Reactivation of tuberculosis is associated with a shift from type 1 to type 2 cytokines

The pattern of cytokines produced by T cells from mice with latent tuberculosis and during reactivation of tuberculosis was determined. A type 1 cytokine pattern was observed in T cells isolated from the lung of mice with latent disease. Reactivation of mycobacterial growth, by activation of the hypothalamic-pituitary-adrenal (HPA) axis, resulted in a shift from a type 1 to a type 2 cytokine pattern in both CD4 and CD8 T cells. Classification of the T cells based on their differential expression of CD45 and CD44 showed that the phenotypically different populations of CD4 and CD8 cells exhibited a type 1 cytokine pattern at latency and that reactivation of latent tuberculosis was associated with a shift in cytokines produced by these populations to a type 2 cytokine response. Control of mycobacterial growth resulted in a return to the type 1 cytokine pattern found during latent disease.     

Identification of the mycobacterial carbohydrate structure that binds the C-type lectins DC-SIGN, L-SIGN and SIGNR1

Mycobacterium tuberculosis represents a worldwide health risk and although macrophages are primarily infected, dendritic cells (DC) are important in inducing cellular immune responses against M. tuberculosis. Recent studies have demonstrated that M. tuberculosis targets the DC-specific C-type lectin DC-SIGN to inhibit the immuno-stimulatory function of DC through the interaction of the mycobacterial mannosylated lipoarabinomannan (ManLAM) to DC-SIGN, which prevents DC maturation and induces the immuno-suppressive cytokine IL-10. This may contribute to survival and persistence of M. tuberculosis. Here, we have identified the specific pathogen-derived carbohydrate structure on ManLAM that is recognized by DC-SIGN. We have synthesized the mannose-cap oligosaccharides man-ara, (man)2-ara and (man)3-ara, and demonstrate that these neoglycoconjugates are specifically bound by DC-SIGN. Moreover, we demonstrate that the human and murine DC-SIGN homologue L-SIGN and SIGNR1, respectively, also interact with mycobacteria through ManLAM. Both homologues have the highest affinity for the (man)3-ara structure, similar to DC-SIGN. This study provides information about the specific carbohydrate structures on pathogens that are recognized by DC-SIGN, and may provide strategies to develop vaccines against these pathogens. Moreover, the identification of SIGNR1 as a receptor for ManLAM will enable in vivo studies to investigate the role of DC-SIGN in M. tuberculosis pathogenesis.     

Concentrations of CXCL8, CXCL9 and sTNFR1 in plasma of patients with pulmonary tuberculosis undergoing treatment

Background: Chemokines are a class of cytokines with chemotactic properties shown to be induced by M. tuberculosis or its antigens in vitro and in experimental infection in vivo. A few studies have also demonstrated the expression of chemokines in clinical samples of patients with active tuberculosis (TB). In the present work, we measured the concentration of chemokines in plasma samples of HIV-negative patients with pulmonary tuberculosis at different stages of chemotherapy. For comparison, we also evaluated the levels of sTNFR1 and TNF-α. Methods: Cytokines and chemokines were measured by ELISA in healthy individuals and patients with active pulmonary TB at different stages of treatment. Results: The concentrations of CXCL8, CXCL9 and sTNFR1 were elevated in patients with active pulmonary TB but returned to background levels at 4–6 months of chemotherapy. The concentration of CCL11 was elevated in patients with active pulmonary tuberculosis when compared to control and remained elevated throughout the specific therapy. There was no difference in the plasma concentration of CCL2 and CXCL10 between pulmonary TB patients and control subjects. Conclusion: Measurement of the CXCL8, CXCL9 and sTNFR1 may be useful to assess response to treatment in pulmonary TB patients. Received 12 September 2005; returned for revision 18 November 2005; returned for final revision 30 January 2006; accepted by M. Parnham 23 May 2006     

HLA-DRB1~* 1501与我国北方汉族儿童结核病的易感性研究

为了研究中国北方儿童结核病与HLAⅠ类基因的关联,我们采用PCR-SSO方法检测了97例北方汉族结核患儿和91例正常对照的HLA-DRBl,DQAl,DQBl等位基因.发现中国北方汉族儿童结核病与HLA-DRBl·1501有显著关联.进一步比较DR分子结构发现β链第86位氨基酸对结核病的易感性可能有重要意义.     

The microbiological diagnosis of tuberculous meningitis of Haydarpasa-1 study

We aimed to provide data on the diagnosis of tuberculous meningitis (TBM) in this largest case series ever reported. The Haydarpasa-1 study involved patients with microbiologically confirmed TBM in Albania, Croatia, Denmark, Egypt, France, Hungary, Iraq, Italy, Macedonia, Romania, Serbia, Slovenia, Syria and Turkey between 2000 and 2012. A positive culture, PCR or Ehrlich-Ziehl-Neelsen staining (EZNs) from the cerebrospinal fluid (CSF) was mandatory for inclusion of meningitis patients. A total of 506 TBM patients were included. The sensitivities of the tests were as follows: interferon-γ release assay (Quantiferon TB gold in tube) 90.2%, automated culture systems (ACS) 81.8%, LÖwenstein Jensen medium (L-J) 72.7%, adenosine deaminase (ADA) 29.9% and EZNs 27.3%. CSF-ACS was superior to CSF L-J culture and CSF-PCR (p <0.05 for both). Accordingly, CSF L-J culture was superior to CSF-PCR (p <0.05). Combination of L-J and ACS was superior to using these tests alone (p <0.05). There were poor and inverse agreements between EZNs and L-J culture (Κ = −0.189); ACS and L-J culture (Κ = −0.172) (p <0.05 for both). Fair and inverse agreement was detected for CSF-ADA and CSF-PCR (Κ = −0.299, p <0.05). Diagnostic accuracy of TBM was increased when both ACS and L-J cultures were used together. Non-culture tests contributed to TBM diagnosis to a degree. However, due to the delays in the diagnosis with any of the cultures, combined use of non-culture tests appears to contribute early diagnosis. Hence, the diagnostic approach to TBM should be individualized according to the technical capacities of medical institutions particularly in those with poor resources.     

抑制Coronin-1基因表达的siRNA载体的构建和筛选

目的:构建和筛选能高效、特异性抑制Coronin-1基因表达的siRNA表达载体。方法:提取鼠巨噬细胞总RNA,RT-PCR扩增Coronin-1基因目标序列,克隆入pSEB-HUS真核表达质粒,构建pSEB-HUS-C重组质粒。将设计、合成的3条siRNA及阴性对照分别克隆入pSEB-HUS-C,构建重组pSEB-HUS-C1、pSEB-HUS-C2、pSEB-HUS-C3及pSEB-HUS-CN质粒。将干涉质粒瞬时转染A549细胞,通过绿色荧光信号观察、实时定量PCR和Western blot法检测其对Coro-nin-1基因表达的影响。结果:经双酶切及测序证实,所构建siRNA表达载体目的基因大小、序列与预期相符。经瞬时转染A549细胞后,其中的pSEB-HUS-C3能明显抑制Coronin-1 mRNA的表达和Coronin-1蛋白的合成,抑制率分别是75.9%和75.1%。结论:成功构建并筛选出高效、特异性抑制Coro-nin-1表达的siRNA表达载体,为进一步研究Coronin-1在巨噬细胞中的作用奠定基础。     

Worsening and unmasking of tuberculosis in HIV-1 infected patients after initiating highly active anti-retroviral therapy in Uganda

Objectives

To determine the proportion of patients developing active tuberculosis (TB) versus that of patients who experience worsening of TB, after initiating highly active anti retroviral therapy (HAART).

Methods

Charts of HAART naïve patients with or without clinically activeTB who consecutively commenced HAART at Mulago Hospital Infectious Diseases Institute were reviewed. Patients were assessed for worsening of TB on treatment or development of new active TB (unmasking of TB) after initiating HAART.

Results

Of 271 patients without activeTB at baseline who initiated HAART, 16 (5.9%) developed activeTB within 6 months (early unmasking) and 10 (2.7%) after 6 months (late unmasking). Seven of 10 late unmasking patients had a past history of treatment for aTB disease episode. Of 45 patients who commenced HAART with coexisting active TB, 13 (29%) experienced worsening of TB symptoms, signs and/or radiological features. Nine of these 45 commenced HAART during the intensive phase of TB treatment, of whom 2 (22%) experienced worsening of TB. Thirty six of 45 started HAART during the continuation phase of TB treatment of whom 11 (31%), experienced worsening of TB. The median time from initiation of HAART to worsening of TB in patients on concurrent active TB treatment was 5 weeks, and 18 weeks to unmasking of new active tuberculosis.

Conclusion

Unmasking of TB was commonest in the first 6 months of HAART and declined in the subsequent months with most in the late unmasking group being TB recurrences. Worsening of TB occurred even after HAART was delayed to the continuation phase of TB treatment.     

HIV coinfections with tuberculosis among HIV-1 infected individuals in old cross river state,Nigeria

ABSTRACT

Human immunodeficiency virus (HIV) and Tuberculosis (TB) are two main global public health threats that dent development in low and middle-income countries. This study evaluated the HIV/TB co-infection rate among HIV-1 infected individuals in old Cross River State, Nigeria. A total of 417 HIV-infected individuals participated in this study, 241 (57.8%) from Calabar, Cross River State, Nigeria and 176 (42.2%) from Uyo, Akwa-Ibom State, Nigeria. The age range of the 417 HIV-1 positive individuals who participated in the study was 4–72 years with an average age of 39.1 years. Plasma samples were analyzed for HIV and TB using fourth-generation Enzyme-Linked immunosorbent Assay. The CD4 count was enumerated using the Partec CyFlow® Counter. Plasma viral loads (PVL) were determined using the Abbott Real-Time HIV-1 assay. Results showed that 230 (55.2%) of the participants were in the 31–45 years age range. The majority (67.4%) of the HIV-1 infected individuals were females and 32.6% were males. An overall prevalence of HIV/TB coinfection in Old Cross River State, Nigeria was 1.4%, with Akwa Ibom State (0.6%) and Cross River State (1.2%). A higher prevalence of HIV/TB coinfection was observed among females (1.8%) than in males (0.7%). Higher prevalences of HIV/TB coinfections was observed in patients above 45 years of age (2.2%), married (2.3%), tertiary education (1.8%) followed by those with secondary education (1.4%), traders and civil servants (3.1%), patients with CD4 counts 200–349 and ≥500 cells/μl (1.9%), and those with viral load <40 copies/mL (2.7%). This study confirmed the presence of HIV/TB co-infection in old Cross River State, Nigeria. Although the prevalence rate of HIV/TB coinfection was low, its presence alone among HIV-1 infected individuals makes it a major source of concern. This finding highlights the need for a well-structured approach to the management of co-infection, and this includes both the social and medical aspects of the problem.     

Th1和Th17细胞在活动期肺结核病患者外周比例失衡的临床研究

结核病是全球性严重危害人类健康的传染性疾病之一,深入解析结核分枝杆菌(Mycobacterium tuberculosis,Mtb)感染过程中宿主免疫应答特征是研发和评价有效的结核疫苗的重要前提。本研究以Th1、Th17和Th22细胞免疫应答为切人点,检测抗原特异性和非特异性活化后的Th1、Th17和Th22型细胞因子分泌细胞的比例。结果显示,和正常人群相比,结核病患者的T细胞在非特异性刺激下的Th1型细胞免疫应答能力明显高于健康人,即分泌IFN-γ和TNF-α的细胞数量明显增多,但IL-17型应答能力则低于健康人群;经M.tb特异性抗原ESAT6-CFP10刺激后,活动期肺结核病患者分泌IFN-γ的T细胞比例高于健康人,而分泌TNF-α的T细胞的比例与健康人相比却显著下降,分析Th1型与Th17型细胞之比显示结核病患者要高于健康人,而Th22型细胞应答在活动期结核病患者中的水平和正常人群相近。上述研究结果显示活动性肺结核病患者外周Th1型和Th17型细胞和正常人相比存在应答失衡,这一比例失调有望为评价结核疫苗效果和抗结核免疫应答的免疫学特征提供新依据。     

Kinetics and cellular sources of cathelicidin during the course of experimental latent tuberculous infection and progressive pulmonary tuberculosis

In spite of advances in immunology on mycobacterial infection, there are few studies on the role of anti‐microbial peptides in tuberculosis. The cathelin‐related anti‐microbial peptide (CRAMP) is the only cathelicidin isolated from mice. In this work we investigated the cellular sources and the production kinetics of this molecule during experimental tuberculosis, using two well‐characterized models of latent or chronic infection and progressive disease. The lung of non‐infected control mice expressed CRAMP at very low levels. In both models of experimental tuberculosis the main cells immunolabelled for CRAMP were bronchial epithelial cells, macrophages and pneumocytes types II and I. After intratracheal infection with a high bacilli dose (H37Rv strain) in Balb/c mice to produce progressive disease, a high CRAMP gene expression was induced showing three peaks: very early after 1 day of infection, at day 21 when the peak of protective immunity in this model is raised, and at day 28 when the progressive phase starts and the immunoelectronmicroscopy study showed intense immunolabelling in the cell wall and cytoplasm of intracellular bacilli, as well as in cytoplasmic vacuoles. Interestingly, at day 60 post‐infection, when advanced progressive disease is well established, characterized by high bacillary loads and extensive tissue damage, CRAMP gene expression decreased but strong CRAMP immunostaining was detected in vacuolated macrophages filled with bacilli. Thus, cathelecidin is highly produced during experimental pulmonary tuberculosis from diverse cellular sources and could have significant participation in its pathogenesis.     

Changing patterns and trends of multidrug-resistant tuberculosis at referral centre in Northern India: A 4-year experience

Purpose: India has a high burden of drug-resistant tuberculosis (TB), although there is little data on multidrug-resistant tuberculosis (MDR-TB). Although MDR-TB has existed for long time in India, very few diagnostic laboratories are well-equipped to test drug sensitivity. The objectives of this study were to determine the prevalence of MDR-TB, first-line drug resistance patterns and its changing trends in northern India in the 4 years. Materials and Methods: This was a prospective study from July 2007 to December 2010. Microscopy, culture by Bactec460 and p-nitro-α-acety lamino-β-hydroxypropiophenone (NAP) test was performed to isolate and identify Mycobacterium tuberculosis (M. tb) complex (MTBC). Drug sensitivity testing (DST) was performed by 1% proportional method (Bactec460) for four drugs: Rifampicin, isoniazid, ethambutol and streptomycin. Various clinical and demographical profiles were evaluated to analyse risk factors for development of drug resistance. Results: We found the overall prevalence rate of MDR-TB to be 38.8%, increasing from 36.4% in 2007 to 40.8% in 2010. we found that the prevalence of MDR-TB in new and previously treated cases was 29.1% and 43.3% (P < 0.05; CI 95%). The increasing trend of MDR-TB was more likely in pulmonary TB when compared with extra-pulmonary TB (P < 0.05; CI 95%). Conclusions: we found a high prevalence (38.8%) of MDR-TB both in new cases (29.1%) and previously treated cases (43.3%).This study strongly highlights the need to make strategies for testing, surveillance, monitoring and management of such drug-resistant cases.     

Mice lacking the multidrug resistance protein 1 have a transiently impaired immune response during tuberculosis

A T helper (Th) 1 immune response is important for host defense against tuberculosis. The multidrug resistance protein (Mrp) 1 is constitutively present at low levels on Th2 lymphocytes, and is expressed on Th1 lymphocytes upon activation. To determine the role of Mrp1 in the pathogenesis of tuberculosis, Mrp1 deficient (-/-) and normal wild type mice were intranasally infected with Mycobacterium tuberculosis. At 2 weeks after infection, Mrp1(-/-) mice had reduced levels of the Th1 cytokine interferon-gamma and an impaired granuloma formation in their lungs. At 5 weeks postinfection, M. tuberculosis outgrowth was enhanced in lungs and livers of Mrp1(-/-) mice. A more prolonged observation of these mice, up to 4 months, revealed no differences in survival or mycobacterial outgrowth. These data suggest that Mrp1 plays an early but dispensable role in the protective immune response to pulmonary tuberculosis.     

结核感染干扰素释放试验中的QFT-G试验在肺结核诊断中的应用评价

目的 探讨结核感染干扰素释放试验中的QFT-G试验在肺结核诊断中的应用评价.方法 采用QFT-G检测外周血结核特异性γ-干扰素的含量,同时与FQ-PCR检测痰中结核分枝杆菌核酸结核菌素、纯化蛋白质衍生物(PPD)皮试进行平行比较分析.结果 QFT-G、FQ-PCR和PPD三种方法的敏感度分别为:91.4％、84.2％、78.9％,特异性分别为86.9％、80.8％和54.8％,阳性预测值分别为91.0％、92.2％和71.1％,阴性预测值分别为66.2％、75.4％和46.3％,准确性分别为87.8％、85.0％和59.6％.结论 QFT-G较FQ-PCR和PPD有较高的敏感度、特异性和准确性,是一种诊断肺结核的有价值的检测方法.     

抑制Mcl-1表达调控结核感染的小鼠腹腔巨噬细胞凋亡的机制初探

目的:应用Mcl-1-shRNA质粒抑制不同毒力结核分枝杆菌(MTB)菌株感染的小鼠腹腔巨噬细胞中Mcl-1的表达,通过观察Bcl-2和Bax表达的变化探讨其调控机制。方法:制备不同毒力MTB菌株悬液,分别感染BALB/c小鼠,再用Mcl-1-shRNA质粒处理感染小鼠模型,并同时设立对应的对照组,于处理后1 d、3 d、5 d和7d处死小鼠并收集腹腔巨噬细胞。应用流式细胞术检测不同处理时间、不同毒力菌株感染时小鼠腹腔巨噬细胞的凋亡率,real-time PCR和Western blot检测Bcl-2和Bax的表达。结果:Mcl-1-shRNA质粒处理后,不同毒力MTB菌株感染的小鼠巨噬细胞凋亡率均比对照组有不同程度的增高,其中以BCG和H37Ra组最明显(P 0. 05); Bcl-2的mRNA和蛋白水平显著减少,而Bax的mRNA和蛋白的表达均显著增加,以BCG感染组较为显著,且二者mRNA的比值与菌株毒力呈负相关(P 0. 05)。结论:抑制Mcl-1的表达可显著促进不同毒力MTB菌株感染的小鼠腹腔巨噬细胞凋亡,其调控机制可能与Bcl-2和Bax蛋白的表达及MTB菌株毒力密切相关。     

Evaluation of the usefulness of phage amplification technology in the diagnosis of patients with paucibacillary tuberculosis

The present study was undertaken to assess the performance of the Fast Plaque TB(TM) (FPTB) test in the diagnostically difficult group of paucibacillary tuberculosis (TB) and to compare its results with the conventional bacteriological methods. The study was conducted on a total of 139 patients, who were negative for TB in sputum-smear examination. Bronchoalveolar lavage (BAL) or pleural biopsy specimens collected from these patients were subjected to smear examination, LJ culture and FPTB test. The smear, culture and the FPTB positivity rates were compared between patients with pulmonary and pleuro-pulmonary involvement. The FPTB test was found to register an overall sensitivity of 58.8% and specificity of 97.9%. The positive and negative predictive values of the test were 98.1 and 56.5, respectively. Among patients with paucibacillary TB, on head-to-head comparison, we found that the sensitivity and specificity values of the FPTB test were marginally better than smear-microscopy and inferior to culture on LJ media.     

Osteoarticular tuberculosis in Tehran,Iran: a 2-year study

Osteoarticular tuberculosis (TB) accounts for 1–5% of all TB cases and 10–18% of those with extrapulmonary infection. Diagnosis is difficult, because the lungs are rarely involved and there are no specific signs or symptoms. The purpose of this study was to assess the frequency and clinical and laboratory findings in osteoarticular TB in two referral hospitals in Tehran, Iran. The hospital dataset of patients admitted with osteoarticular TB during 2003–2005 was reviewed. Patients’ demographic data, clinical presentation and radiological and pathological findings were analysed. Weight loss (50%), fever (36%) and night sweats (38.5%) were the most common constitutional symptoms. Knee, ankle, hip and shoulder joints were the most frequent sites for TB arthritis. In osteomyelitis, long and short bones were equally affected. In TB spondylitis, the lumbar (22.7%) and thoracic (50%) vertebrae were the most commonly involved sites. The most frequently reported complications were sphincter disorder (39.1%), paraplegia (28.9%) and kyphosis (19.3%). TB osteomyelitis must always be borne in mind in countries where TB has high prevalence.     

氟喹诺酮类药物在结核病治疗中的研究进展

氟喹诺酮类药物是临床治疗中常用的抗生素种类,其抗菌谱较广,且具有较强的杀菌效果。近年来,有许多国内研究发现氟喹诺酮类药物对于肺结核杆菌的灭菌效果较好,因此,该药物越来越广泛地应用于肺结核的临床治疗,取得了较好效果的同时也存在着一定的不合理用药情况,影响治疗效果和患者身体健康。本文就近几年来我国对于氟喹诺酮类药物治疗结核病的相关研究进展进行综述,主要包括氟喹诺酮类药物治疗初治结核病、复治结核病和耐多药结核病的应用效果以及不同种类氟喹诺酮类药物对结核病的疗效,为结核病的治疗提供一定的参考和依据。