We investigated the role of reactive oxygen species (ROS) in dendritic cell (DC) differentiation by 10-kDa Mycobacterium tuberculosis secretory Ag (MTSA) and survival of mycobacteria therein. Compared with GM-CSF, MTSA induced lower ROS production during DC differentiation from precursors. This result correlated with higher superoxide dismutase 1 expression in MTSA stimulated precursors as compared with GM-CSF stimulation. Furthermore, a negative regulation of protein kinase C (PKC) activation by ROS was observed during DC differentiation. ROS inhibited the rapid and increased phosphorylation of PKCα observed during DC differentiation by MTSA. In contrast, ROS inhibition increased the weak and delayed PKCα phosphorylation by GM-CSF. Similar to DC differentiation, upon activation with either M. tuberculosis cell extract (CE) or live Mycobacterium bovis bacillus Calmette-Guérin (BCG), DCs differentiated with MTSA (MTSA-DCs) generated lower ROS levels when compared with DCs differentiated with GM-CSF (GM-CSF-DCs). Likewise, a negative regulation of PKCα phosphorylation by ROS was once again observed in DCs activated with either M. tuberculosis CE or live M. bovis BCG. However, a reciprocal positive regulation between ROS and calcium was observed. Compared with MTSA-DCs, stimulation of GM-CSF-DCs with M. tuberculosis CE induced a 2-fold higher ROS-dependent calcium influx. However, pretreatment of MTSA-DCs with H2O2 increased calcium mobilization. Finally, lower ROS levels in MTSA-DCs correlated with increased intracellular survival of M. bovis BCG when compared with survival in GM-CSF-DCs. Although inhibiting ROS in GM-CSF-DCs increased M. bovis BCG survival, H2O2 treatment of MTSA-DCs decreased survival of M. bovis BCG. Overall our results suggest that DCs differentiated with Ags such as MTSA may provide a niche for survival and/or growth of mycobacteria following sequestration of ROS.

BackgroundDendritic cells (DCs) play major roles in mediating immune responses to mycobacteria. A crucial aspect of this is the priming of T cells via chemokines and cytokines. In this study we investigated the roles of chemokines RANTES and IP-10 in regulating protective responses from Mycobacterium tuberculosis (M. tb) 10 kDa Culture Filtrate Protein-10 (CFP-10) differentiated DCs (CFP10-DCs).Methods and FindingsInfection of CFP10-DCs with mycobacteria down-modulated RANTES and IP-10 levels. Pathway specific microarray analyses showed that in addition to RANTES and IP-10, mycobacteria infected CFP10-DCs showed reduced expression of many Th1 promoting chemokines and chemokine receptors. Importantly, T cells co-cultured with RANTES and IP-10 conditioned CFP10-DCs mediated killing of mycobacteria from infected macrophages. Similarly, T cells recruited by RANTES and IP-10 conditioned CFP10-DCs mediated significant killing of mycobacteria from infected macrophages. IFN-gamma treatment of CFP10-DCs restored RANTES and IP-10 levels and T cells activated by these DCs mediated significant killing of virulent M. tb inside macrophages. Adoptive transfer of either RANTES and IP-10 or IL-12 and IFN-gamma conditioned CFP10-DCs cleared an established M. tb infection in mice. The extent of clearance was similar to that obtained with drug treatment.ConclusionsThese results indicate that chemokine and cytokine secretion by DCs differentiated by M. tb antigens such as CFP-10 play major roles in regulating protective immune responses at sites of infection.

We report that stimulation of Mycobacterium tuberculosis (M. tuberculosis) secretory antigen (MTSA)-differentiated dendritic cells (DCs) and MTSA-matured DCs with M. tuberculosis cell extract (CE) down-regulated proinflammatory responses to CE-primed T (CE-T) cells by increasing surface expression of CD86 after CE stimulation. CE stimulation also decreased interleukin (IL)-12p40 and interferon (IFN)- gamma levels and increased IL-10 and transforming growth factor- beta 1 (TGF- beta 1) levels from these DCs. Blocking either CD86, IL-10, or TGF- beta with monoclonal antibodies before CE stimulation restored the attenuated T helper 1 (Th1) responses of CE-T cells. Conversely, treatment of these DCs with IL-12p70 and/or IFN- gamma completely restored Th1 responses from CE-T cells. These results indicate that M. tuberculosis secretory antigens down-regulate proinflammatory Th1 responses to mycobacteria by differentially modulating the cytokine profiles and surface densities of costimulatory molecules on DCs. Of importance, this down-regulation is independent of the maturation status of MTSA-activated DCs and can be rescued after treatment of DCs with IFN- gamma or IL-12.

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Purpose: To analyze the demographics and clinical outcomes of posterior chamber phakic intraocular (IOL) implantation for refractive amblyopia in children and adolescents. Methods: A prospective interventional study was performed on children and adolescents with amblyopia at a tertiary eye care center from January 2021 to August 2022. Twenty-three eyes of 21 anisomyopic and isomyopic amblyopia patients operated for posterior chamber phakic IOL (Eyecryl phakic IOL) as a treatment for amblyopia were included in the study. Patient demographics, pre- and postoperative visual acuity, cycloplegic refraction, anterior and posterior segment examination, intraocular pressure, pachymetry, contrast sensitivity, endothelial count, and patient satisfaction scores were evaluated. Patients were followed up at day 1, 6 weeks, 3 months, and 1 year after surgery, and visual outcomes and complications were documented. Results: The mean age of patients was 14.16 ± 3.49 years (range: 10–19 years). The mean intraocular lens power was − 12.20 diopter spherical (DS) in 23 eyes and − 2.25 diopter cylindrical (DC) in four patients. The mean unaided distant visual acuity (UDVA) and best-corrected visual acuity (BCVA) were 1.39 ± 0.25 and 0.40 ± 0.21 preoperatively on the log of minimum angle of resolution (logMAR) chart. Postoperatively, the visual acuity improved by 2.6 lines in 3 months period and maintained till 1 year. Postsurgery, contrast sensitivity in the amblyopic eyes significantly improved, and the average endothelial loss recorded was 5.78% at 1 year, which was statistically insignificant. Patient satisfaction score was statistically significant, with 4.736/5 recorded on the Likert scale. Conclusion: Posterior chamber phakic IOL is a safe, effective, and alternative method for treating amblyopia patients who are noncompliant with glasses, contact lenses, and keratorefractive procedures.