Inflammation and intracellular exposure of dolutegravir, darunavir, tenofovir and emtricitabine in people living with HIV.

AIMS: Antiretroviral (ARV) therapy reduces inflammation and immune activation in people with HIV, but not down to the levels observed in people without HIV. Limited drug penetration within tissues has been argued as a potential mechanism of persistent inflammation. Data on the inflammation role on ARV plasma/intracellular (IC) pharmacokinetics (PK) through to expression of cytochrome P450 3A/membrane transporters are limited. The aim of this study was to investigate the correlation between inflammation markers (IM) and plasma/IC PK of ARV regimen in HIV-positive patients. METHODS: We included ART-experienced patients switching to three different ARV regimens. Plasma and IC ARV drug concentration means at the end of dosing interval (T0 ), IM on samples concomitantly with ARV PK determination: sCD14, CRP, IL-6 and LPS were analysed. RESULTS: Plasma and IC drug concentrations were measured in 60 samples. No significative differences between CRP, sCD14, IL-6 and LPS values in the three arms were observed. A significant inverse correlation between tenofovir plasma concentration and sCD14 (rho = -0.79, P < .001), and between DRV IC/plasma ratio and Log10 IL-6 concentrations (rho = -0.36, P = .040), and a borderline statistically significant positive trend between DRV plasma concentration and sCD14 (rho = 0.31, P = .070) were suggested. Furthermore, a borderline statistically significant inverse trend between DTG IC concentrations and sCD14 (rho = -0.34, P = .090) was observed in 24 patients on DTG-based triple therapy. CONCLUSIONS: Our preliminary data support the hypothesis of lower DRV and DTG IC concentrations and lower TFV plasma exposure in patients with higher plasma IM suggesting an interplay between HIV drug penetration and persistent inflammation in cART-treated HIV-positive patients.

A Novel Platform for Immune Tolerance Induction in Hemophilia A Mice.

Hemophilia A (HA) is an X-linked bleeding disease caused by factor VIII (FVIII) deficiency. We previously demonstrated that FVIII is produced specifically in liver sinusoid endothelial cells (LSECs) and to some degree in myeloid cells, and thus, in the present work, we seek to restrict the expression of FVIII transgene to these cells using cell-specific promoters. With this approach, we aim to limit immune response in a mouse model by lentiviral vector (LV)-mediated gene therapy encoding FVIII. To increase the target specificity of FVIII expression, we included miRNA target sequences (miRTs) (i.e., miRT-142.3p, miRT-126, and miRT-122) to silence expression in hematopoietic cells, endothelial cells, and hepatocytes, respectively. Notably, we report, for the first time, therapeutic levels of FVIII transgene expression at its natural site of production, which occurred without the formation of neutralizing antibodies (inhibitors). Moreover, inhibitors were eradicated in FVIII pre-immune mice through a regulatory T cell-dependent mechanism. In conclusion, targeting FVIII expression to LSECs and myeloid cells by using LVs with cell-specific promoter minimized off-target expression and immune responses. Therefore, at least for some transgenes, expression at the physiologic site of synthesis can enhance efficacy and safety, resulting in long-term correction of genetic diseases such as HA.

Primary HIV infection features colonic damage and neutrophil inflammation yet containment of microbial translocation.

INTRODUCTION: Impairment of the gastrointestinal barrier leads to microbial translocation and peripheral immune activation, which are linked to disease progression. Data in the setting of primary HIV/SIV infection suggest that gut barrier damage is one of the first events of the pathogenic cascade, preceding mucosal immune dysfunction and microbial translocation. We assessed gut structure and immunity as well as microbial translocation in acutely and chronically-infected, combination antiretroviral therapy (cART)-naive individuals. METHODS: Fifteen people with primary HIV infection (P-HIV) and 13 with chronic HIV infection (C-HIV) c-ART-naive participants were cross-sectionally studied. Gut biopsies were analysed in terms of gut reservoirs (total, integrated and unintegrated HIV DNA); tight junction proteins (E-cadherin, Zonula Occludens-1), CD4 + expression, neutrophil myeloperoxidase (histochemical staining); collagen deposition (Masson staining). Flow cytometry was used to assess γδ T-cell frequency (CD3 + panγδ+Vδ1+/Vδ2+). In plasma, we measured microbial translocation (LPS, sCD14, EndoCAb) and gut barrier function (I-FABP) markers (ELISA). RESULTS: P-HIV displayed significantly higher tissue HIV DNA, yet neutrophil infiltration and collagen deposition in the gut were similar in the two groups. In contrast, microbial translocation markers were significantly lower in P-HIV compared with C-HIV. A trend to higher mucosal E-cadherin, and gut γδ T-cells was also observed in P-HIV. CONCLUSION: Early HIV infection features higher HIV DNA in the gut, yet comparable mucosal alterations to those observed in chronic infection. In contrast, microbial translocation is contained in primary HIV infection, likely because of a partial preservation of E-cadherin and mucosal immune subsets, namely γδ T-cells.

Gamma-Delta T-Cell Phenotype and Function in DAA-Treated HIV-HCV Co-Infected and HCV-Mono-Infected Subjects.

HIV-HCV co-infected subjects are at risk of liver fibrosis which may be linked to immune imbalances. Direct-acting antivirals (DAAs) represent the mainstay of HCV treatment in co-infected individuals, yet their effects on immune cell populations playing a role in fibrogenesis is unknown. We assessed γδ T-cell phenotype and function, Treg and Th17 frequencies, as well as γ-globulins and B-cell activation in 47 HIV-HCV co-infected and 35 HCV mono-infected individuals prior to and following DAA treatment (SVR12). Γδ T-cell activation decreased in both groups yet persisted at higher levels in the HIV-HCV co-infected subjects. No differences were registered in terms of γδT-cell function. Of note, the Vδ2/Th17 ratio, inversely linked to liver damage, increased significantly in the two groups upon treatment, yet a negative correlation between the Vδ2/Th17 ratio and liver function enzymes was found in the co-infected subjects alone. B-cell activation and γ-globulin levels decreased in both settings, yet B-cell activation remained higher in the HIV-HCV co-infected individuals. In HIV-HCV co-infected and HCV mono-infected participants, the effect of DAA was limited to γδ T- and B-cell activation as well as γ-globulin concentrations and the Vδ2/Th17 ratio, with no changes in γδ T-cell function and Treg frequencies. Importantly, γδ T- and B-cell activation remained at higher levels in the co-infected individuals than in those with HCV mono-infection alone. The persistence of such alterations within these cell subsets may be associated with the risk of hepatic and extrahepatic complications.

Predictors of low ovarian reserve in cART-treated women living with HIV.

ABSTRACT: Ovarian dysfunction and lower circulating anti-Müllerian hormone (AMH) feature women living with HIV (WLWH). Because treated human immunodeficiency virus (HIV) infection is characterized by a pro-inflammatory/oxidative phenotype resulting in residual comorbidity, we sought to investigate possible associations between plasma AMH and markers of inflammation, immune activation/senescence/exhaustion, oxidative stress as well as comorbidities in a cohort of combined anti-retroviral therapy (cART)-treated WLWH versus age-matched HIV-uninfected, healthy women.Eighty WLWH on effective cART aged 25 to 50 years and 66 age-matched healthy women were enrolled. We measured: plasma AMH, IL-6, reactive oxygen species modulator 1 (ROMO1) (ELISA); plasma tumor necrosis factor α, IL-10, soluble vascular cell adhesion molecule 1, osteopontin (Luminex); CD4/CD8 activation (CD38/CD69), apoptosis (CD95), exhaustion (PD1), maturation (CD45RA/CD45R0/CD127/CCR7), recent thymic emigrants (CD31/CD103) (flow cytometry). Mann Whitney and chi-squared tests were used. Univariate and multivariate logistic regression analyses were used to assess factors associated with low AMH (≤1 ng/mL).Compared to healthy women, WLWH were more frequently non-Caucasian, drug/alcohol abusers, with history of late menarche, lower hormonal contraceptive use, with higher gravidity and lower parity. WLWH showed significantly lower AMH (P = .004) as well as higher ROMO1 (P = .0003) and tumor necrosis factor α (P < .0001). The multivariate analyses revealed ROMO1 (adjusted odds ratio [AOR]: 1.42, P = .03) and HIV infection (AOR: 8.1, P = .0001) as independently associated with low AMH. The logistic regression model with both HIV status and ROMO1 (a marker of oxidative stress) confirmed HIV as the only predictor of low AMH (AOR: 17, P = .0003).Despite effective cART, WLWH showed lower AMH compared to age-matched peers, indicating pre-mature ovarian ageing. Both HIV and oxidative stress are independently associated with low AMH, emphasizing the impact of HIV-associated oxidative stress on reproductive aging.

Plasmacytoid Dendritic Cells Depletion and Elevation of IFN-γ Dependent Chemokines CXCL9 and CXCL10 in Children With Multisystem Inflammatory Syndrome.

Background: SARS-CoV-2 occurs in the majority of children as COVID-19, without symptoms or with a paucisymptomatic respiratory syndrome, but a small proportion of children develop the systemic Multi Inflammatory Syndrome (MIS-C), characterized by persistent fever and systemic hyperinflammation, with some clinical features resembling Kawasaki Disease (KD). Objective: With this study we aimed to shed new light on the pathogenesis of these two SARS-CoV-2-related clinical manifestations. Methods: We investigated lymphocyte and dendritic cells subsets, chemokine/cytokine profiles and evaluated the neutrophil activity mediators, myeloperoxidase (MPO), and reactive oxygen species (ROS), in 10 children with COVID-19 and 9 with MIS-C at the time of hospital admission. Results: Patients with MIS-C showed higher plasma levels of C reactive protein (CRP), MPO, IL-6, and of the pro-inflammatory chemokines CXCL8 and CCL2 than COVID-19 children. In addition, they displayed higher levels of the chemokines CXCL9 and CXCL10, mainly induced by IFN-γ. By contrast, we detected IFN-α in plasma of children with COVID-19, but not in patients with MIS-C. This observation was consistent with the increase of ISG15 and IFIT1 mRNAs in cells of COVID-19 patients, while ISG15 and IFIT1 mRNA were detected in MIS-C at levels comparable to healthy controls. Moreover, quantification of the number of plasmacytoid dendritic cells (pDCs), which constitute the main source of IFN-α, showed profound depletion of this subset in MIS-C, but not in COVID-19. Conclusions: Our results show a pattern of immune response which is suggestive of type I interferon activation in COVID-19 children, probably related to a recent interaction with the virus, while in MIS-C the immune response is characterized by elevation of the inflammatory cytokines/chemokines IL-6, CCL2, and CXCL8 and of the chemokines CXCL9 and CXL10, which are markers of an active Th1 type immune response. We believe that these immunological events, together with neutrophil activation, might be crucial in inducing the multisystem and cardiovascular damage observed in MIS-C.