移植肾急性排斥时血栓素A2、前列环素代谢改变及其对肾小球滤过率的影响

用放射免疫法检测２１例移植肾急性排斥时，肾移植患者尿液以及血浆血栓素Ｂ２（ＴＸＢ２）和６－酮－前列腺素Ｆ１α（６－ｋｅｔｏ－ＰＧＦ１α）浓度，并检测４例不可逆急性排斥和７例慢性排斥移植肾切除后肾组织ＴＸＢ２和６－ｋｅｔｏ－ＰＧＦ１α含量。发现急性排斥出现时，尿中ＴＸＢ２和６－ｋｅｔｏ－ＰＧＦ１α含量均明显升高，ＴＸＢ２增多出现较早；血浆ＴＸＢ２浓度也显著增加，６－ｋｅｔｏ－ＰＧＦ１α浓度下降。尿液和血浆中ＴＸＢ２／６－ｋｅｔｏ－ＰＧＦ１α比值增大。正常肾组织标本中，肾髓质ＴＸＢ２和６－ｋｅｔｏ－ＰＧＦ１α含量为皮质的４～５倍，皮髓质中ＴＸＢ２／６－ｋｅｔｏ－ＰＧＦ１α比值水平相同。不可逆急性排斥肾组织中，肾皮质ＴＸＢ２／６－ｋｅｔｏ－ＰＧＦ１α比值明显高于髓质。急性排斥时，尿液中ＴＸＢ２和６－ｋｅｔｏ－ＰＧＦ１α比值变化与肾皮质平行。分析表明，急性排斥时，尿液ＴＸＢ２／６－ｋｅｔｏ－ＰＧＦ１α比值增大和移植肾肾小球滤过率负相关。     

移植肾急性排斥时血栓素A2,前列环素代谢改变及其对肾小球？…

用放射免疫法检测２１例移植肾急性排斥时，肾移植患者尿液以及血浆血栓素Ｂ２和６－酮－前列腺素Ｆ１α浓度，并检测４例不可逆急性排斥和７例慢性排斥移植肾切除后肾组织ＴＸＢ２和Ｙ－ｋｅｔｏ－ＰＧＦ１α含量。发现急性排斥出现时，尿中ＴＸＢ２和６－ｋｅｔｏ－ＰＧＦ１α含量均明显升高，ＴＸＢ２增多出现较早；血浆ＴＸＢ２浓度也显著增加，６－ｋｅｔｏｐ－ＰＧＦ１α深度下降。     

猪血管内皮细胞α-Gal对人外周血淋巴作用初探

超急性排斥反应克服后，异种移植物将面临血管性和细胞性排斥。血管内皮是免疫排斥的主要场所，异种移植免疫中血管内皮细胞的作用以及其携带的αＧａｌ在超急性排斥后免疫反应中的作用研究尚少。通过混合淋巴细胞反应（ＭＬＲ），以同种异体ＭＬＲ为对照，对异种（内江猪→人）外周血淋巴细胞（ＰＢＬＣ）混合反应和内江猪血管内皮细胞（ＰＡＥＣ）人ＰＢＬＣ混合反应进行研究，同时考察α半乳糖基酶消化后ＰＡＥＣ对人ＰＢＬＣ的刺激性。结果表明：异种ＭＬＲ增殖较相应的同种异体ＭＬＲ弱，可能与在异种周围淋巴细胞ＭＬＲ中以间接呈递为主有关；ＰＡＥＣ作为刺激细胞的ＭＬＲ增殖极强，可能与ＰＡＥＣ本身是抗原呈递细胞且抗原呈递以直接呈递为主有关；αＧａｌ引起增殖，可能与其在体内数量多、与生物大分子结合可成为全抗原有关，提示该抗原可能也是超急性排斥后排斥反应的靶抗原；去除ＰＡＥＣ表面αＧａｌ后，仍可诱发人Ｔ细胞反应，提示可能还存在其它超急性排斥后抗原。     

高压氧对大鼠急性胰腺炎血浆血栓素A2和前列环素的影响

观察高压氧（ＨＢＯ）实验性治疗后对大鼠急性胰腺炎血浆血栓素Ａ２和前列环素的影响。分析对照组，急性胰谈组（ＡＰ）和急性胰腺炎氧治疗组（ＡＰ＋ＨＢＯ）。采用十二指肠闭裨法制作 急性胰腺炎模型。结果：与对照组比较，急怀胰腺炎模型后３小时和６小时血浆ＴＸＢ２和６－Ｋ－ＰＧＦ１α水平明显升高，出现ＴＸＢ２和６－Ｋ－ＰＧＦ１α比率的变化，ＨＢＯ治疗对急胰腺炎血浆ＴＸＢ２水平的升高有明显的抑制，而对６－Ｋ－ＰＧ     

动脉硬化闭塞症与血管内皮细胞活性因子关系的研究

目的　探讨动脉硬化闭塞症（ＡＳＯ） 与内皮素１（ＥＴ１） 、一氧化氮（ＮＯ） 等血管内皮细胞活性因子的相互关系。方法　选择９６ 例ＡＳＯ 患者，采用密度梯度法检测循环内皮细胞计数（ＣＥＣ），采用放射免疫测定法检测血浆ＥＴ１ 、血栓素Ｂ２（ＴＸＢ２）、６酮前列腺素Ｆ１α（６ＫＰＧＦ１α） ，降钙素基因相关肽（ＣＧＲＰ） 、血管紧张素Ⅱ（ＡＴⅡ）、肿瘤坏死因子（ＴＮＦ），采用酶联免疫检测细胞间粘附分子１（ＩＣＡＭ１）、Ｐ选择素（ＰＳ）。采用Ｇｒｉｓｓ 法检测血浆ＮＯ水平。结果　所有患者均存在高ＥＴ１、ＴＸＢ２ 、ＡＴⅡ、粘附分子、ＣＥＣ血症，存在低ＮＯ、ＣＧＲＰ、６ＫＰＧＦ１α血症，且以Ⅲ期病人最明显。结论　ＡＳＯ 的发生发展同血管内皮细胞活性因子密切相关，ＥＴ１、ＮＯ 等血管活性因子的检测有助于ＡＳＯ发病机制的探讨和病情严重程度的判别。     

川芎嗪改善大鼠胰腺保存的机理

通过测定保存不同时间后未移植胰腺组织内６－酮－前列腺素Ｆ１α及血栓素Ｂ２含量，对川芎嗪改善胰腺保存效果的作用机制作出初步探讨。６－ｋｅｔｏ－ＰＧＦ１α及ＴＸＢ２测定结果显示：加川芎嗪对６－ｋｅｔｏ－ＰＧＦ１α无明显影响，对ＴＸＢ２则能显著性降低，６ｋｅｔｏ－ＰＧＦ１α；ＴＸＢ２的比值在加川芎嗪组最大，认为川芎嗪主要通过抑制ＴＸＡ２合成，升高ＰＧＩ２：ＴＸＡ２的比２值，发挥ＰＧＩ２的保护作用，抑制Ｔ     

猪到人异种移植抗原与移植免疫研究

同种异体组织和器官移植的供体来源有限，使异种移植再度成为研究热点。猪被认为是理想的供体之一。异种移植的主要障碍为异种抗原被人血清中天然存在的抗体结合、激活补体导致的超急排斥反应。现已认识的主要靶抗原为αＧａｌ，它的表达受α１，３半乳糖基转移酶（αＧＴ）的控制。针对αＧａｌ克服超急排斥反应的方法有：免疫吸附去除针对αＧａｌ的天然抗体，酶处理去除内皮细胞（ＰＡＥＣ）表面的αＧａｌ，基因工程获得不表达αＧＴ从而无αＧａｌ的动物。除αＧａｌ以外，还有一些抗原可与人血清抗体结合：如ＴＮＦ诱导ＰＡＥＣ表达的ｇｐ６５和ｇｐ１００，猪组织表达的人血型抗原Ａ，但这些抗原的作用还不清楚。猪ＭＨＣ（即ＳＬＡ）的作用尚有争论，异种移植细胞免疫中，直接途径和间接途径均存在。     

阿魏酸钠对犬心脏停跳复苏后脑组织TXB_2、6-keto-PGF_(1a)及MDA含量的影响

研究了阿魏酸钠对犬心脏停跳１０分钟复苏后４小时脑组织中血栓素Ｂ２（ＴＸＢ２）、６－酮－前列腺素Ｆ１ａ（６－ｋｅｔｏ－ＰＧＦ１ａ）及丙二醛（ＭＤＡ）含量的影响。１７只犬随机分为非缺血对照组（Ａ组）、缺血再灌注常规治疗组（Ｂ组）及缺血再灌注阿魏酸钠治疗组（Ｃ组）。结果发现，Ｂ组ＴＸＢ２、ＭＤＡ含量及ＴＸＢ２／６－ｋｅｔｏ－ＰＧＦ１ａ比值均较 Ａ组明显升高（Ｐ＜０． ０１）。 Ｃ组 ＴＸＢ２、ＭＤＡ含量及 ＴＸＢ２／６－ｋｅｔｏ－ＰＧＦ１ａ比值升高幅度均较Ｂ组明显低（Ｐ＜０．０１）。表明阿魏酸钠可明显抑制犬心脏停跳复苏后脑组织花生四烯酸代谢及脂质过氧化反应。     

硬膜外阻滞下妊高征剖宫产患者血浆TXB2,6—keto—PGF1α的变化

观察硬膜外阻滞对９例中－重度妊高征剖宫产患者血浆血栓素Ａ２（ＴＸＡ２）、前列环素（ＰＧＩ２）的代谢产物－ＴＸＢ２、６－ｋｅｔｏ－ＰＧＦ１α的影响。结果表明，硬膜外阻滞后ＴＸＢ２无显著性改变，６－ｋｅｔｏ－ＰＧＦ１α于切皮前、剖宫前显著升高，ＴＸＢ２／６－ｋｅｔｏ－ＰＧＦ１α比值显著下降。提示硬膜外阻滞对妊高征剖宫产患者是一种安全、有效的麻醉方法，并可能有助于妊高征的治疗。     

内毒素体外诱生人单核细胞分泌PGF1α／TXB2的研究

分离健康人外周血单核细胞，行短期体外培养后，以大肠杆菌内毒素刺激单核细胞，放免法测定细胞培养上清液中前列腺素Ｆ１α血栓素Ｂ２含量。结果表明内毒素体外能诱生人外周血单核细胞合成ＰＧＦ１α／ＴＸＢ２，内毒素刺激量为１０μｇ／ｍｌ时单核细胞合成ＰＧＦ１α／ＴＸＢ２最旺盛，刺激２小时左右合成速率达到高峰，随后下降。     

Production of human monoclonal antibodies against Galalpha(1-3)Gal

The hyperacute rejection observed in models of pig-to-human xenotransplantation is mainly because of the presence of natural antibodies in human blood with specificity for the Galα(1–3)Gal (Gal) carbohydrate moiety present on the surface of porcine endothelial cells. Human monoclonal anti-Gal antibodies could be of use both in the study of the basic mechanisms of hyperacute rejection as well as in its clinical prevention. In the present study we prepared 42 heterohybridomas (human–mouse) secreting antibodies with specificity for the Gal epitope. All of the antibodies produced were of the IgM isotype, according to a dot-blot assay. Twenty-seven antibodies were further characterized, and shown to be specific for Gal by different methods, including an enzyme-linked immunosorbent assay, in which the plates were sensitized with mouse laminin as a source of Gal. Specificity was also confirmed using purified Gal carbohydrate in a hemagglutination inhibition assay. The antibodies were shown to mediate lysis of Gal-expressing rabbit erythrocytes in the presence of complement. However, the heterohybridomas themselves were shown to express Gal, a result of the mouse P3x63Ag8.653 hybridoma cells used during hybridoma generation. The presence of this epitope on the surface of anti-Gal-producing cells, and on the antibody itself, represents a limitation to the production of high affinity anti-Gal antibodies.     

帕瑞昔布钠对急性心肌梗死大鼠心功能的影响

目的 评价帕瑞昔布钠对急性心肌梗死大鼠心功能的影响.方法 成年雄性SD大鼠24只,体重230～250 g,随机分为3组(n=8):假手术组(S组)、急性心肌梗死组(AMI组)和帕瑞昔布钠组(P组).AMI组和P组采用结扎左冠状动脉前降支的方法制备大鼠急性心肌梗死模型,S组冠状动脉穿线但不结扎;24 h后P组腹腔注射帕瑞昔布钠8 mg/kg,1次/d,连续3 d,AMI组用生理盐水替代.术后第4天测定并记录左心室收缩压(LVSP)、左心室舒张末期压(LVEDP)、左心室收缩压最大上升速率(+dp/dtmax)和左心室收缩压最大下降速率(-dp/dtmax);采集颈总动脉血样3 ml,采用放射免疫法测定血浆血栓素A2(TXA2)和前列腺素I2(PGI2)的浓度,并计算PGI2/TXA2;采血后取左心室心肌组织,测定梗死面积,计算心肌梗死体积.结果 与S组比较,AMI组和P组LVSP、±dp/dtmax、血浆PGI2浓度和PGI2/TXA2降低,LVEDP和血浆TXA2浓度升高(P＜0.05).与AMI组比较,P组LVSP、±dp/dtmax、血浆PGI2浓度和PGI2/TXA2升高,LVEDP和血浆TXA2浓度降低(P＜0.05).AMI组和P组心肌梗死体积比较差异无统计学意义(P＞0.05).结论 帕瑞昔布钠可改善急性心肌梗死大鼠左心室功能,其机制与调节PGI2/TXA2相对平衡有关.     

Expression of human soluble complement receptor 1 by a pig endothelial cell line inhibits lysis by human serum

The importance of complement activation and naturally occurring anti-pig antibodies in the hyperacute rejection (HAR) observed in models of pig-to-human xenotransplantation is well established. To overcome this, much effort has been dedicated to preparing transgenic pigs by knocking out Galalpha(1-3)Gal expression in these animals, or knocking in the expression of human complement regulatory proteins (CRPs), such as CD59 or decay accelerating factor. A soluble form of another membrane CRP, complement receptor type 1 (CR1), has also been shown to inhibit complement activation. Here, we show that transfection of a pig endothelial cell line with a truncated form of human soluble complement receptor 1 (sCR1) almost completely protected these cells from complement-mediated lysis by human AB serum. Pigs genetically manipulated to express human sCR1 may represent an additional strategy to inhibit HAR of pig-to-human transplanted organs.     

Modified glycan models of pig-to-human xenotransplantation do not enhance the human-anti-pig T cell response

Genetically modified porcine models of pig-to-human xenotransplantation offer the most immediate answer to a growing shortage of available solid organs. Recently a modified porcine glycan model has been discovered that reduces human antibody binding to levels comparable with allograft standards. As this background provides an answer to the problem of acute humoral xenograft rejection (AHXR), it is important to consider the impact these modifications have on measures of cell-mediated rejection. The objective of this study was to examine the impact of currently relevant glycan knockout models of pig-to-human xenotransplantation in a lymphocyte proliferation assay. To accomplish these goals, genetically modified pigs were created through CRISPR/Cas9-directed silencing of the GGTA1, and CMAH genes. Peripheral blood mononuclear cells (PBMCs) and spleen cells were obtained from these animals and used as a source of stimulation for human responders in one-way mixed lymphocyte reactions. The response was tested in the presence and absence of clinically available immunomodifiers. Conclusions: Clinically relevant glycan knockout models of pig-to-human xenotransplantation do not enhance the human-anti-pig cellular response. Currently available and conventional immunosuppression has the capacity to mediate the human xenogeneic T cell response to these knockout cells.     

Humoral Human Xenoreactivity Against Isolated Pig Pancreatic Islets

It is widely believed that the hyperacute rejection of vascularized xenografts in the pig-to-human combination is triggered by the binding of human preformed natural antibodies (PNAbs) to the Galα.(1,3)Gal epitope in pig endothelium and the subsequent activation of complement. However, it remains poorly defined whether xenogeneic pig pancreatic islets are damaged by antibody and complement-mediated mechanisms. We examined the expression of Galα(1,3)Gal on isolated adult pig islets and the presence of PNAbs in normal human sera directed against islets, using immunofluorescence staining and confocal laser scanning microscopy. The pig islets were not stained with Galα(1,3)Gal-specific lectin GSIB4; however, the exocrine cells reacted strongly with GSIB4, indicating that the Galα(1,3)Gal epitope was highly expressed on exocrine cells, but not on islets. Human sera showed weak reactivity of IgM and IgG class PNAbs to the islets, but strong reactivity to the exocrine cells. Furthermore, we investigated the cytotoxic effect of human serum on pig islets using an in vitro model of pig-to-human islet transplantation. The incubation of pig islets with normal human sera for 45 min resulted in less than 10% specific lysis despite the binding of PNAbs, whereas exposure of porcine aortic endothelial cells to the same human sera caused 56% complement-mediated lysis, determined using a MTT cytotoxic assay. These results support the view that pig islets might not undergo early antibody and complement-mediated rejection in humans. Received: July 8, 1999 / Accepted: May 30, 2000     

A recombinant soluble chimeric complement inhibitor composed of human CD46 and CD55 reduces acute cardiac tissue injury in models of pig-to-human heart transplantation

BACKGROUND: Inasmuch as complement plays a critical role in many pathological processes and in xenograft rejection, efficient complement inhibitors are of great interest. Because the membrane-associated complement inhibitors are very effective, recombinant soluble molecules have been generated. METHODS: We tested the efficacy of complement activation blocker-2 (CAB-2), a recombinant soluble chimeric protein derived from human decay accelerating factor (DAF, CD55) and membrane cofactor protein (MCP, CD46), in two models of pig-to-human xenotransplantation in which tissue injury is complement mediated. The in vitro model consisted of porcine aortic endothelial cells and human serum, and the ex vivo model consisted of a porcine heart perfused with human blood. RESULTS: In vitro, addition of CAB-2 to serum inhibited cytotoxicity and the deposition of C4b and iC3b on the endothelial cells. Ex vivo, addition of CAB-2 to human blood prolonged organ survival from 17.3 +/- 6.4 min in controls to 108 +/- 55.6 min with 910 nM (100 microg/ml) CAB-2 and 219.8 +/- 62.7 min with 1820 nM (200 microg/ml) CAB-2. CAB-2 also retarded the onset of increased coronary vascular resistance. The complement activity of the perfusate was reduced by CAB-2, as was the generation of C3a and SC5b-9. The myocardial tissues had similar deposition of IgG, IgM, and Clq; however, CAB-2 reduced the deposition of C3, C4, and C9. Hearts surviving >240 min demonstrated trace to no deposition of C9 and normal histologic architecture. CONCLUSION: These results indicate that CAB-2 can function as an inhibitor of complement activation and markedly reduce tissue injury in models of pig-to-human xenotransplantation and thus may represent a useful therapeutic agent for xenotransplantation and other complement-mediated conditions.     

Lack of antibody production against Hanganutziu-Deicher (H-D) antigens with N-glycolylneuraminic acid in patients with porcine exposure history

The significance of non-alphagalactosyl antigens remains unclear in pig-to-primate xenotransplantation. Hanganutziu-Deicher (H-D) antigens with terminal N-glycolylneuraminic acid (NeuGc) are widely expressed on endothelial cells of mammalian species, with the exception of humans. As baboons and monkeys also express H-D antigens, a pig-to-non-human primate experimental model cannot resolve the question of whether H-D antigens can elicit a potent humoral response in human recipients. The purpose of this study was to elucidate the clinical significance of H-D antigens by examining the sera from patients who have been previously exposed to porcine tissue. After the digestion of porcine aortic endothelial cells (PAEC) by neuraminidase, NeuGc and N-acetylneuraminic acid (NeuAc) were quantitated by HPLC. IgG and IgM antibody levels against H-D antigens were measured by NeuGc-GM3-coated ELISA plates in the sera of patients who had undergone ex vivo kidney perfusion 1 to 3 weeks and 2 years previously (n=2) or had been injected with fetal porcine islets 2 months previously (n= 10). HPLC determined that 9.7x 10(7) NeuAc and 6.3x 10(7) NeuGc residues per cell were released from PAEC by neuraminidase, while 25.7x 10(7) NeuAc and an undetectable level of NeuGc were released from human aortic endothelial cells (HAEC). No significant elevation of IgG or IgM antibody levels against NeuGc-GM3 was observed in sera from patients with a history of porcine exposure. Considering the active production of antibody against the foreign galactosyl antigens after pig-to-human xenotransplantation, some production of antibodies against the equally foreign H-D antigens would be expected, because large amounts of NeuGc terminated saccharides are present in the pig endothelial cell surface. However, no production of antibodies directed to H-D antigens could be found in patients exposed to porcine tissue. Further studies are warranted to explain why H-D antigens do not elicit a significant antibody production.