Pretransplant conditioning with donor-specific transfusions using heated blood and cyclosporine. Preservation of the transfusion effect in the absence of sensitization

Previous studies from our laboratory showed that pretransplant conditioning with fresh donor-specific blood (DST) combined with cyclosporine (CsA) resulted in long-term prolongation of ACI heterotopic cardiac allografts in LEW recipients treated with subtherapeutic doses of CsA. The concomitant administration of CsA profoundly reduced but did not eliminate the DST-induced sensitization. The purpose of the present study was to investigate in the ACI-to-LEW cardiac allograft model whether heat-treatment of the blood would further reduce the sensitizing potential of DST while maintaining their benefits in our protocol. Fresh heparinized ACI blood was heated at 45 degrees C for 60 min. Then 1.5 ml was administered i.v. to LEW rats on day -8 with respect to grafting (day 0). Controls received heat-treated BUF blood. Donor heat-treated blood (HT-DST), unlike fresh blood, did not induce a humoral cytotoxic response and resulted in the prolongation of cardiac allograft survival (13.2 +/- 2.7 vs. 7.2 +/- 1.0; P less than 0.01). Treatment of HT-DST recipients with postoperative subtherapeutic doses of CsA (2.5 mg/kg/day x 30) extended graft survival (46.6 +/- 22.0 vs. 7.7 +/- 2.0 days; P less than 0.01). The combined pretransplant administration of HT-DST and CsA followed by posttransplant subtherapeutic doses of CsA led to long-term prolongation of cardiac grafts (122.0 +/- 73.0 vs. 31.7 +/- 22.0 days; P less than 0.01). These studies demonstrate that heat-treatment of allogeneic blood eliminates the humoral responses to DST and actually enhances their beneficial effects in terms of graft survival. Such effects can be dramatically increased by CsA. The possible mechanism of these phenomena are discussed.     

Brief cyclosporine treatment prevents intrathymic (IT) tolerance induction and precipitates acute rejection in an IT rat cardiac allograft model

BACKGROUND: Intrathymic (IT) alloantigen combined with administration of rabbit anti-rat anti-lymphocyte serum (ALS) intraperitoneally induces donor-specific tolerance to rat cardiac transplants. The purpose of this study was to examine the effect of a brief course (4 days) of cyclosporine (CsA) on the development of IT tolerance. METHODS: Buffalo (BUF) (RT1b) rats were given 25x10(6) fully MHC-mismatched Lewis (LEW) (RT1l) splenocytes by IT injection plus 1.0 ml of ALS intraperitoneally. Twenty-one days later, IT donor-specific LEW (group 1) or third-party (ACI, RT1a) (group 2) hearts were heterotopically transplanted to the abdominal aorta A third group of BUF (group 3) were given daily CsA (10 mg/kg) by oral gavage for 4 days before administration of IT LEW cells and ALS. Rejection as defined by the cessation of a palpable heartbeat was confirmed by histology. Cytokine profiles of allografts from all groups were then analyzed using a multi-probe RNase protection assay. RESULTS: Sixty-seven percent of IT/ALS-treated BUF recipients not pretreated with CsA accepted LEW heart grafts for greater than 90 days. However, 86% of animals treated with CsA for 4 days before IT injection and ALS rejected allografts at 10.7+/-3.2 days. Third-party allografts (ACI) were uniformly rejected (7.0+/-0.0 days). Histology confirmed cellular rejection in CsA-treated allografts and cytokine analysis detected increased interleukin (IL)-3, IL-5, and tumor necrosis factor-alpha when compared to increased IL-2 and interferon-gamma in rejecting untreated controls. CONCLUSIONS: CsA can prevent the induction of intrathymic alloantigen tolerance. These results support the development of a CsA-sensitive, but IL-2-independent, active regulatory mechanism after intrathymic exposure to donor-specific alloantigen and depletion of mature peripheral T cells.     

Small bowel transplantation in the rat. Effect of pretransplant blood transfusions and cyclosporine on host survival

We investigated the effect of pretransplant conditioning as a way to reduce the need for the aggressive immunosuppressive therapy reportedly required in small bowel (SB) allograft recipients. LEW rats were conditioned with (1) a donor-specific blood transfusion (DST) on day -8 and a concurrent 5-day course of CsA (10 mg/kg/day); (2) a nonspecific blood transfusion and CsA; (3) CsA alone. A 10-cm segment of the host native bowel was then replaced with an equivalent segment of SB obtained from ACI rats. Postoperative treatment consisted of CsA at 2.5 mg/kg/day for 30 days. Rats conditioned with a nonspecific transfusion and CsA or with CsA alone survived for 14.1 +/- 5.8 and 18.3 +/- 5.7 days, respectively. In contrast, rats conditioned with DST and CsA survived for 60.3 +/- 36.2 days (P less than 0.001 vs. both controls). Biopsies taken from long-term survivors showed a normal bowel architecture. The function of the allografts was studied in a group of animals totally deprived of their native bowel and transplanted with a 30-cm segment of ACI SB. CsA-DST-treated recipients survived an average of 90 +/- 43 days and grew at a rate comparable to isografted animals. Treated allograft recipients had maltose absorption indistinguishable from isografted controls at all times tested. In contrast, maltose absorption was severely impaired in recipients rejecting their grafts. This study demonstrates that long-term survival of SB allograft recipients can be achieved with good functional results with low doses of CsA in recipients conditioned with DST and CsA.     

A comparison between pancreas and heart allotransplantation after administration of donor-specific antigen and cyclosporine

These experiments compared the effect of a five-day course (days - 1 to +3) of cyclosporine therapy coupled with pretransplant (day - 1) administration of donor-specific antigen (whole blood or splenocytes) on either pancreatic or heart allograft survival in the Buffalo to Lewis rat donor-recipient combination. CsA therapy alone significantly (P less than 0.001) prolonged both heart (16.2 +/- 1.6 days) and pancreas (12.5 +/- 1.5 days) graft survival when compared with nonimmunosuppressed control heart and pancreas grafts (7.7 +/- 1.8 and 7.9 +/- 1.0 days, respectively). Pretransplant transfusion with either 2 ml of BUF whole blood or 2 x 10(8) red cell-free splenocytes on day -1 also resulted in a significant (P less than 0.001) prolongation of heart survival (14.0 +/- 1.2 and 14.0 +/- 1.6 days, respectively) but did not improve pancreas allograft survival (9.4 +/- 1.5 and 8.5 +/- 1.0 days, respectively). Combination CsA and antigen therapy further improved heart graft survival to 26.5 +/- 6.1 days (whole blood) and 28.8 +/- 5.8 days (splenocytes) but did not improve pancreas graft survival over that of CsA therapy alone. Extension of CsA therapy by adding two additional 3-day cycles on days 10-12 and 17-19 further improved heart graft survival both after CsA alone (35.2 +/- 3.2 days) and after CsA coupled with whole-blood transfusion (45.3 +/- 8.6 days), but did not have a salutary effect on pancreas allograft survival. Portal vein administration of donor antigen was equally effective as systemic inoculation in prolonging heart graft survival when the splenocytes were given alone (11.6 +/- 1.7 days). Conversely, pancreas allograft survival was not beneficially effected by portal antigen administration whether or not CsA was given. These data demonstrate the ability of pretransplant donor-specific antigen administration and short-term CsA therapy to significantly prolong rat heart allograft survival across a strong MHC histocompatibility barrier-but, surprisingly, they also demonstrate the failure of this regimen to have a salutary effect on pancreas allograft survival.     

Suppression of antibody response and prolongation of skin graft survival by multiple blood transfusions in the rat

The effect of blood transfusions (BT) on antibody response and skin graft survival was studied in the strongly MHC-incompatible BN and LEW combination. One-to-three BT induced high titer antibodies. Additional BT, however, led to a decrease of antibody titers. After 15 BT the recipients either had no detectable antibodies, or they had very low antibody titers. This suppression of response was shown to be distinct from a simple loss of antibody activity caused by lack of further antigenic challenge. In multiple transfused rats, humoral nonreactivity persisted in spite of rechallenge with antigen; in animals that lost their antibodies as a result of lack of further stimulation, an additional BT boosted strong antibody production. In LEW recipients of multiple BN transfusions, not only the specific anti-BN response but also reactivity to third-party BUF blood was suppressed. However, whereas the donor-specific response (anti-BN) was largely inhibited after a ten-week interval, the response to third-party BUF blood recovered. The state of humoral nonreactivity could be transferred by spleen cells to nontransfused syngeneic animals. In LEW rats that received three injections of 5 X 10(7) "suppressor" spleen cells, the antibody response to BN blood was strongly impaired as compared with animals that received normal spleen cells. BN or (BN X LEW)F1 skin grafts survived significantly better in multiple transfused LEW rats than in nontransfused controls. This was even more pronounced when ALS was given additionally. Third-party grafts (BUF) survived only slightly better than controls. It is concluded that multiple BT (1) result in humoral anti-donor nonreactivity secondary to an initial antibody response, (2) induce strong specific and weak nonspecific suppressor cell activity, and (3) increase skin graft survival.     

Effects of pre- and postengraftment donor-specific transfusions and cyclosporine on the enhancement of experimental allograft survival.

The tolerogenic effects of immediate pretransplant donor-specific transfusion (DST) with cyclosporine (CsA) have been well described. The purpose of this study was to determine if these effects could be improved upon by the administration of post-transplant DSTs. When added to a 29-day course of CsA, a single DST given 24 hr pretransplant improved graft survival compared to CsA alone (84.9 +/- 12.3 vs 40.0 +/- 8.8 days; P less than 0.05). The administration of an additional DST on post-transplant Days 7, 14, and 21 further improved this survival to 152 +/- 28 days, with 45% of grafts surviving greater than 200 days, until sacrifice. The donor specificity of this effect was demonstrated by the increased survival of second ACI grafts transplanted into Lewis recipients with existing "tolerant" ACI allografts (long-term survivors, or LTS); third-party Buffalo rat hearts transplanted into LTS rats in a similar manner were rejected normally. Loss of graft antigenicity was not seen, as retransplanted ACI hearts obtained from LTS Lewis rats were rejected in a first-order manner. From this we conclude that (1) the addition of multiple post-transplant DSTs improve the enhancement seen with preoperative DST and CsA, (2) loss of graft antigenicity does not contribute to this improved enhancement, and (3) this effect appears to be donor antigen specific.     

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.     

Humoral presensitization in rat heart allotransplantation

In these experiments an attempt was made to create a rat model of the past-positive, current-negative lymphocyte crossmatch (PPCNCx) phenomenon currently of concern in clinical renal transplantation. Lewis rats were sensitized with three to four serial ACI heart fragment (HF) or skin grafts. Subsequent ACI heart graft survival in the presence of high titer LEW-anti-ACI antibody was markedly shortened with 4 of 10 surviving for 24 hr or less in HF-sensitized rats and 11 of 11 surviving less than 24 hr following skin graft sensitization. Thirteen sensitized LEW rats were transplanted with ACI hearts 18 months later when their anti-ACI antibody was nil. Control rats (N = 5) had graft survival of 4.4 +/- 0.6 days; cyclosporine (CsA) therapy prolonged this to 8.0 +/- 3.6 days (N = 4), while combined cyclosporine and cyclophosphamide (Cy) resulted in an MST of 4.3 +/- 0.4 (N = 3). LEW-anti-ACI antibody was present on Day 3 or 4 in the control rats but was absent at the time of rejection in the CsA-Cy treated rats. Adoptive transfer of splenocytes from sensitized LEW rats into naive LEW hosts produced animals with humoral immune memory but no anti-ACI antibody at the time of transplantation. Nonimmunosuppressed adoptively transferred LEW recipients of ACI hearts rejected their grafts in an accelerated fashion (MST of 4.5 +/- 0.5 days) and displayed an anamnestic antibody production with first appearance on Day 3 or 4. Immunosuppression with CsA or Cy prolonged graft survival (greater than 30 days) in all cases and Cy prevented an anamnestic humoral response.(ABSTRACT TRUNCATED AT 250 WORDS)     

Intragraft delivery of 16, 16-dimethyl PGE2 induces donor-specific tolerance in rat cardiac allograft recipients

The stable prostaglandin E2 analogue, 16,16-dimethyl PGE2 (di-M-PGE2) was continuously infused by osmotic pump directly into rat heterotopic cardiac allografts. Intragraft delivery of 20 micrograms/kg/day di-M-PGE2 for 2 weeks completely prevented graft rejection for more than 150 days (n = 10), while untreated Buffalo recipients rejected Lewis cardiac allografts within 8 days after transplantation (mean survival time = 7.4 +/- 0.5 days, n = 5). When given for only 1 week, 20 micrograms/kg/day had a partial effect, since 60% of recipients accepted grafts long-term and 40% experienced rejection by day 14 (n = 5). In contrast, systemic intravenous administration of 20 micrograms/kg/day di-M-PGE2 for 2 weeks could not prolong graft survival (MST = 7.0 +/- 0.0 days, n = 3), and the higher dose of 200 micrograms/kg/day resulted in death by day 2 (n = 5). Long-term BUF recipients of LEW cardiac allografts accepted LEW donor strain skin grafts for more than 35 days while rejecting third-party Wistar Furth skin grafts in a normal fashion (MST = 7.3 +/- 0.5 days, n = 3), indicating the induction of donor-specific tolerance. Long-surviving LEW cardiac allografts retransplanted into naive BUF recipients were rejected within 7 days (MST = 6.7 +/- 0.5 days, n = 3), indicating no change in graft immunogenicity. Therefore, a 14-day infusion of di-M-PGE2 directly into a strongly MHC-mismatched cardiac allograft uniformly has resulted in long-term engraftment and the development of recipient donor-specific tolerance.     

Induction of donor-specific unresponsiveness to rat cardiac allograft by donor leukocytes and cyclosporine

Many recent reports have emphasized the importance of donor antigens in the induction of allograft tolerance. This study examines the effect of pretransplant infusion of 10(8) donor leukocytes (DL) combined with peritransplant cyclosporine (CsA) on W/F cardiac allograft survival in Lewis rats. Peritransplant recipient treatment consisted of CsA 20 mg/kg given i.m. on days 0, +1, and +2 relative to heart transplantation. Lewis recipients, 5-8 per group, were pretreated with 10(8) DL with or without peritransplant CsA. A single DL transfusion on day -3 or day -7 prior to transplantation significantly prolonged the mean survival time (MST) of W/F hearts from 7.0 +/- 0.9 days in controls to 12.2 +/- 4.5 days and 12.4 +/- 1.0 days (P less than 0.01), respectively. Two DL infusions on days -7 and -3 or on days -14 and -7 prolonged the MST to 10.6 +/- 1.3 days (P less than 0.02) and 16.4 +/- 2.8 days (P less than 0.001), respectively. The administration of peritransplant CsA alone significantly prolonged W/F heart allograft survival to 43.1 +/- 2.7 days. When pretransplant DL transfusion on day -3 was combined with CsA treatment, 4/8 animals maintained their grafts indefinitely (greater than 100 days). Similarly, DL infusion on day -7 with peritransplant CsA led to indefinite graft survival in 3/5 animals. Administration of DL on days -7 and -3 combined with CsA resulted in indefinite graft survival (greater than 100 days) in 4/6 animals. Transfusion of DL on day -3 alone or in combination with peritransplant CsA, had no effect on a third-party (ACI) heart allograft survival prolongation compared with appropriate controls. To define the underlying mechanisms responsible for donor-specific unresponsiveness in this model, pooled sera and unseparated spleen cells were passively transferred from recipients of long-term cardiac allografts to syngeneic rats receiving donor-type (W/F) or third-party (ACI) cardiac allografts. Transfer of serum (1 ml on days 0, and 1, 0.5 ml on days +2, +3, and +4) from ungrafted recipients of DL on days -14 and -7 led to significant donor graft survival of 9.8 +/- 0.4 days (P less than 0.02) in unmodified hosts. Similarly, passive transfer of serum obtained at 20 and 100 days after transplantation significantly prolonged the MST of donor-type hearts in syngeneic untreated hosts to 11.3 +/- 0.8 and 10.0 +/- 1.1 days, respectively.(ABSTRACT TRUNCATED AT 400 WORDS)     

Induction of tolerance to hind limb allografts in rats receiving cyclosporine A and antilymphocyte serum: effect of duration of the treatment

BACKGROUND: This study assessed the ability of antilymphocyte serum (ALS) and cyclosporine A (CsA) to induce tolerance for hind limb composite tissue allograft in rats without chronic immunosuppression. METHODS: Hind limb transplantations were performed in Lewis-Brown-Norway (LBN, RT1(1+n)) and Lewis (LEW, RT1(1)) rats. Treatment consisted of ALS only (0.4 mL/kg), CsA only (16 mg/kg), and a combination of CsA and ALS, and it was administered 12 hr before surgery at three different intervals (7, 14, and 21 days). Long-term survivors were tested for tolerance by standard skin grafting from the recipient (LEW), the donor (LBN), and the third party (ACI, RT1 ) 60 days after cessation of the treatment and by mixed lymphocyte reaction at 100 days. T-cell lines were analyzed with flow cytometry. RESULTS: Single use of ALS in all treatment intervals did not prolong allograft survival. Single use of CsA extended survival up to 23 days in the 21-day protocol group. CsA and ALS caused indefinite survival in two of six rats in the 14-day protocol and in all six rats in the 21-day protocol (>420 days). The six long-term survivors in the 21-day protocol accepted the skin grafts from the donor (LBN) and the recipient (LEW) and rejected third-party grafts (ACI). Tolerant animals showed a donor-specific hematopoietic chimerism of 35% to 42% in the peripheral blood. Mixed lymphocyte reaction assay demonstrated tolerance to the host and donor alloantigens and increased response to the third party. CONCLUSIONS: Administration of CsA and ALS for 21 days induced donor-specific tolerance in the recipients of the rat hind limb composite tissue allografts. The mechanism of tolerance should be investigated further.     

Living-donor renal transplantation in SEOPF. The impact of histocompatibility, transfusions, and cyclosporine on outcome

The impact of haplotype match (HM), pretransplant transfusions, and cyclosporine use were examined for living-donor renal transplants performed among 49 centers in the South-Eastern Organ Procurement Foundation (SEOPF) from November 1983 to June 1988 with follow-up through March 1989. During this period, 750 2-HM, 1246 1-HM, and 120 0-HM living-donor transplants were performed at 46, 47, and 27 centers, respectively. Demographic comparisons of the HM categories demonstrated the greatest use of cyclosporine and donor-specific transfusions in the 0-HM group, and the greatest use of random blood transfusions (RBT) or no blood transfusions (NBT) in the 2-HM group. By univariate and multivariate (Cox regression) analyses, actuarial graft survival was significantly associated with haplotype match, although excellent 3-year graft survival was seen for 0-HM as well as 1-HM and 2-HM first transplant recipients: 74 +/- 5%, 80 +/- 2%, and 85 +/- 2%, respectively. Comparisons were also made among patients receiving DST +/- CsA, RBT +/- CsA, and NBT +/- CsA for each HM group by univariate and multivariate analyses. For 0-HM recipients, DST + CsA was most frequently used and associated with the best long-term survival (86 +/- 5% at 3 years) by univariate analysis. For 1-HM recipients, there were no apparent differences in graft survival between DST and RBT groups +/- CsA by univariate analysis, but the absence of transfusion (NBT +/- CsA) was associated with the poorest 3-year survival (79 +/- 4%). This was confirmed by multivariate analysis, where DST (P less than 0.06) and RBT (P less than 0.02) were each significantly associated with graft survival, and provided relative benefits (vs. NBT) of 0.56 and 0.44, respectively; CsA use was not significantly associated with outcome or a significant benefit. For 2-HM recipients, the poorest results were seen with DST + CsA (78 +/- 6% at 3 years) by univariate analysis; multivariate analysis suggested no benefit with DST or RBT, and an increased risk of graft loss with CsA. These results indicate that the use of pretransplant transfusions and CsA therapy may have differential benefits depending upon HM in living-donor renal transplantation.     

The mechanism of synergistic interaction between anti-interleukin 2 receptor monoclonal antibody and cyclosporine therapy in rat recipients of organ allografts

Untreated anephric LEW rats die ca. 9 days following transplantation of LBNF1 kidney allografts. Although treatment with ART-18, a mouse antirat IL-2R mAb (300 micrograms/kg/day x 10 days), prolonged graft survival to ca. 3 weeks, the severely impaired renal function was comparable to untreated controls (creatinine levels 3-5 mg/dl). In contrast, simultaneous infusion of ART-18 and a very low dose of CsA (0.75 mg/kg x 10 days), marginally effective on its own, resulted in survival of greater than 45 days; the grafts exhibited relatively good function comparable to that in rats treated with full-dose (15 mg/kg/day) CsA. This beneficial biological effect did not depend upon elevated CsA trough levels in animals conditioned with both modalities. The CD4:CD8 ratio at the graft site was lowest (0.3-0.4) in recipients treated with ART-18 + CsA. Synergy between the two agents has been demonstrated by adoptive transfer studies in which nonspecific suppression has been conferred selectively by cells infiltrating kidney grafts in rats given ART-18 and CsA in concert but not separately (LBNF1 and WF test cardiac allograft survival ca. 12 days). In contrast, suppression in the recipient spleens was donor-specific; both CD4 and CD8 cells prolonged test graft survival. Immunohistological evaluation of renal allografts revealed that therapy with ART-18 or low-dose CsA alone failed to deplete IL-2R+ cells and prevent production of IL-2, IFN-g, and TNF. In contrast, the frequency of infiltrating IL-2R+ cells and elaboration of endogenous cytokines in non-uremic hosts receiving combination therapy was greatly depressed, stressing again synergistic interaction between ART-18 and CsA. Additionally, markedly reduced class II antigen induction, XL-fibrin deposition, and glomerulitis may also contribute to prolonged survival and satisfactory function of kidney allografts in this animal group.     

The requirement for the renal transplant to induce allograft unresponsiveness by the combination of extracted histocompatibility antigen and cyclosporine

The impact of the presence of the allograft on the induction of unresponsiveness by the immunosuppressive combination of cyclosporine (CsA) and 3M KCl-extracted histocompatibility Antigen (Ag) was assessed by comparing the outcome of renal transplants when the regimen was used pretransplantation versus peri- and posttransplantation. WFu rat hosts, which had been pretreated with either one (-11, -10, -9) or three (-25, -24, -23, -18, -17, -16, -11, -10, -9) cycles of CsA and BUF Ag (-11 or -25, -18, -11) prior to implantation of BUF renal allografts, failed to display the prolonged graft survival achieved with this regimen administered in the peri- (-1, 0, +1) and immediate posttransplant (+7, 8, 9, 14, 15, 16) period. However, OX-8 positive, putative T-suppressor (Ts) cells in the spleens of pretreated hosts were able to transfer slightly prolonged BUF allograft survival to virgin, secondary syngeneic hosts. The OX-8-positive cells induced by pretreatment were apparently inhibited from prolonging BUF allografts in primary hosts by cellular elements vulnerable to splenectomy, total-body irradiation, or a three-day peritransplant course of CsA therapy. Therefore the presence of the renal graft at the time of peri and immediate posttransplant administration of Ag-CsA facilitates the induction of unresponsiveness, possibly due to continued release of histocompatibility antigen to stimulate, and/or to providing an important peripheral environment promoting differentiation and maturation of, Ts cells.     

Prolongation of heterotopic heart allograft survival by local delivery of continuous low-dose cyclosporine therapy

The effectiveness of local versus systemic low-dose CsA (2 mg/kg/day) therapy delivered by osmotic pump for a 14-day continuous infusion was examined in the rat model. Systemic subtherapeutic CsA treatment of WFu recipients either by oral gavage or intravenously using an osmotic pump resulted in quick rejection of BUF heart allografts within a median survival time (MST) of 8 days in comparison with untreated controls (MST = 7 days). In contrast, direct local subtherapeutic CsA delivery to BUF heart allografts produced significantly (P less than 0.01) prolonged heart allograft survivals up to MST of 40 days. Splenic T cells, isolated on days 10 to 12 from locally immunosuppressed WFu recipients, revealed a nonspecifically reduced proliferative response toward alloantigens. Coculture experiments demonstrate that these T cells have the capacity to inhibit normal T cell proliferative responses in a nonspecific fashion either by their suppressor function or more likely by carrying CsA to the culture plate. In contrast, T cells isolated from WFu recipients three weeks after transplantation and tested in vitro demonstrated the presence in alloantigen specific T suppressor cells that coincided with a decreased frequency of alloantigen-specific T cytotoxic cells and may explain the extended heart allograft survival beyond the time of CsA delivery. CsA therapy delivered directly to the graft resulted in high CsA levels within the heart graft (1108 ng/0.1 g) but subtherapeutic levels in other tissues. These results demonstrate that local drug delivery is effective in inhibiting the rejection process within the graft itself, as manifested by prolonged heart allograft survival. Further, subtherapeutic CsA therapy facilitates development of Ts cells that may be responsible for the survival of heart allografts beyond the CsA delivery time.     

Differential response of kidney and pancreas rejection to cyclosporine immunosuppression.

Immunological interferences between kidney and pancreas transplants were investigated in a genetically defined rat model of combined kidney and pancreas transplantation. Kidney and whole-pancreas grafts were transplanted microsurgically either as individual grafts or in a combined technique. Whole pancreas grafts were grafted into streptozotocin diabetic recipients (55 mg/kg bodyweight i.v.) three days after induction of diabetes. The exocrine secretion was suppressed by duct ligation. Rejection of the grafts was defined by recurrence of diabetes in pancreas-grafted recipients and renal failure after kidney transplantation. There were marked differences in the efficacy of identical short-term cyclosporine immunosuppression (15 mg/kg intramuscularly for 14 days): DA kidneys survived indefinitely in LEW rats (MST greater than 100 days), while DA pancreas allografts underwent prolonged but not permanent survival (P less than 0.01) either as individual grafts (MST 27.3 +/- 1,9 days) or when transplanted simultaneously together with the kidney (44 +/- 16 days) (P less than 0.01). LEW rats carrying a DA kidney for 100 days also rejected a subsequent donor-specific pancreas transplant within 30 days. The histological alterations in the kidney were more pronounced than after cyclosporine-induced DA kidney long-term survival alone. By contrast to the rejecting subsequently transferred pancreas, a metachronous second DA kidney was permanently accepted (greater than 100 days) without further immunosuppression after removal of the first graft, while unrelated LEW. 1U kidneys were acutely rejected. In summary, the results indicate that there are not only quantitative differences of kidney and pancreas allograft survival but also differences concerning the state of immunological unresponsiveness induced by identical cyclosporine immunosuppression. While CsA induces donor-specific immunological unresponsiveness after kidney transplantation, pancreas transplants are all eventually rejected after some differential prolongation of survival. Further investigations on the effects of different MHC and minor alloantigens may provide more insight into the complex immunological situation of individual and combined kidney and pancreas transplantation.     

Immunologic consequences of combined pancreas-spleen transplantation in the rat

A rat model of combined pancreas-spleen transplantation (PST) was used in order to characterize the immunologic consequences of PST when compared to pancreas transplantation (PT) alone. Weakly MHC disparate Fischer (F344) PST grafts survived significantly longer in LEW recipients than did F344 PT grafts (17.6 +/- 3.4 vs 12.1 +/- 1.0 days, respectively, P less than 0.001). However, graft versus host disease (GVHD) occurred regularly in the PST recipients. Similarly, in haploidentical LBN to LEW donor-recipient pairs, PST graft survival was also modestly but significantly increased over that of the PT controls (10.6 +/- 1.0 vs 8.5 +/- 0.8 days, respectively, P less than 0.001). Conversely, in the ACI to LEW combination where MCH differences are very strong, PST graft survival was not longer than PT controls (7.5 +/- 0.8 vs 7.0 +/- 0.6 days, respectively, P greater than 0.2). GVHD was not observed in either of the latter two experiments. Short-term immunosuppression with cyclosporine further improved the outcome in LEW recipients of F344 grafts by inducing long-term graft survivals in approximately one-fourth of the PST recipients. Host splenectomy did not improve graft survival in PST recipients but did increase the risk of GVHD in LEW recipients of F344 PST grafts. Graft irradiation prior to transplantation with 500 rad not only abrogated the GVHD potential of the F344 PST graft but also eliminated the graft survival prolonging effect of the donor spleen. Donor spleen cells injected at the time of PT in F344 to LEW transplants resulted in graft prolongation not different from spleen intact PST recipients.(ABSTRACT TRUNCATED AT 250 WORDS)     

Nutritional immunomodulation leads to enhanced allograft survival in combination with cyclosporine A and rapamycin, but not FK506

BACKGROUND: Recently, specific immunonutrients were found to increase experimental allograft survival when combined with cyclosporine A (CsA). This study compared the effect on rat cardiac allograft survival when nutritional immunomodulation was used with CsA, rapamycin (Rapa), or tacrolimus (FK506). METHODS: Intra-abdominal ACI to Lewis cardiac allografts were performed and assessed daily by palpation. Study groups included untreated controls and those receiving CsA, Rapa, or FK506. Rats were fed ad libitum with Impact diet (fortified with fish oil, arginine, and RNA) or standard rat food. Further study groups were transplanted that received a donor-specific transfusion in addition to immunosuppression and diet. RESULTS: Allograft survival was extended by combining Impact with CsA (45.3+/-19 days) and Rapa (165.3+/-52 days), but not FK506 (12.4+/-3.2 days). Mean graft survival in the Rapa/Impact group met criteria for functional tolerance. The addition of a donor-specific transfusion did not lead to graft survival advantages over similar groups not receiving a donor-specific transfusion. CONCLUSIONS: The use of immunonutrients improves transplant outcome in animals treated with short courses of CsA and Rapa, but not FK506. These findings highlight the potential differences in the effects of nutritional immunomodulation with different immunosuppressive drugs in the treatment of transplant patients.     

Donor-specific antigen and cyclosporine in rat islet allografts

Combination therapy with one dose of 3 M KCl extracted donor-soluble antigen (Ag) and a short course of cyclosporine (CsA) has proven to prolong the survival of kidney allografts by enhancing specific T-suppressor populations. This regimen is tested in rat islet allografts in this study (Lewis to ACI). A 3-day perioperative course of 10 mg/kg/day CsA on Days -1, 0, and 1 did not prolong graft survival (MST = 10.7 +/- 2.5 days vs 9.4 +/- 1.2 days in controls). When this course of CsA therapy was combined with a single dose of donor antigen on Day -1, the survival time was prolonged slightly but significantly (MST = 14.0 +/- 5.8 days). Three cycles of a 3-day course of CsA therapy at 7-day intervals, a total of nine doses of 10 mg/kg/day CsA, were effective in delaying rejection of islet allografts (MST = 26.4 +/- 30.3). Moreover, combined therapy with donor antigen and three cycles of a 3-day course of CsA prolonged the survival of islet allografts (MST = 57.7 +/- 51.4 days) with 50% of recipients still normoglycemic at 60 days after transplantation. These findings indicate that the combination therapy of donor antigen with a short course of CsA has a powerful effect to prevent the rejection of islet allografts, as shown in kidney allografts, in rats.     

Prolongation of second heart transplants in rats.

Second clinical kidney grafts often survive longer than first grafts. Using a rat cardiac allograft model, we examined the conditions under which survival of a second graft can be longer than first graft survival. In the BUF-to-LEW combination, following rejection of the first transplant, second cardiac allografts from the same donor strain implanted immediately survived longer than the first grafts (P = 0.047). Although the mean survival time of first grafts was 9.0 days, second grafts implanted in the same animals survived 19.5 days. In contrast, when ACI donors were used for the same LEW recipients, the second grafts were rejected in 3 days compared with 6.6 days for first grafts. Donor-specific spleen cell transfusions in these combinations resulted in prolonged survival in the BUF to LEW combination, but had no effect when the donor strain was ACI. Second grafts from BUF had prolonged survival following rejection of the first graft. Thus the histocompatibility difference was a determining factor of whether or not prolongation would be obtained. Another factor was timing of the second transplant. If 7 days were allowed to elapse following rejection of the first graft before implantation of the second, the enhancement effect was lost. Moreover, in the LEW-to-ACI combination in which second grafts were rejected rapidly, removal of the first graft after 7 days (before rejection), resulted in prolonged survival of the second graft. There is, therefore, a window of time before rejection of first grafts and shortly thereafter, when the enhancement effect can be obtained. Passive transfer of serum in the BUF-to-LEW combination resulted in enhancement, but transfer of splenic cells was ineffective. We conclude that graft rejection can result in induction of enhancement during a specific period, after which this effect is lost; and that enhancement can be obtained only in certain strain combinations. This suggests that human patients with heart transplants that reject might benefit from a second graft, even from a donor with a mismatch similar to the first graft.