Background: The different exacerbation patterns exhibited by patients colonized with Pseudomonas aeruginosa (PA) are not fully understood, and may be influenced by the stage of chronic colonization. We aimed to determine the implications of intermittent versus chronic PA colonization in biological and clinical outcomes while attempting to improve PA diagnosis.Methods: Longitudinal study of patients with BE colonized by PA, including sequential measurements of: sputum microbiology and 16SrRNA gene sequencing by llumina Miseq platform; PA biofilms by Gram-staining and FISH+CLSM; serum AntiPAIgG through ELISA; mucus rheology and the Quality-of-Life Bronchiectasis questionnaire (QoL-B); lung function and the number of mild and hospitalized exacerbations during long-term follow-up. Microbiology of sputum included comparison between standard of care (SOC) and methods recommended by ESCMID for biofilms.Findings: We collected 235 sputa and 262 serum samples from 80 patients, 61 with chronic and 19 with intermittent PA colonization. ESCMID methods achieved better PA detection than SOC. Chronic colonization was associated with higher PA load, mucoid PA and biofilms, but lower quality of life and fewer hospitalized exacerbations than intermittent colonization. Our data suggest a role for high AntiPAIgG in reducing severe exacerbations. Mucoid PA was an independent predictor of chronic PA associated with increased mucus viscoelasticity and with a pathobiome characterized by damaged respiratory biodiversity indicative of late chronic colonization.Interpretation: Since PA biofilms constitute the mechanism by which chronic PA colonization occurs, diagnosis of biofilm infections is a priority to prevent the mucoid pathobiome and to improve patient’s QoL.Funding Information: ISCIII-FEDER (Code: PI18/00145) grant awarded to AT and LFB; Intramural Ciber project 2018 (ES18PI01X1-2021) awarded to AT and LFB; ISCIII-FOS (FI19/00090) grant awarded to RLA, CB 06/06/0028/CIBER de enfermedades respiratorias (Ciberes), Ciberes is an initiative of ISCIII, 2.603/IDIBAPS, ICREA Academy award to AT, SGR/Generalitat de Catalunya, SEPAR grants 2016 (Code:208) and 2019 (Code: 628) awarded to LFB. Funders did not play any role in project design, data collection, data analysis, interpretation, or writing of the paper.Declaration of Interests: A. Torres has received grants from MedImmune, Cubist, Bayer, Theravance and Polyphor, and personal fees as an advisory board member from Bayer, Roche, The Medicines CO and Curetis. He has received bureau fees for keynote speaker presentations from GSK, Pfizer, Astra Zeneca and Biotest Advisory Board, but these were not associated with the study described in this paper. All others have nothing to declare. Ethics Approval Statement: The study was carried out in compliance with the Declaration of Helsinki (current version: Fortaleza, Brazil, October 2013) and with the requirements of the 2007 Spanish Biomedical Research Act. Written informed consent was obtained from all patients and the study was approved by the Internal Review Board of the Hospital Clínic of Barcelona (registry number HCB/0236).

Systems vaccinology has seldomly been used in therapeutic HIV-1 vaccine research. Our aim was to identify early gene ‘signatures’ that predicted virus load control after analytical therapy interruption (ATI) in participants of a dendritic cell-based HIV-1 vaccine trial (DCV2). mRNA and miRNA were extracted from frozen post-vaccination PBMC samples; gene expression was determined by microarray method. In gene set enrichment analysis, responders showed an up-regulation of 14 gene sets (TNF-alpha/NFkB pathway, inflammatory response, the complement system, Il6 and Il2 JAK-STAT signaling, among others) and a down-regulation of 7 gene sets (such as E2F targets or interferon alpha response). The expression of genes regulated by three (miR-223-3p, miR-1183 and miR-8063) of the 9 differentially expressed miRNAs was significantly down-regulated in responders. The deregulation of certain gene sets related to inflammatory processes seems fundamental for viral control, and certain miRNAs may be important in fine-tuning these processes.

People living-with HIV (PLHIV) presented less richness and diversity in the intestinal microbiota than non-infected individuals. Immunopathogenesis of HIV-1 infection has been extensively studied, however the influence of microbiota in those parameters that are key in the pathogenesis are less well characterized. The aim of the current study was to characterize the faecal microbiota composition in a cohort of PLHIV and its relationship with the clinical, metabolic, nutritional, inflammation, bacterial translocation and immunological markers. Baseline faecal samples from seventy-six HIV infected individuals with undetectable viral load (uVL) from a prospective, randomized feeding trial (NCT03846206) were collected and processed. The gut microbiota was determined by sequencing the V3-V4 region of the 16S rRNA gene. After analysing alpha diversity, women presented less diversity (Fisher alpha, P<0.005) and richness (Observed, P<0.005) than men. In addition, less alpha and beta diversity was observed in individuals with ≥15 years with uVL (Fisher alpha, P=0.039). Clinical (years with uVL, Body Mass Index, liver enzymes), metabolic (total and LDL cholesterol, B-apolipoprotein), nutrients (fats, minerals and vitamins) and immunological parameters (Treg cells and immune activation) were associated with the intestinal microbiota composition. This observational study detailed the impact of the gender and the time with uVL on the gut microbiota in PLHIV. The associations among the gut bacteria composition and the clinic, metabolic and immunological markers suggest that the microbiota could influence HIV-1 immunopathogenesis.Funding: This study was funded by the SPANISH AIDS Research Network (RIS) projects RD16/0025/0002 and RD16/0025/0013 and co-funded by the European Regional Development Fund (ERDF).Declaration of Interest: None to declare. Ethical Approval: The study was approved by the Institutional Review Board of the Hospital Clinic (Barcelona, Spain)

OBJECTIVE:The health effects of a supplemented Mediterranean diet (SMD) with extra-virgin olive oil (EVOO) and nuts are well documented in non-HIV-infected individuals. We hypothesised that the benefits of an SMD could be mediated by changes in the gut microbiota, even in those with an altered intestinal microbiota such as people living with HIV. DESIGN:Individuals living with HIV (n = 102) were randomised to receive an SMD with 50 g/day of EVOO and 30 g/day of walnuts (SMD group) or continue with their regular diet (control group) for 12 weeks. METHODS:Adherence to the Mediterranean diet was assessed using the validated 14-item MD-Adherence-Screener (MEDAS) from the PREDIMED study. A sub-study classifying the participants according to their MEDAS scores was performed. RESULTS:The lipid profile was improved in the SMD group vs. that in the control group (delta-total cholesterol and delta-B-lipoprotein). The immune activation (CD4+HLADR+CD38+ and CD8+HLADR+CD38+ cells) and IFN-γ-producing T-cells significantly decreased at week 12 compared to the baseline in the SMD group but not in the control group. The gut microbiota in those from the high-adherence group presented significantly high diversity and richness at the end of the intervention. Succinivibrio and Bifidobacterium abundances were influenced by the adherence to the MD and significantly correlated with Treg cells. CONCLUSION:The Mediterranean diet improved metabolic parameters, immune activation, Treg function, and the gut microbiota composition in HIV-1-infected individuals. Further, Mediterranean diet increased the Bifidobacterium abundances after the intervention, and it was associated to a beneficial profile.

Therapeutic vaccines based on dendritic cells offer a good approach to HIV-specific T-cell responses and partial control of the viral load after antiretroviral therapy interruption. The aim of the present study was to identify mRNA expression profiles and to assess the impact of the gut microbiome composition for predicting the viral load control after antiretroviral therapy interruption. We enrolled 29 patients to receive either placebo or a monocyte-derived dendritic cell vaccine. Patients with a decrease in their viral load of >0.5 log10 copies/mL by 12 weeks after antiretroviral therapy interruption were considered responders. In total, 66 genes were considered differentially expressed between responders and non-responders. Enrichment analysis revealed several upregulated pathways involved in the host defense response to a virus via the type I interferon signaling pathway. Regarding the gut microbiota, responders showed enriched levels of Bacteroidetes (p < 0.005) and Verrucomicrobia (p = 0.017), while non-responders were enriched with Tenericutes (p = 0.049) and Actinobacteria (p < 0.005). We also found important differences at the genus level. However, we did not discover any effect of the dendritic cell vaccine on the transcriptome or the gut microbiota. An alternative analysis did characterize that the microbiota from responders were associated with the metabolic production of short-chain fatty acids, which are key metabolites in the regulation of intestinal homeostasis. The evidence now consistently shows that short-chain fatty acid depletion occurs in HIV-infected individuals receiving antiretroviral treatment.

Atherosclerosis leads to cardiovascular disease (CVD). It is still unclear whether cholesterol-HDL (cHDL) concentration plays a causal role in atherosclerosis development. However, an important factor in early stages of atheroma plaque formation is cholesterol efflux capacity to HDL (the ability of HDL particles to accept cholesterol from macrophages) in order to avoid foam cell formation. This is a key step in avoiding the accumulation of cholesterol in the endothelium and a part of reverse cholesterol transport (RCT) to eliminate cholesterol through the liver. Cholesterol efflux capacity to serum or plasma in macrophage cell models is a promising tool that can be used as biomarker for atherosclerosis. Traditionally, [3H]-cholesterol has been used in cholesterol efflux assays. In this study, we aim to develop a safer and faster strategy using fluorescent labelled-cholesterol (NBD-cholesterol) in a cellular assay to trace the cholesterol uptake and efflux process in THP-1-derived macrophages. Finally, we optimize and standardize the NBD-cholesterol efflux method and develop a high-throughput analysis using 96-well plates.