A Subset of Latency-Reversing Agents Expose HIV-Infected Resting CD4⁺ T-Cells to Recognition by Cytotoxic T-Lymphocytes

Resting CD4⁺ T-cells harboring inducible HIV proviruses are a critical reservoir in antiretroviral therapy (ART)-treated subjects. These cells express little to no viral protein, and thus neither die by viral cytopathic effects, nor are efficiently cleared by immune effectors. Elimination of this reservoir is theoretically possible by combining latency-reversing agents (LRAs) with immune effectors, such as CD8⁺ T-cells. However, the relative efficacy of different LRAs in sensitizing latently-infected cells for recognition by HIV-specific CD8⁺ T-cells has not been determined. To address this, we developed an assay that utilizes HIV-specific CD8⁺ T-cell clones as biosensors for HIV antigen expression. By testing multiple CD8⁺ T-cell clones against a primary cell model of HIV latency, we identified several single agents that primed latently-infected cells for CD8⁺ T-cell recognition, including IL-2, IL-15, two IL-15 superagonists (IL-15SA and ALT-803), prostratin, and the TLR-2 ligand Pam₃CSK₄. In contrast, we did not observe CD8⁺ T-cell recognition of target cells following treatment with histone deacetylase inhibitors or with hexamethylene bisacetamide (HMBA). In further experiments we demonstrate that a clinically achievable concentration of the IL-15 superagonist ‘ALT-803’, an agent presently in clinical trials for solid and hematological tumors, primes the natural ex vivo reservoir for CD8⁺ T-cell recognition. Thus, our results establish a novel experimental approach for comparative evaluation of LRAs, and highlight ALT-803 as an LRA with the potential to synergize with CD8⁺ T-cells in HIV eradication strategies.United States. National Institutes of Health (AI111860

A Randomized Clinical Trial of Nutrition Education for Improvement of Diet Quality and Inflammation in Iranian Obese Women

Background. Obesity is considered as a low grade inflammation condition. The aim of this study was to investigate the effect of nutritional education on diet quality and biomarkers of inflammation in Iranian obese women. Method. Sixty obese women voluntarily participated in this randomized clinical trial and were randomly assigned to intervention or control group (n=30). Intervention group was instructed to attend nutrition education sessions (1 hr/wk, for 3 months) in small groups. Diet quality scores were measured by Healthy Eating Index (HEI). Anthropometric indices and serum concentration of hs-CRP, TNF-α, and adiponectin were measured at the baseline and end of the intervention. Results. There were no significant differences in anthropometric indices of participants between the two groups at the end of intervention (P>0.05). However, the total HEI score was significantly higher in the educated group compared to the control group after intervention (P<0.05). The educated group also showed significant lower concentration of TNF-α and hs-CRP and higher levels of adiponectin than the control group at the end of study (P<0.05). Conclusions. Our results provide limited evidence that higher dietary quality contributes to reduced inflammation in obese women. This effect could be independent of the weight loss

Stimulation of Liver X Receptor Has Potent Anti-HIV Effects in a Humanized Mouse Model.

Previous studies demonstrated that liver X receptor (LXR) agonists inhibit human immunodeficiency virus (HIV) replication by upregulating cholesterol transporter ATP-binding cassette A1 (ABCA1), suppressing HIV production, and reducing infectivity of produced virions. In this study, we extended these observations by analyzing the effect of the LXR agonist T0901317 [N-[4-(1,1,1,3,3,3-hexafluoro-2-hydroxypropan-2-yl)phenyl]-N-(2,2,2-trifluoroethyl)benzenesulfonamide] on the ongoing HIV infection and investigating the possibility of using LXR agonist for pre-exposure prophylaxis of HIV infection in a humanized mouse model. Pre-exposure of monocyte-derived macrophages to T0901317 reduced susceptibility of these cells to HIV infection in vitro. This protective effect lasted for up to 4 days after treatment termination and correlated with upregulated expression of ABCA1, reduced abundance of lipid rafts, and reduced fusion of the cells with HIV. Pre-exposure of peripheral blood leukocytes to T0901317 provided only a short-term protection against HIV infection. Treatment of HIV-exposed humanized mice with LXR agonist starting 2 weeks postinfection substantially reduced viral load. When eight humanized mice were pretreated with LXR agonist prior to HIV infection, five animals were protected from infection, two had viral load at the limit of detection, and one had viral load significantly reduced relative to mock-treated controls. T0901317 pretreatment also reduced HIV-induced dyslipidemia in infected mice. In conclusion, these results reveal a novel link between LXR stimulation and cell resistance to HIV infection and suggest that LXR agonists may be good candidates for development as anti-HIV agents, in particular for pre-exposure prophylaxis of HIV infection

A Subset of Latency-Reversing Agents Expose HIV-Infected Resting CD4+ T-Cells to Recognition by Cytotoxic T-Lymphocytes.

Resting CD4+ T-cells harboring inducible HIV proviruses are a critical reservoir in antiretroviral therapy (ART)-treated subjects. These cells express little to no viral protein, and thus neither die by viral cytopathic effects, nor are efficiently cleared by immune effectors. Elimination of this reservoir is theoretically possible by combining latency-reversing agents (LRAs) with immune effectors, such as CD8+ T-cells. However, the relative efficacy of different LRAs in sensitizing latently-infected cells for recognition by HIV-specific CD8+ T-cells has not been determined. To address this, we developed an assay that utilizes HIV-specific CD8+ T-cell clones as biosensors for HIV antigen expression. By testing multiple CD8+ T-cell clones against a primary cell model of HIV latency, we identified several single agents that primed latently-infected cells for CD8+ T-cell recognition, including IL-2, IL-15, two IL-15 superagonists (IL-15SA and ALT-803), prostratin, and the TLR-2 ligand Pam3CSK4. In contrast, we did not observe CD8+ T-cell recognition of target cells following treatment with histone deacetylase inhibitors or with hexamethylene bisacetamide (HMBA). In further experiments we demonstrate that a clinically achievable concentration of the IL-15 superagonist \u27ALT-803\u27, an agent presently in clinical trials for solid and hematological tumors, primes the natural ex vivo reservoir for CD8+ T-cell recognition. Thus, our results establish a novel experimental approach for comparative evaluation of LRAs, and highlight ALT-803 as an LRA with the potential to synergize with CD8+ T-cells in HIV eradication strategies

CTLs Eliminate Defective HIV Proviruses Without Impacting Infectious Latent Reservoirs

Background: The “shock-and-kill” paradigm of combining latency-reversing agents (LRAs) to “shock” latent HIV reservoirs awake, then “kill” them with immune effectors is currently the predominant strategy in the field of HIV cure research. However, the majority of “shock-and-kill” studies have been performed using primary cell models of HIV latency, which are imperfect representations of natural viral reservoirs found in people living with HIV. Thus, a need remains for a rigorous investigation of the efficacy of this cure strategy in natural reservoirs. Here, we treat ex vivo CD4+ T-cells from HIV+ individuals on long-term ARV therapy (\u3e5 years), with LRAs and autologous HIV-specific CTL clones (targeting non-escaped epitopes), and assessed the impact on total and intact-inducible proviruses. Method: HIV-specific CTL clones targeting known HIV epitopes were isolated from ARV-treated subjects by limiting dilution, and killing activities were confirmed by flow cytometric assays. We developed an HIV eradication (HIVE) assay to test the abilities of these CTLs to reduce viral reservoirs in combination with HDACi’s, PKC activators, or an IL-15 super agonist with Pam3CSK4. In short, resting CD4+ T-cells from HIV+ leukapheresis samples are co-cultured with LRAs + CTLs for 5 days with ARVs, and activation/memory phenotypes are monitored. CD4+ T-cells are isolated after treatment, and total/intact-inducible reservoirs are measured by cell-associated HIV DNA (ddPCR) and by quantitative viral outgrowth assay (QVOA). Results: Combinations of bryostatin and IL-15SA+Pam3CSK4 with HIV-specific CTL generally led to significant decreases in cell-associated HIV DNA, with the greatest effects observed for bryostatin (up to 50% reductions, p \u3c 0.01). Critically, these decreases in HIV DNA were not associated with measurable reductions in intact-inducible virus, regardless of the CTL clone or LRA combination used (powered to detect ~50% reductions with 95% confidence). Even when combined with PMA/ionomycin, CTLs were unable to drive reductions in intact-inducible virus. CTLs degranulated (CD107a) in response to autologous activated CD4+ T-cells that had been infected with virus from positive QVOA wells, ruling out a role for immune escape in our observation. Conclusions: Recently, it has been demonstrated that some defective proviruses can be expressed as antigens, enabling CTL recognition. Data from our ex vivo experiments are consistent with the preferential depletion of defective proviruses by CTLs, leading to reductions in HIV DNA without impacting intact proviruses. Understanding and overcoming the mechanisms limiting CTL against the intact-inducible reservoir may be key to successful CTL-based shock and kill interventions

Extracellular MicroRNA Signature in Chronic Kidney Disease.

MicroRNAs (miRNAs) are noncoding RNAs that regulate posttranscriptional gene expression. In this study we characterized the circulating and urinary miRNA pattern associated with reduced glomerular filtration rate, using Affymetrix GeneChip miR 4.0 in 28 patients with chronic kidney disease (CKD). Top miRNA discoveries from the human studies were validated in an Alb/TGF beta mouse model of CKD, and in rat renal proximal tubular cells (NRK52E) exposed to TGF beta 1. Plasma and urinary levels of procollagen III N-terminal propeptide and collagen IV were elevated in patients with decreased estimated glomerular filtration rate (eGFR). Expression of 384 urinary and 266 circulatory miRNAs were significantly different between CKD patients with eGFR >= 30 vs. <30 ml.min(-1) . 1.73 m(-2). Pathway analysis mapped multiple miRNAs to TGF beta signaling-related mRNA targets. Specifically, Let-7a was significantly downregulated, and miR-130a was significantly upregulated, in urine of patients with eGFR <30; miR-1825 and miR-1281 were upregulated in both urine and plasma of patients with decreased eGFR; and miR-423 was significantly downregulated in plasma of patients with decreased eGFR. miRNA expression in urine and plasma of Alb/TGF beta mice generally resembled and confirmed most, although not all, of the observations from the human studies. In response to TGF beta 1 exposure, rat renal proximal tubular cells overexpressed miR-1825 and downregulated miR-423. Thus, miRNA are associated with kidney fibrosis, and specific urinary and plasma miRNA profile may have diagnostic and prognostic utility in CKD

Extracellular microRNA signature in chronic kidney disease

MicroRNAs (miRNAs) are noncoding RNAs that regulate posttranscriptional gene expression. In this study we characterized the circulating and urinary miRNA pattern associated with reduced glomerular filtration rate, using Affymetrix GeneChip miR 4.0 in 28 patients with chronic kidney disease (CKD). Top miRNA discoveries from the human studies were validated in an Alb/TGF beta mouse model of CKD, and in rat renal proximal tubular cells (NRK52E) exposed to TGF beta 1. Plasma and urinary levels of procollagen III N-terminal propeptide and collagen IV were elevated in patients with decreased estimated glomerular filtration rate (eGFR). Expression of 384 urinary and 266 circulatory miRNAs were significantly different between CKD patients with eGFR >= 30 vs. <30 ml.min(-1) . 1.73 m(-2). Pathway analysis mapped multiple miRNAs to TGF beta signaling-related mRNA targets. Specifically, Let-7a was significantly downregulated, and miR-130a was significantly upregulated, in urine of patients with eGFR <30; miR-1825 and miR-1281 were upregulated in both urine and plasma of patients with decreased eGFR; and miR-423 was significantly downregulated in plasma of patients with decreased eGFR. miRNA expression in urine and plasma of Alb/TGF beta mice generally resembled and confirmed most, although not all, of the observations from the human studies. In response to TGF beta 1 exposure, rat renal proximal tubular cells overexpressed miR-1825 and downregulated miR-423. Thus, miRNA are associated with kidney fibrosis, and specific urinary and plasma miRNA profile may have diagnostic and prognostic utility in CKD