Cross-host evolution of severe acute respiratory syndrome coronavirus in palm civet and human

The genomic sequences of severe acute respiratory syndrome coronaviruses from human and palm civet of the 2003/2004 outbreak in the city of Guangzhou, China, were nearly identical. Phylogenetic analysis suggested an independent viral invasion from animal to human in this new episode. Combining all existing data but excluding singletons, we identified 202 single-nucleotide variations. Among them, 17 are polymorphic in palm civets only. The ratio of nonsynonymous/synonymous nucleotide substitution in palm civets collected 1 yr apart from different geographic locations is very high, suggesting a rapid evolving process of viral proteins in civet as well, much like their adaptation in the human host in the early 2002–2003 epidemic. Major genetic variations in some critical genes, particularly the Spike gene, seemed essential for the transition from animal-to-human transmission to human-to-human transmission, which eventually caused the first severe acute respiratory syndrome outbreak of 2002/2003.     

Memory T Cells Mediate Cardiac Allograft Vasculopathy and are Inactivated by Anti-OX40L Monoclonal Antibody

Purpose

Cardiac allograft vasculopathy (CAV) is a major complication limiting the long-term survival of cardiac transplants. The role of memory T cells (T_mem) in the pathogenesis of CAV remains elusive. This study investigated the role of T_mem cells in the development of CAV and the therapeutic potential of targeting the OX40/OX40L pathway for heart transplant survival.

Methods

T_mem cells were generated in Rag-1^-/- C57BL/6 (B6) mice by homeostatic proliferation (HP) of CD40L null CD3⁺ T cells from B6 mice. Rag-1^-/- B6 mice (H-2^b) harboring T_mem cells received cardiac allografts from BALB/c mice (H-2^d), and were either untreated or treated with anti-OX40L monoclonal antibody (mAb) (0.5 mg/mouse/day) for 10 days.

Results

Six weeks after HP, the majority of transferred CD40L^-/- T cells in Rag-1^-/- B6 mice were differentiated to CD44^high and CD62L^low T_mem cells. BALB/c heart allografts in Rag-1^-/- B6 recipient mice in the presence of these T_mem cells developed a typical pathological feature of CAV; intimal thickening, 100 days after transplantation. However, functionally blocking the OX40/OX40L pathway with anti-OX40L mAb significantly prevented CAV development and reduced the T_mem cell population in recipient mice. Anti-OX40L mAb therapy also significantly decreased cellular infiltration and cytokine (IFN-γ, TNF-α and TGF-β) expression in heart allografts.

Conclusions

T_mem cells mediate CAV in heart transplants. Functionally blocking the OX40/OX40L pathway using anti-OX40L mAb therapy prevents T_mem cell-mediated CAV, suggesting therapeutic potential for disrupting OX40-OX40L signaling in order to prevent CAV in heart transplant patients.     

克山病病区粮食和酱块对鸡骨关节和心脏的影响

目的探讨克山病病区低硒玉米和酱块对鸡骨关节和心脏病理损伤的影响。方法将“京白905”鸡雏80只,按体重随机分为4组,以克山病病区玉米及酱块为原料,配比不同氨基酸合成饲料喂养。饲养4个月后处死,光镜下观察鸡骨关节和心脏病理改变。结果鸡骨关节病变实验组检出率分别为33．33％、22．22％、22．22％,对照组检出率33．33％。组间差异无统计学意义（x2=0．825,P〉0．05）。心肌病变实验组检出率分别为6．67％、11．11％、11．11％,对照组无病变。组间差异无统计学意义（x2=0．543,P〉0．05）。结论克山病病区粮食可导致鸡骨关节和心肌出现病理性损伤。克山病病区酱块可能为保护因素。     

过氧化还原蛋白6对细菌脂多糖诱导的小鼠急性肺损伤氧化应激的保护作用

目的探讨过氧化还原蛋白Peroxiredoxin(Prdx)6对细菌脂多糖(LPS)诱导的小鼠急性肺损伤氧化应激的作用及机制。方法应用PCR法对Prdx6基因敲除小鼠行基因型鉴定并应用免疫组织化学法测定Prdx6蛋白在肺脏的表达。将雄性Prdx6基因敲除型小鼠（18只）按随机数字表法分入Prdx6敲除型对照组（9只）、Prdx6敲除型LPS 24 h组（9只）;将雄性野生型C57BL/6J小鼠（18只）按随机数字表法分入野生型对照组（9只）、野生型LPS 24 h组（9只）。各LPS组小鼠气管滴注LPS(5 mg/kg)制备急性肺损伤模型,于给药后24 h分别行肺脏病理检测,BCA法测定BALF内蛋白浓度,比色法测定肺脏总超氧化物歧化酶(T-SOD)活力和过氧化氢、羰基化蛋白和总抗氧化能力(TAOC),TBA法测定丙二醛的表达。结果Prdx6基因敲除小鼠肺组织免疫组织化学法未检测到Prdx6蛋白表达。两种属小鼠LPS组肺脏病理可见炎症细胞浸润、肺泡间隔增厚及肺泡内出血,Prdx6敲除型较野生型小鼠病理损伤更重。LPS刺激后,野生型LPS 24 h组BALF内的蛋白浓度为(441±54) mg/L,高于野生型对照组的(168±20) mg/L(t=-4.71,P＜0.01);Prdx6敲除型LPS 24 h组为(770±66)mg/L,高于野生型LPS 24 h组（t=-3.69,P＜0.01）。野生型LPS 24 h组T-SOD为(16.0±1.2) U/mg,低于野生型对照组的(26.5±3.9) U/mg(t=-6.22,P＜0.01);Prdx6敲除型LPS24 h组为(14.5±5.3)U/mg,与野生型LPS 24 h组差异无统计学意义（t=-0.56,P=0.60）。野生型LPS 24 h组肺脏过氧化氢和丙二醛[分别为(52.3±7.8)nmol/g和(3.3±0.5)nmol/mg]高于野生型对照组[分别为(29.5±3.2)nmok/g和(1.6±0.8)nmol/mg],差异有统计学意义（t值分别为-4.25和-5.94,均P＜0.01）,Prdx6敲除型LPS 24 h组[分别为(73.5±12.4)nmol/g和(5.9±0.9)nmol/mg],均高于野生型LPS24 h组（t值分别为-3.01和-6.01,均P＜0.05）。野生型LPS24 h组肺脏蛋白羰基为(6.9±1.2)nmol/mg,与野生型对照组的(6.1±0.9)nmol/mg差异无统计学意义(t=-1.62,P=0.15);Prdx6敲除型LPS 24 h组为(8.9±0.9)nmol/mg,高于野生型LPS 24 h组(t=-2.76,P＜0.05)。野生型LPS 24 h组肺脏TAOC为(4.7±0.6) U/mg,低于野生型对照组的(6.5±0.4) U/mg(t =3.35,P＜0.01);Prdx6敲除型LPS 24 h组为(3.9 ±0.4) U/mg,低于野生型LPS24 h组(t =2.44,P=0.04)。Prdx6敲除型对照组与其野生型对照组以上参数表达差异无统计学意义。结论在LPS诱导的肺损伤中,Prdx6基因缺失增加了活性氧的产生,加重了氧化应激反应,从而使肺损伤恶化。     

二十二碳六烯酸增加大鼠冠状动脉平滑肌细胞大电导钙离子激活钾离子流的机制

Objective To investigate the mechanism of enhanced large conductance calciumactivated potassium channel currents (BK) in coronary smooth muscle cells (SMCs) by docosahexaenoic acid (DHA). Methods Coronary SMCs were isolated by enzyme digestion. Potassium channels in coronary SMCs were identified by applications of different potassium blockers. Effects of DHA and its metabolite 16,17-epoxydocosapentaenoic acid (16,17-EDP) on BK channels in the absence and presence of cytochrome P450 epoxygenase inhibitor SKF525A were studied by patch clamp in whole-cell configuration. Results BK channels were widely distributed in SMCs, and BK currents in normal SMCs accounted for (64.2±2.7)%of total potassium currents(n =20). DHA could activate BK channels, and its 50% effective concentration (EC50) was (0.23±0.03)μmol/L, however, the effect of DHA on BK channels was abolished after SMCs were incubated with cytochrome P450 epoxygenase inhibitor SKF525A. 16,17-EDP, a metabolite of DHA, could reproduce the effects of DHA on BK channels, and its EC50 was (19.7± 2.8) nmol/L.Conclusion DHA and metabolites can activate BK channels and dilate coronary arteries through activating cytochrome P450 epoxygenase pathway.     

转化生长因子-β1对大鼠脑膜间皮细胞结缔组织因子表达的影响

目的 观察转化生长因子-β1（TGF-β1）对大鼠软脑膜间皮细胞（RLMCs）结缔组织生长因子（ CTCF）表达的影响.方法 体外培养RLMCs,并将其分为4组：（1）0组（正常对照组）：用无血清培养基（FSM）培养细胞;（2）1组：用含TGF-β11 ng/ml培养细胞;（3）2组：用含TGF-β1 2 ng/ml培养细胞;（4）3组：用含TGF-β14 ng/ml培养细胞;各组分别在TCF-β1刺激6、12及24 b后,采用逆转录-聚合酶链反应（RT-PCR）方法测定CTCF mRNA水平;蛋白免疫印迹（Westem blot）测定CTCF蛋白表达.数据以平均值±标准差（-χ±s）表示,各个浓度点组间比较采用单因素方差分析（One-way ANOVA）,两两比较采用最小显著法（LSD）检验,以P〈0.05有统计学意义.结果 在6、12及24 h分别以TCF-β1 1ng/mL、2 ng/mL、4 ng/mL处理,CTGF mRNA表达水平与对照组比较,差异有显著性（F6h=46.549、F12h= 287.098、F24h=109.202,P均〈0.001）,呈剂量依赖性,以TGF-β1处理12 h组CTGF mRNA表达差异明显.western blotting：正常对照组RLMCs细胞和不同浓度的TGF-β1刺激后均表达CTGF蛋白,在TGF-β1刺激6、12及24 h后,各组均随着浓度的增加而增加,差异有显著性（F6h= 52.988、F12h= 95.331、F24h=157.107,P均〈0.001）,呈剂量依赖性,以TGF-β1处理6h组CTGF蛋白表达差异明显.结论 TGF-β1能诱导RLMCs中CTGF通路激活.TGF-β1刺激RLMCs中CTGF mRNA和蛋白的表达,而且随浓度增加而显著.CTGF可能作为神经系统疾病中抑制脑膜纤维化的靶点而进一步研究.     

不稳定型心绞痛患者介人术后对比剂肾病的危险因素

目的探讨不稳定型心绞痛患者冠状动脉(冠脉)介入诊断及治疗术后对比剂肾病(CIN)的发生率及危险因素。方法连续入选2007年1～8月因不稳定型心绞痛入院并接受择期冠脉介入手术的患者232例。排除标准:(1)血压<120/70 mm Hg;(2)心功能(NYHA)分级>Ⅲ级。术中使用低渗非离子型对比剂碘普罗胺。介入术后24～72 h血肌酐(Scr)较基础值增高25%或44.2 μmol/L定义为CIN。分析CIN发生率与各项危险因素的相关性。结果 CIN总发生率为14.7%。冠脉钙化、术前Scr水平≥132.6 μmol/L、内生肌酐清除率(Ccr)<60 ml/min、NYHA Ⅲ级、糖尿病及年龄≥70岁的患者CIN发生率均明显增高。多变量回归分析显示,基线Scr≥132.6μmol/L、Ccr<60 ml/min、NYHA Ⅲ级与CIN的相关性最强。结论不稳定型心绞痛患者接受冠脉介入术后CIN是常见的并发症;高龄、肾功能不全、心功能不全、糖尿病及造影发现冠脉钙化是CIN发生的危险因素。     

主动脉内球囊反搏临床应用时机的选择与效果分析

目的:探讨主动脉内球囊反搏(IABP)在危重心脏病患者中应用时机的选择对治疗效果的影响。方法:分析48例危重心脏病患者并发心源性休克或低心排血量综合征(低心排)时应用IABP辅助治疗的临床效果以及影响预后的因素。结果:48例患者在应用IABP后血流动力学均有一定的改善;其中早期安装IABP的31例患者,死亡3例,病死率9.7%;手术或介入治疗后常规药物治疗(晚期)12～24 h后安装IABP的17例患者中有8例死亡,病死率47.1%。结论:IABP是一种简单有效的循环辅助手段,对危重心脏病患者改善血流动力学有一定效果,但安装时机的选择对治疗效果有明显的影响。IABP的安装应宁早勿晚。     

静脉移植骨髓间充质干细胞治疗心肌梗塞的可行性研究

目的：探讨经静脉注射骨髓间充质干细胞（MSCs）对心肌梗塞大鼠模型心功能的影响及其在体内的分布情况。方法：在心肌梗塞后1周将标记的MSCs注射到大鼠舌下静脉内,在细胞移植后不同时间点取心、肝、脾、肺、肾脏器,进行组织学检查,观察移植细胞的分布。选取心肌梗塞模型大鼠通过心脏超声评价移植后3周、6周心功能改变情况。结果：静脉注射MSCs后其组织结构未发现明显异常改变,早期主要分布在正常心肌组织内,1周后主要分布在梗塞及交界区内,正常心肌组织内很少见细胞存留。超声检查发现在细胞移植后实验组（12只）左心室没有进一步扩大．左心功能明显好于对照组（12只）：[左心室收缩末期内径（0．92±0．16）：（1．078±0．15）cm;左心室舒张末期内径（0．66±0．13）：（0．79±0．11）cm;左心室短轴缩短率（28．4±4．2）：（24．3±3．1）％;左室射血分数（52．7±4）：（42．89±4．2）％,P均〈0．05]。结论：静脉注射移植MSCs后细胞可以分布到重要组织器官内,移植细胞有向梗塞心肌组织内迁移的趋势,能明显延缓左心室重构及其所导致的心功能恶化。     

适于HIV-1 p24抗原检测试剂的单抗制备与筛选

目的制备抗HIV-1p24单克隆抗体(单抗,McAb),并利用双抗体夹心ELISA法建立HIV-1p24抗原检测试剂。方法以基因工程原核重组抗原HIV-1p24蛋白免疫BALB/c小鼠,利用常规杂交瘤技术和间接ELISA法制备单克隆抗体,单抗经纯化和HRP标记后,利用双抗体夹心ELISA筛选检测p24蛋白的最佳配伍单抗以期建立HIV-1p24抗原检测试剂。结果成功筛选到16株稳定分泌抗HIV-1p24单抗的杂交瘤细胞株,并从中筛选出最佳单抗配伍组合“16G12+2F2b-HRP”,该组合对p24蛋白的检测灵敏度为20pg/mL,对感染性克隆pNL4-3表达的HIV病毒培养上清检测呈阳性,对100份HIV阴性人血清无非特异性反应,显示出良好的检测灵敏度和特异性。结论本研究初步成功地建立了HIV-1p24抗原的检测试剂,并为最终建立HIV第四代诊断试剂(HIV Ag/Ab Assay)奠定了坚实的基础。