Induction of prostacyclin receptor expression in human erythroleukemia cells

AbstractWe have identified both high-affinity (KD = 36±3 nM) and low-affinity (KD = 2.1±0.8 μM) prostacyclin (PGI2)-receptor sites on human erythroleukemia (HEL) cells using the radiolabelled prostacyclin analogue, [3H]iloprost. The addition of the phorbol ester, TPA, to the culture medium caused a 5–10-fold increase in the number of both the low- and the high-affinity sites, without any change in their affinity constants. Iloprost stimulated HEL cell membrane adenylate cyclase activity 5-fold. This stimulation was potentiated in the presence of GTP, indicating a conventional PGI2 receptor-Gs-adenylate cyclase system. HEL cells represent a source of prostacyclin receptor mRNA which may be of value in expression cloning of this receptor

Alteration of human macrophages microRNA expression profile upon infection with Mycobacterium tuberculosis

Background: Mycobacterium tuberculosis (Mtb) has evolved multiple mechanisms to manipulate its cellular niche for its own advantage. Many efforts have been made to understand basal mechanisms of mycobacterial infections. However, the underlying molecular regulation is not fully understood. Recently, a new class of non-coding, small RNAs, called microRNAs (miRNAs), has emerged as important regulators in biological processes, and their involvement in mycobacterial infection has been identified, thus opening a new field of research. Methods: This study aimed to determine by TaqMan Low Density Array the host genome-wide miRNA expression profile of primary human monocyte-derived macrophages (MDM) infected with two members of the Mtb complex: virulent Mtb H37Rv and the non-virulent vaccine strain Mycobacterium bovis Bacillus Calmette-Guerin (BCG) in comparison with chemically-inactivated Mtb bacilli. Results: The findings of this study showed that infection of MDM with H37Rv or BCG results in a signature of miRNA expression mostly overlapping between the two mycobacteria. A substantially different signature emerged from infection with killed virulent bacilli, suggesting an active influence of live intracellular bacteria on cellular miRNA metabolism. Specifically, Mtb induced miRNA signature is composed of miRNAs well established in immune regulation, miR-155 and miR-146a, as well as a set of miRNAs newly associated with Mtb infection: miR-145, miR-222*, miR-27a and miR-27b. All of these miRNAs are predicted to target important immune-related genes. Conclusions: This study signifies the miRNA host response upon intracellular mycobacterial infection in macrophages, providing new aspects of regulation in host-pathogen interactions, at post-transcriptional levels

Inhibition of HIV-1 Infection by Human alpha-Defensin-5, a Natural Antimicrobial Peptide Expressed in the Genital and Intestinal Mucosae

α-defensin-5 (HD5) is a key effector of the innate immune system with broad anti-bacterial and anti-viral activities. Specialized epithelial cells secrete HD5 in the genital and gastrointestinal mucosae, two anatomical sites that are critically involved in HIV-1 transmission and pathogenesis. We previously found that human neutrophil defensins (HNP)-1 and -2 inhibit HIV-1 entry by specific bilateral interaction both with the viral envelope and with its primary cellular receptor, CD4. Despite low amino acid identity, human defensin-5 (HD5) shares with HNPs a high degree of structural homology. T lymphocytes at low micromolar concentration under serum-free and low-ionic-strength conditions similar to those occurring in mucosal fluids. Blockade of HIV-1 infection was observed with both primary and laboratory-adapted strains and was independent of the viral coreceptor-usage phenotype. Similar to HNPs, HD5 inhibits HIV-1 entry into the target cell by interfering with the reciprocal interaction between the external envelope glycoprotein, gp120, and CD4. At high concentrations, HD5 was also found to downmodulate expression of the CXCR4 coreceptor, but not of CCR5. Consistent with its broad spectrum of activity, antibody competition studies showed that HD5 binds to a region overlapping with the CD4- and coreceptor-binding sites of gp120, but not to the V3 loop region, which contains the major determinants of coreceptor-usage specificity.These findings provide new insights into the first line of immune defense against HIV-1 at the mucosal level and open new perspectives for the development of preventive and therapeutic strategies

Inhibition of HIV-1 infection by human α-defensin-5, a natural antimicrobial peptide expressed in the genital and intestinal mucosae.

BACKGROUND: α-defensin-5 (HD5) is a key effector of the innate immune system with broad anti-bacterial and anti-viral activities. Specialized epithelial cells secrete HD5 in the genital and gastrointestinal mucosae, two anatomical sites that are critically involved in HIV-1 transmission and pathogenesis. We previously found that human neutrophil defensins (HNP)-1 and -2 inhibit HIV-1 entry by specific bilateral interaction both with the viral envelope and with its primary cellular receptor, CD4. Despite low amino acid identity, human defensin-5 (HD5) shares with HNPs a high degree of structural homology. METHODOLOGY/PRINCIPAL FINDINGS: Here, we demonstrate that HD5 inhibits HIV-1 infection of primary CD4(+) T lymphocytes at low micromolar concentration under serum-free and low-ionic-strength conditions similar to those occurring in mucosal fluids. Blockade of HIV-1 infection was observed with both primary and laboratory-adapted strains and was independent of the viral coreceptor-usage phenotype. Similar to HNPs, HD5 inhibits HIV-1 entry into the target cell by interfering with the reciprocal interaction between the external envelope glycoprotein, gp120, and CD4. At high concentrations, HD5 was also found to downmodulate expression of the CXCR4 coreceptor, but not of CCR5. Consistent with its broad spectrum of activity, antibody competition studies showed that HD5 binds to a region overlapping with the CD4- and coreceptor-binding sites of gp120, but not to the V3 loop region, which contains the major determinants of coreceptor-usage specificity. CONCLUSION/SIGNIFICANCE: These findings provide new insights into the first line of immune defense against HIV-1 at the mucosal level and open new perspectives for the development of preventive and therapeutic strategies

HD5 inhibits HIV-1 envelope-mediated fusion induced by clinical and laboratory HIV-1 isolates with different coreceptor usage.

<p>Fusion assays were performed using the human T-cell line (PM1) chronically infected with two prototypic laboratory-adapted strains (HIV-1_IIIB and HIV-1_BaL) and four primary clinical isolates (R5: HIV-1_J6366 and HIV-1_J2615; X4: HIV-1_J57 and HIV-1_J287) cocultured with NIH-3T3 cells expressing membrane-bound CD4 and either CCR5 or CXCR4 in the presence or absence of increasing concentrations of HD5 (filled circles) for 2 hrs at 37°C in medium without FCS. For comparison, inhibition by HNP1 (open squares) used at the same concentrations is shown. The data are normalized with respect to fusion observed in the absence of inhibitors. The data represent mean values (±SD) from 3 experiments.</p

HD5 binds to both gp120 and CD4, and blocks the interaction of CD4 with gp120.

<p>(A) Effect of HD5 on HIV-1 envelope-mediated fusion after pre-incubation with effector cells (expressing the viral envelope) or target cells (expressing CD4 and the coreceptor). Prior to the fusion reaction, HD5 was pre-incubated for 20 minutes with effector cells or target cells followed by washing to remove the unbound defensin. Control fusion was performed as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045208#pone-0045208-g002" target="_blank">Figure 2</a> with addition of HD5 at the time of culture between effectors and targets. (B) Binding of HD5 to HIV-1 gp120. Competition of synthetic HD5 with the anti-gp120 mAb IgG1b12 (used at 5 µg/mL) for binding to plastic-immobilized recombinant gp120_BaL in ELISA. (C) Binding of HD5 to human CD4. Competition of synthetic HD5 with the anti-CD4 mAb Leu3a for binding to plastic-immobilized recombinant sCD4 in ELISA. The plates were coated with sCD4 at 5 µg/mL; HD5 was added prior to Leu3a and kept in the wells throughout the reaction period. (D) Inhibition of gp120/CD4 binding by HD5. Competition of HD5 with recombinant HIV-1 gp120_BaL for binding to plastic-immobilized sCD4 in ELISA. The plates were coated with sCD4 at 5 µg/mL; HD5 was added prior to Leu3a or gp120_BaL and kept in the wells throughout the reaction period. Error bars indicate SD of mean values obtained from 3 repeated assays.</p