Acute cell-mediated rejection in orthotopic pig-to-mouse corneal xenotransplantation

Abstract: Background:  To investigate the role of T cells and natural killer (NK) cells in mediating corneal xenograft rejection in a pig-to-mouse model.
Methods:  Pig corneas were orthotopically transplanted into BALB/c, C57BL/6, nude, severe combined immunodeficiency (SCID), and NOD/SCID/γc^null (NOG) mice. Graft survival was clinically assessed by slit-lamp biomicroscopy and median survival times (MST) were calculated. The rejected grafts were histologically evaluated using antibodies against CD4, CD8, NK1.1, and F4/80.
Results:  The pig corneal xenografts were acutely rejected by BALB/c and C57BL/6 mice (MST 9.0 days), while nude, SCID and NOG mice rejected pig corneas in a more delayed fashion (MST 16.0, 16.4, and 16.9 days, respectively). The majority of infiltrating cells found in rejected grafts in C57BL/6 mice were macrophages and CD4⁺ T cells, while CD8⁺ T cells and NK cells were rarely found. The grafts in nude mice had markedly decreased inflammatory infiltration with small numbers of macrophages and CD4⁺ T cells. Infiltration was even more modest in grafts in SCID and NOG mice.
Conclusions:  T cells play an important role in acute rejection of pig corneal xenografts in mice, although acute rejection is not solely the result of T-cell-mediated immunity. NK cells are less likely to be involved in the rejection process.     

Chimeric pig hearts resist hyperacute rejection in ex vivo perfusion model

With surrogate tolerogenesis. the recipient immune system is engrafted within the donor pig before organ transplant. Chimeric pig hearts may resist hyperacute rejection by inducing accommodation. This hypothesis was tested using an ex vivo isolated piglet heart perfusion model. Processed sheep marrow was infused into fetal pigs at 45 days gestation. Heart explants from chimeric or nonchimeric pigs were suspended in a Langendorff apparatus and perfused with plasma from unsensitized sheep or sensitized sheep. Nonchimeric hearts perfused with plasma from unsensitized functioned for 240 min (N = 3). Nonchimeric hearts perfused with sensitized plasma deteriorated rapidly, functioning at 19+/-12 min (N = 6); Immunohistochemistry of heart graft revealed extensive deposition of IgG, IgM in the microvascular. In contrast, chimeric hearts perfused with sensitized plasma functioned for 183+/-46 min (N = 3)(p <.001); Deposition of IgG, IgM had substantially less. Heart grafts procured from chimeric pigs survived in the presence of antidonor IgG, IgM, and complement, demonstrating that chimeric pig hearts resist hyperacute rejection.     

Demonstration of the functional importance of the Gal epitope in an ex vivo model of xenotransplantation

Abstract: The galactose a 1-3 galactose terminal disaccharide (Gal epitope) has been identified as the major porcine xenoantigen recognised by xenoantibody in human plasma. Elimination or suppression of the epitope or antibody will be a major factor in overcoming hyperacute rejection. Inhibition of the antibody by depletion or elimination of the epitope by gene knockout may reveal the importance of other xenoantibodies, and in addition elimination of the epitope may unmask or produce other xenoantibody combinations. This study aims to determine the relative importance of anti-Gal antibody and Gal epitope elimination in a functional model of xenotransplantation, ex vivo perfusion of mouse hearts with human plasma on a Langendorff apparatus. Perfusion of mouse hearts with human plasma depleted of anti-Gal antibody demonstrates a protective effect compared to hearts perfused with undepleted plasma with prolongation of survival time from 24.1 to 44.5 min. Similarly, elimination of the epitope is also protective. Hearts from Gal knockout mice, which were generated by gene targeting of the al,3 galactosyltransferase gene, and hearts from appropriate control mice were perfused with human plasma. Gal knockout mice hearts demonstrated an increase in survival time from 10.2 to 33.8 min compared to control hearts. This was accompanied by a decrease in C3c and IgM, but little change in IgG deposition. The protective effect is incomplete, probably due to the effect of antibodies against non-Gal xenoantigens. There was no functional evidence for generation of neo-antigens in the Gal KO mice that were I recognised by naturally occurring human xenoantibodies.     

Improved ex vivo training model for percutaneous renal surgery

Percutaneous endourological procedures require an advanced level of skills. To facilitate the training of the proper technique, some years ago we developed a porcine ex vivo model for training percutaneous endourological procedures. When dilating the percutaneous tract silicone and gelatine were frequently damaged thus inhibiting proper working with the endoscopes. To circumvent these problems we improved our ex vivo model in order to be as close to the clinical situation as possible. The kidney with the ureter was dissected off the retroperitoneal organ package of the freshly slaughtered pigs. The kidneys were put into bags cut into parts of the thoracic/abdominal wall of these pigs. The renal pelvis can be filled with saline to simulate hydronephrosis; stones can be implanted for PCNL. Our new model allows for even better training of all percutaneous endourological procedures (e.g. percutaneous nephrostomy, PCNL, endopyelotomy). Especially puncturing is extremely close to the situation in humans as the porcine thoracic/abdominal wall in principal has the same anatomy as the human one. The new model has been already used with great success in hands-on courses. Concerning “tissue feeling”, the anatomic relations and the great variety of procedures that can be trained, it is superior to non-biological models. Nevertheless, it is easily available and inexpensive. Presented in parts at the 11th International Symposium on Urolithiasis, Nice, 2008.     

一种新型的经皮肾镜手术离体动物模型

目的 介绍一种新型的X线和B超定位下经皮肾镜手术(PCNL)培训的离体动物模型.方法 模型由带有输尿管的新鲜成年猪肾、2根肋骨的全层胸壁、2根长钉和1块木板组成.输尿管预置导管后,胸壁对折包裹猪肾,2根肋骨覆盖肾上极,用长钉将模型固定在木板上.可以切开肾盂预置石子以训练碎石操作.X线定位:经输尿管导管注入造影剂,逆行肾盂造影显示肾集合系统,确定目标肾盏后,C-臂探头向术者方向旋转30°,使穿刺针、目标肾盏长轴和C-臂长轴在同一平面上,穿刺针头和针尾呈牛眼征.穿刺针确定方向后,C-臂调整到垂直位,观察穿刺深度并按原方向穿人目标肾盏.B超定位:经输尿管导管注入生理盐水制造人工肾积水,B超引导下确定目标肾盏.穿刺完成后,进行扩张、碎石等训练.训练结束后,解剖猪肾,对穿刺针和扩张效果进行检测.结果 126名泌尿外科医生接受培训,其中104名(82.5 %)成功完成模型的经皮肾镜的穿刺、扩张、碎石等操作,114名(90.5%)医生认为该模型对临床手术有帮助或非常有帮助.结论 该模型可以较好地模拟临床PCNL操作过程,为初学者提供一个轻松的学习环境,可反复检查操作训练,为临床手术积累经验.

Abstract：

Objective To lessen the learning curve of percutaneous renal lithotripsy(PCNL),we introduced a novel ex-vivo learning and training model for PCNL under fluoroscopy and ultrasonography-guided access. Methods The model was set up nailing an adult porcine kidney with＞3 cm ureter (freshly removed from the slaughtering factory),with a full thickness flap of the thoracic wall or abdomen wall with two ribs,to a board.The porcine kidney was placed within the flap with the catheterized ureter outside.The kidney was enclosed by the flap so as to create a roodel for percutaneous renal surgery;with the ribs overlying the upper portion of the kidney.The model was fixed to the board by two nails.Artificial stone material was implanted in the renal pelvis.Fluoroscopy guidance access:Retrograde pyelography via injection of contrast medium into the ureteral catheter images the collecting system. After the long axis of the target calyx is identified,the C-arm is rotated 30 degrees toward the surgeon,placing the C-arm axis in the same posterior plane of the kidney.The needle is advanced in the plane of the fluoroscopic beam,and the appropriate needle placement is determined by obtaining a bull's-eye sign on the fluoroscope screen. Rotating the C-arm to a vertical position monitors the depth of the needle penetration. Ultrasonography guidance access:The renal pelvis can he filled with normal saline through a catheter to simulate hydronephrosis and the target calyx is identified under ultrasonography guidance.The tract dilation and stone disintegration were followed.After training,the kidney can be opened to examine the target calyx and the complication of dilation. Results Altogether,126 urologists attended a urologic endoscopic technique training course.Of the 126 trainees,104 (82.5%) successfully performed the whole percutaneous procedure.At the end of training,114 (90.5 %) of the 126 attendees rated the porcine kidney model for simulation of percutaneous renal surgery as very helpful or helpful.Conclusions This biological training model simulates realistically the clinical procedure of PCNL with respect to trainee experience in a low stress environment that provides an opportunity for repetitive performances in order to learn basic technical skills for the clinical procedure of PCNL in the future.     

Normothermic ex vivo renal perfusion

Results of a preliminary investigation to (1) ascertain the feasibility of prolonged normothermic perfusion of the kidney, (2) develop a satisfactory perfusion solution, and (3) promote altered immunogenicity of donor renal tissue are reported. Initially, increased perfusion pressures, decreased flow rates, and severe weight gains were encountered. Addition of protein and manipulation of oxygen concentration in the perfusate greatly improved these parameters, but did not appear to improve viability of transplanted organs.     

Acute humoral renal allograft rejection

In kidney transplantation, it is well established that donor-specific antibodies can cause substantial graft injury. Hyperacute rejection, now virtually eliminated by routine pretransplant cytotoxic crossmatch testing, represents the prototype of humoral rejection. However, there is now increasing evidence that alloantibody-mediated immune reactions may also cause acute rejection. Acute humoral rejection, which is frequently associated with severe graft dysfunction and immunologic graft loss, represents a particular diagnostic and therapeutic challenge. Reliable detection of antibody-mediated graft injury is required to govern the application of antihumoral therapeutic strategies. This review focuses on new approaches in the diagnosis and treatment of acute humoral rejection. Special attention is given to a novel diagnostic marker, the complement split product C4d.     

Aurintricarboxylic acid inhibits endothelial activation, complement activation, and von Willebrand factor secretion in vitro and attenuates hyperacute rejection in an ex vivo model of pig-to-human pulmonary xenotransplantation

Abstract: Background: In the xenotransplantation of vascularized organs, such as the lung, a large area of endothelial cell layer is a big hurdle to be overcome. We investigated the potential protective effect of aurintricarboxylic acid (ATA), a known inhibitor of platelet adhesion, on endothelial damage induced by xenogeneic serum. We also assessed its role in hyperacute xenograft rejection using a porcine ex vivo lung perfusion model. Methods: Porcine endothelial cells were incubated with human serum and other inflammatory stimuli. For the evaluation of von Willebrand factor (vWF) secretion and tissue factor (TF) expression, we used human endothelial cells. E‐selectin expression, complement activation, TF expression and platelet activation were investigated by flow cytometry. In an ex vivo porcine lung perfusion model, the porcine lungs were perfused with fresh human whole blood: unmodified blood (n = 5), ATA‐treated blood (n = 5), and ATA and lepirudin‐treated blood (n = 5). Results: Aurintricarboxylic acid significantly inhibited TNF‐α‐ or lipopolysaccharide‐induced endothelial E‐selectin expression in a dose‐dependent manner. ATA also prevented human serum induced‐E‐selectin expression and human monocytic cell adhesion to porcine endothelial cells. Moreover, ATA abolished thrombin‐induced vWF secretion as well as complement activation. However, ATA induced endothelial TF expression and platelet activation in vitro. In ex‐vivo experiments, ATA treatment improved pulmonary function and attenuated sequestration of leukocytes. Although ATA did not influence thrombin generation, we were able to minimize its activity by adding lepirudin to the blood with ATA. Conclusions: Our study demonstrated in vitro protective effect of ATA on the inhibition of endothelial activation and vWF secretion and confirmed detrimental effect of ATA on induction of endothelial TF and platelet activation. The combination of ATA and lepirudin may act beneficially by preventing coagulation perturbation while maintaining improved xenograft survival.     

Evaluation of phagocytic cell function in an ex vivo model of hemodialysis

An ex vivo model of hemodialysis was used to evaluate the effect of dialysis membranes on phagocytic cell function. Blood was withdrawn continuously from healthy, non-uremic donors, heparinized, and pumped, single pass, through membrane modules under conditions which simulated normal dialysis conditions. The membrane modules contained membranes of cellulose, DEAE-substituted cellulose, or polysulfone. Blood was obtained from the module outlets for determination of complement activation, phagocyte elastase release, zymosan-induced phagocyte chemiluminescence, and monocyte interleukin-1 production. Significantly less complement activation occurred with the polysulfone and DEAE-substituted cellulose membranes than with cellulose membranes. Normal monocyte interleukin-1 production was not stimulated by any of the membranes used. In contrast, the cellulosic, but not the polysulfone, membranes primed the oxidative burst of the phagocytes and caused them to release elastase. DEAE-substituted cellulose had a lesser effect on elastase release than did cellulose and elastase release correlated significantly with the degree of complement activation. However, the correlation between complement activation and priming of phagocyte oxidative burst was weak, suggesting that membranes affect phagocyte oxidative metabolism through more than one mechanism. We conclude that some dialysis membranes stimulate the bacteriacidal functions of normal phagocytic cells, in part through complement-dependent mechanisms.     

Absence of Gal epitope prolongs survival of swine lungs in an ex vivo model of hyperacute rejection

Nguyen B‐NH, Azimzadeh AM, Schroeder C, Buddensick T, Zhang T, Laaris A, Cochrane M, Schuurman H‐J, Sachs DH, Allan JS, Pierson RN. Absence of Gal epitope prolongs survival of swine lungs in an ex vivo model of hyperacute rejection. Xenotransplantation 2011; 18: 94–107. © 2011 John Wiley & Sons A/S. Abstract: Background: Galactosyl transferase gene knock‐out (GalTKO) swine offer a unique tool to evaluate the role of the Gal antigen in xenogenic lung hyperacute rejection. Methods: We perfused GalTKO miniature swine lungs with human blood. Results were compared with those from previous studies using wild‐type and human decay‐accelerating factor‐transgenic (hDAF^+/+) pig lungs. Results: GalTKO lungs survived 132 ± 52 min compared to 10 ± 9 min for wild‐type lungs (P = 0.001) and 45 ± 60 min for hDAF^+/+ lungs (P = 0.18). GalTKO lungs displayed stable physiologic flow and pulmonary vascular resistance (PVR) until shortly before graft demise, similar to autologous perfusion, and unlike wild‐type or hDAF^+/+ lungs. Early (15 and 60 min) complement (C3a) and platelet activation and intrapulmonary platelet deposition were significantly diminished in GalTKO lungs relative to wild‐type or hDAF^+/+ lungs. However, GalTKO lungs adsorbed cytotoxic anti‐non‐Gal antibody and elaborated high levels of thrombin; their demise was associated with increased PVR, capillary congestion, intravascular thrombi and strong CD41 deposition not seen at earlier time points. Conclusions: In summary, GalTKO lungs are substantially protected from injury but, in addition to anti‐non‐Gal antibody and complement, platelet adhesion and non‐physiologic intravascular coagulation contribute to Gal‐independent lung injury mechanisms.     

Successful ex vivo renal artery reconstruction and renal autotransplantation

Background : The increasing experience with renal allotransplantation has led to continuing development in vascular surgical techniques. These improvements have enabled complex ex vivo renal artery surgery and renal autotransplantation to be performed. The aims of the present study were to describe the results achieved with renal autotransplantation and ex vivo renal artery reconstruction (RAR) at the Newcastle Transplant Unit, John Hunter Hospital, and to review the current indications for such surgery. Methods : A retrospective review was performed of patients who required renal autotransplantation with or without RAR at John Hunter Hospital, between 1991 and 1999. Data were obtained from the Newcastle Transplant Unit and the Medical Record Department of John Hunter Hospital. Results : Two patients required ex vivo RAR and renal autotransplantation for severe fibromuscular dysplasia (FMD) complicated by stenoses and renal artery branch aneurysms. The third patient required autotransplantation for bilateral retroperitoneal fibrosis. There was one postoperative complication of pelviureteric junction obstruction that was treated successfully with a temporary ureteric stent. All patients demonstrated normal graft function and were normotensive on follow up, which ranged from 2.5 to 5 years. Conclusion : The present review confirms the long‐term benefits of ex vivo RAR and renal autotransplantation that have been demonstrated by previous studies. In transplant units experienced with this surgery it has been shown to be a successful and durable technique for the treatment of a variety of vascular, urologic and other diseases.     

异种移植免疫排斥的研究进展

异种移植是解决人体器官严重短缺的重要思路.随着对异种移植排斥和人畜共患感染性疾病的深入研究,以及α-1,3-半乳糖苷转移酶基因敲除猪的成功构建,以猪为供体的异种移植与临床应用之间的距离正在逐渐缩短.阻碍异种移植发展的主要障碍仍是免疫排斥反应.本文试就目前异种免疫排斥的研究进展进行综述,希望对未来的临床异种移植研究提供参...     

Acute antibody-mediated rejection in paediatric renal transplant recipients

Acute antibody-mediated rejections (aAMR) after renal transplantation are defined by rapidly deteriorating graft function, detection of donor-specific antibodies (DSA) and characteristic histological features. In adults, anti-rejection strategies comprise intravenous immunoglobulin (IVIG), steroid pulses, plasmapheresis and rituximab. Data of children with aAMR are scarce. We report four episodes of aAMR in three children (aged 10, 10 and 11 years respectively) occurring early after renal transplantation. Pre-transplant complement-dependent cytotoxicity crossmatches were negative; in the case of re-transplantation repeated antigens were excluded. Basic immunosuppression comprised cyclosporine A, MMF and steroids. All four rejection episodes were histologically proven and associated with acute renal failure. De novo DSAs were detected in two aAMRs; one patient was additionally tested positive for AT1-receptor antibodies. All aAMRs were treated with steroid pulses, tacrolimus, MMF, IVIG, plasmapheresis and one single dose of rituximab. Despite therapy one graft was lost; in the remaining three cases kidney function re-established within 1–8 weeks. At follow-up, 14, 15 and 22 months’ post-rejection their GFRs were 65, 88 and 105 ml/min/1.73 m² respectively. A combined therapy of steroid pulses, IVIG, plasmapheresis and rituximab is potentially effective in the treatment of aAMR in children.