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Primary Cell Cultures (primary + cell_culture)
Selected AbstractsPossible Role of Natural Immune Response against Altered Fibroblasts in the Development of Post-Operative AdhesionsAMERICAN JOURNAL OF REPRODUCTIVE IMMUNOLOGY, Issue 6 2006Zeynep Alpay Problem Post-operative adhesion tissue fibroblasts (ATF) differ from normal peritoneal fibroblasts (NPF). Natural immune response participates in the elimination of altered cells. In this study, we investigated NPF and ATF expression patterns of immune response-related markers, and lymphokine-activated killer (LAK) cell-mediated fibroblast elimination in vitro. Method of study Primary cell cultures of both NPF and ATF obtained from the same four patients were used in the experiments. The expression of CD54, CD40 and CD120b, and allogeneic LAK cell-mediated ATF and NPF elimination were studied by flow cytometry. Results Average expression of CD54 in ATF was greater by 12.3-fold compared with NPF (P = 0.021), with ratios of 2.4 and 1.9-fold for CD40 (P < 0.001) and CD120b (P = 0.013), respectively. Average LAK cell-mediated fibroblast killing was 1.8 ± 0.8-fold greater in ATF over NPF (P = 0.008). Furthermore, LAK cell-mediated fibroblast elimination correlated significantly with the increased CD40, CD54 and CD120b expression (R > 0.956; P < 0.05 for each). Conclusions These results demonstrate that ATF are more susceptible to lymphocyte-mediated elimination than NPF and the development of adhesions despite this could be explained by either impaired or overwhelmed autologous natural immune response against reactive fibroblasts. [source] Glucocorticoids Inhibit Diastrophic Dysplasia Sulfate Transporter Activity in Otosclerosis by Interleukin-6THE LARYNGOSCOPE, Issue 9 2006Yutaka Imauchi MD Abstract Hypothesis/Objective: Otosclerosis is a bone remodeling disorder localized to the otic capsule and associated with inflammation. In vitro, increased activity of the diastrophic dysplasia sulf/te transporter (DTDST), which is implicated in bone metabolism, has been reported. Because glucocorticoids modulate the bone turnover and inhibit inflammatory processes, we investigated the effect of dexamethasone (Dex) on interleukin-6 and DTDST in otosclerosis. Study Design: The authors conducted a prospective, case,control study. Materials and Methods: Primary cell cultures were obtained from stapes and external auditory canals in otosclerosis (n = 21) and control patients (n = 18). Assays with [3H]Dex evaluated specific binding sites in otosclerotic and control stapes. The effects of Dex (10,9 to 10,6 M) and RU486 (10,7 M), a glucocorticoid antagonist, were studied on DTDST activity by sulfate uptake. IL-6 secretion was measured in culture media before and after Dex (10,7 M, 24 hours). The effect of IL-6 (10,7 M, 24 hours) was assessed on DTDST activity in control stapes. Results: The number of specific Dex-binding sites was similar in all stapedial cultures. Dex inhibited DTDST activity (19.4 ± 1.02 vs. 29.4 ± 3.94 pmol/,g prot/5 minutes) only in otosclerotic stapes. This effect was dose-dependent, antagonized by RU 486 and only observed 24 hours after Dex exposure. Interleukin (IL)-6 stimulated DTDST activity in normal stapes, whereas Dex inhibited IL-6 production only in otosclerotic stapes. Conclusion: Dex inhibits the DTDST activity, at least in part, through a reduction of IL-6 secretion only in otosclerotic cells. This effect is mediated through the glucocorticoid receptors and may lead to the reduction of bone turnover. [source] Primary cell cultures from anaplastic thyroid cancer obtained by fine-needle aspiration used for chemosensitivity testsCLINICAL ENDOCRINOLOGY, Issue 1 2008Alessandro Antonelli Summary Objective, Anaplastic thyroid cancer (ATC) is often inoperable and chemotherapy and radiotherapy are the main treatments. Until now, ,primary ATC cell cultures' (ANA) have been developed from surgical biopsies. We investigated the possibility of obtaining ANA from fine-needle aspiration (FNA-ANA) and testing their sensitivity to chemotherapeutic agents, which could enable treatments to be more effective and avoid unnecessary surgical procedures. Design and patients, The aim of this study was to obtain FNA-ANA from three ATC patients and to evaluate the chemosensitivity of FNA-ANA to chemotherapeutic agents. Measurements and results, FNA-ANA from ATC patients were cultured in RPMI 1640 and propagated in Dulbecco's modified Eagle's medium (DMEM). Chemosensitivity was evaluated by inhibiting the proliferation (analysing the number of viable cells by the cleavage of tetrazolium salts), by increasing the concentration of four different chemotherapeutic agents: bleomycin, cisplatin, gemcitabine and etoposide. The chemotherapeutic agents significantly inhibited (> 50%) FNA-ANA proliferation. Another ANA for each patient was obtained from a surgical biopsy specimen; the results for the chemosensitivity tests were similar to those obtained using FNA-ANA. Conclusions, Our study demonstrates the possibility of obtaining FNA-ANA, and opens the way to the use of FNA-ANA as a means of testing the chemosensitivity to different chemotherapeutic agents (and possibly the radiosensitivity) in each patient, avoiding unnecessary surgical procedures and the administration of inactive chemotherapeutics. [source] Acute activation of Erk1/Erk2 and protein kinase B/akt proceed by independent pathways in multiple cell typesFEBS JOURNAL, Issue 17 2005Doris Chiu We used two inhibitors of the signaling enzyme phosphatidylinositol 3-kinase (PtdIns3K), wortmannin and LY294002, to evaluate the potential involvement of PtdIns3K in the activation of the MAP kinases (MAPK), Erk1 and Erk2. In dose,response studies carried out on six different cell lines and a primary cell culture, we analyzed the ability of the inhibitors to block phosphorylation of protein kinase B/akt (PKB/akt) at Ser473 as a measure of PtdIns3K activity, or the phosphorylation of Erk1/2 at activating Thr/Tyr sites as a measure of the extent of activation of MAPK/Erk kinase (MEK/Erk). In three different hemopoietic cell lines stimulated with cytokines, and in HEK293 cells, stimulated with serum, either wortmannin or LY294002, but never both, could partially block phosphorylation of Erks. The same observations were made in a B-cell line and in primary fibroblasts. In only one cell type, the A20 B cells, was there a closer correlation between the PtdIns3K inhibition by both inhibitors, and their corresponding effects on Erk phosphorylation. However, this stands out as an exception that gives clues to the mechanism by which cross-talk might occur. In all other cells, acute activation of the pathway leading to Erk phosphorylation could proceed independently of PtdIns3K activation. In a biological assay comparing these two pathways, the ability of LY294002 and the MEK inhibitor, U0126, to induce apoptosis were tested. Whereas LY294002 caused death of cytokine-dependent hemopoietic cells, U0126 had little effect, but both inhibitors together had a synergistic effect. The data show that these two pathways are regulating very different downstream events involved in cell survival. [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. [source] Efficacy of systemic morpholino exon-skipping in duchenne dystrophy dogs,ANNALS OF NEUROLOGY, Issue 6 2009Toshifumi Yokota PhD Objective Duchenne muscular dystrophy (DMD) is caused by the inability to produce dystrophin protein at the myofiber membrane. A method to rescue dystrophin production by antisense oligonucleotides, termed exon-skipping, has been reported for the mdx mouse and in four DMD patients by local intramuscular injection. We sought to test efficacy and toxicity of intravenous oligonucleotide (morpholino)-induced exon skipping in the DMD dog model. Methods We tested a series of antisense drugs singly and as cocktails, both in primary cell culture, and two in vivo delivery methods (intramuscular injection and systemic intravenous injection). The efficiency and efficacy of multiexon skipping (exons 6,9) were tested at the messenger RNA, protein, histological, and clinical levels. Results Weekly or biweekly systemic intravenous injections with a three-morpholino cocktail over the course of 5 to 22 weeks induced therapeutic levels of dystrophin expression throughout the body, with an average of about 26% normal levels. This was accompanied by reduced inflammatory signals examined by magnetic resonance imaging and histology, improved or stabilized timed running tests, and clinical symptoms. Blood tests indicated no evidence of toxicity. Interpretation This is the first report of widespread rescue of dystrophin expression to therapeutic levels in the dog model of DMD. This study also provides a proof of concept for systemic multiexon-skipping therapy. Use of cocktails of morpholino, as shown here, allows broader application of this approach to a greater proportion of DMD patients (90%) and also offers the prospect of selecting deletions that optimize the functionality of the dystrophin protein. Ann Neurol 2009 [source] Genetic immunity and influenza pandemicsFEMS IMMUNOLOGY & MEDICAL MICROBIOLOGY, Issue 1 2006Sergey N. Rumyantsev Abstract In addition to the great number of publications focused on the leading role of virus mutations and reassortment in the origin of pandemic influenza, general opinion emphasizes the victim side of the epidemic process. Based on the analysis and integration of relevant ecological, epidemiological, clinical, genetic and experimental data, the present article is focused on the evolution of ,virus , victim' ecological systems resulting in the formation of innate (i.e. genetic, constitutional) immunity in the involved species and populations. This kind of immunity functions today as the greatest natural barrier to the pandemic spread of influenza among humans and ecologically related kinds of animals. Global influenza pandemics can arise when the worldwide population contains at least a minimum number of people susceptible to a known or mutant influenza virus. Special attention is paid in this article to individual tests for the presence of this barrier, including the implications of specific findings for public health policy. Such tests could be based on in vitro observation of the action of relevant virus strains on primary cell cultures or on their cellular or molecular components extracted from individuals. The resources of the Human Genome Project should also be utilized. [source] Targeting the epidermal growth factor receptor by erlotinib (TarcevaÔ) for the treatment of esophageal cancerINTERNATIONAL JOURNAL OF CANCER, Issue 7 2006Andreas P. Sutter Abstract Esophageal cancer is the sixth most common cause of cancer-related death worldwide. Because of very poor 5-year survival new therapeutic approaches are mandatory. Erlotinib (TarcevaÔ), an inhibitor of epidermal growth factor receptor tyrosine kinase (EGFR-TK), potently suppresses the growth of various tumors but its effect on esophageal carcinoma, known to express EGFR, remains unexplored. We therefore studied the antineoplastic potency of erlotinib in human esophageal cancer cells. Erlotinib induced growth inhibition of the human esophageal squamous cell carcinoma (ESCC) cell lines Kyse-30, Kyse-70 and Kyse-140, and the esophageal adenocarcinoma cell line OE-33, as well as of primary cell cultures of human esophageal cancers. Combining erlotinib with the EGFR-receptor antibody cetuximab, the insulin-like growth factor receptor tyrosine kinase inhibitor tyrphostin AG1024, or the 3-hydroxy-3-methylglutaryl coenzyme. A reductase (HMG-CoAR) inhibitor fluvastatin resulted in additive or even synergistic antiproliferative effects. Erlotinib induced cell cycle arrest at the G1/S checkpoint. The erlotinib-mediated signaling involved the inactivation of EGFR-TK and ERK1/2, the upregulation of the cyclin-dependent kinase inhibitors p21Waf1/CIP1 and p27Kip1, and the downregulation of the cell cycle promoter cyclin D1. However, erlotinib did not induce immediate cytotoxicity or apoptosis in esophageal cancer cells. The inhibition of EGFR-TK by erlotinib appears to be a promising novel approach for innovative treatment strategies of esophageal cancer, as it powerfully induced growth inhibition and cell cycle arrest in human esophageal cancer cells and enhanced the antineoplastic effects of other targeted agents. © 2005 Wiley-Liss, Inc. [source] Interstitial Cajal-like cells (ICLC) in human resting mammary gland stroma.JOURNAL OF CELLULAR AND MOLECULAR MEDICINE, Issue 4 2005Transmission electron microscope (TEM) identification Abstract We have previously shown the existence of ICLC in human resting mammary gland stroma by means of methylene blue (vital) staining and c-kit immunopositivity (immunofluorescence and immunohistochemistry). In addition, we reported the phenotype characteristics of these ICLC in vitro (primary cell cultures). Since the identification of ICLC outside the gut requires, at this moment, the obligatory use of TEM, we used this technique and provide unequivocal evidence for the presence of ICLC in the intralobular stroma of human resting mammary gland. According to the,platinum standard' (10 TEM criteria for the certitude diagnosis of ICLC), we found interstitial cells with the following characteristics: 1. location: among the tubulo-alveolar structures, in the non-epithelial space; 2. caveolae:,2.5% of cell volume; 3. mitochondria:,10% of cell volume; 4. endoplasmic reticulum: either smooth or rough, ,2,3% of cell volume; 5. cytoskeleton: intermediate and thin filaments, as well as microtubules are present; 6.myosin thick filaments: undetectable; 7. basal lamina: occasionally found; 8. gap junctions: occasionally found; 9. close contacts with targets: nerve fibers, capillaries, immunoreactive cells by ,stromal synapses'; 10. characteristic cytoplasmic processes: i) number: frequently 2,3; ii) lenght: several tens of ,m; iii) thickness: uneven caliber, 0.1,0.5 ,m, with dilations, but very thin from the emerging point; iv) aspect: moniliform, usually with mitochondria located in dilations; y) branching: dichotomous pattern; vi) Ca2+ release units: are present; vii) network labyrinthic system: overlapping cytoplasmic processes. It remains to be established which of the possible roles that we previously suggested for ICLC (e.g. juxta- and/or paracrine secretion, uncommited progenitor cells, immunological surveillance, intercellular signaling, etc.) are essential for the epithelium/stroma equilibrium in the mammary gland under normal or pathological conditions. [source] Curcumin downregulates H19 gene transcription in tumor cellsJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 5 2008Renata Novak Kujund Abstract Curcumin (diferuloymethane), a natural compound used in traditional medicine, exerts an antiproliferative effect on various tumor cell lines by an incompletely understood mechanism. It has been shown that low doses of curcumin downregulate DNA topoisomerase II alpha (TOP2A) which is upregulated in many malignances. The activity of TOP2A is required for RNA polymerase II transcription on chromatin templates. Recently, it has been reported that CTCF, a multifunctional transcription factor, recruits the largest subunit of RNA polymerase II (LS Pol II) to its target sites genome-wide. This recruitment of LS Pol II is more pronounced in proliferating cells than in fully differentiated cells. As expression of imprinted genes is often altered in tumors, we investigated the potential effect of curcumin treatment on transcription of the imprinted H19 gene, located distally from the CTCF binding site, in human tumor cell lines HCT 116, SW 620, HeLa, Cal 27, Hep-2 and Detroit 562. Transcription of TOP2A and concomitantly H19 was supressed in all tumor cell lines tested. Monoallelic IGF2 expression was maintained in curcumin-treated cancer cells, indicating the involvement of mechanism/s other than disturbance of CTCF insulator function at the IGF2/H19 locus. Curcumin did not alter H19 gene transcription in primary cell cultures derived from normal human tissues. J. Cell. Biochem. 104: 1781,1792, 2008. © 2008 Wiley-Liss, Inc. [source] N -(Indazolyl)benzamido Derivatives as CDK1 Inhibitors: Design, Synthesis, Biological Activity, and Molecular Docking StudiesARCHIV DER PHARMAZIE, Issue 5 2009Demetrio Raffa Abstract A series of N -1H -indazole-1-carboxamides has been synthesized and their effects on both CDK1 / cyclin B and the K-562 (human chronic myelogenus leukemia) cell line were evaluated. Using a computational model, we have observed that all the most active compounds 9e, f, i,n exhibited the same binding mode of purvanalol A in the ATP-binding cleft. Although they were able to moderately inhibit the leukemic cell line K-562 and to show inhibitory activity against the Cdc2-Cyclin B kinase in the low micromolar range, they turned out to be non-cytotoxic against HuDe (IZSL) primary cell cultures from human derm. These preliminary results are quite encouraging in view of the low toxicity demonstrated by the above-mentioned compounds. [source] Verotoxin 1 binding to intestinal crypt epithelial cells results in localization to lysosomes and abrogation of toxicityCELLULAR MICROBIOLOGY, Issue 2 2003D. E. Elaine Hoey Summary Verotoxins (VTs) are important virulence factors of enterohaemorrhagic Escherichia coli (EHEC), a group of bacteria associated with severe disease sequelae in humans. The potent cytotoxic activity of VTs is important in pathogenicity, resulting in the death of cells expressing receptor Gb3 (globotriaosylceramide). EHEC, particularly serotype O157:H7, frequently colonize reservoir hosts (such as cattle) in the absence of disease, however, the basis to avirulence in this host has been unclear. The objective of this study was assessment of interaction between VT and intestinal epithelium, which represents the major interface between the host and enteric organisms. Bovine intestinal epithelial cells expressed Gb3 in vitro in primary cell cultures, localizing specifically to proliferating crypt cells in corroboration with in situ immunohistological observations on intestinal mucosa. Expression of receptor by these cells contrasts with the absence of Gb3 on human intestinal epithelium in vivo. Despite receptor expression, VT exhibited no cytotoxic activity against bovine epithelial cells. Sub-cellular localization of VT indicated that this toxin was excluded from endoplasmic reticulum but localized to lysosomes, corresponding with abrogation of cytotoxicity. VT intracellular trafficking was unaffected by treatment of primary cell cultures with methyl-,-cyclodextrin, indicating that Gb3 in these cells is not associated with lipid rafts but is randomly distributed in the membrane. The combination of Gb3 isoform, membrane distribution and VT trafficking correlate with observations of other receptor-positive cells that resist verocytotoxicity. These studies demonstrate that intestinal epithelium is an important determinant in VT interaction with major implications for the differential consequences of EHEC infection in reservoir hosts and humans. [source] 3241: Effect of glutaredoxin 2 gene knockout on lens epithelial cells against oxidative stressACTA OPHTHALMOLOGICA, Issue 2010M LOU Purpose The mitochondrial glutaredoxin 2 (Grx2) is known to possess both dethiolase and peroxidase activities, and has shown an ability to protect cells from oxidative stress-induced apoptosis in the human lens epithelial cells. In this study, we further studied the function of Grx2 by using Grx2 knockout mouse lens epithelial (MLE) cells as a model. Methods Primary culture of MLE cells was established from the lenses of wild-type (WT) and Grx2-knockout (Grx2 KO) mice. Cells were probed for ,A-crystallin and Grx2 by Western blot analysis while cell viability was examined by WST-8 assay. Glutathione (GSH) level, Grx2 and Complex I activities, and lactate dehydrogenase (LDH) release were determined by spectrophotometric assays. Reactive oxygen species was detected using DCF-DA fluorescein with a cell sorter. Apoptosis was quantified by flow cytometry. Results Both primary cell cultures were confirmed to be lens epithelial cells by the presence of ,A-crystallin. Western blotting showed normal expression of Grx2 in WT cells but absent in Grx2 KO cells. Both cell types showed similar morphology and growth rate with same level of GSH pool and complex 1 activity in the mitochondrial fraction. However, KO cells were more sensitive to oxidative stress (100 ,M H2O2 for 6 h) and exhibited lower cell viability and more LDH leakage in comparison with the WT cells. In addition, knockdown of Grx2 weakened the cell's ability to detoxify H2O2 and enhanced the H2O2-induced inactivation of complex I in the electron transport chain. Conclusion Grx2 can protect MLE cells from H2O2-induced cell injury, and the mechanism of this protection is likely associated with its ability to detoxify H2O2 and its preservation of complex I activity in the mitochondria. [source] | ||||||||||||||||