Abstract Studies using first trimester trophoblast cells may be limited by the inability to obtain patient samples and/or adequate cell numbers. First trimester trophoblast cell lines have been ...generated by SV40 transformation or similar methods, however, this approach is known to induce phenotypic and karyotypic abnormalities. The introduction of telomerase has been proposed to be a viable alternative for the immortalization of primary human cells. To investigate whether telomerase-induced immortalization might be a more feasible approach for the generation of first trimester trophoblast cell lines, we isolated primary trophoblast cells from a 7-week normal placenta and infected the cells with human telomerase reverse transcriptase (hTERT), the catalytic subunit of telomerase. Although this hTERT-infected first trimester trophoblast cell line, which we have named Swan 71, has been propagated for more than 100 passages, it still has attributes that are characteristic of primary first trimester trophoblast cells. The Swan 71 cells are positive for the expression of cytokeratin 7, vimentin and HLA-G, but do not express CD45, CD68 or the Fibroblast Specific Antigen (FSA), CD90/Thy-1. In addition, we also demonstrated that the Swan 71 cells secrete fetal fibronectin (FFN) as well as low levels of human Chorionic Gonadotrophin (hCG). Moreover, the Swan 71 cells exhibit a cytokine and growth factor profile that is similar to primary trophoblast cells and are resistant to Fas, but not TNF-α-induced apoptosis. This suggests that the Swan 71 cells may represent a valuable model for future in vitro trophoblast studies.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
This study analyzed gene expression patterns and global genomic alterations in hepatocellular carcinomas (HCC), hepatoblastomas (HPBL), tissue adjacent to HCC and normal liver tissue derived from ...normal livers and hepatic resections. We found that HCC and adjacent non‐neoplastic cirrhotic tissue have considerable overlap in gene expression patterns compared to normal liver. Several genes including Glypican 3, spondin‐2, PEG10, EDIL3 and Osteopontin are over‐expressed in HCC vs. adjacent tissue whereas Ficolin 3 is the most consistently under‐expressed gene. HCC can be subdivided into three clusters based on gene expression patterns. HCC and HPBL have clearly different patterns of gene expression, with genes IGF2, Fibronectin, DLK1, TGFb1, MALAT1 and MIG6 being over‐expressed in HPBL versus HCC. In addition, specific areas of the genome appear unstable in HCC, with the same regions undergoing either deletion or increased gene dosage in all HCC. In conclusion, a set of specific genes and areas of genomic instability are found across the board in liver neoplasia. (HEPATOLOGY 2006.)
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
Myeloid-derived suppressor cells (MDSC) contribute significantly to the malignant characters conferred by hypoxic tumor microenvironments. However, selective biomarkers of MDSC function in this ...critical setting have not been defined. Here, we report that miR-210 expression is elevated by hypoxia-inducible factor-1α (HIF1α) in MDSC localized to tumors, compared with splenic MDSC from tumor-bearing mice. In tumor MDSC, we determined that HIF1α was bound directly to a transcriptionally active hypoxia-response element in the miR-210 proximal promoter. miR-210 overexpression was sufficient to enhance MDSC-mediated T-cell suppression under normoxic conditions, while targeting hypoxia-induced miR-210 was sufficient to decrease MDSC function against T cells. Mechanistic investigations revealed that miR-210 modulated MDSC function by increasing arginase activity and nitric oxide production, without affecting reactive oxygen species, IL6, or IL10 production or expression of PD-L1. In splenic MDSC, miR-210 regulated Arg1, Cxcl12, and IL16 at the levels of both mRNA and protein, the reversal of which under normoxic conditions decreased T-cell-suppressive effects and IFNγ production. Interestingly, miR-210 overexpression or targeting IL16 or CXCL12 enhanced the immunosuppressive activity of MDSC in vivo, resulting in increased tumor growth. Taken together, these results provide a preclinical rationale to explore miR-210 inhibitory oligonucleotides as adjuvants to boost immunotherapeutic responses in cancer patients.
Abstract The renin-angiotensin system (RAS) is thought to regulate placentation, however, the expression and localization of RAS pathways in early gestation human placenta is not known. Here we ...describe the expression of prorenin ( REN ), (pro)renin receptor ( ATP6AP2 ), angiotensinogen ( AGT ), angiotensin-converting enzyme 1 and 2 ( ACE; ACE2 ), angiotensin II type 1 and 2 receptors ( AGTR1; AGTR2 ) and angiotensin 1–7 receptor ( MAS1 ), as well as the angiogenic factor, vascular endothelial growth factor ( VEGF ), and transforming growth factor-β1 ( TGF-β1 ), in early gestation (6–16 weeks) and term (>37 weeks) human placentae. We also describe the location of all of the key RAS proteins in the early gestation placentae. The highest levels of REN, ATP6AP2, AGT, AGTR1 and ACE2 mRNAs were found in early gestation, whereas ACE1 mRNA was highest at term. AGTR2 and MAS1 mRNA expression were low to undetectable in all samples. REN , ATP6AP2 and AGTR1 mRNA levels were correlated with VEGF expression, but not with TGF-β1 mRNA. In early gestation placentae, prorenin, (pro)renin receptor and the angiotensin II type 1 receptor (AT1 R) were localized to extravillous trophoblast cells, suggesting they play a key role in trophoblast migration. ACE2 in syncytiotrophoblasts could regulate release of Ang 1-7 into the maternal circulation contributing to the vasodilation of the maternal vasculature. ACE was only found in fetal vascular endothelium and may specifically target the growing fetal placental vessels. Because REN , ATP6AP2 and AGTR1 show strong correlations with expression of VEGF this pathway is likely to be important in placental angiogenesis.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
RATIONALE:The recent emergence of hydrogen sulfide (H2S) as a potent cardioprotective signaling molecule necessitates the elucidation of its cytoprotective mechanisms.
OBJECTIVE:The present study ...evaluated potential mechanisms of H2S-mediated cardioprotection using an in vivo model of pharmacological preconditioning.
METHODS AND RESULTS:H2S (100 μg/kg) or vehicle was administered to mice via an intravenous injection 24 hours before myocardial ischemia. Treated and untreated mice were then subjected to 45 minutes of myocardial ischemia followed by reperfusion for up to 24 hours, during which time the extent of myocardial infarction was evaluated, circulating troponin I levels were measured, and the degree of oxidative stress was evaluated. In separate studies, myocardial tissue was collected from treated and untreated mice during the early (30 minutes and 2 hours) and late (24 hours) preconditioning periods to evaluate potential cellular targets of H2S. Initial studies revealed that H2S provided profound protection against ischemic injury as evidenced by significant decreases in infarct size, circulating troponin I levels, and oxidative stress. During the early preconditioning period, H2S increased the nuclear localization of Nrf2, a transcription factor that regulates the gene expression of a number of antioxidants and increased the phosphorylation of protein kinase C and STAT-3. During the late preconditioning period, H2S increased the expression of antioxidants (heme oxygenase-1 and thioredoxin 1), increased the expression of heat shock protein 90, heat shock protein 70, Bcl-2, Bcl-xL, and cyclooxygenase-2 and also inactivated the proapoptogen Bad.
CONCLUSIONS:These results reveal that the cardioprotective effects of H2S are mediated in large part by a combination of antioxidant and antiapoptotic signaling.
Abstract Interleukin (IL)-17-producing helper T cells may play a pivotal role in the pathogenesis of multiple sclerosis. Here, we examined the effects of IL-17 on microglia, which are known to be ...critically involved in multiple sclerosis. Treatment with IL-17 upregulated the microglial production of IL-6, macrophage inflammatory protein-2, nitric oxide, adhesion molecules, and neurotrophic factors. We also found that IL-17 was produced by microglia in response to IL-23 or IL-1β. Because microglia produce IL-1β and IL-23, these cytokines may act in an autocrine manner to induce IL-17 expression in microglia, and thereby contribute to autoimmune diseases, such as MS, in the central nervous system.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
Summary
There is a need for more detailed elucidation of T‐cell immunity in chikungunya infection. CD8 T cells are one of main actors against viruses. Here, we analysed CD8+ T lymphocytes from ...patients in the acute and chronic phases of chikungunya disease (CHIKD). Our results demonstrate that CD8+ T cells expressed higher ex vivo granzyme B, perforin and CD107A expression in patients in the acute phase of CHIKD compared with healthy individuals and higher ex vivo expression of CD69, interleukin‐17A, interleukin‐10 and CD95 ligand, and co‐expression of CD95/CD95 ligand. These results elucidate the importance of these lymphocytes, demonstrating immune mechanisms mediated in human chikungunya infection.
“These data provide new insights into the functional competence of CD8+ T cells and suggest that modulation of the expression profile in CD8+ T cells may be related to severe chronicity of human chikungunya infection.”
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BFBNIB, DOBA, FZAB, GIS, IJS, IZUM, KILJ, NLZOH, NUK, OILJ, PILJ, PNG, SAZU, SBCE, SBMB, SIK, UILJ, UKNU, UL, UM, UPUK
The deep cerebellar nuclei (DCN) are the main output centers of the cerebellum, but little is known about their development. Using transcription factors as cell type-specific markers, we found that ...DCN neurons in mice are produced in the rhombic lip and migrate rostrally in a subpial stream to the nuclear transitory zone (NTZ). The rhombic lip-derived cells express transcription factors Pax6, Tbr2, and Tbr1 sequentially as they enter the NTZ. A subset of rhombic lip-derived cells also express reelin, a key regulator of Purkinje cell migrations. In organotypic slice cultures, the rhombic lip was necessary and sufficient to produce cells that migrate in the subpial stream, enter the NTZ, and express Pax6, Tbr2, Tbr1, and reelin. In later stages of development, the subpial stream is replaced by the external granular layer, and the NTZ organizes into distinct DCN nuclei. Tbr1 expression persists to adulthood in a subset of medial DCN projection neurons. In reeler mutant mice, which have a severe cerebellar malformation, rhombic lip-derived cells migrated to the NTZ, despite reelin deficiency. Studies in Tbr1 mutant mice suggested that Tbr1 plays a role in DCN morphogenesis but is not required for reelin expression, glutamatergic differentiation, or the initial formation of efferent axon pathways. Our findings reveal underlying similarities in the transcriptional programs for glutamatergic neuron production in the DCN and the cerebral cortex, and they support a model of cerebellar neurogenesis in which glutamatergic and GABAergic neurons are produced from separate progenitor compartments.
Increasing significance of tumor-stromal interaction in development and progression of cancer implies that signaling molecules in the tumor microenvironment (TME) might be the effective therapeutic ...targets for hepatocellular carcinoma (HCC). Here, the role of microRNA miR-199a-3p in the regulation of TME and development of HCC has been investigated by several in vitro and in vivo assays. Expression of miR-199a-3p was observed significantly low in HCC tissues and its overexpression remarkably inhibited in vivo tumor growth and metastasis to lung in NOD-SCID mice. In vitro restoration of miR-199a-3p expression either in endothelial cells (ECs) or in cancer cells (CACs) significantly diminished migration of ECs in co-culture assay. Again incubation of miR-199a-3p transfected ECs with either conditioned media (CM) of CACs or recombinant VEGF has reduced tube formation, in ECs and it was also dropped upon growth in CM of either anti-VEGF antibody-treated or miR-199a-3p-transfected CACs. In addition, bioinformatics and luciferase-reporter assays revealed that miR-199a-3p inhibited VEGF secretion from CACs and VEGFR1 and VEGFR2 expression on ECs and thus restricted cross talk between CACs and ECs. Again, restoration of miR-199a-3p in hepatic stellate cells (HSCs) reduced migration and invasion of CACs in co-culture assay, while it was enhanced by the overexpression of HGF suggesting miR-199a-3p has hindered HSC-CACs cross talk probably by inhibiting HGF and regulating matrix metalloproteinase MMP2, which were found as targets of miR-199a-3p subsequently by luciferase-reporter assay and gelatin zymography, respectively. Thus, these findings collectively highlight that miR-199a-3p restricts metastasis, invasion and angiogenesis in HCC and hence it may be considered as one of the powerful effective therapeutics for management of HCC patients.
Autophagy is a basic cellular homeostatic process important to cell fate decisions under conditions of stress. Dysregulation of autophagy impacts numerous human diseases including cancer and chronic ...obstructive lung disease. This study investigates the role of autophagy in idiopathic pulmonary fibrosis.
Human lung tissues from patients with IPF were analyzed for autophagy markers and modulating proteins using western blotting, confocal microscopy and transmission electron microscopy. To study the effects of TGF-β(1) on autophagy, human lung fibroblasts were monitored by fluorescence microscopy and western blotting. In vivo experiments were done using the bleomycin-induced fibrosis mouse model.
Lung tissues from IPF patients demonstrate evidence of decreased autophagic activity as assessed by LC3, p62 protein expression and immunofluorescence, and numbers of autophagosomes. TGF-β(1) inhibits autophagy in fibroblasts in vitro at least in part via activation of mTORC1; expression of TIGAR is also increased in response to TGF-β(1). In the bleomycin model of pulmonary fibrosis, rapamycin treatment is antifibrotic, and rapamycin also decreases expression of á-smooth muscle actin and fibronectin by fibroblasts in vitro. Inhibition of key regulators of autophagy, LC3 and beclin-1, leads to the opposite effect on fibroblast expression of á-smooth muscle actin and fibronectin.
Autophagy is not induced in pulmonary fibrosis despite activation of pathways known to promote autophagy. Impairment of autophagy by TGF-β(1) may represent a mechanism for the promotion of fibrogenesis in IPF.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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