HIV-1-DNA/RNA and immunometabolism in monocytes: contribution to the chronic immune activation and inflammation in people with HIV-1.

BACKGROUND: Among people living with HIV-1 (PHIV), immunological non-responders (INR) experience incomplete immune recovery despite suppressive antiretroviral treatment (ART), facing more severe non-AIDS events than immunological responders (IR) due to higher chronic immune activation and inflammation (cIA/I). We analyzed the HIV-1 reservoir and immunometabolism in monocytes as a source of cIA/I. METHODS: Cross-sectional study in which 110 participants were enrolled: 25 treatment-naïve; 35 INR; 40 IR; and 10 healthy controls. Cell-associated HIV-1-DNA (HIV-DNA) and -RNA (HIV-RNA) were measured in FACS-isolated monocytes using digital droplet PCR. Intact, 5' deleted, and 3' deleted proviruses were quantified by the intact proviral DNA assay. Systemic inflammation, monocyte immunophenotype, and immunometabolism were characterized by immunoassays, flow cytometry, and real-time cellular bioenergetics measurements, respectively. FINDINGS: Monocytes from INR harbor higher HIV-RNA and HIV-DNA levels than IR. HIV-RNA was found in 14/21 treatment-naïve [2512 copies/106 TBP (331-4666)], 17/33 INR [240 (148-589)], and 15/28 IR [144 (15-309)], correlating directly with sCD163, IP-10, GLUT1high cells and glucose uptake, and inversely with the CD4+/CD8+ ratio. HIV-DNA was identified in all participants with detectable HIV-RNA, with intact provirus in 9/12 treatment-naïve [13 copies/106 monocytes (7-44)], 8/14 INR [46 (18-67)], and 9/13 IR [9 (7-24)]. INR presented glucose metabolism alterations and mitochondrial impairment; decreased coupling efficiency and BHI, and increased mitochondrial dysfunction inversely correlating with the CD4+/CD8+ ratio. INTERPRETATION: HIV-RNA, more than HIV-DNA, in monocytes and their altered metabolism are factors associated with the higher cIA/I that characterize INR. FUNDING: This work was supported by the European Regional Development Fund, ISCIII, grant PI20/01646. Other funding sources: Instituto de Salud Carlos III through the Subprogram Miguel Servet (CP19/00159) to AGV, PFIS contracts (FI19/00304) to EMM, (FI21/00165) to ASA, and (FI19/00083) to CGC, and a mobility grant (MV21/00103) to EMM, from the Ministerio de Ciencia e Innovación, Spain. AJM was granted by a CSL Centenary Award.

Effervescence tablets based on magnetic ionic liquids as simple microdevices for the in situ dispersive liquid-liquid microextraction of urinary biomarkers.

BACKGROUND: Magnetic ionic liquids (MILs) have been explored in dispersive liquid-liquid microextraction (DLLME). Their usage allows to substitute centrifugation and/or filtration steps by a quick magnetic separation. Besides, effervescence-assisted DLLME is one of the most known options to improve the dispersion of the extractant in the sample, while allowing to avoid the consumption of external energy during dispersion. Despite these interesting features, only one study incorporates MILs containing the tetrachloroferrate anion in effervescence tablets. These MILs are highly viscous and liquid at room temperature, thus compromising the stability of the tablets when used as extraction microdevices in effervescence-assisted DLLME, and only allowing their use in the conventional MIL-DLLME mode. RESULTS: A new class of effervescence tablets containing a Ni(II)-based MIL, that is solid at room temperature, is here proposed. This type of tablets permits their use, for first time, in the in situ DLLME mode, occurring through the transformation of a water-soluble MIL into a water-insoluble MIL microdroplet. This way, the tablet formulation included: the MIL, the metathesis reagent lithium bis[(trifluoromethyl)sulfonyl]imide, NaH2PO4 and K2CO3 as effervescence precursors salts, and Na2SO4 as salting-out and desiccating agent. The method is combined with high-performance liquid-chromatography and both fluorescence and ultraviolet detection, for the determination of monohydroxylated polycyclic aromatic hydrocarbons (OH-PAHs) and benzophenones (BPs), as biomarkers in urine. The method simply involved the addition of the effervescence tablet to the sample, thus taken place simultaneously the effervescence process and the metathesis reaction, without requiring any external energy consumption. The method presented limits of detection down to 10 ng L-1 for OH-PAHs and to 0.60 µg L-1 for BPs, inter-day relative standard deviations lower than 17 %, and average relative recoveries of 94 % in urine. The determined OH-PAHs contents in urine were between 0.40 and 16 µg L-1, and between 17.8 and 334 µg L-1 for BPs. SIGNIFICANCE: We have developed the first MIL-based effervescence tablets that are completely solid, thus improving the stability and robustness of these microdevices with respect to previously reported tablets involving MILs, while permitting to perform into the in situ DLLME mode (thus gaining in extraction efficiency). This approach including the MIL-based effervescence tablets constitutes an alternative on-site platform for the analysis of urine, as satisfactory precision, accuracy, and sensitivity are achieved despite not involving any external energy input within the analytical sample preparation setup. This method also constitutes the first application of MIL-based effervescence tablets for bioanalysis.

Plasma concentrations of IL-6, MIP-1ß, IP-10, and PTX-3 as predictors of the immunological response to antiretroviral treatment in people with HIV.

Despite effective antiretroviral therapy (ART), 15-30% of people with HIV experience poor CD4+ T-cell recovery, termed immunologic non-responders (INR). This study aims to evaluate whether pre-ART plasma levels of interleukin-6 (IL-6), interferon gamma-induced protein-10 (IP-10), macrophage inflammatory protein-1-ß (MIP-1ß), and/or pentraxin-3 (PTX-3) could predict subsequent immunologic recovery. Seventy-four participants were enrolled and classified as INR and immunologic responders (IR) based on CD4+/CD8+ ratio increase over 24 months after starting ART. The results showed no significant differences in cytokine levels between INR and IR. Therefore, IL-6, IP-10, MIP-1ß, and PTX-3 were unsuitable as predictive markers of poor immune recovery.

The Genetic Diagnostics of Hemochromatosis: Disparities in Low- Versus High-Income Countries.

This study provides a comprehensive overview of hereditary hemochromatosis (HH), a genetic condition characterized by iron overload due to excessive iron absorption. It elucidates diverse inheritance patterns and clinical manifestations by exploring mutations in critical genes such as HFE (hemochromatosis), HJV (hemojuvelin), HAMP (hepcidin antimicrobial peptide), TfR2 (transferrin receptor 2), and FP (ferroportin). The significance of early screening, diagnosis, and personalized management strategies based on genetic classification is emphasized, particularly in terms of high-income vs. low-income countries. Addressing challenges in diagnosis, genetic testing accessibility, and healthcare disparities, the study highlights the importance of early detection, cost-effective screening strategies, and enhancing healthcare outcomes globally. Advanced genetic testing in high-income countries facilitates early diagnosis and management, reducing complications such as liver disease and cardiomyopathy. In contrast, low-income populations face several barriers, including limited access to genetic testing, high costs, and inadequate healthcare infrastructure. Cost-effective serum ferritin (SF) and transferrin saturation (TS) tests and emerging point-of-care (POC) tests offer affordable diagnostic options for low-resource settings. Additionally, the ongoing development of hepcidin measurement methods holds promise for enhancing diagnostic capabilities. Implementing these strategies can aid healthcare providers in improving global HH management and reducing the burden of iron overload complications. Furthermore, the study underscores the need for public health initiatives to raise awareness about HH, promote routine screenings, and advocate for equitable healthcare policies. Collaborative efforts between governments, healthcare organizations, and research institutions are crucial in addressing the global burden of HH. By fostering international cooperation and resource-sharing, it is possible to bridge the gap between high-income and low-income countries, ensuring all individuals have access to the necessary diagnostic and treatment options. This holistic approach can ultimately lead to better health outcomes and improved quality of life for individuals affected by HH worldwide. This comprehensive examination of HH not only illuminates the genetic and clinical aspects of the condition but also provides a roadmap for addressing the multifaceted challenges associated with its diagnosis and management.

Long-term effects on immunological, inflammatory markers, and HIV-1 reservoir after switching to a two-drug versus maintaining a three-drug regimen based on integrase inhibitors.

Objective: To compare the long-term effects on immune parameters, inflammation, and HIV-1 reservoir after switching to a two-drug (2DR) versus maintaining an integrase inhibitor (InSTI)-based three-drug regimen (3DR). Methods: Cross-sectional study in which HIV-1 treatment-naïve people started and maintained an InSTI-based 3DR or, at different times, switched to 2DR (dolutegravir or darunavir/cobicistat + lamivudine). CD4+ and CD8+ T-cell activation and exhaustion, plasma concentrations of hs-CRP, D-dimer, P-selectin, IL-1ß, IL-6, TNF-α, IFN-γ, IP-10, sTNFR-I/II, MIP-1α/ß, I-FABP, LBP, sCD14, sCD163, MCP-1, and cellular-associated HIV-1-DNA and -RNA were quantified by flow cytometry, different immunoassays, and droplet digital PCR, respectively. The U de Mann-Whitney test evaluated differences between 3DR and 2DR. Immune recovery was evaluated using a general linear model for repeated measures adjusted for different co-variables. Results: Fifty participants per group were included. The median time on 3DR was 82 months for the 3DR group and 30 months for the 2DR group, after which it switched to 2DR for a median of 57 months. We did not find differences between both groups in any of the parameters analyzed. Specifically, some values in 3DR and 2DR were hs-CRP, 0.92 mg/L (0.45-2.23) vs. 1.23 (0.61-2.38); D-dimer, 190.0 µg/L (150.0-370.0) vs. 190.0 (150.0-397.5); IL-6, 2.8 pg/mL (1.3-5.3) vs. 3.2 (2.1-4.7); sCD14, 4.5 ng/mL (3.3-6.2) vs. 5.0 (3.6-6.1), respectively, all p ≥ 0.399. Conclusion: In the long term, switching to 2DR does not negatively affect immunologic parameters, inflammatory markers, or HIV-1 reservoir. Clinical trial registration: identifier NCT04076423.

Exploring a new generation of bimetallic magnetic ionic liquids with ultra-low viscosity in microextraction that enable direct coupling with high-performance liquid-chromatography.

BACKGROUND: The incorporation of bimetallic magnetic ionic liquids (MILs) in microextraction methods is an emerging trend due to the improved magnetic susceptibility offered by these solvents, which relies on the presence of metallic components in both the cation and the anion. This feature favors easy magnetic separation of these solvents in analytical sample preparation strategies. However, reported liquid-phase microextraction methods based on bimetallic MILs still present an important drawback in that the MILs are highly viscous, making a dispersive solvent during the microextraction procedure necessary, while also requiring a tedious back-extraction step prior to the chromatographic analysis. RESULTS: We propose for the first time a new generation of ultra-low viscosity bimetallic MILs composed of two paramagnetic Mn(II) complexes characterized by their easy usage in dispersive liquid-liquid microextraction (DLLME). The approach does not require dispersive solvent and the MIL-DLLME setup was directly combined with high-performance liquid chromatography (HPLC) and fluorescence detection (FD), without any back-extraction step. The approach was evaluated for the determination of five monohydroxylated polycyclic aromatic hydrocarbons, as carcinogenic biomarkers, in human urine. Optimum conditions of the MIL-DLLME method included the use of a low MIL volume (75 µL), a short extraction time (5 min), and no need of any dispersive solvent neither NaCl. The method presented limits of detection down to 7.50 ng L-1, enrichment factors higher than 17, and provided inter-day relative standard deviation lower than 11%. Analysis of urine samples was successfully performed, with biomarker content found at levels between 0.24 and 7.8 ng mL-1. SIGNIFICANCE: This study represents the first liquid-phase microextraction method using the new generation of low-viscous bimetallic MILs. The proposed MIL-DLLME approach represents 2 important advances with respect to previous methods employing bimetallic MILs: 1) no dispersive solvent is required, and 2) direct injection of the MIL in the HPLC is possible after minor dilution (no back extraction steps are required). Therefore, the microextraction strategy is simple, rapid, and consumes very small amounts of energy.

HIV-1-specific T-cell responses and exhaustion profiles in people with HIV after switching to dual therapy vs. maintaining triple therapy based on integrase inhibitors.

BACKGROUND: Dual therapy (DT) has shown comparable results to triple therapy (TT) in efficacy and other immunological aspects. However, there are still some concerns about DT, including several immunological features. Therefore, we evaluated whether HIV-1-specific memory T-cell responses and exhaustion phenotypes are adversely influenced after simplification to DT. METHODS: HIV-1-specific CD4+ and CD8+ T-cell responses were assessed by intracellular cytokine and degranulation marker staining, and polyfunctionality indexes after stimulation with a Gag peptide pool. Exhaustion phenotypes were evaluated by PD-1, TIM-3, and LAG-3 expression in CD4+ and CD8+ T cells. RESULTS: Forty participants in the TRIDUAL trial (ClinicalTrials.gov: NCT03447873) who were randomized to continue integrase inhibitor-based TT (n = 20) or to switch to DT (dolutegravir or darunavir/cobicistat plus lamivudine) (n = 20). After 96 weeks, the magnitude of CD4+ and CD8+ T-cell responses was similar in both treatment arms (p = 0.221 and p = 0.602, respectively). The CD4+ polyfunctionality index decreased in the TT arm (p = 0.013) and remained stable in the DT arm, while the polyfunctionality of CD8+ T cells was unchanged in both arms. There was a significant decrease in the expression of PD-1, TIM-3, and the co-expression of PD-1+TIM-3+LAG-3+, and PD-1 +TIM-3 + in both CD4+ and CD8+ T cells. However, the decrease in the expression of exhaustion markers did not improve HIV-1-specific T-cell responses. CONCLUSIONS: Our results suggest that simplification to DT does not negatively influence the HIV-1-specific T-cell response or the exhaustion phenotype after 96 weeks of follow-up.

Decay kinetics of HIV-1-RNA in seminal plasma with dolutegravir/lamivudine versus dolutegravir plus emtricitabine/tenofovir alafenamide in treatment-naive people living with HIV.

BACKGROUND: This was a substudy of a Phase IV, randomized clinical trial (ClinicalTrials.gov identifier: NCT04295460) aiming to compare the activity of dolutegravir/lamivudine versus dolutegravir plus tenofovir alafenamide/emtricitabine (DTG + TAF/FTC) in the male genital tract. METHODS: Participants were asymptomatic adults without sexually transmitted diseases, treatment-naive people living with HIV (PLWH), with CD4+ T cell counts >200 cells/mm3 and plasma HIV-1-RNA levels >5000 and <500 000 copies/mL, randomized (1:1) to DTG + TAF/FTC or dolutegravir/lamivudine. Blood plasma (BP) and seminal plasma (SP) were collected at baseline and Weeks 4, 8, 12 and 24. HIV-1-RNA was measured in BP and SP using the Cobas 6800 system (Roche Diagnostics) with a lower detection limit of 20 copies/mL. The primary efficacy endpoint was the proportion of subjects with undetectable SP HIV-1-RNA at Week 12 by intention-to-treat analysis. RESULTS: Fifteen participants in the DTG + TAF/FTC and 16 in the dolutegravir/lamivudine arms were analysed, with basal SP viral load of 4.81 (4.30-5.43) and 4.76 (4.09-5.23), P = 0.469, respectively. At Week 12, only one participant in each treatment arm had a detectable SP HIV-1-RNA (DTG + TAF/FTC, 141 copies/mL; dolutegravir/lamivudine, 61 copies/mL). Based on the estimated means, there was no significant difference in the decay of HIV-1-RNA in both BP and SP over time between the two arms of treatment (F = 0.452, P = 0.662, and F = 1.147, P = 0.185, respectively). CONCLUSIONS: After 12 weeks of treatment, there were no differences in the percentage of undetectable SP HIV-1-RNA in naive PLWH who started dolutegravir/lamivudine compared with DTG + TAF/FTC.

Humoral and cellular immunity to SARS-COV-2 after vaccination with mRNA vaccines in PLWH with discordant immune response. Influence of the vaccine administered.

Background: Data on SARS-CoV-2 mRNA vaccine immunogenicity in people living with human immunodeficiency virus (PLWH) and discordant immune response (DIR) are currently limited. Therefore, we compare the immunogenicity of these vaccines in DIR and immunological responders (IR). Methods: A prospective cohort that enrolled 89 participants. Finally, 22 IR and 24 DIR were analyzed before vaccination (T0), one (T1) and six months (T2) after receiving BNT162b2 or mRNA-1273 vaccine. Additionally, 10 IR and 16 DIR were evaluated after a third dose (T3). Anti-S-RBD IgG, neutralizing antibodies (nAb), neutralization activity, and specific memory B cells were quantified. Furthermore, specific CD4+ and CD8+ responses were determined by intracellular cytokine staining and polyfunctionality indexes (Pindex). Results: At T1, all participants developed anti-S-RBD. 100% IR developed nAb compared to 83.3% DIR. Spike-specific B cells were detected in all IR and 21/24 DIR. Memory CD4+ T cells responded in 5/9 IR and 7/9 DIR, mainly based on the expression of IFN-γ and TNF-α, with a higher Pindex in DIR. Memory CD8+ T cells responded in only four participants in each group. At T2, anti-S-RBD and nAb titers were higher in DIR than in IR. In both groups, there was an increase in specific B memory cells, higher in DIR. Six IR and five DIR maintained a specific memory CD4+ response. Memory CD8+ response was preserved in IR but was lost in DIR. In a multivariate linear regression analysis, receiving mRNA-1273 instead of BNT162b2 played a prominent role in the results. Conclusions: Our data suggest that PLWH with DIR can mount an immune response similar to those with higher CD4+, provided they receive the mRNA-1273 vaccine instead of others less immunogenic.

Magnetic Ionic Liquids in Analytical Microextraction: A Tutorial Review.

Magnetic ionic liquids (MILs) are materials of special interest in analytical chemistry and, particularly, in analytical microextraction. These solvents possess several of the properties derived from their inherent nature of ionic liquids, combined with their magnetism, that permits their manipulation with an external magnetic field. This feature allows for performing typical steps of the microextraction procedure in a simpler manner with the aid of a strong magnet. Although there are several important reviews summarizing the most innovative advances in this field, there is a gap of information, as they do not provide useful details and tips related to the experimental set up of these procedures. This tutorial review fills this gap by providing a guide for the proper handling of MILs, their manipulation with magnets, and their proper hyphenation with the most used analytical techniques. Attention is paid to dispersive liquid-liquid microextraction, stir-bar dispersive liquid microextraction, aqueous biphasic systems, and single-drop microextraction, for being the analytical microextraction techniques mostly employed with MILs. This review also introduces a classification of the MILs employed in analytical microextraction in three classes (denoted as A, B, and C) as a function of the MIL nature (metal-containing anion, metal-containing cation, and radical-containing ion), and discuss about the prospect and future trends regarding new MIL families in microextraction together with new directions expected in these procedures.

Clinical, laboratory data and inflammatory biomarkers at baseline as early discharge predictors in hospitalized SARS-CoV-2 infected patients.

BACKGROUND: The SARS-CoV-2 pandemic has overwhelmed hospital services due to the rapid transmission of the virus and its severity in a high percentage of cases. Having tools to predict which patients can be safely early discharged would help to improve this situation. METHODS: Patients confirmed as SARS-CoV-2 infection from four Spanish hospitals. Clinical, demographic, laboratory data and plasma samples were collected at admission. The patients were classified into mild and severe/critical groups according to 4-point ordinal categories based on oxygen therapy requirements. Logistic regression models were performed in mild patients with only clinical and routine laboratory parameters and adding plasma pro-inflammatory cytokine levels to predict both early discharge and worsening. RESULTS: 333 patients were included. At admission, 307 patients were classified as mild patients. Age, oxygen saturation, Lactate Dehydrogenase, D-dimers, neutrophil-lymphocyte ratio (NLR), and oral corticosteroids treatment were predictors of early discharge (area under curve (AUC), 0.786; sensitivity (SE) 68.5%; specificity (S), 74.5%; positive predictive value (PPV), 74.4%; and negative predictive value (NPV), 68.9%). When cytokines were included, lower interferon-γ-inducible protein 10 and higher Interleukin 1 beta levels were associated with early discharge (AUC, 0.819; SE, 91.7%; S, 56.6%; PPV, 69.3%; and NPV, 86.5%). The model to predict worsening included male sex, oxygen saturation, no corticosteroids treatment, C-reactive protein and Nod-like receptor as independent factors (AUC, 0.903; SE, 97.1%; S, 68.8%; PPV, 30.4%; and NPV, 99.4%). The model was slightly improved by including the determinations of interleukine-8, Macrophage inflammatory protein-1 beta and soluble IL-2Rα (CD25) (AUC, 0.952; SE, 97.1%; S, 98.1%; PPV, 82.7%; and NPV, 99.6%). CONCLUSIONS: Clinical and routine laboratory data at admission strongly predict non-worsening during the first two weeks; therefore, these variables could help identify those patients who do not need a long hospitalization and improve hospital overcrowding. Determination of pro-inflammatory cytokines moderately improves these predictive capacities.

Anti-CD4 autoantibodies in immunological nonresponder people with HIV: cause of CD4 + T-cell depletion?

OBJECTIVE: We aimed to evaluate the anti-CD4 IgG role in the poor immune recovery of immunological nonresponder people with HIV (INR). DESIGN: INR display low CD4 + T-cell increase despite long-term undetectable viremia. Among other factors, autologous anti-CD4 IgG-dependent cellular cytotoxicity (ADCC) by natural killer (NK) cells has been proposed to cause CD4 + T-cell depletion. METHODS: Plasma anti-CD4 IgG levels were quantified and purified by chromatography columns for the subsequent use in a coculture of CD4 + T and NK cells. We analyzed NK cell degranulation markers (CD107a, perforin and granzyme B) and IFN-γ release, and CD4 + T-cell death. Binding affinity of anti-CD4 IgG for CD4 + T cells was also assessed. RESULTS: A total of 168 individuals were enrolled (INR, 56; immunological responders, 40; treatment-naive, 39; and healthy controls, 33). The highest anti-CD4 IgG levels were found in treatment-naive people with HIV (PWH), followed by participants on treatment. There were no correlations between anti-CD4 IgG levels and CD4 + T-cell counts. In a 15-participant subgroup (naive, immunological responders, and INR), anti-CD4 IgG induced a slight NK-cell expression of degranulation markers and IFN-γ; however, the percentage of CD4 + T-cell death was negligible. Consistently, no significant changes in NK cell polyfunctionality were observed. In addition, purified anti-CD4 IgG showed scarce binding affinity for CD4 + T cells. These results were similar in all analyzed participant groups. CONCLUSION: Our results suggest that autologous anti-CD4 IgG neither trigger CD4 + T-cell death by ADCC nor are responsible for CD4 + lymphocyte depletion in INR.

Deciphering the quality of SARS-CoV-2 specific T-cell response associated with disease severity, immune memory and heterologous response.

SARS-CoV-2 specific T-cell response has been associated with disease severity, immune memory and heterologous response to endemic coronaviruses. However, an integrative approach combining a comprehensive analysis of the quality of SARS-CoV-2 specific T-cell response with antibody levels in these three scenarios is needed. In the present study, we found that, in acute infection, while mild disease was associated with high T-cell polyfunctionality biased to IL-2 production and inversely correlated with anti-S IgG levels, combinations only including IFN-γ with the absence of perforin production predominated in severe disease. Seven months after infection, both non-hospitalised and previously hospitalised patients presented robust anti-S IgG levels and SARS-CoV-2 specific T-cell response. In addition, only previously hospitalised patients showed a T-cell exhaustion profile. Finally, combinations including IL-2 in response to S protein of endemic coronaviruses were the ones associated with SARS-CoV-2 S-specific T-cell response in pre-COVID-19 healthy donors' samples. These results could have implications for protective immunity against SARS-CoV-2 and recurrent COVID-19 and may help for the design of new prototypes and boosting vaccine strategies.

Immunological and inflammatory changes after simplifying to dual therapy in virologically suppressed HIV-infected patients through week 96 in a randomized trial.

OBJECTIVES: To evaluate whether simplification of antiretroviral treatment to dual therapy (DT) negatively impacts immune recovery (IR), immune activation and inflammation (IA/I), and HIV reservoir. METHODS: An open-label, single-centre, randomized controlled trial conducted in adult virologically suppressed HIV-infected patients on triple therapy (TT) with elvitegravir-cobicistat, emtricitabine and tenofovir alafenamide or dolutegravir (DTG), abacavir, and lamivudine (3TC). Participants were randomized to continue TT or switch to DTG, or darunavir/cobicistat (DRVc) plus 3TC. IR was assessed by CD4+/CD8+ ratio at 48 and 96 weeks. Changes in immune activation, proliferation, exhaustion, senescence, and apoptosis in CD4+ and CD8+ T cells, plasma sCD14, hsCRP, D-dimers, ß2-microglobulin, IL-6, TNF-α and IP-10 levels, cell-associated HIV-DNA (CA-DNA), and unspliced HIV-RNA (usRNA) were also analysed. RESULTS: One hundred and fifty-one participants were enrolled. Fourteen patients did not complete the follow up. In the ITT and PP analysis, the IR was similar between the treatment arms. In the ITT analysis, the median increase in CD4+/CD8+ ratio was 0.10, 0.04, and 0.07 at week 48, and 0.09, 0.05, and 0.08 at week 96 for TT, DTG/3TC, and DRVc/3TC, respectively. After adjusting for confounding factors, the slopes of changes in CD4+/CD8+ ratio over time were independent of treatment (F = 1.699; p = 0.436) and related only to baseline values (F = 756.871; p = 0.000). There were no differences in IA/I, CA-DNA, or usRNA between treatment arms. DISCUSSION: Both IR and IA/I, CA-DNA, and usRNA were similar in the three treatment groups, regardless of maintaining TT or simplifying to DTG/3TC or DRVc/3TC in virologically suppressed HIV-infected patients.

Mimicking human ingestion of microplastics: Oral bioaccessibility tests of bisphenol A and phthalate esters under fed and fasted states.

Notwithstanding the fact that microplastic fragments were encountered in the human stool, little effort has been geared towards elucidating the impact of chemical additives upon the human health. In this work, standardized bioaccessibility tests under both fasting and fed conditions are herein applied to the investigation of human oral bioaccessibility of plastic additives and monomers (i.e. eight phthalate esters (PAEs) and bisphenol A (BPA)) in low-density polyethylene (LDPE) and polyvinyl chloride (PVC) microplastics. The generation of phthalate monoesters is evaluated in the time course of the bioaccessibility tests. Maximum gastric and gastrointestinal bioaccessibility fractions are obtained for dimethyl phthalate, diethyl phthalate and BPA, within the range of 55-83%, 40-68% and 37-67%, respectively, increasing to 56-92% and 41-70% for dimethyl phthalate and diethyl phthalate, respectively, whenever their hydrolysis products are considered. Bioaccessibility fractions of polar PAEs are dependent upon the physicochemical characteristics of the microplastics, with greater bioaccessibility for the rubbery polymer (LDPE). With the method herein proposed, oral bioaccessible pools of moderately to non-polar PAEs can be also accurately assessed for risk-assessment explorations, with values ranging from 1.8% to 32.2%, with again significantly larger desorption percentages for LDPE. Our results suggested that the highest gastric/gastrointestinal bioaccessibility of the eight PAEs and BPA is reached under fed-state gastrointestinal extraction conditions because of the larger amounts of surface-active biomolecules. Even including the bioaccessibility factor within human risk assessment/exposure studies to microplastics, concentrations of dimethyl phthalate, di-n-butyl phthalate and BPA exceeding 0.3% (w/w) may pose severe risks after oral uptake in contrast to the more hydrophobic congeners for which concentrations above 3% (w/w), except for diethylhexyl phthalate, would be tolerated.

Elevated Anti-SARS-CoV-2 Antibodies and IL-6, IL-8, MIP-1ß, Early Predictors of Severe COVID-19.

Viral and host immune kinetics during acute COVID-19 and after remission of acute symptoms need better characterization. SARS-CoV-2 RNA, anti-SARS-CoV-2 IgA, IgM, and IgG antibodies, and proinflammatory cytokines were measured in sequential samples from hospitalized COVID-19 patients during acute infection and six months following diagnosis. Twenty four laboratory confirmed COVID-19 patients with mild/moderate and severe COVID-19 were included. Most were males (83%) with a median age of 61 years. Twenty one percent were admitted to the intensive care unit (ICU) and eight of them (33.3%) met the criteria for severe COVID-19 disease. A delay in SARS-CoV-2 levels' decline during the first six days of follow up, and viral load persistence until month 3 were related to severe COVID-19, but not viral load levels at the diagnosis. Higher levels of anti-SARS-CoV-2 IgA, IgM, IgG and the cytokines IL-6, IL-8 and MIP-1ß at the diagnosis time were related to the severe COVID-19 outcome. Higher levels of MIP-1ß, IL-1ß, MIP-1α and IFN-γ were observed at month 1 and 3 during mild/moderate disease, compared to severe COVID-19. IgG persisted at low levels after six months of diagnosis. In conclusion, higher concentrations of IgA, IgM, and IgG, and IL-6, IL-8 and MIP-1ß are identified as early predictors of COVID-19 severity, whereas no significant association is found between baseline SARS-COV-2 viral load and COVID-19 severity.

Dendritic cell deficiencies persist seven months after SARS-CoV-2 infection.

Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV)-2 infection induces an exacerbated inflammation driven by innate immunity components. Dendritic cells (DCs) play a key role in the defense against viral infections, for instance plasmacytoid DCs (pDCs), have the capacity to produce vast amounts of interferon-alpha (IFN-α). In COVID-19 there is a deficit in DC numbers and IFN-α production, which has been associated with disease severity. In this work, we described that in addition to the DC deficiency, several DC activation and homing markers were altered in acute COVID-19 patients, which were associated with multiple inflammatory markers. Remarkably, previously hospitalized and nonhospitalized patients remained with decreased numbers of CD1c+ myeloid DCs and pDCs seven months after SARS-CoV-2 infection. Moreover, the expression of DC markers such as CD86 and CD4 were only restored in previously nonhospitalized patients, while no restoration of integrin ß7 and indoleamine 2,3-dyoxigenase (IDO) levels were observed. These findings contribute to a better understanding of the immunological sequelae of COVID-19.

An automatic flow-through system for exploration of the human bioaccessibility of endocrine disrupting compounds from microplastics.

This article reports on the first attempt towards investigating the leaching rates in the human gastrointestinal (GI) tract of plastic-borne contaminants that can be ingested accidentally using physiologically relevant body fluids. Oral bioaccessibility under fasted and fed states was determined in dynamic mode exploiting an automatic flow setup. The flow system is able to mimic the fast uptake of the released species from the polymeric matrix by absorption in the human digestive system by the in-line removal of the leached species. Complex GI extractants based on the Unified Bioaccessibility Method (UBM, fasted state) and Versantvoort test (fed-state) were brought through a microplastic-loaded metal microcolumn for semi-continuous leaching of plasticizers (phthalic acid ester congeners) and monomer/antioxidant species (bisphenol A, BPA) followed by in-line solid-phase extraction and clean-up of GI extracts prior to liquid chromatography analysis. The temporal extraction profiles were fitted to a first-order kinetic model for the estimation of maximum bioaccessibility pools and apparent leaching rates. Among all studied contaminants, only BPA, dimethylphthalate and diethylphthalate were appreciably released under dynamic GI conditions from high-density polyethylene pellets (average size of 110 µm), with average bioaccessibility values spanning from 51 to 84% and 48 to 87% for UBM and Versantvoort methods, respectively. No statistically significant differences in oral bioaccessibility pools were found under fed- and fasted-state dynamic extractions. The apparent kinetic constants under the fed state were greater by ≥30% as a consequence of the effect of the larger amounts of bile salts and digestive enzymes in the Versantvoort test on the leaching rates. The estimated average daily intake, in which bioaccessibility data are contemplated, indicated that plastic materials exceeding 0.3% (w/w) BPA might pose real risks to human health.

Mesenchymal stromal cells in human immunodeficiency virus-infected patients with discordant immune response: Early results of a phase I/II clinical trial.

Between 15% and 30% of HIV-infected subjects fail to increase their CD4+ T-cell counts despite continuous viral suppression (immunological nonresponders [INRs]). These subjects have a higher morbidity and mortality rate, but there are no effective treatments to reverse this situation so far. This study used data from an interrupted phase I/II clinical trial to evaluate safety and immune recovery after INRs were given four infusions, at baseline and at weeks 4, 8, and 20, with human allogeneic mesenchymal stromal cells from adipose tissue (Ad-MSCs). Based on the study design, the first 5 out of 15 INRs recruited received unblinded Ad-MSC infusions. They had a median CD4+ nadir count of 16/µL (range, 2-180) and CD4+ count of 253 cells per microliter (171-412) at baseline after 109 (54-237) months on antiretroviral treatment and 69 (52-91) months of continuous undetectable plasma HIV-RNA. After a year of follow-up, an independent committee recommended the suspension of the study because no increase of CD4+ T-cell counts or CD4+ /CD8+ ratios was observed. There were also no significant changes in the phenotype of different immunological lymphocyte subsets, percentages of natural killer cells, regulatory T cells, and dendritic cells, the inflammatory parameters analyzed, and cellular associated HIV-DNA in peripheral blood mononuclear cells. Furthermore, three subjects suffered venous thrombosis events directly related to the Ad-MSC infusions in the arms where the infusions were performed. Although the current study is based on a small sample of participants, the findings suggest that allogeneic Ad-MSC infusions are not effective to improve immune recovery in INR patients or to reduce immune activation or inflammation. ClinicalTrials.gov identifier: NCT0229004. EudraCT number: 2014-000307-26.

Microscale extraction versus conventional approaches for handling gastrointestinal extracts in oral bioaccessibility assays of endocrine disrupting compounds from microplastic contaminated beach sand.

The unified bioaccessibility method (UBM) was harnessed to assess in vitro oral bioaccessibility pools of dialkyl phthalate congeners (with methyl, -ethyl, -butylbenzyl, -n-butyl, -2-ethylhexyl, and -n-octyl moieties) and bisphenol A at the 17 µg g-1 level in beach sand contaminated with polyethylene microplastics. A variety of sample preparation approaches prior to the analysis of the UBM gastrointestinal extracts, including traditional methods (protein precipitation, liquid-liquid extraction, and solid-phase extraction) and dispersive liquid-liquid microextraction (DLLME) were comprehensively evaluated for clean-up and analyte enrichment. DLLME was chosen among all tested approaches on account of the high extraction efficiency (73-95%, excluding bis(2-ethylhexyl)phthalate and di-n-octyl phthalate), high sample throughput (∼7 min per set of samples), and environmental friendliness as demonstrated by the analytical eco-scale score of 83, and the green analytical procedure index pictogram with green/yellow labeling. The release of the less hydrophobic plastic-laden compounds (dimethyl phthalate, diethyl phthalate and bisphenol A) from the contaminated sample into the body fluids was significant, with bioaccessibility values ranging from 30 to 70%, and from 43 to 74% in gastric and gastrointestinal fluids, respectively, and with relative standard deviation < 17% in all cases. The majority of the compounds were leached during gastric digestion, likely as the combined action of the low pH and the gastric enzymes. The risk exposure analysis revealed that accumulation/concentration in the body fluids is potentially relevant for dimethyl phthalate, diethyl phthalate and bisphenol A, with relative accumulation ratios ranging from 1.1 ± 0.1 to 2.6 ± 0.4. The average daily intake values for the suite of compounds, corrected with the bioaccessibility fraction, ranged from 60 to 430 ng kg of body weight-1·day-1, in all cases, far below the tolerable daily intakes, thus indicating the lack of children health risk by ingestion of microplastic-laden sand with elevated concentrations of plasticizers.