Mean Survival Time Of Allografts Research Articles

Inhibition of RelA expression via RNA interference induces immune tolerance in a rat keratoplasty model.

RelA, the most important regulator of NF-kB activity, and its mechanisms in keratoplasty immune rejection have not been fully investigated. In the present study, lentivirus-mediated silencing of RelA expression in a bone marrow-derived dendritic cell (BMDC) model was tested. The BMDCs were transfected with RelA-shRNA to induce an immature, maturation-resistant and tolerogenic phenotype, while not significantly changing IFN-γ, IL-10 and IL-17 expression. A fully allogeneic rat cornea transplant model was established for in vivo studies. The allograft mean survival time (MST) of lv-shRelA-DC injection groups were significantly longer than the untreated BMDC group and control group. The corneal opacity and neovascularization scale of the lv-shRelA-DC injection groups were slight compared to pair control others. Postoperative flow cytometric analysis revealed that the percentage of Treg positive cells was dramatically increased in animals that received an lv-shRelA-DC injection. ELISA and qRT-PCR analyses of serum showed that IFN-γ and IL-17 expression were suppressed by lv-shRelA-DC treatement. In vivo experiments demonstrated that IL-10 induced immunosuppression was partly attributed to injection of lv-shRelA-DC throughout the experiment, differing from the general anti-inflammatory factors. Luciferase and Chromatin IP evaluation showed that RelA knockdown in BMDCs significantly reduces DNA binding to IFN-γ, IL-10 and the IL-17 promoter and inhibited of transcriptional activity. Taken together, this study illustrates a significant role of RelA in mediating the corneal neovascularization by affecting IL-17 expression. Our comprehensive analysis shows that the significant role of RelA provides a novel and feasible therapeutic approach for the prevention of corneal allograft rejection.

Pretreatment of Transfused Donor Splenocytes and Allografts With Mitomycin C Attenuates Acute Rejection in Heart Transplantation in Mice

ObjectiveThe aim of this study was to investigate the effect of pretreatment of donor splenocytes and grafts with mitomycin C (MMC) on heart allograft survival, as well as to demonstrate the mechanism of function. MethodsDonor splenocytes from Balb/C mice were incubated with MMC (40 μg/mL) in vitro and then transfused into recipients (C57BL/6 mice). The heart allograft was perfused with MMC before harvest. Graft survival and histopathology were examined. Lymphocyte activation, regulatory T cells, and donor splenocyte apoptosis were examined with the use of flow cytometry. ResultsMMC incubation in vitro induced apoptosis of donor splenocytes (15.5 ± 2.3% vs 23.2 ± 4.2%; P < .01). Either intravenous injection of MMC-treated donor splenocytes or transplantation of allograft pretreated with MMC prolonged heart allograft mean survival time from 7 ± 0.8 days to 20.5 ± 1.9 days or 10 ± 0.9 days, respectively (both P < .01). A combination of MMC-pretreated donor splenocyte transfusion and allografts showed the best effect on prolongation of graft survival (28.5 ± 1.8 days). Activation of CD4+ T cells in spleen and peripheral lymph nodes of recipients was significantly inhibited by either MMC-splenocyte transfusion or the combination treatment. Meanwhile, the percentage of CD4+CD25+Foxp3+ regulatory T cells in the spleen was increased in the MMC-splenocyte transfusion group (15.5 ± 1.1% vs 18.2 ± 0.9%; P < .05). ConclusionsBoth injection of MMC-conditioned donor splenocytes and MMC-conditioned allograft have effects on prolongation of heart allograft survival in mice, and MMC-conditioned donor splenocytes might play an essential role. MMC pretreatment induced regulatory T cells likely through induction of donor splenocyte apoptosis, and thus it inhibited T-cell activation.

Effects of long-term administration of low-dose FTY720 on survival of murine cardiac allograft

This study examined the effect of long-term administration of low-dose FTY720 on survival of murine cardiac allograft and the possible mechanism. Murine models of abdominal heterotopic heart transplantation were established. Low-dose FTY720 (0.3 mg/kg) was administrated to the animals 4 days before the transplantation of cardiac allografts until the occurrence of rejection or the observation terminals. The animals without FTY720 treatment and those with syngeneic cardiac grafts transplanted served as controls. The mean survival time (MST) of grafts, and T lymphocyte subsets in grafts, peripheral blood and lymphoid organs were measured by histopathological examination or flow cytometry, and compared among groups. The results showed that the MST of allografts in FTY720-treated mice was more than 40 days, significantly longer than that in the untreated group (MST=8 days, P<0.01). After the long-term administration of FTY720, the proportion of CD4(+) and CD8(+) lymphocytes in peripheral blood was diminished significantly, but the proportion of CD4(+) lymphocytes was increased in mesenteric lymph nodes (MLNs) and spleen. Immunofluorescence staining revealed that the infiltration of CD4(+) and CD8(+) lymphocytes in allografts was significantly inhibited after long-term administration of low-dose FTY720. It was concluded that low-dose long-term administration of FTY720 could promote T lymphocytes in lymphatic organs and decrease their infiltration in allografts, resulting in the inhibition of rejection and the long-term survival of allografts.

The Effects of AEB071 (Sotrastaurin) with Tacrolimus on Rat Heterotopic Cardiac Allograft Rejection and Survival

AEB071 (sotrastaurin) is a specific inhibitor of protein kinase C that prevents T-cell activation. Our previous study demonstrated that AEB071 monotherapy could prevent acute cardiac allograft rejection in rats. Herein, we investigated the effects of AEB071 combined with various doses of tacrolimus (Tac) on the allograft rejection and survival in a rat heart transplantation model. Heterotopic cardiac transplantation from Brown-Norway to Lewis rats was performed. Cardiac allograft survival was assessed by monitoring heartbeats in six recipients of each experimental group. Another four recipient rats were selectively sacrificed in each group at d 7 post-transplantation for histologic examination. Serum transaminases, blood urea nitrogen, and creatinine concentrations were measured. AEB071 monotherapy prolonged allograft mean survival time (MST) compared with the untreated control group. Also a combination of AEB071 and Tac prolonged MST compared with monotherapy groups with higher dose of Tac. In the cardiac graft histology, AEB071 combined with Tac 0.6 mg/kg/d significantly decreased the rejection grade as indicative of decreased inflammatory cell infiltration into the graft. No experimental group was found with any abnormal histologic or serologic evidence of liver and kidney toxicity. AEB071 combined with a smaller dosage of Tac may be clinically possible to establish calcineurin inhibitor (CNI) minimization protocol in solid organ transplantation.

Potent Farnesyltransferase Inhibitor ABT-100 Abrogates Acute Allograft Rejection

Farnesyltransferase inhibitors (FTIs) inhibit the function of Ras, a GTPase involved in carcinogenesis and T cell activation. We evaluated the in vitro and in vivo immunomodulatory properties of a rationally designed FTI, ABT-100. The effects of ABT-100 on human peripheral blood mononuclear cell (PBMC) proliferation and the expression of the T cell activation markers CD25 and CD69 were studied. In a Wistar to Lewis rat heterotopic cardiac transplant model, ABT-100 was orally dosed alone or with a subtherapeutic course of cyclosporine (CsA). The degree of graft immune cell infiltrate was determined. ABT-100 potently inhibited PBMC proliferation, but did not decrease expression of CD25 and CD69 during activation. Treatment with 25, 50 and 100 mg/kg ABT-100 BID increased allograft mean survival time (MST) to 12.8+/-3 days, 13.5+/-5 days and 13.8+/-3 days, respectively (vs 6.5+/-3 days for controls, p<0.001 by log rank). A subtherapeutic course of CsA increased MST to 12.7+/-3 days (p<0.001 vs control). Combination with ABT-100 at 25 and 100 mg/kg BID improved MST to 18.7+/-5 days and 19.5+/-4 days (both p<0.001 vs control and respective monotherapy groups). ABT-100 treatment at 100 mg/kg BID significant decreased the amount of graft infiltrate (2.5+/-4 mononuclear cells/high power field (hpf) vs 29+/-11 cells/hpf, p<0.001). This is the first report that a specific FTI delays the development of acute rejection and supports the strategy of inhibiting Ras to impart immunomodulation. The antirejection and anticarcinogenic effects make FTIs a potentially useful adjunct in the antirejection regimens of malignancy-prone organ transplant recipients.

Anti-transferrin receptor monoclonal antibody: a novel immunosuppressant.

Transferrin receptor is a widely distributed cell surface receptor present on most proliferating and highly specialized quiescent cells. Expression of transferrin receptor on the surface of immune cells is up-regulated during T-cell activation after the interaction of the antigen-MHC with the T cell receptor. The role of transferrin receptor in T-cell activation has not been well-established. Since transferrin receptor is physically associated with the CD3 zeta-chain, blockade of transferrin receptor has the potential to interfere with the T-cell signals important in transplant rejection. Anti-transferrin receptor monoclonal antibody (mAb) was administered in vivo and in vitro to determine whether this agent was effective in prolonging allograft survival and altering cell-mediated immunity. Using donor C57BL/6J (H2b) hearts transplanted to CBA/J (H2k) recipients, anti-transferrin receptor mAb at the time of transplantation prolonged cardiac allograft mean survival time to 25.7+/-0.9 days compared with untreated (13.3+/-0.6 days, P < 0.05) or isotype-matched (10.7+/-0.4 days, P < 0.05) controls. Anti-transferrin receptor mAb administered in vivo failed to suppress the subsequent allogeneic responses. However, when added to culture, anti-transferrin receptor mAb suppressed the allogeneic cytotoxic T lymphocyte response by 79-100% but not the mixed lymphocyte response. These studies are the first to suggest that transferrin receptor is a potential therapeutic target for clinical transplantation. Future studies will determine the most efficacious dose and time for maximal immunosuppression and the mechanisms responsible for the immunosuppression exhibited by antitransferrin receptor mAb.

BLOCKADE OF MULTIPLE COSTIMULATORY RECEPTORS INDUCES HYPORESPONSIVENESS

T-lymphocyte activation requires engagement of the T cell receptor with antigen-major histocompatibility complex, and simultaneous ligation of costimulatory pathways via the lymphocyte receptors CD2 and CD28/ CTLA4. Anti-CD2 monoclonal antibody (mAb) blocks the interaction of the antigen-presenting cell receptor CD48 with its ligand CD2, whereas CTLA4Ig binds with high affinity to the antigen-presenting cell ligands B7-1 and B7-2, blocking their interaction with CD28/CTLA4. We tested the immunosuppressive effects of simultaneously blocking both costimulatory pathways. Using donor C57BL/6J (H2b) hearts transplanted to CBA/J (H2k) recipients, anti-CD2 mAb plus CTLA4Ig administered at the time of transplantation prolonged cardiac allograft mean survival time to >120 days compared with untreated controls (12.2+/-0.5 days, P<0.01), anti-CD2 mAb alone (24.8+/-1.0 days, P<0.01), or CTLA4Ig alone (55.0+/-2.0 days, P<0.01). Retransplantation of these recipients with donor-specific and third-party grafts demonstrated that hyporesponsiveness and tolerance were achieved. In vitro stimulation of lymphocytes from tolerant recipients with donor-specific alloantigen resulted in normal cytotoxic T lymphocyte and mixed lymphocyte reaction responses, showing that clonal deletion or anergy did not occur, but that graft adaptation or suppression likely helped to maintain long-term graft survival. In vitro combinations of anti-CD2 mAb and CTLA4Ig suppressed the generation of allogeneic cytotoxic T lymphocytes (58%) and the mixed lymphocyte reaction (36%); CTLA4Ig was more effective in this regard and the two agents were not synergistic. Anti-CD2 mAb and CTLA4Ig suppressed mitogen-driven proliferation in differential fashions, suggesting that they affected independent signaling pathways. Anti-CD2 mAb and CTLA4Ig also inhibited interleukin (IL)-2, IL-4, and IL-2 receptor (CD25). These data indicate that anti-CD2 mAb plus CTLA4Ig induces hyporesponsiveness and tolerance. The mechanism is likely related to the initial disruption of independent pathways of T-lymphocyte activation leading to antigen-specific long-term graft survival.

Local efficacy of cyclosporine in corneal transplant therapy.

Studies were conducted to evaluate the efficacy of direct injections of cyclosporine (CsA) into the anterior chamber for the prevention of corneal allograft rejection in Dutch Belted rabbits. The mean survival time (MST) of grafts progressively increased from 50 to 89 days as the CsA concentration in the dose was increased from 1 to 10 mg/mL. Injection of 30 microL of 20 mg/mL CsA in olive oil prolonged graft survival to beyond 125 days without any signs of rejection. By comparison, the MST of allografts in control animals which received no therapy was 32 +/- 5 days, and the MST in animals administered a placebo of olive oil only was 31 +/- 4 days. The observed concentration dependence of the MST on CsA concentration is likely related to the time over which the drug delivery rate provides sufficient drug to achieve a therapeutic concentration in the aqueous humor; these studies suggest that the minimum delivery rate to the anterior chamber is between 200 and 325 ng/day. The efficacy of CsA was due to local delivery, and was likely not a systemic effect, because CsA was not detected in the systemic circulation at any time. This indicates that direct delivery of CsA to the eye can be useful in prolonging corneal graft survival, while minimizing systemic side effects. Separate experiments revealed that episodes of advanced rejection could not be reversed by a 30 microL dose of 20 mg/mL CsA to the anterior chamber, indicating the importance of avoiding long periods of subtherapeutic dosing.

IMMUNOSUPPRESSIVE PROPERTIES OF THE BENZOTHIAZEPINE CALCIUM ANTAGONISTS DILTIAZEM AND CLENTIAZEM, WITH AND WITHOUT CYCLOSPORINE, IN HETEROTOPIC RAT HEART TRANSPLANTATION

In vitro studies have shown that calcium channel blockers (CCB) exert inhibitory effects on immunocompetent cells but conflicting results have been reported as to the translation of these effects into significant in vivo immunosuppression. In this study we evaluated the effects of the benzothiazepine-like calcium blockers diltiazem and clentiazem, given alone or associated with cyclosporine on survival improvement of heterotopic rat heart transplants. Inbred male rats of the Lewis strain were used as recipients and Wistar-Furth as donors. Following abdominal implantation of the graft, recipients were randomly divided into 9 groups (n = 5). Group 1 were control isografts (Lew-Lew); group 2 were control allografts (WFu-Lew), and group 3 were allografts treated with low-dose oral cyclosporine 2 mg/kg/day. Groups 4 and 5 were allografts treated with oral diltiazem 0.25 and 2.50 mg/kg/day. Groups 6 and 7 were treated with oral clentiazem, 0.25 and 2.50 mg/kg/day. Groups 8 and 9 consisted of allografts receiving low-dose cyclosporine with either diltiazem or clentiazem 2.50 mg/kg/day, all drugs were administered daily by gavage. Graft function was monitored by transabdominal palpation, and rejection was considered to be complete when no contraction of the graft could be detected. Mean survival time of untreated allografts was 6.4 +/- 0.5 days. Cyclosporine alone increased the mean survival time to 10.6 +/- 2.7 days (P < 0.05 vs. group 2). At all doses studied, diltiazem and clentiazem significantly increased mean survival time of allografts, clentiazem being slightly more potent than diltiazem. In addition, the observed beneficial effects of the benzothiazepine-like calcium channel blockers were dose-dependent. When combined with cyclosporine, diltiazem and clentiazem interacted synergistically (mean survival time increased to 16.8 +/- 3.4 days for diltiazem and 15.8 +/- 1.4 days for clentiazem). These results demonstrate that the benzothiazepine-like calcium channel blockers, as opposed to phenylalkylamines or dihydropyridines, afford significant immunosuppression when used alone. These observations, and the fact that they beneficially interact with cyclosporine, suggest that the benzothiazepine-like calcium antagonists should be considered the drugs of choice when CCBs are to be selected in transplanted patients.

THE CHYLOESOPHAGEAL FISTULA A New Approach to Thoraic Duct Drainage

The main reason for the virtual abandonment of external thoracic duct drainage as an immunosuppressive measure is not its lack of efficacy, but the time-consuming technical problems of maintaining cannula patency and replacing the large obligatory losses of fluid and protein. In an effort to overcome these problems we have devised a method of diverting thoracic duct lymph internally into the esophagus of the sheep, our hypothesis being that fluid and protein should be resorbed, but lymphocytes and antibodies destroyed. By isolating that part of the venous system into which the thoracic duct drains and anastomosing this conduit to the cervical esophagus a chyloesophageal fistula was created. A mean patency of 19 days was demonstrated radiologically and there was a reproducible peripheral blood lymphopenia of over 50% of preoperative values at 4 weeks. Although plasma albumin levels fell from 37 g/L to 29 g/L at 1 week, they remained stable thereafter. No parenteral fluid or protein was administered, yet the animals remained well with no significant weight loss or overt signs of dehydration or hypoproteinemia. Skin allograft mean survival time was prolonged from 9 to 11.8 days (P less than 0.01).

Effect of rifampin on the immune response in mice.

In an investigation of the effect of rifampin on the immune response in mice, the cellular immunity was evaluated with the split-heart allograft technique. The survival time of the heart in animals treated with rifampin at a dose of 20 mg/kg per day from the day of the transplantation until the graft was rejected was longer (33.7 days, P less than 0.001) than that of animals not treated with antibiotics (14.5 days). When rifampin was given at a dose of 5 mg/kg per day for the same period, the mean survival time of allografts was 19.5 days. The number of demonstrable plaques of hemolysis and the humoral antibodies to sheep erythrocytes were also reduced by a human therapeutic dose (20 mg/kg per day). However, the suppression of the humoral immune response was probably of more limited biological significane, suggesting a differential sensitivity to rifampin. In contrast to rifampin, benzylpenicillin had no noteworthy inhibiting effect on the cellular or humoral immune response.