Novel vaccination strategies and approaches against human tuberculosis

Tuberculosis (TB) remains one of a major health problem worldwide. Tuberculosis vaccine research has made an extraordinary progress over the past few years. However, there is still no replacement for the Bacillus Calmette‐Guérin vaccine, the only TB vaccine licensed for human use. Therefore, the discovery and development of new TB vaccines remains a priority. This article discusses current strategies used to diversify TB vaccines and includes discussion of the status of efforts to improve protection against Mycobacterium tuberculosis (M tb) infection or TB disease by developing new and safe TB vaccines. This article also highlights the current research efforts in immune‐enhancing approaches to improve vaccination efficacy. The development of more effective TB vaccines might have significant impact on global TB control.     

重组结核分支杆菌16000蛋白抗原(rPA16)的免疫原性研究

目的：研究结核分枝杆菌重组16000蛋白（rPA16)诱发家兔产生抗体的能力并评价其血清学诊断的价值。方法：家兔按注射的抗原及佐剂的不同分组，皮下多点注射分别进行免疫，6次免疫后采血，双向琼脂扩散试验和酶联免疫吸附试验（ELISA）跟踪兔血清抗体效价的时间效应并检测兔血清与所试各类分支杆菌PPD有无交叉反应；同时检测rPA16与各分支杆菌PPD的免疫羊血清的反应，并用rPA16抗原检测人血标本，分析该抗原的临床检测的敏感性和特异性。结果：兔血清抗体效价结果显示：5个月后，加佐剂的抗原组的兔免疫血清仍能检测到抗体，不加佐剂组2个月后血清抗体检测即为阴性，ELISA检测兔免疫血清比双向琼脂扩散方法要更敏感，rPA16抗原加佐剂引起的兔血清抗体滴度最高可达1：6400，不加佐剂组的血清抗体滴度仅为1：400，ELISA法分析抗rPA16的兔血清的特异性表明其特异笥较好，仅与牛、人型结核分支杆菌的PPD呈阳性反应，与其他所试分支杆菌PPD反应均为阴性，同时又分析了rPA16抗原的特异性，与人型PPD相比，该抗原只与所试各类分支杆菌，BCG、牛分支杆菌免疫的血清呈阳性反应，而人型PPD则基本上与结核分支杆菌免疫的血清均呈阳性反应，ELISA检测血清标本结果：肺结核病组中rPA16和PPD检测敏感性分别为64.8%和75.2%；健康组，卡介苗接种阳转健康组，rPA16和PPD检测特异性分别为96.2%,88.5%和94.3%,66.7%。结论：rPA16具有较强的免疫原性和一定的特异性，可能成为结核病血清学诊断试剂之一。     

结核分枝杆菌稳定L型变异的聚合酶链反应研究

目的：探讨结核分枝杆菌稳定L型变异的分子机制。方法：采用结核分枝杆菌聚合酶链反应(PCR)诊断试剂扩增结核分枝杆菌稳定L型纯培养物的染色体DNA，琼脂糖凝胶电泳、聚丙烯酰胺凝胶电泳(polyscrylamid gel electrophoresis，PAGE)薄层凝胶扫描分析。结果：结核分枝杆菌稳定L型比细菌型多一DNA条带。结论：结核分枝杆菌稳定L型的形成可能与基因突变有关，在特异性PCR反应中可形成与其亲代细菌型有差异的产物。     

Interleukin 17-Producing γδT Cells Increased in Patients with Active Pulmonary Tuberculosis

Although it has been known that y8 T cells may play an important role in the immune response to infection of Mycobacterium tuberculosis (M. tb), the mechanisms by which the T8 T cells participate in the innate and/or acquired immunity to tuberculosis (TB) have not been full elucidated. In the present study, 27 patients with active pulmonary TB and 16 healthy donors (HD) were performed. We found that proportion of IL-17-producing cells among lymphocyte was similar between TB patients and HD, whereas the proportions of γδ T cells in IL-17- producing cells (59.2%) and IL-17-producing cells in γδ T cells (19.4%) in peripheral blood were markedly increased in TB patients when compared to those in HD (43.9% and 7.7%, respectively). In addition, the proportions of IFN-γ-producing γδ T cells in TB patients were obviously lower than that in HD. Upon re-stimulated with M. tb heat-treated antigen (M. tb-HAg) in vitro, fewer IL-17-producing γδ T cells were generated from HD and TB patients, whereas IFN-γ-producing γδ T cells were increased in TB patients compared to that in HD. Our findings in TB patients and healthy human were consistent with other murine investigation that the IL-17- producing γδ T cells were main source of IL-17 in mouse model of BCG infection, suggesting that γδ T cells might be involved in the formation of tubercular granuloma in pulmonary TB patients, but need further identification. Cellular & Molecular Immunology. 2008;5(3):203-208.     

Intravesical instillation therapy with bacillus Calmette-Guérin for superficial bladder cancer: Study of the mechanism of bacillus Calmette-Guérin immunotherapy

AIM: In order to clarify the initial step of the mechanism by which bacillus Calmette-Guérin (BCG) exhibits antitumor activity via the immune response induced in the bladder submucosa after intravesical BCG therapy for human bladder cancer, various cytokines secreted in the urine after BCG instillation were measured. METHODS: After transurethral resection of bladder cancer, a 6-week course of BCG instillation was performed. At the first and sixth weeks' dosings, spontaneously excreted urine was collected before and 4, 8, and 24 h after BCG instillation. The urinary cytokines were determined by Sandwich enzyme-linked immunosorbent assay using monoclonal antibodies against granulocyte-macrophage colony-stimulating factor (GM-CSF), tumor necrosis factor (TNF)-alpha, granulocyte colony-stimulating factor (G-CSF), interleukin (IL)-1beta, IL-8, interferon (IFN)-gamma, and IL-12. RESULTS: After the BCG therapy, various cytokines, such as GM-CSF, TNF-alpha, G-CSF, IL-1beta, IL-8, IFN-gamma, and IL-12 were secreted, comprising the immune response cascade. The mean urinary excretions of GM-CSF and TNF-alpha 4 h after the sixth week's instillation were significantly higher than the pre-instillation levels. There were no significant increases in the urinary IFN-gamma or IL-12 levels between 4 and 24 h after the sixth week's instillation. The TNF-alpha level 4 h after the sixth week's instillation had a strong tendency towards the absence of recurrence, with a mean follow-up of 54.1 months. The Kaplan-Meier curve showed the 2, 5, and 10-year recurrence-free survival rates were 72.4%, 65.8%, and 56.4%, respectively. CONCLUSIONS: We suggested that the urinary levels of TNF-alpha might be essential in antitumor activity after BCG therapy and might play an important role in the prevention of bladder tumor recurrence.     

等位基因特异PCR技术检测结核分支杆菌rpoB基因突变研究

目的 建立并评价等位特异多聚酶链反应检测结核分支杆菌耐利福平基因rpoB突变的方法。方法 设计与rpoB基因突变后的碱基配对的引物用于扩增突变基因，建立AS－PCR检测rpoB基因突变的方法，并对58株结核菌进行了检测。结果 AS－PCR检测结核菌耐利福平相关基因rpoB的敏感性为77.3%，特异性达91.7%。AS－PCR检测rpoB基因，有1628A→T、1657C→T，G和1673C→T突变分别占耐利福平结核菌株的18.2%，22.7%和36.4%。检测rpoB突变与MIC测定RFP耐药率和PCR－SSCP检测的总符合率分别为86.2%和74.1%。检测试验可在3－4h内完成。结论 AS－PCR方法是检测结核菌耐利福平基因突变的敏感和快速方法，可在临床实验室使用并为临床医师提供治疗依据。     

结核分枝杆菌分子生物学检测方法新进展

应用分子生物学技术检测结核分枝杆菌，克服了传统检测方法的诸多不足，能够对难以培养、生长缓慢的结核分枝杆菌进行更加敏感、特异、准确、快速的鉴定和药物敏感性评价。本文概述了目前国内外几种结核分枝杆菌分子生物学检测方法的原理、应用现状及评价。     

结核分枝杆菌Mtb8.4基因疫苗与人白细胞介素12表达质粒联合免疫的细胞免疫应答观察

目的 观察结核分枝杆菌Mtb8.4基因疫苗与人白细胞介素12(hIL-12)联合免疫小鼠所诱导的细胞免疫应答.方法 15只C57BL/6N小鼠随机分为Mtb8.4基因疫苗 hIL-12质粒组(联合免疫组)、Mtb8.4基因疫苗组、卡介苗(BCG)组、空载体组和PBS组,基因疫苗、空载体和PBS经肌内注射法免疫各组小鼠,每隔3周免疫1次,共免疫3次,BCG组经尾部皮下注射1×106 CFU BCG免疫1次.ELISA法检测小鼠脾细胞培养上清中细胞因子水平;乳酸脱氢酶(LDH)释放法检测免疫小鼠特异性细胞毒性T淋巴细胞(CTL)杀伤活性.结果 联合免疫组能诱导较强的抗原特异性Th1型细胞免疫应答,免疫小鼠脾细胞培养上清液IFN-γ和IL-2水平(分别为1 493.340±8.128pg/ml、747.489±48.676pg/ml)显著高于Mtb8.4基因疫苗组,与BCG组相当,IL-4分泌减少,特异性CTL杀伤活性增强.结论 hIL-12表达质粒能够增强Mtb8.4基因疫苗所诱导的细胞免疫应答.     

Tuberculosis in children: a 13-year follow up of 1714 patients in a Belgian home care centre

From May 1970 to September 1983, 1714 children with different forms of primary tuberculosis were referred to the paediatric home care centre (Enfants soignés au Foyer, E.S.F.) of the Brussels University Hospital St.-Pierre. They were subdivided in five groups: asymptomatic (33%), symptomatic (28%), dubious tuberculous infections (35%), high-risk contacts (3%) and unestablished diagnosis (1%). They were aged from 10 days to 19 years, and 82% of them were migrants of low socio-economic level. Fifty percent of the symptomatic infections, mainly pulmonary, appeared in children under 3 years of age. An adult source of contamination was identified in 33% of the case (48% of the symptomatic children). Diagnosis was based on tuberculin screening with a 2IU intradermal test. Gastric aspirates yieldedMycobacterium tuberculosis in 15% of our patients, 11% of them showing resistance to one or more tuberculostatic drugs. Treatment was given to 1359 patients with excellent results. Therapy was shortened during the last 2 years of the study from 12 to 6 months for the asymptomatic patients and from 12 to 9 months for the symptomatic infections. Few complications were observed. Tuberculosis remains a serious cause of morbidity particularly in migrant children. Correct diagnosis and treatment of the disease is very important.     

协同刺激分子CD28在结核杆菌抗原体外激活人外周血γδT细胞中的作用

目的 :为了探讨CD2 8协同刺激分子在结核杆菌 (Mtb)低分子多肽抗原体外激活人外周血γδ T细胞中的作用。方法 :采用激发型抗CD2 8单抗模拟第二信号 ,Mtb低分子多肽抗原作为刺激原 ,对纯化的人外周血T细胞进行体外刺激和培养 ;用流式细胞仪检测γδ T细胞上CD2 8分子的表达、γδ T细胞的增殖效应及活化的γδ T细胞上CD6 9分子的表达。结果 :人外周血γδ T细胞中有 5 0 %左右表达CD2 8分子 ;抗CD2 8单抗协同Mtb抗原可刺激γδ T细胞的活化和增殖 ;但抗CD2 8单抗或Mtb抗原单独刺激则无作用。活化的γδ T细胞表面表达CD6 9分子。结论 :Mtb抗原在选择性活化人外周血γδ T细胞时需要第二信号的参与 ;CD2 8在Mtb抗原激活γδ T细胞时可提供协同刺激信号 ;CD6 9可作为γδ T细胞的早期活化标志。