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Introduction: Pleural tuberculosis (PlTB), the most common site of extrapulmonary TB, is characterized by a paucibacillary nature and a compartmentalized inflammatory response in the pleural cavity, both of which make diagnosis and management extremely challenging. Although transcriptional signatures for pulmonary TB have already been described, data obtained by using this approach for extrapulmonary tuberculosis and, specifically, for pleural tuberculosis are scarce and heterogeneous. In the present study, a set of candidate genes previously described in pulmonary TB was evaluated to identify and validate a transcriptional signature in clinical samples from a Brazilian cohort of PlTB patients and those with other exudative causes of pleural effusion. Methods: As a first step, target genes were selected by a random forest algorithm with recursive feature elimination (RFE) from public microarray datasets. Then, peripheral blood (PB) and pleural fluid (PF) samples from recruited patients presenting exudative pleural effusion were collected during the thoracentesis procedure. Transcriptional analysis of the selected top 10 genes was performed by quantitative RT-PCR (RT-qPCR). Results: Reanalysis of the public datasets identified a set of candidate genes (CARD17, BHLHE40, FCGR1A, BATF2, STAT1, BTN3A1, ANKRD22, C1QB, GBP2, and SEPTIN4) that demonstrated a global accuracy of 89.5% in discriminating pulmonary TB cases from other respiratory diseases. Our validation cohort consisted of PlTB (n = 35) patients and non-TB (n = 34) ones. The gene expressions of CARD17, GBP2, and C1QB in PF at diagnosis were significantly different between the two (PlTB and non-TB) groups (p < 0.0001). It was observed that the gene expressions of CARD17 and GBP2 were higher in PlTB PF than in non-TB patients. C1QB showed the opposite behavior, being higher in the non-TB PF. After anti-TB therapy, however, GBP2 gene expression was significantly reduced in PlTB patients (p < 0.001). Finally, the accuracy of the three above-cited highlighted genes in the PF was analyzed, showing AUCs of 91%, 90%, and 85%, respectively. GBP2 was above 80% (sensitivity = 0.89/specificity = 0.81), and CARD17 showed significant specificity (Se = 0.69/Sp = 0.95) in its capacity to discriminate the groups. Conclusion: CARD17, GBP2, and C1QB showed promise in discriminating PlTB from other causes of exudative pleural effusion by providing accurate diagnoses, thus accelerating the initiation of anti-TB therapy.
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Derrame Pleural , Tuberculosis Pleural , Tuberculosis Pulmonar , Humanos , Tuberculosis Pleural/diagnóstico , Tuberculosis Pleural/genética , Exudados y Transudados , Derrame Pleural/diagnóstico , Derrame Pleural/genética , Derrame Pleural/metabolismo , Brasil , Butirofilinas , Antígenos CDRESUMEN
BACKGROUND: Cytopathological examination of pleural effusions is a fast and minimally invasive method for verification of the presence of neoplastic cells. We report our 2-year experience using a categorised diagnostic system and reporting risks of malignancy (ROMs) for each defined category. METHODS: Cytological reports of patients between November 2016 and October 2018 were collected, with results primarily classified into a five-tiered classification scheme. Immunohistochemistry markers used in cytology and their results were also recorded. Final agreement to histology and overall test performance was calculated for cases with available concomitant (up to 3 months) pleural biopsies. RESULTS: A total of 519 samples from 385 patients were collected, being 29 (5.6%) classified as non-diagnostic, 291 (56%) as negative, 28 (5.4%) as atypical, 30 (5.8%) as suspicious and 141 (27.2%) as positive. Most requested markers were calretinin, TTF1, Ber-EP4 and Gata-3, being conclusive in 45 (76.3%) cases. Total cyto-histological agreement was achieved in 49 (80.3%) specimens, with an overall sensitivity and specificity of 69.4% and 93.3%, respectively. Positive predictive value was 96.2% and negative predictive value was of 56%. ROM for each diagnostic category was 50% for non-diagnostic, 44% for negative, 50% for atypical, 83.3% for suspicious and 96.2% for positive. CONCLUSIONS: Our 2-year retrospective study has shown a high specificity and positive predictive value for pleural cytology. The use of a five-tiered system has also shown to be highly effective, with a concordantly progressive higher ROM for the assigned diagnostic categories.
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Citodiagnóstico , Derrame Pleural Maligno/diagnóstico , Derrame Pleural/diagnóstico , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Biomarcadores de Tumor/genética , Calbindina 2/genética , Niño , Preescolar , Proteínas de Unión al ADN/genética , Femenino , Humanos , Inmunohistoquímica , Lactante , Masculino , Persona de Mediana Edad , Derrame Pleural/genética , Derrame Pleural/patología , Derrame Pleural Maligno/genética , Derrame Pleural Maligno/patología , Factores de Transcripción/genética , Adulto JovenRESUMEN
The ability of Mycobacterium tuberculosis (Mtb) to persist in its human host relies on numerous immune evasion strategies, such as the deregulation of the lipid metabolism leading to the formation of foamy macrophages (FM). Yet, the specific host factors leading to the foamy phenotype of Mtb-infected macrophages remain unknown. Herein, we aimed to address whether host cytokines contribute to FM formation in the context of Mtb infection. Our approach is based on the use of an acellular fraction of tuberculous pleural effusions (TB-PE) as a physiological source of local factors released during Mtb infection. We found that TB-PE induced FM differentiation as observed by the increase in lipid bodies, intracellular cholesterol, and expression of the scavenger receptor CD36, as well as the enzyme acyl CoA:cholesterol acyl transferase (ACAT). Importantly, interleukin-10 (IL-10) depletion from TB-PE prevented the augmentation of all these parameters. Moreover, we observed a positive correlation between the levels of IL-10 and the number of lipid-laden CD14+ cells among the pleural cells in TB patients, demonstrating that FM differentiation occurs within the pleural environment. Downstream of IL-10 signaling, we noticed that the transcription factor signal transducer and activator of transcription 3 was activated by TB-PE, and its chemical inhibition prevented the accumulation of lipid bodies and ACAT expression in macrophages. In terms of the host immune response, TB-PE-treated macrophages displayed immunosuppressive properties and bore higher bacillary loads. Finally, we confirmed our results using bone marrow-derived macrophage from IL-10-/- mice demonstrating that IL-10 deficiency partially prevented foamy phenotype induction after Mtb lipids exposure. In conclusion, our results evidence a role of IL-10 in promoting the differentiation of FM in the context of Mtb infection, contributing to our understanding of how alterations of the host metabolic factors may favor pathogen persistence.
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Acetil-CoA C-Acetiltransferasa/inmunología , Regulación Enzimológica de la Expresión Génica/inmunología , Interleucina-10/inmunología , Mycobacterium tuberculosis/inmunología , Derrame Pleural/inmunología , Factor de Transcripción STAT3/inmunología , Esterol O-Aciltransferasa , Tuberculosis Pleural/inmunología , Regulación hacia Arriba/inmunología , Acetil-CoA C-Acetiltransferasa/genética , Animales , Femenino , Células Espumosas , Humanos , Interleucina-10/genética , Masculino , Ratones , Ratones Noqueados , Mycobacterium tuberculosis/genética , Derrame Pleural/genética , Derrame Pleural/patología , Factor de Transcripción STAT3/genética , Tuberculosis Pleural/genética , Tuberculosis Pleural/patologíaRESUMEN
INTRODUCTION: Tuberculosis (TB) and malignant diseases are the most common causes of lymphocytic pleural effusion in adults. Serum and pleural fluid cytokine levels have been analyzed to help in the differential diagnosis, but with limited results. PURPOSE: This study investigates transcription levels of selected cytokine genes in pleural effusion of patients under investigation for TB. METHODS: This was a prospective study that included adult patients under investigation for pleural effusion in Brazil. The expression of 19 cytokine genes was analyzed by RT-qPCR. RESULTS: The majority of cytokine-related genes expressed in pleural fluid of TB patients were similar in non-TB patients, except for RORA and RORC genes, which showed a statistically higher level in TB. All cytokines in the Th17 pattern were induced in TB patients' pleural fluid. Patients with malignant pleural effusion expressed higher levels of IFN-α1, IFN-ß1, TNF-α, IL-4 and IL-6, and suppression of TGFß-1. CONCLUSION: There is still a lot to understand about the cytokine roles in the pro- and anti-inflammatory environment of exudative pleural effusions. The data presented here showed an increased expression of Th17 pattern cytokines genes in TB patients that could be used as markers to differentiate tuberculous pleuritis from other common causes of exudative pleural effusion.
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Citocinas/genética , Exudados y Transudados/metabolismo , Derrame Pleural/genética , Neoplasias Pleurales/genética , Neumonía/genética , ARN Mensajero/metabolismo , Tuberculosis Pleural/genética , Adenocarcinoma/complicaciones , Adenocarcinoma/genética , Adenocarcinoma/secundario , Adolescente , Adulto , Anciano , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Niño , Neoplasias del Colon/genética , Neoplasias del Colon/patología , Femenino , Neoplasias Gastrointestinales/genética , Neoplasias Gastrointestinales/patología , Humanos , Leucemia Linfocítica Crónica de Células B/complicaciones , Leucemia Linfocítica Crónica de Células B/genética , Leucemia Linfocítica Crónica de Células B/patología , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Linfoma/complicaciones , Linfoma/genética , Linfoma/patología , Masculino , Mesotelioma/complicaciones , Mesotelioma/genética , Mesotelioma/patología , Persona de Mediana Edad , Derrame Pleural/diagnóstico , Derrame Pleural/etiología , Neoplasias Pleurales/complicaciones , Neoplasias Pleurales/patología , Neoplasias Pleurales/secundario , Neumonía/complicaciones , Neumonía/diagnóstico , Estudios Prospectivos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Toracocentesis , Transcriptoma , Tuberculosis Pleural/complicaciones , Tuberculosis Pleural/diagnóstico , Adulto JovenRESUMEN
OBJECTIVE: The objective of this study was to establish the value of different markers in differentiating reactive mesothelial cells from metastatic adenocarcinomatous cells in serous effusions (SE). METHODS: Forty-five SE were processed for morphological examination (Papanicolaou stain), assessment of ploidy, AgNOR counting and immunocytochemical assay of carcinoembryonic antigen (CEA), epithelial membrane antigens (EMA), Ber-EP4 and Leu-M1. Ploidy was established in an image-analyser in smears stained by the Feulgen stain method. AgNOR dots were counted in the smears stained with the silver nitrate assay for non-histone proteins present in the nucleolar organizer region. CEA, EMA, Ber-EP4 and Leu-M1 were evaluated by immunocytochemistry using the streptavidin-biotin complex method. RESULTS: All the smears with positive cytology were aneuploid. Three false negatives by morphological studies were aneuploid, with AgNOR values in two of them corresponding to the neoplastic group. CEA and Leu-M1 showed a low specificity; EMA and Ber-EP4 showed moderate sensitivity. CONCLUSIONS: The assessment of ploidy and the study of AgNOR were better methods than immunocytochemistry for distinguishing between reactive mesothelial cells and adenocarcinomatous cells in serous fluid.