The potential of genetically-engineered pigs in providing an alternative source of organs and cells for transplantation

There is a critical shortage of organs, cells, and corneas from deceased human donors worldwide. There are also shortages of human blood for transfusion. A potential solution to all of these problems is the transplantation of organs, cells, and corneas from a readily available animal species, such as the pig, and the transfusion of red blood cells from pigs into humans. However, to achieve these ends, major immunologic and other barriers have to be overcome. Considerable progress has been made in this respect by the genetic modification of pigs to protect their tissues from the primate immune response and to correct several molecular incompatibilities that exist between pig and primate. These have included knockout of genes responsible for the expression of major antigenic targets for primate natural anti-pig antibodies, insertion of human complement- and coagulation-regulatory transgenes, and knockdown of swine leukocyte antigens that stimulate the primate＇s adaptive immune response. As a result of these manipulations, the administration of novel immunosuppressive agents, and other innovations, pig hearts have now functioned in baboons for 6-8 months, pig islets have maintained normoglycemia in diabetic monkeys for 〉 1 year, and pig corneas have maintained transparency for several months. Clinical trials of pig islet trans- plantation are already in progress. Future developments will involve further genetic manipulations of the organ- source pig, with most of the genes that are likely to be beneficial already identified.

Acute antibody-mediated rejection after intestinal transplantation

AIM To investigate the incidence, risk factors and clinical outcomes of acute antibody-mediated rejection(ABMR) after intestinal transplantation(ITx).METHODS A retrospective single-center analysis was performed to identify cases of acute ABMR after ITx, based on the presence of donor-specific antibody(DSA), acute tissue damage, C4 d deposition, and allograft dysfunction.RESULTS Acute ABMR was identified in 18(10.3%) out of 175 intestinal allografts with an average occurrence of 10 d(range, 4-162) after ITx. All acute ABMR cases were presensitized to donor human leukocyte antigens class Ⅰand/or Ⅱ antigens with a detectable DSA. A positive cross-match was seen in 14(77.8%) cases and twelve of 18 patients(66.7%) produced newly-formed DSA following ITx. Histological characteristics of acute ABMR include endothelial C4 d deposits, interstitial hemorrhage, and severe congestion with focal fibrin thrombin in the lamina propria capillaries. Multivariate analysis identified a liver-free graft and high level of panel reactive antibodyas a significant independent risk factor. Despite initial improvement after therapy, eleven recipients(61.1%) lost transplant secondary to rejection. Of those, 9(50%) underwent graft removal and 4(22.2%) received second transplantation following acute ABMR. At an average follow-up of 32.3 mo(range, 13.3-76.4), 8(44.4%) recipients died.CONCLUSION Our results indicate that acute ABMR is an important cause of intestine graft dysfunction, particularly in a liver-exclusive graft and survivors are at an increased risk of developing refractory acute rejection and chronic rejection. More effective strategies to prevent and manage acute ABMR are needed to improve outcomes.

Biliary wound healing, ductular reactions, and IL-6/gp130 signaling in the development of liver disease

Basic and translational wound healing research in the biliary tree lag significantly behind similar studies on the skin and gastrointestinal tract. This is at least partly attributable to lack of easy access to the biliary tract for study. But clinical relevance, more interest in biliary epithelial cell （BEC） pathophysiology, and widespread availability of BEC cultures are factors reversing this trend. In the extra-hepatic biliary tree, ineffectual wound healing, scarring and stricture development are pressing issues. In the smallest intra-hepatic bile ducts either impaired BEC proliferation or an exuberant response can contribute to liver disease. Chronic inflammation and persistent wound healing reactions in large and small bile ducts often lead to liver cancer. General concepts of wound healing as they apply to the biliary tract, importance of cellular processes dependent on IL-6/gp130/STAT3 signaling pathways, unanswered questions, and future directions are discussed.

Role of the IL-33-ST2 axis in sepsis

Sepsis remains a major clinical problem with high morbidity and mortality. As new inflammatory mediators are characterized, it is important to understand their roles in sepsis. Interleukin 33(IL-33) is a recently described member of the IL-1 family that is widely expressed in cells of barrier tissues. Upon tissue damage, IL-33 is released as an alarmin and activates various types of cells of both the innate and adaptive immune system through binding to the ST2/IL-1 receptor accessory protein complex. IL-33 has apparent pleiotropic functions in many disease models, with its actions strongly shaped by the local microenvironment. Recent studies have established a role for the IL-33-ST2 axis in the initiation and perpetuation of inflammation during endotoxemia, but its roles in sepsis appear to be organism and model dependent. In this review, we focus on the recent advances in understanding the role of the IL-33/ST2 axis in sepsis.

A comparison of three methods of decellularization of pig corneas to reduce immunogenicity

·AIM: To investigate whether decellularization using different techniques can reduce immunogenicity of the cornea, and to explore the decellularized cornea as a scaffold for cultured corneal endothelial cells(CECs).Transplantation of decellularized porcine corneas increases graft transparency and survival for longer periods compared with fresh grafts.·METHODS: Six-month-old wild-type pig corneas were cut into 100-200 μm thickness, and then decellularized by three different methods: 1) 0.1% sodium dodecyl sulfate(SDS); 2) hypoxic nitrogen(N2); and 3) hypertonic NaCl. Thickness and transparency were assessed visually. Fresh and decellularized corneas were stained with hematoxylin/eosin(H&E), and for the presence of galactose-α1,3-galactose(Gal) and N-glycolylneuraminic acid(NeuGc, a nonGal antigen). Also, a human IgM/IgG binding assay was performed. Cultured porcine CECs were seeded on the surface of the decellularized cornea and examined after H&E staining.· RESULTS: All three methods of decellularization reduced the number of keratocytes in the stromal tissue by 】80% while the collagen structure remained preserved. No remaining nuclei stained positive for Gal or NeuGc, and expression of these oligosaccharides on collagen was also greatly decreased compared to expression on fresh corneas. Human IgM/IgG binding to decellularized corneal tissue was considerably reduced compared to fresh corneal tissue. The cultured CECs formed a confluent monolayer on the surface of decellularized tissue.· CONCLUSION: Though incomplete, the significant reduction in the cellular component of the decellularized cornea should be associated with a significantly reduced in vivo immune response compared to fresh corneas.

Surgical closure of large splenorenal shunt may accelerate recovery from hepato-pulmonary syndrome in liver transplant patients

Dear editor,Hepatopulmonary syndrome(HPS)is not uncommon in the setting of liver disease,especially in liver cirrhosis patients.The prevalence of HPS in liver cirrhosis patients varies from 4%to 47%.[1-3]About the definition of HPS,it is a pulmonary vascular disorder with evidence of intrapulmonary arterial venous shunt.[4]Pulmonary dyspnea and polycythemia are common presentations of HPS.Dyspnea,cyanosis and clubbed fingers were present in most of all cases.Spider nevi is another common clinical feature of patients with HPS.

Experiences relating to management of biliary tract complications following liver transplantation in 96 cases

OBJECTIVE: To investigate best diagnosing methods and therapy for patients with biliary tract complications after liver transplantation and analyze related factors. METHODS: A review was made of data collected from 96 patients, and confirmed by retrospective case notes examination. RESULTS: A total of 94 patients (97 grafts) survived more than 2 days after transplantation; of whom, 92 had an end-to-end biliary anastomosis with a T tube. The average follow-up was 5.8 months (range: 0.3 - 10.2 months). Among the 94 patients, eight (8.5%, 8/94) had complications: leakage during T-tube removal (2 patients), leakage at an earlier stage (2), simultaneous stricture and leak (2) and just stricture (2). Six patients with biliary tract complications had predisposing factors including hepatic artery stenosis (2 patients, including one hepatic artery stenosis combined with severe rejection, hepatic artery thrombosis (3), and donor-recipient bile duct mismatch (1). There was no difference in cold ischemic time. With hepatic artery thrombosis and/or stenosis > 50%, five patients were re-transplanted; without hepatic artery thrombosis and/or stenosis 50%, re-transplantation is needed as early as possible.

肝移植供肝获取的现代方法与实践

目的 :介绍目前美国供肝切取的现代方法。方法 :3 0例供体 ,17例 ( 5 6.7% )因脑血管意外 ,13例 ( 4 3 .3 % )因外伤致脑死亡。 19例血流动力学相对稳定 ,11例 ( 3 6.7% )需用 2～ 3种升压药维持血压。先探测有无变异供肝动脉 ,游离肝周韧带及切断胆总管后 ,自腹主动脉插管灌注UW液 2 0 0 0ml。 3 0例中 ,2 8例切取供肝后再行门静脉插管灌注UW液 10 0 0ml;2例于术中行腹主动脉、门静脉双插管 ,分别灌注UW液 2 0 0 0ml、10 0 0ml。修整供肝时注意变异的供肝动脉 ,并予以重建。结果 :3 0例供肝中 ,2 8例用于受体 ,其中 1例移植后肝原发性无功能、1例移植后肝功能不良 ,均行两次肝移植 ,1例于移植后 18h大出血死亡 ;余 2 5例移植后如期恢复。结论 :美国获取供肝的条件趋于放宽 ,供肝切取的方法趋于快速 ,简捷。

肝移植术肝动脉栓塞的原因及血管重建的方式

目的分析肝移植术中和术后肝动脉栓塞(hepatic artery thrombosis,HAT)的原因,并探讨不同血管重建方式的效果. 方法回顾性总结Pittssburgh大学器官移植中心和南京大学附属鼓楼医院共21例肝移植术中、术后HAT,其中12例(57.1%) 施行了肝动脉重建术(hepatic artery reconstruction, HAR).HAR方法有直接吻合(4例)、供体肝动脉-受体腹主动脉架桥术(aortohepatic interposition graft,AHIG)(6例)、髂动脉架桥术(interposition graft,IG)(2例). 结果肝动脉血流重建成功率为58.3%(7/12);其余5例平均3.8(3～6)d内再次发生HAT.成人HAR成功率为62.5%(5/8),儿童为50.0%(2/4).HAR结果与HAT发生时间、 HAT原因和HAR方式无明显相关性 (P>0.05). 结论肝移植术HAT可能原因有机械因素、受体肝动脉细小、供体肝动脉感染、供体DIC等,尽量减少或避免上述危险因素是最好的预防方法.肝移植术HAT时HAR方式包括直接吻合、AHIG、IG等,成功取决于能否及时获得诊断.有趋势表明机械因素所致HAT,HAR方式采用直接吻合和AHIG有较好的临床结果.

晚期移植肝功能不全的原因

总结复习晚期移植肝功能不全各方面的相关文献 ,综合分析肝移植术后晚期移植肝功能不全的原因。结果显示晚期移植肝功能不全是比较常见的远期并发症 ,其病因主要包括有排异、胆道或血管并发症、原发病的复发、感染、以及其他原因等。随着肝移植存活率的不断提高 ,晚期移植肝功能不全将日益受到重视。

转基因猪对异种移植的影响研究

对于器官衰竭的患者来说,器官移植是最好的治疗手段。尽管在过去的50年做了很大的努力,但这个问题仍未完全解决,事实上还有很多新的问题。纵观20世纪,有人尝试用动物器官作为临床移植器官的来源[1-2]。这些动物一般为非人类灵长动物（nonhuman primates,NHPs）。通过一些使用NHPs作为供者的实验发现,

Experiences relating to management of biliary tract complications following liver transplantation in 96 cases

To investigate best diagnosing methods and therapy for patients with biliary tract complications after liver transplantation and analyze related factors Methods A review was made of data collected from 96 patients, and confirmed by retrospective case notes examination Results A total of 94 patients (97 grafts) survived more than 2 days after transplantation; of whom, 92 had an end to end biliary anastomosis with a T tube The average follow up was 5 8 months (range: 0 3-10 2 months) Among the 94 patients, eight (8 5%, 8/94) had complications: leakage during T tube removal (2 patients), leakage at an earlier stage (2), simultaneous stricture and leak (2) and just stricture (2) Six patients with biliary tract complications had predisposing factors including hepatic artery stenosis (2 patients, including one hepatic artery stenosis combined with severe rejection, hepatic artery thrombosis (3), and donor recipient bile duct mismatch (1) There was no difference in cold ischemic time With hepatic artery thrombosis and/or stenosis 】50%, five patients were re transplanted; without hepatic artery thrombosis and/or stenosis 【50%, three patients required endoscopic stenting and radiological percutaneous drainage of bile collection with or without balloon dilation All patients survived Conclusions Biliary strictures occur later than leaks after surgery Without hepatic artery thrombosis and/or stricture, there is no need for surgery; with hepatic artery thrombosis and/or stricture 】50%, re transplantation is needed as early as possible

Liver-inclusive intestinal transplantation results in decreased alloimmune-mediated rejection but increased infection

Background and aims:A co-transplanted liver allograft has been thought to protect other organs from rejection-mediated injury;however,detailed analyses of co-transplanted liver on intestinal allograft outcomes have not been conducted to date.The aimof the study was to compare immune-mediated injury,causes of graft failure and clinical outcomes between recipients who underwent either a liver-inclusive intestinal transplant(LITx)or liver-exclusive intestinal transplant(LETx).Methods:Between May 2000 and May 2010,212 adult patients undergoing LITx(n¼76)and LETx(n¼136)were included.LITx underwent either liver combined intestinal or full multivisceral transplantation.LETx underwent either isolated intestinal or modified multivisceral transplantation.Results:During 44.9631.4 months of follow-up,death-censored intestinal graft survival was significantly higher for LITx than LETx(96.9%,93.2%and 89.9%vs 91.4%,69.3%and 60.0%at 1,3 and 5 years;p¼0.0001).Incidence of graft loss due to rejection was higher in LETx than in LITx(30.9%vs 6.6%;p<0.0001),while infection was the leading cause of graft loss due to patient death in LITx(25.0%vs 5.1%;p<0.0001).Despite similar immunosuppression,the average number(0.87 vs 1.42,p¼0.02)and severity of acute cellular rejection episode(severe grade:7.9%vs 21.3%;p¼0.01)were lower in LITx than in LETx.Incidence of acute antibody-mediated rejection was also significantly lower in LITx than in LETx(3.6%vs 15.2%;p¼0.03).Incidence of chronic rejection was reduced in LITx(3.9%vs 24.3%;p¼0.0002).Conclusions:Intestinal allografts with a liver component appear to decrease risk of rejection but increase risk of infection.Our findings emphasize that LITx has characteristic immunologic and clinical features.Lower immunosuppression may need to be considered for patients who undergo LITx to attenuate increased risk of infection.

Vitamin B-reath easier:vitamin B6 derivatives reduce IL-33 to limit lung inflammation

It has been known for some time that low PLP blood levels are common in asthmatic patients,and supplementation with vitamin B6 may reduce the severity of asthma symptoms[12].In the current study,a comparison of PLP levels in the plasma of asthmatic patients(n=52)and healthy controls(n=58)confirmed lower PLP concentrations in asthmatic patients,which correlated with reduced lung function and increased circulating eosinophils,suggestive of increased type 2 inflammation.Using several mouse models of acute lung inflammation,they confirmed that systemic or local administration of PLP reduced lung inflammation and eosinophil density,suggesting that PLP concentration may be directly controlling immune responses that lead to the development of allergic airway disease.ILC2s constitutively express ST2,and IL-33 profoundly promotes ILC2 expansion and secretion of IL-5 and IL-13 to mediate allergic reactions and support host defenses against parasitic worms.PLP reduces the number of type 2 innate lymphoid cells(ILC2s)and their expression of IL-5 and IL-13.Conversely,diet-induced vitamin B6 deficiency in mice increased papain-induced lung inflammation,including increasing the proportion of IL5+and IL-13+ILC2s.The authors revealed that PLP treatment decreased IL-33 levels in the bronchoalveolar lavage fluid(BALF)and lung and targeting IL-33 with an antibody during papain-induced lung inflammation did not reduce inflammation beyond that of PLP treatment alone.An important clue to the regulation of IL-33 was that PLP treatment did not modify IL-33 mRNA levels.Instead,pyridoxal(PL)treatment of a human alveolar basal epithelial cell line(A549)stably expressing full-length IL-33 with a hemagglutinin(HA)tag established that PL exposure potently decreased intracellular IL-33 protein levels.The researchers used this cell line to explore the underlying mechanisms and verified that PL conversion to PLP by pyridoxal kinase(PDXK)controlled the stability of IL-33.Consistent with these data,the IL-33 concentration and papain-induced lung inflammation were augmented significantly in PDXK-deficient mice.The author’s use of degradative pathway inhibitors and truncated IL-33 constructs pinpointed a mechanism involving protective ubiquitylation of the IL-33 N-terminal domain that is inhibited by vitamin B6.A comprehensive database search for proteome-wide known and predicted ubiquitin ligase/deubiquitinase-substrate interactions showed that mouse double minute 2 homolog(MDM2)was a likely E3 ubiquitin ligase interacting with IL-33.Recent studies have shown that MDM2,while best known for its regulation of p53,mediates the ubiquitination and stability of numerous nuclear proteins,including Foxp3,HDACs,and STATs[13,14,15].In the current work,MDM2 interacted with IL-33 via a RING domain to facilitate IL-33 stability via ubiquitination of lysines to control IL-33 homeostasis,and this ubiquitination could be inhibited by vitamin B6(Fig.2).It will be important for future studies to establish how precisely PLP suppresses the functional interactions between IL-33 and MDM2.

Carbamylated erythropoietin regulates immune responses and promotes long-term kidney allograft survival through activation of PI3K/AKT signaling

Modulation of alloimmune responses is critical to improving transplant outcome and promoting long-term graft survival.To determine mechanisms by which a nonhematopoietic erythropoietin(EPO)derivative,carbamylated EPO(CEPO),regulates innate and adaptive immune cells and affects renal allograft survival,we utilized a rat model of fully MHC-mismatched kidney transplantation.CEPO administration markedly extended the survival time of kidney allografts compared with the transplant alone control group.This therapeutic effect was inhibited when the recipients were given LY294002,a selective inhibitor of the phosphoinositide 3-kinase(PI3K)/protein kinase B(AKT)signaling pathway or anti-EPO receptor(EPOR)antibody,in addition to CEPO.In vitro,CEPO inhibited the differentiation and function of dendritic cells and modulated their production of proinflammatory and anti-inflammatory cytokines,along with activating the PI3K/AKT signaling pathway and increasing EPOR mRNA and protein expression by these innate immune cells.Moreover,after CD4^(+)T cells were exposed to CEPO the Th1/Th2 ratio decreased and the regulatory T cell(Treg)/Th17 ratio increased.These effects were abolished by LY294002 or anti-EPOR antibody,suggesting that CEPO regulates immune responses and promotes kidney allograft survival by activating the PI3K/AKT signaling pathway in an EPOR-dependent manner.The immunomodulatory and specific signaling pathway effects of CEPO identified in this study suggest a potential therapeutic approach to promoting kidney transplant survival.

不同比例缩小体积肝移植的大动物研究

目的研究不同比例缩小体积肝移植的结果,确定猪能耐受的最小体积的肝移植。方法远系繁殖猪70头分为原位全肝移植作为对照组和3组不同比例缩小体积原位肝移植(按照缩小体积的移植肝占受体切除肝脏重量的百分比;1组:60%;2组:30%;3组:20%);实验采用原位经典肝移植方式(静脉转流)。术后第3天和第5天取肝标本。结果对照组和3组不同比例缩小体积肝移植的移植物与受体肝脏重量百分比(GIWRW)分别为87·4%±8·3%、59·9%±5·2%、33·6%±4·9%和22·1%±3·4%;移植物与受体体重百分比(GIWBW)分别2·4%±0·4%、1·43%±0·17%、0·81%±0·09%和0·53%±0·06%。对照组、1组和2组存活率达100%;3组存活率仅为53%。4个组的动物处死后肝脏移植物重量均有显著的增加。结论安全的缩小体积肝移植,应以移植物与受体肝脏重量百分比不小于33%,同时移植肝与受体重量百分比不小于0·8%为限。

血小板与异种移植术后凝血功能紊乱的关系

器官短缺是目前阻碍器官移植发展的主要因素,异种移植却可以提供无限的、可供选择的器官、组织,从而克服这个困难。随着α-1,3-半乳糖苷转移酶基因敲除猪(α-1,3-galactosyltransferase gene-knockout,GTKO)与其他转基因猪[组织因子途径抑制因子(tissue factor pathway inhibitor,TFPI)、内皮细胞C蛋白受体(endothelial protein C receptor,EPCR)、CD39等]的成功构建和免疫抑制药物的发展,阻碍异种移植发展的主要因素不再是传统观念上的超急性免疫排斥反应,而是凝血功能紊乱导致的血栓性微血管病(thrombotic microangiopathy,TMA)和消耗性凝血病(consumptive coagulopathy,CC)。虽然,异种移植术后凝血功能紊乱的准确机制尚未研究清楚,但血小板在其中的作用是不容忽视的。因此本文对血小板在异种移植术后凝血功能紊乱中的作用及几种抑制血小板激活的途径进行综述,对未来的临床异种移植研究提供参考。