Ketty Gianesin

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.

Pediatric Human Immunodeficiency Virus infection and cancer in the Highly Active Antiretroviral Treatment (HAART) era

Highly active antiretroviral therapy (HAART) changed the natural history of pediatric HIV infection. This review focuses on trends of HIV-associated cancers in childhood in the HAART era and analyses potential pathogenetic mechanisms. HAART reduced AIDS-defined-malignancies (ADM), but incidence of several non-ADM is increasing. HIV-associated immune activation and inflammation, promoting tumorigenesis, can only partially be reduced by HAART. In addition, HIV-infected children may undergo accelerated immune senescence that favors cancer development. How HAART affects this condition is an open question. Lastly, there is no evidence that prenatal exposure to HAART increases the risk of cancer in childhood, but long-term studies are needed.

Immune senescence and cancer in elderly patients: Results from an exploratory study☆

BACKGROUND The challenge of immune senescence has never been addressed in elderly cancer patients. This study compares the thymic output and peripheral blood telomere length in ≥70year old cancer patients. PATIENTS AND METHODS Fifty-two elderly cancer patients and 39 age-matched controls without personal history of cancer were enrolled. All patients underwent a Comprehensive Geriatric Assessment (CGA), from which a multidimensional prognostic index (MPI) score was calculated. Peripheral blood samples were studied for naïve and recent thymic emigrant (RTE) CD4(+) and CD8(+) cells by flow cytometry. T-cell receptor rearrangement excision circle (TREC) levels, telomere length and telomerase activity in peripheral blood cells were quantified by real-time PCR. RESULTS The percentages of CD8(+) naïve and CD8(+) RTE cells and TREC levels were significantly lower in cancer patients than in controls (p=0.003, p=0.004, p=0.031, respectively). Telomere lengths in peripheral blood cells were significantly shorter in cancer patients than in controls (p=0.046) and did not correlate with age in patients, whereas it did in controls (r=-0.354, p=0.031). Short telomere (≤median)/low TREC (≤median) profile was associated with higher risk of cancer (OR=3.68 [95% CI 1.22-11.11]; p=0.021). Neither unfitness on CGA nor MPI score were significantly related to thymic output or telomere length in either group. CONCLUSIONS Immune senescence is significantly worse in elderly cancer patients than in age-matched controls. The low thymic output and the shorter telomeres in peripheral blood cells of cancer patients may reflect a pre-existing condition which facilitates the onset of malignancies in elderly people.

Protein kinase CK2 regulates AKT, NF-κB and STAT3 activation, stem cell viability and proliferation in acute myeloid leukemia

Protein kinase CK2 sustains acute myeloid leukemia cell growth, but its role in leukemia stem cells is largely unknown. Here, we discovered that the CK2 catalytic α and regulatory β subunits are consistently expressed in leukemia stem cells isolated from acute myeloid leukemia patients and cell lines. CK2 inactivation with the selective inhibitor CX-4945 or RNA interference induced an accumulation of leukemia stem cells in the late S–G2–M phases of the cell cycle and triggered late-onset apoptosis. As a result, leukemia stem cells displayed an increased sensitivity to the chemotherapeutic agent doxorubicin. From a molecular standpoint, CK2 blockade was associated with a downmodulation of the stem cell-regulating protein BMI-1 and a marked impairment of AKT, nuclear factor-κB (NF-κB) and signal transducer and activator of transcription 3 (STAT3) activation, whereas FOXO3a nuclear activity was induced. Notably, combined CK2 and either NF-κB or STAT3 inhibition resulted in a superior cytotoxic effect on leukemia stem cells. This study suggests that CK2 blockade could be a rational approach to minimize the persistence of residual leukemia cells.

Cross talk between EBV and telomerase: the role of TERT and NOTCH2 in the switch of latent/lytic cycle of the virus.

Epstein–Barr virus (EBV)-associated malignancies, as well as lymphoblastoid cell lines (LCLs), obtained in vitro by EBV infection of B cells, express latent viral proteins and maintain their ability to grow indefinitely through inappropriate activation of telomere-specific reverse transcriptase (TERT), the catalytic component of telomerase. Our previous studies demonstrated that high levels of TERT expression in LCLs prevent the activation of EBV lytic cycle, which is instead triggered by TERT silencing. As lytic infection promotes the death of EBV-positive tumor cells, understanding the mechanism(s) by which TERT affects the latent/lytic status of EBV may be important for setting new therapeutic strategies. BATF, a transcription factor activated by NOTCH2, the major NOTCH family member in B cells, negatively affects the expression of BZLF1, the master regulator of viral lytic cycle. We therefore analyzed the interplay between TERT, NOTCH and BATF in LCLs and found that high levels of endogenous TERT are associated with high NOTCH2 and BATF expression levels. In addition, ectopic expression of TERT in LCLs with low levels of endogenous telomerase was associated with upregulation of NOTCH2 and BATF at both mRNA and protein levels. By contrast, infection of LCLs with retroviral vectors expressing functional NOTCH2 did not alter TERT transcript levels. Luciferase reporter assays, demonstrated that TERT significantly activated NOTCH2 promoter in a dose-dependent manner. We also found that NF-κB pathway is involved in TERT-induced NOTCH2 activation. Lastly, pharmacologic inhibition of NOTCH signaling triggers the EBV lytic cycle, leading to the death of EBV-infected cells. Overall, these results indicate that TERT contributes to preserve EBV latency in B cells mainly through the NOTCH2/BAFT pathway, and suggest that NOTCH2 inhibition may represent an appealing therapeutic strategy against EBV-associated malignancies.

Epstein-Barr Virus Load in Children Infected With Human Immunodeficiency Virus Type 1 in Uganda

BACKGROUND Epstein-Barr Virus (EBV) is involved in a wide range of malignancies, particularly in immunocompromised subjects. In Africa, EBV primary infection occurs during early childhood, but little is known about the EBV load in Human Immunodeficiency Virus type 1 (HIV-1)-infected children. METHODS Blood samples from 213 HIV-1-infected children, 140 of whom were receiving antiretroviral therapy (ART), were collected at the Nsambya Hospital in Kampala, Uganda, and obtained for dried blood spot analysis. Nucleic acids were extracted and analyzed for quantification of EBV types 1 and 2; 16S ribosomal DNA (rDNA), a marker of microbial translocation; and HIV-1 RNA. RESULTS Ninety-two of 140 children (66%) receiving ART and 57 of 73 ART-naive children (78%) had detectable EBV DNA levels. Coinfection with both EBV types was less frequent in ART-treated children than in ART-naive children (odds ratio, 0.54 [95% confidence interval {CI}, .30-.98]; P = .042). Mean EBV DNA levels (±standard deviation) were lower in the former (3.99 ± 0.59 vs 4.22 ± 0.54 log10 copies/mL; P = .006) and tended to be inversely associated with ART duration. EBV DNA levels were higher in children with an HIV-1 RNA load of > 3 log10 copies/mL of blood (regression coefficient, 0.32 [95% CI, .05-.59]; P = .020) and correlated with circulating 16S rDNA levels (rs = 0.25 [95% CI, .02-.46]; P = .031). CONCLUSIONS These findings suggest that ART, by limiting HIV-1 replication, microbial translocation, and related immune activation, prevents superinfection with both EBV types and keeps EBV viremia down, thus potentially reducing the risk of EBV-associated lymphomas.