Genetic and transcriptomic analyses support a switch to lytic phase in Epstein Barr virus infection as an important driver in developing Systemic Lupus Erythematosus.

To investigate the molecular mechanisms through which Epstein-Barr virus (EBV) may contribute to Systemic Lupus Erythematosus (SLE) pathogenesis, we interrogated SLE genetic risk loci for signatures of EBV infection. We first compared the gene expression profile of SLE risk genes across 459 different cell/tissue types. EBV-infected B cells (LCLs) had the strongest representation of highly expressed SLE risk genes. By determining an SLE risk allele effect on gene expression (expression quantitative trait loci, eQTL) in LCLs and 16 other immune cell types, we identified 79 SLE risk locus:gene pairs putatively interacting with EBV infection. A total of 10 SLE risk genes from this list (CD40, LYST, JAZF1, IRF5, BLK, IKZF2, IL12RB2, FAM167A, PTPRC and SLC15A) were targeted by the EBV transcription factor, EBNA2, differentially expressed between LCLs and B cells, and the majority were also associated with EBV DNA copy number, and expression level of EBV encoded genes. Our final gene network model based on these genes is suggestive of a nexus involving SLE risk loci and EBV latency III and B cell proliferation signalling pathways. Collectively, our findings provide further evidence to support the interaction between SLE risk loci and EBV infection that is in part mediated by EBNA2. This interplay may increase the tendency towards EBV lytic switching dependent on the presence of SLE risk alleles. These results support further investigation into targeting EBV as a therapeutic strategy for SLE.

The Interaction of Human and Epstein-Barr Virus miRNAs with Multiple Sclerosis Risk Loci.

Although the causes of Multiple Sclerosis (MS) still remain largely unknown, multiple lines of evidence suggest that Epstein-Barr virus (EBV) infection may contribute to the development of MS. Here, we aimed to identify the potential contribution of EBV-encoded and host cellular miRNAs to MS pathogenesis. We identified differentially expressed host miRNAs in EBV infected B cells (LCLs) and putative host/EBV miRNA interactions with MS risk loci. We estimated the genotype effect of MS risk loci on the identified putative miRNA:mRNA interactions in silico. We found that the protective allele of MS risk SNP rs4808760 reduces the expression of hsa-mir-3188-3p. In addition, our analysis suggests that hsa-let-7b-5p may interact with ZC3HAV1 differently in LCLs compared to B cells. In vitro assays indicated that the protective allele of MS risk SNP rs10271373 increases ZC3HAV1 expression in LCLs, but not in B cells. The higher expression for the protective allele in LCLs is consistent with increased IFN response via ZC3HAV1 and so decreased immune evasion by EBV. Taken together, this provides evidence that EBV infection dysregulates the B cell miRNA machinery, including MS risk miRNAs, which may contribute to MS pathogenesis via interaction with MS risk genes either directly or indirectly.

Transcribed B lymphocyte genes and multiple sclerosis risk genes are underrepresented in Epstein-Barr Virus hypomethylated regions.

Epstein-Barr Virus (EBV) infection appears to be necessary for the development of Multiple Sclerosis (MS), although the specific mechanisms are unknown. More than 200 single-nucleotide polymorphisms (SNPs) are known to be associated with the risk of developing MS. About a quarter of these are also highly associated with proximal gene expression in B cells infected with EBV (lymphoblastoid cell lines-LCLs). The DNA of LCLs is hypomethylated compared with both uninfected and activated B cells. Since methylation can affect gene expression, and so cell differentiation and immune evasion, we hypothesised that EBV-driven hypomethylation may affect the interaction between EBV infection and MS. We interrogated an existing dataset comprising three individuals with whole-genome bisulfite sequencing data from EBV transformed B cells and CD40L-activated B cells. DNA methylation surrounding MS risk SNPs associated with gene expression in LCLs (LCLeQTL) was less likely to be hypomethylated than randomly selected chromosomal regions. Differential methylation was independent of genomic features such as promoter regions, but genes preferentially expressed in EBV-infected B cells, including the LCLeQTL genes, were underrepresented in the hypomethylated regions. Our data does not indicate MS genetic risk is affected by EBV hypomethylation.

Gastric Cancer Screening in Common Variable Immunodeficiency.

Individuals with common variable immunodeficiency (CVID) have an increased risk of gastric cancer, and gastrointestinal lymphoma, yet screening for premalignant gastric lesions is rarely offered routinely to these patients. Proposed screening protocols are not widely accepted and are based on gastric cancer risk factors that are not applicable to all CVID patients. Fifty-two CVID patients were recruited for screening gastroscopy irrespective of symptoms or blood results and were compared to 40 controls presenting for gastroscopy for other clinical indications. Overall, 34% of CVID patients had intestinal metaplasia (IM), atrophic gastritis or moderate to severe non-atrophic gastritis, which can increase the risk of gastric cancer, compared to 7.5% of controls (p < 0.01). Focal nodular lymphoid hyperplasia, a precursor lesion for gastrointestinal lymphoma, was seen in eight CVID patients (16%), one of whom was diagnosed with gastrointestinal lymphoma on the same endoscopy. High-risk gastric pathology was associated with increased time since diagnosis of CVID, smoking, Helicobacter pylori, a low-serum pepsinogen I concentration, and diarrhea, but not pepsinogen I/II ratio, iron studies, vitamin B12 levels or upper gastrointestinal symptoms. There was a lower rate of detection of IM when fewer biopsies were taken, and IM and gastric atrophy were rarely predicted by the endoscopist macroscopically, highlighting the need for standardized biopsy protocols. The prevalence of premalignant gastric lesions in patients with CVID highlights the need for routine gastric screening. We propose a novel gastric screening protocol to detect early premalignant lesions and reduce the risk of gastric cancer and gastric lymphoma in these patients.

Prominent subcutaneous oedema as a masquerading symptom of an underlying inflammatory myopathy.

The inflammatory myopathies are a group of immune-mediated inflammatory muscle disorders that typically present with marked proximal muscle weakness. We report four cases of inflammatory myopathies with marked subcutaneous oedema as their main complaint. Three of the four patients had normal or low levels of creatine kinase, an enzyme often markedly elevated in these disorders. Magnetic resonance imaging of the muscles, followed by a muscle biopsy were used to make a definitive diagnosis.

miRNAs and HIV: unforeseen determinants of host-pathogen interaction.

Our understanding of the complexity of gene regulation has significantly improved in the last decade as the role of small non-coding RNAs, called microRNAs (miRNAs), has been appreciated. These 19-22 nucleotide RNA molecules are critical regulators of mRNA translation and turnover. The miRNAs bind via a protein complex to the 3' untranslated region (3' UTR) of mRNA, ultimately leading to mRNA translational inhibition, degradation, or repression. Although many mechanisms by which human immunodeficiency virus-1 (HIV-1) infection eventually induces catastrophic immune destruction have been elucidated, the important role that miRNAs play in HIV-1 pathogenesis is only now emerging. Accumulating evidence demonstrates that changes to endogenous miRNA levels following infection is important: in maintaining HIV-1 latency in resting CD4(+) T cells, potentially affect immune function via changes to cytokines such as interleukin-2 (IL-2) and IL-10 and may predict disease progression. We review the roles that both viral and host miRNAs play in different cell types and disease conditions that are important in HIV-1 infection and discuss how miRNAs affect key immunomodulatory molecules contributing to immune dysfunction. Further, we discuss whether miRNAs may be used as novel biomarkers in serum and the potential to modulate miRNA levels as a unique approach to combating this pathogen.

Role of miR-155 in the regulation of lymphocyte immune function and disease.

MicroRNAs (miRNAs) have emerged as critical regulators of gene expression within cells. One particular miRNA, miR-155, is highly expressed within lymphocytes (both B and T cells) and mediates a number of important roles. These include shaping the transcriptome of lymphoid cells that control diverse biological functions vital in adaptive immunity. The use of mice engineered to be deficient in miR-155, as well as the identification of endogenous targets of miR-155 in T cells by transcriptome-wide analysis, has helped to unravel the crucial role that this miRNA plays in fine tuning the regulation of lymphocyte subsets such as B cells, CD8(+) and CD4(+) T cells ranging from T helper type 1 (Th1), Th2, Th17 and regulatory T cells. In this review, we summarize what we have learned about miR-155 in the regulation of lymphocyte responses at the cellular and molecular levels and in particular, we focus on the recent findings showing that miR-155 shapes the balance between tolerance and immunity.

The microRNA-9/B-lymphocyte-induced maturation protein-1/IL-2 axis is differentially regulated in progressive HIV infection.

The fine control of T-cell differentiation and its impact on HIV disease states is poorly understood. In this study, we demonstrate that B-lymphocyte-induced maturation protein-1 (Blimp-1/Prdm1) is highly expressed in CD4(+) T cells from chronically HIV-infected (CHI) patients compared to cells from long-term nonprogressors or healthy controls. Stimulation through the T-cell receptor in the presence of IL-2 induces Blimp-1 protein expression. We show here that Blimp-1 levels are translationally regulated by microRNA-9 (miR-9). Overexpression of miR-9 induces Blimp-1 repression, restoring IL-2 secretion in CD4(+) T cells via reduction in the binding of Blimp-1 to the il-2 promoter. In CHI patients where IL-2 expression is reduced and there is generalized T-cell dysfunction, we show differential expression of both miR-9 and Blimp-1 in CD4(+) cells compared with levels in long-term nonprogressors. These data identify a novel miR-9/Blimp-1/IL-2 axis that is dysregulated in progressive HIV infection.

Differential regulation of the Let-7 family of microRNAs in CD4+ T cells alters IL-10 expression.

MicroRNAs (miRNAs) are ∼22-nt small RNAs that are important regulators of mRNA turnover and translation. Recent studies have shown the importance of the miRNA pathway in HIV-1 infection, particularly in maintaining latency. Our initial in vitro studies demonstrated that HIV-1-infected HUT78 cells expressed significantly higher IL-10 levels compared with uninfected cultures. IL-10 plays an important role in the dysregulated cytotoxic T cell response to HIV-1, and in silico algorithms suggested that let-7 miRNAs target IL10 mRNA. In a time course experiment, we demonstrated that let-7 miRNAs fall rapidly following HIV-1 infection in HUT78 cells with concomitant rises in IL-10. To show a direct link between let-7 and IL-10, forced overexpression of let-7 miRNAs resulted in significantly reduced IL-10 levels, whereas inhibition of the function of these miRNAs increased IL-10. To demonstrate the relevance of these results, we focused our attention on CD4(+) T cells from uninfected healthy controls, chronic HIV-1-infected patients, and long-term nonprogressors. We characterized miRNA changes in CD4(+) T cells from these three groups and demonstrated that let-7 miRNAs were highly expressed in CD4(+) T cells from healthy controls and let-7 miRNAs were significantly decreased in chronic HIV-1 infected compared with both healthy controls and long-term nonprogressors. We describe a novel mechanism whereby IL-10 levels can be potentially modulated by changes to let-7 miRNAs. In HIV-1 infection, the decrease in let-7 miRNAs may result in an increase in IL-10 from CD4(+) T cells and provide the virus with an important survival advantage by manipulating the host immune response.

Interleukin-27 treated human macrophages induce the expression of novel microRNAs which may mediate anti-viral properties.

Interleukin-27 (IL-27) is a pleiotropic cytokine which plays important and diverse roles in the immune system. We have previously demonstrated that IL-27 induces potent anti-viral effects against HIV-1, HIV-2, SIV, HSV-2, KSHV and influenza viruses in macrophages. This induction occurred in an interferon (IFN) independent manner and involved down regulation of SPTBN1. MicroRNAs (miRNAs) are critical regulators of mRNA translation and turnover. There have been reports that some miRNAs inhibit viral replication. In this study, we hypothesized that IL-27 could induce the expression of novel miRNAs in macrophages which may have functional relevance in terms of anti-viral activity and primary monocytes were differentiated into macrophages using either M-CSF (M-Mac) or a combination of M-CSF and IL-27 (I-Mac) for seven days. Following this, total RNA was extracted from these cells and deep sequencing was performed, in parallel with gene expression microarrays. Using the novel miRNA discovery software, miRDeep, seven novel miRNAs were discovered in these macrophages. Four of which were preferentially expressed in I-Mac (miR-SX1, -SX2, -SX3 and -SX6) whilst three were detected in both M-Mac and I-Mac (miR-SX4, -SX5 and -SX7). The expression of six of the seven novel miRNAs was highly correlated with qRT-PCR using specific primer/probes designed for the novel miRNAs. Gene expression microarray further demonstrated that a number of genes were potentially targeted by these differentially expressed novel miRNAs. Finally, several of these novel miRNAs (miR-SX1, -SX4, -SX5, -SX6 and -SX7) were shown to target the open reading frames of a number of viruses (including HSV-1, HSV-2 and HHV-8) which may partially explain the anti-viral properties observed.

The interaction between Epstein-Barr virus and multiple sclerosis genetic risk loci: insights into disease pathogenesis and therapeutic opportunities.

Multiple sclerosis (MS) is a chronic neurodegenerative autoimmune disease, characterised by the demyelination of neurons in the central nervous system. Whilst it is unclear what precisely leads to MS, it is believed that genetic predisposition combined with environmental factors plays a pivotal role. It is estimated that close to half the disease risk is determined by genetic factors. However, the risk of developing MS cannot be attributed to genetic factors alone, and environmental factors are likely to play a significant role by themselves or in concert with host genetics. Epstein-Barr virus (EBV) infection is the strongest known environmental risk factor for MS. There has been increasing evidence that leaves little doubt that EBV is necessary, but not sufficient, for developing MS. One plausible explanation is EBV may alter the host immune response in the presence of MS risk alleles and this contributes to the pathogenesis of MS. In this review, we discuss recent findings regarding how EBV infection may contribute to MS pathogenesis via interactions with genetic risk loci and discuss possible therapeutic interventions.

Biologics in peripheral ulcerative keratitis.

Over the past two decades biologic therapies have seen a rapid uptake in the management of ocular inflammation. Peripheral ulcerative keratitis (PUK), once a harbinger of blindness and mortality in refractory rheumatological disease, is now increasingly being treated with these agents. We conducted a review to evaluate the evidence base for this application and to provide a road map for their clinical usage in PUK, including dosage and adverse effects. A literature search across Medline, Embase and Cochrane Database of Systematic Reviews was undertaken to identify all patients with PUK that were treated with a biologic in a peer viewed article. Overall, whilst the evidence base for biologic use in PUK was poor, reported cases demonstrate an increasingly powerful and effective role for biologics in refractory PUK. This was particularly the case for rituximab in PUK secondary to granulomatous with polyangiitis.

The conundrum of neuropsychiatric systemic lupus erythematosus: Current and novel approaches to diagnosis.

Recognising neuropsychiatric involvement by systemic lupus erythematosus (SLE) is of growing importance, however many barriers to this exist at multiple levels of our currently available diagnostic algorithms that may ultimately delay its diagnosis and subsequent treatment. The heterogeneous and non-specific clinical syndromes, serological and cerebrospinal fluid (CSF) markers and neuroimaging findings that often do not mirror disease activity, highlight important research gaps in the diagnosis of neuropsychiatric SLE (NPSLE). Formal neuropsychological assessments or the more accessible screening metrics may also help improve objective recognition of cognitive or mood disorders. Novel serum and CSF markers, including autoantibodies, cytokines and chemokines have also shown increasing utility as part of diagnosis and monitoring, as well as in distinguishing NPSLE from SLE patients without SLE-related neuropsychiatric manifestations. Novel neuroimaging studies also expand upon our existing strategy by quantifying parameters that indicate microarchitectural integrity or provide an assessment of neuronal function. Some of these novel markers have shown associations with specific neuropsychiatric syndromes, suggesting that future research move away from considering NPSLE as a single entity but rather into its individually recognized neuropsychiatric manifestations. Nevertheless, it is likely that a composite panel of these investigations will be needed to better address the gaps impeding recognition of neuropsychiatric involvement by SLE.

Isolated anti-Ro52 identifies a severe subset of Sjögren's syndrome patients.

Introduction: Serum autoantibodies targeting the SSA/Ro proteins are a key component of the classification criteria for the diagnosis of Sjögren's syndrome (SS). Most patients' serum reacts with both Ro60 and Ro52 proteins. Here we compare the molecular and clinical characteristics of patients diagnosed with SS with anti-Ro52 in the presence or absence of anti-Ro60/La autoantibodies. Methods: A cross-sectional study was performed. Patients in the SS biobank at Westmead Hospital (Sydney, Australia) that were positive for anti-Ro52 were included and stratified based on the absence (isolated) or presence (combined) of anti-Ro60/La, measured by line immunoassay. We examined clinical associations and the serological and molecular characteristics of anti-Ro52 using ELISA and mass spectrometry in serological groups. Results: A total of 123 SS patients were included for study. SS patients with isolated anti-Ro52 (12%) identified a severe serological subset characterised by higher disease activity, vasculitis, pulmonary involvement, rheumatoid factor (RhF) and cryoglobulinaemia. Serum antibodies reacting with Ro52 in the isolated anti-Ro52 subset displayed less isotype switching, less immunoglobulin variable region subfamily usage and a lower degree of somatic hypermutation than the combined anti-Ro52 subset. Conclusions: In our cohort of SS patients, isolated anti-Ro52 represents a severe subset of SS, and is associated with the presence of cryoglobulinaemia. We therefore provide clinical relevance to the stratification of SS patients by their sero-reactivities. It is possible that the autoantibody patterns may be immunological epiphenomena of the underlying disease process, and further work is required to unearth the mechanisms of the differential clinical phenotypes.

Use of adrenaline to manage suspected anaphylaxis following COVID-19 vaccination: An Australian retrospective cohort study.

BACKGROUND: The rate of anaphylaxis following COVID-19 vaccinations is estimated to be 2-11 cases per million doses administered. However, adrenaline is occasionally used in individuals who are later diagnosed with immunisation stress-related responses, as their initial presenting signs and symptoms can appear similar to that of anaphylaxis. This study aims to describe the clinical profile of individuals who had received adrenaline following a COVID-19 vaccine and their subsequent revaccination outcomes. METHODS: We examined notifications of cases who had received adrenaline following a COVID-19 vaccine in New South Wales, Australia. The cases were classified into Brighton Collaboration Case Definition (BCCD) for anaphylaxis, their clinical presentation, management and subsequent revaccination outcomes were compared. RESULTS: From 22 February 2021 to 30 September 2021, there were 222 cases where adrenaline was administered. Of these, 32 (14 %) fulfilled Level 1 BCCD, 59 (27%) Level 2, 2 (1%) Level 3, 97 (44%) Level 4 and 32 (14 %) Level 5. The most commonly reported symptoms were sensation of throat closure (n = 116, 52%), difficulty breathing (n = 82, 37%) and nausea (n = 55, 25 %). Of the 176 (79%) individuals who proceeded to further vaccination, 89 (51%) received the same vaccine formulation and only 14 (8%) experienced another allergic adverse event with 9 (5%) receiving adrenaline. CONCLUSION: Less than one in five individuals who received adrenaline met Level 1 BCCD criteria for anaphylaxis. Many reactions that were treated with adrenaline had little to no diagnostic certainty of anaphylaxis and in such cases repeat vaccination had a high likelihood of being tolerated. Increased awareness and education on objective signs and symptoms of anaphylaxis is required to ensure appropriate use of adrenaline.

Clinical and laboratory features of COVID-19 illness and outcomes in immunocompromised individuals during the first pandemic wave in Sydney, Australia.

People with immunocompromising conditions are at increased risk of SARS-CoV-2 infection and mortality, however early in the pandemic it was challenging to collate data on this heterogenous population. We conducted a registry study of immunocompromised individuals with polymerase chain reaction (PCR)-confirmed SARS-CoV-2 infection from March-October 2020 in Sydney, Australia to understand clinical and laboratory outcomes in this population prior to the emergence of the Delta variant. 27 participants were enrolled into the study including people with a haematologic oncologic conditions (n = 12), secondary immunosuppression (N = 8) and those with primary or acquired immunodeficiency (i.e. HIV; N = 7). All participants had symptomatic COVID-19 with the most common features being cough (64%), fever (52%) and headache (40%). Five patients demonstrated delayed SARS-CoV-2 clearance lasting three weeks to three months. The mortality rate in this study was 7% compared to 1.3% in the state of New South Wales Australia during the same period. This study provides data from the first eight months of the pandemic on COVID-19 outcomes in at-risk patient groups.

Standardized testing and written communication improve patient understanding of beta-lactam allergy testing outcomes: A multicenter, prospective study.

Background: Historical penicillin allergy is commonly reported, but the lack of standardized allergy clinic practices may diminish the ability to delabel beta-lactam allergy appropriately. Objective: We sought to improve beta-lactam allergy testing and patient understanding of their antibiotic allergy status by standardizing testing and communication practices between 7 adult and pediatric hospital centers. Methods: Phase 1 prospectively described the beta-lactam allergy testing practices at each center. Following this, practice was standardized to achieve a defined panel of skin testing reagents, pro forma result letters for patients and referring doctors, and provision of medical alert jewelry to those with confirmed allergy. Testing outcomes and patient perception regarding allergy status 8 weeks postassessment were compared before (phase 1) and after standardization (phase 2). Primary outcomes were the percentage of participants delabeled after testing, and concordance rates between participant perception of their allergy status and their status as determined by the treating physician at 8-week follow-up. Results: Of 195 adult and pediatric participants (median age, 50 years; 21.5% <18 years; 36.9% males), 75% were delabeled of their beta-lactam allergy. No patient experienced anaphylaxis related to any beta-lactam delabeling testing. In phase 1, 75% of participants received written results, 52% were informed verbally, and 48% received results in more than 1 form. All phase 2 participants received written results (P < .01), 61% received verbal results from a physician as well (P > .05). At 8-week follow-up, 54% of phase 1 participants had concordant perceptions of their allergy status as the testing team versus 91.6% in phase2 (P < .001). Of the 17 participants who were delabeled and treated with a beta-lactam antibiotic during the 8-week follow-up period, there were no reported allergic reactions, although 1 participant experienced anaphylaxis following exposure to amoxicillin-clavulanic acid 1 year after delabeling. Conclusions: Standardization of testing and written patient information improved short-term patient perception of beta-lactam allergy status.