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S100A8 and S100A9 are members of the Alarmin family; these proteins are abundantly expressed in neutrophils, form a heterodimer complex, and are secreted in plasma on pathogen infection or acute inflammatory diseases. Recently, both proteins were identified as novel biomarkers of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and were shown to play key roles in inducing an aggressive inflammatory response by mediating the release of large amounts of pro-inflammatory cytokines, called the "cytokine storm." Although co-infection with SARS-CoV-2 in people living with HIV-1 may result in an immunocompromised status, the role of the S100A8/A9 complex in HIV-1 replication in primary T cells and macrophages is still unclear. Here, we evaluated the roles of the proteins in HIV replication to elucidate their functions. We found that the complex had no impact on virus replication in both cell types; however, the subunits of S100A8 and S100A9 inhibit HIV in macrophages. These findings provide important insights into the regulation of HIV viral loads during SARS-CoV-2 co-infection.

S100A8 and S100A9 are members of the Alarmin family; these proteins are abundantly expressed in neutrophils, form a heterodimer complex, and are secreted in plasma on pathogen infection or acute inflammatory diseases. Recently, both proteins were identified as novel biomarkers of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and were shown to play key roles in inducing an aggressive inflammatory response by mediating the release of large amounts of pro-inflammatory cytokines, called the “cytokine storm.” Although co-infection with SARS-CoV-2 in people living with HIV-1 may result in an immunocompromised status, the role of the S100A8/A9 complex in HIV-1 replication in primary T cells and macrophages is still unclear. Here, we evaluated the roles of the proteins in HIV replication to elucidate their functions. We found that the complex had no impact on virus replication in both cell types; however, the subunits of S100A8 and S100A9 inhibit HIV in macrophages. These findings provide important insights into the regulation of HIV viral loads during SARS-CoV-2 co-infection.

Cytosolic foreign DNA is detected by pattern recognition receptors and mainly induces type I IFN production. We found that transfection of different types of DNA into various untreated cells induces type III IFN (IFN-λ1) rather than type I IFN, indicating the presence of uncharacterized DNA sensor(s). A pull-down assay using cytosolic proteins identified that Ku70 and Ku80 are the DNA-binding proteins. The knockdown studies and the reporter assay revealed that Ku70 is a novel DNA sensor inducing the IFN-lambda1 activation. The functional analysis of IFNL1 promoter revealed that positive-regulatory domain I and IFN-stimulated response element sites are predominantly involved in the DNA-mediated IFNL1 activation. A pull-down assay using nuclear proteins demonstrated that the IFN-λ1 induction is associated with the activation of IFN regulatory factor-1 and -7. Thus, to our knowledge, we show for the first time that Ku70 mediates type III IFN induction by DNA.

Cilia on the ventral surface of the hypotrich ciliate Euplotes are clustered into polykinetids or compound ciliary organelles, such as cirri or oral membranelles, used in locomotion and prey capture. A single polykinetid may contain more than 150 individual cilia; these emerge from basal bodies held in a closely spaced array within a scaffold or framework structure that has been referred to as a basal-body "cage". Cage structures were isolated free of cilia and basal bodies; the predominant component of such cages was found on polyacrylamide gels to be a 45-kDa polypeptide. Antisera were raised against this protein band and used for immunolocalizations at the light and electron microscope levels. Indirect immunofluorescence revealed the 45-kDa polypeptide to be localized exclusively to the bases of the ventral polykinetids. Immunogold staining of thin sections of intact cells further localized this reactivity to filaments of a double-layered dense lattice that appears to link adjoining basal bodies into ordered arrays within each polykinetid. Scanning electron microscopy of isolated cages reveals the lower or "basal" cage layer to be a fine lacey meshwork supporting the basal bodies at their proximal ends; adjoining basal bodies are held at their characteristic spacing by filaments of an upper or "medial" cage layer. The isolated cage thus resembles a miniature test-tube rack, able to accommodate varying arrangements of basal-body rows, depending on the particular type of polykinetid. Because of its clear and specific localization to the basal-body cages in Euplotes, we have termed this novel 45-kDa protein "cagein".

Objective: IL-27 is a novel anti-HIV cytokine that inhibits HIV-1 replication in both CD4 T cells and monocyte-derived macrophages (MDM) as IFN-a does. To elucidate the mechanism of the antiviral activity, we compared the activity and the gene expression profile of IL-27-treated cells with that of IFN-a-treated cells. Methods: CD4 T cells and monocytes were isolated from peripheral blood mononuclear cells of healthy donors. CD4 T cells were stimulated with phytohemagglutinin, and MDM were induced from monocytes using macrophage-colony stimulating factor. HIV-1 replication was monitored by p24 antigen capture assay. The gene expression profiles were analysed using DNA microarray analysis. The increase in the expression of IFN-inducible genes (IFIG) was confirmed by the Quantigene plex assay. Results: Both cytokines preferentially inhibited HIV-1 replication in MDM compared with CD4 T cells. Quantitative real time polymerase chain reaction, enzyme-linked immunosorbent assay and neutralization assay using anti-IFN indicated that IFN-a, IFNb and IFN-g had no significant impact on IL-27-mediated HIV inhibition. DNA microarray analysis illustrated that IFN-a induced 33 and 18 IFIG in MDM and CD4 T cells, respectively. IL-27 induced 28 IFIG in MDM and five IFIG in CD4 T cells. The quantitative assay confirmed that IL-27 activated genes of RNA-dependent kinase, oligoadenylate synthetase, myxovirus protein, and apolipoprotein B messenger RNA-editing enzyme-catalytic polypeptide-like 3G. Conclusion: IL-27 differentially regulates the gene expression between CD4 T cells and MDM. IL-27 significantly induces antiviral genes in MDM as does IFN-a, suggesting that IL-27 inhibits HIV replication in MDM via mechanism(s) similar to that of IFN-a. 2008 Wolters Kluwer Health | Lippincott Williams & Wilkins AIDS 2008, 22:39‐45

The existence of viral latency limits the success of highly active antiretroviral therapy. With the therapeutic intention of reactivating latent virus to induce a cure, in this study we assessed the impact of cell synchronizers on HIV gene activation in latently infected U1 cells and investigated the molecular mechanisms responsible for such effect. Latently infected U1 cells were treated with 10 drugs including hydroxyurea (HU) and HIV-1 replication monitored using a p24 antigen capture assay. We found that HU was able to induce HIV-1 replication by 5-fold. HU has been used in the clinical treatment of HIV-1-infected patients in combination with didanosine; therefore, we investigated the impact of HU on HIV-1 activation in the presence of the proinflammatory cytokines, interleukin 6 (IL-6) and tumor necrosis factor-alpha (TNF-alpha). IL-6 or TNF-alpha alone induced HIV replication by 18- and approximately 500-fold, respectively. Of interest, in the presence of HU, IL-6-mediated HIV-1 activation was enhanced by90-fold, whereas TNF-alpha-mediated activation was inhibited by30%. A reporter gene assay showed that HU and IL-6 synergized to activate HIV promoter activity via the Sp1 binding site. Electrophoretic mobility shift and supershift assays revealed increased binding of the Sp1 and Sp3 transcription factors to this region. Western blot analysis showed that HU and IL-6 co-stimulation resulted in increased levels of Sp1 and Sp3 proteins. In contrast, treatment with HU plus TNF-alpha down-regulated the expression of NF-kappaB. These findings suggest that Sp1/Sp3 is involved in controlling the HU/IL-6-induced reactivation of HIV-1 in latently infected cells.

In order to study the mechanisms responsible for resistance to CDDP, 5 human tumor cell lines were made resistant to CDDP by repeated in vitro exposures. After cloning it was found that the cell lines developed were between 3.3-fold and 17-fold more resistant to CDDP than the parental cell lines at the IC90. These lines were also resistant to carboplatin and tetraplatin; however, resistance to tetraplatin was lower than to the other platinum complexes. Sensitivity was also assessed to Adria, MTX, 5-FU, chlorambucil, 4-HC, 4-HIF, BCNU, Thiotepa, HN2, Mito C and L-PAM, and no consistent cross-resistance was observed. As compared with the parental lines, non-protein sulfhydryl content was elevated in 3 resistant lines, and protein sulfhydryl was elevated in all 5 lines, as was glutathione-S-transferase activity. Measurements of platinum in whole cells and nuclei after exposure of the cultures to 25 microM CDDP for either 1 or 6 hr showed that nuclear levels reflected those in whole cells and that, per mg protein, platinum levels were lower in resistant cells at both time points. Formation of DNA cross-links, determined by alkaline elution, was lower in resistant cell lines than in parental cell lines, but did not correlate with the absolute cell kill observed. These results indicate that cellular resistance to CDDP often involves decreases in drug accumulation and increases in protein sulfhydryl content. Possible strategies for overcoming these mechanisms are discussed.

We examined the ability of lonidamine, which has been described as an inhibitor of cellular respiration and glycolysis, to enhance the cytotoxicity of alkylating agents to MCF-7 human breast-carcinoma cells. Lonidamine was increasingly cytotoxic to MCF-7 cells with increasing time of exposure. With a 12-h exposure, the IC50 for lonidamine was about 365 microM, and with a 24-h exposure it was about 170 microM. A drug concentration of 250 microM was chosen for use in the drug combination studies. Lonidamine appeared to have a dose-modifying effect on cisplatin (CDDP), producing increasingly supra-additive cell kill with increasing CDDP concentration. When simultaneously incubated with lonidamine for 1 h, 500 microM CDDP yielded a cell kill that was 2 log greater than additive cytotoxicity. Extending the exposure to lonidamine for 12 h after CDDP treatment led to a small, additional aliquot of cell kill of about 2.5-fold over the CDDP concentration range. Lonidamine also appeared to have a dose-modifying effect on melphalan cytotoxicity in the melphalan concentration range of 100-500 microM. Between concentrations of 10 and 100 microM melphalan, the drug combination survival after 1 h exposure fell within the envelope of additivity for the two agents. However, maintaining the presence of lonidamine for an additional 12 h increased the effect such that the combination was supra-additive over the entire concentration range of melphalan. Simultaneous exposure to 4-hydroperoxycyclophosphamide (4-HC) and lonidamine for 1 h resulted in greater than additive cell kill, and extending the lonidamine exposure period such that lonidamine was present during and 12 h after 4-HC treatment further increased this effect. Lonidamine had a moderate effect on the cytotoxicity of carmustine (BCNU) with a 1 h simultaneous exposure; however, this treatment combination reached greater than additive cytotoxicity only at the highest concentration of BCNU tested. Extending the lonidamine exposure time for an additional 12 h resulted in supra-additive cell kill over the BCNU concentration range. Therefore, when lonidamine was present during exposure to the alkylating agent and its presence was then extended for an additional 12 h, a synergistic cell kill was produced with all four alkylating agents tested.