Isolation and characterization of extracellular vesicles secreted by pre-pubertal Sertoli cells

Recent studies have shown that extracellular vesicles (Ev) are an important mechanism of intercellular communication. In fact, Ev may contain proteins, DNA and mRNA. In particular, the latter play an important role in various biological processes including regulation and cell differentiation [1]. Sertoli cells (SC), previously considered as a mere “sustentacular cell”, were re-evalued in their functions and elevated to the rank of a “sentinel” in spermatogenesis due to production of trophic, differentiation and immune-modulators factors. Porcine pre-pubertal SC, isolated by our method [2], upon 48 hours culture, SC were stimulated with recombinant a-follitropin (rFSH) or FSH + testosterone (T) to evaluate both the presence in the medium of SC-derived Ev (SC-Ev) and SC-Ev content, in terms of mRNA for Anti-Müllerian hormone (AMH), inhibin B, Androgen Binding Protein (ABP) and FSH-receptor (FSH-r), by RT-PCR. SEM analysis highlighted the presence of SC-Nv in culture medium with mean diameters < 149 nm. We have also demonstrated, within the SC-Ev, significant increase in mRNA for AMH, ABP and FSH-r after both FSH and FSH+T stimulation, as compared to unstimulated SC-Ev. Differently from unstimulated SC-Ev, mRNA inhibin B levels were unchanged in FSH-stimulated SC-Ev, and increased after FSH+T stimulation. Interestingly, an opposite trend was shown in mRNA secretion, in control SC monolayer where, we demonstrated a decrease of AMH and FSH-r mRNA (after both stimulations with FSH or FSH + T) and an increase of inhibin B mRNA. On the contrary, mRNA ABP levels, in SC monolayer, decreased after stimulation with FSH but were unchanged in the presence of FSH+T. For the first time in the Literature, our work has shown the presence of SC-Nv containing AMH, inhibin B, ABP and FSH-r mRNA regulated by FSH with or without T. This result may suggest that other testicular cells could produce factors that, until now, were considered an exclusive SC secretory product.This work was supported by Mr.Gary Harlem (Altucell Inc. 3 Astor Court, Dix Hills, New York, NY) and Merck-Serono (London, UK)

The conformational state of hERG1 channels determines integrin association, downstream signaling, and cancer progression

Ion channels regulate cell proliferation, differentiation, and migration in normal and neoplastic cells through cell-cell and cell-extracellular matrix (ECM) transmembrane receptors called integrins. K+ flux through the human ether-\ue0-gogo- related gene 1 (hERG1) channel shapes action potential firing in excitable cells such as cardiomyocytes. Its abundance is often aberrantly high in tumors, where it modulates integrin-mediated signaling. We found that hERG1 interacted with the \u3b21 integrin subunit at the plasma membrane of human cancer cells. This interaction was not detected in cardiomyocytes because of the presence of the hERG1 auxiliary subunit KCNE1 (potassium voltage-gated channel subfamily E regulatory subunit 1), which blocked the \u3b21 integrin-hERG1 interaction. Although open hERG1 channels did not interact as strongly with \u3b21 integrins as did closed channels, current flow through hERG1 channelswas necessary to activate the integrin-dependent phosphorylation of Tyr397 in focal adhesion kinase (FAK) in both normal and cancer cells. In immunodeficient mice, proliferation was inhibited in breast cancer cells expressing forms of hERG1 with impaired K+ flow, whereas metastasis of breast cancer cells was reduced when the hERG1/\u3b21 integrin interaction was disrupted. We conclude that the interaction of \u3b21 integrins with hERG1 channels in cancer cells stimulated distinct signaling pathways that depended on the conformational state of hERG1 and affected different aspects of tumor progression

Prolongation of skin allograft survival in rats by the transplantation of microencapsulated xenogeneic neonatal porcine Sertoli cells

Skin rejection remains a major hurdle in skin reconstructive transplantation surgery. In fact, 85% of the grafted patients experience at least one episode of acute skin rejection in the first year. It has been observed that Sertoli cells (SC), when co-transplanted with allo- or xenogeneic cell/tissues, can induce graft acceptance in the absence of systemic immunosuppression. A method aimed at significantly prolonging skin allografts in rats transplanted with barium alginate-based microencapsulated xenogeneic porcine SC (SC-MCs) is described. Results demonstrated that intraperitoneal (IP) transplantation of SC-MCs with high cellular viability and function can significantly prolong allogeneic skin grafts when compared to transplantation controls receiving only empty alginate capsules (E-MCs). Lymphocytic infiltration at the skin graft site was not observed in 80% of the SC-MCs transplanted rats and these recipient animals showed a significant increased expression of T regulatory (Tregs) cells when compared to E-MCs transplantation controls. The findings of this report further substantiate the positive therapeutic effects of SC on transplantation technology mediated by Sertoli cell-induced alterations of the host's immune system and indicate new perspectives and new strategies for successful skin tissue allografts. (C) 2012 Elsevier Ltd. All rights reserved

NAMI-A is highly cytotoxic toward leukaemia cell lines: evidence of inhibition of KCa 3.1 channels

We report here that the established anticancer ruthenium( III ) complex NAMI-A induces potent and selective cytotoxic e \ufb00 ects in a few leukaemia cell lines. These results sound very surprising after 20 years of intense studies on NAMI-A, commonly considered as a \u201c non-cytotoxic \u201d antimetastatic agent. In addition, evidence is given for selective inhibition of KCa 3.1 channels. The implications of these \ufb01 ndings are discussed