Okadaic acid (OA) is a specific and potent protein phosphatase inhibitor and tumor promoter. The present study establishes the role of reactive oxygen species (ROS) and mitogen activated protein kinases in cell death induced by okadaic acid. The study showed that okadaic acid is cytotoxic at 10 nM with an IC50 of 100 nM in U-937 cells. The CVDE assay and mitochondrial dehydrogenase assay showed a time dependent cytotoxicity. The phase contrast visualization of the OA treated cells showed the apoptotic morphology and was confirmed with esterase staining for plasma membrane integrity. OA activated caspases-7, 9 and 3, PARP cleavage and induced nuclear damage in a time and dose dependent manner. Compromised mitochondrial membrane potential, release of cytochrome-c and apoptosis inducing factor confirms the involvement of mitochondria. A time dependent decrease in glutathione levels and a dose dependent increase in ROS with maximum at 30 min were observed. ROS scavenger-N-acetyl cysteine, mitochondrial stabilizer-cyclosporin-A, and broad spectrum caspase inhibitor Z-VAD-FMK inhibited the OA induced caspase-3 activation, DNA damage and cell death but caspase-8 inhibitor had no effect. OA activated p38 MAPK and JNK in a time dependent manner, but not ERK½. MAP kinase inhibitors SB203580, SP600125 and PD98059 confirm the role of p38 MAPK and JNK in OA induced caspase-3 activation and cell death. Over all, our results indicate that OA induces cell death by generation of ROS, and activation of p38 MAPK and JNK, and executed through mitochondrial mediated caspase pathway.

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T-2 toxin is the most toxic trichothecene and a frequent contaminant in many agriculture products. Dietary ingestion represents the most common route of T-2 toxin exposure in humans. T-2 toxin exposure leads to many pathological conditions like nervous disorders, cardiovascular alterations, immune depression and dermal inflammation. However, the neuronal toxicity of T-2 toxin in vitro remains unclear. In the present study, we investigated the mechanism of T-2 toxin-induced apoptosis in human neuroblastoma cells (IMR-32). T-2 toxin was cytotoxic at a low concentration of 10 ng/ml. The 50% inhibitory concentration (IC50) of T-2 toxin was found to be 40 ng/ml as assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, crystal violet dye exclusion test and lactate dehydrogenase (LDH) leakage. T-2 toxin increased intracellular reactive oxygen species generation as early as 15 min and peaked at 60 min as analyzed by flow cytometry. Annexin V + propidium iodide staining showed time-dependent increase in percent apoptotic cells. DNA gel electrophoresis showed oligonucleosomal DNA fragmentation typical of apoptotic cells. Additionally, casapse-3 activation and PARP cleavage indicated involvement of mitochondrial mediated caspase-dependent pathway of apoptosis. Cell cycle analysis revealed time-dependent increase in sub-G1 population of cells and significant up-regulation of CDK2, CDK6, cyclin A and p21 messenger RNA (mRNA) levels. Exposure to T-2 toxin induced the phosphorylation of extracellular signal-regulated kinase (ERK), p38-mitogen-activated protein kinase and c-jun N-terminal kinases (JNK). Analysis of human phospho-mitogen-activated protein kinase (MAPK) antibody array revealed time-dependent increase in phosphorylation. Upstream of ERK pathway Grb2, Ras and Raf and downstream transcription factors c-fos and c-jun were significantly up-regulated. Z-VAD-FMK and MAPK inhibitors (PD 98059, SB 203580 and ZM 336372) exposure prior to T-2 toxin treatment significantly decreased percent of apoptotic cells compared to only T-2 toxin-exposed cells. Results of the present study show that T-2 toxin at nanogram concentrations can induce apoptosis in human neuronal cells through multiple signal transduction pathways. The study provides possible leads for developing therapeutic approaches to prevent T-2 toxin-induced neurotoxicity.

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Aldose reductase (ALR) enzyme plays a significant role in conversion of excess amount of glucose into sorbitol in diabetic condition, inhibitors of which decrease the secondary complication of diabetes mellitus. To understand the structural interaction of inhibitors with ALR enzyme and develop more effective ALR inhibitors, a series of substituted 5-phenylbenzoate containing N-substituted rhodanine derivatives were synthesized and evaluated for their in vitro ALR inhibitory activity. Docking studies of these compounds were carried out, which revealed that the 5-phenylbenzoate moiety deeply influenced the key π-π stacking while 4-oxo-2-thioxothiazolidines contributed in hydrogen bond interactions. The phenyl ring of benzylidene system occupied in specific pocket constituted from Phe115, Phe122, Leu300 and Cys303 while the rhodanine ring forms a tight net of hydrogen bond with Val47 at anionic binding site of the enzyme. The structural insights obtained from the docking study gave better understanding of rhodanine and macromolecular interaction and will help us in further designing and improving of ALR inhibitory activity of rhodanine analogs.

Mesenchymal stem/stromal cells (MSC) are well known for immunomodulation; however, the mechanisms involved in their benefits in the ischemic retina are unknown. This study tested the hypothesis that MSC induces upregulation of transcription factor forkhead box protein P3 (Foxp3) in T cells to elicit immune modulation, and thus, protect against retinal damage. Induced MSCs (iMSCs) were generated by differentiating the induced pluripotent stem cells (iPSC) derived from urinary epithelial cells through a noninsertional reprogramming approach. In in-vitro cultures, iMSC transferred mitochondria to immune cells via F-actin nanotubes significantly increased oxygen consumption rate (OCR) for basal respiration and ATP production, suppressed effector T cells, and promoted differentiation of CD4+CD25+ T regulatory cells (Tregs) in coculture with mouse splenocytes. In in-vivo studies, iMSCs transplanted in ischemia-reperfusion (I/R) injured eye significantly increased Foxp3+ Tregs in the retina compared to that of saline-injected I/R eyes. Furthermore, iMSC injected I/R eyes significantly decreased retinal inflammation as evidenced by reduced gene expression of IL1β, VCAM1, LAMA5, and CCL2 and improved b-wave amplitudes compared to that of saline-injected I/R eyes. Our study demonstrates that iMSCs can transfer mitochondria to immune cells to suppress the effector T cell population. Additionally, our current data indicate that iMSC can enhance differentiation of T cells into Foxp3 Tregs in vitro and therapeutically improve the retina’s immune function by upregulation of Tregs to decrease inflammation and reduce I/R injury-induced retinal degeneration in vivo.

The live attenuated SA14‐14‐2 Japanese encephalitis (JE) vaccine is a historical vaccine that protects against JE. Despite its extensive use, the mechanism of protective immunity conferred by the SA14‐14‐2 vaccine is not well established. Here, we used mouse models to understand the mechanism of the development of humoral immunity against the vaccine. The vaccine induces robust GC responses within a week postimmunization. In lethal virus challenge, we show that CD4+ T cells alone, but not CD8+ T cells, are sufficient to confer vaccine‐mediated protection. However, the CD4‐mediated protection was potentiated in the presence of vaccine‐primed CD8+ T cells. Employing CD8‐deficient mice, we show that both the protective traits of CD4+ T cells and the quality of antibody response to the vaccine are impaired in absence of CD8+ T cells. We further demonstrate that the poor protective immune response induced by the vaccine in absence of CD8+ T cells is mainly due to the impaired differentiation and function of follicular Th cells, leading to suboptimal GC reaction. Our study highlights an unprecedented role of CD8+ T cells in the establishment of humoral responses to the vaccine. By elucidating underlying cellular determinants of vaccine‐induced protective immunity, our work has implications for rational design of vaccines against JE virus and related flaviviruses.

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