Analysis of TCR activation kinetics in primary human T cells upon focal or soluble stimulation.?

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Peritubular cells may modulate Leydig cell–mediated testosterone production through a nonclassic pathway

Objective: To evaluate whether paracrine signals are responsible for hormone-independent Leydig cell (Lc) steroidogenesis in the testis. Design: Testicular peritubular cells (PTc), Sertoli cells (Sc), and Lc were isolated and cultured, and their effect on each other was evaluated in terms of lactate production by Sc and testosterone (T) production by Lc. Setting: Research institution. Animal(S): Wistar rats. Intervention(S): Testes were surgically removed, and a new, easily adoptable procedure for PTc was developed; culture media from Sc, PTc, and Lc cultures were used for treating pure populations of these cells. Cells were also cocultured together. Main Outcome Measure(s): To assess culture or coculture supernatants for presence of metabolites and Lc messenger RNA analysis. Result(s): Although PTc secreted factor(s) did not augment production of Sc lactate, essential for germ cell survival, they significantly augmented T secretion by Lc, independent of StAR gene expression. Coculture studies showed that T production by Lc was significantly stimulated when Lc were cocultured with PTc, even in the absence of hormones. Conclusion(s): Testicular peritubular cell-derived factor(s) can potentially augment T production by Lc in a nonclassic manner even in a gonadotropin-deficient environment

Role of Corneal Stromal Cells on Epithelial Cell Function during Wound Healing

Following injury, corneal stromal keratocytes transform into repair-phenotype of activated stromal fibroblasts (SFs) and participate in wound repair. Simultaneously, ongoing bi-directional communications between corneal stromal-epithelial cells also play a vital role in mediating the process of wound healing. Factors produced by stromal cells are known to induce proliferation, differentiation, and motility of corneal epithelial cells, which are also subsequently the main processes that occur during wound healing. In this context, the present study aims to investigate the effect of SFs conditioned medium (SFCM) on corneal epithelial cell function along with substance P (SP). Antibody microarrays were employed to profile differentially expressed cell surface markers and cytokines in the presence of SFCM and SP. Antibody microarray data revealed enhanced expression of the ITGB1 in corneal epithelial cells following stimulation with SP whereas SFCM induced abundant expression of IL-8, ITGB1, PD1L1, PECA1, IL-15, BDNF, ICAM1, CD8A, CD44 and NTF4. All these proteins have either direct or indirect roles in epithelial cell growth, movement and adhesion related signaling cascades during tissue regeneration. We also observed activation of MAPK signaling pathway along with increased expression of focal adhesion kinase (FAK), paxillin, vimentin, β-catenin and vasodilator-stimulated phosphoprotein (VASP) phosphorylation. Additionally, epithelial-to-mesenchymal transition (EMT) regulating transcription factors Slug and ZEB1 expression were enhanced in the presence of SFCM. SP enriched the expression of integrin subunits α4, α5, αV, β1 and β3 whereas SFCM increased α4, α5, αV, β1 and β5 integrin subunits. We also observed increased expression of Serpin E1 following SP and SFCM treatment. Wound healing scratch assay revealed enhanced migration of epithelial cells following the addition of SFCM. Taken together, we conclude that SFCM-mediated sustained activation of ZEB1, Slug in combination with upregulated migration-associated integrins and ERK (Extracellular signal-regulated kinase)-FAK-paxillin axis, may lead to induce type 2 EMT-like changes during corneal epithelial wound healing

Analysis of the Differential Gene and Protein Expression Profiles of Corneal Epithelial Cells Stimulated with Alternating Current Electric Fields

In cells, intrinsic endogenous direct current (DC) electric fields (EFs) serve as morphogenetic cues and are necessary for several important cellular responses including activation of multiple signaling pathways, cell migration, tissue regeneration and wound healing. Endogenous DC EFs, generated spontaneously following injury in physiological conditions, directly correlate with wound healing rate, and different cell types respond to these EFs via directional orientation and migration. Application of external DC EFs results in electrode polarity and is known to activate intracellular signaling events in specific direction. In contrast, alternating current (AC) EFs are known to induce continuous bidirectional flow of charged particles without electrode polarity and also minimize electrode corrosion. In this context, the present study is designed to study effects of AC EFs on corneal epithelial cell gene and protein expression profiles in vitro. We performed gene and antibody arrays, analyzed the data to study specific influence of AC EFs, and report that AC EFs has no deleterious effect on epithelial cell function. Gene Ontology results, following gene and protein array data analysis, showed that AC EFs influence similar biological processes that are predominantly responsive to organic substance, chemical, or external stimuli. Both arrays activate cytokine–cytokine receptor interaction, MAPK and IL-17 signaling pathways. Further, in comparison to the gene array data, the protein array data show enrichment of diverse activated signaling pathways through several interconnecting networks

Analysis of TCR activation kinetics in primary human T cells upon focal or soluble stimulation

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TCR-mediated Erk activation does not depend on Sos and Grb2 in peripheral human T cells.

Sos proteins are ubiquitously expressed activators of Ras. Lymphoid cells also express RasGRP1, another Ras activator. Sos and RasGRP1 are thought to cooperatively control full Ras activation upon T-cell receptor triggering. Using RNA interference, we evaluated whether this mechanism operates in primary human T cells. We found that T-cell antigen receptor (TCR)-mediated Erk activation requires RasGRP1, but not Grb2/Sos. Conversely, Grb2/Sos—but not RasGRP1—are required for IL2-mediated Erk activation. Thus, RasGRP1 and Grb2/Sos are insulators of signals that lead to Ras activation induced by different stimuli, rather than cooperating downstream of the TCR