocytes were undetected at 7 days. Both PH and AAF treatment were necessary for successful colonization and sustained proliferation. Withdrawal of CsA treatment at 10 days after transplantation resulted in rapid rejection of established hepatocytes. This study demonstrates that rapid colonization of the rat spleen with allogeneic hepatocytes can be achieved, and that the viability and structural integrity of these transplanted cells can be maintained for at least 14 days using cyclosporine immunosuppression.     

The combined effect of perioperative donor spleen cells or KCl-extracted antigen and cyclosporine on renal allograft survival in the rat

The effects of using perioperative cyclosporine in conjunction with pretreatment with donor spleen cells or 3M KCl solubilized extracts of donor antigen were investigated in a LEW-to-DA rat renal allograft model. Cyclosporine given orally in a dose of 10 mg/kg/day around the time of transplantation (days -1, 0, +1), did not prolong renal allograft survival (median survival time [MST]--10 days). However when used in combination with pretreatment with either 10(8) donor spleen cells (1 day before transplantation), or 10(5) donor spleen cells (7 days before transplantation), pretreatment regimens that were in themselves ineffective, DA recipients accepted Lewis renal allografts indefinitely (MST greater than 100 days). Soluble antigen was prepared by 3M KCl extraction from donor spleen cells. Absorption assays were used to quantify the amount of class I major histocompatibility complex antigen in the preparation, and amounts of antigen equivalent to that expressed by 10(6)-10(8) donor spleen cells were used for pretreatment. These soluble antigen preparations given either 1 or 7 days before transplantation with or without perioperative cyclosporine did not prolong allograft survival of either homozygous or heterozygous donors (MST 10 days).     

Prolongation of skin allograft survival by combined feeding of donor spleen cells and cyclosporine in mice

BACKGROUND: Oral immune tolerance is a method for inducing donor-specific immunotolerance and prolonging graft survival. OBJECTIVES: We studied the effect of feeding donor spleen cells in combination with cyclosporine (CsA) on skin allograft survival in mice. METHODS: Tail skins from BALB/c (H-2d) female mice were transplanted onto C57BL/6 (H-2b) female mice. The animals were divided into four groups, each with eight mice: group I, untreated controls; group II, treated with spleen cells; group III, treated with CsA; and group IV, treated with spleen cells and CsA. All grafts were inspected daily. Rejection was diagnosed when the graft loss was >80% to 90%. The immune responses of C57BL/6 toward donor mice were examined by delayed-type hypersensitivity (DTH). RESULTS: Survival times of allogeneic skin grafts in groups I, II, III, and IV were 9.9 +/- 0.6, 13.1 +/- 0.6, 14.7 +/- 0.9, and 20.0 +/- 0.7 days, respectively. When compared with group I, the survival times of groups II, III, and IV were prolonged significantly (P < .01). The survival time for group IV was prolonged significantly compared with groups II and III (P < .01). The DTH responses of group IV were decreased significantly in contrast to groups II and III (P < .01). CONCLUSIONS: Feeding donor spleen cells prolonged the survival of skin allografts in mice; combination with CsA led to further prolongation of skin allograft survival.     

The effect of donor-specific blood transfusion, cyclosporine, and dietary prostaglandin precursors on rat cardiac allograft survival. II. Effectiveness of a 24-hour induction period with DST and CsA in inducing long-term graft survival

We investigated the effect of donor-specific transfusion given 24 hours pretransplant, a short course of low-dose cyclosporine, and dietary enrichment with the prostaglandin precursor linoleic acid (LA) to see which of the modalities could act synergistically on cardiac allograft survival in a stringent animal model. ACI male rats (RT1a) were used as blood and heart donors, and Lewis male rats (RT1l) were used as recipients. DST alone (1 ml) given 24 hr pretransplant or LA alone started 24 hr pretransplant and given daily p.o. until rejection prolonged cardiac allograft survival slightly but significantly, from 6 to 8 days. CsA alone started at the time of transplant at a dose of 5 mg/kg/day s.c. and given daily for 14 days prolonged cardiac survival to 11.8 days. However, when CsA was started 24 hr pretransplant and continued for two weeks, there was a significantly prolonged allograft survival to 55 days. CsA given together with DST 24 hr pretransplant and continued for two weeks posttransplant significantly prolonged cardiac allograft survival to 80 days and resulted in permanent tolerance in some animals. The addition of LA to a DST and CSA treatment regimen did not further improve allograft survival. CsA blood levels were determined in a separate group of Lewis rats. Three dosages of CsA were administered s.c. for 2 weeks: 2.5 mg/kg/day, 5 mg/kg/day, and 10 mg/kg/day. One injection of the three CsA doses did not achieve what are considered therapeutic levels in man. After 5 days, all three doses of CsA achieved significant blood levels. Significant blood levels were still present one week, but not 3 weeks after CsA was stopped. We conclude that DST given 24 hr before transplant and a 2-week course of low-dose CsA started one day pretransplant have strong synergism in inducing long-term graft survival in this rat model. Linoleic acid started 24 hr pretransplant, together with DST and CsA, did not contribute significantly to graft survival compared with the group given CsA and DST alone. Prolonged heart allograft survival was not due to persistently high CsA levels after the drug was discontinued.     

Effect of ultraviolet-B-irradiated donor-specific blood transfusions and peritransplant immunosuppression with cyclosporine on rat cardiac allograft survival

We have previously demonstrated that pretreatment of ACI recipients with ultraviolet-irradiated donor-specific blood transfusion (UV-DST) leads to permanent cardiac allograft survival without further host immunosuppression (ACI rats are weak responders to Lewis lymphocytes in mixed-lymphocyte reaction). This study examines the effect of UV-DST and the timing of transfusions on ACI cardiac allograft survival in Lewis recipients with and without the addition of peritransplant cyclosporine (CsA) (20 mg/kg i.m.) given on days 0, +1, and +2 in relation to the time of transplantation. The mean survival time (MST) of ACI cardiac allografts in Lewis recipients was significantly increased to 33.6 +/- 5.7 days (P less than 0.001) by CsA treatment alone as compared to 6.5 +/- 0.5 days survival in control. When DST was given on day -3 combined with CsA, graft survival was increased to 42.0 +/- 9.3 days (P less than 0.01), as compared to 5.8 +/- 1.3 days when DST alone was used. When DST was irradiated with ultraviolet B (UV-DST) and administered on day -3 combined with peritransplant CsA, the MST was increased to 68.83 +/- 16.1 days as compared to an MST of 10.0 +/- 1.0 days in controls treated with UV-DST alone. When UV-DST was given on day -7 and combined with peritransplant CsA immunosuppression, the results were similar. However, when UV-DST was peritransplant CsA course, 4 of 6 recipients maintained their ACI heart allografts indefinitely (greater than 300 days) in contrast to the effect of UV-DST alone (MST of 13.5 days). Third-party (W/F) UV-irradiated blood transfusions were ineffective in prolonging ACI cardiac allografts in Lewis rats, regardless of whether the transfusions were given alone or in combination with peritransplant immunosuppression with CsA. In conclusion, these results demonstrate that UV-DST combined with a brief peritransplant immunosuppression with CsA induces prolonged heart allograft survival in a histoincompatible, strong responder host, and that such effect is donor specific. The use of UV-DST combined with peritransplant CsA immunosuppression offers a promising approach to achieving organ transplant unresponsiveness, and decreased sensitization to the donor blood elements, which eventually may have important clinical implications.     

环孢素对共刺激阻断剂延长移植肾存活效应的影响

目的:探讨环孢素(CsA)对共刺激阻断剂CTLA4Ig延长移植肾存活效应的影响。方法:肾移植大鼠分为对照组(第1组)、CTLA4Ig组(第2组)、CsA CTLA4Ig组(第3组)、CTLA4Ig IL-2组(第4组)和CsA CTLA4Ig IL-2组(第5组),观察术后血肌酐(Scr)、移植肾病理改变、移植肾存活时间。结果:与第1组、第4组相比,第2组、第3组、第5组移植肾存活时间显著延长(P<0.01),其中,第3组移植肾存活时间最长(66.1±10.6)d;术后15天,第2组Scr显著低于第3组、第5组(P<0.05);术后30天,第3组、第5组Scr显著低于第2组(P<0.01);术后30天,第3组、第5组移植肾淋巴细胞浸润明显少于第2组。结论:CsA可增强CTLA4Ig延长移植肾存活的效应,对外源性IL-2逆转CTLA4Ig的效应具有抵抗作用。