A 3D Human Lung Tissue Model for Functional Studies on Mycobacterium tuberculosis Infection

Tuberculosis (TB) still holds a major threat to the health of people worldwide, and there is a need for cost-efficient but reliable models to help us understand the disease mechanisms and advance the discoveries of new treatment options. In vitro cell cultures of monolayers or co-cultures lack the three-dimensional (3D) environment and tissue responses. Herein, we describe an innovative in vitro model of a human lung tissue, which holds promise to be an effective tool for studying the complex events that occur during infection with Mycobacterium tuberculosis (M. tuberculosis). The 3D tissue model consists of tissue-specific epithelial cells and fibroblasts, which are cultured in a matrix of collagen on top of a porous membrane. Upon air exposure, the epithelial cells stratify and secrete mucus at the apical side. By introducing human primary macrophages infected with M. tuberculosis to the tissue model, we have shown that immune cells migrate into the infected-tissue and form early stages of TB granuloma. These structures recapitulate the distinct feature of human TB, the granuloma, which is fundamentally different or not commonly observed in widely used experimental animal models. This organotypic culture method enables the 3D visualization and robust quantitative analysis that provides pivotal information on spatial and temporal features of host cell-pathogen interactions. Taken together, the lung tissue model provides a physiologically relevant tissue micro-environment for studies on TB. Thus, the lung tissue model has potential implications for both basic mechanistic and applied studies. Importantly, the model allows addition or manipulation of individual cell types, which thereby widens its use for modelling a variety of infectious diseases that affect the lungs.     

Differences between tuberculosis cases infected with Mycobacterium africanum, West African type 2, relative to Euro-American Mycobacterium tuberculosis: an update

Mycobacterium africanum (MAF) is a common cause of human pulmonary tuberculosis in West Africa. We previously described phenotypic differences between MAF and Mycobacterium tuberculosis (MTB) among 290 patients. In the present analysis, we compared 692 tuberculosis patients infected with the two most common lineages within the (MTB) complex found in the Gambia, namely MAF West African type 2 (39% prevalence) and Euro-American MTB (55% prevalence). We identified additional phenotypic differences between infections with these two organisms. MAF patients were more likely to be older and HIV infected. In addition, they had worse disease on chest X-ray, despite complaining of cough for an equal duration, and were more likely severely malnourished. In this cohort, the prevalence of MAF did not change significantly over a 7-year period.     

脂阿拉伯甘露糖联合38kD蛋白在结核病诊断中的应用

本研究旨在评价脂阿拉伯甘露糖(LAM)联合38kD蛋白的混合抗原在结核病血清学诊断中的应用价值。选取128例活动性结核病患者(活动性肺结核107例,肺外结核21例)、51例潜伏性结核感染者和68例健康对照者的血清,检测抗LAM和38kD混合抗原的IgG抗体(LAM-38kD-IgG)。结果显示,LAM-38kD-IgG检测的总敏感度为46.88%,特异度为98.53%,阳性预测值为98.36%,阴性预测值为49.63%。其中肺结核组敏感度为46.73%,肺外结核组敏感度为47.62%(P0.05);初治组为45.54%,复治组为51.85%(P0.05)。涂片阳性肺结核组敏感度为52.54%,高于涂片阴性组(39.58%),但差异无统计学意义(P0.05)。LAM-38kD-IgG检测在对照组中的特异度为98.53%。其特异度远高于结核菌素试验(TST)的41.46%(P0.01)。LAM-38kD-IgG水平与结核病病情有一定的相关性。结果提示,应用LAM和38kD混合抗原的酶联免疫吸附试验(ELISA)在诊断活动性结核病方面具有中等敏感度、极高特异度及阳性预测值,不仅可与结核病的其他诊断方法联用以提高诊断的准确性,而且特别适用于细菌学检查阴性结核病和肺外结核病的诊断。     

结核分枝杆菌H37有毒株与无毒株的代谢相关基因pabB和lpdA启动子活性分析

【目的】通过分析结核分枝杆菌无毒株H37Ra的全基因组序列,并与H37Rv基因组序列比较,发现pabB和lpdA预测的启动子区发生了突变。我们利用报告基因,确认启动子突变与其基因转录水平的关系,探索结核分枝杆菌H37Ra毒力丧失的内在原因。【方法】利用生物信息学方法预测这两对基因的启动子区,采用PCR技术克隆这两对基因的启动子,与分枝杆菌启动子探针载体pMC210相连,DNA测序证实连接片段正确后,用电穿孔法将重组质粒转化至耻垢分枝杆菌mc2155。利用Quantitative Real-Time RT-PCR检测报告基因lacZ转录水平的差异,进一步验证这两对基因启动子的突变对相应基因转录水平的影响。【结果】Quantitative Real Time PCR检测结果显示H37RapabB启动子活性是H37Rv pabB启动子活性的6倍(p0.05),而H37Rv lpdA启动子的活性是H37RalpdA启动子的2倍(p0.05)。【结论】pabB,lpdA的启动子在H37Ra中的突变对其启动子的活性产生了影响,其中lpdA启动子的突变可能与结核分枝杆菌H37Ra的毒力丧失有关。     

A Cas12a-based CRISPR interference system for multigene regulation in mycobacteria

Mycobacteria are responsible for a heavy global disease burden, but their relative genetic intractability has long frustrated research efforts. The introduction of clustered regularly interspaced short palindromic repeats (CRISPR) interference (CRISPRi) has made gene repression in mycobacteria much more efficient, but limitations of the prototypical Cas9-based platform, for example, in multigene regulation, remain. Here, we introduce an alternative CRISPRi platform for mycobacteria that is based on the minimal type V Cas12a enzyme in combination with synthetic CRISPR arrays. This system is simple, tunable, reversible, can efficiently regulate essential genes and multiple genes simultaneously, and works as efficiently in infected macrophages as it does in vitro. Together, Cas12a-based CRISPRi provides a facile tool to probe higher-order genetic interactions in mycobacteria including Mycobacterium tuberculosis (Mtb), which will enable the development of synthetically lethal drug targets and the study of genes conditionally essential during infection.     

Mass-spectrometry based minisequencing method for the rapid detection of drug resistance in Mycobacterium tuberculosis

A MALDI TOF MS based minisequencing method has been developed and applied for the analysis of rifampin (RIF)- and isoniazid (INH)-resistant M. tuberculosis strains. Eight genetic markers of RIF resistance-nucleotide polymorphisms located in RRDR of rpoB gene, and three of INH resistance including codon 315 of katG gene and − 8 and − 15 positions of the promoter region of fabG1-inhA operon were worked out. Based on the analysis of 100 M. tuberculosis strains collected from the Moscow region in 1997–2005 we deduced that 91% of RIF-resistant and 94% of INH-resistant strains can be identified using the technique suggested. The approach is rapid, reliable and allows to reveal the drug resistance of M. tuberculosis strains within 12 h after sample isolation.     

Coyotes as Sentinels for Monitoring Bovine Tuberculosis Prevalence in White-Tailed Deer

Abstract: Mycobacterium bovis, the causative agent of bovine tuberculosis (bTB), is endemic in free-ranging white-tailed deer (Odocoileus virginianus) in 5 counties (Alcona, Alpena, Montmorency, Oscoda, and Presque Isle) in the northeastern Lower Peninsula of Michigan, USA. The presence of a wildlife reservoir of tuberculosis in Michigan and the incidence of bTB in cattle (Bos taurus) resulted in Michigan losing its bTB accredited-free status. Subsequent wildlife surveillance programs identified relatively high disease prevalence in coyotes (Canis latrans), generating interest in their potential to serve as a sentinel species to detect bTB prevalence in white-tailed deer. Our goal was to develop an empirical basis for generating hypotheses about the spatial epidemiology of bTB infection in coyotes for future surveillance, management, and modeling efforts. Though variation in coyote home-range size may confound attempts to spatially correlate the incidence of disease in the sentinel and host species at a fine scale, overlap zones (OZs) between adjacent coyote home ranges may be the appropriate sample unit for spatially correlating disease prevalence in coyotes and white-tailed deer. Because overlapping home ranges are generally configured around resource rich (e.g., small mammals and white-tailed deer) timber management patches, the OZ concentrates spatial interaction between adjacent groups in a relatively small area. Furthermore, there is a direct relationship between interaction probabilities and the spatial dispersion of those patches. The latter finding provides a useful metric to incorporate into future efforts to develop spatially explicit models of bTB dynamics. Modeling efforts can then be used as a foundation to predict the epidemiological ramifications of alterations in intensively managed forested landscapes. (JOURNAL OF WILDLIFE MANAGEMENT 71(5):1545-1554; 2007)     

A Frightening Device for Deterring Deer Use of Cattle Feeders

Abstract: The presence of bovine tuberculosis (TB) in cattle can negatively impact a state's economy and cattle industry. In Michigan, USA, wild white-tailed deer (Odocoileus virginianus) are a reservoir for reinfecting cattle herds. Although direct TB transmission between deer and cattle is rare, infected deer may contaminate cattle feed. To mitigate this risk, we designed and evaluated a deer-resistant cattle feeder (DRCF) device for deterring deer from feeders. The device delivered negative stimuli to condition deer to avoid cattle feeders. We tested the device by conducting a comparative change experiment at a high-density captive white-tailed deer operation in northeastern lower Michigan using pretreatment and treatment periods and random allocation of DRCF protection to 3 of 6 feeders during the treatment period. We used animal-activated cameras to collect data on deer use of feeders. Deer use was similar at protected and unprotected feeders during the pretreatment period but was lower at protected feeders during the treatment period. Deer-resistant cattle feeders were 100% effective during the first 2 treatment weeks, 94% during the first 5 weeks, but effectiveness then dropped to 61% during the final week. Excluding problems associated with low battery power and infrared sensors, DRCFs were 99% effective at deterring deer. Our results suggest that DRCFs can effectively limit deer use of cattle feed, potentially with minimal impact on feeding behavior of cattle, thus reducing potential transmission of bovine TB through contaminated feed. By employing DRCFs in bovine TB endemic areas, especially at times that deer are food stressed, agencies and producers can practically and economically reduce the potential for bovine TB to be transmitted from deer to cattle.     

Splenic lesions: FNA findings in 48 cases

OBJECTIVE: To study fine needle aspiration (FNA) cytological findings of splenic lesions and assess the role of FNA in the diagnosis of splenomegaly or splenic tumours. METHODS: This study consisted of 48 cases, 25 males and 23 females. The ages ranged between 3 and 71 years. Most of these cases were aspirated under ultrasonographic guidance and a small number were also aspirated directly by using a 22- to 23-gauge needles. The smears were stained with Wright-Giemsa and Papanicolaou methods. Special stains were used whenever necessary. RESULTS: In this study 14 cases were diagnosed as lymphoma-leukaemia, 7 cases as tuberculosis, 12 cases as kala-azar, 2 cases as hydatid cyst, 3 cases as storage diseases, 3 cases as simple cyst, 2 cases as myeloproliferative disorders, 2 cases as malignant tumours and 3 cases as hamartomas (these were wrongly diagnosed as malignant tumours). CONCLUSION: Splenic aspiration is a safe procedure and is very useful in the diagnosis of parasitic and infectious diseases, especially in endemic countries like Iran.