Studies on Immune Regulation of Epstein-Barr Virus

Epstein-Barr virus (EBV) is a gammaherpes virus that infects >90% of the adult population worldwide. During childhood infection is generally sub-clinical, however if delayed until adolescence infectious mononucleosis (IM) may develop. The virus has also been aetiologically linked with a number of tumours including B-cell lymphoma following organ transplantation: post-transplant lymphoproliferative disease (PTLD). The symptoms of IM are caused by an expansion of immune cells in response to infection whilst in the transplant situation immunosuppressive drug therapy allows the outgrowth of the tumour. Understanding the immuno-regulatory mechanisms involved in such EBV-associated diseases is crucial for devising new treatment strategies. We undertook 3 separate studies (1-3) investigating different aspects of the immune response to EBV. In a recently reported phase II trial using allogenic, EBV-specific cytotoxic T-cell (CTL) to treat PTLD, tumour response was significantly increased with a high degree of donor/recipient HLA-allele matching suggesting that further refinement of the matching procedure may be important. In study 1 we investigated the epitope specificity and T-cell receptor (TCR) clonality of the infused CTL to identify potential areas for refinement. We found the protein specificity of the CTL to be polyclonal with dominant recognition of Epstein-Barr nuclear antigen-3 proteins and sub-dominant recognition of Latent membrane protein (LMP)-1 and LMP-2 proteins. Where possible, specificity was confirmed at the peptide level. No single TCR family was preferentially used by CTLs. The CTL epitope specificity did not differ between treatment responders and non-responders however the response was improved in those with several CTL HLA-restricted epitope matches and those infused with CTL containing polyclonal TCR families as opposed to monoclonal. CTL/recipient matching based on HLA matching alone was improved when also matched via HLA- restricted epitiope specificity. Therefore mapping CTL peptide epitope specificity prior to CTL infusions may enhance patient responses. In recent years, interest has developed in genetic variation within components of the immune system. Of particular interest are cytokine/cytokine receptor genes and genes of the human leukocyte antigen (HLA), both of which act to regulate the immune response. Variation within these genes could potentially alter the immune response leading to disease. In study 2 we investigated single nucleotide polymorphisms (SNPs) in several cytokine genes (TNF, IL-1, -6, -10) in both IM and PTLD cases and compared with relevant control groups. We found that the frequency of two TNF promoter alleles was significantly increased in PTLD patients compared to controls whilst the frequency of a TNF receptor II allele was increased in IM and EBV seropositive individuals, suggesting a role for this allele in susceptibility to EBV infection. The frequency of a second TNF receptor II allele was increased in both PTLD and IM subjects compared to controls highlighting the possible significance of TNF and its receptor in the development of EBV associated disease. In study 3 we analyzed two microsatellite markers and two SNPs located near the HLA class I locus in IM, PTLD and control subjects to further determine whether the HLA genes may affect development of EBV-associated diseases. Alleles of both microsatellite markers were significantly associated with development of IM. Specific alleles of the two SNPs were also more frequent in IM patients. Moreover IM cases possessing the associated microsatellite allele had significantly fewer lymphocytes, increased neutrophils, and displayed higher EBV titres and milder IM symptoms relative to IM cases lacking the allele. The results indicate that HLA class I polymorphisms may predispose patients to development of IM upon primary EBV infection and suggest that genetic variation in T cell responses can influence the course of EBV infection

Early Virological and Immunological Events in Asymptomatic Epstein-Barr Virus Infection in African Children

Epstein-Barr virus (EBV) infection often occurs in early childhood and is asymptomatic. However, if delayed until adolescence, primary infection may manifest as acute infectious mononucleosis (AIM), a febrile illness characterised by global CD8+ T-cell lymphocytosis, much of it reflecting a huge expansion of activated EBV-specific CD8+ T-cells. While the events of AIM have been intensely studied, little is known about how these relate to asymptomatic primary infection. Here Gambian children (14–18 months old, an age at which many acquire the virus) were followed for the ensuing six months, monitoring circulating EBV loads, antibody status against virus capsid antigen (VCA) and both total and virus-specific CD8+ T-cell numbers. Many children were IgG anti-VCA-positive and, though no longer IgM-positive, still retained high virus loads comparable to AIM patients and had detectable EBV-specific T-cells, some still expressing activation markers. Virus loads and the frequency/activation status of specific T-cells decreased over time, consistent with resolution of a relatively recent primary infection. Six children with similarly high EBV loads were IgM anti-VCA-positive, indicating very recent infection. In three of these donors with HLA types allowing MHC-tetramer analysis, highly activated EBV-specific T-cells were detectable in the blood with one individual epitope response reaching 15% of all CD8+ T-cells. That response was culled and the cells lost activation markers over time, just as seen in AIM. However, unlike AIM, these events occurred without marked expansion of total CD8+ numbers. Thus asymptomatic EBV infection in children elicits a virus-specific CD8+ T-cell response that can control the infection without over-expansion; conversely, in AIM it appears the CD8 over-expansion, rather than virus load per se, is the cause of disease symptoms

Evaluation of the antibody response to the EBV proteome in EBV-associated classic Hodgkin lymphoma

The humoral immune response to Epstein–Barr virus (EBV) in classical Hodgkin lymphoma (cHL) stratified by EBV tumor status is unclear. We examined IgG and IgA antibody responses against 202 protein sequences representing 86 EBV proteins using a microarray and sera from 139 EBV‐positive cHL cases, 70 EBV‐negative cHL cases and 141 population‐based controls frequency matched to EBV‐positive cHL cases on sex and age by area (UK, Denmark and Sweden). We leveraged existing data on the proportion of circulating B‐cells infected by EBV and levels of serum CCL17, a chemokine secreted by cHL tumor cells, from a subset of the cHL cases in the UK. Total IgG but not IgA response level was significantly different between EBV‐positive cHL cases and controls. The distinct serological response included significant elevations in 16 IgG antibodies and 2 IgA antibodies, with odds ratioshighest vs. lowest tertile > 3 observed for the following EBV proteins: LMP1 (oncogene), BcLF1 (VCAp160, two variants) and BBLF1 (two variants). Our cHL IgG signature correlated with the proportion of circulating EBV‐infected B‐cells, but not serum CCL17 levels. We observed no differences in the anti‐EBV antibody profile between EBV‐negative cHL cases and controls. BdRF1(VCAp40)‐IgG and BZLF1(Zta)‐IgG were identified as the serological markers best able to distinguish EBV‐positive from EBV‐negative cHL tumors. Our results support the hypothesis that differences in the EBV antibody profile are specific to patients with EBV‐positive cHL and are not universally observed as part of a systematically dysregulated immune response present in all cHL cases

HLA class I and II diversity contributes to the etiologic heterogeneity of non-Hodgkin lymphoma subtypes

A growing number of loci within the human leukocyte antigen (HLA) region have been implicated in non-Hodgkin lymphoma (NHL) etiology. Here, we test a complementary hypothesis of "heterozygote advantage" regarding the role of HLA and NHL, whereby HLA diversity is beneficial and homozygous HLA loci are associated with increased disease risk. HLA alleles at class I and II loci were imputed from genome-wide association studies (GWAS) using SNP2HLA for: 3,617 diffuse large B-cell lymphomas (DLBCL), 2,686 follicular lymphomas (FL), 2,878 chronic lymphocytic leukemia/small lymphocytic lymphomas (CLL/SLL), 741 marginal zone lymphomas (MZL), and 8,753 controls of European descent. Both DLBCL and MZL risk were elevated with homozygosity at class I HLA-B and -C loci (OR DLBCL=1.31, 95% CI=1.06-1.60; OR MZL=1.45, 95% CI=1.12-1.89) and class II HLA-DRB1 locus (OR DLBCL=2.10, 95% CI=1.24-3.55; OR MZL= 2.10, 95% CI=0.99-4.45). Increased FL risk was observed with the overall increase in number of homozygous HLA class II loci (p-trend<0.0001, FDR=0.0005). These results support a role for HLA zygosity in NHL etiology and suggests that distinct immune pathways may underly the etiology of the different NHL subtypes

Multiorgan MRI findings after hospitalisation with COVID-19 in the UK (C-MORE): a prospective, multicentre, observational cohort study

Introduction: The multiorgan impact of moderate to severe coronavirus infections in the post-acute phase is still poorly understood. We aimed to evaluate the excess burden of multiorgan abnormalities after hospitalisation with COVID-19, evaluate their determinants, and explore associations with patient-related outcome measures. Methods: In a prospective, UK-wide, multicentre MRI follow-up study (C-MORE), adults (aged ≥18 years) discharged from hospital following COVID-19 who were included in Tier 2 of the Post-hospitalisation COVID-19 study (PHOSP-COVID) and contemporary controls with no evidence of previous COVID-19 (SARS-CoV-2 nucleocapsid antibody negative) underwent multiorgan MRI (lungs, heart, brain, liver, and kidneys) with quantitative and qualitative assessment of images and clinical adjudication when relevant. Individuals with end-stage renal failure or contraindications to MRI were excluded. Participants also underwent detailed recording of symptoms, and physiological and biochemical tests. The primary outcome was the excess burden of multiorgan abnormalities (two or more organs) relative to controls, with further adjustments for potential confounders. The C-MORE study is ongoing and is registered with ClinicalTrials.gov, NCT04510025. Findings: Of 2710 participants in Tier 2 of PHOSP-COVID, 531 were recruited across 13 UK-wide C-MORE sites. After exclusions, 259 C-MORE patients (mean age 57 years [SD 12]; 158 [61%] male and 101 [39%] female) who were discharged from hospital with PCR-confirmed or clinically diagnosed COVID-19 between March 1, 2020, and Nov 1, 2021, and 52 non-COVID-19 controls from the community (mean age 49 years [SD 14]; 30 [58%] male and 22 [42%] female) were included in the analysis. Patients were assessed at a median of 5·0 months (IQR 4·2–6·3) after hospital discharge. Compared with non-COVID-19 controls, patients were older, living with more obesity, and had more comorbidities. Multiorgan abnormalities on MRI were more frequent in patients than in controls (157 [61%] of 259 vs 14 [27%] of 52; p&lt;0·0001) and independently associated with COVID-19 status (odds ratio [OR] 2·9 [95% CI 1·5–5·8]; padjusted=0·0023) after adjusting for relevant confounders. Compared with controls, patients were more likely to have MRI evidence of lung abnormalities (p=0·0001; parenchymal abnormalities), brain abnormalities (p&lt;0·0001; more white matter hyperintensities and regional brain volume reduction), and kidney abnormalities (p=0·014; lower medullary T1 and loss of corticomedullary differentiation), whereas cardiac and liver MRI abnormalities were similar between patients and controls. Patients with multiorgan abnormalities were older (difference in mean age 7 years [95% CI 4–10]; mean age of 59·8 years [SD 11·7] with multiorgan abnormalities vs mean age of 52·8 years [11·9] without multiorgan abnormalities; p&lt;0·0001), more likely to have three or more comorbidities (OR 2·47 [1·32–4·82]; padjusted=0·0059), and more likely to have a more severe acute infection (acute CRP &gt;5mg/L, OR 3·55 [1·23–11·88]; padjusted=0·025) than those without multiorgan abnormalities. Presence of lung MRI abnormalities was associated with a two-fold higher risk of chest tightness, and multiorgan MRI abnormalities were associated with severe and very severe persistent physical and mental health impairment (PHOSP-COVID symptom clusters) after hospitalisation. Interpretation: After hospitalisation for COVID-19, people are at risk of multiorgan abnormalities in the medium term. Our findings emphasise the need for proactive multidisciplinary care pathways, with the potential for imaging to guide surveillance frequency and therapeutic stratification

Studies on immune regulation of Epstein-Barr virus

Epstein-Barr virus (EBV) is a gammaherpes virus that infects >90% of the adult population worldwide. During childhood infection is generally sub-clinical, however if delayed until adolescence infectious mononucleosis (IM) may develop. The virus has also been aetiologically linked with a number of tumours including B-cell lymphoma following organ transplantation: post-transplant lymphoproliferative disease (PTLD). The symptoms of IM are caused by an expansion of immune cells in response to infection whilst in the transplant situation immunosuppressive drug therapy allows the outgrowth of the tumour. Understanding the immuno-regulatory mechanisms involved in such EBV-associated diseases is crucial for devising new treatment strategies. We undertook 3 separate studies (1-3) investigating different aspects of the immune response to EBV. In a recently reported phase II trial using allogenic, EBV-specific cytotoxic T-cell (CTL) to treat PTLD, tumour response was significantly increased with a high degree of donor/recipient HLA-allele matching suggesting that further refinement of the matching procedure may be important. In study 1 we investigated the epitope specificity and T-cell receptor (TCR) clonality of the infused CTL to identify potential areas for refinement. We found the protein specificity of the CTL to be polyclonal with dominant recognition of Epstein-Barr nuclear antigen-3 proteins and sub-dominant recognition of Latent membrane protein (LMP)-1 and LMP-2 proteins. Where possible, specificity was confirmed at the peptide level. No single TCR family was preferentially used by CTLs. The CTL epitope specificity did not differ between treatment responders and non-responders however the response was improved in those with several CTL HLA-restricted epitope matches and those infused with CTL containing polyclonal TCR families as opposed to monoclonal. CTL/recipient matching based on HLA matching alone was improved when also matched via HLA- restricted epitiope specificity. Therefore mapping CTL peptide epitope specificity prior to CTL infusions may enhance patient responses. In recent years, interest has developed in genetic variation within components of the immune system. Of particular interest are cytokine/cytokine receptor genes and genes of the human leukocyte antigen (HLA), both of which act to regulate the immune response. Variation within these genes could potentially alter the immune response leading to disease. In study 2 we investigated single nucleotide polymorphisms (SNPs) in several cytokine genes (TNF, IL-1, -6, -10) in both IM and PTLD cases and compared with relevant control groups. We found that the frequency of two TNF promoter alleles was significantly increased in PTLD patients compared to controls whilst the frequency of a TNF receptor II allele was increased in IM and EBV seropositive individuals, suggesting a role for this allele in susceptibility to EBV infection. The frequency of a second TNF receptor II allele was increased in both PTLD and IM subjects compared to controls highlighting the possible significance of TNF and its receptor in the development of EBV associated disease. In study 3 we analyzed two microsatellite markers and two SNPs located near the HLA class I locus in IM, PTLD and control subjects to further determine whether the HLA genes may affect development of EBV-associated diseases. Alleles of both microsatellite markers were significantly associated with development of IM. Specific alleles of the two SNPs were also more frequent in IM patients. Moreover IM cases possessing the associated microsatellite allele had significantly fewer lymphocytes, increased neutrophils, and displayed higher EBV titres and milder IM symptoms relative to IM cases lacking the allele. The results indicate that HLA class I polymorphisms may predispose patients to development of IM upon primary EBV infection and suggest that genetic variation in T cell responses can influence the course of EBV infection.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Epstein-Barr Virus Can Establish Infection in the Absence of a Classical Memory B-Cell Population

Epstein-Barr virus (EBV) is a ubiquitous human herpesvirus that persists in the body for life after primary infection. The primary site of EBV persistence is the memory B lymphocyte, but whether the virus initially infects naïve or memory B cells is still disputed. We have analyzed EBV infection in nine cases of X-linked hyper-immunoglobulin M (hyper-IgM) syndrome who, due to a mutation in CD40 ligand gene, do not have a classical, class-switched memory B-cell population (IgD(−) CD27(+)). We found evidence of EBV infection in 67% of cases, which is similar to the infection rate found in the general United Kingdom population (60 to 70% for the relevant age range). We detected EBV DNA in peripheral blood B cells and showed in one case that the infection was restricted to the small population of nonclassical, germinal center-independent memory B cells (IgD(+) CD27(+)). Detection of EBV small RNAs, latent membrane protein 2, and EBV nuclear antigen 3C expression in peripheral blood suggests full latent viral gene expression in this population. Analysis of EBV DNA in serial samples showed variability over time, suggesting cycles of infection and loss. Our results demonstrate that short-term EBV persistence can occur in the absence of a germinal center reaction and a classical memory B-cell population

Gammaherpesviruses and canine lymphoma: no evidence for direct involvement in commonly occurring lymphomas

Lymphoma is the most common haematopoietic malignancy in dogs, but little is known about the aetiology of this heterogeneous group of cancers. In humans, the Epstein-Barr virus (EBV) is associated with several lymphoma subtypes. Recently, it was suggested that EBV or an EBV-like virus is circulating in dogs. We therefore investigated whether EBV, or a novel herpesvirus, is associated with canine lymphoma using both serological and molecular techniques. In an assay designed to detect antibodies to EBV viral capsid antigens, 41 % of dogs were positive. Dogs with cancers, including lymphoma, were more frequently positive than controls, but no particular association with B-cell lymphoma was noted. EBV-specific RNA and DNA sequences were not detected in lymphoma tissue by in situ hybridization or PCR, and herpesvirus genomes were not detected using multiple degenerate PCR assays with the ability to detect novel herpesviruses. We therefore found no evidence that herpesviruses are directly involved in common types of canine lymphoma although cannot exclude the presence of an EBV-like virus in the canine population

NHS capital build projects Identity guidelines

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Human cord blood-derived cells can differentiate into hepatocytes in the mouse liver with no evidence of cellular fusion

BACKGROUND &amp; AIMS: Studies have indicated that stem cells have unexpected plasticity and can differentiate down a multitude of nonhematopoietic cell lineages in rodents. Our aim was to identify whether human cord blood cells, which are a rich source of stem cells, would be able to differentiate into hepatocytes when infused into nonobese diabetic-severe combined immunodeficient (NOD-SCID) mice. We also wanted to test whether such differentiated cells were the result of cellular fusion or true stem cell transdifferentiation.METHODS: Unsorted mononuclear cell preparations of human cord blood were infused into sublethally irradiated NOD-SCID mice. After death, immunohistologic analysis of murine livers was performed using human specific hepatocyte, biliary, and endothelial markers. Fluorescent in situ hybridization (FISH) for mouse and human DNA was also performed.RESULTS: We show that human cord blood cells have the ability to engraft into NOD-SCID liver and become mature hepatocytes. We were unable to identify any biliary or endothelial differentiation. Furthermore, we do not detect any evidence of cell fusion in any of the human cells found in the mouse liver, suggesting that human cord blood cells are capable of true transdifferentiation into hepatocytes in vivo.CONCLUSIONS: We conclude that hepatocytes can derive from human cord blood cells when infused into NOD-SCID mice in the absence of fusion. The demonstration that human stem cell differentiation can occur in this murine model permits comprehensive study of human stem cell plasticity in vivo.</p