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Identification of key intermediates for the spatio-temporal regulation of TLR2 and TLR3 signalling
Toll-like receptors (TLRs) are the first line of host defence and one of the most potent triggers of immune responses. They play a pivotal role in a multitude of pathologies including cancer, chronic inflammatory diseases, and infection. Pioneering studies have shown that the localization of TLRs at the cell surface and at the endosomes is not as rigid as previously envisaged, and that cell surface TLRs may travel to the endosome to engage signalling. However, how the endosomal machinery directs TLR trafficking and modulates spatiotemporal signalling remains incompletely mapped, hindering its biological and therapeutic manipulation. In Chapter 4, through use of flow cytometry and advanced microscopy approaches, including LSM Confocal, Airyscan and Total internal reflection fluorescence microscopy in both endogenously expressing TLR cell lines (E.G. RPE1), in addtion to overexpression models (E.G. HEK293-Blue-TLR2), I provide novel insight on a common endocytic route mediated by Clathrin, shared by TLR2 and TLR3 following activation, including identification of a candidate targeting loci. Complimenting trafficking assays with functional readouts, my results in Chapter 5 further identify a common endosomal sorting machinery, the endosomal sorting complexes required for transport (ESCRT) that promotes TLR2 signalling, while terminating TLR3 signalling by inducing its degradation, signifying a signalling divergence from a common machinery. Promisingly, these observations were mirrored in overexpression assays in addition to endogenous and biologically relevant models such as human derived blood macrophages. Furthermore, in Chapter 6, through trafficking and signalling approaches, I go on to reveal a novel relationship between TLRs and the Retromer complex, which may be able to differentially regulate different TLRs and allows us behind the curtain that provides greater molecular details to TLR biology and regulation for targeting. Together, my results shine a light on how TLR trafficking is a novel regulatory intersection for TLR signalling, and identify new interacting partners of TLRs and signalling regulators. These findings will help pave the way for future selective targeting of the molecular machineries regulating TLR-driven immunity, utilising differential TLR regulation in bacterial and viral pathways to help define new targeted therapeutics.
Risk of repeat self-harm among individuals presenting to healthcare services: development and validation of a clinical risk assessment model (OxSET).
BACKGROUND: A self-harm episode is a major risk factor for repeat self-harm. Existing tools to assess and predict repeat self-harm have major methodological limitations, and few are externally validated. OBJECTIVE: To develop and validate a risk assessment model of repeat self-harm up to 6 months after an episode of non-fatal self-harm that resulted in an emergency visit to hospital or specialised care. METHODS: Using Swedish national registers, we identified 53 172 people aged≥10 years who self-harmed during 2008-2012. We allocated 37 523 individuals to development (2820 or 7.5% repeat self-harm incidents within 6 months) and 15 649 to geographic validation (1373 repeat episodes) samples, based on region of residence. In a temporal validation of people who self-harmed during 2018-2019, we identified 25 036 individuals (2886 repeat episodes). We fitted a multivariable accelerated failure time model to predict risk of repeat self-harm. FINDINGS: In the external validations (n=40 685), rates of repeat self-harm were 8.8%-11.5% over 6 months. The final model retained 17 factors. Calibration and discrimination were similar in both validation samples, with observed-to-expected ratio=1.15 (95% CI=1.09 to 1.21) and c-statistic=0.72 (95% CI=0.70 to 0.73) in the geographical validation. At 6 months and a 10% risk cut-off, sensitivity was 51.5% (95% CI=48.8% to 54.2%) and specificity was 80.7% (95% CI=80.1% to 81.4%) in geographic validation; corresponding values were 56.9% (95% CI=55.1% to 58.7%) and 76.0% (95% CI=75.5% to 76.6%) in temporal validation. Discrimination was slightly worse at the 1-month prediction horizon (c-statistics of 0.66-0.68). CONCLUSIONS: Using mostly routinely collected data, simple risk assessment models and tools can provide acceptable levels of accuracy for repeat of self-harm. CLINICAL IMPLICATIONS: This risk model (OXford SElf-harm repeat tool) may assist clinical decision-making.
Cytomegalovirus-specific CD8+ T cells do not develop in all renal transplant patients at risk of virus infection.
Cytomegalovirus (CMV) is an important pathogen in immunosuppressed renal transplant patients. At greatest risk are CMV IgG seronegative recipients (R-) of kidneys from CMV IgG seropositive donors (D+), although not all develop CMV disease. The aims of the study were to determine whether D+/R- patients who do or do not go on to develop CMV disease differ in their CD8+ T cell responses to CMV. Responses to the immunodominant NLVPMVATV peptide from the CMV structural protein pp65 in HLA-A2+ renal transplant patients were quantified using HLA tetramers/pentamers. Most D+/R+ patients had detectable tetramer+ cells while most D-/R- patients did not. Around 50% of D+/R- patients had some CD8+ tetramer+ cells and there was a strong correlation between % tetramer+ cells and the occurrence of a CMV infection post-transplantation (P<0.005). 18/41 (44%) of CMV negative patients receiving a kidney from a CMV+ donor failed to develop a detectable CMV infection, or significant numbers of tetramer+ cells. There was no relationship between CMV infection and acute cellular rejection. There was a tendency for patients who were given pre-emptive antiviral therapy to have lower levels of tetramer+ cells but this was not statistically significant. Hence the results show that CMV- patients receiving a kidney from a CMV+ donor do not inevitably acquire CMV infection. Those without CMV disease did not show any T cell response while most patients with detectable CMV developed specific CD8+ T cells.
Clusters of post-acute COVID-19 symptoms: a latent class analysis across 9 databases and 7 countries.
OBJECTIVE: Prior evidence has suggested the multisystem symptomatic manifestations of post-acute COVID-19 condition (PCC). Here we conducted a network cluster analysis of 24 WHO proposed symptoms to identify potential latent subclasses of PCC. STUDY DESIGN AND SETTING: Individuals with a positive test of or diagnosed with SARS-CoV-2 after 09/2020 and with at least one symptom within ≥90 to 365 days following infection were included. Sub-analyses were conducted among people with ≥3 different symptoms. Summary characteristics were provided for each cluster. All analyses were conducted separately in 9 databases from 7 countries, including data from primary care, hospitals, national health claims and national health registries, allowing to compare clusters across the different healthcare settings. RESULTS: 787,078 persons with PCC were included. Single-symptom clusters were common across all databases, particularly for joint pain, anxiety, depression and allergy. Complex clusters included anxiety-depression and abdominal-gastrointestinal symptoms. CONCLUSION: Substantial heterogeneity within and between PCC clusters was seen across healthcare settings. Current definitions of PCC should be critically reviewed to reflect this variety in clinical presentation.
Ethnic disparities in COVID-19 mortality and cardiovascular disease in England and Wales between 2020-2022.
An increased risk of COVID-19 mortality risk among certain ethnic groups is well-reported, however data on ethnic disparities in COVID-19-related cardiovascular disease (CVD) are lacking. We estimated age-standardised incidence rates and adjusted hazard ratios for 28-day mortality and 30-day CVD by sex for individual ethnicity groups from England and Wales, using linked health and administrative data. We studied 6-level census-based ethnicity group classification, 10-level classification (only for Wales), and 19-level classification as well as any ethnicity sub-groups comprising >1000 individuals each (only for England). COVID-19 28-day mortality and 30-day CVD risk was increased in most non-White ethnic groups in England, and Asian population in Wales, between 23rd January 2020 and 1st April 2022. English data show mortality decreased during the Omicron variant's dominance, whilst CVD risk [95% confidence interval] remained elevated for certain ethnic groups when compared to White populations (January-April 2022): by 120% [28-280%] in White and Asian men and 58% [32-90%] in Pakistan men, as compared to White British men; and by 75% [13-172%] in Bangladeshi women, 55% [19-102%] in Caribbean women, and 82% [31-153%] in Any Other Ethnic Group women, as compared to White British women. Ethnically diverse populations in the UK remained disproportionately affected by CVD throughout and beyond the COVID-19 pandemic.
Effect of a 2-week interruption in methotrexate treatment versus continued treatment on COVID-19 booster vaccine immunity in adults with inflammatory conditions (VROOM study): a randomised, open label, superiority trial
This article consists of a citation of a published article describing research funded by the Efficacy and Mechanism Evaluation programme under project number NIHR134607, and is provided as as part of the complete record of research outputs for this project. The original publication is available at: https://doi.org/10.1016/S2213-2600(22)00186-2 Background Immunosuppressive treatments inhibit vaccine-induced immunity against SARS-CoV-2. We evaluated whether a 2-week interruption of methotrexate treatment immediately after the COVID-19 vaccine booster improved antibody responses against the S1 receptor-binding domain (S1-RBD) of the SARS-CoV-2 spike protein compared with uninterrupted treatment in patients with immune-mediated inflammatory diseases. Methods We did an open-label, prospective, two-arm, parallel-group, multicentre, randomised, controlled, superiority trial in 26 hospitals in the UK. We recruited adults from rheumatology and dermatology clinics who had been diagnosed with an immune-mediated inflammatory disease (eg, rheumatoid arthritis, psoriasis with or without arthritis, axial spondyloarthritis, atopic dermatitis, polymyalgia rheumatica, and systemic lupus erythematosus) and who were taking low-dose weekly methotrexate (≤25 mg per week) for at least 3 months. Participants also had to have received two primary vaccine doses from the UK COVID-19 vaccination programme. We randomly assigned the participants (1:1), using a centralised validated computer randomisation program, to suspend methotrexate treatment for 2 weeks immediately after their COVID-19 booster (suspend methotrexate group) or to continue treatment as usual (continue methotrexate group). Participants, investigators, clinical research staff, and data analysts were unmasked, while researchers doing the laboratory analyses were masked to group assignment. The primary outcome was S1-RBD antibody titres 4 weeks after receiving the COVID-19 booster vaccine dose, assessed in the intention-to-treat population. This trial is registered with ISRCT, ISRCTN11442263; following the pre-planned interim analysis, recruitment was stopped early. Findings Between Sept 30, 2021 and March 3, 2022, we recruited 340 participants, of whom 254 were included in the interim analysis and had been randomly assigned to one of the two groups: 127 in the continue methotrexate group and 127 in the suspend methotrexate group. Their mean age was 59·1 years, 155 (61%) were female, 130 (51%) had rheumatoid arthritis, and 86 (34%) had psoriasis with or without arthritis. After 4 weeks, the geometric mean S1-RBD antibody titre was 22 750 U/mL (95% CI 19 314-26 796) in the suspend methotrexate group and 10 798 U/mL (8970-12 997) in the continue methotrexate group, with a geometric mean ratio (GMR) of 2·19 (95% CI 1·57-3·04; p<0·0001; mixed-effects model). The increased antibody response in the suspend methotrexate group was consistent across methotrexate dose, administration route, type of immune-mediated inflammatory disease, age, primary vaccination platform, and history of SARS-CoV-2 infection. There were no intervention-related serious adverse events. Interpretation A 2-week interruption of methotrexate treatment for people with immune-mediated inflammatory diseases resulted in enhanced boosting of antibody responses after COVID-19 vaccination. This intervention is simple, low-cost, and easy to implement, and could potentially translate to increased vaccine efficacy and duration of protection for susceptible groups. Funding National Institute for Health and Care Research. Funding This publication was funded by the Efficacy and Mechanism Evaluation programme as a part of award number NIHR134607. This article reports on one component of the research award Vaccine Response On/Off Methotrexate (VROOM): does temporarily suspending methotrexate treatment for two weeks enhance COVID-19 vaccine response? A randomised controlled trial. For more information about this research please view the award page [https://fundingawards.nihr.ac.uk/award/NIHR134607] DOI https://doi.org/10.1016/S2213-2600(22)00186-2
Regulatory T cells attenuate chronic inflammation and cardiac fibrosis in hypertrophic cardiomyopathy.
Hypertrophic cardiomyopathy (HCM) is a common, serious, genetic heart muscle disorder. Although the biophysical mechanisms by which gene variants in sarcomeric proteins disrupt cardiomyocyte function are largely understood, the cellular and molecular pathways leading to the complex, variable, and adverse remodeling of the non-myocyte compartment are unexplained. Here, we report that postmortem and explanted human HCM hearts exhibited chronic focal leukocyte infiltration and prominent activation of immune cells. Gene set enrichment analysis (GSEA) revealed that active immune responses were present in the mid- and late-stage HCM human hearts and in mouse hearts from several HCM mouse models. The alpha cardiac actin 1-E99K (Actc1E99K) HCM mouse model was selected for the study because it closely recapitulates the features of progressive remodeling and fibrosis seen in advanced disease in patients. Genetic depletion of lymphocytes in recombination activating gene 1-knockout (Rag-1KO) mice led to marked exacerbation of adverse cardiac remodeling in the Actc1E99K mice. Detailed characterization of cardiac regulatory T cells (Treg cells) demonstrated a time-dependent increase in Actc1E99K hearts with altered immunosuppressive profiles. Adoptive transfer of splenic Treg cells reduced cardiac fibrosis and improved systolic dysfunction in Actc1E99K mice with or without lymphocytes. In addition, low-dose interleukin-2 (IL-2)/anti-IL-2 complex (IL-2/c), which specifically induced Treg cell expansion in vivo, ameliorated cardiac fibrosis and reduced macrophage infiltration and activation in Actc1E99K mice. These data contribute to our understanding of HCM and support the use of Treg cells as a clinically testable therapeutic strategy for cardiac fibrosis in the HCM heart.
A novel dominant-negative variant of IRF8 in a mother and son: Clinical, phenotypic and biological characteristics.
BACKGROUND: The few reported patients with pathogenic IRF8 variants have manifested 2 distinct phenotypes: (1) an autosomal recessive severe immunodeficiency with significant neutrophilia and absence of or significant decrease in monocytes and dendritic cells and (2) a dominant-negative form with only a decrease in conventional type 2 dendritic cells (cDC2s) and susceptibility to mycobacterial disease. OBJECTIVES: Genetic testing of a child with persistent EBV viremia identified a novel IRF8 variant: c.1279dupT (p.∗427Leuext∗42). The variant was also found in his mother, who was subsequently diagnosed with a human papillomavirus-positive tumor. We sought to examine the pathogenicity of the identified IRF8 variant and its phenotypic and functional characteristics. METHODS: Immunophenotypic and functional flow cytometry, natural killer cell cytotoxicity, matrix-assisted laser desorption/ionization-time of flight mass spectrometry, T-cell receptor Vβ spectratyping, Sanger sequencing, RNA-sequencing, Olink proteomics, immunoblotting, molecular cloning, dual-luciferase reporter assay, immunofluorescence microscopy, and image analysis. RESULTS: The 42 amino acid C-terminal extension of the mutant IRF8 (∼4 kDa heavier than wild type) impaired IRF8 nuclear localization in a dominant-negative manner and inhibited IRF1/IRF8-mediated transcriptional activities. Both patients had a decrease in plasmacytoid dendritic cells (pDCs) and in cDC1s, a mild neutrophilia and a mild monocytosis. Their existing pDCs had impaired IFN-α production. On TLR engagement, the production of IL-1β, IL-6, IL-10, and IL-12 by their monocytes and of IL-12 by their myeloid DCs were within normal limits. Natural killer cell development and cytolytic activity were essentially normal. RNA-sequencing and proteomic approaches bolstered the phenotypic and functional findings. CONCLUSIONS: This study defines the pathogenic nature of the c.1279dupT (p.∗427Leuext∗42) IRF8 variant, determines its dominant-negative mechanism of action, and broadens the existing phenotype of human IRF8 immunodeficiency.
The Challenges of Implementing a Health Referral System in South Africa: A Qualitative Study.
INTRODUCTION: Health system strengthening efforts also entails streamlining an existing referral system in a particular context to improve quality of health care offered to people. Conceptually, the referral system in South Africa, is seemingly sound. Nevertheless, gaps exist in its implementation. The aim of this study was to explore health care professionals' perceptions of referral system implementation in the Buffalo City Metropolitan Municipality (BCMM) in the Eastern Cape Province of South Africa. METHODS: This qualitative study included 12 health care professionals as participants. Each participant was interviewed using a semi-structured interview guide; with their consent, the interviews were audio recorded and transcribed verbatim. For data analysis, a thematic content analysis was used. RESULTS: The participants identified many impediments to the effective implementation of the referral system in BCCM. The main obstacles were deteriorating infrastructure, inadequate staffing, lack of transportation, and inadequate medical supplies and medications. CONCLUSION: In mitigation, the participants proposed suggestions such as increasing the capacity of the health workforce, allocating personnel appropriately, increasing the availability of transportation, and providing essential medications to all levels of care. They also suggested involving all stakeholders in the referral process, providing education and training to health professionals on the referral system, and enhancing communication and feedback between the various levels of care. These challenges emphasised in this study highlight the need for targeted interventions to improve the referral system in this setting.
Peer review reports of randomized controlled trials in oncology can be short and superficial.
OBJECTIVES: To evaluate the quality of open peer review reports published alongside articles of randomised controlled trials (RCTs) in oncology. METHODS: We searched and sampled from completed parallel RCT articles published in 2021 in 62 BioMed Central journals operating open peer review and evaluated their first-round peer review report. We assessed and described the peer review report content, clarity, and completeness and explored whether reviewers commented on the manuscript's importance, robustness, interpretation, discussion of results, and RCT reporting. Two investigators evaluated the review reports independently, with conflict resolution involving a third author. RESULTS: We sampled 26 RCTs and evaluated their 59 first peer review reports. Median word count was 276 (range=0-1047). Only 11 reports were constructive (19%), suggesting solutions for the problems noted. Of reviewers commenting on the manuscript's methods section (n=46/59,78%), 74% (n=34/46) addressed the suitability of the methodology. Fewer commented on the adequacy of conclusions (n=15/59; 25%) or the applicability of results (n=5/59; 9%), or whether study limitations had been acknowledged by authors (n=11/59; 18%). Only four (7%) commented on open research practices, including deviations from protocols, completeness of reporting, and sharing of data and materials. CONCLUSIONS: Peer review reports of published RCTs in oncology were short, superficial, and rarely constructive. Although there is indication that reviewers commented on study methodology, little attention was paid to study conclusions, deviation from study protocols, completeness of reporting or data availability. Such review reports would be of limited value to authors for improving their trial study manuscripts, or to editors in deciding on manuscript publication.
Ex vivo model of functioning human lymph node reveals role for innate lymphocytes and stroma in response to vaccine adjuvant.
Immunological processes that underpin human immune responses to therapeutics and vaccine components, such as vaccine adjuvants, remain poorly defined due to a paucity of models that faithfully recapitulate immune activation in lymphoid tissues. We describe precision-cut human lymph node (LN) slices as a functioning, architecturally preserved, full-organ cross-sectional model system. Using single-cell transcriptomics and multiplexed imaging, we explore early inflammatory response to a potent, clinically relevant liposomal vaccine adjuvant containing a TLR4-agonist and QS-21 saponin. Both TLR4 and NLRP3 inflammasome activation are involved in the direct initiation of the inflammatory response to adjuvant by monocytes and macrophages (Mon./Mac.) with secretion of interleukin (IL)-1β, but not IL-18, dependent on TLR4 signaling. Innate lymphoid cells, including natural killer cells, are indirectly activated by Mon./Mac.-produced cytokines, signaling downstream to B cells via interferon-γ secretion. Resident LN stromal populations, primed both directly and indirectly by vaccine adjuvant, are instrumental in mediating inflammatory cell recruitment, particularly neutrophils.
STAT3 phosphorylation in the rheumatoid arthritis immunological synapse.
Targeting the JAK/STAT pathway has emerged as a key therapeutic strategy for managing Rheumatoid Arthritis (RA). JAK inhibitors suppress cytokine-mediated signaling, including the critical IL-6/STAT3 axis, thereby effectively targeting different aspects of the pathological process. However, despite their clinical efficacy, a subset of RA patients remains refractory to JAK inhibition, underscoring the need for alternative approaches. Here, we identify a novel JAK-independent mechanism of STAT3 activation, which is triggered by the formation of the immunological synapse (IS) in naive CD4+ T cells. Our data demonstrates that LCK mediates the TCR-dependent phosphorylation of STAT3 at the IS, highlighting this pathway as a previously unrecognized hallmark of early T cell activation. Furthermore, we show that the synaptic LCK/TCR-STAT3 pathway is compromised in RA. This discovery highlights a new therapeutic target for RA beyond JAK inhibitors, offering potential avenues for treating patients resistant to current therapies.