Therapeutic strategies designed to combat HIV/AIDS by siRNAs show considerable promise, but targeted delivery of these synthetic molecules into virus infected cells in vivo has been a formidable challenge. In addressing this need, using a humanized mouse model, we sought to evaluate the in vivo efficacy of a chimeric construct consisting of an HIV-1 gp120 directed aptamer with viral neutralization capacity fused to an siRNA with proven efficacy against tat/rev viral transcripts. Humanized Rag2-/- γc-/- (RAG-hu) mice with multilineage hematopoiesis were prepared by engrafting with human CD34+ hematopoietic progenitor cells. These animals were infected with HIV-1 NL4-3 virus to generate viremic mice. Subsequently, the viremic mice were injected intravenously with the aptamer-siRNA chimeras or control RNAs. The viral loads and CD4+ T-cell levels were monitored weekly. Our results show that both the aptamer and aptamer-siRNA chimera treatments markedly suppressed HIV-1 replication and prevented viral induced CD4 T-cell depletion. The addition of the siRNA to the aptamer added to the antiviral effect by triggering target specific knockdown of the HIV-1 encoded tat/rev transcripts, thus validating the dual inhibitory function of the chimeras. Moreover, the presence of siRNAs and target specific knockdown of HIV-1 tat/rev transcripts were detected in gp120 aptamer-siRNA chimera treated mice, but not in the aptamer alone-, mutated aptamer-siRNA chimeras- or naked siRNA-treated animal groups, thus validating systemic, HIV-1 infected cell specific aptamer mediated siRNA delivery. Using RACE PCR we verified the in vivo RNAi mediated cleavage of the HIV-1 tat/rev target transcripts. Collectively, this approach resulted in suppression of viral loads in vivo and most importantly resulted in protection of T-cell depletion, making the aptamer-siRNA chimeras attractive therapeutic candidates for the treatment of HIV-1 infection.
The widespread availability of antiretroviral therapy (ART) for people living with HIV (PLWH) has dramatically reduced mortality and improved life expectancy. However, even with suppression of HIV-1 replication, chronic immune activation and elevated inflammation persist. Chronic immune activation has been linked to a pro-inflammatory gut microbiome composition, exacerbated by compromised intestinal barrier integrity that occurs after HIV infection. Individuals living in urban versus rural areas of sub-Saharan Africa have differences in environmental factors such as water source or diet that may impact gut microbiome composition, yet immune phenotype and gut microbiome composition response to ART in PLWH living in rural versus urban areas of sub-Saharan Africa have not been compared. Here, we measured immune phenotypes and fecal microbiome composition in PLWH and healthy participants recruited from the urban Mabvuku polyclinic in the city of Harare, Zimbabwe and the Mutoko District hospital located in a district 146 km from Harare that services surrounding rural villages. PLWH were either ART naïve at baseline and sampled again after 24 weeks of treatment with efavirenz/lamivudine/tenofovir disoproxil fumarate (EFV/3TC/TDF) and the prophylactic antibiotic cotrimoxazole or were ART experienced at both timepoints. Although expected reductions in the inflammatory marker IL-6, T-cell activation, and exhaustion were observed in individuals who had suppressed HIV-1 with treatment, these changes were significant only when considering individuals in the urban and not the rural area. Gut microbiome composition showed more marked differences from healthy controls in the ART experienced compared to ART naïve cohort, and consistent longitudinal changes were also observed in ART naïve PLWH after 24 weeks of treatment, including a reduction in alpha diversity and altered composition. However, gut microbiome composition showed a more pronounced relationship with chronic immune activation and exhaustion phenotypes in the ART naïve compared to ART experienced PLWH, suggesting a particularly significant role for the gut microbiome in disease progression in uncontrolled infection.
ABSTRACT Varicella-zoster virus (VZV) vasculopathy produces stroke, giant cell arteritis, and granulomatous aortitis, and it develops after virus reactivates from ganglia and spreads transaxonally to arterial adventitia, resulting in persistent inflammation and pathological vascular remodeling. The mechanism(s) by which inflammatory cells persist in VZV-infected arteries is unknown; however, virus-induced dysregulation of programmed death ligand 1 (PD-L1) may play a role. Specifically, PD-L1 can be expressed on virtually all nucleated cells and suppresses the immune system by interacting with the programmed cell death protein receptor 1, found exclusively on immune cells; thus, downregulation of PD-L1 may promote inflammation, as seen in some autoimmune diseases. Both flow cytometry and immunofluorescence analyses to test whether VZV infection of adventitial cells downregulates PD-L1 showed decreased PD-L1 expression in VZV-infected compared to mock-infected human brain vascular adventitial fibroblasts (HBVAFs), perineural cells (HPNCs), and fetal lung fibroblasts (HFLs) at 72 h postinfection. Quantitative RT-PCR analyses showed no change in PD-L1 transcript levels between mock- and VZV-infected cells, indicating a posttranscriptional mechanism for VZV-mediated downregulation of PD-L1. Flow cytometry analyses showed decreased major histocompatibility complex class I (MHC-I) expression in VZV-infected cells and adjacent uninfected cells compared to mock-infected cells. These data suggest that reduced PD-L1 expression in VZV-infected adventitial cells contribute to persistent vascular inflammation observed in virus-infected arteries from patients with VZV vasculopathy, while downregulation of MHC-I prevents viral clearance. IMPORTANCE Here, we provide the first demonstration that VZV downregulates PD-L1 expression in infected HBVAFs, HPNCs, and HFLs, which, together with the noted VZV-mediated downregulation of MHC-I, might foster persistent inflammation in vessels, leading to pathological vascular remodeling during VZV vasculopathy and persistent inflammation in infected lungs to promote subsequent infection of T cells and hematogenous virus spread. Identification of a potential mechanism by which persistent inflammation in the absence of effective viral clearance occurs in VZV vasculopathy and VZV infection of the lung is a step toward targeted therapy of VZV-induced disease.
We describe an analysis of speech during time-critical, team-based medical work and its potential to indicate process delays. We analyzed speech intention and sentence types during 39 trauma resuscitations with delays in one of three major lifesaving interventions: intravenous/intraosseous (IV/IO) line insertion, cardiopulmonary and resuscitation (CPR), and intubation. We found a significant difference in patterns of speech during delays vs. speech during non-delayed work. The speech intention during CPR delays, however, differed from the other LSIs, suggesting that context of speech must be considered. These findings will inform the design of a clinical decision support system (CDSS) that will use multiple sensor modalities to alert medical teams to delays in real time. We conclude with design implications and challenges associated with speech-based activity recognition in complex medical processes.
Table S10. Full list of diet differences in MSM compared to non-MSM un-normalized data. Diet components significantly increased with MSM highlighted in orange OTUs significantly decreased in MSM highlighted in green (Kruskal-Wallis test). (XLSX 18 kb)
The envelope glycoprotein of human immunodeficiency virus (HIV) consists of an exterior glycoprotein (gp120) and a trans-membrane domain (gp41) and has an important role in viral entry into cells. HIV-1 entry has been validated as a clinically relevant anti-viral strategy for drug discovery. In the present work, several 2'-F substituted RNA aptamers that bind to the HIV-1(BaL) gp120 protein with nanomole affinity were isolated from a RNA library by the SELEX (Systematic Evolution of Ligands by EXponential enrichment) procedure. From two of these aptamers we created a series of new dual inhibitory function anti-gp120 aptamer-siRNA chimeras. The aptamers and aptamer-siRNA chimeras specifically bind to and are internalized into cells expressing HIV gp160. The Dicer-substrate siRNA delivered by the aptamers is functionally processed by Dicer, resulting in specific inhibition of HIV-1 replication and infectivity in cultured CEM T-cells and primary blood mononuclear cells (PBMCs). Moreover, we have introduced a 'sticky' sequence onto a chemically synthesized aptamer which facilitates attachment of the Dicer substrate siRNAs for potential multiplexing. Our results provide a set of novel inhibitory agents for blocking HIV replication and further validate the use of aptamers for delivery of Dicer substrate siRNAs.
Table S6. Table of significantly different OTUs between only HIV-negative MSM and non-MSM. OTUs significantly increased with MSM highlighted in orange OTUs significantly decreased in MSM highlighted in green (Kruskal-Wallis test). (XLSX 39 kb)
