Gabriella d’Ettorre

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

Circulating levels of interleukin-7 in antiretroviral-naïve and highly active antiretroviral therapy-treated HIV-infected patients

Abstract Interleukin (IL)-7 is a critical cytokine regulating T-lymphocyte development, regeneration, and function. Purpose: This study analyzes the endogenous IL-7 production in HIV-infected patients receiving highly active antiretroviral treatment (HAART). Method: Plasma levels of IL-7 were measured by enzyme-linked immunosorbent assay (ELISA) in 11 patients with untreated advanced HIV disease, in 8 patients who successfully responded to HAART, and in 9 individuals with virological and immunological treatment failure. Results: We found that in the patients with advanced HIV disease and no treatment IL-7 concentrations were elevated and were inversely related to both CD4 + and CD8 + T-cell counts. When IL-7 was assessed in treated patients, this cytokine was below the detection limit of the assay in all participants who responded to HAART. On the contrary, patients with evidence of HAART failure had increased concentrations of IL-7 that were comparable to those found in the untreated group with progressive disease. Conclusion: These data suggest that IL-7 may play a role in the immune reconstitution of T-cells during HIV infection, especially in the context of potent antiretroviral treatments.

Maraviroc as intensification strategy in HIV-1 positive patients with deficient immunological response: an Italian randomized clinical trial.

Background Immunological non-responders (INRs) lacked CD4 increase despite HIV-viremia suppression on HAART and had an increased risk of disease progression. We assessed immune reconstitution profile upon intensification with maraviroc in INRs. Methods We designed a multi-centric, randomized, parallel, open label, phase 4 superiority trial. We enrolled 97 patients on HAART with CD4+<200/µL and/or CD4+ recovery ≤25% and HIV-RNA<50 cp/mL. Patients were randomized 1:1 to HAART+maraviroc or continued HAART. CD4+ and CD8+ CD45+RA/RO, Ki67 expression and plasma IL-7 were quantified at W0, W12 and W48. Results By W48 both groups displayed a CD4 increase without a significant inter-group difference. A statistically significant change in CD8 favored patients in arm HAART+maraviroc versus HAART at W12 (p=.009) and W48 (p=.025). The CD4>200/µL and CD4>200/µL + CD4 gain ≥25% end-points were not satisfied at W12 (p=.24 and p=.619) nor at W48 (p=.076 and p=.236). Patients continuing HAART displayed no major changes in parameters of T-cell homeostasis and activation. Maraviroc-receiving patients experienced a significant rise in circulating IL-7 by W48 (p=.01), and a trend in temporary reduction in activated HLA-DR+CD38+CD4+ by W12 (p=.06) that was not maintained at W48. Conclusions Maraviroc intensification in INRs did not have a significant advantage in reconstituting CD4 T-cell pool, but did substantially expand CD8. It resulted in a low rate of treatment discontinuations. Trial Registration ClinicalTrials.gov NCT00884858 http://clinicaltrials.gov/show/NCT00884858

Darunavir/ritonavir and raltegravir coadministered in routine clinical practice: potential role for an unexpected drug interaction

The present study aimed to investigate potential drug interactions between darunavir and raltegravir in patients treated for HIV infection. We enrolled HIV-infected subjects on darunavir-containing regimens that underwent measurement of plasma darunavir trough concentration (12±3 h after dosing). Two groups of patients were compared: those taking darunavir plus a nucleoside/nucleotide backbone (group 1) or a backbone+raltegravir (group 2). Interindividual pharmacokinetic variability was evaluated through the coefficient of variation (CV(inter)). We obtained 156 plasma samples from 63 patients, of which 44 in group 1 and 19 in group 2. Overall, darunavir geometric mean concentration was 2.90 mg/L (95% CI 2.34-3.60) while ritonavir geometric mean concentration was 0.21 mg/L (95% CI 0.17-0.27). We observed a high inter-individual variability in darunavir (CV(inter) 59%) and ritonavir (CV(inter) 103%) plasma levels. Darunavir concentration correlated with concomitant ritonavir levels (r=0.476, p<0.001). Patients in group 1 had a higher darunavir geometric mean concentration than those in group 2 [3.44 mg/L (95% CI 2.79-4.23) versus 1.95 mg/L (95% CI 1.19-3.20), p=0.017]. However, the proportion of subjects with concomitant HIV-RNA <50 copies/mL was higher in group 2 (78.9% versus 47.7%, p=0.028). In a multivariable model, raltegravir co-administration was independently related to a lower darunavir concentration (mean difference -0.25 log(10) mg/L, 95% CI -0.46/-0.04, p=0.020) after adjusting for time from last drug intake and concomitant drugs used. In conclusion, a potential drug interaction between darunavir and raltegravir was observed, although this did not seem virologically significant. For the distinct metabolic pathways of these drugs, its mechanism remains to be determined.

Matrix Metalloproteinase-9 and Tissue Inhibitors of Matrix Metalloproteinase-1 in Plasma of Patients Co-Infected with HCV and HIV

BACKGROUND Accelerated progression of hepatic fibrosis has been shown in patients co-infected with hepatitis C virus (HCV) and HIV. Liver fibrosis is a dynamic process in which the altered balance between matrix metalloproteinases (MMPs) and their specific inhibitors (TIMPs) may play a major role. METHOD The involvement of MMP-9 and TIMP-1 in HCV liver disease progression in patients co-infected with HIV was evaluated. Plasma concentrations of human MMP-9 and TIMP-1 were assessed in 76 HIV-infected patients; 27 were co-infected with HCV and 49 were HCV negative. 18 healthy donors were included as controls. RESULTS Patients with HIV infection exhibited a striking increase in TIMP-1 levels; this is more evident in patients with advanced CD4 depletion. There was no elevation in the plasma concentrations of the MMP-9. The highest levels of TIMP-1 were found in the HIV/HCV co-infected patients. The values of TIMP-1 in HIV-infected patients with chronic HCV hepatitis were significantly higher than in HIV-positive individuals without HCV infection, even including those with low CD4 count. No significant differences were seen in the MMP-9 levels. CONCLUSION These findings suggest that the altered balance between circulating MMP-9 and TIMP-1 during HIV infection may play an important role in exacerbating liver fibrosis progression in patients co-infected with HCV.Abstract Background: Accelerated progression of hepatic fibrosis has been shown in patients co-infected with hepatitis C virus (HCV) and HIV. Liver fibrosis is a dynamic process in which the altered balance between matrix metalloproteinases (MMPs) and their specific inhibitors (TIMPs) may play a major role. Method: The involvement of MMP-9 and TIMP-1 in HCV liver disease progression in patients co-infected with HIV was evaluated. Plasma concentrations of human MMP-9 and TIMP-1 were assessed in 76 HIV-infected patients; 27 were co-infected with HCV and 49 were HCV negative. 18 healthy donors were included as controls. Results: Patients with HIV infection exhibited a striking increase in TIMP-1 levels; this is more evident in patients with advanced CD4 depletion. There was no elevation in the plasma concentrations of the MMP-9. The highest levels of TIMP-1 were found in the HIV/HCV co-infected patients. The values of TIMP-1 in HIV-infected patients with chronic HCV hepatitis were significantly higher than in HIV-positive individuals without HCV infection, even including those with low CD4 count. No significant differences were seen in the MMP-9 levels. Conclusion: These findings suggest that the altered balance between circulating MMP-9 and TIMP-1 during HIV infection may play an important role in exacerbating liver fibrosis progression in patients co-infected with HCV.

Safety and efficacy of treatment switch to raltegravir plus tenofovir/emtricitabine or abacavir/lamivudine in patients with optimal virological control: 48-week results from a randomized pilot study (Raltegravir Switch for Toxicity or Adverse Events, RASTA Study)

Abstract Background: The Raltegravir Switch for Toxicity or Adverse Events (RASTA) Study is a 2-arm randomized pilot study exploring the safety and efficacy at 48 weeks of a treatment switch to raltegravir associated with tenofovir/emtricitabine or abacavir/lamivudine in patients with regimens with optimal virological control. Methods: Patients treated with stable protease inhibitor (PI)-, non-nucleoside reverse transcriptase inhibitor (NNRTI)-, or nucleoside reverse transcriptase inhibitor (NRTI)-based regimens, with HIV-RNA levels < 50 copies/ml for ≥ 3 months and a CD4 cell count > 200 cells/μl were eligible. Enrollment of 40 patients was planned: at baseline patients were randomized 1:1 to switch to raltegravir plus tenofovir/emtricitabine (arm A) or abacavir/lamivudine (arm B). Laboratory parameters, raltegravir plasma levels, self- reported adherence, quality of life parameters, neurocognitive performance, bone composition, and body fat distribution were monitored. Virological failure was defined as HIV-RNA > 50 copies/ml on 2 consecutive determinations. Results: After 48 weeks, 5/40 (12.5%) regimen discontinuations occurred: 2 were for low-level viremia virological failure (both in arm A, at weeks 24 and 48) and 3 were for adverse events (neurological disturbances and skin rash in arm B; proximal tubulopathy in arm A). Overall, a significant CD4 increase was observed at weeks 36 and 48, and a significant decrease in total cholesterol, non-high density lipoprotein cholesterol, and triglycerides was observed at each study visit. Physical health/satisfaction in therapy scores and neuropsychological performance improved. The lumbar column Z-score improved, with no modification in other bone composition and fat distribution parameters. Conclusions: The investigated switch strategy was associated with rare virological failure. Improvements in lipid levels, quality of life measures, neuropsychological performance, and bone composition suggest good tolerability of raltegravir-based regimens.

HIV-associated progressive multifocal leukoencephalopathy: longitudinal study of JC virus non-coding control region rearrangements and host immunity

John Cunningham virus (JCV), the etiological agent of progressive multifocal leukoencephalopathy (PML), contains a hyper-variable non-coding control region usually detected in urine of healthy individuals as archetype form and in the brain and cerebrospinal fluid (CSF) of PML patients as rearranged form. We report a case of HIV-related PML with clinical, immunological and virological data longitudinally collected. On admission (t0), after 8-week treatment with a rescue highly active antiretroviral therapy (HAART), the patient showed a CSF-JCV load of 16,732 gEq/ml, undetectable HIV-RNA and an increase of CD4+ cell count. Brain magnetic resonance imaging (MRI) showed PML-compatible lesions without contrast enhancement. We considered PML-immune reconstitution inflammatory syndrome as plausible because of the sudden onset of neurological symptoms after the effective HAART. An experimental JCV treatment with mefloquine and mirtazapine was added to steroid boli. Two weeks later (t1), motor function worsened and MRI showed expanded lesions with cytotoxic oedema. CSF JCV-DNA increased (26,263 gEq/ml) and JCV viremia was detected. After 4 weeks (t2), JCV was detected only in CSF (37,719 gEq/ml), and 8 weeks after admission (t3), JC viral load decreased in CSF and JCV viremia reappeared. The patient showed high level of immune activation both in peripheral blood and CSF. He died 4 weeks later. Considering disease progression, combined therapy failure and immune hyper-activation, we finally classified the case as classical PML. The archetype variant found in CSF at t0/t3 and a rearranged sequence detected at t1/t2 suggest that PML can develop from an archetype virus and that the appearance of rearranged genotypes contribute to faster disease progression.

ISG15 expression correlates with HIV-1 viral load and with factors regulating T cell response

Given the multifactorial nature of action of type I interferon (IFN) in HIV-1 infection and the need to firmly establish the action of key components of IFN pathways, we compared the IFN stimulated gene (ISG)15 expression with that of other well-characterized ISGs, evaluating its relationship with immunosuppressive factors regulating T-cell response in HIV-1 patients. PBMC from 225 subjects were included: healthy donors (n=30), naïve (n=93) and HAART treated HIV-1 subjects (n=102). Levels of ISG15-mRNA, ISG56-mRNA, APOBEC3G/3F-mRNA, TRAIL-mRNA, IDO-mRNA, proviral load andISG15 (rs15842 and rs1921) SNPs were evaluated by using TaqMan assays. We found that ISG15, ISG56, APOBEC3G/3F levels were increased in untreated HIV-1 patients compared to healthy donors, being ISG15 the highest ISG expressed. The amount of ISG15 correlated with viral load and with CD4+ T cell counts whereas no relationship was found between all ISGs analyzed and proviral load or HIV-1 tropism. ISG15 expression was reduced following long-term antiretroviral therapy. In addition, ISG15 levels were correlated with those of TRAIL and IDO in HIV-1 viremic patients. Lastly, ISG15 SNPs had no influence on ISG15 levels. We demonstrates that ISG15 is elevated in viremic HIV-1 patients and is associated with high TRAIL and IDO levels.

Interleukin-32 isoforms: Expression, interaction with interferon-regulated genes and clinical significance in chronically HIV-1-infected patients

Given the growing evidence for a role of interleukin-32 (IL-32) in the immune response to HIV-1 infection and its interplay with type I and III interferons (IFNs), we studied the gene expression of IL-32 isoforms (α and nonα) in untreated chronically HIV-1-infected patients and in gender- and age-matched healthy individuals. To further characterize both the anti-HIV properties of IL-32 and the cytokine’s relationship with host antiviral innate immune responses, we evaluated whether IL-32 can induce ex vivo the expression of antiviral IFN-induced genes (ISGs), namely myxovirus resistance A (MxA), and apolipoprotein B mRNA-editing enzyme catalytic (APOBEC)3G and APOBEC3F. We also investigated whether in vivo IL-32 (α and nonα) mRNA levels were correlated with those of MxA and APOBEC3G/3F. Results indicated that IL-32 (α and nonα) mRNA levels were significantly higher in HIV-1-infected patients than in healthy individuals. Furthermore, IL-32 (α and nonα) mRNA levels correlated negatively with HIV RNA levels, but not with the CD4+ T-cell count. Our ex vivo studies disclosed that ISGs mRNA levels were increased after IL-32γ treatment of peripheral blood mononuclear cells. Interestingly, significant positive correlations were found between transcript levels of both IL-32α and IL-32nonα and those of MxA and APOBEC3G/3F in untreated chronically HIV-1-infected patients. Overall, our results demonstrated that IL-32 isoforms are highly expressed during chronic HIV-1 infection and that IL-32 could have a central role in the antiviral immune response against HIV-1.

Presepsin as a potential marker for bacterial infection relapse in critical care patients. A preliminary study.

Abstract Background: Systemic bacterial infection carries a high risk of mortality in critical care patients. Improvements in diagnostic procedures are required for effective management of sepsis. Recently, the soluble CD14 subtype, or presepsin, has been suggested as a reliable marker of sepsis, and we set out to compare its diagnostic performance with that of procalcitonin (PCT). We focused on a cohort of septic patients who, during their hospitalization, relapsed after a period of clinical relief from symptoms. Methods: In total 21 adult patients were studied during their hospitalization in the Critical Care Unit of Policlinico Umberto I hospital; 74 plasma samples were collected at multiple time points, and presepsin levels were measured using a PATHFAST® analyzer. Results: Presepsin and PCT were significantly lower in healthy controls than in sepsis or severe sepsis (p<0.001), both enabled a significant difference to be detected between systemic inflammatory response syndrome (SIRS) and severe sepsis (p<0.05). The area under the curve (AUC) calculated from the receiver operating characteristic (ROC) curve analysis was 0.888 for presepsin and 0.910 for PCT. In those patients in whom a clinical recurrence of sepsis was observed, while PCT levels normalized during the transient remission phase, presepsin levels (>1000 pg/mL) remained high. Conclusions: This study confirms the importance of monitoring a combination of several biomarkers in order to obtain a reliable diagnosis. Maximal presepsin levels could alert clinicians not to suspend antibiotic treatments and to carefully monitor septic patients’ state of health, even after clinical symptoms have disappeared and PCT levels returned to normal.