The Transcription Factor NFATp Plays a Key Role in Susceptibility to TB in Mice

In T cells, the transcription factor nuclear factor of activated T cells p (NFATp) is a key regulator of the cytokine genes tumor necrosis factor (TNF) and interferon-γ (IFN-γ). Here, we show that NFATp-deficient (NFATp^(−/−)) mice have a dramatic and highly significant increase in mortality after Mycobacterium tuberculosis (MTb) infection as compared to mortality of control animals after MTb infection. Animals deficient in NFATp have significantly impaired levels of TNF and IFN-γ transcription and protein expression in naïve or total CD4+ T cells, but display wild-type levels of TNF mRNA or protein from MTb-stimulated dendritic cells (DC). The rapid mortality and disease severity observed in MTb-infected NFATp^(−/−) mice is associated with dysregulated production of TNF and IFN-γ in the lungs, as well as with increased levels of TNF, in their serum. Furthermore, global blocking of TNF production by injection of a TNF neutralizaing agent at 6 weeks, but not 12 weeks, post-MTb-infection further decreased the survival rate of both wild-type and NFATp^(−/−) mice, indicating an early role for TNF derived from cells from the monocyte lineage in containment of infection. These results thus demonstrate that NFATp plays a critical role in immune containment of TB disease in vivo, through the NFATp-dependent expression of TNF and IFN-γ in T cells

Anti-vascular endothelial growth factor treatment normalizes tuberculosis granuloma vasculature and improves small molecule delivery

Tuberculosis (TB) causes almost 2 million deaths annually, and an increasing number of patients are resistant to existing therapies. Patients who have TB require lengthy chemotherapy, possibly because of poor penetration of antibiotics into granulomas where the bacilli reside. Granulomas are morphologically similar to solid cancerous tumors in that they contain hypoxic microenvironments and can be highly fibrotic. Here, we show that TB-infected rabbits have impaired small molecule distribution into these disease sites due to a functionally abnormal vasculature, with a low-molecular-weight tracer accumulating only in peripheral regions of granulomatous lesions. Granuloma-associated vessels are morphologically and spatially heterogeneous, with poor vessel pericyte coverage in both human and experimental rabbit TB granulomas. Moreover, we found enhanced VEGF expression in both species. In tumors, antiangiogenic, specifically anti-VEGF, treatments can “normalize” their vasculature, reducing hypoxia and creating a window of opportunity for concurrent chemotherapy; thus, we investigated vessel normalization in rabbit TB granulomas. Treatment of TB-infected rabbits with the anti-VEGF antibody bevacizumab significantly decreased the total number of vessels while normalizing those vessels that remained. As a result, hypoxic fractions of these granulomas were reduced and small molecule tracer delivery was increased. These findings demonstrate that bevacizumab treatment promotes vascular normalization, improves small molecule delivery, and decreases hypoxia in TB granulomas, thereby providing a potential avenue to improve delivery and efficacy of current treatment regimens

Metaplastic ossification in the cartilage of the bronchus of a patient with chronic multi-drug resistant tuberculosis: a case report

<p>Abstract</p> <p>Introduction</p> <p>Pulmonary ossification has been rarely observed in pulmonary fibrosis and in some chronic respiratory diseases such as chronic obstructive pulmonary disease. We report here a metaplastic ossification in the bronchial cartilage of a patient with multi-drug resistant tuberculosis.</p> <p>Case presentation</p> <p>We report the case of a 41-year-old Asian man from Korea with chronic multi-drug resistant tuberculosis with a rare focus of bone formation from the cartilage of a bronchus subtending an active cavity. The patient had a large multi-lobed, thick-walled cavitary tuberculosis lesion in his left upper lobe. Severe infiltration of his lymphocytes and epithelioid cells, along with some giant cells and neutrophils, was observed in the patient's bronchial wall. Desquamated bronchial epithelium and acid-fast bacilli were found inside his bronchus. A small focus of bony metaplasia was found in the cartilage of his bronchial wall. Histopathological examination confirmed calcification and showed hematopoietic cells forming in his marrow cavity.</p> <p>Conclusions</p> <p>Chronic inflammation in the lungs of our patient, caused by underlying tuberculosis, probably played a role in the development of osseous metaplasia from the associated cartilage of the bronchial wall.</p

Bacterial loads measured by the Xpert MTB/RIF assay as markers of culture conversion and bacteriological cure in pulmonary TB

Introduction Biomarkers are needed to monitor tuberculosis (TB) treatment and predict treatment outcomes. We evaluated the Xpert MTB/RIF (Xpert) assay as a biomarker for TB treatment during and at the end of the 24 weeks therapy. METHODS: Sputum from 108 HIV-negative, culture-positive pulmonary TB patients was analyzed using Xpert at time points before and during anti-TB therapy. Results were compared against culture. Direct Xpert cycle-threshold (Ct), a change in the Ct (delta Ct), or a novel "percent closing of baseline Ct deficit" (percent closing) were evaluated as classifiers of same-day and end-of-treatment culture and therapeutic outcomes. RESULTS: Xpert was positive in 29/95 (30.5%) of subjects at week 24; and positive one year after treatment in 8/64 (12.5%) successfully-treated patients who remained free of tuberculosis. We identified a relationship between initial bacterial load measured by baseline Xpert Ct and time to culture conversion (hazard ratio 1.06, p = 0.0023), and to the likelihood of being among the 8 treatment failures at week 24 (AUC = 72.8%). Xpert Ct was even more strongly associated with culture conversion on the day the test was performed with AUCs 96.7%, 99.2%, 86.0% and 90.2%, at Day 7, Week 4, 8 and 24, respectively. Compared to baseline Ct measures alone, a combined measure of baseline Ct plus either Delta Ct or percent closing improved the classification of treatment failure status to a 75% sensitivity and 88.9% specificity. CONCLUSIONS: Genome loads measured by Xpert provide a potentially-useful biomarker for classifying same day culture status and predicting response to therapy

Clinical Research and Development of Tuberculosis Diagnostics: Moving From Silos to Synergy

The development, evaluation, and implementation of new and improved diagnostics have been identified as critical needs by human immunodeficiency virus (HIV) and tuberculosis researchers and clinicians alike. These needs exist in international and domestic settings and in adult and pediatric populations. Experts in tuberculosis and HIV care, researchers, healthcare providers, public health experts, and industry representatives, as well as representatives of pertinent US federal agencies (Centers for Disease Control and Prevention, Food and Drug Administration, National Institutes of Health, United States Agency for International Development) assembled at a workshop proposed by the Diagnostics Working Group of the Federal Tuberculosis Taskforce to review the state of tuberculosis diagnostics development in adult and pediatric populations

Automated quantitative assay of fibrosis characteristics in tuberculosis granulomas

IntroductionGranulomas, the pathological hallmark of Mycobacterium tuberculosis (Mtb) infection, are formed by different cell populations. Across various stages of tuberculosis conditions, most granulomas are classical caseous granulomas. They are composed of a necrotic center surrounded by multilayers of histocytes, with the outermost layer encircled by fibrosis. Although fibrosis characterizes the architecture of granulomas, little is known about the detailed parameters of fibrosis during this process.MethodsIn this study, samples were collected from patients with tuberculosis (spanning 16 organ types), and Mtb-infected marmosets and fibrotic collagen were characterized by second harmonic generation (SHG)/two-photon excited fluorescence (TPEF) microscopy using a stain-free, fully automated analysis program.ResultsHistopathological examination revealed that most granulomas share common features, including necrosis, solitary and compact structure, and especially the presence of multinuclear giant cells. Masson’s trichrome staining showed that different granuloma types have varying degrees of fibrosis. SHG imaging uncovered a higher proportion (4%~13%) of aggregated collagens than of disseminated type collagens (2%~5%) in granulomas from matched tissues. Furthermore, most of the aggregated collagen presented as short and thick clusters (200~620 µm), unlike the long and thick (200~300 µm) disseminated collagens within the matched tissues. Matrix metalloproteinase-9, which is involved in fibrosis and granuloma formation, was strongly expressed in the granulomas in different tissues.DiscussionOur data illustrated that different tuberculosis granulomas have some degree of fibrosis in which collagen strings are short and thick. Moreover, this study revealed that the SHG imaging program could contribute to uncovering the fibrosis characteristics of tuberculosis granulomas

The Myxobacterial Antibiotic Myxovalargin: Biosynthesis, Structural Revision, Total Synthesis, and Molecular Characterization of Ribosomal Inhibition

Resistance of bacterial pathogens against antibiotics is declared by WHO as a major global health threat. As novel antibacterial agents are urgently needed, we re-assessed the broad-spectrum myxobacterial antibiotic myxovalargin and found it to be extremely potent against Mycobacterium tuberculosis. To ensure compound supply for further development, we studied myxovalargin biosynthesis in detail enabling production via fermentation of a native producer. Feeding experiments as well as functional genomics analysis suggested a structural revision, which was eventually corroborated by the development of a concise total synthesis. The ribosome was identified as the molecular target based on resistant mutant sequencing, and a cryo-EM structure revealed that myxovalargin binds within and completely occludes the exit tunnel, consistent with a mode of action to arrest translation during a late stage of translation initiation. These studies open avenues for structure-based scaffold improvement toward development as an antibacterial agent