Humoral immune responses during acute rejection in rat lung transplantation

The detailed responses of humoral immunity during acute rejection remain obscure in lung transplantation (LTx). In order to clarify the reactions of alloantibodies (allo-Abs) during acute rejection, we demonstrated the time-course of changes in anti-donor Ab reaction in the peripheral blood and deposition in the grafts using a rat LTx model. Lewis (LEW) rats served as recipients for Brown Norway (BN) lung allografts (MHC fully incompatible combination). The left lung was transplanted orthotopically using a cuff technique. Syngeneic transplants (LEW to LEW) served as control. No immunosuppression therapy was administered in this model. We evaluated the alloreactivity against donor in rat recipients by detecting allo-Abs with a flow cytometric cross-match (FCXM) technique. Recipient serum samples were incubated with donor lymphocytes and stained with anti-rat immunoglobulin (Ig), to determine the titers of circulating allo-Abs in the peripheral blood with a three-color FCXM technique. We also examined the deposition of anti-donor Abs (IgG and IgM) in the grafts with an immunofluorescent method. All allografts were completely rejected and lost their aeration within 6 days after LTx. Strong allo-Abs responses of both IgG and IgM were observed in the peripheral blood during acute rejection. The level of IgM allo-Abs had already significantly increased on day 2 at the time of mild rejection; however, IgG Abs did not elevate until day 6, when the grade of rejection was severe. Circulating IgM levels started decreasing on day 8, whereas IgG Abs continued elevating. On the other hand, no evident deposition of allo-Abs in the grafts was observed until day 6. We have shown in this study that circulating IgM allo-Abs was detected at the time of mild allograft rejection, interestingly, before evident deposition in the graft. It might be suggested that allograft rejection progressed without antibody deposition until severe rejection.     

Humoral rejection after pediatric heart transplantation: a case report

Humoral rejection was observed 2 years after heart transplantation in a 10-year-old African American girl with sickle cell disease. Hemodynamic compromise developed, and the patient started treatment with extracorporeal membrane oxygenation within 24 hours of admission. With cellular rejection initially believed to be the cause, administration of thymoglobulin and high-dose steroids was initiated. Human leukocyte antigen antibody analysis revealed high titers of donor-specific class I and II antibodies. Aggressive treatment for antibody-mediated rejection was started with plasmapheresis and administration of intravenous immune globulin and rituximab. The patient displayed clinical signs of infection and was treated with antimicrobial, antiviral, and antifungal agents. Computed tomography of the chest suggested asperigillous infection. The patient underwent a left upper lobectomy. The patient recovered and has done well, now 4 years after having received the heart transplant. Antibody-mediated rejection should be considered early in heart transplant patients presenting with hemodynamic compromise and may respond to aggressive antibody and B cell-directed therapy. Vigilance for secondary infections, especially during treatment for rejection, is crucial.     

Specificity of lymphocytotoxic antibodies formed after cardiac transplantation and correlation with rejection episodes.

Eighty-two patients have been studied to determine the class and specificity of lymphocytotoxic antibodies produced during the 6 months following cardiac transplantation. Weekly serum samples were monitored for panel-reactive lymphocytotoxic antibodies (PRA) and donor reactive lymphocytotoxic antibodies using dithiothreitol to determine immunoglobulin class. Sera containing donor-reactive antibodies were further analyzed in a cytotoxic inhibition assay to determine whether the antibodies were directed against HLA or non-HLA determinants. A total of 67 (82%) of the patients produced detectable PRA following transplantation, no correlation was found between PRA and the incidence and severity of rejection. In 33 cases where an HLA specificity was defined, the antibody was not directed against the donor HLA phenotype. In contrast, the 32 (53%) recipients who had formed donor-reactive antibodies within 6 months of operation had required significantly more antirejection therapy (methylprednisolone) than the crossmatch-negative recipients (P less than 0.01). This was the case for both IgG and IgM responses. Of 24 positive donor responses, 18 were found to be specific for HLA antigens. These were both IgM (6 cases) and IgG (12 cases) HLA-specific antibodies, and their occurrence was strongly correlated with rejection (P less than 0.001).     

Humoral immune responses in the pathogenesis of bronchiolitis obliterans syndrome after lung transplantation

Lung transplantation is recognized as a viable treatment option in a variety of end-stage pulmonary diseases. However, the long-term survival is limited by the development of bronchiolitis obliterans syndrome (BOS). Bronchiolitis obliterans syndrome occurs in more than half of lung transplant recipients who survive more than 5 years and is the leading cause of death in the late posttransplantation period. The specific etiology and pathogenesis of BOS are not well understood. The current premise is that BOS represents a common lesion in which different inflammatory insults such as ischemia-reperfusion, rejection, and infection can lead to a similar histological and clinical outcome. However, the observation that early development of BOS is predicted by the frequency and severity of acute rejection episodes indicates that alloimmune-dependent mechanisms play a crucial role in the pathogenesis of BOS. The evidence presented in this review will demonstrate that BOS is the result of indolent humoral immune responses developed against major histocompatibility complex molecules expressed by airway epithelial cells of the lung allograft. Currently, treatment of BOS is rarely successful. Therefore, a better understanding of the immunopathogenesis of BOS is of paramount importance toward improving long-term graft function and patient survival after lung transplantation.     

Immunohistochemical analysis of accelerated graft atherosclerosis in cardiac transplantation

HHT was performed between minimally genetic mismatched inbred strains of rats. There was no evidence of rejection and immunosuppressive therapy was not instituted. Immunohistochemical analysis using peroxidase conjugated monoclonal anti-rat ASMA of cardiac arterioles in which AGAS developed revealed a decreased peroxidase signal. The data suggest that modulation of actin expression in subintimal cells of cardiac arterioles may play a critical role in the pathologic development of AGAS.     

Relationship between CMV and graft rejection after heart transplantation

Abstract  This study, which included 153 heart transplant patients, was designed to determine whether the cytomegalovirus (CMV) status of both donor and recipient may influence graft rejection. The follow-up was 1 year and they all received the same triple-drug immunosup-pressive regimen with induction (antilymphocyte serum). There was no difference in the total rejection rate, but an increase in repeated rejection rate was shown in transplant recipients with hearts from CMV seropositive donors ( P < 0.05). These data strongly suggest the impact of CMV in enhancement but not in induction of rejection. To prevent iterative rejection in the CMV seropositive donor group, antiviral therapy could be proposed during enhancement of antirejection therapy.     

Endothelial cell chimerism associated with graft rejection after human lung transplantation

Endotheliitis is a major sign of graft rejection. Recipient-derived endothelial cells found in two series of liver and kidney transplants were related to graft rejection. Here, we assessed the presence and the number of chimeric endothelial cells in lung transplants, and their relation with graft rejection. In six males grafted with female lungs out of 193 lung transplantations, endothelial chimerism was studied by combined XY-fluorescent in situ hybridization with CD31 and CD45 immunostainings and blood group antigens. On samples graded according to the revised working formulation for lung allograft rejection, we found chimeric macrophages (73.1 to 87.2%) in all cases and chimeric endothelial cells (1.3 to 2.1%) in four patients. Another method using ABO blood group also showed endothelial cells positive for recipient-type blood group antigens in three patients. By both methods, presence of chimeric endothelial cells was related to pathological signs of acute rejection (P<0.05).     

巴利昔单抗联合三联免疫抑制方案预防心脏移植后急性排斥反应

目的 评价巴利昔单抗联合常规三联免疫抑制方案预防心脏移植后排斥反应的临床效果及安全性.方法 2004年6月至2011年1月接受心脏移植的受者共214例.所有受者均在术前1h和术后4d时各应用1次巴利昔单抗,剂量为20 mg/次,三联免疫抑制方案为环孢素A(CsA)+吗替麦考酚酯(MMF)+皮质激素.术后采用心内膜活检诊断排斥反应,并按照国际心肺移植协会(ISHLT)的分级标准评价急性排斥反应的严重程度.术后对受者随访1年,收集心内膜活检及发生排斥反应的资料,观察术后并发症的发生情况及死亡情况.结果 受者接受心内膜活检的时间为术后(20.1±7.3) d(1～60 d),结果显示,Ⅰa 级63例(29.4％),Ⅰb 级8例(3.7％),Ⅱ级12例(5.6％);术后1年,143例受者接受心内膜活检,结果显示,Ⅰa 级29例(20.3％),Ⅰb 级1例(0.7％)及Ⅱ级11例(7.7％).住院期间,共有5例受者死亡,死亡原因包括移植心脏功能衰竭3例,多器官功能衰竭1例及猝死1例;出院后至术后1年内,有2例受者死亡,死亡原因包括严重排斥反应1例和多器官功能衰竭1例.术后1个月内,发生感染7例(3.3％),发生急性肾功能不全11例(5.1％),无受者发生肝功能衰竭.结论 应用巴利昔单抗联合常规三联免疫抑制方案是预防心脏移植术后早期急性排斥反应安全、有效的方式.     

Incidence of graft rejection after pancreas and kidney transplantation]

The possibility of an immunological follow-up of the pancreas through the renal transplant after simultaneous pancreaticorenal transplantation (S.P.R.T.) is controversial. Fifty patients have received a neopren-injected extraperitoneal segmental pancreatic transplant and a contralateral renal transplant, after immunological preparation with blood transfusions, without tissue matching but with a negative anti-T lymphocyte cross-match. Immunosuppression consisted in a three- or four-drug therapy during the first 10 days, then a long-term two-drug therapy (ciclosporine and azathioprine). Sixteen rejection episodes were noted in 16 patients during the first 3 postoperative months. No concomitant alteration of the pancreatic function occurred (no pancreatic histology). No isolated pancreatic rejection has been noted so far. One patients presented with 2 episodes of simultaneous rejection 15 and 26 months after transplantation. The actuarial survival rate at 2 years of the patients, kidneys and pancreata respectively is 96%, 92% and 80%. The absence of long-term alteration of the pancreatic function probably proves the absence of undetected pancreatic rejection. In our experience, the follow-up of the renal function allows screening and treating rejection episodes before a possible functional alteration of the pancreatic transplant occurs. In our opinion, extraperitoneal segmental pancreatic transplantation, a simple procedure with satisfactory metabolic results in the long term, is a good technique for S.P.R.T.     

Prophylactic photopheresis and chronic rejection: effects on graft intimal hyperplasia in cardiac transplantation

BACKGROUND: Despite the decreased incidence of acute rejection episodes and improvements in short and intermediate term graft survival with current immunosuppressive agents, there has been little progress in decreasing the morbidity and mortality from chronic rejection. This phenomenon may, in part, be related to the development of a humoral immune response with increases in anti-HLA antibodies, which presents as accelerated graft arteriopathy with intimal hyperplasia. METHODS: Based on prior experimental work, a pilot, prospective, randomized study was performed in 23 primary cardiac transplant recipients to determine whether the addition of prophylactic photopheresis to a cyclosporine, azathioprine and prednisone regimen was safe and resulted in decreased levels of panel reactive antibodies (PRA) and transplant arteriopathy. RESULTS: There was no difference between the two groups in regard to infection or acute rejection incidence. The photopheresis group had a significant reduction in PRA levels at two time points within the first 6 postoperative months. Coronary artery intimal thickness was significantly reduced in the photopheresis group at 1-yr (0.23 vs. 0.49 mm, p < 0.04) and 2-yr (0.28 vs. 0.46 mm, p < 0.02) follow-up compared with the control group. CONCLUSION: In this small pilot study, photopheresis is a safe, well-tolerated immunomodulatory technique that is capable of decreasing the severity of chronic rejection manifesting as post-transplant graft intimal hyperplasia.     

Adiponectin and graft vascular disease after cardiac transplantation

Graft vascular disease (GVD) is a form of accelerated atherosclerosis that involves the transplanted heart, affecting more than 40% of patients after follow-up of 5 years. According to some authors, adiponectin concentration is a prognostic factor for progression of coronary atherosclerosis. Following this line of research, the objective of the present study was to analyze the relationship between adiponectin concentration and development of GVD in 52 cardiac transplant recipients. Patients were divided into 2 groups according to findings at intravascular ultrasound: group 1 with 21 patients without GVD, and group 2 with 31 patients with GVD. Patients with GVD were further divided into 2 additional groups according to results of coronary angiography: group 3 with 35 patients without GVD, and group 4 with 17 patients with any degree of GVD. No significant differences (P=.50) were observed insofar as adiponectin concentration between groups 1 and 2, and groups 3 and 4. Significant differences in adiponectin concentration were observed when the sex of the patient was considered (P=.002), with higher concentrations detected in women. Serious cardiac events were more common in patients with GVD (P=.001). Mean time between transplantation and diagnosis of GVD was 67 months in group 1, 107 months in group 2, 71 months in group 3, and 101 months in group 4. Significant differences were observed between groups 1 and 2 (P=.030).     

The correlation between humoral antibodies and rejection after renal transplantation

Sera from 36 renal transplant patients were selected at random and screened for cytotoxic antibodies to a panel of 50 normal lymphocytes and 3 lymphoblasts. 11 of these patients had received more than one kidney transplant. None of the primary renal transplants demonstrated cytotoxic antibodies to the panel of normal cells. However, 4 of the retransplant patients were found to possess cytoxins. These 4 sera were cytotoxic to the normal cell panel (10%, 20%, 50% and 70% of panel lysed) as well as to certain lymphoblasts. 30% of the sera negative to the normal cell panel demonstrated cytoxins to the lymphoblasts; 15% positive to only the B-lymphoblasts, 5% positive to only the T-lymphoblasts and 10% showed cytotoxic antibodies to both T and B-lymphoblasts and 10% showed cytotoxic antibodies to both T and B-lymphoblasts. No correlation between cytotoxicity and inhibition of MLR was obvious. Of the 14 sera showing cytotoxicity to lymphoblasts, 9 exhibited MLR inhibition. However, inhibition of MLR was not found to be restricted to B-cell cytotoxic sera. These sera were tested by radioimmunoassay. A good correlation was found between acute rejection and percent increase in direct binding of patients sera to radiolabelled B-lymphoblast membranes. Of the 36 sera tested the six sera obtained from patients in acute rejection bound the iodinated lysate to a significantly greater amount than the controls. As yet, no characterization of the antigen specificities has been found.     

Humoral immune response after kidney transplantation is enhanced by acute rejection and urological obstruction and is down-regulated by mycophenolate mofetil treatment

The anti-allograft immune response may have a cellular and a humoral component. Lymphocytotoxic antibodies (Ab) and anti-human leucocyte antigen (HLA) Ab present before kidney transplantation carry an enhanced risk of acute rejection. Current immunosuppressive drugs act predominantly upon the cellular immune pathway which may leave unopposed the humoral mechanisms of anti-allograft response. We studied the production of lymphocytotoxic Ab and anti-HLA Ab after kidney transplantation under different drug therapies. Two hundred and sixty-four consecutive kidney transplant recipients treated with different immunosuppressive drugs, either stable and or with previous acute rejection or acute urologic obstruction, entered this study. Lymphocytotoxic Ab and anti-HLA Ab were evaluated by complement-dependent cytotoxicity and by ELISA. Ab donor-specificity was determined by flow cytometry. Both lymphocytotoxic Ab and anti-HLA Ab were significantly increased in acute rejection whatever the immunosuppressive regimen and almost significantly in urologic obstruction treated with azathioprine (AZA) groups. The presence of antidonor-specific Ab was associated with a significantly higher rate of graft loss. Mycophenolate mofetil (MMF) therapy significantly down-regulated Ab synthesis in all patients groups when compared with AZA. The development of humoral antidonor response post-transplantation is associated with a dismal graft prognosis. This is the first report that acute urologic obstruction may be followed by unspecific lymphocytotoxic and anti-HLA Ab synthesis, surmising that a protracted obstruction may promote renal fibrosis through antibody mediation. The significant down-regulation of the humoral response by MMF when compared with AZA may herald a lower risk to mount a chronic rejection process.     

Acute rejection after cardiac transplantation: detection by interstitial myocardial pH

Intramyocardial pH was assessed as a potential marker for clinical evaluation and treatment of acute rejection following cardiac transplantation. Fifteen cats underwent forty operative procedures. Following intra-abdominal heterotopic heart transplantation, serial laparotomies were performed in the early (days 0 to 2), intermediate (days 5 to 7), and late (days 7 to 16) postoperative periods. Rejection was assessed by serial clinical examinations, ECG analyses, B-mode echocardiography, histological and ultrastructural analyses, and measurements of interstitial myocardial pH. Intramyocardial pH was measured by a new miniature (0.6 X 3.0 mm) fiberoptic pH transducer. At confirmed rejection, concomitant laparotomy and thoracotomy were performed and pH sensors were implanted in both native (anatomical) and graft hearts. Nine animals at rejection were given methylprednisolone and changes in graft and native heart pH were measured. The pH during absence of rejection, mild acute rejection, and severe acute rejection averaged 7.430 +/- 0.019, 7.233 +/- 0.040 (p less than .02), and 6.860 +/- 0.066 (p less than .02), respectively (mean +/- standard error of the mean). A progressive decline in pH was noted in each heart. In animals receiving steroids, graft heart pH increased over 90 minutes from 6.852 +/- 0.065 to 7.043 +/- 0.077 (p less than .05). Although pH decline may be secondary to either inflammatory or ischemic etiology, histological and ultrastructural analyses demonstrate a predominant inflammatory response with progressive mononuclear cell infiltration, interstitial edema, vascular wall edema, infiltration by polymorphonuclear neutrophil leukocytes, vacuolation of sarcoplasmic reticulum, and disarray of myocytes associated with falling pH. Degree of pH change correlated closely with degree of histological rejection, presence of ECG voltage decline, and change in wall thickness by ultrasound.     

Peculiar mechanisms of graft recovery through anti-inflammatory responses after rat lung transplantation from donation after cardiac death

BackgroundAlthough lung transplantation from donation after cardiac death (DCD), especially uncontrolled DCD, is limited by warm ischemic periods, the molecular mechanism of warm ischemia–reperfusion-injury (IRI) has not been well elucidated. The purpose of this study was to clarify the particular longitudinal mechanisms of molecular factors involved in warm IRI.MethodsCold ischemic-time (CIT)-group lungs were retrieved and subjected to 3-h of cold preservation, whereas warm ischemic-time (WIT)-group lungs were retrieved after 3-h of warm ischemia. Orthotopic rat lung transplantation was performed and the grafts were reperfused for 1 or 4-h. The graft functions, gene expression, and activation of inflammatory molecules in the grafts were analyzed. Exhaled-carbon-monoxide-concentration (ExCO-C) was measured during reperfusion.ResultsOnly the WIT-group showed obvious primary graft dysfunction at 1-h reperfusion, but the graft function was recovered during 4-h reperfusion. Most of pro-inflammatory cytokines and stress-induced molecules showed different expression and activation patterns between CIT and WIT groups. In the WIT-group, the expressions of anti-inflammatory molecules, IL-10 and HO-1, were significantly increased at 1-h reperfusion compared to the CIT-group, and these high levels were maintained through 4-h reperfusion. Furthermore, ExCO-C levels in the WIT-group increased immediately after reperfusion compared to the CIT-group.ConclusionsThis study indicates that warm IRI may involve a different mechanism than cold IRI and anti-inflammatory pathways may play important roles in the graft recovery after lung transplantation from uncontrolled DCD.