RESUMEN
The establishment of latent infection and poorly characterized viral reservoirs in tissues represent major obstacles to a definitive cure for HIV. Non-human primate (NHP) models of HIV infection are critical to elucidate pathogenic processes and an essential tool to test novel therapeutic strategies. Thus, the availability of novel assays to measure residual viral replication and reservoirs in NHP models may increase their utility in the search for an HIV cure. We developed a tat/rev induced limiting dilution assay to measure the frequency of CD4+ T cells that express multiply-spliced(ms)_SIV RNA in presence and absence of stimulation. We validated the assay using cell lines and cells from blood and lymph nodes of SIV infected macaques. In vitro, SIV/SHIV TILDA detects only cells expressing viral proteins. In SIV/SHIV-infected macaques, CD4+ T cells that express msSIV/SHIV RNA (TILDA data) were detected also in the setting of very low/undetectable viremia. TILDA data were significantly higher after stimulation and correlated with plasma viral load (pVL). Interestingly, TILDA data from early cART initiation correlated with peak and AUC pVL post-cART interruption. In summary, we developed an assay that may be useful in characterizing viral reservoirs and determining the effect of HIV interventions in NHP models.
Asunto(s)
Infecciones por VIH/genética , VIH-1/genética , Síndrome de Inmunodeficiencia Adquirida del Simio/genética , Virus de la Inmunodeficiencia de los Simios/genética , Animales , Linfocitos T CD4-Positivos/virología , Modelos Animales de Enfermedad , Infecciones por VIH/patología , Infecciones por VIH/virología , VIH-1/patogenicidad , Humanos , Macaca mulatta/virología , Macrófagos/metabolismo , Macrófagos/virología , Primates/virología , ARN Viral/genética , Síndrome de Inmunodeficiencia Adquirida del Simio/patología , Síndrome de Inmunodeficiencia Adquirida del Simio/virología , Virus de la Inmunodeficiencia de los Simios/patogenicidad , Carga Viral/genética , Latencia del Virus/genética , Replicación Viral/genética , Productos del Gen rev del Virus de la Inmunodeficiencia Humana/genética , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/genéticaRESUMEN
A complete set (YES, NOT, AND, and ANDNOT) of molecular scale logic gates based on ligase deoxyribozymes was constructed. The activity of these gates was visualized through the formation of cascades with downstream phosphodieseterase YES gates, which performed fluorogenic cleavage.
Asunto(s)
ADN Ligasas/química , ADN Catalítico/química , Lógica , Secuencia de Bases , Catálisis , ADN Ligasas/metabolismo , ADN Catalítico/metabolismo , Datos de Secuencia Molecular , Oligonucleótidos/química , Oligonucleótidos/metabolismoRESUMEN
We report herein a novel system for the parallel processing of molecular recognition events utilizing arrays of oligonucleotide-based fluorescent sensors to characterize hydrophobic molecules in solution. The binding domains of the sensors were based on three-way junctions that utilize double helical stems as framework regions to reliably fold regardless of variations in or around the binding domain. A reporting domain was introduced by the specific substitution of a single phosphodiester group with a phosphorothioate, followed by selective functionalization with a fluorophore. The sensors were organized into cross-reactive arrays to yield characteristic fingerprints for samples containing hydrophobic molecules. The fingerprints can be used to characterize steroids in solution, including complex biologically important fluids. Arrays have the potential for clinical applications such as the detection of gross errors in steroidogenesis.