Methylation regulation of Antiviral host factors, Interferon Stimulated Genes (ISGs) and T-cell responses associated with natural HIV control.

GWAS, immune analyses and biomarker screenings have identified host factors associated with in vivo HIV-1 control. However, there is a gap in the knowledge about the mechanisms that regulate the expression of such host factors. Here, we aimed to assess DNA methylation impact on host genome in natural HIV-1 control. To this end, whole DNA methylome in 70 untreated HIV-1 infected individuals with either high (>50,000 HIV-1-RNA copies/ml, n = 29) or low (<10,000 HIV-1-RNA copies/ml, n = 41) plasma viral load (pVL) levels were compared and identified 2,649 differentially methylated positions (DMPs). Of these, a classification random forest model selected 55 DMPs that correlated with virologic (pVL and proviral levels) and HIV-1 specific adaptive immunity parameters (IFNg-T cell responses and neutralizing antibodies capacity). Then, cluster and functional analyses identified two DMP clusters: cluster 1 contained hypo-methylated genes involved in antiviral and interferon response (e.g. PARP9, MX1, and USP18) in individuals with high viral loads while in cluster 2, genes related to T follicular helper cell (Tfh) commitment (e.g. CXCR5 and TCF7) were hyper-methylated in the same group of individuals with uncontrolled infection. For selected genes, mRNA levels negatively correlated with DNA methylation, confirming an epigenetic regulation of gene expression. Further, these gene expression signatures were also confirmed in early and chronic stages of infection, including untreated, cART treated and elite controllers HIV-1 infected individuals (n = 37). These data provide the first evidence that host genes critically involved in immune control of the virus are under methylation regulation in HIV-1 infection. These insights may offer new opportunities to identify novel mechanisms of in vivo virus control and may prove crucial for the development of future therapeutic interventions aimed at HIV-1 cure.

TL1A-DR3 Plasma Levels Are Predictive of HIV-1 Disease Control, and DR3 Costimulation Boosts HIV-1-Specific T Cell Responses.

Relative control of HIV-1 infection has been linked to genetic and immune host factors. In this study, we analyzed 96 plasma proteome arrays from chronic untreated HIV-1-infected individuals using the classificatory random forest approach to discriminate between uncontrolled disease (plasma viral load [pVL] >50,000 RNA copies/ml; CD4 counts 283 cells/mm3, n = 47) and relatively controlled disease (pVL <10,000 RNA copies/ml; CD4 counts 657 cells/mm3, n = 49). Our analysis highlighted the TNF molecule's relevance, in particular, TL1A (TNFSF15) and its cognate DR3 (TNFSRF25), both of which increased in the relative virus control phenotype. DR3 levels (in plasma and PBMCs) were validated in unrelated cohorts (including long-term nonprogressors), thus confirming their independence from CD4 counts and pVL. Further analysis in combined antiretroviral treatment (cART)-treated individuals with a wide range of CD4 counts (137-1835 cells/mm3) indicated that neither TL1A nor DR3 levels reflected recovery of CD4 counts with cART. Interestingly, in cART-treated individuals, plasma TL1A levels correlated with regulatory T cell frequencies, whereas soluble DR3 was strongly associated with the abundance of effector HLA-DR+CD8+ T cells. A positive correlation was also observed between plasma DR3 levels and the HIV-1-specific T cell responses. In vitro, costimulation of PBMC with DR3-specific mAb increased the magnitude of HIV-1-specific responses. Finally, in splenocytes of DNA.HTI-vaccinated mice, costimulation of HTI peptides and a DR3 agonist (4C12) intensified the magnitude of T cell responses by 27%. These data describe the role of the TL1A-DR3 axis in the natural control of HIV-1 infection and point to the use of DR3 agonists in HIV-1 vaccine regimens.

[Evaluation of the Sysmex UF-1000i flow cytometer for screening of urinary tract infection]. / Evaluación del citómetro UF-1000i como método de cribado en el diagnóstico de infecciones del tracto urinario.

INTRODUCTION: The urine culture is a huge workload in the Microbiology Laboratory and remains the gold standard for the diagnosis of urinary tract infections. Considering the high prevalence of negative results, the implementation of a reliable screening method could lead to cost saving in the workload, and speed up reporting of negative results. METHODS: We evaluated the usefulness of the flow cytometer UF-1000i in the screening for negative samples than could be excluded from culture. We divided the samples into two groups, Group 1, males and women of childbearing age who were considered positive with a growth ≥ 104 CFU/ml, and Group 2, considered positive with ≥ 105 CFU/ml growth. RESULTS: On comparing the culture and screening data in the ROC curve, the best sensitivity and specificity points were 53.1 bact/µl for Group 1, and 128.3 bact/µl for Group 2. In Group 1, the sensitivity was 92.2% and a specificity of 60%, a reduction in urine cultures of 46%, with 2.1% false negative (42 samples). In Group 2, the sensitivity was 86%, with a specificity of 87.7%, a culture reduction of 57.5%, and 5.1% false negatives (74 samples). CONCLUSION: The incorporating of the UF-1000i cytometer to the screening of urine samples depends on the characteristics of the patients and the definition of positive urine culture. In our case, with only studying bacteriuria, the data on the reduction of workload and the false negatives seriously question this incorporation.

Epigenetic landscape in the kick-and-kill therapeutic vaccine BCN02 clinical trial is associated with antiretroviral treatment interruption (ATI) outcome.

BACKGROUND: The BCN02-trial combined therapeutic vaccination with a viral latency reversing agent (romidepsin, RMD) in HIV-1-infected individuals and included a monitored antiretroviral pause (MAP) as an efficacy read-out identifying individuals with an early or late (< or > 4weeks) viral-rebound. Integrated -omics analyses were applied prior treatment interruption to identify markers of virus control during MAP. METHODS: PBMC, whole-genome DNA methylation and transcriptomics were assessed in 14 BCN02 participants, including 8 Early and 4 Late viral-rebound individuals. Chromatin state, histone marks and integration analysis (histone-3 acetylation (H3Ac), viral load, proviral levels and HIV-specific T cells responses) were included. REDUC-trial samples (n = 5) were included as a control group for RMD administration alone. FINDINGS: DNA methylation imprints after receiving the complete intervention discriminated Early versus Late viral-rebound individuals before MAP. Also, differential chromatin accessibility and histone marks at DNA methylation level were detected. Importantly, the differential DNA methylation positions (DMPs) between Early and Late rebounders before MAP were strongly associated with viral load, proviral levels as well as the HIV-specific T-cell responses. Most of these DMPs were already present prior to the intervention and accentuated after RMD infusion. INTERPRETATION: This study identifies host DNA methylation profiles and epigenetic cascades that are predictive of subsequent virus control in a kick-and-kill HIV cure strategy. FUNDING: European Union Horizon 2020 Framework Programme for Research and Innovation under Grant Agreement N°681137-EAVI2020 and N°847943-MISTRAL, the Ministerio de Ciencia e Innovación (SAF2017_89726_R), and the National Institutes of Health-National Institute of Allergy and Infectious Diseases Program Grant P01-AI131568.

Evolution of the gut microbiome following acute HIV-1 infection.

BACKGROUND: In rhesus macaques, simian immunodeficiency virus infection is followed by expansion of enteric viruses but has a limited impact on the gut bacteriome. To understand the longitudinal effects of HIV-1 infection on the human gut microbiota, we prospectively followed 49 Mozambican subjects diagnosed with recent HIV-1 infection (RHI) and 54 HIV-1-negative controls for 9-18 months and compared them with 98 chronically HIV-1-infected subjects treated with antiretrovirals (n = 27) or not (n = 71). RESULTS: We show that RHI is followed by increased fecal adenovirus shedding, which persists during chronic HIV-1 infection and does not resolve with ART. Recent HIV-1 infection is also followed by transient non-HIV-specific changes in the gut bacterial richness and composition. Despite early resilience to change, an HIV-1-specific signature in the gut bacteriome-featuring depletion of Akkermansia, Anaerovibrio, Bifidobacterium, and Clostridium-previously associated with chronic inflammation, CD8+ T cell anergy, and metabolic disorders, can be eventually identified in chronically HIV-1-infected subjects. CONCLUSIONS: Recent HIV-1 infection is associated with increased fecal shedding of eukaryotic viruses, transient loss of bacterial taxonomic richness, and long-term reductions in microbial gene richness. An HIV-1-associated microbiome signature only becomes evident in chronically HIV-1-infected subjects.

Low nadir CD4+ T-cell counts predict gut dysbiosis in HIV-1 infection.

Human immunodeficiency virus (HIV)-1 infection causes severe gut and systemic immune damage, but its effects on the gut microbiome remain unclear. Previous shotgun metagenomic studies in HIV-negative subjects linked low-microbial gene counts (LGC) to gut dysbiosis in diseases featuring intestinal inflammation. Using a similar approach in 156 subjects with different HIV-1 phenotypes, we found a strong, independent, dose-effect association between nadir CD4+ T-cell counts and LGC. As in other diseases involving intestinal inflammation, the gut microbiomes of subjects with LGC were enriched in gram-negative Bacteroides, acetogenic bacteria and Proteobacteria, which are able to metabolize reactive oxygen and nitrogen species; and were depleted in oxygen-sensitive methanogenic archaea and sulfate-reducing bacteria. Interestingly, subjects with LGC also showed increased butyrate levels in direct fecal measurements, consistent with enrichment in Roseburia intestinalis despite reductions in other butyrate producers. The microbiomes of subjects with LGC were also enriched in bacterial virulence factors, as well as in genes associated with beta-lactam, lincosamide, tetracycline, and macrolide resistance. Thus, low nadir CD4+ T-cell counts, rather than HIV-1 serostatus per se, predict the presence of gut dysbiosis in HIV-1 infected subjects. Such dysbiosis does not display obvious HIV-specific features; instead, it shares many similarities with other diseases featuring gut inflammation.

Gut Microbiota Linked to Sexual Preference and HIV Infection.

The precise effects of HIV-1 on the gut microbiome are unclear. Initial cross-sectional studies provided contradictory associations between microbial richness and HIV serostatus and suggested shifts from Bacteroides to Prevotella predominance following HIV-1 infection, which have not been found in animal models or in studies matched for HIV-1 transmission groups. In two independent cohorts of HIV-1-infected subjects and HIV-1-negative controls in Barcelona (n = 156) and Stockholm (n = 84), men who have sex with men (MSM) predominantly belonged to the Prevotella-rich enterotype whereas most non-MSM subjects were enriched in Bacteroides, independently of HIV-1 status, and with only a limited contribution of diet effects. Moreover, MSM had a significantly richer and more diverse fecal microbiota than non-MSM individuals. After stratifying for sexual orientation, there was no solid evidence of an HIV-specific dysbiosis. However, HIV-1 infection remained consistently associated with reduced bacterial richness, the lowest bacterial richness being observed in subjects with a virological-immune discordant response to antiretroviral therapy. Our findings indicate that HIV gut microbiome studies must control for HIV risk factors and suggest interventions on gut bacterial richness as possible novel avenues to improve HIV-1-associated immune dysfunction.

Evaluación del citómetro UF-1000i como método de cribado en el diagnóstico de infecciones del tracto urinario / Evaluation of the Sysmex UF-1000i flow cytometer for screening of urinary tract infection

INTRODUCCIÓN: El cultivo de orina supone una enorme carga de trabajo en el Laboratorio de Microbiología y sigue siendo técnica de referencia para el diagnóstico de las infecciones urinarias. Considerando la elevada prevalencia de resultados negativos, la implementación de un método de cribado fiable y rápido podría suponer un ahorro en costes de carga de trabajo y adelantar los resultados negativos. MÉTODO: Evaluamos la utilidad del citómetro de flujo UF-1000i® (bioMérieux, España) para cribado de muestras negativas que se pueden excluir del cultivo. Dividimos las muestras en 2 grupos: grupo 1, hombres y mujeres en edad fértil, que se consideran positivas con un crecimiento ≥ 104 UFC/ml, y grupo 2, consideradas positivas con crecimiento ≥ 105 UFC/ml. RESULTADOS: Enfrentando los datos del cultivo y del cribado en curva ROC, los puntos de mejor sensibilidad y especificidad fueron de 53,1 bacterias/μl para el grupo 1, y de 128,35 bacterias/μl para el grupo 2. En el grupo 1 la sensibilidad fue del 92,2%, la especificidad del 60%, la reducción de cultivos de orina del 46%, con el 2,1% de falsos negativos (42 muestras). En el grupo 2, la sensibilidad fue del 86%, la especificidad del 87,7%, la reducción de cultivos del 57,5%, con el 5,1% de falsos negativos (74 muestras). CONCLUSIÓN: La incorporación del citómetro UF-1000i al cribado de las muestras de orina depende mucho de las características de los pacientes y de la definición de cultivo de orina positivo. En nuestro caso, con el estudio exclusivo de la bacteriuria, los datos de reducción de carga de trabajo y de falsos negativos cuestionan seriamente esta incorporación

INTRODUCTION: The urine culture is a huge workload in the Microbiology Laboratory and remains the gold standard for the diagnosis of urinary tract infections. Considering the high prevalence of negative results, the implementation of a reliable screening method could lead to cost saving in the workload, and speedup reporting of negative results. METHODS: We evaluated the usefulness of the flow cytometer UF-1000i in the screening for negative samples than could be excluded from culture. We divided the samples into two groups, Group 1, males and women of childbearing age who were considered positive with a growth ≥ 104 CFU/ml, and Group 2,considered positive with ≥ 105 CFU/ml growth. RESULTS: On comparing the culture and screening data in the ROC curve, the best sensitivity and specificity points were 53.1 bact/l for Group 1, and 128.3 bact/l for Group 2. In Group 1, the sensitivity was 92.2%and a specificity of 60%, a reduction in urine cultures of 46%, with 2.1% false negative (42 samples). In Group 2, the sensitivity was 86%, with a specificity of 87.7%, a culture reduction of 57.5%, and 5.1% false negatives (74 samples). CONCLUSION: The incorporating of the UF-1000i cytometer to the screening of urine samples depends on the characteristics of the patients and the definition of positive urine culture. In our case, with only studying bacteriuria, the data on the reduction of workload and the false negatives seriously question this incorporation