Complete absence of the αGal xenoantigen and isoglobotrihexosylceramide in α1,3galactosyltransferase knock-out pigs

Puga Yung GL, Li Y, Borsig L, Millard A‐L, Karpova MB, Zhou D, Seebach JD. Complete absence of the αGal xenoantigen and isoglobotrihexosylceramide in α1,3galactosyltransferase knock‐out pigs. Xenotransplantation 2012; 19: 196–206. © 2012 John Wiley & Sons A/S. Abstract: Background: Anti‐Galα1,3Galβ‐R natural antibodies are responsible for hyperacute rejection in pig‐to‐primate xenotransplantation. Although the generation of pigs lacking the α1,3galactosyltransferase (GalT) has overcome hyperacute rejection, antibody‐mediated rejection is still a problem. It is possible that other enzymes synthesize antigens similar to Galα1,3Gal epitopes that are recognized by xenoreactive antibodies. The glycosphingolipid isoglobotrihexosylceramide (iGb₃) represents such a candidate expressing an alternative Galα1,3Gal epitope. The present work determined whether the terminal Galα1,3Gal disaccharide is completely absent in Immerge pigs lacking the GalT using several different highly sensitive methods. Methods: The expression of Galα1,3Gal was evaluated using a panel of antibodies and lectins by flow cytometry and fluorescent microscopy; GalT activity was detected by an enzymatic assay; and ion trap mass spectroscopy of neutral cellular membranes extracted from aortic endothelial was used for the detection of sugar structures. Finally, the presence of iGb₃ synthase mRNA was tested by RT‐PCR in pig thymus, spleen, lymph node, kidney, lung, and liver tissue samples. Results: Aortic endothelial cells derived from GalT knockout pigs expressed neither Galα1,3Gal nor iGb₃ on their surface, and GalT enzymatic activity was also absent. Lectin staining showed an increase in the blood group H‐type sugar structures present in GalT knockout cells as compared to wild‐type pig aortic endothelial cells (PAEC). Mass spectroscopic analysis did not reveal Galα1,3Gal in membranes of GalT knockout PAEC; iGb₃ was also totally absent, whereas a fucosylated form of iGb₃ was detected at low levels in both pig aortic endothelial cell extracts. Isoglobotrihexosylceramide 3 synthase mRNA was expressed in all pig tissues tested whether derived from wild‐type or GalT knockout animals. Conclusions: These results confirm unequivocally the absence of terminal Galα1,3Gal disaccharides in GalT knockout endothelial cells. Future work will have to focus on other mechanisms responsible for xenograft rejection, in particular non‐Galα1,3Gal antibodies and cellular responses.     

Long-term effects of PERV-specific RNA interference in transgenic pigs

Semaan M, Kaulitz D, Petersen B, Niemann H, Denner J. Long‐term effects of PERV‐specific RNA interference in transgenic pigs. Xenotransplantation 2012; 19: 112–121. © 2012 John Wiley & Sons A/S. Abstract: Background: Porcine endogenous retroviruses (PERVs) represent a risk of xenotransplantation using porcine cells, tissues, or organs, as they are integrated in the porcine genome and have been shown to be able to infect human cells in vitro. To increase viral safety by RNA interference, transgenic pigs expressing a PERV‐specific small hairpin (sh)RNA targeted to a highly conserved sequence in the pol gene (pol2) were generated in which expression of PERVs was reduced (Xenotransplantation, 15, 2008, 38). However, it remains to be shown how long expression of the shRNA and the RNA interference is effective in reducing PERV expression. Methods: To analyze the long‐term duration of RNA interference, expression of the PERV‐specific pol2 shRNA and inhibition of PERV expression was studied repeatedly in fibroblasts and peripheral blood mononuclear cells (PBMCs) of transgenic pigs over a period of 3 yr, when animals were sacrificed and expression was studied in different organs. Expression of the PERV‐specific shRNA was measured using a newly developed real‐time PCR, and expression of PERV was measured using a PERV‐specific real‐time PCR. Results: Over a period of 3 yr, PERV‐specific shRNA and green fluorescent protein (GFP) as reporter of the vector system were consistently expressed in transgenic animals. PERV expression was significantly reduced during the entire period. Levels of PERV and shRNA expression were different in the various organs. PERV expression was highest in the spleen and the lungs and lowest in liver and heart. However, in all organs of the transgenic pigs, PERV expression was inhibited compared with the vector control animals. Conclusions: Transgenic pigs expressing PERV‐specific shRNA maintained their specific RNA interference long term, suggesting that PERV expression in the xenotransplants will be suppressed over extended periods of time.     

Anti‐gal antibodies in α1,3‐galactosyltransferase gene‐knockout pigs

Fang J, Walters A, Hara H, Long C, Yeh P, Ayares D, Cooper DKC, Bianchi J. Anti‐gal antibodies in α1,3‐galactosyltransferase gene‐knockout pigs. Xenotransplantation 2012; 19: 305–310. © 2012 John Wiley & Sons A/S. Abstract Serum anti‐galactose‐α1,3‐galactose (Gal) IgM and IgG antibody levels were measured by ELISA in α1,3‐galactosyltransferase gene‐knockout (GTKO) pigs (78 estimations in 47 pigs). A low level of anti‐Gal IgM was present soon after birth, and rose to a peak at 4–6 m, which was maintained thereafter even in the oldest pigs tested (at >2 yr). Anti‐Gal IgG was also present at birth, peaked at 3 m, and after 6 m steadily decreased until almost undetectable at 20 m. No differences in this pattern were seen between pigs of different gender. Total IgM followed a similar pattern as anti‐Gal IgM, but total IgG did not decrease after 6m. The data provide useful baseline data for future experimental studies in GTKO pigs, e.g., relating to the antibody response to WT pig allografts.     

Identification of human preformed antibody targets in GTKO pigs

Burlak C, Wang ZY, Chihara RK, Lutz AJ, Wang Y, Estrada JL, Tector AJ. Identification of human preformed antibody targets in GTKO pigs. Xenotransplantation 2012; 19: 92–101. © 2012 John Wiley & Sons A/S. Abstract: Background: Human preformed antibodies continue to recognize porcine xenografts, despite the advent of α‐galactosyltransferase knockout (GTKO) pigs. This study examined the potential reactivity of human preformed IgG and IgM antibodies toward antigens in the GTKO pig liver. Methods: Human serum was analyzed for the concentration of IgG, IgM, anti‐αgal antibody, anti‐non‐αgal antibody and cytotoxicity toward domestic and GTKO fibroblasts and liver sinusoidal endothelial cells (LSEC). We detected preformed antibodies in human serum directed toward GTKO pig liver cells and tissue samples using advanced proteomic techniques. The targets of preformed antibodies were identified by MALDI TOF TOF mass spectrometry and validated by confocal microscopy, immunoblot, and immunoprecipitation. Results: Human serum used in this study contained 2.06 μg/ml IgG and 0.013 μg/ml IgM directed toward GTKO fibroblasts. Human IgG and IgM bound to GTKO LSEC in a dose‐dependent manner and were cytotoxic. We detected 357 protein spots recognized by human IgG and 233 by human IgM. Two hundred and nineteen proteins were common to both human IgG and IgM. Mass spectrometry identified numerous immunoreactive proteins, of which 19 were membrane proteins on liver cells. The most significant to this study were α‐enolase, CFTR, and E‐cadherin, which were abundant in GTKO pig tissues and expressed on the surface of GTKO LSEC. Human IgG captured α‐enolase, CFTR, and E‐cadherin by immunoprecipitation validating the proteomic identification. Conclusion: These experiments indicate that several membrane antigens in GTKO pigs could be recognized directly by human IgG or IgM. Further studies on the contribution of these antigens to antibody‐mediated xenograft rejection are necessary.     

猪肺挫伤数码断层图像与CT诊断的比较

目的比较分析病理数码断层图像(pathologicaldigitizedsectionalimage,PDSI)与CT扫描在猪肺挫伤诊断中的差异。方法采用BIMⅡ型生物撞击机制作小香猪肺挫伤模型,并于伤后6小时行CT断层摄影(层厚5mm),吸气末结扎气管后,行肺动脉冲洗及灌注。截取猪胸部并冰冻后,在-25℃低温实验室中用TK26350型数控铣床(铣切精度为1mm)逐层铣切,并逐层摄取猪胸部的连续断层图像数据,运用3Ddoctor软件分割数码断层图像并与对应层面CT扫描结果进行对比分析。结果CT扫描25层,采集PDSI167幅,对应的PDSI层面与CT图像基本吻合。PDSI上出血呈褐色改变。CT第14～18层提示肺中叶局限性挫伤出血,大小约为2.5cm×1.8cm×2.0cm,PDSI上相应部位出血区大小约为2.0cm×0.7cm×1.9cm。CT提示右肺中叶及下叶后外侧基底段为可疑的损伤区域,而在PDSI上,该区域呈形状不规则的大片深褐色出血改变,范围从第52层至114层。结论首次获得猪肺挫伤的数码断层图像集,CT表现与实际肺损伤之间存在差异,实际伤情较CT提示的重。PDSI的采集和研究对促进CT诊断水平有意义。     

分阶段添加细胞诱导因子诱导豚鼠脂肪间充质干细胞定向分化内耳毛细胞

背景：感音神经性耳聋主要是由内耳毛细胞的缺失或受损造成,用脂肪间充质干细胞来再生修复内耳毛细胞是治疗听力损失的有效方法。目的：探讨体外定向诱导豚鼠脂肪间充质干细胞向内耳毛细胞样细胞分化的可行性。方法：体外分离培养豚鼠脂肪间充质干细胞至第3代,流式细胞仪检测细胞免疫表型,分阶段加入表皮生长因子、碱性成纤维细胞生长因子、胰岛素样生长因子1、全反式维甲酸、脑源性神经营养因子、神经营养因子3,观察诱导分化的效果。结果与结论：豚鼠脂肪间充质干细胞体外培养呈梭形,漩涡状贴壁生长,第3代细胞表面标记CD29与CD44表达呈现阳性,CD34与CD45表达呈现阴性。应用细胞因子诱导后细胞早期nestin和GFAP表达阳性,继续诱导10 d后表达毛细胞特异性标记物MyosinⅦa和Math1,说明细胞因子可定向诱导豚鼠脂肪间充质干细胞向内耳毛细胞分化。     

Methicillin-resistant Staphylococcus aureus in food products: cause for concern or case for complacency?

The widespread use of antimicrobial agents, in combination with insufficient infection control measures, is the main driver of the current pandemic of antimicrobial resistance in human pathogens. The use of antimicrobials in food animal production also contributes, because resistant organisms and resistance genes can spread from animals to humans by direct contact or through the food chain. An important, traditionally human, pathogen, methicillin-resistant Staphylococcus aureus (MRSA), is currently endemic in many hospitals around the world and has also emerged in the community. Recently, a new reservoir of MRSA has been identified in food production animals and people in contact with these animals. This involves a specific clone, multilocus sequence type 398 (ST398), which has spread extensively among animals. ST398 has also been found in up to 11.9% of retail meat samples in several surveys from different parts of the world, posing a potential threat to human health.     

肿节风颗粒对小型猪腮腺放射损伤所致活性氧簇的清除作用

目的探讨肿节风对小型猪腮腺经15 Gyγ射线照射后所致活性氧簇(ROS)的清除作用。方法 45只雄性小型猪随机分成空白组、单照组、药照组3个大组,每组分成a、b、c三个平行组。药照组在照射前1周开始持续给药至取出腮腺标本,空白组和单照组给予等量的生理盐水。在麻醉条件下单照组和药照组均给予15 Gyγ射线照射双侧腮腺,空白组给予0 Gyγ射线照射。a、b、c三个平行组分别于照射后10、40、90 d取双侧腮腺,称重后分装,以猪活性氧簇(ROS)酶联免疫分析试剂盒检测其ROS的含量。结果药照组ROS的含量在各个时点均低于单照组,差异均有统计学意义(P<0.01),药照a组和药照b组、药照c组的差异均无统计学意义(P>0.01),药照b组和药照c组差异有统计学意义(P<0.01);在放射后10 d时肿节风对ROS的清除作用较强,药照组和与空白组的差异无统计学意义(P>0.01);放射后40和90 d肿节风对ROS均有有一定的清除作用,药照组与空白组的差异有统计学意义(P<0.01)。结论肿节风对小型猪腮腺放射损伤所致ROS有一定的清除作用,尤其对放射后10 d腮腺ROS的清除作用明显;清除放射所产生的ROS可能是肿节风防护腮腺放射损伤的主要机制。