Integrin α2β1 Expression Regulates Matrix Metalloproteinase-1-Dependent Bronchial Epithelial Repair in Pulmonary Tuberculosis.

Pulmonary tuberculosis (TB) is caused by inhalation of Mycobacterium tuberculosis, which damages the bronchial epithelial barrier to establish local infection. Matrix metalloproteinase-1 plays a crucial role in the immunopathology of TB, causing breakdown of type I collagen and cavitation, but this collagenase is also potentially involved in bronchial epithelial repair. We hypothesized that the extracellular matrix (ECM) modulates M. tuberculosis-driven matrix metalloproteinase-1 expression by human bronchial epithelial cells (HBECs), regulating respiratory epithelial cell migration and repair. Medium from monocytes stimulated with M. tuberculosis induced collagenase activity in bronchial epithelial cells, which was reduced by ~87% when cells were cultured on a type I collagen matrix. Matrix metalloproteinase-1 had a focal localization, which is consistent with cell migration, and overall secretion decreased by 32% on type I collagen. There were no associated changes in the specific tissue inhibitors of metalloproteinases. Decreased matrix metalloproteinase-1 secretion was due to ligand-binding to the α2β1 integrin and was dependent on the actin cytoskeleton. In lung biopsies, samples from patients with pulmonary TB, integrin α2β1 is highly expressed on the bronchial epithelium. Areas of lung with disrupted collagen matrix showed an increase in matrix metalloproteinases-1 expression compared with areas where collagen was comparable to control lung. Type I collagen matrix increased respiratory epithelial cell migration in a wound-healing assay, and this too was matrix metalloproteinase-dependent, since it was blocked by the matrix metalloproteinase inhibitor GM6001. In summary, we report a novel mechanism by which α2β1-mediated signals from the ECM modulate matrix metalloproteinase-1 secretion by HBECs, regulating their migration and epithelial repair in TB

Strategies for early detection of renal injury in HIV-infected patients : the new troponin

Tese de mestrado, Doenças Infecciosas Emergentes, Faculdade de Medicina, Universidade de Lisboa, 2011Background: HIV-infected patients have a known increased risk of kidney disease. For that reason, a biomarker that enables reliable detection of early and mild kidney dysfunction would be advantageous. Cystatin C is considered to be a better marker of kidney function than creatinine. Objectives: This study aimed to evaluate if serum cystatin C was a better marker than creatinine in a HIV-infected population. Material and Methods: This was an observational study of HIV-infected patients that attend the Infectious Diseases Department, at the Hospital Santa Maria, Lisbon. Patients with known kidney disease or HIV-2 infection were excluded. Clinical and demographic data was recorded and serum creatinine and cystatin C levels were determined. Results: A total of 242 patients were included. Cystatin C is an independent factor of age, gender, ethnicity and body mass index. Of the 17 patients with elevated levels of cystatin C but with an estimated creatinine clearance within normal range, 11 were on antiretroviral therapy with tenofovir and/or atazanavir. Fourteen patients were smokers and 10 patients had hepatitis C virus co-infection, which are known causes of inflammation. In a matched control group of 27 patients, with cystatin C within normal range, the majority of patients also were on an antiretroviral regimen with tenofovir and/or atazanavir. However, none of them had hepatitis C virus co-infection. Moreover, a statistical association was found between high levels of cystatin C and alanine transaminase. Differences between biomarkers’ levels and patients on combinations with tenofovir, ritonavir boosted atazanavir or both were also studied. Although it was not found any statistically significant difference in creatinine and estimated creatinine clearance, with cystatin C, patients that were on atazanavir or tenofovir and had a higher level of cystatin C, compared with patients that never were on these drugs. Conclusion: Cystatin C levels may be increased in inflammatory conditions. Therefore, in HIV-infected patients, where chronic systemic inflammation is present, often associated to other inflammation sources, such as chronic hepatitis C virus co-infection, the use of cystatin C to monitor kidney function may overestimate kidney impairment. In what concerns to nephrotoxicity, it was found that patients on atazanavir had higher levels of cystatin C. It is important to develop prospective studies, in order to assess the real long-time impact of the more recent antiretroviral drugs on kidney function.Introdução: Os doentes infectados por VIH possuem um risco aumentado para o desenvolvimento de doença renal. Por esta razão, seria importante utilizar um marcador que permitisse uma detecção precoce de alterações da função renal. A cistatina C tem sido referida na literatura como um melhor marcador da função renal, comparativamente com a creatinina. Objectivos: O objectivo deste estudo é analisar se a cistatina C sérica constitui um melhor marcador da função renal que a creatinina, para a população de doentes infectados por VIH. Material e métodos: Este é um estudo observacional de doentes infectados por VIH, seguidos no Serviço de Doenças Infecciosas do Hospital de Santa Maria, Lisboa. Os doentes com doença renal documentada e infecção por VIH-2 foram excluídos deste estudo. Foram recolhidos dados clínicos e demográficos e foram determinados os níveis séricos de creatinina e cistatina C. Resultados: Um total de 242 doentes foi incluído neste estudo. Os níveis de cistatina C mostraram-se independentes da idade, sexo, etnia e índice de massa corporal. Dos 17 doentes com níveis elevados de cistatina C mas com clearance da creatinina dentro dos valores de referência, 11 doentes estavam sob terapêutica antirretroviral com tenofovir e/ou atazanavir potenciado com ritonavir. Catorze doentes eram fumadores e 10 estavam co-infectados por vírus da hepatite C, que constitui um factor conhecido de inflamação. Num grupo controlo de 27 doentes, com cistatina C dentro dos valores normais, a maioria dos doentes também se encontrava sob terapêutica antirretroviral com tenofovir e/ou atazanavir. No entanto, nenhum possuía co-infecção por vírus da hepatite C. Foram também analisadas diferenças entre os níveis dos marcadores e doentes em combinações com tenofovir, atazanavir potenciado com ritonavir ou com ambos. Embora não tenha sido encontrada nenhuma diferença estatisticamente significativa nos níveis de creatinina e clearence da creatinina estimado, com a cistatina C, doentes sob atazanavir ou tenofovir e atazanavir possuíam níveis mais elevados deste marcador, comparativamente com os doentes que nunca estiveram sob estes antirretrovirais. Conclusão: Os níveis de cistatina C podem aumentar com factores inflamatórios. Por esta razão, no caso dos doentes infectados por VIH, onde existe uma inflamação sistémica crónica, muitas vezes associada a outras fontes de inflamação, como a co-infecção por vírus da hepatite C, a utilização exclusiva da cistatina C para a monitorização da função renal pode conduzir a uma sobrestimação de uma lesão renal. Em termos de nefrotoxicidade, verificou-se que doentes sob atazanavir possuíam níveis mais elevados de cistatina C. É de extrema importância, o desenvolvimento de novos estudos prospectivos, de forma a permitir a análise do impacto a longo prazo destes antirretrovirais mais recentes na função renal

Hypoxia and tissue destruction in pulmonary TB.

BACKGROUND: It is unknown whether lesions in human TB are hypoxic or whether this influences disease pathology. Human TB is characterised by extensive lung destruction driven by host matrix metalloproteinases (MMPs), particularly collagenases such as matrix metalloproteinase-1 (MMP-1). METHODS: We investigated tissue hypoxia in five patients with PET imaging using the tracer [18F]-fluoromisonidazole ([18F]FMISO) and by immunohistochemistry. We studied the regulation of MMP secretion in primary human cell culture model systems in normoxia, hypoxia, chemical hypoxia and by small interfering RNA (siRNA) inhibition. RESULTS: [18F]FMISO accumulated in regions of TB consolidation and around pulmonary cavities, demonstrating for the first time severe tissue hypoxia in man. Patlak analysis of dynamic PET data showed heterogeneous levels of hypoxia within and between patients. In Mycobacterium tuberculosis (M.tb)-infected human macrophages, hypoxia (1% pO2) upregulated MMP-1 gene expression 170-fold, driving secretion and caseinolytic activity. Dimethyloxalyl glycine (DMOG), a small molecule inhibitor which stabilises the transcription factor hypoxia-inducible factor (HIF)-1α, similarly upregulated MMP-1. Hypoxia did not affect mycobacterial replication. Hypoxia increased MMP-1 expression in primary respiratory epithelial cells via intercellular networks regulated by TB. HIF-1α and NF-κB regulated increased MMP-1 activity in hypoxia. Furthermore, M.tb infection drove HIF-1α accumulation even in normoxia. In human TB lung biopsies, epithelioid macrophages and multinucleate giant cells express HIF-1α. HIF-1α blockade, including by targeted siRNA, inhibited TB-driven MMP-1 gene expression and secretion. CONCLUSIONS: Human TB lesions are severely hypoxic and M.tb drives HIF-1α accumulation, synergistically increasing collagenase activity which will lead to lung destruction and cavitation.Medical Research CouncilThis is the final version of the article. It first appeared from the British Medical Journal via https://doi.org/10.1136/thoraxjnl-2015-20740

Neutrophil-Derived MMP-8 Drives AMPK-Dependent Matrix Destruction in Human Pulmonary Tuberculosis.

Pulmonary cavities, the hallmark of tuberculosis (TB), are characterized by high mycobacterial load and perpetuate the spread of M. tuberculosis. The mechanism of matrix destruction resulting in cavitation is not well defined. Neutrophils are emerging as key mediators of TB immunopathology and their influx are associated with poor outcomes. We investigated neutrophil-dependent mechanisms involved in TB-associated matrix destruction using a cellular model, a cohort of 108 patients, and in separate patient lung biopsies. Neutrophil-derived NF-kB-dependent matrix metalloproteinase-8 (MMP-8) secretion was up-regulated in TB and caused matrix destruction both in vitro and in respiratory samples of TB patients. Collagen destruction induced by TB infection was abolished by doxycycline, a licensed MMP inhibitor. Neutrophil extracellular traps (NETs) contain MMP-8 and are increased in samples from TB patients. Neutrophils lined the circumference of human pulmonary TB cavities and sputum MMP-8 concentrations reflected TB radiological and clinical disease severity. AMPK, a central regulator of catabolism, drove neutrophil MMP-8 secretion and neutrophils from AMPK-deficient patients secrete lower MMP-8 concentrations. AMPK-expressing neutrophils are present in human TB lung biopsies with phospho-AMPK detected in nuclei. These data demonstrate that neutrophil-derived MMP-8 has a key role in the immunopathology of TB and is a potential target for host-directed therapy in this infectious disease

Tissue specificity of MMP gene expression and secretion in tuberculosis

In tuberculosis (TB), matrix metalloproteinases (MMPs) have a major role in tissue destruction and cavitation. The composition of each extracellular matrix (ECM) gives specificity to tissues and is important in control of local immune responses by acting as a “molecular postcode”. Hence, it is likely to have important implications in TB. During central nervous system (CNS) infection, the effect of TB on blood-brain barrier (BBB) function is unknown. I hypothesised that the ECM environment may determine the MMP response during innate immune activation in pulmonary and CNS TB. I aimed to investigate the effect of adhesion to the lung’s ECM in regulation of MMP expression and activity. I also aimed to develop a BBB cellular model and investigate the role of Mycobacterium tuberculosis (Mtb)-driven MMP secretion on BBB function. In a model of pulmonary TB, human bronchial epithelial cells (NHBEs) and monocytes were exposed to ECM components and stimulated with conditioned medium from Mtb-infected monocytes (CoMtb) or infected with Mtb. Type I collagen (Coll-I) matrix was shown to decrease MMP-1 mRNA accumulation by 48% and collagenolytic activity compared to NHBEs in the absence of matrix. MMP-1 co-localised with integrin α2β1 resulting in enhanced cell migration in wound healing assays. In contrast, soluble Coll-I led to integrin α2β1 occupancy without clustering and caused a 7-fold increase in collagenolytic activity but decreased migration. In Mtb-infected monocytes, adhesion to ECM components increased MMP-1 secretion by over 60%. Fibronectin and Coll-I also increased MMP-10 by 55% and 90% respectively. Surface expression of integrin αVβ3 was upregulated by Mtb-infection and adhesion to Coll-I. Activation of integrin αVβ3 mimicked the effect of Coll-I MMP secretion, while its inhibition impaired monocyte migration. CoMtb-stimulation of the BBB model decreased trans-endothelial electrical resistance from 154±1.2ohm.cm2 to 111.6±4.7ohm.cm2, while Papp increased 3-fold. Coll-IV and tight junction proteins (TJPs) degradation was also detected. MMP concentrations increased 125-fold for MMP-1 (0.35±2 to 43.8±5.1ng/mL) and 619-fold for MMP-9 (0.072±0.014 to 44.6±8.9ng/ml). Treatment with the MMP inhibitor Ro32-3555 prevented BBB disruption. Neutrophil and monocyte transmigration increased 60% and 80% respectively and returned to control levels with MMP blockade. MMP-9 was shown to be responsible for BBB disruption and its inhibition by antibodies prevented BBB disruption. The Hedgehog pathway was downregulated during infection, resulting in decrease TJPs gene expression, which contributes to BBB dysfunction. In summary, the ECM regulates both epithelial and monocyte expression of MMP-1 via integrins signalling. In the CNS, MMP-9 drives tissue destruction and BBB disruption which may be a potential reversible event in CNS TB immunopathologyOpen Acces

Use of flow cytometry (Sysmex® UF-100) to screen for positive urine cultures: in search for the ideal cut-off

Background: Cultures for urinary tract infections (UTI) constitute a large workload in the clinical microbiology laboratory, although up to 80% are usually negative. Several automated methods are available to screen urines for UTI, one being the flow cytometry-based Sysmex® UF-100. Methods: The performance of the UF-100 was evaluated over a 16-month period using urine culture as the reference method. Results: During this period, a total of 5356 urine samples were studied (469 children; 3229 women and 1658 men), of which 706 were culture positive (593 grew Gram negative bacilli). Receiver operating characteristics (ROC) curve analysis showed an area under the curve (AUC) of 0.83 for leukocytes and 0.85 for bacterial count. Applying cut-off values reported in the literature gave sensitivities ranging from 75% to 90%, resulting in 73–174 false negatives (FN). Using a logical combination (leukocytes ≥15×106/L OR bacteria ≥500×106/L) gave a sensitivity of 98%. However, the specificity dropped to 25%, resulting in 15 FN. Conclusions: Screening urine samples for UTI detects a large number of culture positive samples. However, the rather large number of FN observed precludes the use of the UF-100 as a routine screening method to exclude urine samples from culture. Clin Chem Lab Med 2010;48:289–92.Peer Reviewe

Matrix metalloproteinase-9 activity and a downregulated Hedgehog pathway impair blood-brain barrier function in an in vitro model of CNS tuberculosis (vol 7, 16031, 2017)

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