结核分枝杆菌重组蛋白Rv0222的表达及血清学鉴定

目的 构建Mtb重组蛋白Rv0222的重组质粒,评价其用于结核病血清学诊断的价值。方法 通过PCR从Mtb染色体基因组中扩增出Rv0222基因片段,插入pMD18-T载体后,再亚克隆入表达载体pET30a中,诱导表达重组菌,组氨酸标签（His-tag）亲和层析纯化蛋白,通过免疫印迹法分析重组蛋白的免疫反应性,通过间接ELISA方法检测142例血清样本,来自82例结核病患者（痰涂片或痰培养阳性,抗结核治疗症状减轻者）,28例非结核病患者（8例肺癌、15例肺炎和5例肺气肿）和32例健康体检者,检测数据通过专业医学软件MedCalc 11.5.0分析ROC曲线下面积和最佳阳性判定值。 结果 成功构建重组载体,重组蛋白Rv0222经电泳后显示相对分子质量约为27 000,镍柱纯化后蛋白纯度达95%以上,通过蛋白免疫印迹法证实重组蛋白Rv0222与结核病患者血清能特异性结合。酶联免疫检测ROC曲线下面积为0.893,最佳阳性判定值为0.12时,结核病患者组抗体检测敏感度为69.5%（57/82）,非结核病患者和健康体检者抗体检测特异度为90.0%（54/60）,结核病患者与非结核病患者及健康体检者相比,差异有统计学意义（t=25.78,P＜0.0001）。结论 成功构建pET30a-Rv0222表达载体,获得高纯度的重组蛋白Rv0222,ELISA结果显示Rv0222蛋白有望成为结核病血清学诊断的有效抗原之一。     

结核分枝杆菌12种抗原在结核病血清学诊断中应用价值的研究

摘要：目的 研究结核分枝杆菌11种重组蛋白和1种脂阿拉伯甘露糖(LAM)的抗原性,评价其在结核病血清学诊断中的价值。方法 将12种抗原包被于酶标板,通过酶联免疫吸附试验（ELISA）检测470例血清中抗结核抗体IgG水平,分析不同抗原组合对结核检出的灵敏度与特异度。结果 单个抗原检测抗结核抗体的灵敏度介于13.3%（28/210）与40.5%（85/210）之间,特异度介于96.4%（185/192）与99.5%（191/192）之间,4种抗原联合检测的灵敏度和特异度分别达到65.2%（137/210）和92.2%（177/192）。结论 多种抗原联合应用可提高结核病血清学诊断的灵敏度和特异度,有助于结核病的临床诊断。     

Assessing and screening for T-cell epitopes from Mycobacterium tuberculosis RD2 proteins for the diagnosis of active tuberculosis

The Region of D eletion 2 (RD2) of Mycobacterium tuberculosis encodes reserved antigens that contribute to bacterial virulence. Among these antigens, Rv1983, Rv1986, Rv1987, and Rv1989c have been shown to be immunodominant in infected cattle; however, their diagnostic utility has not been evaluated in humans.In this study, we screened 87 overlapping synthetic peptides encoded by five RD2 proteins for diagnosing tuberculosis epitopes in 50 active tuberculosis (TB) cases, 31 non-tuberculosis patients and 36 healthy individuals. A pool of promising epitopes was then assessed for their diagnostic value in 233 suspected TB patients using a whole blood IFN-γ release assay.Only 10 peptides were recognized by more than 10% of active tuberculosis patients. The IFN-γ release responses to Rv1986-P9, P15, P16, Rv1988-P4, P11, and Rv1987-P11 were significantly higher in the active TB group than in the control groups (p < 0.05). The whole blood IFN-γ release assay based on these epitopes yielded a sensitivity of 51% and a specificity of 85% in diagnosing active tuberculosis, and the corresponding results using the T-SPOT.TB assay were 76% and 75%, respectively.In conclusion, these results suggest that the six epitopes from the RD2 of M. tuberculosis have potential diagnostic value in TB.     

Development and evaluation of a novel multiple-antigen ELISA for serodiagnosis of tuberculosis

In this study, we describe the development and evaluation of a novel multiple-antigen ELISA for rapid diagnosis and screening of active tuberculosis (TB). The humoral immune responses of 136 active TB patients and 57 healthy subjects against antigens Rv3425, 38 kDa and lipoarabinomannan (LAM) from Mycobacterium tuberculosis H37Rv were examined by ELISA. Three essential results were obtained. (i) Rv3425 antigen is a potential candidate for serodiagnosis of active TB. Of 136 active TB patients, Rv3425 antigen provided a sensitivity of 31.6%, lower than that of LAM antigen, but higher than that of 38 kDa antigen, with an overall specificity of 100%. (ii) For 62 smear-negative pulmonary TB patients and 15 extra-pulmonary TB patients, the multiple-antigen test provided a sensitivity of 43.5% and 26.7%, respectively, representing an improvement over acid-fast bacilli (AFB) smear-based diagnosis. (iii) Compared with the single-antigen ELISA and the two available commercial kits, the multiple-antigen test offered the highest accuracy (71.0%). In conclusion, the multiple-antigen ELSIA test based on Rv3425, 38 kDa, and LAM antigens is a potentially useful tool for the serodiagnosis and screening of active TB. Combinations of Rv3425 with other mycobacterial antigens may also be worthy of further investigation.     

结核抗体检测联合结核感染T细胞斑点试验的临床诊断价值

目的 探讨结核抗体检测联合结核感染T细胞斑点试验(T-SPOT.TB)在结核病诊断中的应用价值。方法 选择2016年1月1日至2017年4月12日就诊于北京老年医院的961例结核病患者[结核病组;包括364例菌阳肺结核患者(通过细菌学诊断确诊)和597例菌阴肺结核患者(通过临床进行诊断)]和非结核病的1046例患者(对照组)。所有患者同时使用上海奥普生物医药有限公司及新加坡MP生物医学亚太私人有限公司生产的结核抗体金标试剂(分别为TB-DOT、ASSURE TB试剂盒)检测血清中的结核抗体;961例结核病组中的574例及1046例对照组中的664例患者同时使用T-SPOT.TB试剂盒和2种结核抗体金标试剂进行检测。对两组患者采用3种实验室技术进行检测或联合检测(包括串联与并联),对检测结果的敏感度、特异度进行统计学分析。结果 TB-DOT和ASSURE TB检测结核病组和对照组患者的敏感度和特异度分别为52.86%(508/961)、74.95%(784/1046)和45.99%(442/961)、76.00%(795/1046)。TB-DOT 和ASSURE TB对菌阳和菌阴肺结核患者检测的敏感度分别为64.84%(236/364)、62.09%(226/364)和45.56%(272/597)、36.18%(216/597);菌阳肺结核患者TB-DOT和ASSURE TB检测的敏感度均高于菌阴肺结核患者,差异均有统计学意义(χ ²值分别为60.99、33.69,P值均<0.05)。结核病组和对照组患者在2种结核抗体金标试剂和T-SPOT.TB试剂盒的串联联合检测中,敏感度为24.04%(138/574),但特异度达到89.91%(597/664);并联联合检测中,特异度为21.23%(141/664),但敏感度达到89.55%(514/574)。结核病组和对照组在ASSURE TB、TB-DOT和T-SPOT.TB 3种检测方法中,均为阳性和均为阴性两种谱型的构成比分别为24.04%(138/574)、10.09%(67/664)和10.45%(60/574)、21.23%(141/664),差异均有统计学意义(χ ²值分别为43.37和26.32,P值均<0.05)。 结论 两种结核抗体与T-SPOT.TB行串联联合检测能够提高检测特异度,并联联合检测能够提高检测敏感度;串联联合检测时,3种检测方法结果均为阳性和均为阴性两种谱型在临床上对结核病血清学诊断具有辅助诊断价值。     

Auxotrophic complementation as a selectable marker for stable expression of foreign antigens in Mycobacterium bovis BCG

Mycobacterium bovis BCG has the potential to be an effective live vector for multivalent vaccines. However, most mycobacterial cloning vectors rely on antibiotic resistance genes as selectable markers, which would be undesirable in any practical vaccine. Here we report the use of auxotrophic complementation as a selectable marker that would be suitable for use in a recombinant vaccine. A BCG auxotrophic for the amino acid leucine was constructed by knocking out the leuD gene by unmarked homologous recombination. Expression of leuD on a plasmid not only allowed complementation, but also acted as a selectable marker. Removal of the kanamycin resistance gene, which remained necessary for plasmid manipulations in Escherichia coli, was accomplished by two different methods: restriction enzyme digestion followed by re-ligation before BCG transformation, or by Cre-loxP in vitro recombination mediated by the bacteriophage P1 Cre Recombinase. Stability of the plasmid was evaluated during in vitro and in vivo growth of the recombinant BCG in comparison to selection by antibiotic resistance. The new system was highly stable even during in vivo growth, as the selective pressure is maintained, whereas the conventional vector was unstable in the absence of selective pressure. This new system will now allow the construction of potential recombinante vaccine strains using stable multicopy plasmid vectors without the inclusion of antibiotic resistance markers.     

肺结核患儿胃液样本结核分枝杆菌培养阳性率提高方法的探讨

目的:建立提高肺结核患儿胃液样本结核分枝杆菌(Mycobacterium tuberculosis,MTB)培养阳性率的方法,同时,探讨培养液的pH值对MTB抗原-胶体金检测试剂正确判读结果的影响和磷酸盐缓冲液(phosphate buffer solution, PBS)对结核样本前处理液pH值的调节作用。方法:将70份疑似肺结核患儿胃液样本同时用两步培养法、传统培养法和Xpert Ultra进行检测。两步培养法为先将胃液样本直接接种到罗氏培养基上,约20～30 d后收集可疑MTB菌落再行常规的碱处理-中和离心法处理进行分离培养(传统培养法)。比较传统培养法和两步法阳性检出率的差异;以样本的Xpert Ultra检测结果作为菌载量标准,分析两步法在不同菌载量样本中的阳性检出率差异。同时,配制系列pH值梯度的溶液,确定MTB抗原-胶体金检测试剂获得最佳显示结果的最适pH值范围;用系列体积的PBS缓冲液(pH=6.8)中和3%或4%NaOH与等体积样本的混合液,确定pH值至7～9时所需的体积稀释倍数。结果:传统培养法阳性检出率为26.5%(18/68),两步法阳性检出率为23.1%(15...     

Isoniazid treatment of Mycobacterium bovis in cattle as a model for human tuberculosis

Cattle infected with Mycobacterium bovis spoligotype 9 were treated with Isoniazid (INH) from three to 14 weeks post infection, rested for four weeks to allow INH depletion and then challenged with M. bovis spoligotype 35. Post mortem examination (PME) 35 weeks after the initial infection showed partial protection against infectious challenge following INH-attenuated infection compared with the spoligotype 35 challenge controls. Antigen-specific IFN-γ responses decreased over time with INH therapy, following a similar pattern to that observed in the treatment of M. tuberculosis infection in humans. Following cessation of therapy, specific IFN-γ responses increased more strongly in those calves that were visibly lesioned at PME. IFN-γ responses were also used to identify two antigens, TB10.4 and Acr2, that induced anamnestic responses in INH-treated, re-challenged calves, suggesting a role for both antigens in protective immunity. Specific IL-10 responses were observed in all calves following treatment with INH suggesting a role for IL-10 in the resolution of infection.     

Identification of a cytoplasmic linker protein as a potential target for neovascularization

Objective

Atherosclerosis and other cardiovascular diseases are serious threats to human health and become the leading cause of death in the world. Emerging evidence reveals that inhibition of plaque neovascularization could be an effective approach for the treatment of atherosclerosis. This study was conducted to identify cytoplasmic linker protein 170 as a potential target for cardiovascular diseases through modulation of neovascularization.

Methods and results

Immunofluorescence microscopy revealed that cytoplasmic linker protein 170 was ubiquitously expressed in mouse kidney, liver, lung, normal non-atherosclerotic aorta, and atherosclerotic aorta and was partly localized in the vascular endothelium. siRNAs were introduced to human umbilical vein endothelial cells (HUVECs) and the effect of knockdown was confirmed by Western blotting. Vascularization study was assessed with matrigel-based capillary assembly, branching, and in vivo matrigel plug assays. The data showed that siRNA-mediated knockdown of the cytoplasmic linker protein remarkably compromised the assembly and branching of capillary-like blood vessels and neovascularization in vivo. Cell motility and polarity properties were then analyzed using scratch wound repair, boyden chamber, and immunofluorescence assays, and the results revealed that the cytoplasmic linker protein was critical for the motility abilities of HUVECs through its actions on cell polarity.

Conclusion

Both in vitro and in vivo studies demonstrate the significance of the cytoplasmic linker protein for blood vessel formation. Mechanistic investigation reveals that its effect on neovascularization is orchestrated through its regulation of vascular endothelial cell polarity and motility. These findings provide the basis for exploring effective approaches to regulate neovascularization in cardiovascular diseases.     

Identification of TNIK as a novel potential drug target in thyroid cancer based on protein druggability prediction

Thyroid cancer is a common endocrine malignancy; however, surgery remains its primary treatment option. A novel targeted drug for the development and application of targeted therapy in thyroid cancer treatment remain underexplored.We obtained RNA sequence data of thyroid cancer from The Cancer Genome Atlas database and identified differentially expressed genes (DEGs). Then, we constructed co-expression network with DEGs and combined it with differentially methylation analysis to screen the key genes in thyroid cancer. PockDrug-Server, an online tool, was applied to predict the druggability of the key genes. Finally, we constructed protein-protein interaction (PPI) network to observe potential targeted drugs for thyroid cancer.We identified 3 genes correlated with altered DNA methylation level and oncogenesis of thyroid cancer. According to the druggable analysis and PPI network, we predicted TRAF2 and NCK-interacting protein kinase (TNIK) sever as the drug targeted for thyroid cancer. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis indicated that genes in protein-protein interaction network of TNIK enriched in mitogen-activated protein kinase signaling pathway. For drug repositioning, we identified a targeted drug of genes in PPI network.Our study provides a bioinformatics method for screening drug targets and provides a theoretical basis for thyroid cancer targeted therapy.     

分子生物学假阳性致肺癌性淋巴管炎误诊为肺结核的临床诊治过程分析

2019年4月17日,武汉市肺科医院呼吸科收治1例69岁男性因外院结核分子生物学阳性诊断为肺结核的肺癌性淋巴管炎患者。该患者因“发现肺部阴影3个月,间断咳嗽2个月”入院。既往有结肠癌病史。胸部CT扫描显示双肺弥漫性沿支气管血管束分布结节伴磨玻璃影,伴小叶间隔增厚,双肺门及纵隔淋巴结肿大,双侧胸腔积液,在外院行肺泡灌洗液(BALF)GeneXpert MTB/RIF(简称“ GeneXpert”)检出MTB(极低)及TB-PCR检出MTB,诊断为肺结核,转诊至武汉市肺科医院。入院后完善结核相关检查,PPD皮肤试验阴性,γ干扰素释放试验阴性,胸部CT影像学特征不符合肺结核改变,临床怀疑BALF结核病病原分子生物学检测假阳性,建议患者复查BALF、GeneXpert及经气管镜超声引导针吸活检术(EBUS-TBNA),患者拒绝。给予诊断性胸腔穿刺,抽出胸腔积液20ml,送检显示,癌胚抗原(118.4μg/L)明显升高,提示恶性胸腔积液;最后行内科胸腔镜胸膜活检,提示转移性低分化腺癌,结合胸部CT表现,诊断为肺癌性淋巴管炎。患者后因病情恶化死亡。笔者认为,影像学表现为沿支气管血管束分布结节伴小叶间隔增厚及纵隔淋巴结肿大时需鉴别肺癌性淋巴管炎。影像学表现与肺结核不相符时,分子生物学阳性诊断肺结核需谨慎,以避免误诊误治。     

Claudin 7 as a possible novel molecular target for the treatment of pancreatic cancer

Background/objectives

Pancreatic cancer consists of various subpopulations of cells, some of which have aggressive proliferative properties. The molecules responsible for the aggressive proliferation of pancreatic cancer may become molecular targets for the therapies against pancreatic cancer.

Use of Quantiferon-TB-Gold in Tube test for detecting latent tuberculosis in patients considered as candidates for anti-TNF therapy in routine clinical practice

Background/methods

Quantiferon-TB-Gold in Tube^® test (QFT-G-IT) may have advantages if combined with TST when screening for Latent Tuberculosis Infection (LTBI) prior to initiating anti-TNF therapy in an area of intermediate tuberculosis incidence such as Spain. In a small-scale prospective study, we evaluate the use of QFT-G-IT in combination with the screening recommended in Spain (Tuberculin-Skin Test, TST retest, clinical data, and Chest X-Ray (CXR)) for LTBI in patients considered as candidates for anti-TNFα treatment.

Results

From June 2008 to October 2010, 123 patients from a 300-bed hospital in Palma de Mallorca (Spain) were included in the study. The majority of patients were under immunosuppressive therapy. A positive TST and TST booster were found in 22 and 17 patients, respectively. Thus 39 (31.7%) of the 123 patients had a positive TST. QFT-G-IT was positive in 16 patients (13.6%), indeterminate in 4 (3.2%), and negative in 103 (83.7%). One of the two tests was positive and LTBI was diagnosed in 34.1% of patients. The agreement between TST and QFT-G-IT among vaccinated patients was low and not statistically significant (Kappa = 0.15) and was almost perfect among non-BCG vaccinated patients (K = 0.81). TST positive responses were significantly related to BCG-vaccination (p < 0.05) and QFT-G-IT positive response rates were related to older age (p < 0.05).

Conclusion

QFT-G-IT may have advantages when combined with TST in immunosuppressed patients especially in older patients with a negative TST; in BCG vaccinated patients with a positive TST, QFT-G-IT could avoid unnecessary treatments and toxicities related to a false-positive TST result.     

Analysis on epidemic and management of tuberculosis among senior middle school students from Qinghai Tibetans in a school of Daxing District of Beijing from 2010 to 2019

目的 分析北京市大兴区某中学高中部青海省玉树藏族自治州(西部结核病高发地区)集体来京藏族学生肺结核疫情及处置情况,为学校结核病防控工作提供参考依据。方法 回顾性分析2010—2019年大兴区某中学高中部在校学生肺结核疫情及处置情况;其中83.19%(10687/12847)为本地学生,16.81%(2160/12847)为青海省玉树藏族自治州集体来京的藏族学生。对青海藏族学生开展入(返)校胸部X线摄影(简称“胸片”)和单纯结核菌素皮肤试验(简称“PPD试验”)中度及以上阳性学生预防性治疗情况进行总结与分析。采用SPSS 21.0软件对数据进行统计学分析。计数资料采用χ²检验,当理论频数<1,或者分析总数<40时,采用Fisher确切概率法,以P<0.05为差异有统计学意义。结果 2010—2019年该校高中部报告活动性肺结核31例,其中青海藏族学生患者23例,报告发病率为1064.81/10万(23/2160);本地学生肺结核8例,报告发病率为74.86/10万(8/10687),差异有统计学意义(Fisher确切概率法,P=0.000)。23例青海藏族患者全部为住宿生,其中男13例(56.52%),女10例(43.48%),男∶女=1.3∶1。截至2019年9月,对1518名青海藏族学生进行了胸片检查,经临床综合评估确诊肺结核9例,检出率为0.59%(9/1518),占10年间全部青海藏族学生肺结核患者的39.13%(9/23);其中2017年2月在返校藏族学生筛查中,经临床综合评估确诊肺结核6例,检出率为3.03%(6/198)。2016—2019年单纯PPD试验中度及以上阳性学生91名,预防性服药者的发病率[4.17%(3/72)]低于未接受预防性服药者的发病率[10.53%(2/19)],但差异无统计学意义(Fisher确切概率法,P=0.280)。5例患者均于筛查后或服药后1年内发病。2019年1月至2020年5月本报告完稿时未再出现学生肺结核患者。结论 学生是结核病防控的重点人群,对西部结核病高发地区集体来京学生开展入(返)校胸片检查,能够实现关口前移,是预防和控制结核病在校园传播的有效手段。     

2010—2019年北京市大兴区某中学高中部青海藏族集体来京学生肺结核疫情处置分析

目的分析北京市大兴区某中学高中部青海省玉树藏族自治州(西部结核病高发地区)集体来京藏族学生肺结核疫情及处置情况,为学校结核病防控工作提供参考依据。方法回顾性分析2010—2019年大兴区某中学高中部在校学生肺结核疫情及处置情况;其中83.19%(10687/12847)为本地学生,16.81%(2160/12847)为青海省玉树藏族自治州集体来京的藏族学生。对青海藏族学生开展入(返)校胸部X线摄影(简称胸片)和单纯结核菌素皮肤试验(简称PPD试验)中度及以上阳性学生预防性治疗情况进行总结与分析。采用SPSS 21.0软件对数据进行统计学分析。计数资料采用χ²检验,当理论频数<1,或者分析总数<40时,采用Fisher确切概率法,以P<0.05为差异有统计学意义。结果2010—2019年该校高中部报告活动性肺结核31例,其中青海藏族学生患者23例,报告发病率为1064.81/10万(23/2160);本地学生肺结核8例,报告发病率为74.86/10万(8/10687),差异有统计学意义(Fisher确切概率法,P=0.000)。23例青海藏族患者全部为住宿生,其中男13例(56.52%),女10例(43.48%),男∶女=1.3∶1。截至2019年9月,对1518名青海藏族学生进行了胸片检查,经临床综合评估确诊肺结核9例,检出率为0.59%(9/1518),占10年间全部青海藏族学生肺结核患者的39.13%(9/23);其中2017年2月在返校藏族学生筛查中,经临床综合评估确诊肺结核6例,检出率为3.03%(6/198)。2016—2019年单纯PPD试验中度及以上阳性学生91名,预防性服药者的发病率[4.17%(3/72)]低于未接受预防性服药者的发病率[10.53%(2/19)],但差异无统计学意义(Fisher确切概率法,P=0.280)。5例患者均于筛查后或服药后1年内发病。2019年1月至2020年5月本报告完稿时未再出现学生肺结核患者。结论学生是结核病防控的重点人群,对西部结核病高发地区集体来京学生开展入(返)校胸片检查,能够实现关口前移,是预防和控制结核病在校园传播的有效手段。