Epstein-Barr virus (EBV) reactivation and therapeutic inhibitors.

Epstein-Barr virus (EBV) is a ubiquitous human virus which infects almost all humans during their lifetime and following the acute phase, persists for the remainder of the life of the individual. EBV infects B lymphocytes leading to their immortalisation, with persistence of the EBV genome as an episome. In the latent phase, EBV is prevented from reactivating through efficient cytotoxic cellular immunity. EBV reactivates (lytic phase) under conditions of psychological stress with consequent weakening of cellular immunity, and EBV reactivation has been shown to occur in a subset of individuals with each of a variety of cancers, autoimmune diseases, the autoimmune-like disease, chronic fatigue syndrome/myalgic encephalitis and under other circumstances such as being an inpatient in an intensive care unit. Chronic EBV reactivation is an important mechanism in the pathogenesis of many such diseases, yet is rarely tested for in immunocompetent individuals. This review summarises the pathogenesis of EBV infection, EBV reactivation and its role in disease, and methods which may be used to detect it. Known inhibitors of EBV reactivation and replication are discussed, including drugs licensed for treatment of other herpesviruses, licensed or experimental drugs for various other indications, compounds at an early stage of drug development and nutritional constituents such as vitamins and dietary supplements.

Epstein-Barr Virus Induced Gene-2 Upregulation Identifies a Particular Subtype of Chronic Fatigue Syndrome/Myalgic Encephalomyelitis.

Chronic Fatigue Syndrome/Myalgic Encephalomyelitis (CFS/ME) is a chronic multisystem disease characterized by a variety of symptoms, and exhibits various features of an autoimmune-like disease. Subtypes are well recognized but to date are difficult to identify objectively. The disease may be triggered by infection with a variety of micro-organisms, including Epstein-Barr virus (EBV). A subset of CFS/ME patients exhibit up regulation of EBV virus induced gene 2 (EBI2) mRNA in peripheral blood mononuclear cells (PBMC), and these patients appear to have a more severe disease phenotype and lower levels of EBNA1 IgG. EBI2 is induced by EBV infection and has been found to be upregulated in a variety of autoimmune diseases. EBI2 is a critical gene in immunity and central nervous system function; it is a negative regulator of the innate immune response in monocytes. Its heterogeneous expression in CFS/ME could explain the variable occurrence of a variety of immune and neurological abnormalities which are encountered in patients with CFS/ME. The EBI2 subtype occurred in 38-55% CFS/ME patients in our studies. Further work is required to confirm the role of EBV and of EBI2 and its oxysterol ligands in CFS/ME, and to identify the most practical means to identify patients of the EBI subtype. There are two EBI2 antagonists currently in development, and these may hold promise in the treatment of CFS/ME patients of the EBI subtype.

MicroRNAs hsa-miR-99b, hsa-miR-330, hsa-miR-126 and hsa-miR-30c: Potential Diagnostic Biomarkers in Natural Killer (NK) Cells of Patients with Chronic Fatigue Syndrome (CFS)/ Myalgic Encephalomyelitis (ME).

BACKGROUND: Chronic Fatigue Syndrome (CFS/ME) is a complex multisystem disease of unknown aetiology which causes debilitating symptoms in up to 1% of the global population. Although a large cohort of genes have been shown to exhibit altered expression in CFS/ME patients, it is currently unknown whether microRNA (miRNA) molecules which regulate gene translation contribute to disease pathogenesis. We hypothesized that changes in microRNA expression in patient leukocytes contribute to CFS/ME pathology, and may therefore represent useful diagnostic biomarkers that can be detected in the peripheral blood of CFS/ME patients. METHODS: miRNA expression in peripheral blood mononuclear cells (PBMC) from CFS/ME patients and healthy controls was analysed using the Ambion Bioarray V1. miRNA demonstrating differential expression were validated by qRT-PCR and then replicated in fractionated blood leukocyte subsets from an independent patient cohort. The CFS/ME associated miRNA identified by these experiments were then transfected into primary NK cells and gene expression analyses conducted to identify their gene targets. RESULTS: Microarray analysis identified differential expression of 34 miRNA, all of which were up-regulated. Four of the 34 miRNA had confirmed expression changes by qRT-PCR. Fractionating PBMC samples by cell type from an independent patient cohort identified changes in miRNA expression in NK-cells, B-cells and monocytes with the most significant abnormalities occurring in NK cells. Transfecting primary NK cells with hsa-miR-99b or hsa-miR-330-3p, resulted in gene expression changes consistent with NK cell activation but diminished cytotoxicity, suggesting that defective NK cell function contributes to CFS/ME pathology. CONCLUSION: This study demonstrates altered microRNA expression in the peripheral blood mononuclear cells of CFS/ME patients, which are potential diagnostic biomarkers. The greatest degree of miRNA deregulation was identified in NK cells with targets consistent with cellular activation and altered effector function.

The role of parvovirus B19 in the pathogenesis of autoimmunity and autoimmune disease.

Human parvovirus B19 is a single-stranded DNA virus which preferentially targets the erythroblasts in the bone marrow. B19 infection commonly causes erythema infectiosum, arthralgia, fetal death, transient aplastic crisis in patients with shortened red cell survival, and persistent infection in people who are immunocompromised. Less common clinical manifestations include atypical skin rashes, neurological syndromes, cardiac syndromes, and various cytopenias. B19 infection has also been associated with development of a variety of different autoimmune diseases, including rheumatological, neurological, neuromuscular, cardiovascular, haematological, nephrological and metabolic. Production of a variety of autoantibodies has been demonstrated to occur during B19 infection and these have been shown to be key to the pathogenesis of the particular disease process in a significant number of cases, for example, production of rheumatoid factor in cases of B19-associated rheumatoid arthritis and production of anti-glutamic acid decarboxylase (GAD) in patients with B19-associated type 1 diabetes mellitus. B19 infection has also been associated with the development of multiple autoimmune diseases in 12 individuals. Documented mechanisms in B19-associated autoimmunity include molecular mimicry (IgG antibody to B19 proteins has been shown to cross react with a variety of recognised human autoantigens, including collagen II, keratin, angiotensin II type 1 receptor, myelin basic protein, cardiolipin, and platelet membrane glycoprotein IIb/IIIa), B19-induced apoptosis with presentation of self-antigens to T lymphocytes, and the phospholipase activity of the B19 unique VP1 protein.

A review of blood diseases and cytopenias associated with human parvovirus B19 infection.

Parvovirus B19 is a single-stranded DNA virus which preferentially targets the erythroblast resulting in red cell aplasia, which is temporary in immunocompetent persons. Since the discovery of B19 virus in 1975, a wide variety of blood diseases and cytopenias affecting several blood cell lineages have been documented during or following B19 infection. These include cytopenias affecting the erythroid, megakaryoblastoid, myeloid and lymphoid lineages, as well as a variety of bicytopenias, pancytopenia, bone marrow necrosis / fat embolism syndrome, myelodysplastic syndrome, leucoerythroblastopenia, and hemophagocytic lymphohistiocytosis. B19 infection may also complicate and precede the course of acute leukemia, the significance of which remains to be determined. This review describes the current state of knowledge of the abnormalities of individual blood cell lineages encountered during parvovirus B19 infection, over the almost 40 years since its discovery, and reveals some very interesting themes, which improve our understanding of the pathogenesis of B19 infection with particular reference to the bone marrow.

Increased prevalence of two mitochondrial DNA polymorphisms in functional disease: Are we describing different parts of an energy-depleted elephant?

About 20% of the population suffers from "functional syndromes". Since these syndromes overlap greatly in terms of co-morbidity, pathophysiology (including aberrant autonomic activity) and treatment responses, common predisposing genetic factors have been postulated. We had previously showed that two common mitochondrial DNA (mtDNA) polymorphisms at positions 16519 and 3010 are statistically associated with the functional syndromes of migraine, cyclic vomiting syndrome and non-specific abdominal pain. Herein, among individuals with mtDNA haplogroup H (HgH), the presence of these two mtDNA polymorphisms were ascertained in additional functional syndromes: chronic fatigue syndrome, complex regional pain syndrome, sudden infant death syndrome, and major depressive disorder. Polymorphic prevalence rates were compared between disease and control groups, and within each disease group in participants with and without specific clinical findings. In all four conditions, one or both of the polymorphisms was significantly associated with the respective condition and/or co-morbid functional symptomatology. Thus, we conclude that these two mtDNA polymorphisms likely modify risk for the development of multiple functional syndromes, likely constituting a proportion of the postulated common genetic factor, at least among individuals with HgH. Pathophysiology likely involves broad effects on the autonomic nervous system.

The role of parvovirus B19 and the immune response in the pathogenesis of acute leukemia.

In this article, we review the evidence suggesting a possible role for B19 virus in the pathogenesis of a subset of cases of acute leukemia. Human parvovirus B19 infection may complicate the clinical course of patients with acute leukemia and may also precede the development of acute leukemia by up to 180 days. Parvovirus B19 targets erythroblasts in the bone marrow and may cause aplastic crisis in patients with shortened-red cell survival. Aplastic crisis represents a prodrome of acute lymphoblastic leukemia in 2% patients. There is a significant overlap between those HLA classes I and II alleles that are associated with a vigorous immune response and development of symptoms during B19 infection and those HLA alleles that predispose to development of acute leukemia. Acute symptomatic B19 infection is associated with low circulating IL-10 consistent with a vigorous immune response; deficient IL-10 production at birth was recently found to be associated with subsequent development of acute leukemia. Anti-B19 IgG has been associated with a particular profile of methylation of human cancer genes in patients with acute leukemia, suggesting an additional hit and run mechanism. The proposed role for parvovirus B19 in the pathogenesis of acute leukemia fits well with the delayed infection hypothesis and with the two-step mutation model, which describes carriage of the first mutation prior to birth, followed by suppression of hematopoiesis, which allows rapid proliferation of cells harboring the first mutation, acquisition of a second activating mutation, and expansion of cells carrying both mutations, resulting in acute leukemia.

Use of single-nucleotide polymorphisms (SNPs) to distinguish gene expression subtypes of chronic fatigue syndrome/myalgic encephalomyelitis (CFS/ME).

AIMS: We have reported gene expression changes in patients with chronic fatigue syndrome/myalgic encephalomyelitis (CFS/ME) and the fact that such gene expression data can be used to identify subtypes of CFS/ME with distinct clinical phenotypes. Due to the difficulties in using a comparative gene expression method as an aid to CFS/ME disease and subtype-specific diagnosis, we have attempted to develop such a method based on single-nucleotide polymorphism (SNP) analysis. METHODS: To identify SNP allele associations with CFS/ME and CFS/ME subtypes, we tested genomic DNA of patients with CFS/ME (n=108), patients with endogenous depression (n=17) and normal blood donors (n=68) for 504 human SNP alleles located within 88 CFS-associated human genes using the SNP Genotyping GoldenGate Assay (Illumina, San Diego, California, USA). 360 ancestry informative markers (AIM) were also examined. RESULTS: 21 SNPs were significantly associated with CFS/ME compared with depression and normal groups. 148 SNP alleles had a significant association with one or more CFS/ME subtypes. For each subtype, associated SNPs tended to be grouped together within particular genes. AIM SNPs indicated that 4 subjects were of Asian origin while the remainder were Caucasian. Hierarchical clustering of AIM data revealed the relatedness between 2 couples of patients with CFS only and confirmed the overall heterogeneity of all subjects. CONCLUSIONS: This study provides evidence that human SNPs located within CFS/ME associated genes are associated with particular genomic subtypes of CFS/ME. Further work is required to develop this into a clinically useful subtype-specific diagnostic test.

Two new aspects of continuity of care.

OBJECTIVE: To determine whether the original continuity of care framework is still applicable to family medicine today. DESIGN: Qualitative descriptive study. SETTING: Kingston, Ont. PARTICIPANTS: Three groups of first-year family medicine residents (18 in total), 2 groups of family physicians in established comprehensive practices (9 in total), and 2 groups of family physicians working in episodic care settings (10 in total). METHODS: Using focus groups, a semistructured discussion guide, and a qualitative descriptive design, we explored the residents' and practising physicians' conceptions about continuity of care. Qualitative content analysis was used to identify themes. MAIN FINDINGS: Focus group participants consisting of family physicians providing comprehensive care, episodic care physicians, and family medicine residents exposed 2 new dimensions of continuity of care-community continuity of care (the physicians' roles in understanding the lives of their patients, and how this affects their overall health) and continuity of care within the health care team (the continuity between a patient and members of the interprofessional team, including the family physician). Geographic continuity of care (the care of a patient in various settings by the same physician) was not prominently discussed, perhaps reflecting the paucity of family physicians in the hospital setting. CONCLUSION: Both of these new dimensions of continuity of care are consistent with the ongoing evolution of family medicine as a discipline, and have important implications for how family medicine training programs should be designed to best prepare trainees for future practice.

Absence of xenotropic murine leukaemia virus-related virus in UK patients with chronic fatigue syndrome.

BACKGROUND: Detection of a retrovirus, xenotropic murine leukaemia virus-related virus (XMRV), has recently been reported in 67% of patients with chronic fatigue syndrome. We have studied a total of 170 samples from chronic fatigue syndrome patients from two UK cohorts and 395 controls for evidence of XMRV infection by looking either for the presence of viral nucleic acids using quantitative PCR (limit of detection <16 viral copies) or for the presence of serological responses using a virus neutralisation assay. RESULTS: We have not identified XMRV DNA in any samples by PCR (0/299). Some serum samples showed XMRV neutralising activity (26/565) but only one of these positive sera came from a CFS patient. Most of the positive sera were also able to neutralise MLV particles pseudotyped with envelope proteins from other viruses, including vesicular stomatitis virus, indicating significant cross-reactivity in serological responses. Four positive samples were specific for XMRV. CONCLUSIONS: No association between XMRV infection and CFS was observed in the samples tested, either by PCR or serological methodologies. The non-specific neutralisation observed in multiple serum samples suggests that it is unlikely that these responses were elicited by XMRV and highlights the danger of over-estimating XMRV frequency based on serological assays. In spite of this, we believe that the detection of neutralising activity that did not inhibit VSV-G pseudotyped MLV in at least four human serum samples indicates that XMRV infection may occur in the general population, although with currently uncertain outcomes.

Microbial infections in eight genomic subtypes of chronic fatigue syndrome/myalgic encephalomyelitis.

BACKGROUND: The authors have previously reported genomic subtypes of chronic fatigue syndrome/myalgic encephalomyelitis (CFS/ME) based on expression of 88 human genes. AIM: To attempt to reproduce these findings, determine the specificity of this signature to CFS/ME, and test for associations between CFS/ME subtype and infection. METHODS: Expression levels of 88 human genes were determined in blood of 62 new patients with idiopathic CFS/ME (according to Fukuda criteria), six patients with Q-fever-associated CFS/ME from the Birmingham Q-fever outbreak (according to Fukuda criteria), 14 patients with endogenous depression (according to DSM-IV criteria) and 29 normal blood donors. RESULTS: In patients with CFS/ME, differential expression was confirmed for all 88 genes. Q-CFS/ME had similar patterns of gene expression to idiopathic CFS/ME. Gene expression in patients with endogenous depression was similar to that in the normal controls, except for upregulation of five genes (APP, CREBBP, GNAS, PDCD2 and PDCD6). Clustering of combined gene data in CFS/ME patients for this and the authors' previous study (117 CFS/ME patients) revealed genomic subtypes with distinct differences in SF36 scores, clinical phenotypes, severity and geographical distribution. Antibody testing for Epstein-Barr virus, enterovirus, Coxiella burnetii and parvovirus B19 revealed evidence of subtype-specific relationships for Epstein-Barr virus and enterovirus, the two most common infectious triggers of CFS/ME. CONCLUSIONS: This study confirms the involvement of these genes in CFS/ME.

Antibody to parvovirus B19 nonstructural protein is associated with chronic arthralgia in patients with chronic fatigue syndrome/myalgic encephalomyelitis.

Chronic fatigue syndrome/myalgic encephalomyelitis (CFS/ME) is a neuro-immune disease of uncertain pathogenesis. Human parvovirus B19 infection has been shown to occur just prior to development of the onset of CFS/ME in several cases, although B19 seroprevalence studies do not show any significant differences between CFS/ME and controls. In this study, we analysed parvovirus B19 markers in CFS/ME patients (n=200), diagnosed according to Fukuda CDC criteria, and normal blood donors (n=200). Serum from each subject was tested for anti-B19 VP2 IgM and IgG (by Biotrin ELISA), anti-B19 NS1 IgM and IgG (by immunofluorescence), and B19 DNA (by real-time PCR). CFS/ME patients and normal blood donors had a similar B19 seroprevalence (75 % versus 78 %, respectively). Eighty-three CFS patients (41.5 %) as compared with fourteen (7 %) normal blood donors tested positive for anti-B19 NS1 IgG (chi(2)=64.8; P<0.0001; odds ratio=9.42, CI 5.11-17.38). Of these 83 patients, 61 complained of chronic joint pain, while 22 did not. Parvovirus B19 DNA was detected in serum of 11 CFS patients and none of the controls by Taqman real-time PCR (chi(2)=9.35, P<0.002). Positivity for anti-B19 NS1 IgG was associated with higher expression levels of the human CFS-associated genes NHLH1 and GABPA. As NS1 antibodies are thought to indicate chronic or severe courses of B19 infection, these findings suggest that although the seroprevalence of B19 in CFS patients is similar to controls, the immune control of the virus in these patients may not be efficient.

Gene profiling of patients with chronic fatigue syndrome/myalgic encephalomyelitis.

Chronic fatigue syndrome/myalgic encephalomyelitis (CFS/ME) is a multisystem disease, the pathogenesis of which remains undetermined. Following two microarray studies, we reported the differential expression of 88 human genes in patients with CFS; 85 of these genes were upregulated and 3 were downregulated. The top functional categories of these 88 genes were hematologic disease and function, immunologic disease and function, cancer, cell death, immune response, and infection. Clustering of quantitative polymerase chain reaction data from CFS/ME patients revealed seven subtypes with distinct differences in Short Form (SF)-36 scores, clinical phenotypes, and severity. Gene signatures in each subtype implicate five human genes as possible targets for specific therapy. Development of a diagnostic test for subtype status is now a priority. The possibility that these subtypes represent individual host responses to particular microbial infections is being investigated and may provide another route to specific therapies for CFS patients.

Gene expression subtypes in patients with chronic fatigue syndrome/myalgic encephalomyelitis.

Chronic fatigue syndrome/myalgic encephalomyelitis (CFS/ME) is a multisystem disease, the pathogenesis of which remains undetermined. We set out to determine the precise abnormalities of gene expression in the blood of patients with CFS/ME. We analyzed gene expression in peripheral blood from 25 patients with CFS/ME diagnosed according to the Centers for Disease Control and Prevention diagnostic criteria and 50 healthy blood donors, using a microarray with a cutoff fold difference of expression of >or=2.5. Genes showing differential expression were further analyzed in 55 patients with CFS/ME and 75 healthy blood donors, using quantitative polymerase chain reaction. Differential expression was confirmed for 88 genes; 85 were upregulated, and 3 were downregulated. Highly represented functions were hematological disease and function, immunological disease and function, cancer, cell death, immune response, and infection. Clustering of quantitative polymerase chain reaction data from patients with CFS/ME revealed 7 subtypes with distinct differences in Medical Outcomes Survey Short Form-36 scores, clinical phenotypes, and severity.

Preexisting psychological stress predicts acute and chronic fatigue and arthritis following symptomatic parvovirus B19 infection.

BACKGROUND: Psychological stress is thought to be an important factor in the pathogenesis of chronic fatigue syndrome/myalgic encephalomyelitis (CFS/ME). Therefore, we sought to examine this relationship in the context of parvovirus B19 infection. METHODS: Thirty-nine patients with laboratory-documented acute parvovirus B19 infection were asked to complete questionnaires on negative life events, perceived stress, and negative affect relevant to the time of onset of parvovirus infection and during the preceding 12 months. These scores were combined into an overall stress index, which was then examined for associations with particular parvovirus-associated symptoms at acute infection and during the ensuing 1-3 years. Additional characteristics monitored included presence of parvovirus antibodies and nucleic acid, cortisol level, dehydroepiandrosterone level, autoantibodies, levels of a range of serum cytokines, and human leukocyte antigen class I and II alleles. RESULTS: Stress index was significantly associated with development of fatigue during the acute phase of parvovirus B19 infection and also with chronic fatigue and arthritis occurring 1-3 years following acute parvovirus B19 infection. Logistic regression that included all clinical variables indicated that a high stress index at the time of onset of infection was the primary predictor of CFS/ME 1-3 years following acute parvovirus B19 infection (odds ratio, 25.7; 95% confidence interval, 1.7-121.9; P=.005). CONCLUSIONS: We report a highly significant association between psychological stress and development of acute and chronic fatigue and arthritis several years following laboratory-documented acute parvovirus B19 infection.

The impact of interest: how do family medicine interest groups influence medical students?

OBJECTIVE: To describe the knowledge of, and experience with, the Interest Group in Family Medicine (IgFM) at the University of Toronto, among undergraduate medical students; to explore the effects of the IgFM on undergraduate medical students; and to help determine future directions for the IgFM and other family medicine interest groups in Canada. DESIGN: Qualitative descriptive design and focus groups. SETTING: The Faculty of Medicine at the University of Toronto in Ontario. PARTICIPANTS: A total of 45 students in the undergraduate medical school program at the University of Toronto participated in this study. Approximately equal numbers of students from each year were represented in the sample. METHOD: Focus groups were conducted to determine students' knowledge of, experiences with, and perspectives on the IgFM. The focus groups were audiotaped and transcribed verbatim. Transcripts were coded and analyzed for themes using qualitative content analysis. Data were collected until saturation of emerging themes was reached. MAIN FINDINGS: The students were generally knowledgeable about the IgFM and many had attended IgFM events. The IgFM had different effects on students depending on their level of interest in family medicine (FM). For those already interested in FM, the IgFM helped to maintain and support that interest. For students still undecided about their career choices, the IgFM helped to support continued interest in FM by dispelling negative myths about the discipline, providing positive peer influences, and supplying information about careers in FM. For students not interested in FM, the IgFM provided helpful information about the discipline. Students also had many useful suggestions regarding the future direction of the IgFM. CONCLUSION: The IgFM has been successful in increasing medical student exposure to FM and in supporting students' interest in this discipline. Information from this study also provides strategies for future direction to the IgFM and other family medicine interest groups in Canada and the United States.