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Cell Cocultures (cell + coculture)
Selected AbstractsChanges in the expression of claudins and transepithelial electrical resistance of mouse Sertoli cells by Leydig cell cocultureINTERNATIONAL JOURNAL OF ANDROLOGY, Issue 5 2003M. C. Gye Summary In the testis, tight junctions (TJs) between adjacent Sertoli cells are important for the formation of blood,testis barrier (BTB). To verify the role of paracrine interactions between the Sertoli and Leydig cells in the structure and function of BTB in testis, the expression of claudin-1 and -11, and transepithelial electrical resistance (TER) of the mouse Sertoli cells were examined under the Leydig cell coculture. TER of Sertoli cell monolayer was significantly larger under the Leydig cell coculture in comparison with the control culture. Meanwhile, the expression of claudin-1 slightly decreased and claudin-11 significantly increased in the Sertoli cells in the Leydig cell coculture compared with control. Testosterone significantly increased claudin-11 expression in cultured Sertoli cells. Taken together, it suggested that Leydig cell coculture changed the structure and functions of inter-Sertoli TJs in vitro. Interactions between Leydig and Sertoli cells might be involved in the development of functional blood testis barrier in mouse testis. [source] Chronological gene expression of ADAMs during testicular development: Prespermatogonia (gonocytes) express fertilin , (ADAM2)DEVELOPMENTAL DYNAMICS, Issue 3 2003Carolina Rosselot Abstract Immediately after birth, primordial germinal cell-derived prespermatogonia (PSG), located in the center of the testicular cords, migrate between adjacent Sertoli cells to establish contact with the cord basal lamina. PSG migration suggests continued assembly and disassembly of cell,cell contacts by a molecular mechanism that may involve integrins and their ligands, the disintegrin domain of spermatogenic cell-specific plasma membrane proteins called ADAMs. We have analyzed the temporal gene expression of selected ADAMs in intact fetal, early postnatal, and pubertal rat testis and Sertoli,spermatogenic cell cocultures by reverse transcriptase-polymerase chain reaction, in situ hybridization, and immunocytochemistry. We report that several ADAM transcripts are expressed in fetal, neonatal, and prepubertal testes. Cyritestin (ADAM3), ADAM5, ADAM6, and ADAM15 are expressed in day 17 fetal testes. In contrast, no expression of fertilin , (ADAM1) and fertilin , (ADAM 2) was detected in fetal testes. Fertilin , gene expression starts after postnatal day 2, subsequent to the expression of fertilin ,, which occurs on postnatal day 1. After postnatal day 2, all the indicated ADAMs, including the fertilin , and fertilin ,, continue to be expressed. Transcripts of spermatogenic cell-specific fertilin ,, fertilin ,, ADAM3, and ADAM5 were detected during the coculture of PSG with Sertoli cells for up to 72 hr after plating. The presence of fertilin , mRNA and protein in cocultured PSG was visualized by in situ hybridization and immunocytochemistry, respectively. These observations indicate that PSG in coculture with Sertoli cells provide a suitable approach for analyzing cell,cell adhesive responses involving spermatogenic cell-specific ADAMs. Development Dynamics 458,467, 2003. © 2003 Wiley-Liss, Inc. [source] IL-10 inhibits endothelium-dependent T cell costimulation by up-regulation of ILT3/4 in human vascular endothelial cellsEUROPEAN JOURNAL OF IMMUNOLOGY, Issue 1 2007Christian Abstract Effects of IL-10 on endothelium-dependent T cell activation have not been investigated in detail. We confirm expression of the IL-10 receptor and effective signaling via STAT-3 in human umbilical vein endothelial cells (HUVEC). In CD4 T cell cocultures with HUVEC, pretreatment of endothelial cells with IL-10 resulted in significant dose-dependent inhibition of CD4 T cell proliferation, which also occurred when IL-10 was removed after pretreatment before starting cocultures. Th1/Th2 polarization of proliferated T cells, endothelial nitric oxide (NO), or IL-12 production were unchanged. However, IL-10 stimulation resulted in up-regulation of SOCS-3, a negative regulator of cytokine secretion, and induction of the inhibitory surface molecules immunoglobulin-like transcript 3 and 4 (ILT3/ILT4) in EC, potentially involving glucocorticoid-induced leucine zipper (GILZ). Addition of blocking antibodies against ILT3/ILT4 to EC/T cell cocultures resulted in nearly complete reestablishment of T cell proliferation. In contrast, addition of soluble ILT3 or overexpression of ILT3 in cocultures significantly reduced T cell proliferation. No induction of foxp3+ regulatory T cells was seen. In conclusion, the T cell costimulatory potential of human EC is markedly suppressed by IL-10 due to up-regulation of ILT3/ILT4, obviously not involving generation of Treg. This identifies a novel action of IL-10 in EC and a potential therapeutical target for local immunomodulation. [source] Expression and regulation of interleukin-10 and interleukin-10 receptor in rat astroglial and microglial cellsEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 7 2002Annemarie Ledeboer Abstract Activated glial cells crucially contribute to brain inflammatory responses. Interleukin-10 (IL-10) is an important modulator of glial cell responses in the brain. In the present study we describe the expression of IL-10 and the IL-10 receptor (IL-10R1) in primary cocultures of rat microglial and astroglial cells. Using quantitative RT-PCR and ELISA, we show that IL-10 mRNA expression and subsequent IL-10 secretion is time-dependently induced by lipopolysaccharide (LPS). IL-10R1, however, is constitutively expressed in glial cell cocultures, as shown by RT-PCR and immunocytochemistry. Radioligand binding studies using 125I-IL-10 reveal that rat glial cells express a single binding site with an apparent affinity of approximately 600 pm for human IL-10. Observations in enriched cultures of either microglial or astroglial cells indicate that both cell types express IL-10 mRNA and are capable of secreting IL-10. Both cell types also express IL-10R1 mRNA and protein. However, in glial cell cocultures immunoreactive IL-10R1 protein is predominantly observed in astrocytes, suggesting that microglial expression of IL-10R1 in cocultures is suppressed by astrocytes. In addition, exogenous IL-10 is highly potent in down-regulating LPS-induced IL-1, and IL-10 mRNA, and, at a higher dose, IL-10R1 mRNA in untreated and LPS-treated cultures, suggesting that IL-10 autoregulates its expression and inhibits that of IL-1, at the transcriptional level. Together the findings support the concept that IL-10, produced by activated microglial and astroglial cells, modulates glia-mediated inflammatory responses through high-affinity IL-10 receptors via paracrine and autocrine interactions. [source] Rapid method for culturing embryonic neuron,glial cell coculturesJOURNAL OF NEUROSCIENCE RESEARCH, Issue 5 2003Åsa Fex Svenningsen Abstract A streamlined, simple technique for primary cell culture from E17 rat tissue is presented. In an attempt to standardize culturing methods for all neuronal cell types in the embryo, we evaluated a commercial medium without serum and used similar times for trypsinization and tested different surfaces for plating. In 1 day, using one method and a single medium, it is possible to produce robust E17 cultures of dorsal root ganglia (DRG), cerebellum, and enteric plexi. Allowing the endogenous glial cells to repopulate the cultures saves time compared with existing techniques, in which glial cells are added to cultures first treated with antimitotic agents. It also ensures that all the cells present in vivo will be present in the culture. Myelination commences after approximately 2 weeks in culture for dissociated DRG and 3,4 weeks in cerebellar cultures. In enteric cultures, glial wrapping of the enteric neurons is seen after 3 weeks (2 weeks in ascorbic acid), suggesting that basal lamina production is important even for glial ensheathment in the enteric nervous system. No overgrowth of fibroblasts or other nonneuronal cells was noted in any cultures, and myelination of the peripheral nervous system and central nervous system cultures was very robust. © 2003 Wiley-Liss, Inc. 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