Riccardo Freguja

Immune Activation in HIV-Infected Aging Women on Antiretrovirals—Implications for Age-Associated Comorbidities: A Cross-Sectional Pilot Study

Background Persistent immune activation and microbial translocation associated with HIV infection likely place HIV-infected aging women at high risk of developing chronic age-related diseases. We investigated immune activation and microbial translocation in HIV-infected aging women in the post-menopausal ages. Methods Twenty-seven post-menopausal women with HIV infection receiving antiretroviral treatment with documented viral suppression and 15 HIV-negative age-matched controls were enrolled. Levels of immune activation markers (T cell immune phenotype, sCD25, sCD14, sCD163), microbial translocation (LPS) and biomarkers of cardiovascular disease and impaired cognitive function (sVCAM-1, sICAM-1 and CXCL10) were evaluated. Results T cell activation and exhaustion, monocyte/macrophage activation, and microbial translocation were significantly higher in HIV-infected women when compared to uninfected controls. Microbial translocation correlated with T cell and monocyte/macrophage activation. Biomarkers of cardiovascular disease and impaired cognition were elevated in women with HIV infection and correlated with immune activation. Conclusions HIV-infected antiretroviral-treated aging women who achieved viral suppression are in a generalized status of immune activation and therefore are at an increased risk of age-associated end-organ diseases compared to uninfected age-matched controls.

Epstein-Barr Virus load and immune activation in Human Immunodeficiency Virus type 1-infected patients

BACKGROUND Patients infected with HIV-1 are at high risk of developing Epstein-Barr Virus (EBV)-related diseases. Chronic immune activation is a hallmark of HIV-1 pathogenesis and may play a role in B-cell stimulation and expansion of EBV-infected cells. OBJECTIVES The aim of the study was to define the relationship between parameters of immune activation and EBV load in HIV-1-infected subjects. STUDY DESIGN A total of 156 HIV-1-infected patients were studied. EBV types 1 and 2 were quantified on peripheral blood mononuclear cells by multiplex real-time PCR. Plasma levels of cytokines and lipopolysaccharide (LPS) were determined by immunoenzymatic assays. B-cell activation was analyzed by flow cytometry. RESULTS EBV-DNA was detected in 114 patients, and in all but 3 was EBV type 1. The median [interquartile] EBV-DNA load was 43[1-151] copies/10(5) PBMC. EBV-DNA load was higher in patients with detectable HIV-1 plasma viremia, despite good immunological status (CD4>500 cells/μl), than in patients with undetectable HIV-1 plasma viremia regardless of immunological status (46[5-136] copies/10(5) cells vs 17[1-56] copies/10(5) cells, p=0.008). Patients with high EBV-DNA load (>median value) had higher levels of LPS and proinflammatory cytokines (IL-6, IL-10 and TNF-α) than patients with low EBV load. Furthermore, percentages of activated B-cells correlated with EBV-DNA load (r(s)=0.754; p<0.001). CONCLUSIONS Overall, these findings indicate a strong association between HIV-1 viremia, markers of immune activation and EBV load and suggest that persistence of HIV-1 viremia and immune activation, regardless of peripheral CD4 cell depletion/repopulation, may favor expansion of EBV-infected cells and onset of EBV-related malignancies.

Regulatory T cells and chronic immune activation in human immunodeficiency virus 1 (HIV‐1)‐infected children

The function of CD4+ T cells with regulatory activity (Tregs) is the down‐regulation of immune responses. This suppressive activity may limit the magnitude of effector responses, resulting in failure to control human immunodeficiency virus 1 (HIV‐1) infection, but may also suppress chronic immune activation, a characteristic feature of HIV‐1 disease. We evaluated the correlation between viral load, immune activation and Tregs in HIV‐1‐infected children. Eighty‐nine HIV‐1‐infected children (aged 6–14 years) were included in the study and analysed for HIV‐1 plasmaviraemia, HIV‐1 DNA load, CD4 and CD8 cell subsets. Treg cells [CD4+ CD25highCD127lowforkhead box P3 (FoxP3high)] and CD8‐activated T cells (CD8+CD38+) were determined by flow cytometry. Results showed that the number of activated CD8+CD38+ T cells increased in relation to HIV‐1 RNA plasmaviraemia (r = 0·403, P < 0·0001). The proportion of Tregs also correlated positively with HIV‐1 plasmaviraemia (r = 0·323, P = 0·002), but correlated inversely with CD4+ cells (r = −0·312, P = 0·004), thus suggesting a selective expansion along with increased viraemia and CD4+ depletion. Interestingly, a positive correlation was found between the levels of Tregs and CD8+CD38+ T cells (r = 0·305, P = 0·005), and the percentage of Tregs tended to correlate with HIV‐1 DNA load (r = 0·224, P = 0·062). Overall, these findings suggest that immune activation contributes to the expansion of Treg cells. In turn, the suppressive activity of Tregs may impair effector responses against HIV‐1, but appears to be ineffective in limiting immune activation.

Epstein-Barr virus-driven lymphomagenesis in the context of human immunodeficiency virus type 1 infection

Epstein–Barr virus (EBV) is a ubiquitous human γ-herpes virus which establishes a life-long asymptomatic infection in immunocompetent hosts. In human immunodeficiency virus type 1 (HIV-1) infected patients, the impaired immunosurveillance against EBV may favor the development of EBV-related diseases, ranging from lymphoproliferative disorders to B cell non-Hodgkin’s lymphomas (NHL). Antiretroviral therapy (ART) has significantly modified the natural course of HIV-1 infection, resulting in decreased HIV-1 plasmaviremia, increased CD4 lymphocytes, and decreased opportunistic infections, indicating a restoration of immune functions. However, the impact of ART appears to be less favorable on EBV-related malignancies than on other AIDS-defining tumors, such as Kaposi’s sarcoma, and NHL remains the most common cancer during the ART era. EBV-driven tumors are associated with selective expression of latent oncogenic proteins, but uncontrolled lytic cycle with virus replication and/or reactivation may favor cell transformation, at least in the early phases. Several host’s factors may promote EBV reactivation and replication; besides immunodepression, inflammation/chronic immune stimulation may play an important role. Microbial pathogen-associated molecular patterns and endogenous damage-associated molecular patterns, through Toll-like receptors, activate the immune system and may promote EBV reactivation and/or polyclonal expansion of EBV-infected cells. A body of evidence suggests that chronic immune stimulation is a hallmark of HIV-1 pathogenesis and may persist even in ART-treated patients. This review focuses on lymphomagenesis driven by EBV both in the context of the natural history of HIV-1 infection and in ART-treated patients. Understanding the mechanisms involved in the expansion of EBV-infected cells is a premise for the identification of prognostic markers of EBV-associated malignancies.

Premature aging and immune senescence in HIV-infected children.

Objective:Several pieces of evidence indicate that HIV-infected adults undergo premature aging. The effect of HIV and antiretroviral therapy (ART) exposure on the aging process of HIV-infected children may be more deleterious since their immune system coevolves from birth with HIV. Design:Seventy-one HIV-infected (HIV+), 65 HIV-exposed-uninfected (HEU), and 56 HIV-unexposed-uninfected (HUU) children, all aged 0–5 years, were studied for biological aging and immune senescence. Methods:Telomere length and T-cell receptor rearrangement excision circle levels were quantified in peripheral blood cells by real-time PCR. CD4+ and CD8+ cells were analysed for differentiation, senescence, and activation/exhaustion markers by flow cytometry. Results:Telomere lengths were significantly shorter in HIV+ than in HEU and HUU children (overall, P < 0.001 adjusted for age); HIV+ ART-naive (42%) children had shorter telomere length compared with children on ART (P = 0.003 adjusted for age). T-cell receptor rearrangement excision circle levels and CD8+ recent thymic emigrant cells (CD45RA+CD31+) were significantly lower in the HIV+ than in control groups (overall, P = 0.025 and P = 0.005, respectively). Percentages of senescent (CD28−CD57+), activated (CD38+HLA-DR+), and exhausted (PD1+) CD8+ cells were significantly higher in HIV+ than in HEU and HUU children (P = 0.004, P < 0.001, and P < 0.001, respectively). Within the CD4+ cell subset, the percentage of senescent cells did not differ between HIV+ and controls, but programmed cell death receptor-1 expression was upregulated in the former. Conclusions:HIV-infected children exhibit premature biological aging with accelerated immune senescence, which particularly affects the CD8+ cell subset. HIV infection per se seems to influence the aging process, rather than exposure to ART for prophylaxis or treatment.

Polymorphisms of innate immunity genes influence disease progression in HIV-1-infected children.

Toll-like receptors (TLRs) and defensins (DEFs) play a crucial role in the hosts innate immunity and may influence HIV-1 disease progression. We investigated the impact of TLR9 +1174G > A, 1635A > G and DEF&bgr;1 −44C > G, −52G > A single nucleotide polymorphisms on the clinical outcome of 95 HIV-1-infected children. The TLR9 1635AG genotype and TLR9 [G;G] haplotype were associated with rapid disease progression, whereas the DEF&bgr;1 −44CG genotype and DEF&bgr;1 [G;G] haplotype correlated with a better clinical outcome.

The Role of Genetic Variants of Stromal Cell-Derived Factor 1 in Pediatric HIV-1 Infection and Disease Progression

Stromal cell-Derived Factor 1 (SDF1) is the natural ligand of CXCR4, the coreceptor of HIV-1 X4 viruses. This study investigated the role of the single nucleotide polymorphism (SNP) rs1801157 (NM_000609.5:c.*519G>A) of the SDF1 gene in the natural history of mother-to-child transmission of HIV-1 and disease progression of HIV-1-infected children. The study was conducted in 428 children born to HIV-1-seropositive mothers, who had not undergone antiretroviral therapy (ART) during pregnancy, and in 120 HIV-1-infected children for whom the end-point was the onset of AIDS or the initiation of ART; 16 children developed early AIDS (<24 months of life), 13 from 24 to 84 months of age, and 14 had late AIDS (>84 months). The rs1801157 SNP was not associated with risk of perinatal infection in any genetic models tested. By contrast, this SNP influenced disease progression in a time-dependent manner. rs1801157 GA heterozygous children had a higher risk of late AIDS (HR = 6.3, 95%CI 1.9–20.7, p = 0.002) than children with the rs1801157 GG genotype. Children were studied for viral coreceptor usage at birth, after 84 months of age and/or at AIDS onset. While R5 viruses using CCR5 coreceptor were predominant at birth (94%) and at early AIDS (85%), viruses using CXCR4 coreceptor emerged during the course of infection and were detected in 49% of children older than 84 months and in 62% of late AIDS. The rs1801157 SNP did not influence the emergence of R5X4 viruses, but children with the rs1801157 GA genotype and R5X4 viruses were at significantly higher risk of late AIDS than children with rs1801157 GG genotype (OR = 8.0, 95% CI 1.2–52.2, p = 0.029). Our results indicate that the rs1801157 SNP does not influence perinatal infection, but impacts disease progression. This effect is time-dependent and linked to the coreceptor-usage of viral variants that undergo evolution during the course of HIV-1 infection.

Short-term inhibition of TERT induces telomere length-independent cell cycle arrest and apoptotic response in EBV-immortalized and transformed B cells.

Besides its canonical role in stabilizing telomeres, telomerase reverse transcriptase (TERT) may promote tumorigenesis through extra-telomeric functions. The possible therapeutic effects of BIBR1532 (BIBR), a powerful TERT inhibitor, have been evaluated in different cellular backgrounds, but no data are currently available regarding Epstein–Barr virus (EBV)-driven B-cell malignancies. Our aim was to characterize the biological effects of TERT inhibition by BIBR on EBV-immortalized lymphoblastoid cell lines (LCLs) and fully transformed Burkitt’s lymphoma (BL) cell lines. We found that BIBR selectively inhibits telomerase activity in TERT-positive 4134/Late and 4134/TERT+ LCLs and EBV-negative BL41 and EBV-positive BL41/B95.8 BL cell lines. TERT inhibition led to decreased cell proliferation, accumulation of cells in the S-phase and ultimately to increased apoptosis, compared with mock-treated control cells. All these effects occurred within 72 h and were not observed in BIBR-treated TERT-negative 4134/TERT- and U2OS cells. The cell cycle arrest and apoptosis, consequent upon short-term TERT inhibition, were associated with and likely dependent on the activation of the DNA damage response (DDR), highlighted by the increased levels of γH2AX and activation of ATM and ATR pathways. Analyses of the mean and range of telomere lengths and telomere dysfunction-induced foci indicated that DDR after short-term TERT inhibition was not related to telomere dysfunction, thus suggesting that TERT, besides stabilizing telomere, may protect DNA via telomere-independent mechanisms. Notably, TERT-positive LCLs treated with BIBR in combination with fludarabine or cyclophosphamide showed a significant increase in the number of apoptotic cells with respect to those treated with chemotherapeutic agents alone. In conclusion, TERT inhibition impairs cell cycle progression and enhances the pro-apoptotic effects of chemotherapeutic agents in TERT-positive cells. These results support new therapeutic applications of TERT inhibitors in EBV-driven B-cell malignancies.

Polymorphims of innate immunity genes influence disease progression in HIV-1 infected children

Toll-Like Receptors (TLRs) and Defensins play a crucial role in hosts innate immune response. Genetic variations in Defensins and TLRs may affect host-virus interactions and impact HIV1 disease progression, particularly in infants who acquire immune infection when adaptive immune response is still under development.

Impact of monotherapy on HIV-1 reservoir, immune activation, and co-infection with Epstein-Barr virus

Objectives Although monotherapy (mART) effectiveness in maintaining viral suppression and CD4 cell count has been extensively examined in HIV-1-infected patients, its impact on HIV-1 reservoir, immune activation, microbial translocation and co-infection with Epstein-Barr Virus (EBV) is unclear. Methods This retrospective study involved 32 patients who switched to mART; patients were studied at baseline, 48 and 96 weeks after mART initiation. Thirty-two patients who continued combined antiretroviral therapy (cART) over the same period of time were included in the study. Markers of HIV-1 reservoir (HIV-1 DNA and intracellular HIV-1 RNA) were quantified by real-time PCR. Markers of T-(CD3+CD8+CD38+) and B-(CD19+CD80/86+ and CD19+CD10-CD21lowCD27+) cell activation were evaluated by flow cytometry. Plasma levels of microbial translocation markers were quantified by real-time PCR (16S ribosomal DNA and mitochondrial [mt]DNA) or by ELISA (LPS and sCD14). EBV was typed and quantified by multiplex real-time PCR. Results At baseline, no differences were found between mART and cART groups. Three (10%) mART-treated patients had a virological failure vs none in the cART group. Levels of HIV-1 DNA, intracellular HIV-1 RNA and EBV-DNA remained stable in the mART group, while decreased significantly in the cART group. Percentages of T- and B-activated cells significantly increased in the mART-treated patients, while remained at low levels in the cART-treated ones (p = 0.014 and p<0.001, respectively). Notably, levels of mtDNA remained stable in the cART group, but significantly rose in the mART one (p<0.001). Conclusions Long-term mART is associated with higher levels of T- and B-cell activation and, conversely to cART, does not reduce the size of HIV-1 reservoir and EBV co-infection.