Home About us Contact
|
Home About us Contact | ||
|
|
||
Organ Culture (organ + culture)
Kinds of Organ Culture Terms modified by Organ Culture Selected AbstractsEnhanced Transcription of Contractile 5-Hydroxytryptamine 2A Receptors via Extracellular Signal-Regulated Kinase 1/2 after Organ Culture of Rat Mesenteric ArteryBASIC AND CLINICAL PHARMACOLOGY & TOXICOLOGY, Issue 4 2005Yong-Xiao Cao The present study was designed to examine if vascular 5-HT2A receptors are up-regulated during organ culture and if the extracellular signal-regulated protein kinase 1/2 (ERK1/2) pathways are involved. Compared with fresh rat mesenteric artery ring segments, the contractile responses to 5-HT were significantly increased in the segments cultured for 6, 24 or 48 hr (P<0.05, P<0.01, P<0.01, respectively). The 5-HT-induced contraction occurred via 5-HT2A receptors, since the selective 5-HT2A antagonist ketanserin blocked the 5-HT-induced contraction in the fresh segments with a pA2 value 9.5 (slope was 0.98 with 95% confidence intervals from 0.8 to 1.1). A similar result was obtained in the segments cultured for 24 hr with a pA2 value of 9.43 (slope=0.91 and 95% confidence intervals between 0.45 to 2.3). In addition, the enhanced 5-HT2A receptor contraction occurred with a significant increase of 5-HT2A receptor mRNA (P<0.05). Organ culture of the mesenteric artery was found to activate ERK1/2 already within 1 and 3 hr. It is likely that the ERK1/2 pathways were involved as a initial switch, since the selective ERK1/2 pathway inhibitor SB386023 abolished both up-regulation of 5-HT2A mRNA transcription and the enhanced contractile response to 5-HT. These data reveal a role of ERK1/2 in up-regulation of 5-HT2A receptors and suggest a possibility to inhibit the enhanced responses to 5-HT by inhibition of the ERK1/2 pathway. [source] Organ culture, but not hypothermic storage, facilitates the repair of the corneal endothelium following mechanical damageACTA OPHTHALMOLOGICA, Issue 4 2010Jana Nejepinska Abstract. Purpose:, To evaluate the reparative capacity of the mechanically injured endothelium of corneas stored under organ culture (OC) or hypothermic conditions. Methods:, The central endothelium of 12 pairs of human corneas with similar endothelial parameters was damaged to create a 1 mm2 lesion. One cornea from each pair was stored under OC and one under hypothermic conditions. The endothelial cell density (ECD), coefficient of variation, hexagonality and percentage of dead cells were assessed before and after damage and on days 7, 14, 21 and 28 of storage. Results:, The mean ECD of corneas subsequently stored under OC or hypothermic conditions was 2764/mm2. Immediately after damage, a denuded Descemet's membrane with a few remaining dead cells was observed at the injured area. After 7 days of storage under OC conditions, almost no dead cells were observed at the place of injury. A non-significant worsening of the qualitative parameters (polymegatism and pleomorphism) was found. After 14 days, ECD was 1933/mm2 and 2478/mm2 centrally and pericentrally, respectively. Similar values were found after 21 and 28 days of storage. The lesions with remnant dead cells persisted throughout hypothermic preservation. From day 14 the corneas became cloudy and in poor condition, while the pericentral ECD was 2523/mm2. Conclusion:, The reparative capacity of the cornea is maintained under OC but not under hypothermic conditions. For corneas containing dead endothelial cells, OC is therefore the method of choice because it may improve the quality of the stored tissue. [source] Interleukin-1, attenuates endothelin B receptor-mediated airway contractions in a murine in vitro model of asthma: roles of endothelin converting enzyme and mitogen-activated protein kinase pathwaysCLINICAL & EXPERIMENTAL ALLERGY, Issue 9 2004Y. Zhang Summary Background Asthma is a chronic airway disease, known to involve several inflammatory mediators. Little is known about how these mediators interact in order to produce or attenuate even basic features of the disease, like airway hyper-reactivity and remodelling. Endothelin-1 (ET-1) and IL-1, are two mediators suggested to play important roles in the induction of airway inflammation. Objective To investigate the interactions between ET-1 and IL-1,, using a novel in vitro model of asthma, focusing on airway smooth muscle contractility. Methods Isolated murine tracheal segments were cultured from 1 to 8 days in the absence and presence of IL-1,. The subsequent contractile responses to sarafotoxin 6c (S6c) (selective agonist for ETB receptor) and sarafotoxin 6b (S6b) (ETA and ETB receptor agonist) were recorded by a myographs system. In all experiments, ETB receptors were desensitized before the contractile response to S6b was recorded. Thus, the response to S6b is only mediated by ETA receptors in the present study. The mRNA expressions for ET-1 and endothelin (ET) receptors were quantified by real-time PCR. Results Organ culture in the presence of IL-1, attenuated the maximal contraction induced by S6c, but not S6b. This reduction was concentration-dependent and was significant after 2, 4 and 8 days of culture. To investigate the mechanisms behind this, inhibitors for endothelin converting enzyme (ECE) phosphoramidon, c-JUN N-terminal kinase (JNK) SP600125, extracellular-signal-regulated kinase 1/2(ERK 1/2) PD98059 and p38 pathway SB203580 were used. Individually, SP600125 and PD98059, but not SB203580, could partly reverse the reduction induced by IL-1,. An additional effect was obtained when SP600125 and PD98059 were combined. The mRNA expressions for ET-1 and ETB receptor were up- and down-regulated, respectively, by IL-1,. Conclusion Presence of IL-1, in the airways attenuate the contractile response mediated via ETB receptors, an effect dependent on ECE, JNK and ERK 1/2 pathways. [source] Chick limbs with mouse teeth: An effective in vivo culture system for tooth germ development and analysisDEVELOPMENTAL DYNAMICS, Issue 1 2003Eiki Koyama Abstract Mouse tooth germ development is currently studied by three main approaches: in wild-type and mutant mouse lines, after transplantation of tooth germs to ectopic sites, and in organ culture. The in vivo approaches are the most physiological but do not provide accessibility to tooth germs for further experimental manipulation. Organ cultures, although readily accessible, do not sustain full tooth germ development and are appropriate for short-term analysis. Thus, we sought to establish a new approach that would combine experimental accessibility with sustained development. We implanted fragments of embryonic day 12 mouse embryo first branchial arch containing early bud stage tooth germs into the lateral mesenchyme of day 4,5 chick embryo wing buds in ovo. Eggs were reincubated, and implanted tissues were examined by histochemistry and in situ hybridization over time. The tooth germs underwent seemingly normal growth, differentiation, and morphogenesis. They reached the cap, bell, and crown stages in approximately 3, 6, and 10 days, respectively, mimicking in a striking manner native temporal patterns. To examine mechanisms regulating tooth germ development, we first implanted tooth germ fragments, microinjected them with neutralizing antibodies to the key signaling molecule Sonic hedgehog (Shh), and examined them over time. Tooth germ development was markedly delayed, as revealed by poor morphogenesis and lack of mature ameloblasts and odontoblasts displaying characteristic traits such as an elongated cell shape, nuclear relocalization, and amelogenin gene expression. These phenotypic changes began to be reversed upon further incubation. The data show that the limb bud represents an effective, experimentally accessible as well as economical system for growth and analysis of developing tooth germs. The inhibitory effects of Shh neutralizing antibody treatment are discussed in relation to roles of this signaling pathway proposed by this and other groups previously. © 2002 Wiley-Liss, Inc. [source] Platelet-derived growth factor receptors expressed in response to injury of differentiated vascular smooth muscle in vitro: effects on Ca2+ and growth signalsACTA PHYSIOLOGICA, Issue 2 2001A. Lindqvist Vascular smooth muscle cells (VSMCs) in the intact vascular wall are differentiated for contraction, whereas the response to vascular injury involves transition towards a synthetic phenotype, with increased tendency for proliferation. Platelet-derived growth factor (PDGF) is thought to be important for this process. We investigated expression and functional coupling of PDGF receptors (PDGFRs) , and , in rat tail arterial rings kept in organ culture, in order to capture early events in the phenotypic transition. In freshly dissected rings no PDGFR immunoreactivity was found in medial VSMCs, whereas PDGFR , was detected in nerve fibres. After organ culture for 1,4 days PDGFR , and , as well as phospholipase C,2 (PLC,2), known to couple to PDGFR, were expressed in VSMCs within 100 ,m of the cut ends. Calponin, a marker for the contractile phenotype, was decreased near the injured area, suggesting that cells were in transition towards synthetic phenotype. In these cells, which showed functional Ca2+ -release from the sarcoplasmic reticulum, PDGF-AB (100 ng mL,1) had no effect on [Ca2+]i, whereas cultured VSMCs obtained from explants of rat tail arterial rings responded to PDGF-AB with an increase in [Ca2+]i. However, PDGFR within the cultured rings coupled to growth signalling pathways, as PDGF-AB caused a tyrphostin AG1295-sensitive activation of extracellular signal-regulated kinases 1 and 2 and of [3H]-thymidine incorporation. Thus, early expression of PDGFR in VSMC adjacent to sites of vascular injury coincides with signs of dedifferentiation. These receptors couple to growth signalling, but do not activate intracellular Ca2+ release. [source] Establishment of a chick embryo model for analyzing liver development and a search for candidate genesDEVELOPMENT GROWTH & DIFFERENTIATION, Issue 6 2005Yuji Yokouchi The liver plays a crucial role in metabolism. There is considerable interest in how the liver develops, as such knowledge could prove of importance in regenerative medicine. However, our understanding of liver development remains somewhat limited. We have developed a model system using the chick embryo that is cost effective and is easy to manipulate experimentally. We performed four fundamental studies: (i) construction of an atlas of the developing chick liver; (ii) identification of differentiation marker genes in the developing chick embryo; (iii) development of germ-layer specific electroporation; and (iv) establishment of organ culture from the developing chick liver. Using this system, we have been able to demonstrate the functions of candidate genes within a shorter period and in a more cost-effective manner. In parallel with the establishment of this system, we examined the expression patterns of genes known to be required for organ development in the developing chick embryo in order to identify genes also involved in liver development. To date, we have found sixteen genes that are expressed in the developing chick liver (GELD, genes expressed in liver development). This knowledge will be fundamental to the establishment of the basic technology for engineering liver tissue in the future. [source] Developmental expression of Smoc1 and Smoc2 suggests potential roles in fetal gonad and reproductive tract differentiationDEVELOPMENTAL DYNAMICS, Issue 11 2009Dorothy E. Pazin Abstract SMOC1 and SMOC2 are matricellular proteins thought to influence growth factor signaling, migration, proliferation, and angiogenesis. We examined the expression and regulation of Smoc1 and Smoc2 in fetal gonad/mesonephros complexes to discover possible roles for these genes in gonad and mesonephros development. Smoc1 was upregulated at ,E10.75 in a center-to-poles wave in pre-Sertoli and pre-granulosa cells and its expression was greatly reduced in Wt1, Sf1, and Fog2 mutants. After E13.5, Smoc1 was downregulated in an anterior-to-posterior wave in granulosa cells but persisted in Sertoli cells, suggesting a sexually dimorphic requirement in supporting cell lineage differentiation. Smoc2 was expressed in Leydig cells, mesonephroi, and Wnt4 mutant ovaries, but not wildtype ovaries. Using organ culture, we determined that Smoc2 expression was dependent on Hedgehog signaling in testes, mesonephroi, and kidneys. Overall, these results demonstrate that SMOC1 and SMOC2 may mediate intercellular signaling and cell type,specific differentiation during gonad and reproductive tract development. Developmental Dynamics 238:2877,2890, 2009. © 2009 Wiley-Liss, Inc. [source] Shh/BMP-4 signaling pathway is essential for intestinal epithelial development during Xenopus larval-to-adult remodelingDEVELOPMENTAL DYNAMICS, Issue 12 2006Atsuko Ishizuya-Oka Abstract During amphibian larval-to-adult intestinal remodeling, progenitor cells of the adult epithelium actively proliferate and differentiate under the control of thyroid hormone (TH) to form the intestinal absorptive epithelium, which is analogous to the mammalian counterpart. We previously found that TH,up-regulated expression of bone morphogenetic protein-4 (BMP-4) spatiotemporally correlates with adult epithelial development in the Xenopus laevis intestine. Here, we aimed to clarify the role of BMP-4 in intestinal remodeling. Our reverse transcriptase-polymerase chain reaction and in situ hybridization analyses indicated that mRNA of BMPR-IA, a type I receptor of BMP-4, is expressed in both the developing connective tissue and progenitor cells of the adult epithelium. More importantly, using organ culture and immunohistochemical procedures, we have shown that BMP-4 not only represses cell proliferation of the connective tissue but promotes differentiation of the intestinal absorptive epithelium. In addition, we found that the connective tissue-specific expression of BMP-4 mRNA is up-regulated by sonic hedgehog (Shh), whose epithelium-specific expression is directly induced by TH. These results strongly suggest that the Shh/BMP-4 signaling pathway plays key roles in the amphibian intestinal remodeling through epithelial,connective tissue interactions. Developmental Dynamics 235:3240,3249, 2006. © 2006 Wiley-Liss, Inc. [source] Chick limbs with mouse teeth: An effective in vivo culture system for tooth germ development and analysisDEVELOPMENTAL DYNAMICS, Issue 1 2003Eiki Koyama Abstract Mouse tooth germ development is currently studied by three main approaches: in wild-type and mutant mouse lines, after transplantation of tooth germs to ectopic sites, and in organ culture. The in vivo approaches are the most physiological but do not provide accessibility to tooth germs for further experimental manipulation. Organ cultures, although readily accessible, do not sustain full tooth germ development and are appropriate for short-term analysis. Thus, we sought to establish a new approach that would combine experimental accessibility with sustained development. We implanted fragments of embryonic day 12 mouse embryo first branchial arch containing early bud stage tooth germs into the lateral mesenchyme of day 4,5 chick embryo wing buds in ovo. Eggs were reincubated, and implanted tissues were examined by histochemistry and in situ hybridization over time. The tooth germs underwent seemingly normal growth, differentiation, and morphogenesis. They reached the cap, bell, and crown stages in approximately 3, 6, and 10 days, respectively, mimicking in a striking manner native temporal patterns. To examine mechanisms regulating tooth germ development, we first implanted tooth germ fragments, microinjected them with neutralizing antibodies to the key signaling molecule Sonic hedgehog (Shh), and examined them over time. Tooth germ development was markedly delayed, as revealed by poor morphogenesis and lack of mature ameloblasts and odontoblasts displaying characteristic traits such as an elongated cell shape, nuclear relocalization, and amelogenin gene expression. These phenotypic changes began to be reversed upon further incubation. The data show that the limb bud represents an effective, experimentally accessible as well as economical system for growth and analysis of developing tooth germs. The inhibitory effects of Shh neutralizing antibody treatment are discussed in relation to roles of this signaling pathway proposed by this and other groups previously. © 2002 Wiley-Liss, Inc. [source] Inhibition of Notch signaling biases rat thymocyte development towards the NK cell lineageEUROPEAN JOURNAL OF IMMUNOLOGY, Issue 5 2004Jens van den Brandt Abstract Notch receptors are involved in directing the choice between alternative cell fates in developmental scenarios such as thymopoiesis. By pharmacological interference in rat fetal thymus organ culture we show that inhibition of Notch signaling arrests T,cell development at an early double-negative stage and is accompanied by a dramatic increase in the number of NK cells. These cells show an activated phenotype, lack recombination of the TCR, gene locus and express perforin. Similarly, in thymic lobes reconstituted with fetal liver cells, progenitors predominantly develop into NK cells both after pharmacological interference of Notch and after treatment with a recombinant rat Notch1/Fc chimera. Collectively, this identifies the lineage decision of NK/T precursor cells as an important site of Notch action in rat thymocytes. [source] The tyrosine kinase Syk is required for light chain isotype exclusion but dispensable for the negative selection of B,cellsEUROPEAN JOURNAL OF IMMUNOLOGY, Issue 4 2004Josephine Meade Abstract In this study we set out to test whether Syk was required for negative selection of immature B,cells. B,cells expressing a B,cell antigen receptor (BCR) transgene (3,83, anti-H-2Kk) underwent negative selection independently of Syk in both fetal liver organ culture and radiation chimera models. Furthermore, Syk-independent negative selection was not reversed by transgenic overexpression of Bcl-2. Receptor editing was not apparent in Syk-deficient B,cells, presumably as a consequence of the failure of mature edited B,cells to develop in the absence of Syk. Interestingly, light chain isotype exclusion by the BCR transgene failed in the absence of Syk. We observed a dramatic reduction in the overall BCR-mediated tyrosine phosphorylation of cellular proteins in Syk-deficient immature B,cells. However, the tyrosine phosphorylation of a number of substrates including phospholipase,C,2, although reduced, was not completely abrogated. BCR ligation triggered an increase in calcium flux in the absence of Syk. Thus signaling events that mediate negative selection can still occur in the absence of Syk. This may be due to redundancy with zeta-associated protein,70 (ZAP-70), which we demonstrate to be expressed in immature B,cells. [source] Toll-like receptor stimulation induces airway hyper-responsiveness to bradykinin, an effect mediated by JNK and NF-,B signaling pathwaysEUROPEAN JOURNAL OF IMMUNOLOGY, Issue 4 2004Ofir Bachar Abstract Airway infections induce hyper-responsiveness in asthmatic patients. Toll-like receptors (TLR) mediate inflammatory responses to microbes. Occurrence and effects of TLR2, TLR3 and TLR4 were examined in a mouse organ culture model of asthma focusing on the smooth muscle responses to bradykinin. TLR2, TLR3 and TLR4 mRNA, and TLR2 and TLR4 immunoreactivity were detected in the tracheal muscle layer. Tracheal organ culture for 1 or 4,days with lipopolysaccharide (LPS; TLR2/4 agonist) or polyinosinic polycytidylic acid (poly-I-C; TLR3 agonist) enhanced bradykinin- and [des-Arg9]-bradykinin-induced contractions. Simultaneous LPS and poly-I-C treatment resulted in synergistic enhancement of bradykinin-induced contraction. In carbachol-pre-contracted segments TLR stimulationinduced less potent relaxations to bradykinin and [des-Arg9]-bradykinin. The LPS and poly-I-C enhancement of bradykinin-induced contraction was inhibited by the transcriptional inhibitor actinomycin-D, dexamethasone, the proteasome inhibitor MG-132 and the c-Jun N-terminal kinase (JNK) inhibitor SP600125. LPS and poly-I-C induced translocation of NF-,B p65 to the nucleus and up-regulation of kinin B1 and B2 receptor mRNA. In summary, TLR2, TLR3 and TLR4 are expressed in the mouse tracheal smooth muscle. Costimulation of these receptors results in NF-,B- and JNK-mediated transcription of B1 and B2 receptor, inducing hyper-responsiveness to bradykinin. [source] Weak agonist self-peptides promote selection and tuning of virus-specific T cellsEUROPEAN JOURNAL OF IMMUNOLOGY, Issue 3 2003Samuel Abstract Recent progress has begun to define the interactions and signaling pathways that are triggered during positive selection. To identify and further examine self-peptides that can mediate positive selection, we searched a protein-database to find peptides that have minimal homology with the viral peptide (p33) that activates a defined P14 transgenic TCR. We identified four peptides that could bind the restriction element H-2Db and induce proliferation of P14 transgenic splenocytes at high concentration. Two of the four peptides (DBM and RPP) were able to positively select thevirus-specific TCR in fetal thymic organ culture but were unable to induce clonal deletion. Reverse-phase HPLC and mass spectrometry demonstrated that these peptides were presented by H-2Db molecules on thymic epithelial cell lines. We also examined whether the selecting ligands altered T cell responsiveness in vitro. DBM-selected T cells lost their ability to respond to the positively selecting ligand DBM, whereas RPP-selected T cells only retainrd their ability to respond to high concentrations of RPP. These results demonstrate that self-peptides that mediate positiveselection can differentially "tune" the activation threshold of T cells and alter the functional repertoire of T cells. [source] Polyamines and hair: a couple in search of perfectionEXPERIMENTAL DERMATOLOGY, Issue 9 2010Yuval Ramot Please cite this paper as: Polyamines and hair: a couple in search of perfection. Experimental Dermatology 2010; 19: 784,790. Abstract:, Polyamines (spermidine, putrescine and spermine) are multifunctional cationic amines that are indispensable for cellular proliferation; of key significance in the growth of rapidly regenerating tissues and tumors. Given that the hair follicle (HF) is one of the most highly proliferative organs in mammalian biology, it is not surprising that polyamines are crucial to HF growth. Indeed, growing (anagen) HFs show the highest activity of ornithine decarboxylase (ODC), the rate-limiting enzyme of polyamine biosynthesis, while inhibition of ODC, using eflornithine, results in a decreased rate of excessive facial hair growth in vivo and inhibits human scalp hair growth in organ culture. In sheep, manipulation of dietary intake of polyamines also results in altered wool growth. Polyamine-containing nutraceuticals have therefore been proposed as promoters of human hair growth. Recent progress in polyamine research, coupled with renewed interest in the role of polyamines in skin biology, encourages one to revisit their potential roles in HF biology and highlights the need for a systematic evaluation of their mechanisms of action and clinical applications in the treatment of hair disorders. The present viewpoint essay outlines the key frontiers in polyamine-related hair research and defines the major open questions. Moreover, it argues that a renaissance in polyamine research in hair biology, well beyond the inhibition of ODC activity in hirsutism therapy, is important for the development of novel therapeutic strategies for the manipulation of human hair growth. Such targets could include the manipulation of polyamine biosynthesis and the topical administration of selected polyamines, such as spermidine. [source] Reorganization of hair follicles in human skin organ culture induced by cultured human follicle-derived cellsEXPERIMENTAL DERMATOLOGY, Issue 8 2005Walter Krugluger Abstract:, Studies of human hair follicle (HF) induction by follicle-derived cells have been limited due to a lack of suitable test systems. In this study, we established a skin organ culture system which supports HF formation by follicle-derived cells. Long-term skin organ cultures were set up from human retroauricular skin specimens and maintained in culture for up to 8 weeks. In vitro expanded human HF-derived cells from the dermal papilla (DP) and the outer root sheath (ORS) were injected together into the skin specimens and evaluated for their ability to induce reorganization of HFs. Macroscopic analysis of the cultured skin specimens demonstrated the growth of velus-like hair after 4 weeks in culture. Histologic evaluation of the cultured skin specimens after 8 weeks of culture revealed multiple miniaturized HFs with sebaceous glands. In addition, cell clusters of various differentiation stages could be demonstrated in serial sections of the cultured skin specimens. Labeling of HF-derived cells with the fluorescence dye CFDA-1 prior to injection suggested a de novo reorganization of HFs out of the injected cells. In conclusion, the study demonstrated HF formation by HF-derived cells in an in vitro skin organ culture model. [source] Onset of promiscuous gene expression in murine fetal thymus organ cultureIMMUNOLOGY, Issue 3 2006Renato Sousa Cardoso Summary T-cell differentiation and induction of tolerance to self-antigens occurs mainly in the thymus. Thymic stromal cells, specifically medullary thymic epithelial cells, express a diverse set of genes encoding parenchymal organ-specific proteins. This phenomenon has been termed promiscuous gene expression (PGE) and has been implicated in preventing organ-specific autoimmunity by inducing T-cell tolerance to self antigens. Early thymopoiesis and the critical factors involved in T-cell differentiation can be reproduced in vitro by murine fetal thymus organ culture (FTOC), which mimics the natural thymic microenvironment. To evaluate the occurrence of PGE in FTOC, gene expression profiling during in vitro thymic development in BALB/c mice was performed using a set of nylon cDNA microarrays containing 9216 sequences. The statistical analysis of the microarray data (sam program) revealed the temporal repression and induction of 57 parenchymal and seven lymphoid organ-specific genes. Most of the genes analysed are repressed during early thymic development (15,17 days post-coitum). The expression of the autoimmune regulator (AIRE) gene at 16 days post-coitum marks the onset of PGE. This precedes the induction of parenchymal organ genes during the late developmental phase at 20 days post-coitum. The mechanism of T-cell tolerance induction begins during fetal development and continues into adulthood. Our findings are significant because they show a fine demarcation of PGE onset, which plays a central role in induction of T-cell tolerance. [source] Anti-inflammatory role of interleukin-15 in Crohn's diseaseINFLAMMATORY BOWEL DISEASES, Issue 3 2005Manuel A Silva MD Abstract Background: Interleukin (IL)-15 is overexpressed in intestinal tissue with active Crohn's disease (CD). However, its role in the pathogenesis of the disease remains uncertain. We studied the effects of IL-15 on colonic mucosal proinflammatory cytokine response in vitro using organ culture of human colonic explants. Methods: Colonic tissue was obtained from (1) resections in pediatric CD patients (inflamed and noninflamed) and (2) rectal biopsies in patients with CD undergoing colonoscopy (n = 31) and controls (n = 9). In preliminary experiments, explants from the resections were cultured in the presence or absence of a simulated TH1 stimulation using ionomycin (Io) and phorbol-myristate-acetate (PMA), with or without IL-15, or in medium alone. Rectal biopsies were cultured in the same conditions as above, with or without adding a monoclonal anti-IL-15 neutralizing antibody (mAb). Levels of interferon (IFN)-,, tumor necrosis factor (TNF)-,, and IL-2R, were measured by enzyme-linked immunosorbent assay. Results: IL-15, in the absence of Io + PMA, did not induce the expression of IFN-,, TNF-,, or IL-2R,. Only inflamed explants from resections stimulated with Io + PMA expressed IFN-,, TNF-,, and IL-2R,. This TH1 stimulatory effect was inhibited by IL-15 in a dose-dependent fashion. In rectal biopsy explants, inflamed, noninflamed CD, and control tissue responded to stimulation with Io + PMA (P < 0.05) with increased IFN-, and TNF-, (P < 0.05). This response was again inhibited by IL-15. The inhibitory effect of IL-15 was specifically reversed by anti-IL-15 mAb (P < 0.05). The data for the CD group were also analyzed according to the severity of colonic inflammation and medication use. Conclusions: Our results suggest a possible anti-inflammatory role for IL-15 in CD. We postulate that its overexpression in CD potentially represents a protective mechanism against the exaggerated TH1 immune response. [source] An ex vivo swine tracheal organ culture for the study of influenza infectionINFLUENZA AND OTHER RESPIRATORY VIRUSES, Issue 1 2010Sandro F. Nunes Background The threat posed by swine influenza viruses with potential to transmit from pig populations to other hosts, including humans, requires the development of new experimental systems to study different aspects of influenza infection. Ex vivo organ culture (EVOC) systems have been successfully used in the study of both human and animal respiratory pathogens. Objectives We aimed to develop an air interface EVOC using pig tracheas in the study of influenza infection demonstrating that tracheal explants can be effectively maintained in organ culture and support productive influenza infection. Methods Tracheal explants were maintained in the air interface EVOC system for 7 days. Histological characteristics were analysed with different staining protocols and co-ordinated ciliary movement on the epithelial surface was evaluated through a bead clearance assay. Explants were infected with a swine H1N1 influenza virus. Influenza infection of epithelial cells was confirmed by immunohistochemistry and viral replication was quantified by plaque assays and real-time RT-PCR. Results Histological analysis and bead clearance assay showed that the tissue architecture of the explants was maintained for up to 7 days, while ciliary movement exhibited a gradual decrease after 4 days. Challenge with swine H1N1 influenza virus showed that the EVOC tracheal system shows histological changes consistent with in vivo influenza infection and supported productive viral replication over multiple cycles of infection. Conclusion The air interface EVOC system using pig trachea described here constitutes a useful biological tool with a wide range of applications in the study of influenza infection. [source] Comparison of operative procedure variables on pulpal viability in an ex vivo modelINTERNATIONAL ENDODONTIC JOURNAL, Issue 5 2008P. E. Murray Abstract Aim, To measure and compare the responses of pulp tissue to cavity preparation and restoration variables using a novel tooth slice culture model. Methodology, Experimental cavities (265) were continuously cut, under carefully controlled conditions, into the dentine of the labial aspect of 28-day-old Wistar rat incisors, and slices of these teeth maintained in organ culture for up to 2 weeks. The experimental variables examined were: the preparation method, remaining dentine thickness, coolant, drill speed, conditioning with EDTA and filling materials. The reactions of the dentine,pulp complex to the experimental variables were measured using pathohistometric analysis and the correlations between variables were determined using analysis of variance statistical tests. Results, In rank order of surgically induced restorative pulpal injury, from the most to the least injurious were: remaining dentine thickness, absence of coolant during cavity preparation, bur speed, cavity conditioning treatments and the filling material. Conclusions, To reduce pulp injury and to promote pulpal repair activity, the correct use of appropriate materials are important. However, of relatively greater importance is the operative technique adopted, the need to avoid the excess removal of dentine and to minimize trauma during preparation. [source] Cyclooxygenase-2 Expression and Prostaglandin E2 Production in Response to Acidic pH Through OGR1 in a Human Osteoblastic Cell Line,,JOURNAL OF BONE AND MINERAL RESEARCH, Issue 7 2008Hideaki Tomura Abstract Acidosis has been shown to induce depletion of bone calcium from the body. This calcium release process is thought to be partially cell mediated. In an organ culture of bone, acidic pH has been shown to induce cyclooxygenase-2 (COX-2) induction and prostaglandin E2 (PGE2) production, resulting in stimulation of bone calcium release. However, the molecular mechanisms whereby osteoblasts sense acidic circumstances and thereby induce COX-2 induction and PGE2 production remain unknown. In this study, we used a human osteoblastic cell line (NHOst) to characterize cellular activities, including inositol phosphate production, intracellular Ca2+ concentration ([Ca2+]i), PGE2 production, and COX-2 mRNA and protein expression, in response to extracellular acidification. Small interfering RNA (siRNA) specific to the OGR1 receptor and specific inhibitors for intracellular signaling pathways were used to characterize acidification-induced cellular activities. We found that extracellular acidic pH induced a transient increase in [Ca2+]i and inositol phosphate production in the cells. Acidification also induced COX-2 induction, resulting in PGE2 production. These proton-induced actions were markedly inhibited by siRNA targeted for the OGR1 receptor and the inhibitors for Gq/11 protein, phospholipase C, and protein kinase C. We conclude that the OGR1/Gq/11/phospholipase C/protein kinase C pathway regulates osteoblastic COX-2 induction and subsequent PGE2 production in response to acidic circumstances. [source] Ex vivo organ culture of adipose tissue for in situ mobilization of adipose-derived stem cells and defining the stem cell nicheJOURNAL OF CELLULAR PHYSIOLOGY, Issue 3 2010Young-Il Yang In spite of the advances in the knowledge of adipose-derived stem cells (ASCs), in situ location of ASCs and the niche component of adipose tissue (AT) remain controversial due to the lack of an appropriate culture system. Here we describe a fibrin matrix-supported three-dimensional (3D) organ culture system for AT which sustains the ASC niche and allows for in situ mobilization and expansion of ASCs in vitro. AT fragments were completely encapsulated within the fibrin matrix and cultured under dynamic condition. The use of organ culture of AT resulted in a robust outgrowth and proliferation in the fibrin matrix. The outgrown cells were successfully recovered from fibrin by urokinase treatment. These outgrown cells fulfilled the criteria of mesenchymal stem cells, adherence to plastic, multilineage differentiation, and cell surface molecule expression. In vitro label retaining assay revealed that newly divided cells during the culture resided in interstitium between adipocytes and capillary endothelial cells. These interstitial stromal cells proliferated and outgrew into the fibrin matrix. Both in situ mobilized and outgrown cells expressed CD146 and ,-smooth muscle actin (SMA), but no endothelial cell markers (CD31 and CD34). The structural integrity and spatial approximation of CD31,/CD34,/CD146+/SMA+ interstitial stromal cells, adipocytes, and capillary endothelial cells were well preserved during in vitro culture. Our results suggest that ASCs are natively associated with the capillary wall and more specifically, belong to a subset of pericytes. Furthermore, organ culture of AT within a fibrin matrix-supported 3D environment can recapitulate the ASC niche in vitro. J. Cell. Physiol. 224: 807,816, 2010. © 2010 Wiley-Liss, Inc. [source] Localization of insulin-like growth factor binding protein-2 in chondrocytes of bovine articular cartilageJOURNAL OF ORTHOPAEDIC RESEARCH, Issue 2 2003Teresa I. Morales Purpose: Previous work indicated that transforming growth factor (TGF-,) treatment of bovine articular cartilage resulted in an accumulation of insulin-like growth factor binding protein-2 (IGF-BP-2). The purpose of the work presented in this paper was to define the localization of the IGF-BP-2 in freshly excised articular cartilage and in slices cultured in the presence and absence of TGF-,. Method: Newborn calf articular cartilage was dissected and immediately fixed or maintained in organ culture for five days under basal conditions (media without added serum or growth factors) or with basal media containing 15 ng/ml of TGF-,1. Frozen or paraffin embedded sections were prepared, and immunohistochemistry using anti-IGF-BP-2 performed. Results: The paraffin sections provided the best preservation of morphology and consistency of immunohistochemical staining patterns. In fresh cartilage slices, IGF-BP-2 was associated with most of the chondrocytes. The basal cultured cartilage showed positive immunostaining in some areas, but not others: the most consistently stained area was the upper radial zone. In all cases where a positive reaction was observed, it was associated mostly with chondrocytes. On the other hand, all the TGF-, treated samples that were examined in this study were evenly stained, and most chondrocytes were positive in all areas from superficial to deep zones, thus closely resembling the pattern of fresh tissue. Conclusions: It is concluded that IGF-BP-2 is closely cell associated in bovine articular cartilage. Following culture of cartilage slices, TGF-, increases the number of cells with positive immunostaining. These data help to support the postulate that TGF-, exerts at least some of its actions in articular cartilage via cross-talk mechanisms involving the IGF-BP-2 system. © 2002 Orthopaedic Research Society. Published by Elsevier Science Ltd. All rights reserved. [source] Lentivirus vector-mediated gene transfer to the developing bronchiolar airway epithelium in the fetal lambTHE JOURNAL OF GENE MEDICINE, Issue 6 2007Ze-Yan Yu Abstract Background Development of effective and durable gene therapy for treatment of the respiratory manifestations of cystic fibrosis remains a formidable challenge. Obstacles include difficulty in achieving efficient gene transfer to mature airway epithelium and the need to stably transduce self-renewing epithelial progenitor cells in order to avoid loss of transgene expression through epithelial turnover. Targeting the developing airway epithelium during fetal life offers the prospect of circumventing these challenges. Methods In the current study we investigated vesicular stomatitis virus glycoprotein (VSVg)-pseudotyped HIV-1-derived lentivirus vector-mediated gene transfer to the airway epithelium of mid-gestation fetal lambs, both in vitro and in vivo. In the in vitro studies epithelial sheet explants and lung organ culture were used to examine transduction of the proximal and more distal airway epithelium, respectively. For the in vivo studies, vector was delivered directly into the proximal airway. Results We found that even during the early pseudoglandular and canalicular phases of lung development, occurring through mid-gestation, the proximal bronchial airway epithelium was relatively mature and highly resistant to lentivirus-mediated transduction. In contrast, the more distal bronchiolar airway epithelium was relatively permissive for transduction although the absolute levels achieved remained low. Conclusion This result is promising as the bronchiolar airway epithelium is a major site of pathology in the cystic fibrosis airway, and much higher levels of transduction are likely to be achieved by developing strategies that increase the amount of vector reaching the more distal airway after intratracheal delivery. Copyright © 2007 John Wiley & Sons, Ltd. [source] Synovial fibroblasts self-direct multicellular lining architecture and synthetic function in three-dimensional organ cultureARTHRITIS & RHEUMATISM, Issue 3 2010Hans P. Kiener Objective To define the intrinsic capacity of fibroblast-like synoviocytes (FLS) to establish a 3-dimensional (3-D) complex synovial lining architecture characterized by the multicellular organization of the compacted synovial lining and the elaboration of synovial fluid constituents. Methods FLS were cultured in spherical extracellular matrix (ECM) micromasses for 3 weeks. The FLS micromass architecture was assessed histologically and compared with that of dermal fibroblast controls. Lubricin synthesis was measured via immunodetection. Basement membrane matrix and reticular fiber stains were performed to examine ECM organization. Primary human and mouse monocytes were prepared and cocultured with FLS in micromass to investigate cocompaction in the lining architecture. Cytokine stimuli were applied to determine the capacity for inflammatory architecture rearrangement. Results FLS, but not dermal fibroblasts, spontaneously formed a compacted lining architecture over 3 weeks in the 3-D ECM micromass organ cultures. These lining cells produced lubricin. FLS rearranged their surrounding ECM into a complex architecture resembling the synovial lining and supported the survival and cocompaction of monocyte/macrophages in the neo,lining structure. Furthermore, when stimulated by cytokines, FLS lining structures displayed features of the hyperplastic rheumatoid arthritis synovial lining. Conclusion This 3-D micromass organ culture method demonstrates that many of the phenotypic characteristics of the normal and the hyperplastic synovial lining in vivo are intrinsic functions of FLS. Moreover, FLS promote survival and cocompaction of primary monocytes in a manner remarkably similar to that of synovial lining macrophages. These findings provide new insight into inherent functions of the FLS lineage and establish a powerful in vitro method for further investigation of this lineage. [source] CCAAT/ENHANCER binding protein , mediates expression of matrix metalloproteinase 13 in human articular chondrocytes in inflammatory arthritisARTHRITIS & RHEUMATISM, Issue 3 2009Mitsumasa Hayashida Objective To determine the function of CCAAT/enhancer binding protein , (C/EBP,) in the expression of matrix metalloproteinase 13 (MMP-13) in chondrocytes in inflammatory arthritis. Methods Cartilage obtained from patients with rheumatoid arthritis and osteoarthritis was immunostained for expression of C/EBP, or MMP-13. Interleukin-1,, or tumor necrosis factor , (TNF,),stimulated chondrocytes were subjected to Western blotting and real-time reverse transcriptase,polymerase chain reaction (RT-PCR). MMP-13 promoter assays were conducted, and the C/EBP, response element was characterized by deletion and mutation analysis. C-28/I2 cells were treated with TNF, and subjected to chromatin immunoprecipitation (ChIP) assays. Finally, C/EBP,,liver-enriched activator protein (LAP) was overexpressed in C-28/I2 cells or cartilage tissues, and MMP-13 expression was analyzed. Results C/EBP, and MMP-13 expression was colocalized in chondrocytes in arthritic cartilage. MMP-13 promoter activity was stimulated by C/EBP, overexpression in a dose-dependent manner. Luciferase assays revealed that a ,981-bp promoter had the greatest activity, while deletion to ,936 bp strongly diminished promoter activity. Luciferase activity was repressed to basal levels by mutations in potential C/EBP binding sites. The stimulatory effects of C/EBP, overexpression were diminished by mutation. ChIP assays revealed that TNF, treatment enhanced the binding of C/EBP, to the MMP-13 promoter. When C/EBP,-LAP was overexpressed in C-28/I2 cells, endogenous MMP-13 expression was stimulated up to 32-fold as detected by real-time RT-PCR. Furthermore, following adenoviral overexpression of C/EBP,-LAP in organ culture of articular cartilage, stimulation of MMP-13 was also detected by immunohistochemistry. Conclusion C/EBP, directly binds to the MMP-13 promoter region and stimulates the expression of MMP-13 in chondrocytes in inflammatory arthritis. [source] Osteogenic Evaluation of Glutaraldehyde Crosslinked Gelatin Composite with Fetal Rat Calvarial Culture ModelARTIFICIAL ORGANS, Issue 8 2001Hwa-Chang Liu Abstract: The cytotoxicity of the synthetic bone substitute composed of tricalcium phosphate and glutaraldehyde crosslinked gelatin (GTG) were evaluated by osteoblast cell culture. In a previous study, the GTG composites were soaked in distilled water for 1, 2, 4, 7, 14, 28, and 42 days, and then the solutions (or extracts) were cocultured with osteoblasts to evaluate the cytotoxicity of GTG composites by alive cell counting. In this study, the extracts were cocultured with the osteoblasts; thereafter, the concentration of transforming growth factor-, (TGF-,1) and prostaglandin E2 (PGE2) in the medium was analyzed to strictly reflect the biological effects of GTG composites on the growth of osteoblasts. In order to investigate the osteoconductive potential of the GTG composites on new bone formation in a relative short term, a model of neonatal rat calvarial organ culture was designed prior to animal experiments. Three experimental materials of 4, 8, and 12% GTG composites were evaluated by fetal rat calvarial organ culture for their ability for bone regeneration. Deproteinized bovine and porcine cancellous bone matrixes were used as the controlled materials. All the organ culture units were maintained in cultured medium for 5 weeks. Following the culture period, the morphology of tissue was observed under an optical microscope, and the quantitative evaluation of the new generation bone was determined by using a semiautomatic histomorphometeric method. Except in the initial 4 days, the concentration of TGF-,1 of 4% and 8% GTG composites was higher than that of the blank group for all the other experimental time periods. The PGE2 concentration for 4% and 8% GTG composites was lower than that of the blank group. It revealed that the 4% and 8% GTG composites would not lead to inflammation and would promote osteoblast growth. The morphology and activity of the osteoblasts were not transformed or changed by the 2 GTG composites. For the 12% GTG composite, the performance of the in vitro condition was inferior to the blank group and the other 2 GTG composites. Although the concentration of TGF-,1 and PGE2 was gradually back to normal after 14 days, the morphology of the osteoblasts was abnormal with features such as contracted cytoplast structures. The osteoblast was damaged perhaps in the initial stage. We suggested that the 4% and 8% GTG composites should be soaked in distilled water at least for 4 days before medical applications. The 12% GTG composite and the composites with a concentration of glutaraldehyde solution higher than 12% were not recommended as a medical prostheses in any condition. The fetal rat calvaria culture also showed the same results with the analysis of TGF-,1 and PGE2. From the study, we could predict the results of animal experiments in the future. [source] Enhanced Transcription of Contractile 5-Hydroxytryptamine 2A Receptors via Extracellular Signal-Regulated Kinase 1/2 after Organ Culture of Rat Mesenteric ArteryBASIC AND CLINICAL PHARMACOLOGY & TOXICOLOGY, Issue 4 2005Yong-Xiao Cao The present study was designed to examine if vascular 5-HT2A receptors are up-regulated during organ culture and if the extracellular signal-regulated protein kinase 1/2 (ERK1/2) pathways are involved. Compared with fresh rat mesenteric artery ring segments, the contractile responses to 5-HT were significantly increased in the segments cultured for 6, 24 or 48 hr (P<0.05, P<0.01, P<0.01, respectively). The 5-HT-induced contraction occurred via 5-HT2A receptors, since the selective 5-HT2A antagonist ketanserin blocked the 5-HT-induced contraction in the fresh segments with a pA2 value 9.5 (slope was 0.98 with 95% confidence intervals from 0.8 to 1.1). A similar result was obtained in the segments cultured for 24 hr with a pA2 value of 9.43 (slope=0.91 and 95% confidence intervals between 0.45 to 2.3). In addition, the enhanced 5-HT2A receptor contraction occurred with a significant increase of 5-HT2A receptor mRNA (P<0.05). Organ culture of the mesenteric artery was found to activate ERK1/2 already within 1 and 3 hr. It is likely that the ERK1/2 pathways were involved as a initial switch, since the selective ERK1/2 pathway inhibitor SB386023 abolished both up-regulation of 5-HT2A mRNA transcription and the enhanced contractile response to 5-HT. These data reveal a role of ERK1/2 in up-regulation of 5-HT2A receptors and suggest a possibility to inhibit the enhanced responses to 5-HT by inhibition of the ERK1/2 pathway. [source] Date seed oil limit oxidative injuries induced by hydrogen peroxide in human skin organ cultureBIOFACTORS, Issue 2-3 2007Ines Dammak Abstract The skin is chronically exposed to pro-oxidant agents, leading to the generation of reactive oxygen species (ROS). To protect the skin against an over-load of oxidant species, we studied the chemoprotective effect of one new natural product: "date seed oil: DSO". This oil may serve as a potential source of natural antioxidants such as phenols and tocopherols. Here, the antioxidative potential of DSO was compared that of to extra virgin olive oil. Adult human skin was maintained in organ culture in the presence of the DSO and extra virgin olive oil before the addition of hydrogen peroxide (H2O2 ), in order to prevent the tissue from its oxidizing effects. Skin specimens were collected for histology and for melanin studies. In the investigated model system, DSO protects skin against oxidative injuries. It has a significant chemoprotective effect, by inhibition of damage caused by H2O2 compared with specimens without such addition endowing with a radical scavenging ability. The various components from DSO were much more potent antioxidant and more free radical scavengers of the H2O2 than those of olive oil. Our study shows that topical DSO treatment of the skin stimulates events in the epidermis leading to repair skin damage possibly due to antioxidant synergisms. [source] Thyrotropin-releasing hormone and oestrogen differentially regulate prolactin and prolactin receptor expression in female human skin and hair follicles in vitroBRITISH JOURNAL OF DERMATOLOGY, Issue 5 2010E.A. Langan Summary Background, Human skin and scalp hair follicles are both a nonclassical target and an extrapituitary source of prolactin (PRL), which is a potent hair growth modulator. However, how the expression of PRL and PRL receptor (PRLR) is regulated in human skin is unknown. Objectives, To investigate whether two key stimulators of pituitary PRL secretion, thyrotropin-releasing hormone (TRH) and oestrogen, also regulate cutaneous PRL and PRLR expression. Methods, Female scalp skin and/or microdissected hair follicles were treated for 6 days in serum-free organ culture with oestrogen (100 nmol L,1), TRH (1,10 ng mL,1, 3,30 nm) or vehicle control. Quantitative immunohistomorphometry of skin and hair follicle sections was complemented with quantitative polymerase chain reaction for PRL and PRLR in cultured hair follicles and/or female human outer root sheath (ORS) keratinocytes. Results, Oestrogen treatment significantly upregulated PRL and PRLR immunoreactivity in selected skin and hair follicle compartments, at the gene and protein level (P < 0·05). TRH significantly increased PRL immunoreactivity and transcription in hair follicles (P < 0·05); however, while it also increased PRLR transcription in hair follicles, it downregulated PRLR immunoreactivity in the hair follicle ORS (P < 0·05). Conclusions, Our pilot study shows that two key endocrine controls of pituitary PRL secretion, oestrogen and TRH, also regulate PRL and PRLR expression in human skin. This provides novel insights into the regulation of extrapituitary PRL and PRLR expression, and invites exploration of oestrogen and TRH as novel therapeutic agents in the management of skin and hair diseases characterized by aberrant PRLR-mediated signalling. [source] Organ culture, but not hypothermic storage, facilitates the repair of the corneal endothelium following mechanical damageACTA OPHTHALMOLOGICA, Issue 4 2010Jana Nejepinska Abstract. Purpose:, To evaluate the reparative capacity of the mechanically injured endothelium of corneas stored under organ culture (OC) or hypothermic conditions. Methods:, The central endothelium of 12 pairs of human corneas with similar endothelial parameters was damaged to create a 1 mm2 lesion. One cornea from each pair was stored under OC and one under hypothermic conditions. The endothelial cell density (ECD), coefficient of variation, hexagonality and percentage of dead cells were assessed before and after damage and on days 7, 14, 21 and 28 of storage. Results:, The mean ECD of corneas subsequently stored under OC or hypothermic conditions was 2764/mm2. Immediately after damage, a denuded Descemet's membrane with a few remaining dead cells was observed at the injured area. After 7 days of storage under OC conditions, almost no dead cells were observed at the place of injury. A non-significant worsening of the qualitative parameters (polymegatism and pleomorphism) was found. After 14 days, ECD was 1933/mm2 and 2478/mm2 centrally and pericentrally, respectively. Similar values were found after 21 and 28 days of storage. The lesions with remnant dead cells persisted throughout hypothermic preservation. From day 14 the corneas became cloudy and in poor condition, while the pericentral ECD was 2523/mm2. Conclusion:, The reparative capacity of the cornea is maintained under OC but not under hypothermic conditions. For corneas containing dead endothelial cells, OC is therefore the method of choice because it may improve the quality of the stored tissue. [source] | ||||||||||||||||||||||||||||||||||