A short course of mycophenolate immunosuppression inhibits rejection, but not tolerance, of rat liver allografts in association with inhibition of interleukin-4 and alloantibody responses

BACKGROUND: Some immunosuppressive drug therapies inhibit transplant tolerance in animal models, and we have shown that treatment of recipients with methylprednisolone, but not cyclosporine, inhibits spontaneous acceptance of liver transplants. This study investigates the effects of mycophenolate mofetil (MMF) on liver acceptance and rejection. METHODS: Piebald Virol Glaxo rat livers were transplanted into Dark Agouti recipients, which spontaneously tolerate (TOL) the liver, or into Lewis recipients, which reject (REJ) the liver. MMF (40 mg/kg/day subcutaneously) was given for 5 days from days 0 to 4 (early) or from days 3 to 7 (late). In separate experiments, liver grafts were collected for assessment of infiltrate and of interleukin (IL)-2, IL-4, IL-10, and interferon-gamma mRNA expression. RESULTS: TOL liver transplants had a median survival time (MST) of more than 100 days (n=6), and neither early nor late MMF treatment of TOL transplants reduced survival (MST 85 days, P=0.19 and 78 days, P=0.08, respectively). Liver failure in most of these animals was the result of biliary problems, not rejection. There were few consistent differences between treated and untreated TOL animals in infiltrate or liver cytokine expression, although there was a moderate reduction in T-cell infiltrate in MMF-treated TOL animals (P=0.003 on day 5 TOL). In contrast, REJ transplants had an MST of 13 days (n=10), and early MMF treatment led to five of six animals surviving more than 100 days (P=0.0002), whereas late treatment was much less effective, with one of six animals surviving more than 100 days. REJ livers had significantly more IL-4 mRNA expression and immunoglobulin G1 deposition in the graft than TOL livers, and this was inhibited by early, but not late, MMF treatment. CONCLUSIONS: MMF treatment inhibited rejection but not acceptance of liver allografts. Early administration was more effective in preventing rejection and demonstrated a more marked effect on IL-4 expression and alloantibody deposition than on graft T-cell infiltrate and expression of other cytokines.     

Blocking indoleamine dioxygenase activity early after rat liver transplantation prevents long-term survival but does not cause acute rejection

In a well-characterized rat model of liver transplantation, Piebald Virol Glaxo strain livers are accepted long term in fully mismatched Dark Agouti recipients (tolerance; TOL), but rejected in Lewis recipients (rejection; REJ). Spontaneous tolerance induction is associated with increased interferon-gamma expression, and we examined the role of the interferon-gamma-inducible immunomodulatory enzyme indoleamine dioxygenase (IDO) in this model. On day 3 after transplantation, IDO expression in the spleen of TOL recipients was significantly greater than in REJ. The B-cell population accounted for this early IDO increase. Intragraft expression of IDO increased to the same extent in both TOL and REJ. IDO inhibition for 7 days after transplantation reduced survival, but did not cause acute rejection of the liver in the TOL model. In conclusion, the differential IDO expression by B lymphocytes in the spleen of TOL recipients is not critical for preventing acute rejection.     

Spontaneous acceptance of mouse kidney allografts is associated with increased Foxp3 expression and differences in the B and T cell compartments

Spontaneous acceptance of organ allografts can identify novel mechanisms of drug-free transplantation tolerance. Spontaneous acceptance occurs in both mouse kidney transplants and rat liver transplants however the early immune processes of mouse kidney acceptance have not been studied. Acceptance of C57BL/6 strain kidney allografts in fully MHC-incompatible B10.BR recipients was compared with rejection (REJ) of heart allografts in the same strain combination. Graft infiltrate and antibody deposition were examined by immunohistochemical staining. Expression of mRNA was measured by quantitative real-time PCR. Apoptosis was examined by TUNEL staining. The majority of kidney allografts were accepted long-term and induced tolerance (TOL) of donor-strain skin grafts, showing that acceptance was not due to immune ignorance. There was an extensive infiltrate of T cells in the TOL kidney that exceeded the level in REJ hearts but subsequently declined. The main differences were deposition of IgG2a antibody in REJ that was absent in TOL, more B cells infiltrating TOL kidneys and a progressive increase in the ratio of CD8:CD4 cells during rejection. There was also significantly greater Foxp3 mRNA expression in TOL. Kidneys from RAG-/- donors were accepted, showing that donor lymphocytes were not necessary for acceptance. Neutralising antibodies to TGF-β administered from day 0 to day 6 did not prevent TOL. On the basis of cytokine expression and apoptosis there was no evidence for immune deviation or deletion as mechanisms of acceptance. In accord with the findings of spontaneous acceptance of liver allografts in rats, the main difference between mouse kidney TOL and heart REJ was in the B cell compartment. The major difference to rat liver allograft acceptance was that apoptosis of infiltrate did not appear to play a role. Instead, increased Foxp3 expression in TOL kidneys implies that regulatory T cells might be important.     

Gene array analysis of a rat model of liver transplant tolerance identifies increased complement C3 and the STAT-1/IRF-1 pathway during tolerance induction.

This study aimed to define the molecular mechanism during induction of spontaneous liver transplant tolerance using microarrays and to focus on molecular pathways associated with tolerance by meta-analysis with published studies. The differences in the early immune response between PVG to DA liver transplant recipients that are spontaneously tolerant (TOL) and PVG to Lewis liver transplants that reject (REJ) were examined. Spleens from TOL and REJ on days 1 and 3 were compared by 2 color microarray. Forty-six of 199 genes differentially expressed between TOL and REJ had an immunological function. More immune genes were increased in TOL vs. REJ on day 1, including STAT-1, IRF-1 and complement C3. Differential expression of selected genes was confirmed by quantitative RT-PCR. The results were compared to two published high-throughput studies of rat liver transplant tolerance and showed that C3 was increased in all three models, while STAT-1 and IRF-1 were increased in two models. The early increases in immune genes in TOL confirmed previous reports of an active early immune response in TOL. In conclusion, the increase in STAT-1, IRF-1 and complement component C3 in several models of liver transplant tolerance suggests that the STAT-1/IRF-1 apoptotic pathway and C3 may be involved in the tolerogenic mechanism.     

Dynamic changes of indoleamine 2,3-dioxygenase of Kupffer cells in rat liver transplant rejection and tolerance

Aims

To study the dynamic changes and the immunologic role of indoleamine 2, 3-dioxygenase (IDO) in Kupffer cells (KCs) after rat liver transplantation.

Methods

Animals were randomly divided into two groups: a rejection group (REJ; LEW to BN) and a tolerance group (TOL; BN to LEW). Liver morphological changes were observed optically with hematoxylin/eosin staining. KCs were isolated from recipients. mRNA and protein expressions of IDO were detected by real-time polymerase chain reaction and Western blotting at 1, 3, 5, and 7 days after transplantation.

Results

The levels of IDO mRNA and protein in KCs of TOL groups were similar to those in REJ groups at day 1 posttransplantation. However, the expression of IDO mRNA and protein time-dependently increased to much higher levels in the TOL than the in REJ groups at 3, 5, and 7 days posttransplantation (P < .05). The peak was observed at 7 days.

Conclusions

The IDO level of KCs was closely associated with immune tolerance induction. IDO-mediated immune modulation appears to be an attractive means to assess transplant tolerance induction.     

Donor IL-4-treatment induces alternatively activated liver macrophages and IDO-expressing NK cells and promotes rat liver allograft acceptance

Most approaches to transplant tolerance involve treatment of the recipient to prevent rejection. This study investigates donor treatment with IL-4 for its effect on subsequent rat liver allograft survival. Rat orthotopic liver transplants were performed in rejecting (PVG donor to Lewis recipient) or spontaneously tolerant (PVG to DA) strain combinations. Donors were untreated or injected intraperitoneally with IL-4 (30,000 U/day) for 5 days. Tissue infiltrates and gene expression were examined by immunohistochemistry and real-time quantitative PCR. IL-4 induced a marked leukocyte infiltrate in donor livers prior to transplant. Macrophages comprised the major population, although B cells, T cells and natural killer (NK) cells also increased. IL-4-induced liver macrophages had an alternatively activated phenotype with increased expression of mannose receptor but not inducible nitric oxide synthase (NOS2). IL-4 also induced IDO and IFN-gamma expression by NK cells. Donor IL-4-treatment converted rejection to acceptance in the majority of Lewis recipients (median survival time > 96 days) and did not prevent acceptance in DA recipients. Acceptance in Lewis recipients was associated with increased donor cell migration to recipient spleens and increased splenic IL-2, IFN-gamma and IDO expression 24 h after transplantation. Donor IL-4-treatment increased leukocytes in the donor liver including potentially immunosuppressive populations of alternatively activated macrophages and IDO-expressing NK cells. Donor treatment led to long-term acceptance of most livers in association with early immune activation in recipient lymphoid tissues.     

1alpha,25-dihydroxyvitamin D3 shows strong and additive immunomodulatory effects with cyclosporine A in rat renal allotransplants.

BACKGROUND: Vitamin D3 and its metabolites have long been found to exert immunosuppressive effects both in vivo and in vitro. The present study investigated the effect of 1alpha,25-dihydroxycholecalciferol (1,25DHC) on vascularized renal allografts in rats. METHODS: Three days prior to transplantation, two groups of animals were subjected to 1,25DHC (1 microg/kg/day IP) and a low calcium diet, which was continued until the end of the experiments. Recipient organs were removed and single allografts were transplanted in a high responder strain combination (ACI --> Lewis). Following transplantation, low-dose cyclosporine A (3.2 mg/kg/day CsA) administration was started in two experimental groups of recipients (one group receiving 1,25 DHC additionally) whereas the control allograft recipients received no immunosuppression (control III). Graft survival and renal function was monitored until death or the end of experiments and allograft rejection was assessed histologically using the Banff classification. RESULTS: 1,25DHC significantly prolonged allograft survival in comparison to control III (9.6 +/- 1 vs. 5.7 +/- 0.2 days; P=0.009). In addition, a combination of 1,25DHC and low-dose CsA increased allograft survival compared to CsA administration alone (24 +/- 0.9 vs. 13 +/- 0.3 days; P=0.008). 1,25DHC preserved renal creatinine clearance and decreased proteinuria in comparison to control III, and the combination of 1,25DHC and low-dose CsA again showed an additive effect on preservation of renal function. 1,25DHC and low-dose CsA both decreased interleukin (IL)-2 and IL-12 expression levels in serum and allografts, and a combination treatment produced the strongest attenuation of IL-2 and IL-12 expression. In addition, 1,25DHC increased IL-4 and IL-10 expression levels in allografts, whereas CsA alone did not alter IL-4 and IL-10 expression. In contrast, combination of 1,25DHC and low-dose CsA showed a significant increase in IL-10 expression levels whereas IL-4 expression was not elevated. CONCLUSION: Monotherapy with 1,25DHC significantly prolongs survival of renal allografts and preserves graft function in rats. A combination of 1,25DHC and CsA caused an additive effect on graft survival with differential regulation of pro- and anti-inflammatory cytokines, as compared to 1,25DHC administration alone.     

Combined donor leucocyte administration and immunosuppressive drug treatment for survival of rat heart allografts

BACKGROUND: Donor leucocytes (DL) play an important role in rat liver transplant tolerance and their postoperative administration can convert rejection to tolerance. They appear to induce early activation, altered patterns of infiltration and death of recipient alloreactive T cells. The ability of immunosuppressive drugs to combine with DL administration was examined in a rat heart transplant model. METHODS: Immediately after PVG to DA heterotopic heart transplantation, 6 x 10(7) spleen DL were injected. Cyclosporine A (CsA), 1.5 mg/kg/day, or methotrexate (MTX), 0.1 or 0.2 mg/kg/day, were given from day (d) 0 to d4 (early) or from d3 to d7 (delayed). Castanospermine (CAST) was administered from d0 to d7 at 100 or 300 mg/kg/day. In a separate experiment, transplanted hearts and recipient spleens were collected from treatment groups for analysis of infiltrate and cytokine mRNA expression. RESULTS: Delayed treatment with CsA or early treatment with MTX but not CAST combined with DL to result in prolonged graft survival. Recipients treated with DL and delayed CsA had a reduced level of intra-graft interleukin (IL)-2, interferon (IFN)-gamma, IL-4, and IL-4R mRNA expression and reduced infiltrate compared to DL alone. Early MTX plus DL led to almost complete inhibition of all markers of inflammation during treatment followed by a rapid increase after cessation. In combination with DL, CsA was more effective than MTX for induction of donor-specific tolerance at the dose and administration regimens tested. CONCLUSIONS: Delayed CsA or early MTX combine with DL to prolong heart allograft survival. Early and extensive inhibition of rejection by MTX was less effective than delayed and partial inhibition of the response by CsA for induction of transplant tolerance.     

The role of Foxp3+ regulatory T cells in liver transplant tolerance

The liver has long been considered a tolerogenic organ that favors the induction of peripheral tolerance. The mechanisms underlying liver tolerogenicity remain largely undefined. In this study, we characterized Foxp3-expressing CD4+ CD25+ regulatory T cells (Treg) in liver allograft recipients and examined the role of Treg in inherent liver tolerogenicity by employing the mouse spontaneous liver transplant tolerance model. Orthotopic liver transplantation was performed from C57BL/10 (H2b) to C3H/HeJ (H2k) mice. The percentage of CD4+ CD25+ Treg was expanded in the liver grafts and recipient spleens from day 5 up to day 100 posttransplantation, associated with high intracellular Foxp3 and CTLA4 expression. Immunohistochemistry further demonstrated significant numbers of Foxp3+ cells in the liver grafts and recipient spleens and increased transforming growth factor beta expression in the recipient spleens throughout the time courses. Adoptive transfer of spleen cells from the long-term liver allograft survivors significantly prolonged donor heart graft survival. Depletion of recipient CD4+ CD25+ Treg using anti-CD25 monoclonal antibody (250 microg/d) induced acute liver allograft rejection, associated with elevated anti-donor T-cell proliferative responses, CTL and natural killer activities, enhanced interleukin (IL)-2, interferon-gamma, IL-10, and decreased IL-4 production, and decreased T-cell apoptotic activity in anti-CD25-treated recipients. Moreover, CTLA4 blockade by anti-CTLA4 monoclonal antibody administration exacerbated liver graft rejection when combined with anti-CD25 monoclonal antibody. Thus, Foxp3+ CD4+ CD25+ Treg appear to underpin spontaneous acceptance of major histocompatability complex- mismatched liver allografts in mice. CTLA4, IL-4, and apoptosis of alloreactive T cells appear to contribute to the function of Treg and regulation of graft outcome.     

Suppression of allograft rejection with anti-alphabeta T cell receptor antibody in rat corneal transplantation

BACKGROUND: Anti-alphabeta T cell receptor monoclonal antibody (R73) has been reported to be a potent immunosuppressant. The suppressive effects of this antibody on allograft rejection after corneal transplantation are unknown. METHODS: Orthotopic rat penetrating keratoplasty was performed using Lewis rats as recipients and Brown Norway and Fisher rats as donors. The treated groups received R73 intraperitoneally until day 12 after the transplantation. In grafted rats with or without R73 treatment, cytokine expression of the aqueous humor, corneal-infiltrating cells, draining lymph nodes, and splenocytes was determined. Delayed-type hypersensitivity (DTH) responses were compared. RESULTS: All allografts in the untreated controls of Fisher-to-Lewis or BN-to-Lewis rat combinations were rejected within 14 days. In contrast, indefinite survival rates of the postoperative R73-treated group increased to 86% in the Fisher-to-Lewis and 23% in the Brown Norway-to-Lewis combinations, respectively. Interferon-y, interleukin (IL)-2 (T helper [Th]1), and IL-10 (Th2), but not IL-4 (Th2), expression of the eye and DTH responses in the control group were suppressed in the R73-treated group. Both IL-2 and IL-10 expression after mixed lymphocyte culture in the R73-treated group were significantly lower than those of the naive and untreated control group. CONCLUSIONS: alphabeta T cell receptor-targeted therapy prevents allograft rejection in rat corneal transplantation as evidenced by suppression of DTH responses. The cytokine profile after R73 treatment was characterized by low interferon-gamma, IL-2, and IL-10, and high IL-4 expression.     

Posttransplant interleukin-4 treatment converts rat liver allograft tolerance to rejection

BACKGROUND: Previous studies showed that liver transplant rejection in the Piebald Virol Glaxo (PVG)-to-Lewis combination was associated with more intragraft interleukin (IL)-4 mRNA expression than in spontaneously tolerant grafts in the PVG-to-Dark Agouti (DA) combination. There was also immunoglobulin (Ig) G1 antibody deposition, suggesting an IL-4-induced IgG class switch in rejection. The aim of this study was to investigate whether IL-4 treatment converts PVG-->DA liver transplant tolerance to rejection. METHODS: DA (RT1a) rats were recipients of orthotopic PVG (RT1c) liver transplants and DA liver transplants were syngeneic controls. Supernatant from IL-4-transfected Chinese hamster ovary cells (0.5 mL, 30,000 U) or from untransfected cells was injected intraperitoneally on days 3 through 7. Samples were taken for immunohistochemical staining of frozen tissue sections to analyze cellular infiltrate and antibody deposition. RESULTS: IL-4 treatment significantly reduced survival of liver allografts from greater than 100 days in untreated animals to 9 days (P=0.004). Pathologic analysis of IL-4-treated animals showed that death was caused by liver transplant rejection, with a heavy infiltrate of mononuclear cells, disruption of portal tract areas, and infarction. Immunohistochemistry revealed an extensive infiltrate of T cells, CD25-expressing cells, and B cells that was similar to the level in PVG--> Lewis liver allograft recipients that reject the liver. There was also a more extensive monocyte-macrophage infiltrate and more major histocompatibility complex class II expression in IL-4-treated animals compared with untreated animals. There was moderate increase of IgM, little IgG1, and no IgE or IgG2a antibody deposition. CONCLUSIONS: IL-4, a T-helper type 2 cytokine that has previously been shown to inhibit delayed-type hypersensitivity responses such as rejection, was found to promote rejection of liver allografts. There was only slight evidence of a graft-specific antibody response, showing that IL-4 induces liver allograft rejection in association with some, but not all, of the changes accompanying rejection in the PVG-->Lewis strain combination.     

The role of tumor necrosis factor in allograft rejection. III. Evidence that anti-TNF antibody therapy prolongs allograft survival in rats with acute rejection

We have previously demonstrated that TNF-alpha levels are elevated in liver transplant patients experiencing acute rejection. In addition, prophylactic administration of anti-TNF-alpha or anti-TNF-beta antibodies prolonged graft survival in a rat heterotopic cardiac transplant model. This experiment was designed to evaluate anti-TNF therapy in the treatment of acute allograft rejection. Heterotopic cardiac transplants were performed using Buffalo donors and Lewis recipients. Histologic sections of transplanted grafts from untreated animals revealed significant rejection at day 4 with terminal rejection occurring on day 10.8 +/- 0.4. Animals in the experimental groups received antirejection therapy from postoperative days 4-13. Treatment with cyclosporine at 2 mg/kg/day prolonged graft survival to 16.5 +/- 2.0 days (P = 0.01 versus controls). Administration of polyclonal anti-TNF-alpha in combination with polyclonal anti-TNF-beta increased graft survival to 14.6 +/- 0.4 days (P less than 0.001 versus controls). Use of a monoclonal anti-TNF-alpha antibody was even more effective, with graft survival of 17.4 +/- 0.7 days (P less than 0.001 versus controls). Combination immunotherapy with monoclonal anti-TNF-alpha in conjunction with CsA extended survival to greater than 30 days. In contrast, recombinant TNF-alpha (5 micrograms/day, i.p.) markedly accelerated the time to graft failure (7.4 +/- 0.2 days, P less than 0.001 versus controls). Examination of explanted graft tissue on postoperative day 9 from animals treated with anti-TNF showed decreased mononuclear cell infiltrate when compared to untreated animals. Treatment with TNF-alpha markedly increased the inflammatory process. These results suggest that TNF may play a role in the pathogenesis of acute rejection.     

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.     

Prolongation of liver allograft survival after interleukin-10 gene transduction 24-48 hours before donation

BACKGROUND: Interleukin- (IL) 10 may be a potent regulator for controlling of allograft rejection. A single administration of IL-10 is not effective for controlling graft rejection. Gene transfer is an attractive vehicle for prolonging the expression of short-lived proteins. METHODS: Donor or recipient livers were transduced with 1 x 10(10) p.f.u. of replication-deficient adenovirus vectors harboring human IL-10 cDNA (AdCMVhIL-10) via the ileocecal vein before or after rat orthotopic liver transplantation. RESULTS: DA allografts given AdCMVhIL-10 24-48 hr before donation survived for more than 56 days in Lewis recipients, although DA allografts given the adenovirus vector 7 days or 6 hr before, and 3 days after transplantation were rejected within 30 days in recipients. Serum levels of human IL-10 in gene-transferred rats were maximum from day 2 to 7. The serum level of human IL-10 then decreased gradually, and human IL-10 was not detected by ELISA 30 days after gene-transduction. In gene-transduced long-term surviving liver allografts, IL-10 was expressed, and the expression of IL-4 was also up-regulated on posttransplant day 3, despite the expression of Th1 cytokines (IL-2 and interferon-gamma), although in rejected liver allografts, IL-2 and interferon-gamma were expressed without expression of IL-4 and IL-10. CONCLUSIONS: The prolongation of survival of IL-10 cDNA transferred liver allografts might be due to inhibition of the early phase of alloimmune-response by over expression of IL-10, despite the expression of IL-2 and interferon-gamma.     

A rat model of chronic allograft liver rejection

INTRODUCTION: The aim of this study was to develop a rat model of chronic irreversible rejection, which is a major causes of late graft loss and retransplantation after orthotopic liver allotransplantation. METHODS: Allogeneic liver transplantation was performed in a rat combination of Dark Agouti (DA) to Brown Norway (BN). Group A was left without treatment, group B received cyclosporine' (CsA; 1 mg/kg/d) and group C, CsA (4 mg/kg/d). Animals were followed for 6 months. Liver tissue was harvested to construct a time course of histological changes after liver transplantation using histopathological and morphometric techniques. We compared the total histological score of rejection activity index and survival rates. RESULTS: In untreated animals, irreversible acute rejection developed, all animals died within 15 days. In the low-dose CsA group, all animals that survived more than 30 days developed moderate to severe manifestations of chronic liver rejection, with graft infiltration, ductular damage or proliferation, obliterative arteriopathy, and liver fibrosis. No apparent histological alterations were observed in group C. Survival analysis showed significant differences between the three groups. CONCLUSIONS: In the rat strain combination of DA --> BN with low-dose immunosuppression, early mild inflammation was followed by the development of chronic rejection.     

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)     

Tolerance of rat liver allografts induced by short-term selective immunosuppression combining monoclonal antibodies directed against CD25 and CD54 with subtherapeutic cyclosporine

BACKGROUND: Our purpose was to develop and evaluate protocols for selective immunosuppression after liver transplantation using the monoclonal antibodies (mAbs) NDS-61, directed against the interleukin-2 receptor (CD25), and 1A29, directed against the intercellular adhesion molecule-1 (CD54), in combination with subtherapeutic cyclosporine (CsA). METHODS: Orthotopic rat liver transplantation (ORLT) was performed in a DA-to-LEW strain combination. Immunosuppression was administered from day 0 to +13. Functional parameters such as survival time, body weight, and serum bilirubin levels were measured and the liver grafts were evaluated histologically. RESULTS: A stepwise tapering of CsA from 3 to 0.25 mg/kg/day reduced the long-term survival rate. All animals died at a CsA dosage of 0.25 mg/kg/day, which was therefore defined as subtherapeutic. Monotherapy with the anti-CD25 mAb was performed at dosages of 600 and 1800 microg/kg/day. The lower mAb dosage resulted in a long-term survival rate of 12% and was defined as subtherapeutic. The combination therapy of CsA (0.25 mg/kg/day) and anti-CD25 mAb (600 microg/kg/day) produced a synergistic effect and led to a long-term survival rate of 84%. This survival rate was significantly higher than those after either CsA (P<0.005) or anti-CD25 mAb (P<0.001) monotherapy. Both dosages (10 and 30 microg/kg/day) of anti-CD54 mAb monotherapy as well as anti-CD54 mAb combined with a subtherapeutic dosage of CsA were ineffective in preventing acute allograft rejection. The addition of anti-CD54 mAb (30 microg/kg/day) to combined CsA plus anti-CD25 mAb therapy (triple therapy), however, increased the long-term survival rate to 100%. In the triple therapy group there was no rejection process in the liver allografts at any time, and donor-specific tolerance could be shown by donor-specific and third-party heterotopic heart transplantation. CONCLUSIONS: The synergistic action of subtherapeutic CsA plus anti-CD25 mAb NDS-60 could be demonstrated, whereas anti-CD54 mAb only had a positive effect in a triple therapy group. Triple therapy prevented both acute and chronic rejection and induced donor-specific tolerance.     

Long-term survival of orthotopic bowel allografts in the rat treated with short-term low-dose cyclosporine

Orthotopic bowel transplantation continues to be a "difficult" procedure, both experimentally and clinically, despite the advent of cyclosporine. Even with high-dosage CsA administration, long-term survival has been sporadic and short-term mortality high. In the present study, a low-dosage CsA regimen (5 mg/kg/day for 2 weeks postoperatively) produced prolonged survival in BN-Lewis donor/recipient combinations utilizing an orthotopic bowel transplantation model with portal venous anastomosis. The transplanted bowel continued to survive despite the lack of maintenance dosage after initial induction. The recipient animals evinced donor-specific hyporesponsiveness as donor skin grafts were rejected in a slow, indolent fashion. Third-party skin grafts acutely rejected in normal fashion. It was remarkable that the bowel grafts continued to survive in good condition despite the rejection of donor-specific and nonspecific challenging skin grafts placed on the animals. The precise mechanism of this donor-specific hyporesponsiveness is not known.