PURPOSE. Intraocular inflammation in tuberculosis-associated uveitis (TBU) is usually widespread, and responds unpredictably to treatment. Herein, we analyze the intraocular Tcell response in TBU for its surface phenotype, antigenic specificity, and functional characteristics to explain the above observations. METHODS. We isolated T cells from vitreous humor samples of patients with TBU and non-TB uveitis (controls). These were directly stained for surface markers CD4, CD8, CD45RO, CD45RA, CCR7, as well as intracellular cytokines IFN-c, TNF-a, and IL-17 and analyzed on flow cytometry. Antigenic specificity was determined by activating with Mycobacterium tuberculosis-specific antigen Early Secreted Antigenic Target-6 (ESAT-6) or retinal crude extract (RCE). Activation-induced cell death (AICD) characteristics of each T-cell population were analyzed by staining for PI-Annexin V, Fas-FasL, phospho-Akt, and phospho-Erk1/2. RESULTS. Immunophenotyping of vitreous humor samples demonstrated polyfunctional effector and central memory CD4þ T helper cells coexpressing IFN-c, TNF-a, and IL-17. Both ESAT-6 and RCE (autoreactive) specificity was found in T cells extracted from TBU samples; however, the mycobacterial and autoreactive T-cell populations differed in their sensitivity to AICD. Autoreactive T cells appeared to resist AICD through decreased expression of apoptotic markers, FasL and caspase-3, sustained phosphorylation of Akt, and lowered Erk1/2 activity.CONCLUSIONS. Autoreactive T cells are present in TBU eyes and are relatively resistant to AICD. An understanding of this epiphenomenon could be crucial in planning treatment of TBU patients, and interpreting response to anti-TB therapy.
A strong clinical suspicion that translates into early vitrectomy plus intravitreal antifungal antibiotics leads to favorable visual and structural outcomes. A long wait till microbiological confirmation to institute antifungal therapy may result in poorer outcome.
A loop-mediated isothermal amplification (LAMP) assay targeting the mpb64 gene for the diagnosis of intraocular tuberculosis was highly specific (100%), sensitive (85.7%), rapid, and easy to perform. The LAMP assay can be an alternative to conventional PCR for the diagnosis of ocular tuberculosis in resource-limited settings.I ntraocular tuberculosis (TB) is a disease with an extremely paucibacillary nature that is difficult to diagnose with conventional tests such as microscopy and culture. Molecular techniques such as PCR are vital for definitive diagnosis of this condition (1). However, PCR requires expensive instrumentation, which hinders its widespread use. The loop-mediated isothermal amplification (LAMP) assay relies on autocycling strand displacement DNA synthesis in the presence of Bst DNA polymerase under isothermal conditions and can be conducted in the laboratory water bath or heating block (2). We report the application of a LAMP assay targeting the mpb64 gene for rapid detection of intraocular tuberculosis.The study was approved by the institutional review board. A set of six LAMP primers (Table 1) targeting the mpb64 gene in the Mycobacterium tuberculosis H37Rv strain (GenBank accession number NC_018143) were designed using primer explorer V4 software (http://primerexplorer.jp/elamp4.0.0/index.html). The mpb64 gene was chosen since significant number of clinical isolates of M. tuberculosis in India may have low numbers of copies (1, 2) or no copies (3) of the IS6110 gene element. Also, the sensitivity of mpb64 detection was found to be much higher than that of IS6110 detection during PCR analysis of ocular samples in our laboratory (unpublished data). The LAMP assay was standardized according to a previously published protocol (4), except that 2.0 l instead of 1.5 l of 50 mM MgSO 4 was used. Nondenatured DNA was used as the template. The reaction mixture was incubated at 65°C for 1 h and heated at 80°C for 2 min to terminate the reaction in a thermal cycler (Veriti; Applied Biosystems), though the same could be achieved with a water bath. M. tuberculosis H37Rv DNA was used as the positive control and double-autoclaved Milli-Q water as the negative control. LAMP-positive amplicons were confirmed by adding 1 l of (1,000ϫ) SYBR green ⌱ dye (Sigma-Aldrich) to each reaction tube and incubating in the dark for 10 min, as previously described (5). A yellowish green color (under natural light conditions) indicated a positive reaction, while reddish orange indicated a negative reaction (Fig. 1). LAMP products were verified by 2% agarose gel electrophoresis with UV light transillumination. The analytical sensitivity of the LAMP assay was found to be 10 fg per reaction (2 copies/reaction), For clinical verification, we included 14 intraocular samples (10 aqueous humor and 4 vitreous humor) collected from patients with presumed ocular TB (criteria described previously) (1). Twenty aqueous humor samples from patients undergoing cataract surgery were used as negative controls. DNA was extracted from all the sa...
Gelatinous drop-like corneal dystrophy (GDLD) is a rare autosomal recessive form of corneal dystrophy characterised by subepithelial and stromal amyloid deposits. It is relatively common in Japan. It usually presents in the first two decades of life with subepithelial nodular lesions that later coalesce to form mulberry-like opacities. Although various surgical modalities have been attempted, recurrence remains a major challenge.
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