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Messenger RNA Expression (messenger + rna_expression)
Selected AbstractsORIGINAL ARTICLE: Identification of Toll-Like Receptors in the Rat (Rattus norvegicus): Messenger RNA Expression in the Male Reproductive Tract Under Conditions of Androgen VariationAMERICAN JOURNAL OF REPRODUCTIVE IMMUNOLOGY, Issue 4 2009Barnali Biswas Problem, Although the majority of Toll-like receptors (TLRs) are reported in many species, some of them are not yet described in the rat. Further, factors that govern Tlr expression in the male reproductive tract have received little attention. We attempt to identify and characterize Tlrs in the rat and determine the expression profile under conditions that affect male reproductive tract gene expression. Method of study, Rat Tlr5, Tlr10, and Tlr11 transcript sequences were submitted to GenBank and in silico characterization carried out using bioinformatics tools. RT-PCR analyses using gene specific primers for rat Tlr1,13 were carried out with RNA isolated from reproductive tract tissues of various experimental groups. Results,Tlr5, Tlr10, and Tlr11 identified in this study share features that are characteristic of the known TLRs. Abundant Tlr expression was observed in the male reproductive tract of adult and developing rats. Further, Tlr expression was also observed in the epididymides of androgen ablated rats. Conclusion,Tlr5, Tlr10, and Tlr11 are ubiquitously expressed in the rat. Tlrs seem to be expressed during male reproductive tract development and under conditions of androgen ablation, suggesting the preparedness of the male reproductive tract to detect an infection under all conditions of androgen status. [source] Thyroglobulin type-1 domain protease inhibitors exhibit specific expression in the cortical ooplasm of vitellogenic rainbow trout oocytesMOLECULAR REPRODUCTION & DEVELOPMENT, Issue 2 2004Antony W. Wood Abstract The synthesis, uptake, and processing of yolk proteins remain poorly described aspects of oviparous reproductive development. In this study, we report the identification and characterization of two protease inhibitors in rainbow trout ovary whose expression and distribution are directly associated with yolk protein uptake in vitellogenic oocytes. The first transcript, termed "oocyte protease inhibitor-1" (OPI-1), is predicted to encode a 9.1 kDa, 87 amino acid protein containing a single thyroglobulin type-1 (TY) domain, identifying it as a putative TY domain inhibitor. The second transcript, termed OPI-2, is predicted to encode an 18.3 kDa, 173 amino acid protein with two similar, but not identical, TY domains. Messenger RNA expression of both genes was first detected in ovarian tissues at the onset of vitellogenesis, and persisted throughout the vitellogenic growth phase. We did not detect expression of either gene in previtellogenic ovaries, nor in any somatic tissues examined. Expression of OPI-1 mRNA was significantly reduced in atretic follicles as compared to healthy vitellogenic follicles, suggesting a downregulation of inhibitor expression during oocyte atresia. Western immunoblot analyses of whole yolk from vitellogenic oocytes revealed the presence of two immunoreactive proteins that corresponded to the predicted sizes of OPI-1 and OPI-2. We detected strong crossreactivity of this antiserum with specific vesicles in the cortical ooplasm of vitellogenic oocytes, in regions directly associated with vitellogenin processing. The identification of OPI-1 and OPI-2 provides new evidence for the expression of multiple TY domain protease inhibitors likely involved in the regulation of yolk processing during oocyte growth in salmonids. Mol. Reprod. Dev. 69: 205,214, 2004. © 2004 Wiley-Liss, Inc. [source] Differentiation, proliferation and retinoid receptor status of papillary carcinoma of the thyroidPATHOLOGY INTERNATIONAL, Issue 4 2003Weihua Tang Messenger RNA expression of retinoic acid receptors (RAR,, RAR, and RAR,) and retinoid X receptors (RXR,, RXR, and RXR,) was examined using reverse transcription-polymerase chain reaction in 42 papillary thyroid carcinomas (PTCs). A loss of mRNA expression was observed in 18 cases of the 42 PTCs, including three cases for RAR,, 14 cases for RAR,, six cases for RXR, and five cases for RXR,. The expressions of RAR, and RXR, were found in all 42 PTCs. Based on Ki 67/MIB1 labeling index (LI), the 42 PTCs were classified into Group A (20 cases; LI = 0,2%), Group B (17 cases; LI = 2,5%) and Group C (5 cases; LI > 5%). The PTCs of groups B and C showed solid, trabecular or scirrhous arrangements, infiltrative growth, loss of cellular polarity and cohesiveness more frequently, but capsulated growth pattern less frequently, when compared with PTCs of Group A. They also showed more frequent extrathyroidal extension than Group A. However, no significant differences were identified in sex, age, nodal status and tumor size. Loss of expression for one or more retinoid receptors frequently occurred in groups B and C. These results suggest that the loss of retinoid receptors might occur during the loss of differentiation and tumor progression of PTC. [source] Comparison of the antilipolytic effects of an A1 adenosine receptor partial agonist in normal and diabetic ratsDIABETES OBESITY & METABOLISM, Issue 2 2009A. K. Dhalla Introduction and Aims:, Elevated plasma free fatty acid (FFA) concentrations play a role in the pathogenesis of type 2 diabetes (2DM). Antilipolytic agents that reduce FFA concentrations may be potentially useful in the treatment of 2DM. Our previous observation that CVT-3619 lowered plasma FFA and triglyceride concentrations in rats and enhanced insulin sensitivity in rodents with dietary-induced forms of insulin resistance suggested that it might be of use in the treatment of patients with 2DM. The present study was undertaken to compare the antilipolytic effects of CVT-3619 in normal (Sprague Dawley, SD) and Zucker diabetic fatty (ZDF) rats. Results:, ZDF rats had significantly higher fat pad weight, glucose, insulin and FFA concentrations than those of SD rats. EC50 values for forskolin-stimulated FFA release from isolated adipocytes from SD and ZDF rats were 750 and 53 nM, respectively (p < 0.05). Maximal forskolin stimulation of FFA release was significantly (p < 0.01) less in ZDF rats (133 ± 60 ,M) compared with SD rats (332 ± 38 ,M). EC50 values for isoproterenol to increase lipolysis in adipocytes from SD and ZDF rats were 2 and 7 nM respectively. Maximal isoproterenol-stimulated lipolysis was significantly (p < 0.01) lower in adipocytes from ZDF rats (179 ± 23 ,M) compared with SD rats (343 ± 27 ,M). Insulin inhibited lipolysis in adipocytes from SD rats with an IC50 value of 30 pM, whereas adipocytes from ZDF rats were resistant to the antilipolytic actions of insulin. In contrast, IC50 values for CVT-3619 to inhibit the release of FFA from SD and ZDF adipocytes were essentially the same (63 and 123 nM respectively). CVT-3619 inhibited lipolysis more than insulin in both SD (86 vs. 46%, p < 0.001) and ZDF (80 vs. 13%, p < 0.001) adipocytes. In in vivo experiments, CVT-3619 (5 mg/kg, PO) lowered FFA to a similar extent in both groups. Plasma concentrations of CVT-3619 were not different in SD and ZDF rats. There was no significant difference in the messenger RNA expression of the A1 receptors relative to ,-actin expression in adipocytes from SD (0.98 ± 0.2) and ZDF rats (0.99 ± 0.3). Conclusion:, The antilipolytic effects of CVT-3619 appear to be independent of insulin resistance and animal model. [source] An evaluation of the etiology of reduced CYP1A1 messenger RNA expression in the Atlantic tomcod from the Hudson River, New York, USA, using reverse transcriptase polymerase chain reaction analysisENVIRONMENTAL TOXICOLOGY & CHEMISTRY, Issue 5 2001Nirmal K. Roy Abstract Adult Atlantic tomcod, Microgadus tomcod, from the Hudson River, New York State, USA, exhibit reduced inducibility of hepatic cytochrome P4501A1 (CYP1A1) mRNA compared with adult tomcod from the cleaner Miramichi River, New Brunswick, Canada, when treated with coplanar polychlorinated biphenyl (PCB) congeners or 2,3,7,8-tetrachlorodibenzo- p -dioxin. In contrast, little difference in CYP1A1 inducibility is observed between tomcod from these two rivers when treated with polycyclic aromatic hydrocarbons (PAHs). We sought to determine if impaired hepatic CYP1A1 inducibility in Hudson River tomcod results from a multigenerational, genetic adaptation or a single generational, physiological acclimation. Embryos and larvae from controlled experimental crosses of Hudson River and Miramichi River parents were exposed for 24 h to water-borne PCB congener 77 (10 ppm), benzo[a]pyrene (BaP; 10 ppm), or dimethysulfoxide, and CYP1A1 expression was assessed in individual larva using competitive reverse transcriptase polymerase chain reaction (RT-PCR) analysis. The CYP1A1 mRNA was significantly induced in larvae from both populations by BaP (47- and 52-fold) and PCB 77 (9- and 22-fold), although levels of expression were higher in offspring of Miramichi matings. Most important, CYP1A1 mRNA was significantly induced by PCB 77 in larvae from Hudson River parents. Concentrations of dioxin, furan, and PCB congeners were measured in livers and eggs of female tomcod from these two locales to quantify the extent of maternal transfer of contaminants. For both rivers, wet-weight contaminant concentrations were significantly higher (4,7 times) in livers than in eggs of the same females, suggesting that a threshold level of contaminants may have to be reached before CYP1A1 transcription is impaired. We conclude that reduced inducibility of hepatic CYP1A1 mRNA in adult tomcod from the Hudson River is most consistent with single-generational acclimation. [source] Developmental and activity-dependent genomic occupancy profiles of CREB in monkey area V1GENES, BRAIN AND BEHAVIOR, Issue 2 2009J. Lalonde The mammalian neocortex displays significant plastic rearrangement in response to altered sensory input, especially during early postnatal development. It is believed that cyclic AMP-response element-binding (CREB) plays an important role in orchestrating the molecular events that guide neuroplastic change, although the details of its genomic targets during normal postnatal development or in response to sensory deprivation remain unknown. Here, we performed CREB chromatin immunoprecipitation (ChIP) from monkey area V1 tissue and hybridized enriched DNA fragments to promoter microarrays (ChIP chip analysis). Our goal was to determine and categorize the CREB regulon in monkey area V1 at two distinct developmental stages (peak of critical period vs. adulthood) and after 5 days of monocular enucleation (ME) at both ages. Classification of enriched candidates showed that the majority of isolated promoter loci (n = 795) were common to all four conditions. A particularly interesting group of candidates (n = 192) was specific to samples derived from enucleated infant area V1. Gene ontology analysis of CREB targets during early postnatal development showed a subgroup of genes implicated in cytoskeleton-based structural modification. Analysis of messenger RNA expression (quantitative real-time,polymerase chain reaction) of candidate genes showed striking differences in expression profiles between infant and adult area V1 after ME. Our study represents the first extensive genomic analysis of CREB DNA occupancy in monkey neocortex and provides new insight into the multifaceted transcriptional role of CREB in guiding neuroplastic change. [source] CCR2 promotes hepatic fibrosis in mice,HEPATOLOGY, Issue 1 2009Ekihiro Seki Chemokines and chemokine receptors contribute to the migration of hepatic stellate cells (HSCs) and Kupffer cells, two key cell types in fibrogenesis. Here, we investigate the role of CCR2, the receptor for monocyte chemoattractant protein (MCP)-1, MCP-2, and MCP-3, in hepatic fibrosis. Hepatic CCR2, MCP-1, MCP-2, and MCP-3 messenger RNA expression was increased after bile duct ligation (BDL). Both Kupffer cells and HSCs, but not hepatocytes, expressed CCR2. BDL- and CCl4 -induced fibrosis was markedly reduced in CCR2,/, mice as assessed through collagen deposition, ,-smooth muscle actin expression, and hepatic hydroxyproline content. We generated CCR2 chimeric mice by the combination of clodronate, irradiation, and bone marrow (BM) transplantation allowing full reconstitution of Kupffer cells, but not HSCs, with BM cells. Chimeric mice containing wild-type BM displayed increased macrophage recruitment, whereas chimeric mice containing CCR2,/, BM showed less macrophage recruitment at 5 days after BDL. Although CCR2 expressed in the BM enhanced macrophage recruitment in early phases of injury, CCR2 expression on resident liver cells including HSCs, but not on the BM, was required for fibrogenic responses in chronic fibrosis models. In vitro experiments demonstrated that HSCs deficient in CCR2,/, or its downstream mediator p47phox,/, did not display extracellular signal-regulated kinase and AKT phosphorylation, chemotaxis, or reactive oxygen species production in response to MCP-1, MCP-2, and MCP-3. Conclusion: Our results indicate that CCR2 promotes HSC chemotaxis and the development of hepatic fibrosis. (HEPATOLOGY 2009.) [source] Allelic imbalances and homozygous deletion on 8p23.2 for stepwise progression of hepatocarcinogenesis,,HEPATOLOGY, Issue 2 2009Yutaka Midorikawa Early hepatocellular carcinoma (eHCC) originates from the hepatocytes of chronic liver disease and develops into classical hepatocellular carcinoma (HCC). To identify sequential genetic changes in multistep hepatocarcinogenesis, we analyzed molecular karyotypes using oligonucleotide genotyping 50K arrays. First, 1q21.3-44 gain and loss of heterozygosity (LOH) on 1p36.21-36.32 and 17p13.1-13.3 were frequently observed in eHCC, but not in chronic liver diseases, suggesting that such chromosomal aberrations are early, possibly causative events in liver cancer. Next, we detected 25 chromosomal loci associated with liver cancer progression in five HCCs with nodule-in-nodule appearance, in which the inner nodule develops within eHCC lesion. Using these chromosomal regions as independent variables, decision tree analysis was applied on 14 early and 25 overt HCCs, and extracted combination of chromosomal gains on 5q11.1-35.3 and 8q11.1-24.3 and LOH on 4q11-34.3 and 8p11.21-23.3 as distinctive attributes, which can classify early and overt HCCs recursively. In these four altered regions identified as late events of hepatocarcinogenesis, two tumors in 32 overt HCCs analyzed in the present study and one in a set of independent samples of 36 overt HCCs in our previous study harbored a homozygous deletion near the CSMD1 locus on 8p23.2. CSMD1 messenger RNA expression was decreased in HCC without 8p23.2 deletion, possibly due to hypermethylation of the CpG islands in its promoter region. Conclusion: 1q gain and 1p and 17p LOH are early molecular events, whereas gains in 5q and 8q and LOH on 4q and 8p only occur in advanced HCC, and inactivation of the putative suppressor gene, CSMD1, may be the key event in progression of liver cancer. (HEPATOLOGY 2009.) [source] Adiponectin protects LPS-induced liver injury through modulation of TNF-, in KK-Ay obese miceHEPATOLOGY, Issue 1 2004Takayuki Masaki Adiponectin, an adipocytokine, has been identified in adipose tissue, and its receptors are widely distributed in many tissues, including the liver. The present study was performed to clarify the role of adiponectin in lipopolysaccharide (LPS)-induced liver injury using KK-Ay obese mice. We analyzed the effects of adiponectin pretreatment on liver injury induced by D -galactosamine/LPS (GalN/LPS) in KK-Ay obese mice. GalN/LPS treatment induced significant increases in aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels in the blood, apoptotic and necrotic changes in hepatocytes, and/or showed a high degree of lethality. The GalN/LPS-induced liver injury was more pronounced in KK-Ay obese mice than in lean controls. Pretreatment with adiponectin ameliorated the GalN/LPS-induced elevation of serum AST and ALT levels and the apoptotic and necrotic changes in hepatocytes, resulting in a reduction in lethality. In addition, pretreatment with adiponectin attenuated the GalN/LPS-induced increases in serum and hepatic tumor necrosis factor , (TNF-,) levels and increased peroxisome proliferator-activated receptor (PPAR) , messenger RNA expression in the liver. Furthermore, abdominal macrophages from KK-Ay obese mice pretreated with adiponectin in vitro exhibited decreased LPS-induced TNF-, production compared with controls. Finally, adiponectin pretreatment also ameliorated TNF-,-induced liver injury. In conclusion, these findings suggest that adiponectin prevents LPS-induced hepatic injury by inhibiting the synthesis and/or release of TNF-, of KK-Ay obese mice. (HEPATOLOGY 2004;40:177,184.) [source] Effect of porto-systemic shunting on NOS expression after extended hepatectomy in ratsHEPATOLOGY RESEARCH, Issue 1 2009Hironori Hayashi Aim:, Several surgical procedures have been developed for reducing portal vein pressure to prevent postoperative liver injury. Nitric oxide synthase expression (NOS) induced by elevation of portal vein pressure is thought to play an important role in liver regeneration, but the details are not well understood. Methods:, Rats in the control group and in the subcutaneous splenic transposition (SST) group underwent 90% partial hepatectomy. Survival and portal vein pressure were analyzed. The serum IL-6 and TNF-, levels were measured by enzyme-linked immunosorbent assay (ELISA). Hepatocyte proliferation and apoptosis 12 hours after hepatectomy were analyzed immunohistochemically. The protein and messenger RNA expression of inducible and endothelial NOS were analyzed using Western blotting and quantitative reverse transcriptase polymerase chain reaction, respectively. Results:, The survival rate of the SST group was significantly higher. Portal vein pressure, TNF-, level and the apoptotic index were significantly lower in the SST group. Twelve hours after surgery, liver inducible NOS (iNOS) protein expression was significantly lower in the SST group. However, protein expression of endothelial NOS was not significantly different between the groups. Conclusion:, Inducible NOS expression after extended hepatectomy is related to the effects of porto-systemic shunting on the splanchnic circulation. Also, iNOS induction and concomitant nitric oxide generation appear to participate in the cytotoxicity of excessive portal pressure after extended hepatectomy. [source] Tumor necrosis factor-, augments lipopolysaccharide-induced suppressor of cytokine signalling 3 (SOCS-3) protein expression by preventing the degradationIMMUNOLOGY, Issue 1 2010Jargalsaikhan Dagvadorj Summary The regulatory role of tumour necrosis factor-, (TNF-,) on the expression of suppressor of cytokine signalling 3 (SOCS-3) in response to lipopolysaccharide (LPS) was examined using peritoneal macrophages from TNF-,-deficient mice. The LPS-induced SOCS-3 expression was markedly augmented in macrophages from wild-type mice whereas such augmentation was not seen in the cells from TNF-,-deficient mice. However, there was no significant difference in the level of SOCS-3 messenger RNA expression between macrophages from wild-type mice and those from TNF-,-deficient mice. The addition of exogenous TNF-, augmented the LPS-induced SOCS-3 expression in macrophages from TNF-,-deficient mice. The pulse chase analysis suggested augmented degradation of LPS-induced SOCS-3 protein in macrophages from TNF-,-deficient mice. Moreover, MG 132, a 26S proteasome inhibitor, sustained the LPS-induced SOCS-3 expression in those cells. The tyrosine phosphorylation of SOCS-3 was definitely induced in LPS-stimulated macrophages from TNF-,-deficient mice but not wild-type mice. A tyrosine phosphatase inhibitor enhanced the tyrosine phosphorylation of SOCS-3 in wild-type mice and accelerated the degradation. Therefore, it was suggested that TNF-, prevented the degradation of SOCS-3 protein via inhibition of the tyrosine phosphorylation in LPS-stimulated macrophages. [source] Interleukin-6 is responsible for aberrant B-cell receptor-mediated regulation of RAG expression in systemic lupus erythematosusIMMUNOLOGY, Issue 3 2007Sophie Hillion Summary Defective regulation of secondary immunoglobulin V(D)J gene rearrangement promotes the production of autoantibodies in systemic lupus erythematosus (SLE). It remains unclear, however, whether the regulation of the recombination-activating genes RAG1 and RAG2 is effective in SLE. RAG1 and RAG2 messenger RNA expression was analysed before and after in vitro activation of sorted CD19+ CD5, B cells with anti-immunoglobulin M antibodies, in 20 SLE patients and 17 healthy controls. The expression of CDK2 and p27Kip1 regulators of the RAG2 protein, were examined. The levels of interleukin-6 (IL-6) and its influence on RAG regulation were also evaluated in vitro. SLE patients had increased frequency of RAG-positive B cells. B-cell receptor (BCR) engagement induced a shift in the frequency of ,- and ,-positive cells, associated with a persistence of RAG messenger RNA and the maintenance of RAG2 protein within the nucleus. While expression of the RAG2-negative regulator CDK2 was normal, the positive regulator p27Kip1 was up-regulated and enhanced by BCR engagement. This effect was the result of the aberrant production of IL-6 by SLE B cells. Furthermore, IL-6 receptor blockade led to a reduction in p27Kip1 expression, and allowed the translocation of RAG2 from the nucleus to the cytoplasm. Our study indicates that aberrant production of IL-6 contributes to the inability of SLE B cells to terminate RAG protein production. Therefore, we hypothesize that because of constitutive IL-6 signalling in association with BCR engagement, SLE B cells would become prone to secondary immunoglobulin gene rearrangements and autoantibody production. [source] Generation of hepatocytes from cultured mouse embryonic stem cellsLIVER TRANSPLANTATION, Issue 10 2003Xiao Ling Kuai Embryonic stem (ES) cells are pluripotent cells derived from the inner cell mass of fertilized blastocysts in vitro. ES cells can be induced to undergo differentiation into potentially all cell types. The aim of this study is to examine the differentiating potential of mouse ES cells into hepatocytes in the presence of retinoic acid (RA), hepatocyte growth factor (HGF), and ,-nerve growth factor (,-NGF). RA, HGF, and ,-NGF were added to the cell culture. Hepatocyte induction was confirmed morphologically, as well as biochemically, through immunohistochemical assays of ,1 -antitrypsin (,1 -AT) and alfafetaprotein (AFP) expression and reverse-transcriptase polymerase chain reaction tests for the presence of albumin, transthyretin, glucose 6 phosphates, hepatic nuclear factor 4, and SAPK/ERK kinase-1 (SEK1) messenger RNA, produced only by functioning hepatocytes. Fifteen days after the addition of HGF and ,-NGF to the cell culture, many epithelioid cells were noticed. ,1 -AT, AFP, albumin, transthyretin, glucose 6 phosphates, hepatic nuclear factor 4, and SEK1 messenger RNA expression also was detected, indicating successful ES cell differentiation into functioning hepatocytes. However, in the presence of RA alone, only transthyretin messenger RNA was positive, whereas no other expression pertaining to functioning hepatocytes could be detected. In the presence of HGF and ,-NGF, mouse ES cells can differentiate into functioning hepatocytes, whereas RA function is limited. [source] Expression of cyclooxygenase-2 in chronic hepatitis B and the effects of anti-viral therapyALIMENTARY PHARMACOLOGY & THERAPEUTICS, Issue 2 2002A. S. L. Cheng Backgound: Cyclooxygenase-2 may play a role in the development of hepatocellular carcinoma, but the relationship between cyclooxygenase-2 and chronic hepatitis B is unknown. Aim: To investigate the expression and cellular localization of cyclooxygenase-2 in chronic hepatitis B patients and the effects of anti-viral therapy. Methods: Using immunohistochemistry, in situ hybridization, Western blot and reverse transcription polymerase chain reaction, protein and messenger RNA expression and cellular localization of cyclooxygenase-2 in 35 chronic hepatitis B patients were assessed. Fourteen histologically normal and non-viral-infected livers were used as controls. The cyclooxygenase-2 immunoreactivities of paired liver biopsies from 12 patients receiving anti-viral therapy were compared. Results: Immunohistochemistry and in situ hybridization revealed that cyclooxygenase-2 expression was confined to hepatocytes. Patients with chronic hepatitis B had significantly higher cyclooxygenase-2 expression compared with controls. The cyclooxygenase-2 expression of hepatitis B e antigen-positive and -negative chronic hepatitis B patients was not significantly different, although the necro-inflammatory activity of the latter group was significantly lower. Over-expression of cyclooxygenase-2 in patients with chronic hepatitis B was further confirmed by Western blot and reverse transcription polymerase chain reaction. Twelve hepatitis B e antigen-positive chronic hepatitis B patients received anti-viral therapy: lamivudine in seven and interferon in five. Despite hepatitis B e antigen seroconversion, disappearance of hepatitis B virus DNA in serum, normalization of liver enzymes and a significant reduction in necro-inflammatory activity in all 12 patients, no significant change in cyclooxygenase-2 expression was found. Conclusions: Chronic hepatitis B is associated with elevated cyclooxygenase-2 levels in hepatocytes, and the over-expression of this enzyme does not reflect inflammatory activity. Up-regulation of cyclooxygenase-2 persists after successful anti-viral therapy. [source] Omega-3 fatty acid regulates inflammatory cytokine/mediator messenger RNA expression in Porphyromonas gingivalis -induced experimental periodontal diseaseMOLECULAR ORAL MICROBIOLOGY, Issue 4 2007L. Kesavalu Introduction:,Porphyromonas gingivalis is strongly implicated in the etiology of adult periodontitis by inducing inflammatory cytokines, resulting in gingival and periodontal tissue inflammation and alveolar bone resorption. This study tested the hypothesis that supplementing the diet with omega-3 fatty acid (,-3 FA; i.e. fish oil) would exert anti-inflammatory effects in the gingival tissues of P. gingivalis -infected rats. Methods:, Rats were fed either fish oil or corn oil diets ad libitum for 22 weeks and infected with P. gingivalis strain 381 or strain A7A1-28. After sacrifice, rat gingival tissues were excised and the RNA was isolated and analyzed for proinflammatory mediators [interleukin-1, (IL-1,), tumor necrosis factor-, (TNF-,), IL-6], T helper type 1 and type 2 cytokines [interferon-, (IFN-,), IL-4, IL-10), antioxidant enzymes [catalase (CAT), superoxide dismutase (SOD)], and genes critical for eicosanoid mediator production [cyclo-oxygenase-2 (COX-2), 5-lipoxygenase (5-LO)] by reverse transcription-polymerase chain reaction using rat-specific primers. Results:, Rats on the ,-3 FA diet exhibited decreased proinflammatory cytokine gene expression (IL-1,, TNF-,) and enhanced IFN-,, CAT and SOD messenger RNA expression compared to rats fed a corn oil diet, supporting a diet-induced modulation of host inflammatory reactions. Analyses of alveolar bone resorption in the rats related to gene expression profiles demonstrated significant positive correlations with IL-1,, IL-6 and COX-2 and negative correlations with CAT and SOD. Conclusion:, These findings suggest that diets enriched for ,-3 FA modulate the local gingival inflammatory milieu of the host following oral P. gingivalis infection, which impacts on alveolar bone resorption in rats. [source] Quantitative analysis of messenger RNA expression of matrix metalloproteinases (MMP-2 and MMP-9), tissue inhibitor-2 of matrix metalloproteinases (TIMP-2), and steroidogenic enzymes in bovine placentomes during gestation and postpartumMOLECULAR REPRODUCTION & DEVELOPMENT, Issue 7 2007M. Takagi Abstract The relationship between the mRNA expression of proteolytic and steroidogenic enzymes in bovine placentomes was examined. Caruncle and cotyledon tissues were collected every 6 hr after spontaneous parturition until the fetal membranes were released. Based on the time of fetal membrane release after parturition, the specimens were classified as follows: (1) the early group, in which the fetal membranes were released within 6 hr after parturition; and (2) the late group, in which the fetal membranes were released 6,12 hr after parturition. The placentomes from a slaughterhouse were additionally collected as samples for the examination of enzymes during the gestation period. The mRNA expression of steroidogenic enzymes in the cotyledon was observed to be higher than that in caruncle tissues; however, the mRNA expression patterns of P450scc and StAR tended to be similar in both placental tissues. On the other hand, although the expression levels of TIMP-2 mRNA in both caruncle and cotyledon tissues were similar, during gestation and postpartum the expression levels of MMP-2 and MMP-9 mRNA were approximately 10 times higher in caruncle than in cotyledon tissue. Marked contrasting changes in mRNA expression patterns between pre- and postpartum periods were observed for MMP-2 and MMP-9 in caruncle tissues and for MMP-9 and TIMP-2 in cotyledon tissues. The present study provides the first evidence that MMP-2, MMP-9, and TIMP-2 mRNAs are expressed in bovine placentomes during the gestational and postpartum periods and suggests that these enzymes, in conjunction with steriodogenic enzymes, mediate fetal membrane detachment after parturition. Mol. Reprod. Dev. 74: 801,807, 2007. © 2006 Wiley-Liss, Inc. [source] Interleukin-6 involvement in brain arteriovenous malformationsANNALS OF NEUROLOGY, Issue 1 2006Yongmei Chen MD We recently reported that the GG genotype of the interleukin-6 (IL-6),174G>C promoter polymorphism is associated with clinical presentation of intracranial hemorrhage in brain arteriovenous malformation (AVM) patients. In this study, we investigated whether tissue IL-6 expression was associated with IL-6,174G>C genotype, and whether IL-6 was linked to downstream targets involved in angiogenesis and vascular instability. Our results showed that the highest IL-6 protein levels in brain AVM tissue were associated with IL-6,174GG genotype (GG: 57.7 ± 20.2; GC: 35.6 ± 26.6; CC: 13.9 ± 10.2pg/mg; p = 0.001). IL-6 protein levels were increased in AVM tissue from patients with hemorrhagic presentation compared with patients without hemorrhage (55 ± 22 vs 40 ± 27pg/mg; p = 0.038). IL-6 messenger RNA expression strongly correlated with messenger RNA levels of IL-1,, tumor necrosis factor-,, IL-8, matrix metalloproteinase-3 (MMP-3), MMP-9, and MMP-12. We further investigated the plausibility of IL-6 being an upstream cytokine responsible for initiating the angiogenic cascade by cell culture and animal experiments. IL-6 induced MMP-3 and MMP-9 expression and activity in mouse brain and increased proliferation and migration of cerebral endothelial cells. Together, our results suggest that the IL-6 genotype associated with intracranial hemorrhage modulates IL-6 expression in brain AVM tissue, which is consistent with the hypothesis that inflammatory processes induce angiogenic activity possibly contributory to brain AVM intracranial hemorrhage. Ann Neurol 2005 [source] A functional RANKL polymorphism associated with younger age at onset of rheumatoid arthritisARTHRITIS & RHEUMATISM, Issue 10 2010Wenfeng Tan Objective We previously observed the association of the co-occurrence of the HLA,DRB1 shared epitope (SE) and RANKL single-nucleotide polymorphisms (SNPs) with younger age at the onset of rheumatoid arthritis (RA) in 182 rheumatoid factor (RF),positive European American patients with early-onset RA. The aim of this study was to fine-map the 48-kb RANKL region in the extended cohort of 210 European American RF-positive patients with early RA, to seek replication of RA-associated SNPs in additional RA cohorts of 501 European Americans and 298 African Americans, and to explore the functional consequences of RA-associated SNPs. Methods SNP genotyping was conducted using pyrosequencing or TaqMan polymerase chain reaction (PCR) assays. Associations of rs7984870 with RANKL expression in plasma, peripheral blood mononuclear cells, and isolated T cells were quantified using enzyme-linked immunosorbent assay and reverse transcription,PCR. Site-directed mutagenesis of rs7984870 within the 2-kb RANKL promoter was performed to drive the luciferase reporter gene in osteoblast and stromal cell lines. Interaction of DNA and protein was determined by electrophoretic mobility shift assay. Results A single promoter SNP, rs7984870, was consistently significantly associated with earlier age at the onset of RA in 3 independent seropositive (RF or anti,cyclic citrullinated peptide antibody) RA cohorts but not in seronegative RA patients. The C risk allele of rs7984870 conferred 2-fold higher plasma RANKL levels in RF-positive patients with RA, significantly elevated RANKL messenger RNA expression in activated normal T cells, and increased promoter activity after stimulation in vitro via differential binding to the transcription factor SOX5. Conclusion The RANKL promoter allele that increased transcription levels upon stimulation might promote interaction between activated T cells and dendritic cells, predisposing to a younger age at the onset of RA in seropositive European American and African American patients. [source] Transcriptional profiling and biochemical analysis of mechanically induced cartilaginous tissues in a rat modelARTHRITIS & RHEUMATISM, Issue 4 2010Kristy T. Salisbury Palomares Objective To characterize patterns of molecular expression that lead to cartilage formation in vivo in a postnatal setting, by profiling messenger RNA expression across the time course of mechanically induced chondrogenesis. Methods Retired breeder Sprague-Dawley rats underwent a noncritical-sized transverse femoral osteotomy. Experimental animals (n = 45) were subjected to bending stimulation (60° cyclic motion in the sagittal plane for 15 minutes/day) of the osteotomy gap beginning on day 10 after the operation. Control animals (n = 32) experienced continuous rigid fixation. Messenger RNA isolated on days 10, 17, 24, and 38 after surgery was analyzed using a microarray containing 608 genes involved in skeletal development, tissue differentiation, fracture healing, and mechanotransduction. The glycosaminoglycan (GAG) content in the stimulated tissues was compared with that in native articular cartilage as a means of assessing the progression of chondrogenic development of the tissues. Results The majority of the 100 genes that were differentially expressed were up-regulated in response to mechanical stimulation. Many of these genes are associated with articular cartilage development and maintenance, diarthrodial joint development, cell adhesion, extracellular matrix synthesis, signal transduction, and skeletal development. Quantitative real-time polymerase chain reaction results were consistent with the microarray findings. The GAG content of the stimulated tissues increased over time and was no different from that of articular cartilage on day 38 after surgery. Conclusion Our findings indicate that mechanical stimulation causes up-regulation of genes that are principally involved in joint cavity morphogenesis and critical to articular cartilage function. Further study of this type of stimulation may identify key signaling events required for postnatal hyaline cartilage formation. [source] The 423Q polymorphism of the X-linked inhibitor of apoptosis gene influences monocyte function and is associated with periodic feverARTHRITIS & RHEUMATISM, Issue 11 2009Massimo Ferretti Objective Hereditary periodic fever syndromes (HPFs) develop as a result of uncontrolled activation of the inflammatory response, with a substantial contribution from interleukin-1, or tumor necrosis factor , (TNF,). The HPFs include familial Mediterranean fever (FMF), hyperimmunoglobulinemia D with periodic fever syndrome (HIDS), TNF receptor,associated syndrome (TRAPS), and cryopyrinopathies, which are attributable to mutations of the MEFV, MVK, TNFRSF1A, and CIAS1 genes, respectively. However, in many patients, the mutated gene has not been determined; therefore, the condition in these patients with an HPF-like clinical picture is referred to as idiopathic periodic fever (IPF). The aim of this study was to assess involvement of X-linked inhibitor of apoptosis (XIAP), which plays a role in caspase inhibition and NF-,B signaling, both of which are processes that influence the development of inflammatory cells. Methods The XIAP gene (X-linked) was sequenced in 87 patients with IPF, 46 patients with HPF (13 with HIDS, 17 with TRAPS, and 16 with FMF), and 182 healthy control subjects. The expression of different alleles was evaluated by sequencing XIAP -specific complementary DNA mini-libraries and by real-time polymerase chain reaction and Western blot analyses. The functional effect of XIAP on caspase 9 activity was assessed by a fluorimetric assay, and cytokine secretion was evaluated by enzyme-linked immunosorbent assay. Results Sequencing disclosed a 1268A>C variation that caused a Q423P amino acid substitution. The frequency of 423Q-homozygous female patients and 423Q-hemizygous male patients was significantly higher in the IPF group than in the control group (69% versus 51%; odds ratio 2.17, 95% confidence interval 1.23,3.87, P = 0.007), whereas no significant difference was detected in the HPF group (59%) compared with controls. In primary lymphocytes and transfected cell lines, 423Q, as compared with 423P, was associated with higher XIAP protein and messenger RNA expression and lower caspase 9 activation. In lipopolysaccharide-activated monocytes, 423Q was associated with higher secretion of TNF,. Conclusion These results suggest that 423Q is a predisposing factor for IPF development, possibly through its influence on monocyte function. [source] Th17 and natural Treg cell population dynamics in systemic lupus erythematosusARTHRITIS & RHEUMATISM, Issue 5 2009Ji Yang Objective To investigate the relative abundance and activities of Th17 cells and natural Treg cells in systemic lupus erythematosus (SLE). Methods Blood samples were collected from 50 adult patients with SLE. Samples were processed to detect Th17 cells and natural Treg cells by flow cytometry, and related gene expression was assessed by real-time reverse transcription,polymerase chain reaction. Skin biopsy specimens were collected for histologic assessment. The function of Th17 cells in relation to human umbilical vein endothelial cells (HUVECs) was studied in vitro. Th17 cells were also examined in lupus-prone MRL/Mp- lpr/lpr (MRL/lpr) mice. Results We demonstrated the presence of Th17 cells among the peripheral blood mononuclear cells (PBMCs) and in the involved organs of patients with active SLE. Both the percentage of circulating Th17 cells and the ability to produce interleukin-17A (IL-17A) were increased in samples derived from patients with active SLE. The number of Th17 cells increased during SLE flare, especially in patients with vasculitis, and decreased following certain treatments. We observed that IL-17A from patients with SLE could induce adhesion molecule messenger RNA expression in HUVECs and adhesion of T cells to HUVECs. An increase in the percentage of Th17 cells was correlated with natural Treg cell depletion during disease flare. Finally, expansion of the Th17 cell population was detected in MRL/lpr mice. Conclusion SLE flare might be linked to the expansion of the Th17 cell population and the depletion of natural Treg cell subpopulations. Expansion of the Th17 cell population might be related to a distinct cytokine environment in active SLE. Th17 cells and microenvironmental IL-17A are involved in vascular inflammation in SLE. Antagonism of Th17 cells by IL-17A,blocking antibodies should be explored as a treatment of SLE. [source] Epigallocatechin-3-gallate diminishes CCL2 expression in human osteoblastic cells via up-regulation of phosphatidylinositol 3-Kinase/Akt/Raf-1 interaction: A potential therapeutic benefit for arthritisARTHRITIS & RHEUMATISM, Issue 10 2008Sze-Kwan Lin Objective To assess the effects of epigallocatechin-3-gallate (EGCG) on oncostatin M (OSM),induced CCL2 synthesis and the associated signaling pathways in human osteoblastic cells. The therapeutic effect of EGCG on collagen-induced arthritis (CIA) in rats was also studied. Methods CCL2 and c-Fos messenger RNA expression was analyzed by Northern blotting. The modulating effects of EGCG on the activation of Raf-1, Akt, and phosphatidylinositol 3-kinase (PI 3-kinase) were examined by coimmunoprecipitation, Western blotting, and PI 3-kinase activity assay. Interactions between c-Fos and CCL2 promoter were evaluated by electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) assay. The effect of EGCG on CIA in rats was examined clinically and immunohistochemically. Results EGCG inhibited OSM-stimulated CCL2 expression in primary human osteoblasts and MG-63 cells. In MG-63 cells, EGCG alleviated the OSM-induced phosphorylation of Raf-1 at Ser338 but restored the dephosphorylation of Raf-1 at Ser259. EGCG increased the activity of PI 3-kinase, the level of phosphorylated Akt (Ser473), and binding between Raf-1 and active Akt. EMSA and ChIP assay revealed that EGCG attenuated activator protein 1 (AP-1),CCL2 promoter interaction, possibly by reducing c-Fos synthesis. Codistribution of CD68+ macrophages and CCL2+ osteoblasts in osteolytic areas was obvious in the CIA model. Administration of EGCG markedly diminished the severity of CIA, macrophage infiltration, and the amount of CCL2-synthesizing osteoblasts. Conclusion By stimulating PI 3-kinase activity, EGCG promoted Akt/Raf-1 crosstalk, resulting in decreased AP-1 binding to CCL2 promoter, and finally reduced CCL2 production in osteoblasts. EGCG alleviated the severity of CIA, probably by suppressing CCL2 synthesis in osteoblasts to diminish macrophage infiltration. Our data support the therapeutic potential of EGCG on arthritis. [source] Efficient suppression of murine arthritis by combined anticytokine small interfering RNA lipoplexesARTHRITIS & RHEUMATISM, Issue 8 2008Maroun Khoury Objective Blocking tumor necrosis factor (TNF) effectively inhibits inflammation and joint damage in rheumatoid arthritis (RA), but 40% of RA patients respond only transiently or not at all to the current anti-TNF biotherapies. The purpose of this study was to develop an alternative targeted therapy for this subgroup of RA patients. As proof of concept, we tested the efficiency of an RNA interference (RNAi),based intervention that targets proinflammatory cytokines in suppressing murine collagen-induced arthritis (CIA). Methods Two synthetic short interfering RNA (siRNA) sequences were designed for each of the proinflammatory cytokines interleukin-1 (IL-1), IL-6, and IL-18. Their silencing specificity was assessed according to lipopolysaccharide-induced messenger RNA expression in J774.1 mouse macrophages as compared with control siRNA. For in vivo administration, siRNA were formulated as lipoplexes with the RPR209120/DOPE liposome and a carrier DNA and were injected intravenously (0.5 mg/kg) into DBA/1 mice with CIA. Results Weekly injections of anti,IL-1, anti,IL-6, or anti,IL-18 siRNA-based lipoplexes significantly reduced the incidence and severity of arthritis, abrogating joint swelling and destruction of cartilage and bone, both in the preventative and the curative settings. The most striking therapeutic effect was observed when the 3 siRNA were delivered in combination. The siRNA lipoplex cocktail reduced all pathologic features of RA, including inflammation, joint destruction, and the Th1 response, and overall parameters of RA were improved as compared with anti-TNF siRNA lipoplex,based treatment. Conclusion Our results present a novel option for in vivo RNAi-based antiinflammatory immunotherapy. Our findings indicate that intravenous administration of a lipoplex cocktail containing several anticytokine siRNA is a promising novel antiinflammatory therapy for RA, as well as a useful and simple tool for understanding the pathophysiology of RA and for evaluating new therapeutic candidates. [source] Development and characterization of a fusion protein between thermally responsive elastin-like polypeptide and interleukin-1 receptor antagonist: Sustained release of a local antiinflammatory therapeuticARTHRITIS & RHEUMATISM, Issue 11 2007Mohammed F. Shamji Objective Interleukin-1 receptor antagonist (IL-1Ra) has been evaluated for the intraarticular treatment of osteoarthritis. Such administration of proteins may have limited utility because of their rapid clearance and short half-life in the joint. The fusion of a drug to elastin-like polypeptides (ELPs) promotes the formation of aggregating particles that form a "drug depot" at physiologic temperatures, a phenomenon intended to prolong the presence of the drug. The purpose of this study was to develop an injectable drug depot composed of IL-1Ra and ELP domains and to evaluate the properties and bioactivity of the recombinant ELP-IL-1Ra fusion protein. Methods Fusion proteins between IL-1Ra and 2 distinct sequences and molecular weights of ELP were overexpressed in Escherichia coli. Environmental sensitivity was demonstrated by turbidity and dynamic light scattering as a function of temperature. IL-1Ra domain activity was evaluated by surface plasmon resonance, and in vitro antagonism of IL-1,mediated lymphocyte and thymocyte proliferation, as well as IL-1,induced tumor necrosis factor , (TNF,) expression and matrix metalloproteinase 3 (MMP-3) and ADAMTS-4 messenger RNA expression in human intervertebral disc fibrochondrocytes. IL-1Ra immunoreactivity was assessed before and after proteolytic degradation of the ELP partner. Results Both fusion proteins underwent supramolecular aggregation at subphysiologic temperatures and slowly resolubilized at 37°C. Interaction with IL-1 receptor was slower in association but equivalent in dissociation as compared with the commercial antagonist. Anti,IL-1 activity was demonstrated by inhibition of lymphocyte and thymocyte proliferation and by decreased TNF, expression and ADAMTS-4 and MMP-3 transcription by fibrochondrocytes. ELP domain proteolysis liberated a peptide of comparable size and immunoreactivity as the commercial IL-1Ra. This peptide was more bioactive against lymphocyte proliferation, nearly equivalent to the commercial antagonist. Conclusion The ELP-IL-1Ra fusion protein proved to retain the characteristic ELP inverse phase-transitioning behavior as well as the bioactivity of the IL-1Ra domain. This technology represents a novel drug carrier designed to prolong the presence of bioactive peptides following intraarticular delivery. [source] Overexpression of synoviolin in peripheral blood and synoviocytes from rheumatoid arthritis patients and continued elevation in nonresponders to infliximab treatmentARTHRITIS & RHEUMATISM, Issue 7 2006Myew-Ling Toh Objective Synoviolin is a novel E3 ubiquitin ligase that has been implicated in the pathogenesis of rheumatoid arthritis (RA). The purpose of this study was to examine the expression and regulation of synoviolin by tumor necrosis factor , (TNF,), both in vivo and in vitro. Methods A total of 54 RA patients and 23 healthy control subjects were analyzed before, 4 hours after the first infusion, and at week 22 of infliximab treatment. Clinical response was assessed by the American College of Rheumatology criteria for 20% improvement and the Disease Activity Score in 28 joints (DAS28) at 6 months. Synoviolin messenger RNA expression was measured by real-time reverse transcription,polymerase chain reaction in peripheral blood (PB) and fibroblast-like synoviocytes (FLS) and with and without TNF, or interleukin-1, (IL-1,) stimulation. Results Synoviolin expression was increased in whole PB obtained from RA patients as compared with that from healthy controls and was significantly reduced early and late after infliximab treatment in responders, but in not nonresponders. Reduction in synoviolin expression was associated with reduced levels of markers of disease activity, including C-reactive protein levels. Nonresponders to infliximab therapy had significantly higher synoviolin expression at baseline as compared with responders, and this elevation persisted despite infliximab therapy. PB CD14+ monocytes expressed increased synoviolin levels compared with CD3+ lymphocytes, and TNF, or IL-1, induced a further increase in expression in CD3+ cells. TNF, or IL-1, induced sustained synoviolin expression in RA FLS. Conclusion Elevated PB levels of synoviolin were identified in circulating PB mononuclear cells and were associated with nonresponse to infliximab treatment. Sustained up-regulation of synoviolin by IL-1, and TNF, may contribute to prolonged survival of immune cells and dysregulated FLS proliferation, leading to RA chronicity. [source] Halofuginone inhibition of COL1A2 promoter activity via a c-Jun,dependent mechanismARTHRITIS & RHEUMATISM, Issue 10 2002Tracy L. McGaha Objective The naturally occurring compound halofuginone has been shown to antagonize collagen synthesis by fibroblasts both in vitro and in vivo. We previously demonstrated that this inhibitory property was related to the ability of halofuginone to disrupt transforming growth factor , signal transduction. The present study further analyzed the ability of halofuginone to affect transcription factors that can regulate type I collagen gene expression by examining its effect on c-Jun, the negative regulator of collagen gene transcription. Methods The phosphorylation state of c-Jun in the presence of halofuginone was examined via direct Western blotting, and the transcriptional activity of the activator protein 1 (AP-1) binding element via electrophoretic mobility shift assay and luciferase reporter assay. We determined whether the effect of halofuginone on collagen synthesis was dependent on the presence of c-Jun by ectopic expression of a wild-type or dominant-negative c-Jun construct in the presence of halofuginone and assaying ,2(I) collagen promoter strength via luciferase reporter assay. The effect of halofuginone on ,2(I) collagen message levels in fibroblasts when wild-type or dominant-negative c-Jun was overexpressed was determined. We also determined whether halofuginone had an effect on the phosphorylation state of c-Jun in the skin of TSK/+ mice via immunohistochemistry. Results Treatment of fibroblasts with 10,8M halofuginone enhanced basal and mitogen-mediated phosphorylation of c-Jun in culture. This elevated phosphorylation of c-Jun correlated with enhanced DNA binding and transcriptional activation of an AP-1 complex consisting of c-Jun and Fos but lacking the c-Jun antagonist JunB. Overexpression of c-Jun enhanced in a dose-dependent manner the ability of halofuginone to inhibit the activity of a luciferase reporter construct under control of the ,3200-bp to +54-bp COL1A2 promoter, whereas the expression of a dominant-negative c-Jun construct abolished this effect. Northern blotting showed that overexpression of c-Jun enhanced the ability of halofuginone to reduce collagen ,2(I) messenger RNA levels in fibroblasts, whereas expression of the dominant-negative c-Jun abolished this effect. Topical administration of a halofuginone-containing cream for 20 days to TSK mice, which spontaneously develop dermal fibrosis, greatly increased the phosphorylated form of c-Jun in the skin; this was followed by a decrease in skin thickness and type I collagen messenger RNA expression. Conclusion Our findings illustrate the powerful down-regulatory property of c-Jun toward type I collagen and establish that halofuginone exerts its effect on collagen synthesis in a c-Jun,dependent manner. [source] | |||||||||||||||||||||||||