Molecular genetic characterization of XRCC4 function

XRCC4 is a generally expressed protein of 334 amino acids that is involved in the repair of DNA double-strand breaks and in V(D)J recombination, but its function is unknown. In this study, we have used a mutational approach and the yeast two-hybrid method to perform an initial characterization of this protein. We show that the XRCC4 protein is located in the nucleus. We also demonstrate that several potential phosphorylation sites are not required for XRCC4 function in a transient V(D)J recombination assay. In addition, we show that XRCC4 forms a homodimer in vivo with the homodimerization domain being located within amino acids 115-204. Finally, we define a core domain of XRCC4 that functions in V(D)J recombination and comprises amino acids 18-204. Potential functions of XRCC4 are discussed.      

Hepatitis C virus-specific immune responses and quasi-species variability at baseline are associated with nonresponse to antiviral therapy during advanced hepatitis C

Pretreatment hepatitis C virus (HCV)-specific lymphoproliferative (LP) responses, neutralizing antibody (NA) responses, intrahepatic cytotoxic T lymphocyte (CTL) responses, and HCV quasi-species (QS) diversity and complexity were examined in patients with advanced hepatic fibrosis (Ishak fibrosis score of > or = 3) and prior nonresponse to interferon (IFN)- alpha therapy who were enrolled in the initial phase of the Hepatitis C Antiviral Long-Term Treatment against Cirrhosis Trial. Positive baseline HCV E1- and/or E2-specific NA responses (P = .01) and higher baseline HCV QS diversity (P = .01) were more commonly found in patients who did not become sustained virologic responders (SVRs) at week 72 (W72) than they were in those who did. No patients with positive results for both the LP and NA assays achieved a sustained virologic response. Multiple logistic regression analysis revealed that, when the presence of cirrhosis, prior ribavirin therapy, genotype 1 infection, log serum HCV RNA level, and receipt of >80% of the prescribed medication were controlled for, a sustained virologic response (W72) was negatively correlated with positive baseline LP assay results (P = .02) and with 1 or more positive assays (LP, NA, or CTL) (P = .02). No differences were noted in baseline intrahepatic CTL activity between SVRs and non-SVRs. Thus, in patients with advanced hepatic fibrosis due to HCV infection, pretreatment HCV-specific immune responses and increased QS variability appear to hinder viral clearance by pegylated IFN- alpha 2a and ribavirin combination therapy.     

The Role of Iron in Hepatitis C Infection

Iron overload of varying degrees is common among patients with chronic hepatitis C. The clinical significance of this iron overload is uncertain. Studies that have evaluated the effect of hepatic iron stores on the response to anti-viral treatment or on the natural history of chronic hepatitis C have found variable results depending on the technique used to measure hepatic iron stores and the degree of iron overload present among the study population. We have tried to comprehensively analyze the literature regarding the clinical interaction between iron overload and the natural history of chronic hepatitis C. The one clear relationship that emerges is that pre treatment serum ferritin inversely correlates with the odds of achieving sustained virological(SVR) response after combination interferon ribavirin treatment. We have also reviewed the limited literature that reports the effect of therapeutic phlebotomy to reverse iron overload among patients with chronic hepatitis C. A small meta-analysis of 6 prospective randomized trials and a subsequent seventh trial do suggest that phlebotomy to induce iron depletion enhances the likelihood of achieving (SVR) after anti-viral therapy. However, these studies are primarily in patients receiving interferon monotherapy, which is of course now obsolete. Finally, a few small studies suggest that therapeutic phlebotomy to induce iron depletion reduces liver transaminase levels and may improve histology, and perhaps even reduce the risk of hepato-cellular carcinoma. Prospective randomized controlled trials of phlebotomy among patients with advanced hepatitis C and iron overload are needed.     

Severe cholestatic hepatitis caused by thiazolidinediones: risks associated with substituting rosiglitazone for troglitazone

Troglitazone maleate (Rezulin) has been associated with severe hepatotoxicity, which led to its withdrawal from the U.S. market in March 2000. Rosiglitazone maleate (Avandia) is being marketed as a safe alternative in the treatment of type 2 diabetes mellitus. We report a case of severe thiazolidinedione-induced cholestatic hepatitis in a 56-year-old female patient at a university hospital who was given rosiglitazone, 8 mg/day, after she developed milder hepatotoxicity while taking troglitazone. Rosiglitazone was discontinued, and the patient was treated with prednisone, azathioprine, and ursodiol. Clinical evaluation and liver biopsy were performed and liver function tests were monitored. After being switched from troglitazone to rosiglitazone the patient developed a severe cholestatic hepatitis with marked jaundice and moderate increases in serum alkaline phosphatase and -glutamyltranspeptidase but only mild increases in serum aminotransferases. Discontinuation of rosiglitazone and treatment with prednisone, azathioprine, and ursodiol led to improvement, albeit with residual injury, dropout of intrahepatic bile ducts, and persisting elevations of serum alkaline phosphatase. Rosiglitazone is not always a safe alternative in patients who have had hepatotoxicity to troglitazone. It is important to monitor the serum alkaline phosphatase in addition to the serum aminotransferases in patients taking thiazolidinediones.     

Heparin‐induced thrombocytopenia: towards standardization of platelet factor 4/heparin antigen tests

Summary. Background: Laboratory confirmation of heparin‐induced thrombocytopenia (HIT) is based on detection of heparin‐dependent platelet‐activating antibodies. Platelet factor 4 (PF4)/heparin enzyme‐immunoassays (EIA) are a widely available surrogate for platelet‐activating antibodies. Objective: Defining the optical density (OD) reactivity profiles of a PF4/heparin EIA in reference subject and patient populations and the correlation of the EIA results (expressed in OD units) with the prevalence of platelet‐activating antibodies. Patients/methods: Using quantile regression we determined the 97.5th percentile of PF4/heparin‐immunoglobulin G (IgG) EIA reactivities in non‐heparin‐treated individuals [blood donors (n = 935)] and patients before heparin therapy (n = 1207). In patients with suspected HIT, we compared the correlation of EIA‐IgG reactivities (Greifswald laboratory; n = 2821) and the heparin‐induced platelet activation assay (HIPA) with the correlation of reactivities of another EIA‐IgG (McMaster laboratory; n = 1956) with the serotonin‐release assay (SRA). Results: PF4/heparin‐IgG EIA OD reactivities had a lower OD 97.5th percentile in blood donors compared with patient groups before heparin treatment (P < 0.001). The percentage of sera testing positive in the functional assays strongly correlated with PF4/heparin‐IgG EIA OD reactivities in both laboratories with very similar results (correlation coefficient > 0.9) when normalized OD ranges (maximum OD divided by 10) were used instead of absolute OD values. Conclusions: Results of PF4/heparin‐IgG EIA should not be reported as only positive or negative as there is no single acceptable cut‐off value. Instead, reporting PF4/heparin‐IgG EIA OD results in ranges allows for risk‐stratified prediction for presence of platelet‐activating antibodies. Use of normalized OD ranges permits a standardized approach for inter‐laboratory comparisons.     

经冠状动脉移植自体骨髓单个核细胞和间充质干细胞对急性心肌梗死模型心功能的改善

目的:为保护濒死心肌提供机会窗口,对比观察经冠脉移植自体骨髓单个核细胞或间充质干细胞后,实验性急性心肌梗死动物心功能变化及心肌组织核转录因子кB、心肌细胞凋亡情况。方法:实验于2005-03/2006-11在河北省人民医院实验中心完成。选用24只雄性冀中白猪,随机数字表法分为4组:正常对照组、模型组、单个核细胞组、间充质干细胞组,6只/组。①24只猪均以盐酸氯胺酮200mg臀部肌肉注射麻醉后,分别于各自右侧股骨抽取骨髓20mL,采用Fercoll法分离获得骨髓单个核细胞,加入胶体金溶液,培养12～16h待用。分离过程中取出含有骨髓单个核细胞成分的细胞层,常规培养传代,每3d换液1次,贴壁生长细胞即为骨髓间充质干细胞,加入胶体金溶液,培养24h待用。②除正常对照组外,其余各组均经导管球囊封闭第一对角支以远的前降支,复制猪急性心肌梗死模型。单个核细胞组、间充质干细胞组均于造模后立即开通前降支,分别经球囊注入预先分离的骨髓单个核细胞6×108个、间充质干细胞6×108个。模型组造模后于梗死1h开通前降支,经球囊注入磷酸盐缓冲液10mL。③各组分别于术前及术后4周经心脏超声检测心功能,取材行病理学检查、心肌组织核转录因子кB的免疫组织化学检测及心肌细胞凋亡检测。结果:24只雄性白猪均进入结果分析。①心功能变化:术前各组左心室收缩末内径、左心室舒张末内径、左心室射血分数、短轴缩短率基本相似。移植术后4周,正常对照组、单个核细胞组、间充质干细胞组左心室舒张末内径均明显低于模型组(F=4.68,P=0.01),左心室射血分数及短轴缩短率均明显高于模型组(F=5.14,P=0.01;F=3.32,P=0.04),各组左心室收缩末内径差异无显著性意义(F=1.64,P=0.21)。②心肌组织病理学改变:电镜下单个核细胞组、间充质干细胞组在梗死边缘区可见有胶体金颗粒的不成熟的心肌细胞,胞质中散在肌丝结构,肌丝排列紊乱不规则。③心肌组织核转录因子кB阳性率表达:与模型组比较,单个核细胞组、间充质干细胞组的梗死边缘区核转录因子кB阳性率明显降低(F=25.59,P=0.0001);正常心肌区核转录因子кB阳性率亦明显降低(F=18.20,P=0.0001)。④心肌细胞凋亡检测结果:与模型组比较,单个核细胞组、间充质干细胞组在心肌梗死区细胞凋亡率均明显降低(F=6.63,P=0.0027),梗死边缘区细胞凋亡率亦明显降低(F=36.07,P=0.0001)。正常心肌区单个核细胞组细胞凋亡率与模型组基本相似(F=9.69,P=0.004),但间充质干细胞组有所降低。⑤心功能与心肌细胞凋亡及心肌组织NF-кB的相关性:急性心肌梗死4周时,左心室射血分数与心肌细胞凋亡、心肌组织核转录因子кB均呈负相关(r=0.613,P=0.001;r=-0.437,P=0.033)。心肌细胞凋亡与心肌组织核转录因子кB呈正相关(r=0.672,P=0.002)。结论:经冠脉移植骨髓单个核细胞和间充质干细胞均可改善实验性急性心肌梗死动物的心功能,与梗死边缘区核转录因子кB表达降低及心肌细胞凋亡减少有关。骨髓单个核细胞移植的促血管增生作用优于间充质干细胞移植。     

针刺足三里和悬钟治疗缺血性脑卒中

目的:观察针刺足三里、悬钟2穴对缺血性脑卒中脑血管功能的影响,分析其可能的作用机制,并对临床疗效做出评价。方法:选择2004-11/2006-05湖北中医药高等专科学校附属古城医院针灸科、荆州市第五人民医院中医康复科、荆州市第三人民医院中医科3单位缺血性脑卒中患者合适病例160例,采用查随机数字表的方法,将其随机分为对照组和针刺组,各80例。对照组采用现代医学常规干预方法进行治疗:卧床,保持呼吸道通畅,预防感染,控制颅内压、血压,维持水电解质平衡。针刺组在此基础上加针刺足三里、悬钟2穴,采用慢速捻转进针法针刺,留针20～30min,每隔5min行针1次。1次/d。两组患者治疗30d。并以经颅多普勒检测观察缺血性脑卒中患者治疗前后脑血管舒缩反应能力、脑血流自动调节功能、大脑半球侧枝循环代偿功能的变化,同时以治疗前后神经功能缺损程度为指标评价其临床疗效。结果:160例病例全部进入结果分析。①针刺组与治疗前相比,脑血管舒缩反应能力明显加强,差异有显著性意义(t=2.97,P<0.05),且优于对照组(t=2.45,P<0.05)。②针刺组与治疗前相比,脑血流自动调节能力明显改善,差异有非常显著性意义(t=8.01,P<0.01),且优于对照组(t=7.67,P<0.05)。③针刺组与治疗前相比,大脑半球侧枝循环代偿功能得到加强,差异有显著性意义(t=3.15,P<0.05),且优于对照组(t=5.16,P<0.05)。④针刺组与治疗前相比,神经功能缺损积分明显降低,差异有非常显著性意义(t=4.83,P<0.01),且优于对照组(t=5.43,P<0.05)。结论:针刺足三里、悬钟2穴对缺血性脑卒中患者脑血管舒缩反应能力、脑血流自动调节功能、大脑半球侧枝循环代偿功能有明显改善作用,并能促进神经功能的恢复。