Cytokine interactome alterations promote oropharyngeal Epstien-barr virus (EBV) replication and shedding during HIV infection
ABSTRACT: Proteomic analysis of cytokines in unstimulated oropharyngeal secretions. Epstein-barr virus (EBV) is a type 1 carcinogen which causes many cancers in humans. Here we explored the cytokine involvement of the EBV replication process in the oropharynx. Cytokine interactomic profiles were geneerated to understand the involved signalling pathways in HIV infected group and the healthy group. Proteome profilers were used to understand the major cytokine expression levels that are related to infection and immune regulation. Overall design: We analyzed unstimulated oropharyngeal samples (UOPS) from 42 healthy subjects and 72 HIV positive subjects using the R & D Proteome Profiler array panels. No techinical replicates were performed. 14 samples in HIV group without therapy (NHAART group); 58 HIV patients with highly active antiretroviral therapy (HAART group); 42 samples in healthy group
Project description:Proteomic analysis of cytokines in unstimulated oropharyngeal secretions. Epstein-barr virus (EBV) is a type 1 carcinogen which causes many cancers in humans. Here we explored the cytokine involvement of the EBV replication process in the oropharynx. Cytokine interactomic profiles were geneerated to understand the involved signalling pathways in HIV infected group and the healthy group. Proteome profilers were used to understand the major cytokine expression levels that are related to infection and immune regulation. We analyzed unstimulated oropharyngeal samples (UOPS) from 42 healthy subjects and 72 HIV positive subjects using the R & D Proteome Profiler array panels. No techinical replicates were performed. 14 samples in HIV group without therapy (NHAART group); 58 HIV patients with highly active antiretroviral therapy (HAART group); 42 samples in healthy group
Project description:The association between Epstein-Barr virus (EBV) and the development of head and neck cancer was reported by many researchers. The aim of the present study was to detect EBV DNA and EBV antibodies in 110 Polish patients with oropharyngeal and laryngeal cancer compared to 40 healthy individuals.Frozen tumor tissue fragments were tested using nested PCR assay for EBV DNA detection. Sera from all individuals were investigated using ELISA tests to detect the presence of VCA IgM and IgG, EBNA IgG, EA IgG.EBV DNA was detected in 52.7% of the patients (25% in controls). EBVCA were detected in 94.5%, EBNA in 96.4% and EA in 94.5% of patients. The significantly higher level of EA in the patients suggests EBV reactivation. The majority of patients (83%) were infected with wild-type EBV.Our study showed that this variant seems to be associated with oropharyngeal and laryngeal cancer in the Polish population.
Project description:Epstein-Barr virus (EBV) is an oncogenic virus associated with the development of aggressive and poor-prognosis B-cell lymphomas in patients infected with human immunodeficiency virus (HIV+ patients). The most important risk factors for these malignancies include immune dysfunction, chronic immune activation, and loss of T-cell receptor (TCR) repertoire. The combination of all these factors can favor the reactivation of EBV, malignant cell transformation, and clinical progression toward B-cell lymphomas. The overarching aim of this study was to evaluate the frequency, phenotype, functionality, and distribution of TCR clonotypes for EBV-specific T-cell subpopulations in HIV+ patients at different clinical stages and for HIV+ patients with B-cell lymphoma, as well as to establish their association with clinical variables of prognostic value. Factors were studied in 56 HIV+ patients at different clinical stages and in six HIV+ subjects with diagnosed B-cell lymphoma. We found a significant decrease in all subpopulations of EBV-specific CD4+ T cells from HIV+ patients at stage 3 and with B-cell lymphoma. EBV-specific effector CD8+ T cells, particularly effector memory cells, were also reduced in HIV+ patients with B-cell lymphoma. Interestingly, these cells were unable to produce IFN-? and lacked multifunctionality in HIV+ patients. The TCR-V? repertoire, which is key for protection against EBV in healthy individuals, was less diverse in HIV+ patients due to a lower frequency of TCR-V?2+, V?4+, V?7.1+, V?9+, V?13.6+, V?14+, V?17+, V?22+ CD4+, V?14+, and V?17+ CD8+ T cells. HIV+ patients with positive plasma EBV loads (EBV+HIV+) had a noteworthy decrease in the levels of both TNF-?+ and multifunctional TNF-?+/IL-2+ and TNF-?+/IFN-?+ CD8+ T cells. Altogether, our findings demonstrate that HIV+ patients have significant alterations in the immune response to EBV (poor-quality immunity) that can favor viral reactivation, escalating the risk for developing EBV-associated B-cell lymphomas.
Project description:A growing number of studies reveal that oxidative stress is associated with viral infections or cancer development. However, there are few reports assessing the relationships between oxidative stress, viral infection, and carcinogenesis. The present study analyzed the level of total antioxidant status (TAS) as well as the activities of glutathione peroxidase (GPx) and superoxide dismutase (SOD) in patients with oropharyngeal cancer both Epstein-Barr virus (EBV)-positive and EBV-negative in comparison with the control group. The correlations between these parameters and EBV type (wild-type LMP1 (wt-LMP1) or LMP1 with deletion (del-LMP1)), level of antibodies against EBV, the degree of tumor differentiation, and TNM classification were also investigated. Fresh frozen tumor tissue samples collected from 66 patients with oropharyngeal squamous cell carcinoma were tested using nested PCR assay for EBV DNA detection. Spectrophotometric methods were used to measure TAS values as well as SOD and GPx activities in homogenates of tissue, using diagnostic kits produced by Randox Laboratories. Sera from all individuals were investigated using ELISA method to detect the presence of Epstein-Barr virus capsid antigen (EBVCA) IgM and IgG, Epstein-Barr virus nuclear antigen (EBNA) IgG, and early antigen (EA) IgG antibodies. The level of TAS and activities of antioxidant enzymes (GPx and SOD) were significantly decreased in tissues with oropharyngeal cancer, particularly in EBV-positive cases. In 82.3% of patients, wt-LMP1 was detected. Significantly lower TAS, GPx, and SOD values were stated in patients infected with wild-type EBV. The presence of antibodies against early antigen (anti-EA) was detected in over 80% of patients, which suggests reactivation of EBV infection. The correlation between the degree of tumor differentiation and TN classification, especially in EBV-positive patients, was also observed. Determination of these parameters may be useful in evaluating tumor burden in patients with various stages of oropharyngeal cancer and could be an important prognostic factor. Future studies are needed to understand the role of EBV lytic reactivation induced by oxidative stress.
Project description:Mounting evidence indicates that infection with Epstein-Barr virus (EBV) has a major role in the pathogenesis of multiple sclerosis (MS). Defective elimination of EBV-infected B cells by CD8+ T cells might cause MS by allowing EBV-infected autoreactive B cells to accumulate in the brain. Here we undertake a comprehensive analysis of the T-cell response to EBV in MS, using flow cytometry and intracellular IFN-? staining to measure T-cell responses to EBV-infected autologous lymphoblastoid cell lines and pools of human leukocyte antigen (HLA)-class-I-restricted peptides from EBV lytic or latent proteins and cytomegalovirus (CMV), in 95 patients and 56 EBV-seropositive healthy subjects. In 20 HLA-A2+ healthy subjects and 20 HLA-A2+ patients we also analysed CD8+ T cells specific for individual peptides, measured by binding to HLA-peptide complexes and production of IFN-?, TNF-? and IL-2. We found a decreased CD8+ T-cell response to EBV lytic, but not CMV lytic, antigens at the onset of MS and at all subsequent disease stages. CD8+ T cells directed against EBV latent antigens were increased but had reduced cytokine polyfunctionality indicating T-cell exhaustion. During attacks the EBV-specific CD4+ and CD8+ T-cell populations expanded, with increased functionality of latent-specific CD8+ T cells. With increasing disease duration, EBV-specific CD4+ and CD8+ T cells progressively declined, consistent with T-cell exhaustion. The anti-EBNA1 IgG titre correlated inversely with the EBV-specific CD8+ T-cell frequency. We postulate that defective CD8+ T-cell control of EBV reactivation leads to an expanded population of latently infected cells, including autoreactive B cells.
Project description:Current pathophysiological models of schizophrenia focus on neurodevelopmental and immunological mechanisms. We investigated a molecular pathway traditionally linked to the neurodevelopmental hypothesis (neuregulin 1 - ErbB), and pathogen-associated pattern recognition receptors associated with the immune hypothesis (Toll-like receptors, TLRs). We recruited 42 first-episode, drug-naïve patients with schizophrenia and 42 matched healthy control subjects. In monocytes TLR4/TLR5 and ErbB expressions were measured with flow-cytometry. Pro-inflammatory cytokines (IL-1β, IL-6, and TNF-α) and the anti-inflammatory cytokine IL-10 were determined following the stimulation of TLR4/TLR5 and ErbB. Results revealed increased TLR4/TLR5 and decreased ErbB4 expression in schizophrenia relative to the control subjects. The expression of ErbB2 and ErbB3 receptors was unaltered in schizophrenia. TLR4 stimulation resulted in lower pro-inflammatory cytokine production in schizophrenia compared to the control levels, whereas the stimulation of ErbB by neuregulin 1 led to higher pro-inflammatory cytokine levels in patients with schizophrenia relative to the control group. In healthy controls, ErbB activation was associated with a marked production of IL-10, which was dampened in schizophrenia. These results indicate that the stimulation of TLR4 and ErbB induces opposite pro-inflammatory cytokine responses in schizophrenia.
Project description:Cytokine-based diagnostic assays are increasingly used in research and clinical practice. Assays developed for adults such as the interferon-gamma release assay for tuberculosis show inferior performance in children. Limited evidence suggests that release of cytokines is influenced by age but normal ranges of cytokines in children are lacking. Whole blood of healthy children (0-12 years) undergoing elective/diagnostic procedures was stimulated with SEB, PHA, Candida albicans for 24?hours or left unstimulated. Concentrations of eight cytokines were measured by multiplex bead-based immunoassays and associations with age and other factors quantified by regression analysis. 271 children (median age 5.2 years) were included. In unstimulated samples IL-1ra, IP-10 and TNF-? concentrations decreased by up to -60% with age. Following antigen stimulation, an age-associated increase (ranging from +90% to +500%) was observed for all cytokines except IL-1ra (significant for IL-4, IFN-? and TNF-?). Inter-individual variability in cytokine concentrations was large with a coefficient of variation ranging from 42% to 1412%. Despite inter-individual variation age was identified as a strong influencing factor of cytokine concentrations. Age-specific normal values need to be considered for cytokine-based diagnostic purposes. These results are relevant for development of novel cytokine-based diagnostic assays and for optimal dosing of therapeutic agents targeting cytokines.
Project description:CD4+ T cells are essential for immune protection against viruses, yet their multiple roles remain ill-defined at the single-cell level in humans. Using HLA class II tetramers, we studied the functional properties and clonotypic architecture of EBV-specific CD4+ T cells in patients with infectious mononucleosis, a symptomatic manifestation of primary EBV infection, and in long-term healthy carriers of EBV. We found that primary infection elicited oligoclonal expansions of TH1-like EBV-specific CD4+ T cells armed with cytotoxic proteins that responded immediately ex vivo to challenge with EBV-infected B cells. Importantly, these acutely generated cytotoxic CD4+ T cells were highly activated and transcriptionally distinct from classically described cytotoxic CD4+ memory T cells that accumulate during other persistent viral infections, including CMV and HIV. In contrast, EBV-specific memory CD4+ T cells displayed increased cytokine polyfunctionality but lacked cytotoxic activity. These findings suggested an important effector role for acutely generated cytotoxic CD4+ T cells that could potentially be harnessed to improve the efficacy of vaccines against EBV.
Project description:Fungi are important pathogens but challenging to enumerate using next-generation sequencing because of low absolute abundance in many samples and high levels of fungal DNA from contaminating sources.Here, we analyze fungal lineages present in the human airway using an improved method for contamination filtering. We use DNA quantification data, which are routinely acquired during DNA library preparation, to annotate output sequence data, and improve the identification and filtering of contaminants. We compare fungal communities and bacterial communities from healthy subjects, HIV+ subjects, and lung transplant recipients, providing a gradient of increasing lung impairment for comparison. We use deep sequencing to characterize ribosomal rRNA gene segments from fungi and bacteria in DNA extracted from bronchiolar lavage samples and oropharyngeal wash. Comparison to clinical culture data documents improved detection after applying the filtering procedure.We find increased representation of medically relevant organisms, including Candida, Cryptococcus, and Aspergillus, in subjects with increasingly severe pulmonary and immunologic deficits. We analyze covariation of fungal and bacterial taxa, and find that oropharyngeal communities rich in Candida are also rich in mitis group Streptococci,a community pattern associated with pathogenic polymicrobial biofilms. Thus, using this approach, it is possible to characterize fungal communities in the human respiratory tract more accurately and explore their interactions with bacterial communities in health and disease.