Dysregulated expression of microRNAs in aqueous humor from intraocular tuberculosis patients.

BACKGROUND: Systemic Mycobacterium tuberculosis (Mtb) infection alters microRNA's expression that controls cellular processes and modulates host defense mechanisms. However, the role of miRNAs in intraocular tuberculosis (IOTB) remains unknown. Therefore, this study aims to identify dysregulated miRNAs in the aqueous humor (AH) of patients with IOTB. METHODS: AH from intraocular tuberculosis patients (n = 2) and cataract controls (n = 2) were used for small RNA deep sequencing using HiSeq Illumina sequencing platform. Differentially expressed miRNAs and their targets were identified by the bioinformatics approach, and their regulatory functions were predicted by pathway enrichment analysis. The expression of selected miRNAs and their binding targets were further validated by real-time quantitative PCR (RT-qPCR). RESULTS: In total, we identified 56 differentially expressed miRNAs in the AH of intraocular tuberculosis (IOTB) patients compared to controls. We selected four significantly dysregulated miRNAs (miR-423-5p, miR-328-3p, miR-21-5p, and miR-16-5p) based on the RT-qPCR validation and predicted their gene targets. We developed a miRNA-targets regulatory network by combining pathways of interest and genes associated with TB. We identified that these four miRNAs might play an important role in IOTB pathogenesis via tuberculosis-associated pathways; PI3K-Akt signaling, autophagy and MAPK pathway. CONCLUSIONS: For the first time, this study identifies the dysregulation of four miRNAs in the AH of IOTB patients using the ultra-low input small-RNA sequencing approach. Further target prediction and validation identify the role of these miRNAs in tuberculosis pathogenesis via tuberculosis-related pathways. This study identifies miRNAs as potentially ideal biomarkers in the aqueous humor of IOTB patients.

NLRP3 inflammasome activation by mycobacterial ESAT-6 and dsRNA in intraocular tuberculosis.

The molecular basis of intraocular tuberculosis (TB) is not well understood. In this study, we investigated the role of two constituents of viable Mycobacterium tuberculosis - Early Secreted Antigenic Target-6 (ESAT-6), and mycobacterial RNA- in inflammasome activation in the retinal pigment epithelium (RPE), a key site of inflammation in intraocular TB. We found that ESAT-6 induced caspase-1 activation and inflammasome priming in mouse RPE cells, substantially more in wild-type than in Tlr2/3/4/7/9-/-, Myd88-/- or Nlrp3-/- RPE cells. Sub-retinal ESAT-6 injection resulted in greater RPE degeneration in wild-type than in Nlrp3-/- mice. In human ocular TB tissue sections, NLRP3 staining was noted in retina as well as RPE. Mycobacterial RNA, specifically its double stranded component, also induced caspase-1 activation, and the double stranded RNA was immunolocalized to human ocular TB sections. Our observations suggest that inflammasome activation in RPE by viable M. tuberculosis could potentially contribute to human intraocular TB.

Orbital tuberculosis: Clinicopathological correlation and diagnosis using PCR in formalin-fixed tissues.

This article describes the clinicopathological correlation and challenges encountered in the diagnosis of orbital tuberculosis (TB). Retrospective, interventional case series. A chart review of six patients who underwent biopsy for an orbital mass between January 2012 and December 2013 was performed. Institutional review board approval was obtained for the study. Clinical, radiological, and laboratory findings were documented, and response to antitubercular therapy (ATT) was noted. Age at presentation ranged from 18 to 64 years and duration of complaints varied from 2 weeks to 2 months. Pain, redness, and swelling with an orbital mass on computerized tomography (CT) were the common features. None of the patients was clinically suspected to have orbital TB at presentation. History of prior contact with a patient of TB and history of prior TB were present in one case each. Chest radiography (CXR) showed pulmonary consolidation in one case. Tuberculin skin test (TST) readings ranged from nonreactive to 23 mm. The presence of sputum acid fast bacilli (AFB) was tested in three cases and was negative. Polymerase chain reaction (PCR) for MPB64 gene was positive in all six cases. All cases responded to ATT. Clinical presentation of orbital TB can be variable and can simulate conditions like inflammatory disease and malignancy. Biopsy with histopathology plays a key role in diagnosis. Supportive laboratory investigations are necessary to clinch the diagnosis in cases where histopathology is suggestive of TB. In this regard, PCR for Mycobacterium tuberculosis (MTB) on tissue biopsy specimens may be a sensitive diagnostic tool.

Ocular tuberculosis: a clinicopathologic and molecular study.

OBJECTIVE: To analyze the clinical profiles, histopathologic features, and Mycobacterium tuberculosis polymerase chain reaction testing in patients with ocular tuberculosis. DESIGN: Retrospective case series. PARTICIPANTS: Forty-two patients. METHODS: This retrospective study was approved by the Armed Forces Institute of Pathology (AFIP) Institutional Review Board. The AFIP data banks were screened for cases with diagnosis of ocular tuberculosis using key words such as mycobacterium; tuberculosis; and acid-fast bacilli. Files and slides stained with hematoxylin-eosin and acid-fast staining were reviewed by the Division of Ocular Pathology and by the Infectious Diseases and Parasitic Diseases Pathology Branches. When available; blocks and unstained slides were sent to the Doheny Eye Institute; Los Angeles; California; for quantitative polymerase chain reaction (qPCR) analysis to detect Mycobacterium tuberculosis-specific DNA. MAIN OUTCOME MEASURES: Tuberculin skin test (TST) results, as well as the chest radiograph results, were recorded. When acid-fast bacilli were identified in tissue, their locations-ocular or extraocular sites-were recorded. Emphasis was placed on lymph node involvement and any systemic diseases. RESULTS: In the histopathologic specimens, microscopy revealed a paucity of organisms, and often there were only 1 or 2 organisms associated with or near a giant cell or near an area of necrosis. The qPCR analysis was performed on 6 biopsy specimens. These specimens showed necrotizing granulomatous inflammation from 6 different patients; 3 had positive qPCR results. In 2 of the 3 cases with positive qPCR results, acid-fast bacilli were not found in the tissue sections. In 17 patients, TST results were available; 10 had positive results (60%) and 7 had negative results (40%). Fourteen chest radiograph results were submitted, and 8 (57%) of 14 patients had normal chest films. CONCLUSIONS: This study suggests that in dealing with those populations at increased risk of tuberculosis (e.g., immigrants from endemic areas and human immunodeficiency virus-infected patients) or patients receiving biologic therapy, the ophthalmologist should endeavor to entertain this diagnosis and to rely on the support of infectious disease specialists and pulmonologists to help solidify the diagnosis, because the current methods for the diagnosis have limited sensitivity.