The effectiveness of pretreatment with soluble or membrane-bound donor class I major histocompatibility complex antigens in the induction of unresponsiveness to a subsequent rat renal allograft.

We have examined the ability of two physical forms of RT1.A class I molecules to induce immunologic unresponsiveness to renal allografts in the rat. Both preparations of class I MHC antigen were derived from rat liver. Class I MHC antigen was presented either as purified membrane-bound molecules incorporated into protein micelles or as a water-soluble preparation containing soluble RT1.A class I molecules. The amount of RT1.A class I contained in each preparation was compared with the amount of class I antigen expressed by whole viable liver cells by quantitative absorption analysis using F16.4.4.11 mAb. The results demonstrated that DA recipients pretreated with a single dose of 1.75 x 10(10) cellular equivalents or multiple doses of 5 x 10(9) cellular equivalents of purified LEW membrane-bound class I molecules, delivered in aggregated micelle form, accepted their LEW renal allografts indefinitely (MST greater than 100 days). In contrast, no prolongation of graft survival was observed using the liver cell cytosol preparation containing soluble RT1.A class I molecules (MST 10 days) at the concentrations tested (10(8) -3 x 10(8) cell equivalents). However, when preoperative treatment with single (greater than or equal to 5 x 10(7) cellular equivalents of soluble class I MHC antigen) or multiple doses (greater than or equal to 10(7) cellular equivalents per dose) of the liver cell cytosol preparation was combined with a subtherapeutic dose of CsA given postoperatively (day +2, 10 mg/kg), suppression of renal allograft rejection was achieved with long-term survival (MST greater than 100 days). The immunologic unresponsiveness observed in both cases was donor specific.     

Prevention of graft vessel disease by combined FTY720/cyclosporine. A treatment in a rat carotid artery transplantation model

BACKGROUND: Graft vessel disease (GVD) is an important problem often responsible for late graft loss. The effects of FTY720, an immunomodulator with a novel mechanism of action were investigated in combination with cyclosporine A (CsA) in a carotid artery allograft model. METHODS: A segment of the carotid artery of Lewis rats was replaced by a DA allograft. Seven groups of eight rats were treated for 8 weeks with vehicle (P), CsA 0.3 (C0.3), 1 (C1) or 3 (C3) mg x kg(-1).day(-1) or a combination of CsA 1 with FTY 0.01 (C1F0.01), 0.03 (C1F0.03), and 0.1 (C1F0.1) mg x kg(-1).day(-1). Lumen area was estimated by magnetic resonance imaging, peripheral lymphocyte count and drug concentrations were determined at 1 and 8 weeks. Neointima, media, and lumen area were measured morphometrically. Intimal and adventitial infiltration of mononuclear cells, and medial smooth muscle cells number was assessed using a score. RESULTS: FTY720 did not influence CsA blood concentrations. FTY720 but not CsA decreased the PLC dose dependently. Magnetic resonance imaging revealed that treatment groups have larger lumen size than group P. Histological and morphometric evaluation showed that all aspects of GVD were dose dependently suppressed by treatment and lumen narrowing was prevented. CONCLUSIONS: CsA, at clinically relevant blood levels, suppressed GVD only partly. The addition of FTY720 was well tolerated and completely suppressed GVD development. In vivo lumen size did not correlate with the histologically estimated neointimal thickness.     

The effects of cyclosporine on the induction of donor class I and class II MHC antigens in heart and kidney allografts in the rat

We have previously reported 5-30-fold increases in the expression of class I and class II major histocompatibility complex (MHC) antigens in rejecting heart and kidney allografts in the DA-to-PVG rat strain combination. We examine here the effects of immunosuppression with cyclosporine on the induction of donor class I and class II MHC antigens in heart and kidney allografts in this strain combination. Immunohistological studies and quantitative absorption analyses using monoclonal antibodies and assay systems specific for donor class I and class II MHC antigens were used throughout. Heart allografts in cyclosporine-treated rats were examined on day 3,5,7,9,11, and 14 after transplantation, and kidney allografts in cyclosporine-treated rats were examined at day 7. In addition, untreated heart and kidney isografts were studied at days 1,3,5, and 7 after grafting. Immunohistological studies on frozen sections showed that cyclosporine-treated heart and kidney allografts showed no induction of class II MHC antigens, in contrast to untreated heart and kidney allografts. Class I MHC antigen induction did occur in spite of cyclosporine-therapy, but at levels lower than those seen in untreated allografts. Moreover, the pattern and degree of class I induction in the cyclosporine-treated allografts resembled very closely those seen in isografts, and so this induction was, in all probability, a consequence of the transplantation procedure rather than of specific immune responses. We also noted, in the cyclosporine-treated heart allografts, that all donor interstitial dendritic cells had disappeared and been replaced by recipient interstitial dendritic cells by the end of the second week after grafting. In addition, there was no reduction in the class II antigen content of kidney allografts treated for 7 days with cyclosporine. The absence of class II antigen induction in allografts where rejection is effectively suppressed with cyclosporine might be of clinical value in the differential diagnosis between rejection and cyclosporine toxicity in renal transplantation, and between active and inactive cellular infiltrates in heart transplantation.     

Heterotopic transplantation of cryopreserved tracheae in a rat model.

INTRODUCTION: The successful use of cryopreserved tracheal allografts in canine models suggests their use in humans. The grade of genetic difference, the mechanism of revascularisation and the method of cryopreservation are not clearly defined. The purpose of our study was to investigate the rejection of tracheal transplants in a standardised heterotopic rat model using different forms of cryopreservation. METHODS: Tracheae from Brown Norway rats were implanted into the omentum from Brown Norway rats or Lewis rats. We transplanted fresh isografts or allografts and pretreated isografts or allografts. Cryopreservation was performed in a medium containing 10% dimethyl sulphoxide at -80 degrees C for 28 days (I) or -196 degrees C for 84 days (II) or without medium at -80 degrees C for 28 days (III). The transplants were excised after 7 and 21 days, respectively. RESULTS: Histological examinations revealed normal structure and function of isografts after 21 days. In the cryopreserved isograft, the epithelium had disappeared and the tracheal lumen was partially obstructed by a non-compact fibrous tissue. In the fresh allografts, the epithelium was replaced by aggressive fibrous tissue, infiltrating the membranous part of the trachea and occluding the tracheal lumen. The cartilage was vital without any sign of rejection. In the cryopreserved allografts, the tracheal lumen was obstructed by dense fibrous tissue with an inflammatory reaction. The cartilage of cryopreserved allografts (II) and (III) had lost the nuclei corresponding to non-vital tissue. Only in the cryopreserved allografts (I) did we find nodular regeneration at the edges of the cartilaginous bow. CONCLUSIONS: The heterotopic transplantation model allows the study of the mechanisms leading to tracheal obstruction. Cryopreservation was found to have no clear advantage in reducing transplant immunogenicity. Cryopreservation leads to significant damage to the cartilage, the intensity of which is dependent on the mode of cryopreservation.     

Induction of transplantation tolerance in rats by spleen allografts. III. The role of T suppressor cells in the induction of specific unresponsiveness

Heterotopic (WAG x AGUS)F1 spleen allografts survive indefinitely when transplanted to normal AGUS recipients and induce long-term donor-specific unresponsiveness. In this report, we have examined the immune reactivity of spleen graft recipients soon after transplantation, in an attempt to define the immunological mechanisms responsible for the induction of donor-specific unresponsiveness. Unresponsiveness develops as early as one week after splenic transplantation. T cells obtained from the recipient lymph node and spleen exhibit reduced mixed lymphocyte reaction responses to donor (WAG) but respond normally to third-party (PVG) stimulators. In contrast, T cells obtained from the spleen graft are unresponsive to both donor and third-party stimulators. Donor specific T suppressor cells (Ts) appear in the recipient's lymph node and spleen by one week posttransplantation--however, at this time antigen nonspecific suppressor cells predominate in the spleen graft. Only minimal cytotoxic T cell activity could be detected in the spleen graft, with the host spleen and lymph nodes being devoid of cytotoxic T lymphocytes. Sera obtained one or two weeks following splenic transplantation did not contain cytotoxic alloantibodies, and only a very weak response could be detected at one month. These data demonstrate that the unresponsiveness associated with the spontaneous acceptance of spleen allografts is correlated with the early induction of antigen specific Ts in recipient lymphoid tissue and the presence of nonspecific suppressor cells at the graft site.     

T suppressor cells induced by transfusions and cyclosporine. Studies in the murine cardiac allograft model

Pregraft transfusion combined with immunosuppression at the time of grafting improves the survival of clinical and experimental allografts. The mechanisms responsible for this effect were investigated in the murine model of cardiac transplantation, combining transfusions 7 to 30 days prior to transplantation with cyclosporine 100 mg/kg, 7 to 20 days pregraft or on days 0, 4, and 6 after grafting. Pregraft DST, third-party blood, and CsA all improved graft survival in the BALB/c-to-CBA donor-recipient combination. In animals treated with DST at 14 days pregrafting, 4/9 grafts survived for greater than 100 days. In those given C57BL/6 blood, or CsA on days 0, 4, 6 postgraft, 1/9 grafts survived for greater than 100 days. When 10(7) spleen cells from DST-treated CBA mice with long-surviving BALB/c heart grafts were transferred to naive CBA mice that then received a BALB/c heart 24 hr later, the transferred cells prolonged graft survival, with all grafts functioning at greater than 40 days, and 4/7 at greater than 100 days. Selective removal of T cells from the spleen cell population prior to transfer showed that L3T4+ T cells, but not Ly-2+ T cells, were required to maintain BALB/c allografts. Combining a short course of CsA with DST was more effective than either treatment alone. The most effective combined treatment was DST at day -14 with 100 mg/kg CsA given on days 0, 4, and 6 postgrafting (8/10 grafts survived greater than 100 days). This treatment also induced splenic suppressor T cells of the L3T4+ Ly-2- phenotype. These results clearly show that L3T4+ splenic T suppressor cells are induced by donor-specific blood transfusion with or without CsA treatment, and that these cells play a role in maintaining long-term tolerance to allografts in the mouse heart transplant model.     

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

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

The effects of 16, 16 dimethyl prostaglandin E2 therapy alone and in combination with low-dose cyclosporine on rat small intestinal transplantation.

The use of prostaglandin E (PGE) in the setting of allotransplantation both clinically and experimentally has been suggested because PGE has significant immunosuppressive effects and potentially could lessen the toxic effects of cyclosporine. In the present study, we examined the immunosuppressive effects of 16,16 dimethyl prostaglandin E2 (dmPGE2) alone and in combination therapy with low-dose CsA to assess the clinical course, histology and expression of monocyte/macrophage procoagulant activity (PCA) following small intestinal transplantation in a heterotopic model of rat allograft rejection. Therapy with low-dose CsA (1 mg/kg) failed to prevent rejection and all animals reached a terminal state by day 26. In contrast, animals treated with high-dose CsA (10 mg/kg) showed no clinical or histological evidence of rejection and all animals survived. The dmPGE2 (100 micrograms/kg/twice daily) delayed the onset of rejection, but all animals developed severe rejection and subsequently died. Treatment of animals with low-dose CsA (1 mg/kg) in combination with dmPGE2 (100 micrograms/kg twice daily) resulted in a delay in the onset (P = 0.05) and a reduction in the intensity of allograft rejection (P = 0.0001) compared with either agent used alone. Monocyte/macrophage procoagulant activity levels correlated with the degree of rejection in all animals (P = 0.03). There was a statistically significant relationship between PCA levels and the time of onset of rejection and histologic grade of rejection in all groups. The data presented here, therefore, demonstrate a beneficial role for long-term combination therapy with CsA and PGE in small intestinal transplantation and strongly suggest a role for allogeneic induction of PCA in the pathogenesis of rejection.     

大鼠胰十二指肠移植动物模型的制作

目的　建立大鼠腔静脉内分泌引流、肠道外分泌引流的动物模型。方法　雄性SD大鼠为供受者,供体鼠门静脉与受体鼠左肾静脉行袖套吻合,形成腔静脉内分泌引流;供者腹主动脉与受者腹主动脉行端侧吻合;供者十二指肠与受者近端空肠行侧侧吻合。结果　5 0只药物诱导的糖尿病大鼠移植术后超过2 4h者46只,手术时间为(93±7)min。移植前大鼠血糖为(2 8.3±1.7)mmol/L ,移植后43只大鼠血糖降至正常水平。结论　该模型方法简单易行,可作为胰腺移植的理想模型     

Preoperative low-dose irradiation promotes long-term allograft acceptance and induces regulatory T cells in a porcine model of pulmonary transplantation

BACKGROUND: A simplified conditioning protocol including single-dose preoperative thymic and low-dose whole body irradiation with or without subsequent donor bone marrow transplantation (BMTx) can be applied in porcine lung transplantation. We hypothesized that this protocol would prolong allograft survival. METHODS: Left-sided single lung transplantation from major histocompatibility complex (MHC)-mismatched donors was performed in 27 minipigs. Recipients received whole body (1.5 Gy) and thymic irradiation (7 Gy) before transplantation (IRR group, n=6), intravenous immunosuppression with methylprednisolone, cyclosporine, and azathioprine for 27 postoperative days (IS group, n=5) or both (IRR+IS group, n=10). BMTx group recipients were treated with irradiation, immunosuppression and a donor bone marrow infusion on postoperative day 1. Peripheral blood leukocyte phenotype and donor cell chimerism were monitored by flow cytometry. Purified CD25+ T cells from long-term survivors or rejecting animals were used for in vitro MLR suppression assays. RESULTS: Median graft survival was: IRR 12 days, IS 55 days, IRR+IS 239 days, and BMTx 80 days (P<0.0001). Early peripheral blood macrochimerism was substantial in both the IRR+IS and the BMTX group but was lost in all groups after day 80. The frequency and suppressive function of CD4+CD25+ T cells were enhanced in IRR+IS group long-term survivors. CONCLUSION: Although donor bone marrow infusion was not beneficial in our model, a substantial proportion of the animals treated with irradiation and a 28-day course of immunosuppression accepted their lung allografts long term. The mechanism involved in maintaining allograft tolerance may be based on peripheral T-cell regulation.     

Prolonged reversal of diabetes in the rat by transplantation of allogeneic islets from a single donor and cyclosporine treatment

Islets were isolated using neutral red as a dye to exclude lymphatic and acinar impurities in the transplant preparation. In a one donor-one recipient model, intraportally transplanted isogeneic islets survived indefinitely. Mean graft survival could be prolonged from 5 to 90 days, using a 3-dose rejection prophylaxis with CsA in allografts of (DA [RT1a]----Lew (RT 1l). Histological examination revealed intact islets in the grafted normoglycemic animals. Immunohistochemical staining of these islets showed B cells with insulin-rich cytoplasma. Blood glucose levels and the results of i.v. glucose tolerance tests 50 days after transplantation are presented. The different outcomes of our experiments are discussed.     

Multiorgan transplantation in the rat: development of a new microsurgical model.

Although transplantation of multiple abdominal viscera (MOTx) has been performed in humans, reproducible animal models and extensive laboratory work are needed to explore the physiological and immunological aspects of this new transplant procedure. We therefore developed a microsurgical model that allows en bloc transplantation of three major abdominal viscera. Using isogeneic rat strain combinations, 30% of the liver, pancreas, and duodenum and a segment of small bowel (SB) were heterotopically transplanted. A segment of aorta that included the celiac axis and the superior mesenteric artery provided the blood supply to the graft. Venous outflow occurred via the graft suprahepatic vena cava into the recipient's infrahepatic vena cava. The graft SB was anastomosed to the native SB. After a training period (N = 15), we could achieve a success rate of 83% (N = 23). Mean operation time was 105 minutes. Macroscopic and microscopic appearance of transplanted organs was normal. This study documents the feasibility of MOTx in the rat and provides a microsurgical model that should facilitate preclinical experimental research in this area.     

In vitro studies of transplantation antigens present on bone cells in the rat.

In vitro studies on isolated bone cells were undertaken to investigate the presence of transplantation (histocompatibility) antigens. Bone cells were cultured with allogeneic lymphocytes and exposed to cytotoxic sera containing antibodies against transplantation antigens, to determine their antigenic profile. Preliminary results suggest that bone cells may not express lymphocyte stimulating antigens in an active form, at least after the isolation procedure performed. On the other hand, bone cells were killed by cytotoxic antibodies in a specific way, providing evidence for the presence of serologically defined (SD) transplantation antigens on the cell surface. Additional studies with absorbed sera suggest "sharing" of histocompatibility antigens between bone cells and lymphocytes. The relevance of the surface antigens of bone cells to clinical fields such as bone allotransplantation, susceptibility to various orthopaedic diseases and skeletal sarcomata is discussed.