Identification of the antibody to vascular endothelial cells in patients undergoing cardiac transplantation

Acute cardiac dysfunction occurred in four cardiac allograft recipients with negative donor-specific lymphocyte crossmatches. In two recipients the transplanted heart was removed and the patients were maintained on bypass for several hours until a second cardiac allograft was available. In these patients the second transplanted heart also underwent acute dysfunction. The lymphocyte crossmatch was again negative in both second transplants. Two of the four recipients had no detectable antibody to a panel of lymphocytes. Examination of the hearts demonstrated histologic findings consistent with hyperacute rejection. Direct immunofluorescence performed on the transplanted hearts revealed the presence of immunoglobulin and complement deposited on the vascular endothelium. Pathology data was available on 3 of the 4 patients who experienced acute cardiac dysfunction. Pretransplant sera from these four recipients were screened for the presence of antivascular endothelial cell (VEC) antibody. The sera from all four recipients were found to contain antibody against an endothelial cell panel. In addition, donor-specific aorta and vena cava were available from one of the heart donors. The recipient was found to have donor-specific antibody to VEC. Thus, antibody directed against VEC specific antigens appears to be related to hyperacute rejection of heart allografts.     

血管内皮生长因子在大鼠心脏移植急性排斥期的表达

目的　观察血管内皮生长因子(VEGF)在大鼠心脏移植急性排斥期中的表达及其和排斥的关系。方法　大鼠分对照组,CSA组,每组12只。分别静脉给予生理盐水及CSA干预,采用颈部心脏异位移植术式建立移植模型。常规监测排斥反应发生情况。每组5只用于观察移植物存活时间,7只用于动态切取标本。应用逆转录 聚合酶链反应(RT PCR)检测移植物局部VEGF的表达水平。结果　CSA组移植心存活时间(2 0 .4±5 .1)d显著长于对照组(8.6±1.5 )d ,CSA组VEGF的表达强度弱于对照组,其时间变化趋势和高峰时间较对照组推迟并和移植心存活延长时间有相关性。病理观察显示移植心局部的炎性细胞和淋巴细胞浸润和VEGF表达有关。结论　VEGF高表达促进排斥反应的发生缩短移植心存活时间,提示VEGF的表达和移植心的炎性浸润以及急性排斥有密切关系     

The clinical significance of antibody to vascular endothelial cells after renal transplantation

Abstract: Vascular endothelial cells (ECs) are considered to be a primary target for injury in allograft rejection. However, the relationship between serum antibody activity to ECs and rejection episodes has not been examined extensively in renal transplantation. Twenty-two renal transplant recipients were included in this study. Serum antibody activity to vascular endothelial cells (AECA) was measured using a cellular enzyme-linked immunosorbent assay (ELISA) in which human umbilical vein endothelial cells (HUVEC) and human glomerular endothelial cells (HGEC) were preincubated with TNF-α used as target cells. Serum samples were obtained just before transplantation and once a week during the immediate 1–3-month post-transplantation period. There was a significant correlation between the presence of AECA against HGEC and rejection episodes ( P  < 0.05). Patients with multi-episodes of rejection showed significantly higher frequencies of AECA than patients with mono-episodic rejection ( P  < 0.0005). It should be noted that patients suffering from multi-episodes of rejection revealed higher AECA titres before transplantation. These findings imply that the HGEC–ELISA could be used as a prospective, informative test to identify patients with a higher risk of acute rejection in renal transplantation.     

The clinical significance of antibodies to human vascular endothelial cells after cardiac transplantation

BACKGROUND: Vascular endothelial cells are primary targets for injury during both cellular and humoral allograft rejection (AR). In cardiac transplantation, the role of humoral immunity in mediating AR has not been extensively characterized. METHODS: Antibodies against human vascular endothelial cells (AECA) were measured using a cellular ELISA developed from human umbilical vein endothelial cells in 80 consecutive patients after cardiac transplantation. The aim was to determine the incidence of AECA formation after transplantation and their association with different types of AR, graft survival, and development of cardiac allograft vasculopathy (CAV). At least eight serum samples obtained from each patient were examined for AECA and an endomyocardial biopsy was performed at regular intervals during the first year after transplantation. RESULTS: Of the 80 patients examined, 31 were AECA (+) and 49 patients were AECA (-). There were no significant differences between the AECA (+) and (-) groups when examined for age, sex, and pretransplantation ischemia time. A significant correlation was found between the presence of AECA and humoral AR (P<0.015). AECA positivity did not correlate with the presence of cellular AR or the number of rejection episodes. In addition, allograft survival at 2 years after transplantation was significantly better in the AECA (-) group compared with that in the AECA (+) group (89.8% vs. 71.0%, P<0.0004). The persistence of AECA positivity during the first year after transplantation was also associated with a significantly greater incidence of CAV when compared with the patients who were AECA (-) (25.8% vs. 14.3%, P<0.004). CONCLUSIONS: AECA may be important in the mediation of humoral AR, may decrease allograft survival, and may identify a high-risk group for CAV.     

The association of antivascular endothelial cell antibody with hyperacute rejection: a case report

Early graft loss almost always occurs when recipients of a renal allograft develop antibody directed against antigens specific for donor vascular endothelial cells (VECs) and peripheral blood monocytes. In studies involving recipients of human leukocyte antigen identical, living-related grafts exhibiting preformed antibody to the VEC antigens of their donors, the median onset of rejection was 3 days after transplantation. Although preformed antibody to VEC antigens has been related in numerous articles to early graft loss, there has never been a published report of anti-VEC antibody leading to hyperacute rejection. We report a patient who hyperacutely rejected a renal allograft after undergoing a donor-specific transfusion protocol with her mother in which the kidney was removed in less than 24 hours. Nine months later the patient had a retransplantation with an allograft from a cadaveric donor. The cadaveric graft was again hyperacutely rejected, and this kidney was removed immediately. Anti-VEC/monocyte antibody directed against both donors was detected in the patient's pretransplant sera. With the exception of a positive B-lymphocyte crossmatch with her mother, all the standard crossmatches were negative.     

移植肾超急性排斥反应临床探讨

我院施行尸体肾移植术４７２例次，其中术的一发生超急性排斥反应９例（１０次），发生率为２．１％，本文就ＨＡＲ的发生机理，发生原因，诊断与鉴别诊断，处理及预防进行讨论。     

补体抑制治疗与心脏移植超急性排斥反应

心脏移植超急性排斥反应(hyperacute rejection,HAR)与补体系统激活有关,迅速导致移植心脏功能丧失.动物实验中使用眼睛蛇毒因子、可溶性补体受体1、补体调节蛋白以及C5单克隆抗体等补体抑制剂,可以从补体级联反应的不同水平抑制同种或异种心脏移植HAR的发生.抑制补体激活将在临床心脏移植HAR的预防及治疗中显示出重要的作用.     

Lewis rat pancreas, but not cardiac xenografts, are resistant to anti-gal antibody mediated hyperacute rejection

BACKGROUND: The objective of this study was to evaluate the role of anti-Gal Abs and non-anti-Gal Abs in hyperacute rejection (HAR) of concordant pancreas xenografts compared with heart xenografts. In addition, we tested whether rejection of Lewis rat pancreas grafts was T-cell dependent and could be prevented by anti-T-cell treatment. METHODS: To determine the role of anti-Gal Abs in the induction of HAR, Lewis rat pancreas and heart xenografts were transplanted into alpha1,3Galactosyltransferase knockout (GT-Ko) mice treated with normal human serum (NHS) or hyperimmune serum, or into presensitized GT-Ko mice. To investigate whether rejection of pancreas xenograft was mediated by a T-cell dependent response, Lewis rat pancreas grafts were transplanted into streptozotocin (STZ)-induced diabetic GT-Ko mice treated with FK506, anti-CD4 mAbs (GK1.5), and thymectomy. Antidonor-specific IgM and IgG and anti-Gal Abs were analyzed by flow cytometry. Rejected and long-term surviving pancreas xenografts were assessed by functional (blood glucose) and histopathological examination. RESULTS: HAR of Lewis rat pancreas xenografts could not be induced by NHS (0.4 ml), whereas NHS (0.2 ml) resulted in HAR of Lewis heart xenografts. Infusion of Lewis rat-specific hyperimmune serum (0.2 ml) resulted in HAR of Lewis rat pancreas xenografts. In addition, second Lewis rat pancreas grafts were hyperacutely rejected by presensitized GT-Ko mice. Immunohistochemical staining showed a low expression of Galalpha1,3Gal antigen in the endocrine tissue compared with that in the cardiac grafts. The levels of anti-Gal Abs in pancreas xenograft transplantation did not increase in GT-Ko mice after pancreas xenograft transplantation that was significantly increased after heart transplantation. FK506 treatment induced long-term survival of Lewis pancreas xenografts (mean survival time (MST) >90 days). Anti-CD4 treatment delayed rejection of Lewis rat pancreas xenografts with MST of 34.3 days, whereas anti-CD4, in combination with thymectomy, synergistically prolonged survival of pancreas xenograft (MST=70.4 days). CONCLUSION: Pancreas xenograft is resistant to anti-Gal Abs-induced HAR but is susceptible to anti-donor specific Abs. Rejection of Lewis pancreas xenograft in STZ-induced, diabetic, GT-Ko mice is T-cell dependent.     

Serum vascular endothelial growth factor as a surveillance marker for cellular rejection in pediatric cardiac transplantation.

BACKGROUND: Early detection and treatment of acute rejection in cardiac transplant recipients significantly improves long-term survival. Endomyocardial biopsy is used routinely for diagnosing allograft rejection; however, in young children, this procedure carries some risk. We evaluated serum vascular endothelial growth factor (VEGF) as a potential surveillance marker of acute cellular rejection. METHODS: Blood samples (n=62) were analyzed from 23 patients and compared with controls (n=18) using an ELISA for VEGF. Results were correlated with endomyocardial biopsy rejection grades. RESULTS: Mean baseline VEGF levels of the transplant population were consistently higher than controls. Serum VEGF levels were significantly higher during acute cellular rejection when compared with the non-rejecting transplant group (700.7+/-154 pg/ml vs. 190.5+/-29 pg/ml). VEGF decreased two- to eightfold after immunosuppressive therapy in 9 of 11 rejection episodes. CONCLUSIONS: These data suggest that VEGF may play a role in the pathogenesis of acute allograft rejection and it may serve as a reliable serologic surveillance marker.     

Delayed hyperacute xenograft rejection in porcine to canine fetal liver transplantation.

Fetal tissues are generally considered to express weaker antigenic cell-surface molecules than adult tissues. We have reported that transplantation of porcine fetal liver tissue (fragments) is useful for acute and chronic hepatic failure in rats. We further investigated, in the present study, whether transplantation of a porcine fetal liver has the advantage of delayed hyperacute xenograft rejection (HAR) in comparison with that of an adult liver. Porcine fetal liver heterotopically transplanted into dogs was compared. Haematoxylin-eosin (HE) and immunohistochemical studies using IgM, C3, IgG antibodies were performed in serial biopsies of the liver grafts. Lectin binding to target antigen epitopes on pig and dog tissues was studied by flow cytometry. Carbohydrate expression on the liver was also studied by immunohistochemistry. The macroscopic and HE section findings indicate that HAR started 15 min postgraft in fetal and adult liver grafts. Thereafter, vascular changes and parenchymal damage progressed more rapidly in the adult grafts. The final HAR time in adult liver transplantation was determined to be 60 min, while it was determined to be 180 min in fetal liver transplantation. IgM, C3 and IgG were deposited more strongly in the adult grafts than in the fetal grafts up until 60 min after xenografting. Phaseolus vulgaris erythroagglutinin lectin competitively blocked dog sera binding to porcine PBLs. The fetal liver expressed oligosaccharide at a significantly lower level than the adult liver. We conclude that porcine fetal liver xenografts had a significantly delayed HAR.     

Potential various appearances of hyperacute rejection in human liver transplantation

Summary Out of 81 liver transplantations 2 graft failures were diagnosed to be due to hyperacute rejection. In the first patient the operative procedure was difficult requiring 19 units of blood and plasma, but the graft was functioning well from the beginning until day 1–2, when rapid deterioration occurred. The cross-match was positive. The second patient received a third graft after the first graft had failed due to donor reasons and the second ABO-incompatible graft had been rejected. The third graft transplanted in an uncomplicated operation requiring only 10 units of blood and plasma failed within hours. Both incidences are throught to be a consequence of an immunological assault, consistent with hyperacute rejection. Thus two different clinical appearances could be observed: the so-called delayed type in the first patient and the more classical type in the second patient. For establishing diagnosis of hyperacute rejection two prerequisites were considered essential: 1) histological findings of necrosis and patchy deposits of immunoglobulins, namely IgG, IgM, IgA, C-3 complement component, properdine and fibrinogen, and 2) the proof of at least a short period of an initial function of the graft prior to deterioration in order to exclude primary non-function due to other causes. The low frequency of the appearance of the classical hyperacute rejection and hypothetical causes for the more frequent appearance of the delayed type are discussed.

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Hyperakute Abstoßung bei klinischer Lebertransplantation- mögliche unterschiedliche Erscheinungsformen

Zusammenfassung Bei 2 von 81 Lebertransplantationen wurde ein Transplantatversagen beobachtet, das auf eine hyperakute Abstoßung zurückgeführt wurde. Bei der ersten Patientin, die in einer schwierigen, 19 Einheiten Blut und Plasma erfordernden Operation transplantiert wurde, zeigte sich zunächst eine gute Sofortfunktion des Transplantates, bis es am Tag 1–2 zu einem plötzlichen Funktionsverlust kam. Das Cross-match war positiv. Der zweite Patient erhielt ein drittes Transplantat, nachdem das erste eine spenderbedingte Nichtfunktion aufgewiesen hatte und das zweite, blutgruppeninkompatible Organ abgestoßen worden war. Das dritte Transplantat, das in einer unkomplizierten, nur 10 Einheiten Blut und Plasma erfordernden Operation transplantiert wurde, zeigte eine Sofortfunktion. Innerhalb weniger Stunden kam es jedoch zum Funktionsverlust. Beide Transplantatversagen werden auf immunologische, einer hyperakuten Abstoßung entsprechende Vorgänge zurückgeführt. Somit konnten zwei unterschiedliche Erscheinungsformen beobachtet werden: der sogenannte Delayed Type bei der ersten Patientin und die mehr klassische Form bei dem zweiten Patienten. Für die Diagnosestellung hyperakute Abstoßung werden zwei Befunde für erforderlich gehalten: 1. histologische Befunde von Nekrosen und herdförmige Immunglobulinablagerungen, besonders IgG, IgM, IgA, C3-Komplement-Komponente, Properdin und Fibrinogen, und 2. der Nachweis zumindest einer kurzzeitigen initialen Transplantatfunktion, um eine initiale Nichtfunktion aufgrund anderer Ursachen auszuschließen. Die seltene Diagnosestellung bei der klassischen Form einer hyperakuten Abstoßung und hypothetische Gründe für die häufigere Beobachtung des Delayed Type werden diskutiert.

Supported by the Deutsche Forschungsgemeinschaft and the VW-Stiftung     

脾脏在小鼠同种肝移植排斥反应中的作用

目的 观察脾切除在小鼠同种肝移植急性排斥反应过程中的作用.方法 双袖套法建立小鼠原位肝移植模型,随机分为3组,即建模保留脾脏组、建模3 d后切除脾脏与建模同时切除脾脏组,各组于移植术后14 d处死,ELESA法测定血清IgM水平;肝功能检测采用速率法;流式细胞仪检测CD4与CD8T细胞亚群;并同时行肝脏及脾脏的病理形态观察.结果 建模保留脾脏组、建模3 d后切除脾脏与建模同时切除脾脏组血清IgM水平分别为3.0181±0.4627、3.0936±0.4559、3.1953±0.4449,各组间差异无统计学意义(P＞0.05);ALT水平分别为108.6875±20.3657、83.0000±22.7799、76.8000±19.5784,差异有统计学意义(P＜0.05);AST水平分别为:105.3750±29.0583、93.0000±22.7799、93.2000±33.4220,各组间差异无统计学意义(P＞0.05);CD46+/CD8+T细胞分别为:1.9162±0.2778、1.5654±0.4750、1.4616±0.2762,差异有统计学意义(P＞0.05);3组肝脏间质及汇管区淋巴细胞浸润程度依次减弱,供肝灶状坏死程度逐渐减轻,在保留脾脏组中建模后第14天脾脏边缘区及淋巴鞘较建模同时切除的脾脏增宽.结论 在小鼠同种异体肝移植排斥反应中细胞免疫起主要作用,脾切除可部分抑制同种异体肝移植急性排斥反应,保护供体肝脏.     

Complement inhibition with an anti-C5 monoclonal antibody prevents hyperacute rejection in a xenograft heart transplantation model

BACKGROUND: The present study was undertaken to determine whether anti-complement 5 (C5) monoclonal antibodies (mAb) prevent hyperacute rejection (HAR) in a rat-to-presensitized mouse heart transplantation model and whether these mAb, combined with cyclosporine (CsA) and cyclophosphamide (CyP), can achieve long-term graft survival. METHODS: BALB/c mice were presensitized with 2x10(7) splenocytes from Lewis rats 14 days before grafting. Heart grafts from Lewis rats were heterotopically transplanted into BALB/c mice. Presensitized mice were treated with either anti-C5 mAb or a combination of anti-C5 mAb, CsA, and CyP. Controls included: presensitized mice with no treatment, presensitized mice treated with either CsA + CyP or IgG, and nonpresensitized mice with either no treatment or with CsA + CyP treatment. RESULTS: Although typical features of HAR were evident in the presensitized grafts, the mAb completely inhibited complement activation and successfully prevented HAR. Despite complement inactivation, the graft was rejected on postoperative day 6 with acute vascular rejection (AVR) also known as delayed xenograft rejection (DXR). Notably, this type of rejection cannot be effectively overcome by CsA and CyP. CONCLUSIONS: We conclude that (1) anti-C5 mAb prevents HAR, (2) AVR/DXR still occurs when HAR is prevented by complement inactivation, and (3) AVR/DXR cannot be overcome by conventional immunosuppression. These data suggest that anti-C5 mAb may be valuable for preventing HAR in future clinical xenotransplantation and that additional interventions may be required to address AVR/DXR.