Circulating Strains of Human Respiratory Syncytial Virus in Central and South America

Human respiratory syncytial virus (HRSV) is a major cause of viral lower respiratory tract infections among infants and young children. HRSV strains vary genetically and antigenically and have been classified into two broad subgroups, A and B (HRSV-A and HRSV-B, respectively). To date, little is known about the circulating strains of HRSV in Latin America. We have evaluated the genetic diversity of 96 HRSV strains by sequencing a variable region of the G protein gene of isolates collected from 2007 to 2009 in Central and South America. Our results show the presence of the two antigenic subgroups of HRSV during this period with the majority belonging to the genotype HRSV-A2

The significance of autoantibodies to DFS70/LEDGFp75 in health and disease: integrating basic science with clinical understanding

Antinuclear autoantibodies (ANAs) displaying the nuclear dense fine speckled immunofluorescence (DFS-IIF) pattern in HEp-2 substrates are commonly observed in clinical laboratory referrals. They target the dense fine speckled autoantigen of 70 kD (DFS70), most commonly known as lens epithelium-derived growth factor p75 (LEDGFp75). Interesting features of these ANAs include their low frequency in patients with systemic autoimmune rheumatic diseases (SARD), elevated prevalence in apparently healthy individuals, IgG isotype, strong trend to occur as the only ANA specificity in serum, and occurrence in moderate to high titers. These autoantibodies have also been detected at varied frequencies in patients with diverse non-SARD inflammatory and malignant conditions such as atopic diseases, asthma, eye diseases, and prostate cancer. These observations have recently stimulated vigorous research on their clinical and biological significance. Some studies have suggested that they are natural, protective antibodies that could serve as biomarkers to exclude a SARD diagnosis. Other studies suggest that they might be pathogenic in certain contexts. The emerging role of DFS70/LEDGFp75 as a stress protein relevant to human acquired immunodeficiency syndrome, cancer, and inflammation also points to the possibility that these autoantibodies could be sensors of cellular stress and inflammation associated with environmental factors. In this comprehensive review, we integrate our current knowledge of the biology of DFS70/LEDGFp75 with the clinical understanding of its autoantibodies in the contexts of health and disease.NIH/NIMHDNIH/NIMGSRoche Tissue Diagnost, Ventana Med, Tucson, AZ USAInova Diagnost Inc, Dept Res, San Diego, CA USALoma Linda Univ, Dept Basic Sci, Ctr Hlth Dispar & Mol Med, Sch Med, Mortensen Hall 142,11085 Campus St, Loma Linda, CA 92350 USAUniv Fed Sao Paulo, Div Rheumatol, Sao Paulo, BrazilFleury Med & Hlth Labs, Div Immunol, Sao Paulo, BrazilUniv Calgary, Fac Med, Calgary, AB, CanadaLoma Linda Univ, Sch Med, Dept Med, Div Rheumatol, Loma Linda, CA 92354 USAUniv Fed Sao Paulo, Div Rheumatol, Sao Paulo, BrazilNIH/NIMHD: P20MD006988NIH/NIMGS: R25GM060507Web of Scienc

LEDGF/p75 Overexpression Attenuates Oxidative Stress-Induced Necrosis and Upregulates the Oxidoreductase ERP57/PDIA3/GRP58 in Prostate Cancer.

Prostate cancer (PCa) mortality is driven by highly aggressive tumors characterized by metastasis and resistance to therapy, and this aggressiveness is mediated by numerous factors, including activation of stress survival pathways in the pro-inflammatory tumor microenvironment. LEDGF/p75, also known as the DFS70 autoantigen, is a stress transcription co-activator implicated in cancer, HIV-AIDS, and autoimmunity. This protein is targeted by autoantibodies in certain subsets of patients with PCa and inflammatory conditions, as well as in some apparently healthy individuals. LEDGF/p75 is overexpressed in PCa and other cancers, and promotes resistance to chemotherapy-induced cell death via the transactivation of survival proteins. We report in this study that overexpression of LEDGF/p75 in PCa cells attenuates oxidative stress-induced necrosis but not staurosporine-induced apoptosis. This finding was consistent with the observation that while LEDGF/p75 was robustly cleaved in apoptotic cells into a p65 fragment that lacks stress survival activity, it remained relatively intact in necrotic cells. Overexpression of LEDGF/p75 in PCa cells led to the upregulation of transcript and protein levels of the thiol-oxidoreductase ERp57 (also known as GRP58 and PDIA3), whereas its depletion led to ERp57 transcript downregulation. Chromatin immunoprecipitation and transcription reporter assays showed LEDGF/p75 binding to and transactivating the ERp57 promoter, respectively. Immunohistochemical analysis revealed significantly elevated co-expression of these two proteins in clinical prostate tumor tissues. Our results suggest that LEDGF/p75 is not an inhibitor of apoptosis but rather an antagonist of oxidative stress-induced necrosis, and that its overexpression in PCa leads to ERp57 upregulation. These findings are of significance in clarifying the role of the LEDGF/p75 stress survival pathway in PCa

The stress oncoprotein LEDGF/p75 interacts with the methyl CpG binding protein MeCP2 and influences its transcriptional activity

The lens epithelium derived growth factor p75 (LEDGF/p75) is a transcription co-activator that promotes resistance to oxidative stress- and chemotherapy-induced cell death. LEDGF/p75 is also known as the dense fine speckles autoantigen of 70 kD (DFS70), and has been implicated in cancer, HIV-AIDS, autoimmunity, and inflammation. To gain insights into mechanisms by which LEDGF/p75 protects cancer cells against stress, we initiated an analysis of its interactions with other transcription factors and the influence of these interactions on stress gene activation. We report here that both LEDGF/p75 and its short splice variant LEDGF/p52 interact with MeCP2, a methylation-associated transcriptional modulator, in vitro and in various human cancer cells. These interactions were established by several complementary approaches: transcription factor protein arrays, pull down and AlphaScreen® assays, co-immunoprecipitation, and nuclear co-localization by confocal microscopy. MeCP2 was found to interact with the N-terminal region shared by LEDGF/p75 and p52, particularly with the PWWP-CR1 domain. Like LEDGF/p75, MeCP2 bound to and transactivated the Hsp27 promoter (Hsp27pr). LEDGF/p75 modestly enhanced MeCP2-induced Hsp27pr transactivation in U2OS cells, while this effect was more pronounced in PC3 cells. LEDGF/p52 repressed Hsp27pr activity in U2OS cells. Interestingly, siRNA-induced silencing of LEDGF/p75 in U2OS cells dramatically elevated MeCP2-mediated Hsp27pr transactivation, whereas this effect was less pronounced in PC3 cells depleted of LEDGF/p75. These results suggest that the LEDGF/p75-MeCP2 interaction differentially influences Hsp27pr activation depending on the cellular and molecular context. These findings are of significance in understanding the contribution of this interaction to the activation of stress survival genes.status: publishe

Multi-screen immunoblotting analysis to identify candidate stress proteins upregulated by LEDGF/p75 in RWPE-2 cells.

<p>A. Lysates from cells stably overexpressing LEDGF/p75 (RWPE-2–<i>ledgf/p75</i>) and RWPE-2 cells transfected with empty pcDNA3.1 vector (RWPE-2-<i>Vec</i>) were individually analyzed by immunoblotting using the Kinetworks™ KHSP-1.0 screen platform. Validation was performed using commercial antibodies and changes in protein expression were determined in LEDGF/p75-overexpressing RWPE-2 cells in comparison to cells transfected with empty vector. B. Untransfected RWPE-2 cells, cells transfected with empty vector, and cells stably overexpressing LEDGF/p75 were tested in-house for additional validation of upregulation of ERp57 and Hsp90. β-actin was used as loading control.</p

Effects of transient depletion of LEDGF/p75 on ERp57 expression levels in DU145 cells.

<p>A. LEDGF/p75 and ERp57 transcript and protein levels were assessed by qPCR and immunoblotting, respectively. A. Transcript and protein expression levels of LEDGF/p75 and ERp57 in DU145-DR cells, selected for their resistance to DTX, compared to parental DU145 cells. B. Parental DU145 cells with and without siRNA induced transient depletion of LEDGF/p75. C. DU145-DR cells with and without siRNA induced transient depletion of LEDGF/p75. Each graph represents the average of at least 3 independent experiments performed in triplicates (*<i>P<0</i>.<i>05</i>, **<i>P</i> <0.01). <i>P</i> values were determined in comparison to cells transfected with non-specific, scrambled control siRNAs (siSD) using the Student’s <i>t</i>-test.</p

LEDGF/p75 binds to ERp57 promoter in ChIP assays.

<p>A. Schematic diagram of ERp57 promoter. PCR primers targeted <i>ERp57pr</i> regions A (bp –498 to +1), B (bp –898 to – 490), C (bp – 890 to – 1298), D (bp – 1290 to – 1698), and E (bp – 1690 to – 2098). ChIP analysis of LEDGF/p75 binding to <i>ERp57pr</i> in PC3 (B) and U2OS (C) cells. Formaldehyde-fixed cells were precipitated with nonspecific IgG antibody or antibody specific for LEDGF/p75. PCR amplifications of immunoprecipitated DNA were carried out with primer sets specific for <i>ERp57pr</i> regions A to E. Primers for human β-actin or GAPDH were used to control for optimal enzymatic digestion of chromatin.</p

LEDGF/p75 transactivates ERp57 promoter in luciferase-based transcription reporter assays.

<p><i>ERp57pr</i> transactivation by LEDGF/p75 in A. PC3; B. DU145; C. RWPE-2; and D. U2OS cells. Luciferase activity in PCa cells co-transfected with <i>ERp57pr</i> vector and pcDNA-<i>ledgfp/75</i> vector was compared to activity in cells co-transfected with <i>ERp57pr</i> vector and pcDNA empty vector (A-C). Luciferase activity in U2OS cells co-transfected with <i>ERp57pr</i> vector and pCruzHA-<i>ledgfp/75</i> vector was compared to activity in cells co-transfected with <i>ERp57pr</i> vector and pCruzHA empty vector (D). Promoter activity determined as luciferase light units/protein is expressed as fold activation compared to control activity, which was normalized to one. Each graph represents the average of at least 3 independent experiments performed in triplicates (*<i>P<0</i>.<i>05</i>, **<i>P</i> <0.01). P values were determined using the Student’s <i>t</i>-test.</p