DA Heart Research Articles

MicroRNA let 7i regulates DCs maturation targeting IL-10 via JAK1-STAT3 signal pathway subsequently induces prolonged cardiac allograft survival in rats

Abstract Objective In this study, we investigated whether microRNA (MiR) let 7i regulates DCs maturation targeting IL-10 via JAK1-STAT3 signal pathway subsequently prolongs rat cardiac allograft survival. Methods QRT-PCR, ELISA, dual luciferase assay were performed to verify whether IL-10 was the target of let 7i. Tregs were assessed by flow cytometry and immunohistochemical study, and JAK1, STAT3 and pSTAT3 expressions were detected by western bolt. Lewis recipients of DA heart were transfused with PBS, LPS-mDCs, or let 7i inhibitor-mDCs. Allograft survival times were recorded, and histologic study were performed. Results Expression of IL-10 mRNA level and production of IL-10 were increased in let 7i inhibitor-mDCs as compared to LPS-mDCs. Luciferase activity showed that the translational level of the IL-10 luciferase reporter was decreased by let 7i mimic, but increased by let 7i inhibitor. MiR let 7i inhibitor suppressed DC maturation by depressing CD80 and CD86, however， pretreatment of IL-10 SiRNA attenuated the suppression. Expressions of JAK1, STAT3, and pSTAT3 in mDCs were suppressed by let 7i mimic and pretreatment of IL-10 SiRNA, however up regulated by let 7i inhibitor. Lewis recipients transfused with let 7i inhibitor-mDCs significantly prolonged DA cardiac allograft survival. The allografts transfused with let 7i inhibitor-mDCs showed slight cell infiltration and significantly preserved graft structure. Inhibition of let 7i increased Tregs and modulated cytokine profiles both in vivo and in vitro. Conclusion MiR let 7i regulated DCs maturation and function targeting the IL-10 through JAK1-STAT3 pathway. Moreover, transfusion of let 7i inhibitor-mDCs induced prolonged cardiac allograft survival and generated Tregs.

Role of the liver in determining alloimmune response in vitro following donor-specific spleen cell injection

The aim of our study was to investigate the allogeneic influence inside and outside the liver in vitro following donor-specific cell injection (DSI). DA rats (RT1a) were used as donors and WS rats (RT1k) as recipients. WS were sensitized with DA spleen cells, followed 24 h later by total hepatectomy. The liver was transplanted into another WS (sensitized liver-grafted; SL-Grafted). The hepatectomized WS underwent liver transplantation from a naive WS (sensitized liver-removed; SL-Removed). Alloantigens accumulated in the liver in SL-Grafted and in the extrahepatic tissue/organ(s) in SL-Removed. DA hearts were transplanted 10 days after antigen administration. To analyze the immune responses, we measured Th1/Th2 cytokine profiles, and perforin mRNA in various organs, allogeneic mixed lymphocyte reaction (MLR), and donor-specific immunoglobulin. Th1 cytokine levels in the liver of SL-Grafted and in spleen of SL-Removed were highly and rapidly upregulated but decreased thereafter. IFN-γ and perforin mRNAs were significantly higher in SL-Grafted and lower in SL-Removed. MLR was significantly higher in SL-Grafted than SL-Removed and controls. There was no significant difference in the donor-specific immunoglobulin level. Our findings suggest that liver and other organs may behave differently to alloantigen, suggesting the importance of an early Th1 reaction in the liver and spleen.

Experimental heart transplantation: effect of cyclosporine on expression and activity of metzincins.

Metzincins, such as matrix metalloproteases (MMP), and extracellular matrix (ECM) proteins are differentially regulated in inflammation. We hypothesised that metzincins are also dysregulated in experimental acute cardiac allograft rejection. We investigated the Dark Agouti-to-Lewis (DA-to-Lew) rat model of acute cardiac allograft rejection. Cyclosporine (CsA) (7.5 mg/kg/d) was given from transplantation to sacrifice (day +5). At that time, mRNA levels were analysed by Affymetrix genechip and quantitative reverse transcription polymerase chain reaction (qRTPCR). MMP protein and activities were analysed by immunohistology, fluorometry, zymography and Western blots. In untreated rejected DA allografts, mRNA levels of MMP-2/-7/-9/-/12-/14, a disintegrin and metalloprotease (ADAM)-17, tissue inhibitor of metalloprotease (TIMP)-1/-3 were increased, whereas MMP-11/-16/-24 and TIMP-2/-4 were lowered compared to native DA hearts. With respect to these untreated allografts, CsA lowered mRNA levels of MMP-7, TIMP-1/-3 (TIMP-2/-4 remained relatively low) and ADAM17, but augmented mRNA levels of MMP-11/-16/-23 and of many ECM genes. Immunohistology showed increased staining of MMP-2 in acute rejection (AR). Overall MMP activity was augmented in both transplanted groups, but CsA reduced MMP-9 activity and MMP-14 production. Taken together, MMP and TIMP were upregulated during acute AR. CsA ameliorated histology of rejection but showed potential pro-fibrotic effects. Thus, MMP and TIMP may play a role in acute cardiac allograft rejection, and beneficial modification of the MMP-ECM balance requires interventions beyond CsA.

Experimental heart transplantation: effect of cyclosporine on expression and activity of metzincins.

Metzincins, such as matrix metalloproteases (MMP), and extracellular matrix (ECM) proteins are differentially regulated in inflammation. We hypothesised that metzincins are also dysregulated in experimental acute cardiac allograft rejection. We investigated the Dark Agouti-to-Lewis (DA-to-Lew) rat model of acute cardiac allograft rejection. Cyclosporine (CsA) (7.5 mg/kg/d) was given from transplantation to sacrifice (day +5). At that time, mRNA levels were analysed by Affymetrix genechip and quantitative reverse transcription polymerase chain reaction (qRTPCR). MMP protein and activities were analysed by immunohistology, fluorometry, zymography and Western blots. In untreated rejected DA allografts, mRNA levels of MMP-2/-7/-9/-/12-/14, a disintegrin and metalloprotease (ADAM)-17, tissue inhibitor of metalloprotease (TIMP)-1/-3 were increased, whereas MMP-11/-16/-24 and TIMP-2/-4 were lowered compared to native DA hearts. With respect to these untreated allografts, CsA lowered mRNA levels of MMP-7, TIMP-1/-3 (TIMP-2/-4 remained relatively low) and ADAM17, but augmented mRNA levels of MMP-11/-16/-23 and of many ECM genes. Immunohistology showed increased staining of MMP-2 in acute rejection (AR). Overall MMP activity was augmented in both transplanted groups, but CsA reduced MMP-9 activity and MMP-14 production. Taken together, MMP and TIMP were upregulated during acute AR. CsA ameliorated histology of rejection but showed potential pro-fibrotic effects. Thus, MMP and TIMP may play a role in acute cardiac allograft rejection, and beneficial modification of the MMP-ECM balance requires interventions beyond CsA.

Tolerance to rat liver allograft after total lymphoid irradiation is mediated by CD4+CD25+ regulatory T cells

We have previously determined that post transplant total lymphoid Irradiation (TLI) induces tolerance in an experimental model of liver transplantation. The purpose of this study is to characterize the mechanism of TLI induced tolerance.TLI significantly prolonged survival in Lewis recipients of fully‐histoincompatible DA liver allografts with 88% of the recipients surviving long‐term (>100 days). CD4+CD25+Foxp3+ T cells were markedly increased in the liver grafts, PBMC, and spleen by day 35 post‐transplant and remained high in the spleen >100 days post‐transplant. Splenocytes from long‐term surviving recipients have a reduced alloresponse in MLR. Moreover, isolated splenic CD4+CD25+ T cells inhibited the MLR of naive Lewis splenocytes to DA splenocytes. Importantly, adoptive transfer of whole splenocytes into naive Lewis recipients of DA heart grafts significantly prolonged cardiac graft survival (MST whole splenocytes 33 days vs. control 18.5 days:p<0.01), whereas depletion of CD4+CD25+ T cells from splenocytes completely abrogated the prolongation of cardiac allograft survival (MST 16 days; p<0.01, vs. whole splenocytes).TLI induces tolerance to liver allografts via a mechanism involving CD4+CD25+Foxp3+ T cells that accumulate in the liver early after transplant and remain in the spleen >100 days. Studies using TLI to induce tolerance in clinical liver transplantation are currently underway.

In Vivo Assessment of Resting Myocardial Function in High and Low Performing Genetic Models of Aerobic Running Capacity

BACKGROUND: We previously identified two inbred rat strains divergent for treadmill aerobic running capacity (ARC); the low performing Buffalo (BUF) and the high performing DA rats. Subsequent cardiac performance assessment using the Langendorff-Neely working heart preparation demonstrated that isolated DA hearts exhibit increased stroke volume and cardiac output compared to the BUF hearts, with no difference in intrinsic heart rate (HR). In the present study, we investigated in vivo resting cardiac function in BUF and DA rats using echocardiography. METHODS: Male DA (n=11) and BUF (n=12) rats (aged 14-17 weeks) were lightly anesthetized with pentobarbital (40mg/kg, ip), the chest was shaved, and the animal was situated in the supine position on a warming pad. Two-dimensional guided M-mode studies were performed from the parasternal long axis for the determination of left ventricular end-diastolic (LVDd) and end-systolic (LVSd) diameters and fractional shortening (FS). The myocardial performance index (MPI), stroke volume (SV) and cardiac output (CO) were calculated using Doppler pulsed-wave traces of mitral and aortic flow measured at the level of the LV outflow tract from the apical four-chamber view. Cardiac index (CI) was calculated by dividing CO by body weight. RESULTS: BUF rats had significantly greater body weight and a larger SV than the DA rats, but had significantly lower HR and CI (Table). No differences in LVDd, LVSd, FS, MPI, or CO were observed (Table).TableCONCLUSION: After correcting for differences in HR and body weight, in vivo assessment of resting cardiac function revealed that DA rats exhibit greater cardiac performance than BUF rats. Our findings contrast with those of the working heart preparation in that in vivo assessment of cardiac function shows DA rats to have a smaller stroke volume and greater HR than the BUF rats, resulting in an identical CO between the two strains. Nevertheless, cardiac index is significantly greater in DA rats, possibly contributing to the superior ARC observed in this rat strain compared to that of the BUF strain.

Anergic lymphocytes generated by blocking CD28 and ICOS pathways in vitro prolong rat cardiac graft survival

Regulatory cells may play a pivotal role in inducing and maintaining transplantation tolerance. We investigated the mechanism of anergic lymphocytes with regulatory cell potential generated in vitro by ICOS and CD28 co-stimulatory blockades as a source of cellular therapy for treating allograft rejection. Anergic lymphocytes were generated by a mixed lymphocyte reaction consisting of DA splenocytes as the stimulator and Lewis splenocytes as the responder in the presence of anti-ICOS mAb and rCTLA-4Ig. Immunoregulatory effects of these lymphocytes were evaluated by secondary MLR and using other various stimulations. DA heart was transplanted into 7.5 Gy-irradiated Lewis rat after intravenous administration of these cells and/or Lewis spleen lymphocytes. We observed that these lymphocytes were not only anergic to alloantigen and polyclonal stimulations but also exhibited regulative activity to inhibit the alloreactive T-cell response. Our adoptive transfer studies revealed that irradiated recipients that received both anergic lymphocytes and naïve Lewis lymphocytes had significantly prolonged DA cardiac graft survival (mean 17.5 days) compared with a group that received Lewis lymphocytes alone (mean 10.8 days). Furthermore, some of the recipients accepted the graft indefinitely after receiving anergic lymphocytes alone (> 100 days). These results demonstrated that anergic lymphocytes with regulatory activities can be generated through blocking co-stimulatory signals, CD28 and ICOS, simultaneously in vitro, and may advance a new immunomodulatory strategy for preventing allorejection in organ transplantation.

Downregulation of interleukin-12p35 and upregulation of interleukin-12p40 mRNA expression in heart allografts by blood transfusion from granulocyte colony-stimulating factor-treated donors.

Pretransplant treatment of recipients with recombinant human granulocyte colony-stimulating factor (rhG-CSF, 250 microg/kg/day s.c. for 5 days) facilitates heart allograft acceptance in tacrolimus-treated rat recipients. We examined effectiveness of transfusion of in vivo rhG-CSF-treated blood since rhG-CSF induces immunoregulatory cells in human blood. DA heart grafts were transplanted into tacrolimus (2 mg/kg i.m. on day 0)-treated Lewis recipients. Although graft survival prolongation by blood transfusion on day 0 from rhG-CSF-treated syngeneic Lewis was comparable to that in directly rhG-CSF-pretreated recipients (p = 0.22), transfusion of rhG-CSF-treated allogeneic DA blood was much more effective (p = 0.0016). Intragraft cytokine mRNA levels were measured by reverse transcription and real-time polymerase chain reaction at 24 h after transplantation. IL-12p35 expression was downregulated by both treatments. Notably, IL-12p40 was upregulated by rhG-CSF-treated DA blood transfusion but downregulated by transfusion of rhG-CSF-treated isogeneic blood. Differential expression of IL-12 subunits was associated with facilitation of graft acceptance by rhG-CSF-treated donor blood transfusion.

Prolongation of heart allograft survival of rats treated by a Th2 inhibitor

In terms of Th1/Th2 balance in response to signals given during donor antigen presentation, induction of tolerance is more often correlated with Th2-type than with Th1-type reactions. However, in our study, heart allograft survival was prolonged by treatment of rats with a Th2 inhibitor. Suplatast tosilate (IPD; Taiho; Tokyo, Japan) is a novel immunoregulator that suppresses IgE production and eosinophil infiltration through selective inhibition of interleukin (IL)-4 and IL-5 synthesis by Th2-like cells but not IFN-γ production in Th1 cells. Five LEW rats of DA heart grafts were treated with IPD (100 μg/day, p.o.) for 10 days. Heart allograft survival of all IPD-treated cases was prolonged more than 14 days while the beating of heart grafts in control groups was stopped within 9 days. In an in vitro study, the cell proliferation both in Con A blast and in mixed lymphocyte reaction assay was suppressed by IPD in dose-dependent manner. We could at least in part conclude that Th2 inhibition might temporarily suppress heart allograft rejection.

Short-course methotrexate and long-term acceptance of fully allogeneic rat cardiac grafts: a possible mechanism of tolerance

Although a short course of methotrexate (MTX) has a potential immunoregulatory effect on clinical allograft rejection, little data are available about the drug, and the mechanism of hyporeactivity after withdrawal is still unknown. In previous studies, we achieved permanent graft acceptance through administration of a short course of high-dose MTX during heterotopic rat heart transplantation (HHT) in a combination of DA (MHC haplotype; RT1 a) to PVG/c (RT1 c) rats. A 3-week course of MTX (0.25 mg/kg/day) was administered intraperitoneally to the PVG/c recipients of a DA heart graft, and 11 of 16 rats survived longer than 300 days after HHT. The splenic lymphocytes obtained from one recipient showed high reactivity against donor type splenic lymphocytes, but others did not. All serum samples from recipients showed immunosuppressive activity. The serum had anti-donor antibodies. These results showed that tolerance induced by short-course MTX was maintained by a serologic factor believed to be anti-idiotypic antibodies.

Cardiac performance in inbred rat genetic models of low and high running capacity.

1. Previous work demonstrating that DA inbred rats are superior to COP inbred rats in aerobic treadmill running capacity has indicated their utility as genetic models to explore this trait. We tested the general hypothesis that intermediate phenotypes of cardiac function and calcium metabolism are responsible for the difference in capacity between these strains. 2. Logical cardiac trait differences were estimated at a tissue (isolated papillary muscle), cellular (isolated left ventricular cells), and biochemical level of organization. 3. DA hearts were found to give significantly higher values than COP hearts for: (1) maximal developed tension (38.3 % greater), and rates of tension change in contraction (61 %) or relaxation (59 %) of isolated papillary muscle, (2) fractional shortening (50 %), amplitude of the Ca(2+) transient (78.6 %), and caffeine-induced release of Ca(2+) from the sarcoplasmic reticulum (SR; 260 %) in isolated ventricular myocytes, and (3) Na(+),K(+)-ATPase activity of isolated myocytes (17.3 %). 4. Our results suggest that these trait differences may prove useful for further studies into the genes responsible for natural variations in both ventricular function and aerobic endurance capacity. Understanding the genetic basis of aerobic capacity will help define the continuum between health and disease.

Simplified method of heterotopic rat heart transplantation using the cuff technique: Application to sublethal dose protocol of methotrexate on allograft survival

Rodent heterotopic heart transplantation (HHT) models have been developed for the study of transplantation immunology. Most of these transplantations are performed by hand-suture techniques, requiring several months of training. We describe a modified technique of rat HHT in the neck, using a cuff method that can be mastered by beginners within a few weeks. Our main modification of the rat HHT in the neck is that the right superior vena cava of the graft is chosen as an outflow duct, while the pulmonary artery has been taken as an effluent drainage in the ordinary HHT models. The aorta of the donor is anastomosed with the carotid artery of the recipient. Donation can be completed within 5 min and vascular connections in the recipient done within 3 min, resulting in a minimum of ischemia time. Using this minimum surgical intervention model, we tested the immunosuppressive effect of a sublethal dose of methotrexate (MTX), which has been widely used in cancer therapy. Our results showed that high doses of MTX severely suppressed the recipient bone marrow, but prolonged heart allografts for more than 365 days after HHT. In conclusion, the new model simplified the rat HHT procedure and made it possible for the beginner of rodent transplantation to master this skill within a few weeks. Using this minimized intervention technique, we found that the high doses of MTX can significantly prolong the survival of fully mismatched DA heart graft in PVG/c recipient.

LIVER CONTROLS ALLOIMMUNE RESPONSE FOLLOWING DONOR SPLEEN CELLS INJECTION I

273 Introduction: The tolerance of allografts after donor-specific injection (DSI) by intraportal or intravenously have been reported, therefore liver considered as an one of the immune-modulate organs. It is unknown, however, whether the underlying mechanisms after DSI involve the liver alone or a combination of other tissue/organ(s). In this study, we developed the new model to examine the relationship between a liver and extrahepatic tissue/organ(s) in the alloimmune responses induced by antigen inoculation. Methods: WS (RT 1k) rats were sensitized with 5×107 DA (RT 1a) spleen cells which were injected intravenously. One, two or three days after DSI, a total hepatectomy was performed and the liver was transplanted into another WS rat (sensitized-liver-grafted; SLG), the hepatectomized WS rat underwent liver transplantation from a naive WS rat (sensitized-liver-removed; SLR). In SLG, the alloantigens accumulated in liver on just time transplanted liver after DSI; in SLR, in the extrahepatic tissue/organ(s). Following these treatments, the animals were grafted with DA skin or heart (Tx) on 10 days after antigen administration. The immune responses following these treatments were evaluated in terms of allografts survival, Th1/Th2 cytokine profile and perforin mRNA in various organs, T cell response (allogeneic MLR before just Tx and delated-type hypersensitivity (DTH) response after Tx) and B cell response (complement-dependent cytotoxicity (CDC) titer and donor-specific immunogloburin after DSI and Tx). Results: SLG animals on one day vigorously rejected skin or heart allografts (5.5±0.2 days; vs other groups, p<0.005, 4.2±0.3 days; vs other groups, p<0.005) compared with non-treated controls (8.5±0.2, 11.6±0.5). In contrast, SLR animals rejected allografts in a significantly delayed fashion (16.5±1.1; vs other groups, p<0.005, 27.7±2.2, vs other groups, p<0.005). On the other hand, SLG and SLR animals transplanted liver on other days rejected in a manner similar to non-treated controls. When skin grafts from LEW (RT 1l) rats were used as a third party, SLG and SLR animals rejected in a manner similar to non-treated controls; when LEW rats were used as a source of spleen cells, these animals rejected DA skin grafts in a same manner. SLG animals on one day associated with a significant higher IFN-γ and perforin mRNA upregulation, CDC titer and DTH response after cardiac transplantation, SLR animals associated with the decrease of them. Before cardiac transplantation, Th1 cytokine levels in liver of SLG and in spleen of SLR on one day highly upregulated rapidly and decline thereafter, mixed lymphocyte proliferative reaction significantly increased in SLG (11412.4±2898.8 cpm; vs other groups, p<0.005) than SLR (87691.2±1308.2) and non-treated controls (87069.0±1944.5). There was no significant difference about the point of peak switched from a IgM to a IgG response in each group. Conclusion: Our results in vivo and vitro on one day after DSI clearly indicate that two compartments of the immune system (liver and other organs) behave differently to alloantigen, suggested early Th1 reaction in liver or spleen after DSI correlate with T cell clonal activation or suppression but not B cell before cardiac transplantation. The liver may play an important role to determine unresponsiveness or rejection after DSI.

TOLERANCE INDUCTION IN RATS DOES NOT REQUIRE GRAFT RESIDENT DONOR-TYPE ANTIGEN PRESENTING CELLS

498 Background: It has been postulated that donor-type bone marrow-derived antigen presenting cells (APCs) may be important for tolerance induction via a mechanism of posttransplant microchimerism. Our goal was to use chimeric hearts created via BMT to test whether donor-type APCs are required for the induction and maintenance of cardiac allograft tolerance in the rat. Methods: Chimeric PVG hearts were created via DA→PVG BMTs. Elimination of PVG APCs was confirmed by flow cytometry, MLR, immunohistochemistry and RT-PCR. Chimeric and naïve hearts were then used for heterotopic transplants, in which APCs were matched or mismatched to the recipient, while organ parenchyma was always mismatched to the recipient. To induce tolerance, oral cyclosporine (CyA, 20 mg/kg, ×10 days) was given following transplantation. Rejection was monitored by palpation of the graft and histological analysis was performed in a blinded fashion. Results: CyA led to permanent survival of PVG→DA heart grafts with minimal vascular changes. 5/7 chimeric PVG hearts survived long-term in untreated DA recipients, but uniformly demonstrated mild cellular rejection and cardiac allograft vasculopathy (CAV). 4/6 chimeric PVG hearts survived long-term in CyA treated recipients, and two of these long-term surviving hearts that have been examined showed focal rejection and moderate CAV. Table Animals from all experimental groups accepted second naïve PVG hearts (normal histology) without immunosuppression (Group II: 3/3, Group III: 2/2, Group IV: 3/3) and rejected third party F344 hearts (Groups II, IV: 2/2), confirming induction of antigen-specific tolerance in CyA-treated recipients.Conclusions: Donor-type bone marrow-derived APCs are not required for tolerance induction in this rat model. Furthermore, these data support recently published work by Tullius et al showing that donor-specific tolerance can develop despite the presence of vasculopathy in the first graft.

The cellular basis of cardiac allograft rejection: VIII. Mechanisms underlying delayed allograft rejection in PVG C6-deficient rats.

The delayed allograft rejection in C6-deficient PVG C6- rats compared with normal PVG rats has been attributed to the lack of alloantibody activation of the membrane attack complex of complement. As T cells alone have been shown to effect graft rejection, we examined T-cell responses in PVG C6- rats. The cellular infiltrate and its mRNA for cytokines and effector molecules in DA heart allografts to PVG and PVG C6- rats was compared by immunoperoxidase staining and semiquantitative reverse transcriptase polymerase chain reaction. The ability of pure populations of T cells or alloantibody to mediate DA heart graft rejection in irradiated (750 rads) PVG and PVG C6- rats was also compared. The median rejection time of DA heart allografts was 8 days in PVG rats and 17.5 days in PVG C6-. PVG C6- rats sensitized to DA by two skin grafts rejected DA heart grafts in 5-6 days. CD3+, CD4+, CD8+, interleukin-2 receptor-positive T cell, macrophage, and natural killer cell infiltration, as well as class II major histocompatibility complex and intercellular adhesion molecule-1 up-regulation, in grafts was similar in naive PVG and PVG C6- rats. mRNA for T helper 1 cytokine interleukin-2, interferon-gamma, tumor necrosis factor-beta, macrophage molecules tumor necrosis factor-alpha, and inducible nitric oxide synthase, as well as cytotoxic T-cell effector molecules perforin and granzyme A and B, were found to be the same in the grafts from both naive PVG and naive PVG C6- rats. Thus, there appeared to be no difference in the T-cell effector response between the PVG and PVG C6- groups. There were higher alloantibody titers in PVG C6- rats than in PVG hosts. Irradiation ablated rejection and alloantibody responses and reconstitution with naive T cells alone restored rejection in both PVG and PVG C6- rats. Irradiated rats given serum from PVG rats that had rejected DA grafts did not effect rejection of DA grafts even if given naive T cells. Sensitized T cells restored second set. PVG C6- rats have normal T-cell responses and can mediate allograft rejection in the absence of alloantibody. The failure of PVG C6- to reject allografts rapidly may be a result of the poor clearance of alloantisera leading to enhancement of graft survival rather than a critical role for complement and membrane attack complex in acute rejection.

Donor pretreatment with leflunomide has an adverse effect on rat cardiac allograft survival

The effect of leflunomide (Lef) donor pretreatment (DPT) on rat cardiac allograft survival was investigated. In untreated Lewis recipients of untreated DA hearts, median graft survival was 5 days. DPT (Lef 5 or 10 mg/kg per day for 30 days) reduced median grafts survival to 4.0 and 4.5 days, respectively. In immunosuppressed (Lef 5 mg/kg for 10 or 30 days) Lewis recipients of untreated DA hearts, median graft survival was 21 and 33 days, respectively. DPT with Lef 5 mg/kg per day for 5 days or 30 days reduced median graft survival to 12 and 23.5 days, respectively ( p < 0.05). DPT with Lef resulted in earlier graft rejection but the histological appearance at the time of rejection was the same as in untreated controls. DPT with lef resulted in a 40% reduction in MHC class II-psotive cells in the heart. Histological examination of rejecting hearts showed no obvious difference in the nature of the rejection process between DPT and untreated control hearts. The paradox between class II reduction but earlier rejection indicates that DPT is exerting a deleterious effect through some unrecognized property of the graft.

Prolonged rat allograft survival induced by temporary elimination of alpha/beta T cells with monoclonal antibody.

We tested the ability of lewis (LEW; RT-1(1)) recipients to reject DA (RT-1av1) cardiac allografts following the selective elimination of alpha/beta T cells with the mouse monoclonal antibody R73. One group of adult rats (6 weeks old) received 1000 microg R73 i.p. on days 2 and 1 before transplantation, and 100 microg R73 every third day after transplantation up to day 18. Prolonged cardiac graft survival was noted (30, 30, 32, 51, 62, 108, > 500, > 500, > 500 days). Untreated controls (n = 10) rejected their grafts within 7 +/- 1 days. R73 therapy induced a dramatic decrease in alpha/beta T cells from 69% before treatment to 5% within the first 5 days, followed by an increase to 64% by day 8. The T cell increase was paralleled by the appearance of anti-mouse antibody. A second group of adult rats (10 weeks old) received the same treatment. These "older" recipients rejected their grafts within 20 +/- 5 days. Chronic R73 therapy from birth until the day of transplantation (100 microg R73 i.p. twice a week) resulted in graft survival of 37 +/- 9 days in eight animals. Two rats had a graft survival of more than 200 days. When chronic R73 therapy was continued to day 70 after transplantation, DA hearts were accepted well in all animals for more than 100 days. Alpha/beta T cells were virtually absent throughout the whole time of treatment. Antibodies against R73 were not detected. We concluded that selective elimination of alpha/beta T cells has a strong effect on allograft survival.

Heart allograft survival in rats following immunization with soluble peptide MHC class I donor antigens: evidence for the role of indirect recognition in rejection.

The current series of experiments addressed the question of whether indirect priming with donor MHC antigens affects heart allograft survival. LEW (RT-1(1)) rats were immunized with a mixture of two peptides corresponding to the variable region of MHC class I locus Aa antigen (alpha1 and alpha2 domain). The recipients were transplanted with a DA (RT1-1a) heart 1 month after immunization, and graft survival was closely monitored by ECG. All peptide-treated recipients presented with anti-peptide antibodies at the time of transplantation and developed a strongly accelerated graft rejection. These findings indicated that indirect recognition of MHC I donor antigens promotes heart allograft rejection.

Histocompatibility differences and cardiac transplant tolerance produced by intrathymic pretreatment

Control of cardiac transplant rejection without toxic immunosuppressive drugs remains an unreached goal. Our laboratory and others have shown that intrathymic inoculation of donor-specific allogeneic spleen cells can produce tolerance to a subsequent cardiac allograft. The present experiments were designed to investigate whether the degree of donor-recipient histoincompatibility influenced the efficacy of this technique. Four congeneic strains of rats with different degrees of histoincompatibility were studied. Heterotopic cardiac transplantation was done with the following congeneic strain combinations: DA donor into PVG recipient (full major histocompatibility complex and nonmajor histocompatibility complex incompatibility); PVG.RT1a donor into PVG recipient (full major histocompatibility complex incompatibility); PVG.RT1a donor into PVG.R1 recipient (partial major histocompatibility complex incompatibility). Prospective graft recipients underwent intraperitoneal injection of 1 ml antilymphocyte serum and intrathymic injection of 5 x 10(7) prospective donor spleen cells. Three weeks later, heterotopic cardiac transplantation was done with a heart from a donor of the same strain as that used to obtain splenocytes for intrathymic injection. Prolongation of graft survival was observed in pretreated recipients in all strain combinations but was greatest in recipients that differed from donors at fewer histocompatibility loci. Complete graft tolerance was not seen in strain combinations that included nonmajor histocompatibility complex incompatibilities. DA heart survival in PVG recipients was 50.6 days (p < 0.04 versus controls); PVG.RT1a graft survival in PVG hosts was 165.8 days (p < 0.02 versus control) and in PVG.R1 recipients 163.8 days (p < 0.02 versus controls) with four of five grafts in each group surviving indefinitely (more than 200 days).

EVIDENCE OF ALLOREACTIVE T SUPPRESSOR CELLS IN THE MAINTENANCE PHASE OF SPONTANEOUS TOLERANCE AFTER ORTHOTOPIC LIVER TRANSPLANTATION IN THE RAT

In the DA-->PVG rat strain combination liver allografts are spontaneously tolerated and induce a state of systemic tolerance towards donor antigens. Thirty long-term (> 100 days)-surviving liver recipients in this strain combination were used to study humoral and cellular mechanisms of tolerance in the maintenance phase after liver transplantation. In the popliteal lymph node assay splenocytes of liver grafted or native PVG rats displayed a similar proliferative response in (DAxPVG)F1 hybrid rats, indicating an unaffected capacity of these cells to mount graft-versus-host reactions. Using adoptive transfer assays T-splenocytes (1x10(7) cells, MRC Ox 19+, MRC Ox12-, CD 5) of long-term-surviving liver recipients were able to suppress the rejection of DA hearts (donor-specific), of DA and AO kidney grafts in irradiated (300 cGy) PVG recipients. Serum of long-term-surviving liver recipients was able to suppress the rejection of semiallogeneic (DAxPVG)F1 heart grafts after double injection of 1 ml serum, while fully allogeneic DA heart as well as skin and pancreaticoduodenal grafts were resistant to passive enhancement. The results confirm the development of spontaneous tolerance after liver grafting in the DA-->PVG strain combination, and the demonstration of T cells with suppressive capacity in adoptive transfer experiments indicates a role for T suppressor cells in the maintenance phase of spontaneous tolerance after liver grafting.