以大量蛋白尿、水肿为主要表现的乙型肝炎病毒相关性肾炎临床特征分析

[目的]探讨以大量蛋白尿、水肿为主要表现的乙型肝炎病毒相关性肾炎(HBV-GN)的临床特征,以提高对该病的认识。[方法]对我院近5年来收治的5例以大量蛋白尿、水肿为主要表现的HBV-GN患者的临床资料进行回顾分析,并结合文献进行复习。[结果]全部患者均有明显水肿和蛋白尿,3例有腹水,尿蛋白定量为3.58~7.50g/d,(平均5.42g/d),血浆白蛋白为20.4~28.2g/L(平均24.5g/L);4例表现为肾病综合征,1例为肾炎综合征;病理类型4例为膜性肾病,1例为局灶性节段性肾小球硬化。阿德福韦酯与泼尼松联合治疗取得了很好的近期疗效。[结论]以大量蛋白尿、水肿为主要表现的HBV-GN患者大多以肾病综合征、膜性肾病为主要表现,应早期给予抗病毒与糖皮质激素联合治疗。消化内科医师应不断提高对该病的认识,早诊早治,减少误诊。     

蓝氏贾第鞭毛虫α-19贾第素的单克隆抗体制备与亚细胞定位研究

目的 制备蓝氏贾第鞭毛虫(Giardia lamblia,简称贾第虫)α-19贾第素的特异性抗体,并通过免疫荧光对该贾第素的亚细胞定位进行鉴定。方法 经PCR获得α-19贾第素编码区片段,双酶切将目的 片段连入原核表达载体pET-28a(+)-α-19,重组载体转化E.coli Rosetta(DE3),IPTG诱导表达,SDS-PAGE及 Western blot验证表达情况;采用设计的合成抗原肽免疫小鼠经细胞融合和筛选制备特异性单克隆抗体,分别用贾第虫滋养体裂解物和α-19贾第素重组蛋白Western blot验证抗体特异性和结合力,选择特异性最好的单克隆抗体通过免疫荧光技术对α-19贾第素的亚细胞定位进行观察。结果 成功构建了原核表达载体pET-28a(+)-α-19,经SDS-PAGE及 Westernblot分析显示重组载体在大肠杆菌中表达出相对分子量约49 kD的重组蛋白,与理论值相符;Western blot显示单克隆杂交瘤细胞株α-19-3-7-3-5分泌的抗体结合力和特异性俱佳,可用于定位研究;免疫荧光定位结果表明α-19贾第素主要位于贾第虫滋养体的腹鞭毛。结论 制备了α-19贾第素的特异性单克隆抗体,免疫荧光显示α-19贾第素定位于贾第虫滋养体的腹鞭毛。     

海南省西部地区莱姆病血清学调查

目的 了解海南省西部地区关节炎或神经性疾病患者的莱姆病感染状况。方法 在东方市收集莱姆病疑似病例血清400份,儋州市收集莱姆病疑似病例血清500份,均为关节炎或神经性疾病患者。采用间接免疫荧光法(IFA)对血清标本进行莱姆病抗体初筛检测;并采用免疫印迹法(WB)对IFA检测阳性的血清标本进行确诊实验。结果 IFA方法检测血清样本900份,35份阳性,阳性率为3.89％。应用 WB法对上述35份IFA阳性血清样本进行检测,结果15份阳性, 阳性率为1.67％。不同年龄人群均有莱姆病螺旋体感染,其中,≤20岁阳性率最高,为11.76%(4/34),差异有统计学意义(χ²=24.108,P<0.001);莱姆病螺旋体感染在性别和症状间差异无统计学意义 (χ²=0.001,P=0.975;χ²=0.011,P=0.917)。结论 证实了海南省西部地区人群中存在莱姆病,建议当地医生在诊治关节炎和神经性疾病患者时,应排除莱姆病的可能。     

Prevalence,specificity and risk of red blood cell alloantibodies among hospitalised Hubei Han Chinese patients

Background

The prevalence, specificity and risk of red blood cell alloantibodies vary widely among different geographic areas, races, and diseases and according to different methods of study, but no data are available on the Chinese Han population, who were investigated in the present study.

Materials and methods

Antibody screening was conducted among 42,517 hospitalised Hubei Han Chinese individuals using column agglutination technology. Samples that were positive in antibody screening were subjected to antibody identification by the tube test. Clinical data, including gender, age, race, transfusion history and records of alloantibody detection, transfusion reactions or haemolytic disease of the newborn, were collected to analyse the prevalence and specificity of alloantibodies and complications associated with them.

Results

A total of 212 patients with alloantibodies were identified among 42,517 patients, yielding a prevalence of 0.50% in this study. Significantly different prevalence rates were observed according to age and sex. The most frequently identified alloantibodies were anti-E (87/212, 41.0%), anti-D (45/212, 21.2%), anti-M (41/212, 19.3%) and a combination of anti-E and anti-c (13/212, 6.1%). Haemolytic disease was observed in 13 infants with anti-D, three infants with anti-E and one infant with anti-Fy^a alloantibodies. Delayed haemolytic transfusion reactions occurred in four patients with alloantibodies.

Discussion

In hospitalised Hubei Han Chinese individuals, the overall prevalence of alloantibodies was 0.50%, with anti-E, anti-D and anti-M being the most frequently identified alloantibodies. These results indicate that anti-D and anti-E alloantibodies were major risk factors for haemolytic disease of the newborn or delayed haemolytic transfusion reactions in this study population.     

联合检测抗CCP、RF、抗RA33在类风湿关节炎的临床应用

目的:探讨抗环瓜氨酸肽抗体（抗CCP）、类风湿因子（RF）、抗RA33抗体联合检测在类风湿关节炎（RA）诊断中意义。方法:选取在我院确诊RA患者266例、非RA风湿病患者228例（结缔组织病、强直性脊柱炎、骨关节炎、系统性红斑狼疮、干燥综合征）、健康体检者215名,检测3组抗CCP、RF、抗RA33数值,统计各指标阳性率,并进行统计学分析比较;通过Spearman 相关分析探讨各指标之间的关系;ROC曲线比较各指标的诊断性能;通过平行试验与系列实验探讨3项指标联合检测RA的诊断效能。结果:RA患者各指标数值均显著高于非RA自身免疫疾病患者与健康对照组（P ＜0.01）;Spearman相关分析显示RA患者抗CCP与抗RA33之间不存在关系,RF与抗CCP、RF与抗RA33之间存在显著相关性（P ＜0.01）;ROC曲线分析显示,抗CCP、RF、抗RA33的AUC值分别为0.942、0.922、0.653;3项指标中,阳性率:RF＞抗CCP＞抗RA33,敏感度:RF＞抗CCP＞抗RA33,特异度:抗RA33＞抗CCP＞RF;平行试验敏感度增高（98.50%）,但特异度降低（64.91%）,系列实验敏感度为15.41%,特异度为98.68%。结论:抗CCP、RF、抗RA33联合检测可提高诊断RA的敏感度与特异性,适用于RA的早期诊断,临床上值得推广使用。     

含Hp特异性抗体羊奶2种检测方法的比较及初步临床效果

［摘要］ 目的比较含抗Hp特异性抗体羊奶的2种检测方法,并初步观察抗体羊奶的临床效果。 方法用国际标准菌株ATCC26695及ATCC26695、SS1混合菌液制备2种含抗Hp特异性抗体的羊奶,分别用双相免疫扩散试验及酶联免疫吸附测定(enzyme linked immunosorbent assay,ELISA) 法检测羊奶中抗Hp特异性抗体含量,并比较二者检测效果;30名受试者按照随机抽样法分为试验组和对照组,试验组每天饮用抗体羊奶,对照组饮用普通羊奶,连续饮用30 d,用14C尿素呼气试验(14C urea breath test,14C-UBT) 评估效果。 结果2种方法检测结果均证实了抗体羊奶中抗Hp特异性抗体的存在,ELISA结果显示,2种抗体羊奶中抗Hp特异性抗体含量高于对照孔,差异有统计学意义(P＜0.05),且随着稀释倍数增大,抗体含量逐渐降低;2种抗体羊奶间抗体含量差异无统计学意义(P＞0.05)。初步临床试验证明试验组经服用抗体羊奶后有效率为66.6%,与服用前比较差异有统计学意义(P＜0.05)。 结论2种检测方法均可较好地反映抗体羊奶中抗Hp特异性抗体存在,ELISA法简便、快速,能够更为精确地定量检测抗体含量,所获得的结果更为可靠。临床试验表明抗体羊奶能够有效降低患者14C-UBT数值,具有良好的临床应用前景。     

探讨ABO、RhD血型调查与建立Rh表型库在安全输血中的临床意义

目的 调查河北省邯郸市峰峰矿区ABO、RhD血型在人群中的分布情况,分析患者不规则抗体筛查结果,探讨ABO、Rh血型调查与建立Rh表型库在安全输血中的临床意义。 方法 收集我院8 000例住院患者ABO血型及RhD血型的检测结果,对需输血的患者5 000例进行不规则抗体检测,阳性结果标本进一步行抗体特异性鉴定。随机选取3 000袋RhD阳性滤白红细胞悬液样本检测Rh表型,通过电脑建立献血者Rh表型数据库,探讨其在输血中的重要意义及价值。 结果 8 000例患者中ABO血型分布情况从多到少依次为B型血（32.02%）、O型血（28.98%）、A型血（27.01%）、AB型血（11.99%）。不同年龄组间ABO血型分布情况差异有统计学意义（P＜0.05）,不同性别组中ABO血型分布情况差异无统计学意义（P＞0.05）。8 000例患者中RhD阴性48例（0.60%）。男性和女性 Rh血型分布差异无统计学意义（P＞0.05）。5 000例需输血患者的血样标本中不规则抗体阳性者30例（0.60%）,Rh血型系统抗体阳性19例（63.33%）,抗-E检出率最高。不规则抗体的检出率女性显著高于男性,差异有统计学意义（P＜0.05）。3 000例RhD阳性献血者血样标本Rh血型表型共8种,其中CCDee检出率最多,其次为CcDEe,DcDEE和ccDee检出率较少。 结论 ABO血型的分布与地域及年龄有关;临床上除在患者输血前进行不规则抗体筛查,应进一步检测其抗体特异性;建立献血者Rh表型数据库有利于预防因Rh表型不符输血引起的溶血性输血反应,对于临床安全输血工作具有重大意义。     

Target-mediated clearance and bio-distribution of a monoclonal antibody against the Kunitz–type protease inhibitor 2 domain of Tissue Factor Pathway Inhibitor

Introduction

A humanised monoclonal antibody, concizumab, that binds with high affinity to the Kunitz-type protease inhibitor (KPI) 2 domain of human tissue factor pathway inhibitor (TFPI) is in clinical development. It promotes coagulation by neutralising the inhibitory function of TFPI and may provide a subcutaneous prophylaxis option for patients with haemophilia. We aimed to study biodistribution and pharmacokinetics (PK) of concizumab.

Materials and Methods

Blockage of cellular TFPI by concizumab was measured by tissue factor/Factor VIIa-mediated Factor X activation on human EA.hy926 cells. Biodistribution of concizumab was analysed in rabbits by immunohistology, and the PK was measured in rabbits and rats.

Results and Conclusions

Concizumab bound to cell surface TFPI on EA.hy926 cells and neutralised TFPI inhibition of Factor X activation. The antibody cross-reacted with rabbit TFPI, but not with rat TFPI, allowing for comparative PK studies. PK data in rats described a log-linear profile typical for a non-binding antibody, whereas PK data in rabbits revealed a non-linear, dose-dependent profile, consistent with a target-mediated clearance mechanism. Immunohistology in rabbits during target-saturation showed localisation of the antibody on the endothelium of the microvasculature in several organs. We observed a marked co-localisation with endogenous rabbit TFPI, but a negligible sub-endothelial build-up. Concizumab binds and neutralises the inhibitory effect of cell surface-bound TFPI. The PK profile observed in rabbits is consistent with a TFPI-mediated drug disposition. Double immunofluorescence shows co-localisation of the antibody with TFPI on the endothelium of the microvasculature and points to this TFPI as a putative target involved in the clearance mechanism.     

Microfluidic integration for automated targeted proteomic assays

A dearth of protein isoform-based clinical diagnostics currently hinders advances in personalized medicine. A well-organized protein biomarker validation process that includes facile measurement of protein isoforms would accelerate development of effective protein-based diagnostics. Toward scalable protein isoform analysis, we introduce a microfluidic "single-channel, multistage" immunoblotting strategy. The multistep assay performs all immunoblotting steps: separation, immobilization of resolved proteins, antibody probing of immobilized proteins, and all interim wash steps. Programmable, low-dispersion electrophoretic transport obviates the need for pumps and valves. A three-dimensional bulk photoreactive hydrogel eliminates manual blotting. In addition to simplified operation and interfacing, directed electrophoretic transport through our 3D nanoporous reactive hydrogel yields superior performance over the state-of-the-art in enhanced capture efficiency (on par with membrane electroblotting) and sparing consumption of reagents (ca. 1 ng antibody), as supported by empirical and by scaling analyses. We apply our fully integrated microfluidic assay to protein measurements of endogenous prostate specific antigen isoforms in (i) minimally processed human prostate cancer cell lysate (1.1 pg limit of detection) and (ii) crude sera from metastatic prostate cancer patients. The single-instrument functionality establishes a scalable microfluidic framework for high-throughput targeted proteomics, as is relevant to personalized medicine through robust protein biomarker verification, systematic characterization of new antibody probes for functional proteomics, and, more broadly, to characterization of human biospecimen repositories.     

Human broadly neutralizing antibodies to the envelope glycoprotein complex of hepatitis C virus

Hepatitis C virus (HCV) infects ～2% of the world's population. It is estimated that there are more than 500,000 new infections annually in Egypt, the country with the highest HCV prevalence. An effective vaccine would help control this expanding global health burden. HCV is highly variable, and an effective vaccine should target conserved T- and B-cell epitopes of the virus. Conserved B-cell epitopes overlapping the CD81 receptor-binding site (CD81bs) on the E2 viral envelope glycoprotein have been reported previously and provide promising vaccine targets. In this study, we isolated 73 human mAbs recognizing five distinct antigenic regions on the virus envelope glycoprotein complex E1E2 from an HCV-immune phage-display antibody library by using an exhaustive-panning strategy. Many of these mAbs were broadly neutralizing. In particular, the mAb AR4A, recognizing a discontinuous epitope outside the CD81bs on the E1E2 complex, has an exceptionally broad neutralizing activity toward diverse HCV genotypes and protects against heterologous HCV challenge in a small animal model. The mAb panel will be useful for the design and development of vaccine candidates to elicit broadly neutralizing antibodies to HCV.