Fiction Fix 08

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Paired quantitative and qualitative assessment of the replication-competent HIV-1 reservoir and comparison with integrated proviral DNA

HIV-1-infected individuals harbor a latent reservoir of infected CD4⁺ T cells that is not eradicated by antiretroviral therapy (ART). This reservoir presents the greatest barrier to an HIV-1 cure and has remained difficult to characterize, in part, because the vast majority of integrated sequences are defective and incapable of reactivation. To characterize the replication-competent reservoir, we have combined two techniques, quantitative viral outgrowth and qualitative sequence analysis of clonal outgrowth viruses. Leukapheresis samples from four fully ART-suppressed, chronically infected individuals were assayed at two time points separated by a 4- to 6-mo interval. Overall, 54% of the viruses emerging from the latent reservoir showed gp160 env sequences that were identical to at least one other virus. Moreover, 43% of the env sequences from viruses emerging from the reservoir were part of identical groups at the two time points. Groups of identical expanded sequences made up 54% of proviral DNA, and, as might be expected, the sequences of replication-competent viruses in the active reservoir showed limited overlap with integrated proviral DNA, most of which is known to represent defective viruses. Finally, there was an inverse correlation between proviral DNA clone size and the probability of reactivation, suggesting that replication-competent viruses are less likely to be found among highly expanded provirus-containing cell clones

Use of Dried Blood Spots to Elucidate Full-Length Transmitted/Founder HIV-1 Genomes.

BACKGROUND: Identification of HIV-1 genomes responsible for establishing clinical infection in newly infected individuals is fundamental to prevention and pathogenesis research. Processing, storage, and transportation of the clinical samples required to perform these virologic assays in resource-limited settings requires challenging venipuncture and cold chain logistics. Here, we validate the use of dried-blood spots (DBS) as a simple and convenient alternative to collecting and storing frozen plasma. METHODS: We performed parallel nucleic acid extraction, single genome amplification (SGA), next generation sequencing (NGS), and phylogenetic analyses on plasma and DBS. RESULTS: We demonstrated the capacity to extract viral RNA from DBS and perform SGA to infer the complete nucleotide sequence of the transmitted/founder (TF) HIV-1 envelope gene and full-length genome in two acutely infected individuals. Using both SGA and NGS methodologies, we showed that sequences generated from DBS and plasma display comparable phylogenetic patterns in both acute and chronic infection. SGA was successful on samples with a range of plasma viremia, including samples as low as 1,700 copies/ml and an estimated ∼50 viral copies per blood spot. Further, we demonstrated reproducible efficiency in gp160 env sequencing in DBS stored at ambient temperature for up to three weeks or at -20°C for up to five months. CONCLUSIONS: These findings support the use of DBS as a practical and cost-effective alternative to frozen plasma for clinical trials and translational research conducted in resource-limited settings

HER-2, p53, p21 and hormonal receptors proteins expression as predictive factors of response and prognosis in locally advanced breast cancer treated with neoadjuvant docetaxel plus epirubicin combination

BACKGROUND: Neoadjuvant chemotherapy has been considered the standard care in locally advanced breast cancer. However, about 20% of the patients do not benefit from this clinical treatment and, predictive factors of response were not defined yet. This study was designed to evaluate the importance of biological markers to predict response and prognosis in stage II and III breast cancer patients treated with taxane and anthracycline combination as neoadjuvant setting. METHODS: Sixty patients received preoperative docetaxel (75 mg/m(2)) in combination with epirubicin (50 mg/m(2)) in i.v. infusion in D1 every 3 weeks after incisional biopsy. They received adjuvant chemotherapy with CMF or FEC, attaining axillary status following definitive breast surgery. Clinical and pathologic response rates were measured after preoperative therapy. We evaluated the response rate to neoadjuvant chemotherapy and the prognostic significance of clinicopathological and immunohistochemical parameters (ER, PR, p51, p21 and HER-2 protein expression). The median patient age was 50.5 years with a median follow up time 48 months after the time of diagnosis. RESULTS: Preoperative treatment achieved clinical response in 76.6% of patients and complete pathologic response in 5%. The clinical, pathological and immunohistochemical parameters were not able to predict response to therapy and, only HER2 protein overexpression was associated with a decrease in disease free and overall survival (P = 0.0007 and P = 0.003) as shown by multivariate analysis. CONCLUSION: Immunohistochemical phenotypes were not able to predict response to neoadjuvant chemotherapy. Clinical response is inversely correlated with a risk of death in patients submitted to neoadjuvant chemotherapy and HER2 overexpression is the major prognostic factor in stage II and III breast cancer patients treated with a neoadjuvant docetaxel and epirubicin combination

A Proteomic Approach to Study the Effect of Thiotaurine on Human Neutrophil Activation

Thiotaurine, a thiosulfonate related to taurine and hypotaurine, is formed by a metabolic process from cystine and generated by a transulfuration reaction between hypotaurine and thiocysteine. Thiotaurine can produce hydrogen sulfide (H2S) from its sulfane sulfur moiety. H2S is a gaseous signaling molecule which can have regulatory roles in inflammatory process. In addition, sulfane sulfur displays the capacity to reversibly bind to other sulfur atoms. Thiotaurine inhibits PMA-induced activation of human neutrophils, and hinders neutrophil spontaneous apoptosis. Here, we present the results of a proteomic approach to study the possible effects of thiotaurine at protein expression level. Proteome analysis of human neutrophils has been performed comparing protein extracts of resting or PMA-activated neutrophils in presence or in absence of thiotaurine. In particular, PMA-stimulated neutrophils showed high level of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) expression compared to the level of the same glycolytic enzyme in the resting neutrophils. Conversely, decreased expression of GAPDH has been observed when human neutrophils were incubated with 1 mM thiotaurine before activation with PMA. This result, confirmed by Western blot analysis, suggests again that thiotaurine shows a bioactive role in the mechanisms underlying the inflammatory process, influencing the energy metabolism of activated leukocytes and raises the possibility that thiotaurine, acting as a sulfur donor, could modulate neutrophil activation via persulfidation of target proteins, such as GAPDH

High Multiplicity Infection by HIV-1 in Men Who Have Sex with Men

Elucidating virus-host interactions responsible for HIV-1 transmission is important for advancing HIV-1 prevention strategies. To this end, single genome amplification (SGA) and sequencing of HIV-1 within the context of a model of random virus evolution has made possible for the first time an unambiguous identification of transmitted/founder viruses and a precise estimation of their numbers. Here, we applied this approach to HIV-1 env analyses in a cohort of acutely infected men who have sex with men (MSM) and found that a high proportion (10 of 28; 36%) had been productively infected by more than one virus. In subjects with multivariant transmission, the minimum number of transmitted viruses ranged from 2 to 10 with viral recombination leading to rapid and extensive genetic shuffling among virus lineages. A combined analysis of these results, together with recently published findings based on identical SGA methods in largely heterosexual (HSX) cohorts, revealed a significantly higher frequency of multivariant transmission in MSM than in HSX [19 of 50 subjects (38%) versus 34 of 175 subjects (19%); Fisher's exact p = 0.008]. To further evaluate the SGA strategy for identifying transmitted/founder viruses, we analyzed 239 overlapping 5′ and 3′ half genome or env-only sequences from plasma viral RNA (vRNA) and blood mononuclear cell DNA in an MSM subject who had a particularly well-documented virus exposure history 3–6 days before symptom onset and 14–17 days before peak plasma viremia (47,600,000 vRNA molecules/ml). All 239 sequences coalesced to a single transmitted/founder virus genome in a time frame consistent with the clinical history, and a molecular clone of this genome encoded replication competent virus in accord with model predictions. Higher multiplicity of HIV-1 infection in MSM compared with HSX is consistent with the demonstrably higher epidemiological risk of virus acquisition in MSM and could indicate a greater challenge for HIV-1 vaccines than previously recognized