Significant changes in the alloantibody after lung transplantation in the cyclosporine treated rat model

BACKGROUND: The diagnosis of acute rejection after organ transplantation is often complicated by other possibilities, such as infection. Despite many attempts to identify rejection episodes after transplantation, only the detection of the humoral anti-human leukocyte antigen antibody has been effective in measuring alloimmunization, especially detected with flow cytometry cross-match (FCXM). As an initial step towards gaining a better understanding of the correlation between humoral responses and graft rejection in an immunosuppressant recipient, we investigated responses of alloantibodies (allo-Abs) after lung transplantation (LTx) in a rat model treated with adequate or inadequate cyclosporine A (CsA) therapy. METHODS: Orthotopic LTx was performed using a major histocompatibility complex fully incompatible combination (Brown Norway to Lewis rat). CsA was given subcutaneously to recipients at an optimal or a sub-optimal dosage for 3 days after transplantation. A FCXM technique was used to determine the time-course of changes in titers of allo-Abs in serum. The allo-Ab deposition in the grafted lung was detected with an immunofluorescent staining method. RESULTS: Circulating IgM allo-Ab levels were significantly elevated on day 4 in both groups when histological findings revealed early stage of acute rejection. IgM levels in the sub-optimal dosage group were maximal and significantly higher than those in the optimal dosage group on day 4, and levels then decreased after day 8. IgG allo-Ab levels increased significantly on day 8 and continued to increase throughout the observation period. CONCLUSIONS: Our data suggest that the monitoring IgM allo-Abs might be effective for identifying acute rejection in recipients with inadequate immunosuppression therapy.     

Role of antibody in rat renal allograft rejection. II. Failure to enhance renal allografts by sensitization to donor class I antigens presented by donor strain erythrocytes

BN rats were immunized with one or three doses of 1 X 10(8) highly purified LEW erythrocytes (LEW-E) yielding IgM antibody (IgM-BN rats) and IgG antibody (IgG-BN rats) to LEW class I antigens, respectively. LEW kidneys transplanted into IgM-BN rats elicited cytotoxic T cell responses and lymphocytotoxic antibody responses comparable to those elicited by LEW renal grafts in unmodified BN rats. However, LEW kidneys were rejected by IgM-BN hosts in a slightly delayed fashion compared with controls (mean rejection times (MRTs), 9.4 versus 7.1 days); delayed rejection was associated with the absence of anti-LEW IgG hemagglutinins from the recipients' blood and the absence of vasculonecrotic lesions from rejected renal grafts. LEW kidneys inserted into IgG-BN rats were rejected in a slightly accelerated fashion compared with controls (MRT, 6.6 days). Lymphocytotoxins developed in IgG-BN recipients of LEW kidneys in a fashion similar to that of controls, but cytotoxic T cell responses were delayed up to the 6th day after transplantation. These observations confirm our previous finding that cytotoxic T cells do not play a decisive role in acute rejection in this model. The association observed between delayed or accelerated rejection of LEW kidneys by BN rats sensitized with LEW-E and the absence or presence of anti-donor IgG hemagglutinins in the blood of these recipients after transplantation suggests an important role for IgG anti-donor class I antibodies in the rejection of LEW renal allografts by BN rats.     

Down-regulation of the immune response to histocompatibility antigens and prevention of sensitization by skin allografts by orally administered alloantigen.

The effects of oral administration of major histocompatibility antigens on the alloimmune response have not been investigated. Lymphocytes from inbred LEW (RT1u) rats that were pre-fed allogeneic WF (RT1l) splenocytes exhibited significant antigen specific reduction of the mixed lymphocyte response in vitro and delayed-type hypersensitivity response in vivo, when compared with unfed controls. In an accelerated allograft rejection model, LEW rats were presensitized with BN (RT1n) skin allografts 7 days before challenging them with (LEW x BN)F1 or BN vascularized cardiac allografts. While sensitized control animals hyperacutely reject their cardiac allografts within 2 days, animals prefed with BN splenocytes maintained cardiac allograft survival to 7 days, a time similar to that observed in unsensitized control recipients. This phenomenon was antigen-specific, as third-party WF grafts were rejected within 2 days. Immunohistologic examination of cardiac allografts harvested on day 2 from the fed animals had markedly reduced deposition of IgG, IgM, C3, and fibrin. In addition, there were significantly fewer cellular infiltrates of total white blood cells, neutrophils, macrophages, T cells, IL-2 receptor-positive T cells, and mononuclear cells with positive staining for the activation cytokines IL-2 and IFN-g. On day 6 posttransplant, the grafts from fed animals showed immunohistologic changes typical of acute cellular rejection usually seen in unsensitized rejecting controls. Feeding allogeneic splenocytes prevents sensitization by skin grafts and transforms accelerated rejection of vascularized cardiac allografts to an acute form typical of unsensitized recipients. Oral administration of alloantigen provides a novel approach to down-regulate the specific systemic alloimmune response against histocompatibility antigens.     

Chronic renal allograft rejection. Selective involvement of the glomerular endothelium in humoral immune reactivity and intravascular coagulation

To study immune reactive and thrombotic mechanisms involved in chronic renal allograft rejection, Lewis rat kidneys were transplanted into bilaterally nephrectomized Brown Norway recipients tolerant of LEW erythrocyte antigens. Such BN rats fail to produce anti class I MHC alloantibodies after insertion of a LEW kidney. The LEW renal allografts experience a transient rejection episode without proteinuria followed by the development of chronic rejection, clinically characterized by glomerular proteinuria in the presence of stable renal function. Immunohistological studies of such chronically rejected LEW renal allografts showed the occurrence of glomerular and interstitial infiltration of predominantly monocytes and T cells. CD4-positive T cells dominated over CD8-positive T cells in the chronically rejected LEW renal grafts. IgG deposition was found deposited throughout the renal vasculature--this in contrast to IgM, which was observed only in the glomerular vasculature. Glomerular antibodies were not directed to endothelial class II MHC antigens, and showed only weak complement fixation as demonstrated by C3 staining. Selective glomerular IgM deposition was associated with vascular (platelet-containing) thrombi, and focal and segmental fibrinoid necrosis. In contrast, acutely rejected LEW renal grafts in unmodified BN recipients showed IgM deposition as well as thrombus formation throughout the entire renal vasculature. The results demonstrate that the antibody response to endothelial--and, in particular, glomerular endothelial non-MHC antigens--may bring about chronic vascular renal allograft rejection. How the formation of glomerular thrombotic lesions may be assisted by endothelial reactivity to cytokines from local immune reactive cells is discussed.     

Vascular endothelial cell apoptosis induced by anti-donor non-MHC antibodies: a possible injury pathway contributing to chronic allograft rejection.

BACKGROUND: Non-major histocompatibility complex (non-MHC) alloantibodies may play a pathogenic role in chronic rejection but remain poorly characterized. METHODS: The kinetics of alloantibody production and the mechanism by which non-MHC alloantibodies cause graft injury were investigated in a Lewis-to-Fischer 344 (LEW-to-F344) rat model of cardiac transplantation. RESULTS: Flow cytometry detected that all the F344 recipients of LEW allografts produced anti-donor immunoglobulin G (IgG) antibodies reactive with LEW lymphocytes and endothelial cells. A sub-group of recipients that rejected their grafts in 30 to 60 days exhibited markedly increased levels of anti-donor IgG antibodies (n = 6, mean fluorescence intensity [MFI]:23.85 +/- 2.7) than recipients with long-surviving allografts (n = 4, MFI:11.23 +/- 0.81; p = 0.00058). Passive transfer of anti-donor sera induced chronic rejection of LEW heart allografts in an immune non-responsiveness model of F344 rats induced by intrathymic inoculation of donor-specific lymphocytes. Immunoglobulin G antibodies purified from the anti-LEW sera exhibited complement-dependent cytotoxicity against LEW vascular endothelial cells in flow-cytometric cytotoxicity assay. The targeted endothelial cells displayed early (annexin V+) and late (TUNEL+) evidence for programmed cell death. Western blot analysis of poly (ADP-ribose) polymerase (PARP) demonstrated that the 25-kD PARP-cleavage fragment was present at the lysates of the vascular endothelial cells treated with anti-donor IgG antibodies, indicating apoptosis-associated caspase activity in these cells. In situ teminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) staining demonstrated that vascular endothelial cell apoptosis was consistently present in all LEW heart allografts with chronic rejection. CONCLUSIONS: Non-MHC alloantibodies are pathogenic and capable of causing chronic graft injury through an antibody-induced cell apoptosis mechanism. The results emphasize the importance of non-MHC antibodies as a common predisposing factor in the development of chronic rejection.     

The role of flow cytometry-detected IgG and IgM anti-donor antibodies in cardiac allograft recipients

BACKGROUND: At our transplant center, cardiac allograft recipients undergo transplantation following a negative IgG anti-human globulin (AHG) crossmatch (XM). Flow cytometry crossmatching (FCXM) is a more sensitive XM procedure than the AHG XM procedure, yet there is limited information regarding the clinical relevance of FCXM to cardiac allograft outcome. METHODS: FCXM was performed retrospectively using the pretransplant sera from 140 recipients of primary cardiac allografts who underwent transplantation after AHG-IgG-NEG XM. The FCXM results were correlated to posttransplant rejection and patient survival. RESULTS: All of the patients were auto-XM-NEG. Twenty-two of 140 patients (16%) displayed IgG(+) FCXM and had a significantly poorer 1-year survival rate than did 57 of the FCXM-NEG recipients (68% vs. 86%, P<0.02). Moreover, 50% of the IgG(+) FCXM recipients experienced early rejections (< or =14 days postoperatively) compared with only 16% for the FCXM-NEG recipients (P<0.01). The survival rate of 92% for IgM(+) FCXM recipients (n=37) was significantly improved compared with the 86% survival rate for FCXM-NEG control recipients (P<0.05), suggesting a protective role for IgM. Consistent with this interpretation is that the 1-year survival rate of 79% for the IgG, IgM FCXM(+) recipients (n=24) was significantly better than the 68% survival rate for the IgG(+) FCXM recipients (P<0.02). CONCLUSIONS: These data suggest that IgG(+) FCXM identifies a subset of AHG-IgG-NEG XM cardiac allograft recipients who are at risk for early rejections and poor survival. In contrast, the presence of IgM may be beneficial to survival.     

Presensitization accelerates chronic allograft rejection in a heterotopic rat tracheal allograft model with immunosuppression

BACKGROUND: In the field of organ transplantation, the effect of pretransplant humoral allosensitization on the organ transplant outcome has been highlighted. To clarify the correlation of presensitization with chronic rejection that can lead to a poor prognosis in transplant recipients, we examined humoral alloreactivity and allograft rejection in sensitized recipients by using a rat heterotopic tracheal transplant model. METHODS: An MHC fully incompatible combination strain was used in this study. Lewis (LEW) rats sensitized by transplantation with Brown Norway (BN) skin grafts received tracheal segments from BN rats in the dorsal subcutaneous pouch. Four allogenic groups (n=5) were investigated. Group 1, non-sensitized recipients without cyclosporine A (CsA) administration; group 2, non-sensitized recipients with CsA administration; group 3, sensitized recipients without CsA administration; and group 4, sensitized recipients with CsA administration. In the immunosuppressant groups (groups 2 and 4), the recipients were administered a subcutaneous injection of CsA (25 mg x kg(-1) x day(-1)) for 3 days from the day of operation. All recipients were sacrificed 21 days after transplantation. Tracheal segments were extracted from the recipients and histologically evaluated with regard to the following parameters: (1) airway lining epithelial loss, (2) lymphocyte/plasma cell infiltration, and (3) luminal obliteration due to granulation tissue formation and/or fibrosis. In order to analyze alloantibody (allo-Ab) responses, sera samples were tested by the flow cytometric cross-match (FCXM) technique. RESULTS: Histological findings revealed that the chronic rejection score in sensitized recipients treated with CsA was significantly higher than that in non-sensitized recipients treated with CsA (9.0+/-1.2 vs. 3.0+/-0.54, p<0.05). In other words, CsA therapy reduced the rejection score in non-sensitized recipients, but not in sensitized recipients. No significant differences were observed in the level of IgM Abs among the groups. However, donor-specific IgG Abs were induced after presensitization by donor skin grafting prior to tracheal transplantation. Heterotopic tracheal implantation also induced IgG Ab production. This elevation in the Ab levels was inhibited by CsA treatment in non-sensitized recipients. Conversely, the Ab levels were significantly higher in sensitized recipients than in non-sensitized recipients, regardless of CsA administration. CONCLUSIONS: Our data showed that presensitization accelerates chronic allograft rejection with a marked elevation in the level of donor specific IgG Abs. These results suggest that presensitization will be a significant risk factor for the lung transplant recipient, furthermore, the effect of immunosuppression might be insufficient in sensitized recipients.     

Weak humoral posttransplant alloresponse after a well-HLA-matched cadaveric kidney transplantation

BACKGROUND: Screening of donor-specific antibodies or alloantibodies after kidney transplantation has not been performed routinely. The aim of this study was to evaluate the humoral antidonor and alloresponse of immunologically low-risk recipients of cadaveric renal allografts during the first posttransplant year. METHODS: Alloresponse against the donor was analyzed by means of T-cell immunoglobulin (Ig)G and IgM and B cell IgG flow cytometric crossmatch (FCXM) tests with sera from days 0, 21, 90, and 365 posttransplant. In addition, panel reactive anti-human leukocyte antigen (HLA) class I and class II antibodies (PRA I and PRA II) were analyzed using flow cytometric methods. The recipients were treated either with a low initial cyclosporine regimen with single-bolus antithymocyte globulin (ATG) or basiliximab induction or conventional cyclosporine triple therapy. RESULTS: No significant posttransplant anti-HLA class I or class II sensitization was found in the recipients as a whole. Recipients receiving a single-bolus ATG showed significantly higher proportion of PRA I positivity in the day 21 sample compared with the other groups. Flow cytometric donor-specific T- and B-cell IgG alloresponses remained low, but the proportion of T-cell IgM crossmatch-positive recipients increased during the study. Positive T-cell IgM FCXM was found to be associated with acute rejection episodes and cytomegalovirus (CMV) infections. CONCLUSIONS: In immunologically low-risk kidney-graft recipients, positive T-cell IgM FCXM at transplantation was found to be a risk factor for rejection episodes. Conversion of T-cell IgM FCXM to positive was found to be associated with CMV infections.     

Intrathymic immunomodulation in sensitized rat recipients of cardiac allografts: requirements for allorecognition pathways.

BACKGROUND: Intrathymic injection of alloantigen in the form of donor cells, soluble major histocompatibility complex (MHC) molecules, or MHC allopeptides induces donor-specific tolerance in a variety of acute allograft rejection models. We have previously shown that a single intrathymic injection of donor spleen cells into pre-sensitized rats abrogates accelerated (circa 24-hour) rejection and prolongs the survival of cardiac allografts to about 7 days. The present study was designed to investigate the mechanisms by which intrathymic administration of donor cells modifies the course of accelerated rejection. METHODS: Lewis RT1(1) (LEW) rats sensitized by transplantation with Wistar-Furth RT1(u) (WF) skin grafts received WF cardiac allografts 7 days later-a classic model of accelerated rejection. At the time of skin challenge, however, certain animals received intrathymic cell suspensions (either allogeneic or syngeneic) or donor-derived class I and/or class II MHC peptides. RESULTS: Control animals (sensitized by skin grafts but receiving no other treatment) rejected cardiac allografts within 24 hours. Intrathymic injection of WF splenocytes at the time of skin transplantation abrogated rejection at 24 hours and prolonged cardiac allograft survival to 6.6+/-0.6 days (p<0.001), whereas intrathymic administration of syngeneic (LEW) or allogeneic third party Brown Norway RT1(n) cells was ineffective in this regard. Intrathymic injection of gamma-irradiated donor cells marginally extended cardiac allograft survival to 3.0+/-0.9 days (p< 0.001), but the grafts were still rejected in an accelerated fashion. Intrathymic injection of donor-derived class I and/or class II MHC allopeptides at the same time period also failed to prolong cardiac allograft survival beyond 3 days. In the group receiving unmodified donor cells, elevated immunoglobulin M (IgM) and immunoglobulin G (IgG) allo-antibodies were found at the time of cardiac transplantation; this pattern was not observed with any other treatment. CONCLUSION: The superiority of non-modified donor spleen cells over gamma-irradiated donor cells or donor specific allopeptides in modifying the course of accelerated cardiac rejection suggests that direct allorecognition is the dominant pathway initiating rejection in sensitized transplant recipients. Marked alterations in the antidonor IgM and IgG responses are associated with successful abrogation of accelerated rejection by thymic immunomodulatory mechanisms.     

Flow cytometry crossmatching as a predictor of acute rejection in sensitized recipients of cadaveric renal transplants

Flow cytometry crossmatching (FCXM) was developed as a more sensitive assay than the standard complement-dependent cytotoxicity crossmatch (CDCXM) for the detection of anti-donor antibodies, that mediate hyperacute rejection and graft loss in the early post-transplant period in renal transplant recipients. The role of FCXM in predicting long-term clinical outcome in renal allograft recipients is unclear. This study examines the role of FCXM in predicting long-term clinical outcome in highly sensitized recipients of cadaveric renal transplants. All patients (n = 100) with peak panel reactive antibody (PRA) levels > 30%, who received cadaveric renal transplants between 1/1/'90 and 12/31/'95 at our institution, were divided into FCXM + and FCXM - groups. The incidence of acute rejection was determined for each group during the first yr after transplant. Graft survival rates at 1, 2, and 3 yr, and creatinine levels were also compared between groups. FCXM + patients experienced a higher incidence of acute rejection during the first yr after transplant (69 vs. 45%), and a higher percentage of FCXM + patients had more than one episode of acute rejection during the first yr after transplant (34 vs. 8%) when compared to FCXM - patients. There was no statistically significant difference in 1-, 2-, or 3-yr graft survival between FCXM + and FCXM - patients (76 vs. 83, 62 vs. 80, 62 vs. 72%, respectively). These results suggest that sensitized FCXM + cadaveric renal transplant recipients have a higher incidence of acute rejection episodes in the first yr after transplant. Given the association of multiple rejection episodes with poor long-term allograft survival, FCXM may be a useful predictor of long-term clinical outcome in this sub-group of renal transplant recipients.     

Early biological and immune response to semi-identical liver or kidney allograft in miniature swine

In inbred miniature swine, semi-identical liver allograft recipients survive up to 3 months without immunosuppression, whereas similarly mismatched kidney allografts are uniformly rejected within 2 weeks. The early biological and immunological events were assessed in this unique model. SLA(d/d) pigs (MGH, Harvard Medical School, Boston, MA, USA) received liver or kidney allograft from heterozygous SLA(c/d) miniature swine. Survival, graft function, histology, intragraft cytokines, peripheral lymphocyte and platelet count, plasma cortisol level and cellular/humoral anti-donor immune response were assessed. Kidney allografts were uniformly rejected within 2 weeks, whereas liver allografts survived for up to 87 days. After both liver and kidney transplantation, the peripheral lymphocyte count decreased during the first week concomitantly to a significant elevation of plasma cortisol level. Early decrease of peripheral platelet count was observed after liver but not renal transplantation. Up-regulation of transforming growth factor beta1 (TGF-beta1) and interferon-gamma (IFN-gamma) was observed during the first postoperative week in semi-identical liver allografts and IFN-gamma as well as IL-10 in kidney allografts. In liver recipients, labelled autologous lymphocytes accumulated in the liver graft and native spleen, whereas after renal allograft, lymphocytes accumulated in the native spleen and liver but never in the kidney allograft. Specific cellular anti-donor unresponsiveness was observed from the first post-transplant day in both liver and kidney recipients, while the humoral anti-donor response remained intact. In semi-identical liver allograft, recipient rejection is milder and slower than in similarly matched kidney allograft. The intragraft up-regulation of TGF-beta1 in semi-identical liver allograft might be one mediator to explain the modulation of rejection after liver transplant. The rapid, nonspecific accumulation of recipient lymphocytes in the liver allograft but not in kidney allograft might also play a role in the different survival time in this model.     

IgG alloantibody responses to donor-specific blood transfusion in different rat strain combinations as a predictor of renal allograft survival.

Donor-specific blood transfusion prolongs the survival of fully allogeneic ACI (RT1a) renal allografts in PVG (RT1c) recipients from 7-10 days to greater than 100 days. We have observed significant differences in the alloantibody (Ab1) responses to ACI renal allografts in control and DSBT-treated PVG recipients: DSBT is associated with decreased IgG and IgM alloantibody circulating in serum, deposited in the allograft, and produced in culture by splenocytes. In the present studies the effects of DSBT on alloantibody production and renal allograft survival were extended to examine other recipient strains: F344 (RT1lv1), BN (RT1n), W/F (RT1u) and LEW (RT1l). Animals of each recipient strain were injected i.v. with 0.5 ml of ACI blood alone or followed by a renal allograft. Studies on the kinetics of IgM and IgG alloantibody responses were performed by flow cytometry on lymphocytes from donor ACI, PVG, and PVG.R1 (RT1.Aa class I MHC antigen on PVG background) rats. In F344 and PVG rats, DSBT from ACI rats elicited a transient IgM response that peaked at day 7 and was not followed by a switch to IgG. In control PBS transfused F344 recipients, an ACI renal allograft stimulated both IgM and IgG alloantibody production. DSBT pretreatment significantly decreased circulating IgG alloantibody following ACI renal transplantation and prolonged graft survival in F344 recipients. In DSBT-treated F344 recipients that rejected ACI renal allografts acutely, small amounts of IgG (5-12 mode channel shift) were detected in sera harvested 7 days after transplantation, whereas almost no IgG was detected in the sera from DSBT treated F344 rats that accepted their renal allografts indefinitely. In contrast, DSBT alone from ACI to BN, W/F, or LEW strains elicited a transient IgM response that peaked at day 7 and was followed by a strong IgG response that peaked on days 10-14 and remained high through day 21. DSBT failed to prolong ACI renal allograft survival in any of these strains (survival less than 11 days in control and DSBT rats). The alloantibody response to DSBT in all five recipient strains examined was directed primarily to RT1.Aa class I MHC antigens, as determined by binding studies on lymphocytes from ACI, PVG and PVG.R1 rats and alloantibody blocking studies using biotinylated rat monoclonal antibodies to distinct epitopes of the RT1.Aa antigen. The relative magnitude of blocking of R2/10P and R2/15S binding by sera from BN, W/F, and LEW rats was: control allograft recipients greater than DSBT pretreated allograft recipients greater than DSBT alone.(ABSTRACT TRUNCATED AT 400 WORDS)     

负向免疫调节树突状细胞对心脏移植大鼠免疫状态的影响

目的 探讨经体外光化学法(PUVA)处理的供者脾淋巴细胞与受者树突状细胞(DC)共培养后,对移植受者体液免疫、细胞免疫及移植物排斥反应的影响.方法 以DA大鼠为供者,LEW大鼠为受者,建立大鼠腹部异位心脏移植模型.分离供者脾淋巴细胞(SP),制备经PUVA处理的供者脾淋巴细胞(PUVA-SP).在体外分别将供者PUVA-SP和SP与受者骨髓来源的未成熟DC共培养,得到PUVA-SP-DC及SP-DC,流式细胞仪检测上述DC表型.根据受者心脏移植术前1周静脉输注成分的不同,将受者随机分为3组:(1)对照组(n=7):单纯输注磷酸盐缓冲液(PBS);(2)SP-DC组(n=8):输注Sp-DC 5×106个;(3)PUVA-SP-DC组(n=8):输注PUVA-SP-DC 5×106个.每日观察各组移植心的存活状况.移植后第6天,检测受者血清中抗供者特异性IgG水平;通过混合淋巴细胞反应(MLR)检测受者脾脏T淋巴细胞对供者抗原刺激的增殖反应;比较各组受者脾脏体积的大小.结果 供者脾淋巴细胞经PUVA处理后细胞凋亡率为81.93%.正常LEW大鼠DC共刺激分子CD80和CD86阳性率分别为(3.5±0.27)%和(13.0±0.58)%,受者DC与供者SP混合培养后,其CD80和CD86的表达水平为(16.6±0.72)%和(36.5±0.87)%,后者明显高于前者(P<0.01);受者DC与供者PUVA-SP混合培养后,其CD86和CD80的表达率分别为(3.9±0.12)%和(13.4±0.59)%,与正常LEW大鼠DC相当(P>0.05).PUVA-SP-DC组的受者抗供者特异性IgG水平明显低于SP-DC组及对照组(P<0.01).PUVA-SP-DC组受者T淋巴细胞对供者抗原的刺激反应指数为1.66±0.29,明显低于SP-DC及对照组(7.28±0.38、4.19±0.16,P<0.01);而其对无关供者抗原的刺激反应指数为4.37±0.11,与SP-DC及对照组相当(4.51±0.40、4.36±0.14,P>0.05).PUVA-SP-DC组的移植心存活时间比其他两组明显延长(P<0.01),而且其脾脏体积最小.结论 PUVA-SP-DC能够特异性的下调移植受者对供者抗原的细胞免疫及体液免疫反应,从而明显延长移植物存活时间.     

In vivo and in vitro mechanisms of cardiac allograft acceptance in the rat after short treatment with 15-deoxyspergualin

15-Deoxyspergualin (DSG) has been reported to be a useful immunosuppressive agent already used to inhibit acute rejection in clinical transplantation. In the present study, the survival of heart allograft in rats after a short course of DSG treatment and the mechanisms underlying DSG-induced heart allograft acceptance were studied. Male LEW rats were used as recipients. Male ACI and Wistar rats were used as donors and third-party donors, respectively. Survival of ACI heart grafts in LEW recipients treated with a short course of DSG starting on day 4 after grafting was markedly prolonged, with a mean survival time of 16.6±5.8 days and 29.8±3.0 days at doses of 2.5 mg/kg per day and 5 mg/kg per day, respectively. On day 20 after grafting, the mechanism of inducing allograft survival after DSG treatment at a dose of 5 mg/kg per day was analyzed by testing the activation of spleen cells or serum in several assay systems. Spleen cells from DSG-treated rats with surviving heart allografts showed almost no proliferative response against donor strain stimulator cells compared with controls. The cytotoxic activity towards donor strain target cells of spleen cells from DSG-treated rats with surviving heart allografts was lower than that of spleen cells from rats with rejected heart allografts. Adding various concentrations of spleen cells or serum from DSG-treated LEW rats with surviving ACI heart allografts to the mixed lymphocyte reaction when responder cells from normal LEW rats were exposed to irradiated ACI or Wistar (third-party) stimulator cells, revealed strong suppression in a cell-dose-dependent manner and a serum-dose-dependent manner. Moreover, transfer of 2.0x10⁸ spleen cells or 2 ml serum from DSG-treated LEW rats with surviving ACI heart allografts to irradiated grafted host did not prolong the survival either of ACI heart grafts or of thirdparty Wistar heart grafts. These results suggest that proliferative response and cytotoxic activity are decreased and suppressor cells and suppressor humoral factor(s) are induced by treatment with DSG in rats with surviving allografts.Abbreviotions DSG 15-Deoxyspergualin - MLR mixed lymphocyte reaction - CML cell-mediated lympholysis     

The role of indirect recognition of MHC class I and II allopeptides in a fully mismatched miniature swine model of lung transplantation

Considerable evidence suggests that indirect recognition of MHC allopeptides plays an important role in solid-organ rejection. Here, we examine whether immunization with class I or class II allopeptides accelerates rejection in a fully MHC-mismatched lung transplant model in miniature swine. METHODS: Recipients were immunized with either donor-derived class I or class II peptides. Sensitization to the peptides was confirmed by DTH testing and in vitro proliferation assays. Nonimmunized control (n = 6), class I peptide-immunized (n = 3), and class II peptide-immunized (n = 3) swine were transplanted with fully mismatched lungs using only a 12-day course of tacrolimus. RESULTS: One control animal rejected its graft on postoperative day 103, while the others maintained their grafts for over 1 year. In the class I peptide-immunized group, two recipients rejected their grafts (days 14 and 52). The third animal has not rejected the graft (day 120, experiment is ongoing). In contrast, in the class II-peptide immunized group, only one animal rejected its graft on day 52, while the others maintained their grafts over 1 year. Both anti-donor IgM and IgG antibodies were detectable in all acute rejectors, although no alloantibody was detectable in long-term acceptors. Regardless of the fate of the graft, all animals have maintained their proliferative responses to the peptides. However, only acceptors maintained donor-specific hyporesponsiveness in cell-mediated lymphocytotoxity and mixed lymphocyte reaction assays. CONCLUSIONS: Pretransplant sensitization of lung allograft recipients to donor allopeptides accelerates graft rejection. This appears particularly true for class I-derived allopeptides, suggesting that class II molecules may be less antigenic when presented indirectly.     

PG490-88, a new immunosuppressant, effectively prevents acute and chronic rejection in rat renal allografts

PG490-88 is a semisynthetic derivative of the novel compound PG490 (triptolide) purified from a Chinese herb. It has been shown to prolong acute allograft survival in multiple experimental organ transplant models. However, the effect of PG490-88 on prevention of acute and chronic renal allograft rejection has not been determined. Kidneys of ACI or F344 rats were transplanted into bilaterally nephrectomized LEW recipients as the acute or chronic allograft rejection models, respectively. Treatment of LEW recipients with PG490-88 significantly prolonged ACI kidney graft survival in a dose-dependent manner when compared with the untreated allograft controls. LEW recipients of F344 kidney grafts who received PG490-88 for 90 days with a brief course of low-dose FK506 showed normal serum creatinine levels and markedly reduced histological changes of chronic rejection at day 90 after transplantation. These results suggest that PG490-88 significantly prolongs kidney allograft survival in an acute rejection model and prevents chronic allograft rejection in rats.     

Acute Humoral Rejection of Human Lung Allografts and Elevation of C4d in Bronchoalveolar Lavage Fluid

Antibody-mediated rejection is well established for renal allografts but remains controversial for lung allografts. Cardinal features of antibody-mediated rejection in renal allografts include antibodies to donor human leukocyte antigen (HLA) and evidence for antibody action, such as complement activation demonstrated by C4d deposition. We report a lung allograft recipient with circulating antibodies to donor HLA who failed treatment for acute cellular rejection but responded to therapy for humoral rejection. To address the second criteria for antibody-mediated rejection, we determined whether complement activation could be detected by measuring C4d in bronchoalveolar lavage fluid (BALF) by ELISA. Airway allergen challenge of asthmatics activates the complement pathway; therefore, we used BALF from asthmatics pre- and post-allergen challenge to measure C4d. These controls demonstrated that ELISA could detect increases in C4d after allergen challenge. BALF from the index patient had elevated C4d concomitant with graft dysfunction and anti-donor HLA in the absence of infection. Analysis of BALF from 25 additional lung allograft recipients showed that C4d concentrations >100 ng/mL were correlated with anti-HLA antibodies (p = 0.006), but were also observed with infection and in asyptomatic patients. The findings support the occurrence of anti-HLA-mediated lung allograft rejection and suggest that C4d measurement in BALF may be useful in diagnosis.     

Increased risk of antibody-mediated rejection of reduced-size liver allografts

Because of the shortage of liver allografts in children, transplantation of reduced-size liver allografts from adult cadaveric donors or living, related donors is being done more frequently. Reduced-size liver allografts may be used in cases of ABO incompatibility and T-cell warm cross-match positivity. This experimental study in inbred rats was undertaken to determine if reduced-size liver allografts are more sensitive to antibody-mediated rejection than full-size liver allografts. Brown-Norway (BN) (RT1(n)) rats were sensitized by three successive skin grafts at 10-day intervals. Then orthotopic Lewis (LEW) (RT1(1)) liver grafts were transplanted into these BN rats. Full-size liver allografts were compared with reduced-size liver allografts (70% of donor liver). Control groups were composed of full-size and/or reduced-size isografts. Titers of specific antibodies were assayed using a complement-dependent assay before and after orthotopic liver transplantation. Histological and immunofluorescence studies (IgG, IgM, C(3), and fibrinogen deposits) were assessed. Recipients of reduced-size liver allografts died of hyperacute rejection at 36.6 +/- 4.1 h, significantly earlier than recipients receiving full-size liver allografts, which died of accelerated acute rejection at 259.2 +/- 25.2 h (P < 0.001). Either full-size or reduced-size isograft recipients survived indefinitely. A decrease in the titers of donor-specific antibodies was observed in both groups of animals. Slight deposits of IgG, IgM, C(3), and fibrinogen were observed in recipients of reduced-size liver allografts, whereas larger deposits were observed in recipients of full-size liver allografts. Our data demonstrate that there is an increased risk of antibody-mediated rejection of reduced-size liver allografts in sensitized recipients. This may have important clinical implications for partial liver grafting in cases of ABO incompatibility and T-cell warm cross-match positivity.     

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

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

A novel CD154 monoclonal antibody in acute and chronic rat vascularized cardiac allograft rejection

BACKGROUND: The CD40-CD154 interaction is critically important in the cell-mediated immune responses. Blockade of this costimulatory pathway has been shown to prevent acute allograft rejection in murine, as well as nonhuman primate models. However, the role of the CD40-CD154 pathway in the development of chronic rejection and the effects of CD154 targeting on progression of chronic rejection have not been evaluated. METHODS: We examined the effect of AH.F5, a new hamster anti-rat CD154 monoclonal antibody, in a fully allogeneic acute(u) into Lewis [LEW] (RT11) and chronic [WF.1L (RT1l) into LEW (RT1l)] vascularized cardiac allograft rejection model. In the chronic model, the antibody was evaluated for prevention (starting day of transplant) and interruption of progression (starting day 30 or 60 after transplant) of chronic vasculopathy. Graft survival, morphology, and immunohistology were evaluated. RESULTS: In the acute rejection model, anti-CD154 therapy alone prevented acute allograft rejection and resulted in 50% long-term allograft survival (>200 days) and donor-specific tolerance. In recipients treated with anti-CD154 monoclonal antibody in combination with a short course of cyclosporine, 100% of allografts survived long-term and all recipients achieved donor-specific tolerance. In the chronic rejection model, allografts from animals treated with the anti-CD154 antibody had a statistically significant lower score of graft arteriosclerosis and fibrosis in both the prevention and 30-day interruption groups when compared with control allografts. In addition, immunohistochemistry showed a decrease in intragraft mononuclear cell infiltration and activation. CONCLUSION: A new anti-CD154 antibody not only prevents acute allograft rejection, but also inhibits and interrupts the development of chronic rejection. In the acute rejection model cyclosporine acts synergistically with anti-CD154 therapy to prolong allograft survival and induce tolerance. In the chronic rejection model relatively early initiation of therapy is essential to prevent progression of chronic allograft vasculopathy and fibrosis.