Blocking TNF or IL-6 decreases HIV-1 replication significantly in a dose dependent manner.

<p>Virus replication was determined by measuring HIV-1 p24 levels in the culture supernatants in PBMC from six individual donors at day 7 post-infection performed in duplicate. Blocking TNF (at 2 ug/ml or 5 ug/ml) or IL-6 (5 ug/ml) significantly decreased virus replication (*, p<0.05) as compared to the same concentrations of an IgG₁ control antibody. Blocking of MCP-1 alone on average minimally decreased HIV-1 replication, but this did not reach statistical significance. Virus replication was further significantly inhibited in cultures containing a mixture of blocking antibodies to TNF, IL-6, and MCP-1 at a concentration of 2 µg/ml and 5 µg/ml (**, p<0.01). Results are shown as mean±SEM.</p

CDC1551 and HN878 infection differentially regulate TNF, IL-6 and MCP-1 in human PBMC.

<p>PBMC from eight different donors were infected in duplicate with the MTb strain CDC1551, HN878, or HN878 <i>pks1-15::hyg</i> for 2 or 4 days and levels of TNF, IL-6 and MCP-1 were assessed. Production of TNF, IL-6 and MCP-1 was significantly higher in CDC1551 infected cells as compared to HN878 infected cells (*p<0.05; **, p<0.01; ***, p<0.005). Infection of PBMC with HN878 <i>pks1-15::hyg</i> non-significantly increased the production of these cellular factors as compared to infection with HN878. Expression of IL-1β, IL-2 and IL-10 were also on average minimally higher in CDC1551 versus HN878 infected cells, but not significantly.</p

CDC1551 and HN878 differentially induce transcription and nuclear localization of the p65 subunit of NF-κB.

<p>(A) p65 mRNA levels in PBMC from six individual donors infected with CDC1551, HN878, or HN878 <i>pks1-15::hyg</i> for 3, 8 and 24 hours. There was no significant difference in p65 mRNA levels across uninfected cells and the cells infected with CDC1551, HN878, or HN878 <i>pks1-15::hyg</i> after 3 and 8 hours infection. A significant difference in p65 mRNA levels was observed 24 hours after infection in CDC1551 infected cells as compared to HN878 infected cells (*, p<0.05). (B) A representative EMSA analysis using nuclear extracts from PBMC infected with CDC1551, HN878, and HN878 <i>pks1-15::hyg</i> for 24 hours. DNA binding activity of NF-κB was higher in nuclear extracts prepared from cells infected with CDC1551 as compared to HN878 (lanes 2 and 3). Infection with HN878 <i>pks1-15::hyg</i> increased NF-κB DNA binding activity as compared to HN878. (lanes 2, 3 and 4). The gel shown is representative of EMSAs performed using nuclear extracts prepared from PBMC from six individual donors. (C) The induced complex seen on EMSA contains the activating p65 subunit of NF-κB. An antibody to p65 specifically reacted with the complex that is differentially increased by CDC1551 and HN878, specifically inhibiting the formation of the p65 complex in lane 3 relative to the complex in lane 2. When quantified by densitometric analysis the inhibition of the complex after antibody reaction was 57%. (D) Increased nuclear translocation of the p65 NF-κB subunit after infection with CDC1551 as compared to HN878. The histogram shows an average of the densitometric analyses of the p65 band from autoradiographs of the EMSAs from nuclear extracts prepared from the six donors whose PBMC were either uninfected or infected with CDC1551, HN878, or HN878 <i>pks1-15::hyg</i> as indicated. The results are shown as mean±SEM and demonstrate that there was significantly higher p65 levels in CDC1551 than in HN878 infected cells. (*, p<0.05).</p

Considering a participatory approach to social work – Service user research

Service-user involvement in social work research is much vaunted and considered desirable. Yet, it is not common. This is despite the fact that research-funding bodies are increasingly mandating inclusion of service users in the research process. It would seem timely for the profession to look again at participatory research as an approach to working collaboratively with service users in the co-production of research. This article reviews the arguments for service-user collaboration in social work research; it considers the evolution of service-user engagement and its current status in practice. Building on the foundations of social work research methodologies, the article considers the practicalities of participatory research and the potential barriers. The article draws on vignettes of published participatory research to illustrate this type of research in social work

Specific compounds enhance autophagy or inflammatory cytokine release.

<p>(A) J774 murine macrophages were infected with H37Rv, then treated with the indicated compounds. Cells were harvested for Western-blot analysis of LC3 conversion from LC3-I to LC3-II as an indicator of autophagy. Data represents one of three independent experiments. Densitometry for LC3-II to LC3-I ratio for the shown Western blot was performed using ImageJ image analysis software. (B) Cells were infected with H37Rv, then treated with compound after a 4 hour phagocytosis. Supernatants were collected at 24 hours after infection and TNF-α concentration was determined by ELISA. Points represent average for 2 wells +/− standard deviation. Data represents one of two independent experiments.</p

Selected screen hits have dose-dependent activity in J774 cells and bone-marrow derived macrophages.

<p>Selected hit compounds were retested at varying doses in (A) J774 murine macrophages and (B) mouse bone marrow-derived macrophages (BMDM). Cells were infected with <i>M. tuberculosis</i> strain H37Rv at an MOI of 1∶1 and treated with each compound at the indicated doses (µM) after a 4 h phagocytosis period. At day 3 (J774) or day 5 (BMDM) after infection, cells were washed, lysed, and plated for CFU. Each column represents the mean and standard deviation of four biological replicates. Each experiment was repeated three times and a representative experiment is shown. With the exception of fluoxetine at 6.25 µM, all p-values were <0.05 for the comparison of each compound treatment condition with DMSO. p-values were calculated using the Mann Whitney U test.</p

AKT and ABL are important for mycobacterial infection of macrophages.

<p>(A) J774 cells were infected with H37Rv at an MOI of 1 or an MOI of 10. Cells were harvested two hours after infection, and lysates were probed for AKT activation using an antibody to phospho-serine at position 473. (B) J774 cells were infected with H37Rv at an MOI of 1 for 4 hours, then washed and treated with compound. Cells were harvested at 3 hours after treatment and lysates were probed for AKT activation using an antibody to phospho-serine at position 473. (C) <i>akt1</i> and <i>akt2</i> or <i>akt1</i>, <i>akt2</i>, and <i>akt3</i> were silenced with siRNA in J774 cells. Cells were then infected with H37Rv at an MOI and 1, and infection was allowed to progress for 3 days. Cells were then lysed and plated for CFU. (D) <i>abl1</i> was silenced in J774 macrophages using siRNA. Cells were then infected with H37Rv. After 4 hours of phagocytosis, wells were lysed and plated for CFU to determine uptake (day 0). Infection was allowed to progress in the remaining wells; day 3 after infection, cells were lysed and plated for CFU. Each experiment was repeated a minimum of three times, and a representative experiment is shown. Error bars are standard deviation, *p = 0.0195 by Mann Whitney U for (C) and (D).</p

Inhibitors of protein kinases impair mycobacterial growth in macrophages.

<p>(A) J774 cells or (B) BMDM were infected with <i>M. tuberculosis</i> strain H37Rv at an MOI of 1∶1, and treated with each kinase inhibitor at the indicated concentrations (µM) after a 4 h phagocytosis period. At day 3 (J774) or day 5 (BMDM) after infection, cells were washed, lysed, and plated for CFU. Each column represents the mean and standard deviation of four biological replicates and each graph represents one of three independent experiments. For (A) all p<0.001 with the exception of GNF-2 at 2.5 µM, imatinib at 5 µM and gefitinib at 5 µM. For (B) inhibitors were used at the following concentrations: AKTi1/2 5 µM (p<0.03), H-89 5 µM (p<0.03), GNF-2 10 uM (not significant), imatinib 5 µM (p<0.03), gefitinib 5 µM (p<0.03), lapatinib 5 µM (p = <0.06). All p values were calculated using the Mann Whitney U test.</p