异种移植血管内皮细胞组织特异性表达人CD59转基因小鼠的建立

目的建立用于异种移植研究血管内皮细胞组织特异性表达人CD59转基因小鼠。方法采用受精卵显微注射技术，将含有人ICAM-2启动子、人CD59基因第1个内含子、人CD59 cDNA、BGH polyA终止信号的外源基因导入小鼠受精卵的原核中。选取注射后仍健康的受精卵移植入假孕母鼠的输卵管中待分娩。聚合酶链反应（PCR）及Southern blot确定外源基因整合阳性转基因小鼠。流式细胞术用于外源基因蛋白质水平表达检测。免疫组织化学方法观察人CD59在转基因小鼠心脏、肝脏、肾脏等器官表达分布情况。结果产仔130只，9只外源基因整合阳性。整合率6％（9／130）。6只获得蛋白质水平表达。蛋白质水平表达强度为人CD59在人白细胞表达强度的80％至95％，转基因效率5％（6／130）。免疫组织化学检测显示人CD59在转基因小鼠心脏、肝脏、肾脏组织有较强表达，且表达限于血管内皮细胞。结论成功地建立了用于异种移植研究血管内皮细胞组织特异性表达人CD59转基因小鼠。     

Characterization of human CD55 and CD59 transgenic pigs and kidney xenotransplantation in the pig-to-baboon combination

New transgenic pigs expressing combinations of regulators of complement activation and other molecules are needed to resist xenograft hyperacute rejection (HAR) and to further analyze and treat xenograft rejection. Double transgenic pigs for human CD55 (hCD55) and human CD59 (hCD59) using the promoter of the human elongation factor 1 alpha gene were generated, and their kidneys were transplanted into nonimmunosuppressed baboons. hCD55 and hCD59 were mainly expressed by the endothelial cells, and these cells showed increased resistance to complement-mediated lysis. Baboons receiving kidneys from hCD55hCD59 pigs survived for 5 and 6 days, and displayed alterations in coagulation. Thrombocytopenia and platelet microthrombi were present within the kidneys. Nontransgenic kidneys showed HAR in less than 2 days. Kidneys from pigs expressing hCD55hCD59 displayed protection against HAR in the absence of immunosuppression. Rejection was associated with coagulopathy leukocyte infiltration and a rebound of anti-alpha Gal antibodies.     

转人CRP基因在异种移植中的研究

目的：研究转入补体调节蛋白（CRP）DAF、MCP和CD59基因对抑制人补体激活从而克服超急性排斥反应的作用。方法：利用显微注射建立转人衰变加速因子（hDAF)小鼠和猪的模型和转梁hMCP及hCD59真核表达质粒的猪内皮细胞（EC）,研究小鼠和猪EC表达抑制人补体激活的人补体调节蛋白（CRP）对异种移植超急性排斥反应的抑制作用。结果（1）转人DAF基因小鼠心脏用新鲜人血连续丛外灌注,转基因组心脏搏动时间（174．6min)比对照组（106．5min)明显延延长。（2）转hDAF基因猪心脏异位移植给猕猴、移植心最长存活90h,受者死亡前移植心仍有功能,移植心病理检查未见超急性排斥反应病理改变。（3）转DAF基因基因猪EC死亡率在不同浓度血清时均明显低于对照组,在转hDAF基因猪EC上再分别转染hMCP及hCD59真核表达质粒,转hDAF hMCP或hDAF hCD59在不同血清浓度时EC死亡率较单纯hDAF组明显下降（P＜0．05）。结论,转人DAF及MCP、CD59补体调节蛋白基因能克服人对异种器官或组织的超急性排斥反应。     

Expression of 17-1A antigen and complement resistance factors CD55 and CD59 on liver metastasis in colorectal cancer

Despite radical surgery, the prognosis for colorectal cancer patients with liver metastases has not changed markedly. Furthermore, no standard adjuvant therapeutic regimen has been developed. Adjuvant therapy with monoclonal antibodies (e.g., against 17-l A), which has been shown to be effective in preventing metastatic relapse in patients with Dukes’ C colorectal cancer, might be a promising approach for these patients. However, the cytotoxic effects of monoclonal antibodies can be blocked by coexpression of complement resistance factors that inhibit antibody-dependent complement-mediated cytotoxicity. We therefore analyzed immunohistochemically the expression of 17-1A and the membrane-bound complement resistance factors CD55 and CDS9 on metastatic tumor cells in the livers of 71 patients with colorectal carcinoma who had undergone resection of their metastases with curative intent. In 67 (94%) of 71 patients, liver metastases with homogeneous expression of 17-IA was seen. Heterogeneous expression of 17-1A was seen in four patients (6%). Heterogeneous expression of CD55 or CD59 was observed in 8 (11%) of 71 patients and 4 (6%) of 71 patients, respectively. None of the patients showed homogeneous expression of either CD55 or CD59. All patients with CD55 or CD59 expression showed homogeneous 17-1A expression, whereas none of the four patients with heterogeneous 17-1A expression was positive for CD55 or CD59. Our data indicate that 17-IA is widely expressed on liver metastases of patients with colorectal carcinoma. Therefore patients with completely resected liver metastases might be suitable candidates for adjuvant therapy with anti-l7-1A antibody since only a few of these lesions showed coexpression of complement resistance factors. Supported by grants from the Hamburger Krebsgesellschaft and Otto-Stiftung Hamburg, Germany.     

The effect of antibiotics on cell-mediated immunity.

Normal human lymphocytes in culture were stimulated by the addition of phytohemagglutinin, and deoxyribonucleic acid (DNA) synthesis was measured by the incorporation of tritiated thymidine. The effect of 11 commonly used antibiotics on DNA synthesis then was measured by adding each antibiotic to the culture in concentrations ranging from 2 to 64 microng/ml, a range which covers the plasma levels obtained during customary clinical therapy. Severe dose-dependent suppression of DNA synthesis was found in the presence of two preparations of minocycline, oxytetracycline, and the ascorbic acid salt of tetracycline. Less severe but stil significant suppression was found in the presence of chloramphenicol, clindamycin, tetracycline, and ascorbic acid alone. No effect was noted when penicillin, carbenicillin, or cephalothin was added, and slight stimulation was found in the presence of gentamicin. It is postulated that these findings may help to explain fungal and viral superinfection following antibiotic therapy and that they may play a role in the failure of antibiotic therapy to eliminate some infections.     

Prevention of hyperacute rejection in a model of orthotopic liver xenotransplantation from pig to baboon using polytransgenic pig livers (CD55, CD59, and H-transferase)

INTRODUCTION: The search for alternative sources for transplant organs leads us to the search for animals as an inexhaustible source of organs. The objective of this study was to analyze whether livers from polytransgenic pigs expressing the human complement regulatory proteins CD55 (hDAF), CD59, and alfa alpha1,2-fucosyltransferase (H-transferase), protected against hyperacute rejection after orthotopic liver xenotransplantation to a baboon and also to study pig liver function in a nonhuman primate. MATERIALS AND METHODS: Nine liver transplants from pig to baboon were divided into two groups: a control group (n = 4) of genetically unmodified pigs and an experimental group (n = 5) of pigs transgenic for CD55, CD59, and H-transferase as donors. All the donating piglets obtained through hysterectomy were maintained in specific pathogen-free conditions. The selection of transgenic pig donors followed demonstration of transgene expression using monoclonal antibodies (antiCD55, antiCD59) and immunohistological studies on liver biopsies. RESULTS: All animals in the control group developed hyperacute rejection with survival rates less than 16 hours without function of transplanted livers. In the experimental group none of the animals suffered hyperacute rejection. Survival in this group was between 13 and 24 hours. The livers were functional, producing bile and maintaining above 35% prothrombin activity. Only in one case was there primary dysfunction of the xenograft. CONCLUSION: Polytransgenic livers for complement regulatory proteins prevent hyperacute rejection when xenotransplanted into a baboon.     

Cytoprotective effect of prostaglandin I2 on ischemia-induced hepatic cell injury

The protective effect of prostaglandin I2 (PGI2) on ischemia-induced liver cell injury was investigated during 60-min, 75-min, and 90-min liver ischemia. Vehicle-treated rats tolerated the 75-min hepatic ischemia poorly. Only 25% of the rats in this group survived more than 7 days. However, the survival rate of PGI2-treated rats (350 ng/kg/min) significantly improved to 67%. Liver cell organelles were well-preserved by the PGI2 treatment. Adenosine triphosphate levels in the liver of the PGI2-treated rats were significantly higher than those of vehicle-treated rats at 60 min of reoxygenation following 75-min ischemia. Cyclic 3'-5' adenosine monophosphate levels markedly increased during 60-min PGI2 infusion. Cyclic 3'-5' guanosine monophosphate levels also significantly increased during the PGI2 infusion and were still higher than those of vehicle-treated rats at the end of the 75-min ischemia. Although the exact cytoprotective mechanism of PGI2 at the cellular level is still unclear, our results demonstrate that elevated ATP and cyclic nucleotides levels play an important role in liver cell preservation during ischemia.     

Cytoprotective effect of pentagastrin and epidermal growth factor on stress ulcer formation. Possible role of somatostatin.

This study was designed to test the effects of pentagastrin and epidermal growth factor (EGF) on stress-induced ulceration and on the antral content of gastrin and somatostatin (SLI) in rats. Four groups of 14 to 15 rats had been prepared for 7 days by one of the following methods: saline injection (control); injection of pentagastrin (250 micrograms/kg, 3 times/day); injection of EGF (10 micrograms/kg, 3 times/day); or injection of EGF plus pentagastrin. At the end of the treatment period, half of each group of rats were sacrificed (nonstress group). There were no ulcers in the nonstress control groups of rats. Stress was applied by water immersion in the remaining half of the rats. The injections of pentagastrin and/or EGF resulted in substantial increase in antral content of SLI. After 20 hours of stress, the ulcer index was 40.5 +/- 3.3 in the controls, compared to 6.4 +/- 1.2 and 16.2 +/- 2.3 in rats that received pentagastrin or EGF, respectively. Injections of both pentagastrin and EGF resulted in an ulcer index of 26.2 +/- 2.0, which was significantly lower than that in controls, but higher than that in rats treated with either peptide alone. The stress resulted in significant decrease in antral SLI in all groups of rats, whereas SLI content in rats treated with pentagastrin and/or EGF remained significantly higher than that of controls. Antral content of gastrin did not differ significantly in the four groups tested. The ulcer index was inversely correlated with antral SLI content. We confirm and extend previous observations that pentagastrin and EGF prevent stress ulcer formation, and suggest that endogenous SLI may account, at least in part, for their antiulcer activity.     

血红素加氧酶-1在异种移植适应现象中的研究进展

血红素加氧酶（heine oxygenase,HO）是血红素降解过程中的限速酶,HO-1在体内外实验已被证实具有抗炎症、抗凋亡、调节细胞分化作用,对器官移植的缺血再灌注损伤具有保护作用。适应（accommodation）是指受体体内存在抗供体的抗体,但移植物仍然可以被接受的现象,其发生机制目前还不清楚。新近报道在适应的异种移植动物模型中出现HO-1的高表达。HO-1高表达是否与异种移植免疫保护存在关联,是否参与适应机制的产生,目前还没有定论。HO-1是否通过其反应的下游产物产生保护作用,其机制尚不明了。目前,如何将异种移植物引入适应状态是异种移植研究的热点之一。理解HO-1与异种移植,尤其是与适应有关的异种移植之间的关系具有重要的临床应用前景。     

High-level co-expression of complement regulators on vascular endothelium in transgenic mice: CD55 and CD59 provide greater protection from human complement-mediated injury than CD59 alone

Abstract: High-level endothelial expression of the human complement regulatory factor CD59 has been shown to protect transgenic mouse hearts from human complement-mediated injury in an ex vivo perfusion model. In this study we examine whether co-expression of CD55 provides additional protection. CD55/CD59 double-transgenic mice were generated by co-injection of CD55 and CD59 expression constructs driven by the human intercellular adhesion molecule 2 (ICAM-2) promoter. A line was established from one mouse that exhibited strong expression of CD55 and CD59 on vascular endothelium in the heart and other transplantable organs. An ex vivo perfusion model was used to compare hearts from these CD55/CD59 mice with hearts from a previously established line, which expressed CD59 at a similar level to the double transgenic line. CD59 hearts displayed prolonged survival compared to wild-type hearts during perfusion with 40% human plasma and maintained approximately 20% maximum work after 60 min. CD55/CD59 hearts were further protected, with work maintained at 35% of the maximum level after 60 min. The data demonstrate that high-level endothelial co-expression of CD55 and CD59 provides greater protection from human complement-mediated injury in this model than expression of CD59 alone.     

The effect of intravenous hyperalimentation on cell mediated immunity.

The effect of intravenous hyperalimentation (IVH) on cell mediated immunity was examined in 22 patients. Each patient received PHA and PPD skin tests before and after the performance of IVH. In this study both PHA and PPD skin reactivity showed significant increase after IVH, and serum albumin levels had positive correlation with the PPD skin reaction changes. Absence of the established delayed hypersensitivity in the surgical patient, especially those with malignant diseases, is probably secondary to generalized malnutrition, and established cell mediated immunity can be restored by proper nutritional repletion.     

The effect of BK polyomavirus large T antigen on CD4 and CD8 T cells in kidney transplant recipients

Human BK polyomavirus (BKPyV) can affect the machinery of the host cell to induce optimal viral replication or transform them into tumor cells. Reactivation of BKPyV happens due to immunosuppression therapies following renal transplantation which might result in BK polyomavirus nephropathy (BKPyVAN) and allograft loss. The first protein that expresses after entering into host cells and has an important role in pathogenicity is the Large T antigen (LT-Ag). In this review tries to study the molecular and cellular inter-regulatory counteractions especially between CD4 and CD8 T cells, and BKPyV LT-Ag may have role in nephropathy after renal transplantation.     

Cytoprotective effect of trimetazidine on 60 minutes of intestinal ischemia-reperfusion injury in rats

Trimetazidine (TMZ), a potent antioxidant agent, has been used to protect the myocardium, liver and kidney from ischemia reperfusion (IR) injury. We investigated the effect of TMZ, a cellular anti-ischemic agent and a free radical scavenger, on 60 min of warm intestinal IR injury in rats. Sprague-Dawley rats were divided into three groups: a sham-operated group (no IR injury, n = 8), an ischemic control group (control, n = 8), and a TMZ-treated group (3 mg/kg, n = 8). Malondialdehyde (MDA) levels, myeloperoxidase (MPO) activity, and mucosal damage were investigated after 120 min of reperfusion. MDA levels and MPO activity were more elevated and histopathological damage more severe in the control group than in the sham group (P < 0.05). MDA levels and MPO activity were lower and there was less histopathological damage in the TMZ group than in the control group (P < 0.05). Accumulation of lipid peroxidation products and neutrophils in mucosal tissues were significantly inhibited by TMZ treatment. We conclude that pretreatment of rats with TMZ before intestinal ischemia attenuates but does not prevent, histological damage. Received: 7 May 1998 Received after revision: 25 September 1998 Accepted: 12 October 1998