SIRT1, a NAD dependent histone and protein deacetylase, is a member of the histone deacetylase cl... more SIRT1, a NAD dependent histone and protein deacetylase, is a member of the histone deacetylase class III family. We previously showed that SIRT1 mRNA expression is significantly lower in peripheral blood mononuclear cells (PBMCs) of multiple sclerosis (MS) patients during relapses than in stable patients. We have now investigated SIRT1 as a possible biomarker to predict relapse as well as responsiveness to glatiramer acetate (GA) treatment in relapsing-remitting MS (RRMS) patients. Over the course of 2years, a cohort of 15 GA-treated RRMS patients were clinically monitored using the Expanded Disability Status Scale and assessed for MS relapses. Blood samples collected from MS patients were analyzed for levels of SIRT1 and histone H3 lysine 9 (H3K9) acetylation and dimethylation. During relapses, MS patients had a lower expression of SIRT1 mRNA than did stable MS patients. In addition, there was a significant decrease in H3K9 dimethylation (H3K9me2) during relapses in MS patients whe...
The shift from the differentiated contractile smooth muscle cell (SMC) phenotype to a less differ... more The shift from the differentiated contractile smooth muscle cell (SMC) phenotype to a less differentiated, proliferative state and excessive extracellular matrix (ECM) production, both TGF-β regulated events, are key alterations in atherogenesis. 1 The complement system takes part in the initiation and progression of atherosclerosis. First described as a cell cycle regulator, Response Gene to Complement (RGC)-32 is also implicated in tumorigenesis, immune system regulation, scar tissue formation, regulation of lipid and glucose metabolism and atherogenesis. We have shown that RGC-32 modulates C5b-9-induced endothelial cell (EC) proliferation and migration and is involved in EC cytoskeletal organization and cell adhesion. 2 Additionally, RGC-32 promotes vascular SMC proliferation. 3 RGC-32 also mediates some of TGF-βinduced profibrotic effects in a variety of tissues. 4, 5
Organizations should carefully consider how to implement infection prevention recommendations fro... more Organizations should carefully consider how to implement infection prevention recommendations from external agencies in order to create policies that satisfy those recommendations and simultaneously meet the needs of patients, families, and staff. Communicating changes in isolation policies throughout a large enterprise presents specific challenges.
We have previously shown that RGC-32 is involved in cell cycle regulation in vitro. To define the... more We have previously shown that RGC-32 is involved in cell cycle regulation in vitro. To define the in vivo role of RGC-32, we generated RGC-32 knockout mice. These mice develop normally and do not spontaneously develop overt tumors. To assess the effect of RGC-32 deficiency on cell cycle activation in T cells we determined the proliferation rate of CD4 and CD8 from spleens of RGC-32-/- mice compared to wild type (WT) mice. CD4 T cells from RGC-32-/- mice display a significant increase in 3H-thymidine incorporation when compared with WT mice after stimulation with anti-CD3/anti-CD28. In addition, both CD4 and CD8 T cells from RGC-32-/- displayed a significant increase in the proportion of proliferating Ki67+ cells, indicating that in T cells, RGC-32 has an inhibitory effect on cell cycle activation. Furthermore, Akt and FOXO1 phosphorylation induced in CD4 from RGC-32-/- were significantly higher indicating that RGC-32 inhibits cell cycle activation by suppressing FOXO1 activation. To further examine the effect of RGC-32 on T cell functions we investigated the effect of RGC-32 on T regulatory (Treg) cells differentiation. A significantly lower percentage of CD4+ FoxP3+ Treg cells was found in unstimulated cells from RGC-32-/- compared to WT mice. Significantly fewer CD4+ FoxP3+ Treg cells are induced by TGF-β in RGC-32-/- compared to WT mice. Thus, RGC-32 is involved in controlling cell cycle in vivo, and in addition seems to play a role in the differentiation of Treg cells.
SIRT1 is a NAD-dependent histone deacetylase involved in the regulation of transcription, apoptos... more SIRT1 is a NAD-dependent histone deacetylase involved in the regulation of transcription, apoptosis, metabolism and differentiation. We have previously demonstrated that sublytic levels of complement C5b-9 terminal complex increased the survival of oligodendrocytes (OLGs) and induced their dedifferentiation. In this study we investigated the role of SIRT1 in OLGs differentiation and the effect of sublytic levels of C5b-9 on SIRT1 expression. We also investigated the downstream effects of SIRT1 by measuring histone H3 Lysine 9 trimethylation (H3K9me3) and expression of cyclin D1. OLG progenitor cells purified from the brain of rat pups were differentiated in vitro and stimulated with sublytic C5b-9 or C5b6 for 3, 6 and 18 h. The level of SIRT1 mRNA was measured using real-time PCR and SIRT1, cyclin D1 and H3K9me3 protein expression were measured using western blotting. We found a decreased expression of SIRT1 mRNA and protein, a decreased level of H3K9me3 and an increased expression of cyclin D1 during OLG differentiation. Stimulation of OLGs with sublytic C5b-9 for 3h resulted in a significant decrease in SIRT1 mRNA and protein levels while stimulation with C5b6 had no effect. SIRT1 protein level in OLGs after 8h of exposure to C5b-9 were significantly lower than in C5b6 treated cells. H3K9me3 levels also decreased significantly after stimulation with C5b-9 as compared with unstimulated or C5b6-treated OLGs. Cyclin D1 expression increased after stimulation with C5b-9, indicating cell cycle activation. Our data show that C5b-9 stimulation of OLGs reduces SIRT1 expression, contributing to cell cycle activation by decreasing repressive trimethylation of histone H3 lysine 9.
We have previously shown that response gene to complement 32 (RGC-32) mediates transforming growt... more We have previously shown that response gene to complement 32 (RGC-32) mediates transforming growth factor-β (TGF-β)-induced astrocyte reactivity and extracellular matrix production. However, the molecular mechanisms underlying RGC-32 expression and function remain to be elucidated. In the present study, we found that TGF-β induced expression and nuclear translocation of both RGC-32 and SMAD3 in astrocytes. Using specific pathways inhibitors, we have found that RGC-32 induction is mediated by SMAD-dependent and independent mechanisms. We investigated whether RGC-32 interacts with SMAD2 or SMAD3 in astrocytes and if this association plays a role in extracellular matrix synthesis. Co-immunoprecipitation showed that SMAD3, but not SMAD2 physically interacts with RGC-32 in astrocytes. SIS3, an inhibitor of SMAD3 phosphorylation, significantly reduced RGC-32 translocation to the nucleus, indicating that SMAD3 phosphorylation is required for RGC-32 nuclear translocation. On the other hand, silencing RGC-32 with siRNA had no effect on SMAD3 nuclear translocation. Y27632, a selective ROCK inhibitor was also able to significantly inhibit RGC-32 nuclear translocation, while the SMAD3 nuclear translocation was unaffected. The inhibition of SMAD3 phosphorylation also blocked the RGC-32-induced collagen type I expression, indicating a synergistic action between RGC-32 and SMAD3 to induce the expression of collagen type I. Taken together, our data demonstrate for the first time that RGC-32 interacts with SMAD3 to mediate the extracellular matrix production in astrocytes. These data suggest that a similar mechanism might be involved in mediation of gliosis seen in chronic multiple sclerosis lesions.
recent in vivo pathogenesis studies in mice, have shown that intestinal colonization by RG strain... more recent in vivo pathogenesis studies in mice, have shown that intestinal colonization by RG strains isolated from Lupus patients in flare, but not RG strains from healthy adults, induce zonulin-dependent increases in intestinal permeability, RG translocation to mesenteric lymph nodes as well as serum IgG anti-RG antibody and anti-native DNA autoantibody responses. Conclusions As many Lupus patients are known to suffer relapsing illness despite appropriate treatment, we speculate that gut blooms of pathogenic bacteria that impair gut barrier function and stoke systemic inflammation directly contribute to immunopathogenesis. We propose that future therapeutic interventions designed to promote a durable remission consider the potential necessity to target both the immunologic abnormalities of the disease, as well as re-establish stability within the gut microbiota community.
The shift from the differentiated contractile smooth muscle cell (SMC) phenotype to a less differ... more The shift from the differentiated contractile smooth muscle cell (SMC) phenotype to a less differentiated, proliferative state and excessive extracellular matrix (ECM) production, both TGF-β regulated events, are key alterations in atherogenesis. 1 The complement system takes part in the initiation and progression of atherosclerosis. First described as a cell cycle regulator, Response Gene to Complement (RGC)-32 is also implicated in tumorigenesis, immune system regulation, scar tissue formation, regulation of lipid and glucose metabolism and atherogenesis. We have shown that RGC-32 modulates C5b-9-induced endothelial cell (EC) proliferation and migration and is involved in EC cytoskeletal organization and cell adhesion. 2 Additionally, RGC-32 promotes vascular SMC proliferation. 3 RGC-32 also mediates some of TGF-βinduced profibrotic effects in a variety of tissues. 4, 5
Background: Glatiramer acetate (GA) is US-approved for relapsing multiple sclerosis. Objectives: ... more Background: Glatiramer acetate (GA) is US-approved for relapsing multiple sclerosis. Objectives: To describe GA long-term clinical profile. To compare effectiveness of early start (ES) versus delayed start (DS; up to 3 years) with GA. Methods: Phase 3 trial participants entered a randomized placebo-controlled period then an open-label extension (OLE) with GA. Results: Overall, 208 out of 251 (82.9%) randomized participants entered the OLE; 24 out of 101 (23.8%, ES) and 28 out of 107 (26.2%, DS) participants completed the OLE. Median GA treatment was 9.8 (0.1–26.3) years. Annualized change in Expanded Disability Status Scale (EDSS) score was lower with ES versus DS ( p = 0.0858: full study; p = 0.002; Year 5). Participants with improved/stable EDSS was consistently higher with ES versus DS: 40.3% versus 31.6% ( p = 0.1590; full study); 70.8% versus 55.6% ( p = 0.015; Year 5). ES prolonged time-to-6-month confirmed disease worsening (CDW) versus DS (9.8 vs 6.7 years), time-to-12-month CDW (18.9 vs 11.6 years), and significantly reduced time-to-second-6-month CDW ( p = 0.0441). No new safety concerns arose. Conclusion: GA long-term treatment maintained clinical benefit with a similar safety profile to phase 3 results; a key limitation was that only 25% of participants completed the OLE. Early initiation of GA had sustained benefits versus delayed treatment.
Proliferation of endothelial cells (EC) and smooth muscle cells (SMC) is a critical process in at... more Proliferation of endothelial cells (EC) and smooth muscle cells (SMC) is a critical process in atherosclerosis. Here, we investigated the involvement of sublytic C5b-9 effector Response Gene to Complement 32 (RGC-32) in cell cycle activation, phenotypic switch, and production of extracellular matrix (ECM) in SMC. Overexpression of RGC-32 augmented C5b-9-induced cell cycle activation and proliferation of SMC in an ERK1-dependent manner and silencing of RGC-32 inhibited C5b-9-induced cell cycle activation. C5b-9-induced cell cycle activation also required phosphorylation of RGC-32 at threonine 91. We found that ECM components fibronectin and collagens I-V were expressed by SMC in human aortic atherosclerotic tissue. Silencing of RGC-32 in cultured SMC was followed by a significant reduction in TGF-β-induced expression of SMC differentiation markers myocardin, SM22 and α-SMA, and that of collagens I, IV and V. These data suggest that RGC-32 participates in both sublytic C5b-9-induced cell cycle activation and TGF-β-induced ECM production.
The biosynthesis of steroid hormones in endocrine steroidsecreting glands results from a series o... more The biosynthesis of steroid hormones in endocrine steroidsecreting glands results from a series of successive steps involving both cytochrome P450 enzymes, which are mixed-function oxidases, and steroid dehydrogenases. So far, the subcellular distribution of steroidogenic enzymes has been mostly studied following subcellular fractionation, performed in placenta and adrenal cortex. In order to determine in situ the intracellular distribution of some steroidogenic enzymes, we have investigated the ultrastructural localization of the three key enzymes: P450 side chain cleavage (scc) which converts cholesterol to pregnenolone; 3-hydroxysteroid dehydrogenase (3-HSD) which catalyzes the conversion of 3-hydroxy-5ene steroids to 3-oxo-4-ene steroids (progesterone and androstenedione); and P450 c17 which is responsible for the transformation of C 21 into C 19 steroids (dehydroepiandrosterone and androstenedione). Immunogold labeling was used to localize the enzymes in rat adrenal cortex and gonads. The tissues were fixed in 1% glutaraldehyde and 3% paraformaldehyde and included in LR gold resin. In the adrenal cortex, both P450 scc and 3-HSD immunoreactivities were detected in the reticular, fascicular and glomerular zones. P450 scc was exclusively found in large mitochondria. In contrast, 3-HSD antigenic sites were mostly observed in the endoplasmic reticulum (ER) with some gold particles overlying crista and outer membranes of the mitochondria. P450 c17 could not be detected in adrenocortical cells. In the testis, the three enzymes were only found in Leydig cells. Immunolabeling for P450 scc and 3-HSD was restricted to mitochondria, while P450 c17 immunoreactivity was exclusively observed in ER. In the ovary, P450 scc and 3-HSD immunoreactivities were found in granulosa, theca interna and corpus luteum cells. The subcellular localization of the two enzymes was very similar to that observed in adrenocortical cells. P450 c17 could also be detected in theca interna cells of large developing and mature follicles. As observed in Leydig cells, P450 c17 immunolabeling could only be found in the ER. These results indicate that in different endocrine steroid-secreting cells P450 scc , 3-HSD and P450 c17 have the same association with cytoplasmic organelles (with the exception of 3-HSD in Leydig cells), suggesting similar intracellular pathways for biosynthesis of steroid hormones.
Activation of heterotrimeric guanine nucleotide-binding proteins (G proteins) by terminal complem... more Activation of heterotrimeric guanine nucleotide-binding proteins (G proteins) by terminal complement complexes (TCC) was investigated on human lymphoblastoid B-cell line JY2S and its mutant J Y S deficient in glycosylphosphatidylinositol-anchored proteins. TCC assembly achieved by antibody-dependent activation of C7-deficient serum reconstituted with C7 increased specific guanosine-6'-(ythio)triphosphate (GTP+) binding, 4-and &fold, in JY2S and J Y S membranes, respectively, between 2 and 10 min, over the level without C7. TCC also increased GTPase activity 5-and 4-fold in JY2S and JYS, respectively, between 5 and 10 min. Increased GTPase activity was noted first with CSb-7 aseembly, which increased further with CSbd and CSb-9. The presence of G proteins in anti-TCC immunoprecipitates of cell lysates was investigated by demonstration of Ga subunit that can be ADP-ribosylated by pertussis toxin (PTX). Immunoprecipitated TCC complexes contained a PTX-sensitive 41-kDa GidGoa subunit, as shown by SDS-PAGE and Western blotting. These complexes were functionally active as determined by GTPyS binding. W e have further shown that enhanced TCC elimination from the plasma membrane induced by TCC-generated signals was inhibited by PTX. In conclusion the biological activities induced by TCC in nucleated cells may be mediated in part by activation of PTX-sensitive G proteins. Complement activation in infection and inflammation plays a crucial role in host defense by generating inflammatory mediators, such as C3a and C5a,' by opsonization of activating particles through C4b, C3b, and iC3b and lysis of target cells by forming C5b-9 channels. Assembly of the cytolytic C5b-9 complex is accomplished through a sequential interaction of C5-C9 plasma proteins, which results in amphipathic conformational *This work was supported by Grants R01-AI19622 and R01-NS15662 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 4624. $ Recipient of International Fogarty Research Fellowship F05, TWO 0 To whom correspondence should be addressed. The abbreviations used are: C3a, C5a, cleavage products of C3 and C5, respectively; App(NH)p, adenosine-5'-(P,y-imino)triphosphate, BCA, bicinchoninic acid; BSA, bovine serum albumin; C7D, C8D, C9D, human serum deficient in C7, C8, or C9, respectively; CTX, cholera toxin; DAG, diacylglycerol; Dm, DL-dithiotreitol; G proteins, guanine nucleotide-binding regulatory proteins; Gi, guanine nucleotide-binding inhibitory protein; Gs, guanine nucleotide-binding stimulatory protein; GTPyS, guanosine-5'-(y-thio)triphosphate; NHS, normal human serum; PBS, phosphate-buffered saline; PMSF, phenylmethylsulfonyl fluoride; F' TX, pertussis toxin; PAGE, polyacrylamide gel electrophorewhich include C5b-7, C5b-8, and C5b-9; MHC, major histocornpatability sis; TBS, Tris-buffered saline; TCC, terminal complement complexes, complex.
The shift from the differentiated contractile smooth muscle cell (SMC) phenotype to a less differ... more The shift from the differentiated contractile smooth muscle cell (SMC) phenotype to a less differentiated, proliferative state and excessive extracellular matrix (ECM) production, both TGF-β regulated events, are key alterations in atherogenesis. 1 The complement system takes part in the initiation and progression of atherosclerosis. First described as a cell cycle regulator, Response Gene to Complement (RGC)-32 is also implicated in tumorigenesis, immune system regulation, scar tissue formation, regulation of lipid and glucose metabolism and atherogenesis. We have shown that RGC-32 modulates C5b-9-induced endothelial cell (EC) proliferation and migration and is involved in EC cytoskeletal organization and cell adhesion. 2 Additionally, RGC-32 promotes vascular SMC proliferation. 3 RGC-32 also mediates some of TGF-βinduced profibrotic effects in a variety of tissues. 4, 5
foetuses exposed to maternal anti-Ro52 autoantibodies. Recent studies investigating other pathoge... more foetuses exposed to maternal anti-Ro52 autoantibodies. Recent studies investigating other pathogenic autoantibodies (antiinterferon, anti-desmoglein) report that they arise as a result of somatic mutation. The aim of this study was to determine how anti-Ro52 autoantibodies originate. Methods We traced the evolution of two anti-Ro52 autoantibodies isolated from circulating IgG-switched memory B-cells from a mother of two children with cardiac neonatal lupus. Each antibody was expressed as its immune form or preimmune ancestor by reverting somatic mutations to germline sequence. Antibody reactivity against autoantigens Ro52, Ro60, La and dsDNA were tested by ELISA. Results Both anti-Ro52 autoantibodies utilised the same heavy and light chain genes (IGHV3-23 and IGLV1-44) but represented distinct clones based on differing complementarity determining region sequences. Anti-Ro52 autoantibodies exhibited a low frequency (3%-4%) of somatic mutations compared to the average rate of 8% in healthy switched memory B-cells. In contrast to other pathogenic autoantibodies, the preimmune (germlined) anti-Ro52 autoantibodies showed specific binding to Ro52. However, Ro52 reactivity was higher for the mutated post-immune antibodies compared to their preimmune counterparts demonstrating that autoreactivity was enhanced by affinity maturation. Conclusions These data demonstrate that Ro52 reactivity is an intrinsic property of the germline antibody repertoire in a mother of children affected by neonatal lupus and indicate defects in central and peripheral tolerance pathways allow propagation of pathogenic autoantibodies.
SIRT1, a NAD dependent histone and protein deacetylase, is a member of the histone deacetylase cl... more SIRT1, a NAD dependent histone and protein deacetylase, is a member of the histone deacetylase class III family. We previously showed that SIRT1 mRNA expression is significantly lower in peripheral blood mononuclear cells (PBMCs) of multiple sclerosis (MS) patients during relapses than in stable patients. We have now investigated SIRT1 as a possible biomarker to predict relapse as well as responsiveness to glatiramer acetate (GA) treatment in relapsing-remitting MS (RRMS) patients. Over the course of 2years, a cohort of 15 GA-treated RRMS patients were clinically monitored using the Expanded Disability Status Scale and assessed for MS relapses. Blood samples collected from MS patients were analyzed for levels of SIRT1 and histone H3 lysine 9 (H3K9) acetylation and dimethylation. During relapses, MS patients had a lower expression of SIRT1 mRNA than did stable MS patients. In addition, there was a significant decrease in H3K9 dimethylation (H3K9me2) during relapses in MS patients whe...
The shift from the differentiated contractile smooth muscle cell (SMC) phenotype to a less differ... more The shift from the differentiated contractile smooth muscle cell (SMC) phenotype to a less differentiated, proliferative state and excessive extracellular matrix (ECM) production, both TGF-β regulated events, are key alterations in atherogenesis. 1 The complement system takes part in the initiation and progression of atherosclerosis. First described as a cell cycle regulator, Response Gene to Complement (RGC)-32 is also implicated in tumorigenesis, immune system regulation, scar tissue formation, regulation of lipid and glucose metabolism and atherogenesis. We have shown that RGC-32 modulates C5b-9-induced endothelial cell (EC) proliferation and migration and is involved in EC cytoskeletal organization and cell adhesion. 2 Additionally, RGC-32 promotes vascular SMC proliferation. 3 RGC-32 also mediates some of TGF-βinduced profibrotic effects in a variety of tissues. 4, 5
Organizations should carefully consider how to implement infection prevention recommendations fro... more Organizations should carefully consider how to implement infection prevention recommendations from external agencies in order to create policies that satisfy those recommendations and simultaneously meet the needs of patients, families, and staff. Communicating changes in isolation policies throughout a large enterprise presents specific challenges.
We have previously shown that RGC-32 is involved in cell cycle regulation in vitro. To define the... more We have previously shown that RGC-32 is involved in cell cycle regulation in vitro. To define the in vivo role of RGC-32, we generated RGC-32 knockout mice. These mice develop normally and do not spontaneously develop overt tumors. To assess the effect of RGC-32 deficiency on cell cycle activation in T cells we determined the proliferation rate of CD4 and CD8 from spleens of RGC-32-/- mice compared to wild type (WT) mice. CD4 T cells from RGC-32-/- mice display a significant increase in 3H-thymidine incorporation when compared with WT mice after stimulation with anti-CD3/anti-CD28. In addition, both CD4 and CD8 T cells from RGC-32-/- displayed a significant increase in the proportion of proliferating Ki67+ cells, indicating that in T cells, RGC-32 has an inhibitory effect on cell cycle activation. Furthermore, Akt and FOXO1 phosphorylation induced in CD4 from RGC-32-/- were significantly higher indicating that RGC-32 inhibits cell cycle activation by suppressing FOXO1 activation. To further examine the effect of RGC-32 on T cell functions we investigated the effect of RGC-32 on T regulatory (Treg) cells differentiation. A significantly lower percentage of CD4+ FoxP3+ Treg cells was found in unstimulated cells from RGC-32-/- compared to WT mice. Significantly fewer CD4+ FoxP3+ Treg cells are induced by TGF-β in RGC-32-/- compared to WT mice. Thus, RGC-32 is involved in controlling cell cycle in vivo, and in addition seems to play a role in the differentiation of Treg cells.
SIRT1 is a NAD-dependent histone deacetylase involved in the regulation of transcription, apoptos... more SIRT1 is a NAD-dependent histone deacetylase involved in the regulation of transcription, apoptosis, metabolism and differentiation. We have previously demonstrated that sublytic levels of complement C5b-9 terminal complex increased the survival of oligodendrocytes (OLGs) and induced their dedifferentiation. In this study we investigated the role of SIRT1 in OLGs differentiation and the effect of sublytic levels of C5b-9 on SIRT1 expression. We also investigated the downstream effects of SIRT1 by measuring histone H3 Lysine 9 trimethylation (H3K9me3) and expression of cyclin D1. OLG progenitor cells purified from the brain of rat pups were differentiated in vitro and stimulated with sublytic C5b-9 or C5b6 for 3, 6 and 18 h. The level of SIRT1 mRNA was measured using real-time PCR and SIRT1, cyclin D1 and H3K9me3 protein expression were measured using western blotting. We found a decreased expression of SIRT1 mRNA and protein, a decreased level of H3K9me3 and an increased expression of cyclin D1 during OLG differentiation. Stimulation of OLGs with sublytic C5b-9 for 3h resulted in a significant decrease in SIRT1 mRNA and protein levels while stimulation with C5b6 had no effect. SIRT1 protein level in OLGs after 8h of exposure to C5b-9 were significantly lower than in C5b6 treated cells. H3K9me3 levels also decreased significantly after stimulation with C5b-9 as compared with unstimulated or C5b6-treated OLGs. Cyclin D1 expression increased after stimulation with C5b-9, indicating cell cycle activation. Our data show that C5b-9 stimulation of OLGs reduces SIRT1 expression, contributing to cell cycle activation by decreasing repressive trimethylation of histone H3 lysine 9.
We have previously shown that response gene to complement 32 (RGC-32) mediates transforming growt... more We have previously shown that response gene to complement 32 (RGC-32) mediates transforming growth factor-β (TGF-β)-induced astrocyte reactivity and extracellular matrix production. However, the molecular mechanisms underlying RGC-32 expression and function remain to be elucidated. In the present study, we found that TGF-β induced expression and nuclear translocation of both RGC-32 and SMAD3 in astrocytes. Using specific pathways inhibitors, we have found that RGC-32 induction is mediated by SMAD-dependent and independent mechanisms. We investigated whether RGC-32 interacts with SMAD2 or SMAD3 in astrocytes and if this association plays a role in extracellular matrix synthesis. Co-immunoprecipitation showed that SMAD3, but not SMAD2 physically interacts with RGC-32 in astrocytes. SIS3, an inhibitor of SMAD3 phosphorylation, significantly reduced RGC-32 translocation to the nucleus, indicating that SMAD3 phosphorylation is required for RGC-32 nuclear translocation. On the other hand, silencing RGC-32 with siRNA had no effect on SMAD3 nuclear translocation. Y27632, a selective ROCK inhibitor was also able to significantly inhibit RGC-32 nuclear translocation, while the SMAD3 nuclear translocation was unaffected. The inhibition of SMAD3 phosphorylation also blocked the RGC-32-induced collagen type I expression, indicating a synergistic action between RGC-32 and SMAD3 to induce the expression of collagen type I. Taken together, our data demonstrate for the first time that RGC-32 interacts with SMAD3 to mediate the extracellular matrix production in astrocytes. These data suggest that a similar mechanism might be involved in mediation of gliosis seen in chronic multiple sclerosis lesions.
recent in vivo pathogenesis studies in mice, have shown that intestinal colonization by RG strain... more recent in vivo pathogenesis studies in mice, have shown that intestinal colonization by RG strains isolated from Lupus patients in flare, but not RG strains from healthy adults, induce zonulin-dependent increases in intestinal permeability, RG translocation to mesenteric lymph nodes as well as serum IgG anti-RG antibody and anti-native DNA autoantibody responses. Conclusions As many Lupus patients are known to suffer relapsing illness despite appropriate treatment, we speculate that gut blooms of pathogenic bacteria that impair gut barrier function and stoke systemic inflammation directly contribute to immunopathogenesis. We propose that future therapeutic interventions designed to promote a durable remission consider the potential necessity to target both the immunologic abnormalities of the disease, as well as re-establish stability within the gut microbiota community.
The shift from the differentiated contractile smooth muscle cell (SMC) phenotype to a less differ... more The shift from the differentiated contractile smooth muscle cell (SMC) phenotype to a less differentiated, proliferative state and excessive extracellular matrix (ECM) production, both TGF-β regulated events, are key alterations in atherogenesis. 1 The complement system takes part in the initiation and progression of atherosclerosis. First described as a cell cycle regulator, Response Gene to Complement (RGC)-32 is also implicated in tumorigenesis, immune system regulation, scar tissue formation, regulation of lipid and glucose metabolism and atherogenesis. We have shown that RGC-32 modulates C5b-9-induced endothelial cell (EC) proliferation and migration and is involved in EC cytoskeletal organization and cell adhesion. 2 Additionally, RGC-32 promotes vascular SMC proliferation. 3 RGC-32 also mediates some of TGF-βinduced profibrotic effects in a variety of tissues. 4, 5
Background: Glatiramer acetate (GA) is US-approved for relapsing multiple sclerosis. Objectives: ... more Background: Glatiramer acetate (GA) is US-approved for relapsing multiple sclerosis. Objectives: To describe GA long-term clinical profile. To compare effectiveness of early start (ES) versus delayed start (DS; up to 3 years) with GA. Methods: Phase 3 trial participants entered a randomized placebo-controlled period then an open-label extension (OLE) with GA. Results: Overall, 208 out of 251 (82.9%) randomized participants entered the OLE; 24 out of 101 (23.8%, ES) and 28 out of 107 (26.2%, DS) participants completed the OLE. Median GA treatment was 9.8 (0.1–26.3) years. Annualized change in Expanded Disability Status Scale (EDSS) score was lower with ES versus DS ( p = 0.0858: full study; p = 0.002; Year 5). Participants with improved/stable EDSS was consistently higher with ES versus DS: 40.3% versus 31.6% ( p = 0.1590; full study); 70.8% versus 55.6% ( p = 0.015; Year 5). ES prolonged time-to-6-month confirmed disease worsening (CDW) versus DS (9.8 vs 6.7 years), time-to-12-month CDW (18.9 vs 11.6 years), and significantly reduced time-to-second-6-month CDW ( p = 0.0441). No new safety concerns arose. Conclusion: GA long-term treatment maintained clinical benefit with a similar safety profile to phase 3 results; a key limitation was that only 25% of participants completed the OLE. Early initiation of GA had sustained benefits versus delayed treatment.
Proliferation of endothelial cells (EC) and smooth muscle cells (SMC) is a critical process in at... more Proliferation of endothelial cells (EC) and smooth muscle cells (SMC) is a critical process in atherosclerosis. Here, we investigated the involvement of sublytic C5b-9 effector Response Gene to Complement 32 (RGC-32) in cell cycle activation, phenotypic switch, and production of extracellular matrix (ECM) in SMC. Overexpression of RGC-32 augmented C5b-9-induced cell cycle activation and proliferation of SMC in an ERK1-dependent manner and silencing of RGC-32 inhibited C5b-9-induced cell cycle activation. C5b-9-induced cell cycle activation also required phosphorylation of RGC-32 at threonine 91. We found that ECM components fibronectin and collagens I-V were expressed by SMC in human aortic atherosclerotic tissue. Silencing of RGC-32 in cultured SMC was followed by a significant reduction in TGF-β-induced expression of SMC differentiation markers myocardin, SM22 and α-SMA, and that of collagens I, IV and V. These data suggest that RGC-32 participates in both sublytic C5b-9-induced cell cycle activation and TGF-β-induced ECM production.
The biosynthesis of steroid hormones in endocrine steroidsecreting glands results from a series o... more The biosynthesis of steroid hormones in endocrine steroidsecreting glands results from a series of successive steps involving both cytochrome P450 enzymes, which are mixed-function oxidases, and steroid dehydrogenases. So far, the subcellular distribution of steroidogenic enzymes has been mostly studied following subcellular fractionation, performed in placenta and adrenal cortex. In order to determine in situ the intracellular distribution of some steroidogenic enzymes, we have investigated the ultrastructural localization of the three key enzymes: P450 side chain cleavage (scc) which converts cholesterol to pregnenolone; 3-hydroxysteroid dehydrogenase (3-HSD) which catalyzes the conversion of 3-hydroxy-5ene steroids to 3-oxo-4-ene steroids (progesterone and androstenedione); and P450 c17 which is responsible for the transformation of C 21 into C 19 steroids (dehydroepiandrosterone and androstenedione). Immunogold labeling was used to localize the enzymes in rat adrenal cortex and gonads. The tissues were fixed in 1% glutaraldehyde and 3% paraformaldehyde and included in LR gold resin. In the adrenal cortex, both P450 scc and 3-HSD immunoreactivities were detected in the reticular, fascicular and glomerular zones. P450 scc was exclusively found in large mitochondria. In contrast, 3-HSD antigenic sites were mostly observed in the endoplasmic reticulum (ER) with some gold particles overlying crista and outer membranes of the mitochondria. P450 c17 could not be detected in adrenocortical cells. In the testis, the three enzymes were only found in Leydig cells. Immunolabeling for P450 scc and 3-HSD was restricted to mitochondria, while P450 c17 immunoreactivity was exclusively observed in ER. In the ovary, P450 scc and 3-HSD immunoreactivities were found in granulosa, theca interna and corpus luteum cells. The subcellular localization of the two enzymes was very similar to that observed in adrenocortical cells. P450 c17 could also be detected in theca interna cells of large developing and mature follicles. As observed in Leydig cells, P450 c17 immunolabeling could only be found in the ER. These results indicate that in different endocrine steroid-secreting cells P450 scc , 3-HSD and P450 c17 have the same association with cytoplasmic organelles (with the exception of 3-HSD in Leydig cells), suggesting similar intracellular pathways for biosynthesis of steroid hormones.
Activation of heterotrimeric guanine nucleotide-binding proteins (G proteins) by terminal complem... more Activation of heterotrimeric guanine nucleotide-binding proteins (G proteins) by terminal complement complexes (TCC) was investigated on human lymphoblastoid B-cell line JY2S and its mutant J Y S deficient in glycosylphosphatidylinositol-anchored proteins. TCC assembly achieved by antibody-dependent activation of C7-deficient serum reconstituted with C7 increased specific guanosine-6'-(ythio)triphosphate (GTP+) binding, 4-and &fold, in JY2S and J Y S membranes, respectively, between 2 and 10 min, over the level without C7. TCC also increased GTPase activity 5-and 4-fold in JY2S and JYS, respectively, between 5 and 10 min. Increased GTPase activity was noted first with CSb-7 aseembly, which increased further with CSbd and CSb-9. The presence of G proteins in anti-TCC immunoprecipitates of cell lysates was investigated by demonstration of Ga subunit that can be ADP-ribosylated by pertussis toxin (PTX). Immunoprecipitated TCC complexes contained a PTX-sensitive 41-kDa GidGoa subunit, as shown by SDS-PAGE and Western blotting. These complexes were functionally active as determined by GTPyS binding. W e have further shown that enhanced TCC elimination from the plasma membrane induced by TCC-generated signals was inhibited by PTX. In conclusion the biological activities induced by TCC in nucleated cells may be mediated in part by activation of PTX-sensitive G proteins. Complement activation in infection and inflammation plays a crucial role in host defense by generating inflammatory mediators, such as C3a and C5a,' by opsonization of activating particles through C4b, C3b, and iC3b and lysis of target cells by forming C5b-9 channels. Assembly of the cytolytic C5b-9 complex is accomplished through a sequential interaction of C5-C9 plasma proteins, which results in amphipathic conformational *This work was supported by Grants R01-AI19622 and R01-NS15662 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 4624. $ Recipient of International Fogarty Research Fellowship F05, TWO 0 To whom correspondence should be addressed. The abbreviations used are: C3a, C5a, cleavage products of C3 and C5, respectively; App(NH)p, adenosine-5'-(P,y-imino)triphosphate, BCA, bicinchoninic acid; BSA, bovine serum albumin; C7D, C8D, C9D, human serum deficient in C7, C8, or C9, respectively; CTX, cholera toxin; DAG, diacylglycerol; Dm, DL-dithiotreitol; G proteins, guanine nucleotide-binding regulatory proteins; Gi, guanine nucleotide-binding inhibitory protein; Gs, guanine nucleotide-binding stimulatory protein; GTPyS, guanosine-5'-(y-thio)triphosphate; NHS, normal human serum; PBS, phosphate-buffered saline; PMSF, phenylmethylsulfonyl fluoride; F' TX, pertussis toxin; PAGE, polyacrylamide gel electrophorewhich include C5b-7, C5b-8, and C5b-9; MHC, major histocornpatability sis; TBS, Tris-buffered saline; TCC, terminal complement complexes, complex.
The shift from the differentiated contractile smooth muscle cell (SMC) phenotype to a less differ... more The shift from the differentiated contractile smooth muscle cell (SMC) phenotype to a less differentiated, proliferative state and excessive extracellular matrix (ECM) production, both TGF-β regulated events, are key alterations in atherogenesis. 1 The complement system takes part in the initiation and progression of atherosclerosis. First described as a cell cycle regulator, Response Gene to Complement (RGC)-32 is also implicated in tumorigenesis, immune system regulation, scar tissue formation, regulation of lipid and glucose metabolism and atherogenesis. We have shown that RGC-32 modulates C5b-9-induced endothelial cell (EC) proliferation and migration and is involved in EC cytoskeletal organization and cell adhesion. 2 Additionally, RGC-32 promotes vascular SMC proliferation. 3 RGC-32 also mediates some of TGF-βinduced profibrotic effects in a variety of tissues. 4, 5
foetuses exposed to maternal anti-Ro52 autoantibodies. Recent studies investigating other pathoge... more foetuses exposed to maternal anti-Ro52 autoantibodies. Recent studies investigating other pathogenic autoantibodies (antiinterferon, anti-desmoglein) report that they arise as a result of somatic mutation. The aim of this study was to determine how anti-Ro52 autoantibodies originate. Methods We traced the evolution of two anti-Ro52 autoantibodies isolated from circulating IgG-switched memory B-cells from a mother of two children with cardiac neonatal lupus. Each antibody was expressed as its immune form or preimmune ancestor by reverting somatic mutations to germline sequence. Antibody reactivity against autoantigens Ro52, Ro60, La and dsDNA were tested by ELISA. Results Both anti-Ro52 autoantibodies utilised the same heavy and light chain genes (IGHV3-23 and IGLV1-44) but represented distinct clones based on differing complementarity determining region sequences. Anti-Ro52 autoantibodies exhibited a low frequency (3%-4%) of somatic mutations compared to the average rate of 8% in healthy switched memory B-cells. In contrast to other pathogenic autoantibodies, the preimmune (germlined) anti-Ro52 autoantibodies showed specific binding to Ro52. However, Ro52 reactivity was higher for the mutated post-immune antibodies compared to their preimmune counterparts demonstrating that autoreactivity was enhanced by affinity maturation. Conclusions These data demonstrate that Ro52 reactivity is an intrinsic property of the germline antibody repertoire in a mother of children affected by neonatal lupus and indicate defects in central and peripheral tolerance pathways allow propagation of pathogenic autoantibodies.
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