Sara Serafino

Probiotics Reduce Inflammation in Antiretroviral Treated, HIV-Infected Individuals: Results of the “Probio-HIV” Clinical Trial

Background HIV infection results in damage to the gastrointestinal (GI) tract, microbial translocation and immune activation. These are not completely normalized with combined antiretroviral therapy (cART). Moreover, increate morbidity and mortality of cART-treated HIV-infected individuals is associated with inflammation. Methods In order to enhance GI tract immunity, we recruited and treated 20 HIV-infected humans with cART supplemented with probiotics and followed inflammation and immunological parameters (clinical trial number NCT02164344). 11 HIV seronegative subjects were included as control group. The enumeration of CD4+, CD8+, CD38+ and HLA-DR+ lymphocytes were evaluated on peripheral blood; HIV-RNA levels, sCD14, d-dimer, C-reactive protein (CRP) high sensitivity C-reactive protein (hsCRP), IL-6 and Lipopolysaccharide Binding Protein (LBP) were assayed on plasma. Results We observe that cART does not normalize the levels of immune activation in HIV positive patients anyway inflammation and markers of microbial translocation were significantly reduced with probiotic supplementation. Patients show a clear and statistically significant reduction in the levels of immune activation on CD4 T-lymphocytes, for both markers CD38 and HLA-DR and their simultaneous expression, LBP and hsCRP plasma levels after probiotic diet supplementation settling to values comparable to controls. Conclusions Supplementing cART with probiotics in HIV-infected individuals may improve GI tract immunity and there by mitigate inflammatory sequelae, ultimately improving prognosis. Trial Registration ClinicalTrials.gov NCT02164344

Probiotic supplementation promotes a reduction in T-cell activation, an increase in Th17 frequencies, and a recovery of intestinal epithelium integrity and mitochondrial morphology in ART-treated HIV-1-positive patients

HIV infection is characterized by a persistent immune activation associated to a compromised gut barrier immunity and alterations in the profile of the fecal flora linked with the progression of inflammatory symptoms. The effects of high concentration multistrain probiotic (Vivomixx®, Viale del Policlinico 155, Rome, Italy in EU; Visbiome®, Dupont, Madison, Wisconsin in USA) on several aspects of intestinal immunity in ART‐experienced HIV‐1 patients was evaluated.

What happens to cardiovascular system behind the undetectable level of HIV viremia

Despite the combined antiretroviral therapy has improved the length and quality of life of HIV infected patients, the survival of these patients is always decreased compared with the general population. This is the consequence of non-infectious illnesses including cardio vascular diseases. In fact large studies have indicated an increased risk of coronary atherosclerotic disease, myocardial infarction even in HIV patients on cART. In HIV infected patients several factors may contribute to the pathogenesis of cardiovascular problems: life-style, metabolic parameters, genetic predisposition, viral factors, immune activation, chronic inflammation and side effects of antiretroviral therapy. The same factors may also contribute to complicate the clinical management of these patients. Therefore, treatment of these non-infectious illnesses in HIV infected population is an emerging challenge for physicians. The purpose of this review is to focus on the new insights in non AIDS-related cardiovascular diseases in patients with suppressed HIV viremia.

Probiotics Differently Affect Gut-Associated Lymphoid Tissue Indolamine-2,3-Dioxygenase mRNA and Cerebrospinal Fluid Neopterin Levels in Antiretroviral-Treated HIV-1 Infected Patients: A Pilot Study

Recently the tryptophan pathway has been considered an important determinant of HIV-1 infected patients’ quality of life, due to the toxic effects of its metabolites on the central nervous system (CNS). Since the dysbiosis described in HIV-1 patients might be responsible for the microbial translocation, the chronic immune activation, and the altered utilization of tryptophan observed in these individuals, we speculated a correlation between high levels of immune activation markers in the cerebrospinal fluid (CSF) of HIV-1 infected patients and the over-expression of indolamine-2,3-dioxygenase (IDO) at the gut mucosal surface. In order to evaluate this issue, we measured the levels of neopterin in CSF, and the expression of IDO mRNA in gut-associated lymphoid tissue (GALT), in HIV-1-infected patients on effective combined antiretroviral therapy (cART), at baseline and after six months of probiotic dietary management. We found a significant reduction of neopterin and IDO mRNA levels after the supplementation with probiotic. Since the results for the use of adjunctive therapies to reduce the levels of immune activation markers in CSF have been disappointing so far, our pilot study showing the efficacy of this specific probiotic product should be followed by a larger confirmatory trial.

Analysis of Th17 and Tc17 Frequencies and Antiviral Defenses in Gut-Associated Lymphoid Tissue of Chronic HIV-1 Positive Patients

The complex relationship between both the Th1/Th17 and Tc1/Tc17 axis and innate defences in the intestinal mucosa during HIV-1 infection has not been well characterized. This study examined the frequency, phenotype, and functional status of T cell populations in the gut-associated lymphoid tissue and peripheral blood of virologically suppressed HIV-1-infected patients on therapy, focusing on the Th1, Th17, Tc1, and Tc17 cell subsets. We found a persistent immune cell activation (CD38 and HLADR expression) into the GALT despite the higher levels of Th17 and Tc17 in respect to peripheral blood. An upregulation of type I IFN response in GALT compared to the peripheral blood compartment was also recorded. Furthermore, IFN-α/β levels were negatively related to the frequencies of Th1 naïve cells and Tc1 cell subsets (naïve, central memory, and effector memory) in the GALT. In contrast, no relationships between type I IFN response and Th1 or Tc1 cell subsets in peripheral blood compartment and between IFN-α/β and Th17/Tc17 in both GALT and peripheral blood district were recorded. These data indicate that prolonged antiretroviral treatment improves GALT immune function despite the persistence of immune activation and type I IFN response in chronic HIV-1 positive patients.

A pilot study on the effects of probiotic supplementation on neuropsychological performance and microRNA‐29a‐c levels in antiretroviral‐treated HIV‐1‐infected patients

The gut microbiota is involved in the regulation of cognition, mood, anxiety, and pain, and can impact cognitive functions by producing neuroactive substances or releasing bacterial by‐products and metabolites. No information is available on the effects of a probiotic supplementation on brain function of HIV+ subjects. In light of the above considerations, we performed a pilot study in cART‐treated HIV‐1‐positive patients with long‐term virologic suppression. The aims were to analyze the effect of high‐concentration multistrain probiotic supplementation (Vivomixx®; Visbiome®) on several neurocognitive abilities and to evaluate the safety of this supplementation.

Impact of switching from lopinavir/ritonavir to boosted and un-boosted atazanavir on glucose metabolism: the ATAzanavir & GLUcose metabolism (ATAGLU) study

Previous studies have reported that protease inhibitors (PIs) can contribute to glycaemic alterations. However, there are few trials examining the direct effect of a single PI. The objective of the study was to evaluate the modifications of glucose and lipid profiles after a switch from lopinavir/ritonavir (LPV/r) to atazanavir, used as ritonavir-boosted (ATV/r) or un-boosted. We conducted a retrospective observational cohort study on the effect of ATV/(r) on glycaemic metabolism (ATAGLU) in patients with undetectable levels of HIV-RNA who switched from LPV/r. In total, 235 patients treated for 48 weeks with LPV/r plus two nucleoside reverse transcriptase inhibitors (NRTIs) and with undetectable HIV-RNA were included: 134 continued LPV/r after the initial 48 weeks and 101 switched to ATV(/r) (18.3% to ATV; 24.7% to ATV/r). A significant decrease in mean glucose level and insulin resistance was observed in patients who switched to ATV(/r). The mean cholesterol triglyceride levels increased in the LPV/r group and decreased among the patients who switched. A significant increase of CD4 T cells with undetectable levels of HIV-RNA was observed in all groups. The long-term results obtained in this real-life study suggest that patients who have achieved initial suppression on a regimen including LPV/r + two NRTIs can switch to ATV/(r) + two NRTIs with an improvement in lipid and glycaemic metabolism.

Dynamics of HIV DNA and residual viremia in patients treated with a raltegravir-containing regimen.

To the Editors: Current antiretroviral therapy (ART) can reduce plasma HIV-1 RNA levels to below the detection limit of clinical assay (50 copies of HIV-1 RNA), but HIV-1 DNA remains detectable in patients during suppressive ART. HIV-1 persistence despite ART is likely the consequence of a reservoir of latently infected cells established early in infection and cryptic viral replication presumably occurring in anatomical sanctuary sites and facilitated by cell-to-cell transmission. In addition to HIV-1 persistence in T CD4+ cells and other reservoirs, most patients on ART with HIV-1 RNA levels below the detection limit of commercial assays have low-level residual viremia. The origin of residual viremia is uncertain, but it may arise, at least in part, from latently infected cells induced to produce HIV-1. Several studies have addressed the impact of ART on HIV DNA and the relation between the size of the latent reservoir and residual viremia. In particular, the effect of ART on HIV-1 DNA has been studied in patients treated with raltegravir (RAL)-based therapy. Some studies showed that in the presence of RAL, linear HIV-1 cDNA is prevented from integrating into chromatin and is subsequently converted to episomal cDNA. Therefore, an increase in episomal cDNA occurs when active replication is blocked by integrase inhibitors. However, another study found that the switch to an RALcontaining regimen was not associated with a significant change in the levels of HIV-1 2-long terminal repeat (LTR) circles, and no significant changes were observed in HIV-1 total DNA. By contrast, Charpentier et al reported that a RAL-containing regimen in drug-experienced patients was associated with a rapid HIV-1 DNA decrease. All together, these data suggest that the impact of RAL-based therapy on HIV-1 DNA level is still unclear. A number of studies have also evaluated the relationship between HIV1 DNA and residual viremia, finding a positive correlation between these 2 virological parameters. Nevertheless, additional studies are needed to establish whether the association between residual viremia and latent reservoirs persists after long-term suppression of HIV-1 replication. This study analyzed total and integrated HIV-1 DNA levels for up to 196 weeks in patients initiating a RALcontaining regimen, investigating the relationship between residual viremia and the size of the latent reservoir. Specifically, 30 treatmentexperienced HIV-1–infected patients attending Policlinico Umberto I Hospital were retrospectively selected according to the following criteria: (1) receiving a RAL-containing regimen, (2) virological response to RAL-based therapy, (3) follow-up of approximately 196 weeks, and (4) availability of at least 3 peripheral blood mononuclear cell samples collected during follow-up. The main reasons for initiating a RAL-based therapy were virological failure (18 patients) and side effects to the previous therapeutic regimen (12 patients). In addition to RAL, the optimized background therapy contained at least 1 active ARV drug according to the genotypic resistance test. Patients had been infected for a median time of 14 years [interquartile range (IQR), 8–16] and had a long therapeutic history [median: 14 years (IQR, 9–17)]. The median CD4 nadir was 206 cells per microliter (IQR, 113– 700). At baseline, the median CD4 cell count was 380 cells per milliliter (IQR, 152–741). At W196, the median increase in CD4 T-cell count was 124 cells per microliter (IQR, 78–171). At the beginning of the study, the median HIV-1 RNA level was 6777 copies per milliliter (IQR, 1193–14,602). At the end of the follow-up, all participants showed levels of plasma viremia below 37 copies per milliliter (Siemens Healthcare Diagnostic, Tarrytown, NY). Total cell DNA was extracted from frozen peripheral blood mononuclear cells (PBMC) pellets by the DNeasy Blood & Tissue kit (Qiagen, Milan, Italy). Total and integrated HIV1 DNA were quantified by in-house realtime PCR at baseline (W0) and after mean times (6SD) of 52 weeks (68), 96 weeks (612), 144 weeks (616), and 196 weeks (616) from the start of RALbased therapy. Total HIV-1 DNA was quantified using primers (NEC 152 and NEC 131) direct in the LTR gene. Integrated HIV DNA was quantified using an Alu-gag PCR followed by a real-time PCR for LTR gene. At the same time, b-glucuronidase PCR was performed to rule out the presence of inhibitors. To quantify HIV-1 DNA, we used a standard curve (5-fold dilutions of 8E5 cells DNA), and all samples from each patient were tested in triplicate in the same assay. Results were expressed as number of HIV-1 DNA copies/106 PBMC. Plasma HIV-1 RNA loads at W0, W52, and W96 were measured with branched DNA (Siemens Healthcare Diagnostic) showing a dynamic range of 50–500,000 copies per milliliter. At W144 and W196, a more sensitive real-time PCR assay (Versant kPCR; Siemens Healthcare Diagnostic) was used. This assay quantifies the viral load over the range of 37–11,000,000 copies per milliliter and reports qualitative results below the lower limit of quantification as “Target Detected” and Target Not Detected, suggesting the presence or absence of residual viremia. To determine the level of residual viremia, the real-time PCR assay was performed in triplicate for each sample studied to minimize the quantification error associated with the detection of extremely low values. Supported by “Sapienza” University Grant to G.A. Presented at the 24th European Congress of Clinical Microbiology and Infectious Diseases, May 10–13, 2014, Barcelona, Spain. The authors have no conflicts of interest to disclose.

Dominant enrichment of phenotypically activated CD38+HLA‐DR+CD8+ T cells, rather than CD38+HLA‐DR+CD4+ T cells, in HIV/HCV coinfected patients on antiretroviral therapy

HIV infection may enhance immune‐activation, while little is known regarding the role of HCV infection. This study investigates the impact of HCV in HIV coinfected patients with undetectable viraemia under HAART on the levels of peripheral T cells immune‐activation. We determined T lymphocytes subsets to characterize immune‐activation defined as CD38 and/or HLA‐DR expression in chronic monoinfected HCV, HIV, and HIV/HCV coinfected subjects. One hundred and fifty six patients were divided into three groups: (i) 77 HIV+ patients; (ii) 50 HCV+ patients; and (iii) 29 coinfected HIV/HCV patients. The level of CD4+ was significantly higher in HCV+ than in HIV+ or in coinfected HIV/HCV subjects. The frequencies of CD4+CD38+/HLA‐DR‐, CD4+CD38‐/HLA‐DR+ and CD4+CD38+/HLA‐DR+ in HIV+ patients were comparable to those measured in coinfected patients, but statistically higher than those observed in HCV+ subjects. The percentage of CD8+ was comparable in HIV‐1+ patients and coinfected HIV/HCV but the results obtained in both groups were significantly higher compared to the results obtained in HCV patients. The level of CD8+CD38+/HLA‐DR‐ showed values lower in HIV+ patients than in that monoinfected HCV and coinfected HIV/HCV patients. The frequencies of CD8+CD38‐/HLA‐DR+ were higher in HIV+ patients compared to HCV+ and coinfected HIV/HCV patients. HIV/HCV coinfected group showed highest levels of CD8+CD38+/HLA‐DR+. HIV plays a pivotal role to determine the immune activation in the host. The role of HCV needs of further investigations but our data show that HCV mainly influences the immune‐activation of the pool of CD8, but also probably plays a supporting additive effect on CD4 immune‐activation. J. Med. Virol. 88:1347–1356, 2016.

Direct-acting antiviral therapy enhances total CD4+ and CD8+ T-cells responses, but does not alter T-cells activation among HCV mono-infected, and HCV/HIV-1 co-infected patients

AIM Chronic immune activation and poor T-cell immune response are strongly associated with disease progression and pathogenesis of both hepatitis C virus (HCV) and human immunodeficiency virus (HIV)-1 infections. Little is known about the impact of anti-HCV Interferon (IFN)-free direct-acting antiviral (DAA) therapy on the systemic T-cells activation and patterns of peripheral T-cells producing pro-inflammatory cytokines. PATIENTS AND METHODS Forty-five subjects including 18 HCV mono-infected, 17 HCV/HIV-1 co-infected patients under antiretroviral therapy (ART), and 10 healthy controls (HCs) were recruited. Blood samples were collected at baseline (T0) and 12 weeks after the end of DAA therapy (T1). Cell phenotypes (CD3, CD4, CD8), activation markers (CD38 and HLA-DR), and frequency of IFN-γ, interleukin (IL)-17, and IL-22 producing CD4+ and CD8+ T-cells were measured by flow cytometry. Plasma levels of related cytokines were also measured by enzyme-linked immunosorbent assay (ELISA). RESULTS Both HCV, and HCV/HIV-1 patients before and after therapy, showed significant higher percentages of any T-cell subset expressing CD38 and/or HLA-DR compared to HCs. No differences were observed in T-cells activation at T1 compared to T0 in patient groups, and when HCV patients were compared to HCV/HIV-1 group (P>0.05). After therapy, the potential of total circulating T helper (Th) and T cytotoxic (Tc) cells producing IFN-γ, IL-17, and IL-22 were increased. Plasma level of IFN-γ at baseline was showed difference compared to HCs, and significantly reduced after therapy (P<0.05). CONCLUSION Total T-cells immune response enhances after therapy, however, the state of immune activation may remain elevated for a longtime after the end of treatment and contribute to post-Sustained Virologic Response (SVR) consequences.