43 research outputs found
Dynamics of Epstein-Barr Virus (EBV) in human immunodeficiency virus (HIV)-1 infected adults and children
No full textEpstein-Barr Virus (EBV) is involved in a wide range of malignancies, particularly in immunocompromised subjects. Besides immunodepression, chronic immune activation may induce B-cell stimulation leading to an expansion of EBV-infected cells, thus increasing the risk of EBV-related malignancies. The factors that may contribute to HIV-1-induced B cell activation and expansion of EBV-infected cells are largely unknown. In Africa EBV primary infection occurs during infancy and early childhood and EBV-associated lymphomas represent an important cause of morbidity and mortality in children. High levels of EBV represent a risk factor for the onset of EBV-related malignancies. To date, no data are available about EBV-infection and related malignancies in the context of HIV-1 infection in African children; this may be partly due to lack of access of laboratory analyses in developing countries. The use of Dried Blood Spot (DBS) may represent an easy method to collect and store blood samples and allow their reliable transport to specialized laboratories.
My PhD research focused on the following themes:
1) the relationship between markers of immune activation and EBV load in HIV-1 infected patients.
2) EBV infection in HIV-1 infected and uninfected children in African countries.
3) a new application of Dried Blood Spot to type and quantify EBV and to identify B cell clonality in order to diagnose and monitor B-cell malignancies.
1) EBV load and immune activation in HIV-1-infected patients
A total of 156 HIV-1-infected patients were included in this study, 85 of which were under antiretroviral therapy (ART). EBV-DNA was detected in 114 patients, and in all but 3 it was EBV type 1. The median [interquartile] EBV-DNA load was 43[1-151] copies/105 cells and it 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] vs 17[1-56] copies/105 cells, p=0.008). Patients with high EBV-DNA load (>median value) presented 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=0.754; p<0.001). 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 favour the expansion of EBV-infected cells.
2) Association between NHL and blood levels of EBV in children in Tanzania, and dynamics of EBV in HIV-1-infected children in Uganda
A matched case-control study was performed in children with Non-Hodgkin’s Lymphoma (NHL) admitted to three clinical centers in Tanzania, and their age-matched controls. Blood samples were collected on DBS. 21 out of 35 (60%) NHL patients and only 21 out of 70 (30%) controls presented EBV detectable in peripheral blood, thus showing a significant association between NHL and EBV in blood (OR=4.77 [95% CI 1.71–13.33], p=0.003). Furthermore, EBV-DNA levels were higher in cases compared to EBV-positive controls (p=0.024). Overall, these findings indicate that EBV-DNA load in peripheral blood might have diagnostic relevance.
In a second study, blood samples from 213 HIV-1-infected children were collected on DBS at the Nsambya Home Care, Kampala, Uganda. 92 out of 140 (66%) children on ART and 57 out of 73 (78%) ART-naive children were found to be EBV-positive. After adjusting for CD4 Z-score, age and WHO stage, children on ART presented less odds of having detectable EBV than ART-naive children (OR=0.39 [95% CI 0.16-0.94], p=0.036). EBV load was significantly higher in ART-naive children than those on ART (4.22 [3.87-4.58] vs 3.99 [3.55-4.33] log10 copies/ml; p=0.016). Levels of 16S rDNA, a marker of microbial translocation, were significantly higher in ART-naive children than those on ART (2.16 [2.11-2.28] vs 2.09 [2.02-2.22] log10 copies/μl; p=0.007) and correlated with EBV-DNA levels (r=0.382, p=0.016), suggesting that circulating microbial products lead to B cell activation and expansion of EBV-infected B cells. Treatment with ART, likely by limiting HIV-1 load and thus the HIV-1-driven immune activation, may restrict EBV replication and expansion of EBV-infected cells.
3) Detection of clonal B-cell populations from DBS sampling: a tool for the diagnosis and monitoring of B-cell malignancies
Firstly, through blood samples from donors, we ensured that DBS contains sufficient lymphocytes to perform a clonality assay without yielding false positive results. Using Namalwa cells that contain 2 EBV copies/cell, we found a good relationship between the expected and detected EBV-DNA copies (r=0.987, p<0.0001) and we established that a clonal B-cell population on DBS was detected when there were at least 200 clonal cells in the analysed sample. Moreover, very similar clonal results were obtained between DNA from DBS and fresh whole blood from patients with chronic lymphocytic leukemia. This study demonstrated the possibility to perform clonality testing on DBS sampling, thus improving the diagnostic and monitoring options.
Conclusions
In these studies, we found a significant relationship between markers of microbial translocation, levels of pro-inflammatory cytokines and EBV-DNA levels in both HIV-1 infected adults and children, suggesting that cell activation driven by HIV-1 antigens may result in chronic B cell stimulation and expansion of EBV-infected B cells, a risk factor for the development of EBV-related malignancies. Of interest, we found that EBV-DNA levels were higher in patients with a gain in CD4 lymphocytes, but incomplete suppression of HIV-1 viremia than in patients with undetectable plasma viremia, regardless their CD4 cell number.
The relationship between immune activation and EBV levels was also supported by the findings in HIV-1 infected children in Uganda. Moreover, we found that EBV-DNA and 16S rDNA levels were significantly lower in children on ART than in those ART-naive, suggesting that treatment with ART, likely by limiting immune activation, may prevent B cell stimulation and expansion of EBV-infected B cells. These observations may become particularly interesting to plan future therapeutic strategies in HIV-1 infected patients.
We also found that NHLs are strongly associated with EBV load in peripheral blood, suggesting that high levels of EBV in blood might have diagnostic and prognostic relevance for the diagnosis and monitoring of EBV-related B cell malignancies. In this context we assessed that DBS sampling was also suitable to identify the presence of a B cell clonal population. Our results showed it is possible to perform clonality testing on DBS sampling, thereby improving the diagnostic and monitoring capability of B cell lymphomas in resource-limited settings
Post-transplant lymphoproliferative disorders: From epidemiology to pathogenesis-driven treatment
No full textPost-transplant lymphoproliferative disorders (PTLDs) represent the most severe complication of both solid organ and hematopoietic stem cell transplantation. The Epstein–Barr Virus (EBV) is the main driver of PTLD, particularly those occurring early after transplantation. EBV-driven malignancies are associated with selective expression of latent viral proteins, but uncontrolled lytic replication may favor early phases of cell transformation. Besides immunodepression, persistent immune activation and chronic inflammation play an important role in both virus reactivation and expansion of EBV-infected B cells. EBV-induced immortalization requires the expression of telomerase. TERT, the rate-limiting component of the telomerase complex, is central in the switch from the lytic to the latent viral program, and TERT inhibition induces the EBV lytic cycle and cell death. Immunotherapy and combination of EBV lytic cycle inducers with antiviral drugs are promising strategies to improve the treatment of PTLD patients. This review is aimed at providing an update on the intriguing association between EBV and PTLD, mainly focusing on cases arising after kidney and liver transplantation, which account for the vast majority of transplants
Extra-telomeric functions of telomerase in the pathogenesis of Epstein-Barr virus-driven B-cell malignancies and potential therapeutic implications
No full textAbstract
The Epstein-Barr virus (EBV) is a ubiquitous human γ-herpesvirus causally linked to a broad spectrum of both lymphoid and epithelial malignancies. In order to maintain its persistence in host cells and promote tumorigenesis, EBV must restrict its lytic cycle, which would ultimately lead to cell death, selectively express latent viral proteins, and establish an unlimited proliferative potential. The latter step depends on the maintenance of telomere length provided by telomerase. The viral oncoprotein LMP-1 activates TERT, the catalytic component of telomerase. In addition to its canonical role in stabilizing telomeres, TERT may promote EBV-driven tumorigenesis through extra-telomeric functions. TERT contributes toward preserving EBV latency; in fact, through the NOTCH2/BATF pathway, TERT negatively affects the expression of BZLF1, the master regulator of the EBV lytic cycle. In contrast, TERT inhibition triggers a complete EBV lytic cycle, leading to the death of EBV-infected cells. Interestingly, short-term TERT inhibition causes cell cycle arrest and apoptosis, partly by inducing telomere-independent activation of the ATM/ATR/TP53 pathway. Importantly, TERT inhibition also sensitizes EBV-positive tumor cells to antiviral therapy and enhances the pro-apoptotic effects of chemotherapeutic agents. We provide here an overview on how the extra-telomeric functions of TERT contribute to EBV-driven tumorigenesis. We also discuss the potential therapeutic approach of TERT inhibition in EBV-driven malignancies
Beyond Telomeres: Unveiling the Extratelomeric Functions of TERT in B-Cell Malignancies
No full textThe reactivation of telomerase enables cancer cells to maintain the telomere length, bypassing replicative senescence and achieving cellular immortality. In addition to its canonical role in telomere maintenance, accumulating evidence highlights telomere-length-independent functions of TERT, the catalytic subunit of telomerase. These extratelomeric functions involve the regulation of signaling pathways and transcriptional networks, creating feed-forward loops that promote cancer cell proliferation, resistance to apoptosis, and disease progression. This review explores the complex mechanisms by which TERT modulates key signaling pathways, such as NF-κB, AKT, and MYC, highlighting its role in driving autonomous cancer cell growth and resistance to therapy in B-cell malignancies. Furthermore, we discuss the therapeutic potential of targeting TERT’s extratelomeric functions. Unlike telomere-directed approaches, which may require prolonged treatment to achieve effective telomere erosion, inhibiting TERT’s extratelomeric functions offers the prospect of rapid tumor-specific effects. This strategy could complement existing chemotherapeutic regimens, providing an innovative and effective approach to managing B-cell malignancies
Accelerated aging in perinatally HIV-infected children: clinical manifestations and pathogenetic mechanisms
No full textBACKGROUND:
Premature aging and related diseases have been documented in HIV-infected adults. Data are now emerging also regarding accelerated aging process in HIV-infected children.
METHODS:
A narrative review was performed searching studies on PubMed published in English language in 2004-2017, using appropriate key words, including "aging", "children", "HIV", "AIDS", "immunosenescence", "pathogenesis", "clinical conditions".
RESULTS:
Premature immunosenescence phenotype of B and T cells in HIV-infected children is mediated through immune system activation and chronic inflammation. Ongoing inflammation processes have been documented by increased levels of pathogen-associated molecular patterns (PAMPS), increased mitochondrial damage, higher levels of pro-inflammatory cytokines, and a positive correlation between sCD14 levels and percentages of activated CD8+ cells. Other reported features of premature aging include cellular replicative senescence, linked to an accelerated telomeres shortening. Finally, acceleration of age-associated methylation pattern and other epigenetic modifications have been described in HIV-infected children. All these features may favor the clinical manifestations related to premature aging. Lipid and bone metabolism, cancers, cardiovascular, renal, and neurological systems should be carefully monitored, particularly in children with detectable viremia and/or with CD4/CD8 ratio inversion.
CONCLUSION:
Aging processes in children with HIV infection impact their quality and length of life. Further studies regarding the mechanisms involved in premature aging are needed to search for potential targets of treatment
Telomere and Telomerase in Carcinogenesis: Their Role as Prognostic Biomarkers
No full textUnlimited replicative potential is the hallmark of cancer cells. Telomere shortening, which occurs at each
cell division, restricts cell proliferation in normal somatic cells. Maintenance of telomere length, required for the
unlimited cell proliferation displayed by cancer cells, is provided by telomerase activity, expressed in the vast
majority of tumors. Telomere/telomerase interplay has a critical role in tumor initiation and progression. Many
tumor-based studies have demonstrated that neoplastic cells generally have shorter telomeres than their adjacent
non-cancerous mucosa, strongly supporting the concept that telomere erosion is a critical event in carcinogenesis.
Telomerase reverse transcriptase (TERT), the catalytic component of the telomerase complex, is usually absent in
normal somatic cells but is expressed at variable levels in tumors. Specific mutations in its promoter may influence
TERT levels. A body of data indicates that telomere length and levels of TERT/telomerase activity may be prognostic
markers in cancers. Circulating cell-free TERT RNA may also be a promising marker for minimally invasive moni-
toring of disease progression and response to therap
Relationship Between Non-Hodgkin's Lymphoma and Blood Levels of Epstein-Barr Virus in Children in North-Western Tanzania: A Case Control Study.
No full textNon-Hodgkin's Lymphomas (NHL) are common in African children, with endemic Burkitt's lymphoma (BL) being the most common subtype. While the role of Epstein-Barr Virus (EBV) in endemic BL is known, no data are available about clinical presentations of NHL subtypes and their relationship to Human Immunodeficiency Virus (HIV) infection and Epstein Barr Virus (EBV) load in peripheral blood of children in north-western, Tanzania. A matched case control study of NHL subtypes was performed in children under 15 years of age and their respective controls admitted to Bugando Medical Centre, Sengerema and Shirati district designated hospitals in north-western, Tanzania, between September 2010 and April 2011. Peripheral blood samples were collected on Whatman 903 filter papers and EBV DNA levels were estimated by multiplex real-time PCR. Clinical and laboratory data were collected using a structured data collection tool and analysed using chi-square, Fisher and Wilcoxon rank sum tests where appropriate. The association between NHL and detection of EBV in peripheral blood was assessed using conditional logistic regression model and presented as odds ratios (OR) and 95% confidence intervals (CI). A total of 35 NHL cases and 70 controls matched for age and sex were enrolled. Of NHLs, 32 had BL with equal distribution between jaw and abdominal tumour, 2 had large B cell lymphoma (DLBCL) and 1 had NHL-not otherwise specified (NHL-NOS). Central nervous system (CNS) presentation occurred only in 1 BL patient; 19 NHLs had stage I and II of disease. Only 1 NHL was found to be HIV-seropositive. Twenty-one of 35 (60%) NHL and 21 of 70 (30%) controls had detectable EBV in peripheral blood (OR = 4.77, 95% CI 1.71 - 13.33, p = 0.003). In addition, levels of EBV in blood were significantly higher in NHL cases than in controls (p = 0.024). BL is the most common childhood NHL subtype in north-western Tanzania. NHLs are not associated with HIV infection, but are strongly associated with EBV load in peripheral blood. The findings suggest that high levels of EBV in blood might have diagnostic and prognostic relevance in African children
Evaluation of the Efficacy of Ozonated Oil in Liposomes as an Adjuvant in Intravitreal Injection Prophylaxis: OPERA Study
No full textBackground/Purpose:
The prevention of endophthalmitis continues to pose a significant challenge for intravitreal injections (IVIs) due to the increasing prevalence of these procedures. In response, we conducted a prospective, interventional, single-center clinical study to evaluate the collective efficacy of ozonated oil in liposomes, administered via eye drops (Ozodrop IVTTM), and eyelid foam (BlefoxTM) in combination with povidone iodine (PI) 5% as prophylaxis.
Methods:
Both eyes of each patient were included and categorized into two groups. Group 1 received prophylaxis with Ozodrop IVTTM and BlefoxTM for three days, followed by PI 5% before intravitreal (IV) injection. Group 2, comprising contralateral eyes, underwent standard prophylaxis with PI 5% only. Conjunctival and eyelid edge swabs were collected from both eyes at T0 (four days before IV) and T1 (10 minutes before injection, after 30-second treatment with PI 5% and washing with balance salt solution). Swabs were plated on chocolate agar for aerobic bacteria and blood agar for anaerobic bacteria. The primary endpoint assessed the difference in bacterial colonies reduction between the two groups at specified time points. The study employed a paired-eye design. Secondary endpoints included conjunctival hyperemia evaluation and subject satisfaction grade.
Results:
A total of 390 eyes from 195 patients (mean age: 69.6 ± 10.4 years, 48% female) were enrolled. Chocolate agar demonstrated a significant reduction in bacterial load, with decreases of 72.3% in group 1 and 50.3% in group 2 (p<0.001). Testing on blood agar also exhibited reductions, with percentages of 66.7% in group 1 and 49.7% in group 2 (p<0.001). Five (2.66%) patients reported traces, while mild was reported by only one (0.53%) patient regarding conjunctival hyperemia. No other adverse events were reported. A total of 159 (84.57%) patients reported no ocular discomfort, while mild discomfort occurred in 28 (14.89%) patients.
Conclusions:
The combination of ozonated oil in liposomes with PI in IV prophylaxis effectively reduced microbial load, surpassing the efficacy of standard prophylaxis with PI alone. This finding suggests a potential enhancement in intravitreal injection safety through the incorporation of ozonated oil in liposomes as an adjuvant therapy
EPSTEIN-BARR VIRUS-DRIVEN LYMPHOMAGENESIS IN THE CONTEXT OF HUMAN IMMUNODEFICIENCY VIRUS TYPE 1 INFECTION
No full textEpstein–Barr Virus (EBV) is a ubiquitous human γ-herpes virus which establishes a life-long asymptomatic infection in immunocompetent hosts. In 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 (ADC), 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 (PAMPs) and endogenous damage-associated molecular patterns (DAMPs), through Toll-like receptors (TLR), 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
Impact of monotherapy on HIV-1 reservoir, immune activation, and co-infection with Epstein-Barr virus.
No full textAlthough 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.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.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).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
