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Alopecia areata (AA) is an immune-mediated disease that causes non-scarring hair loss. Autoreactive CD8 T cells are key pathogenic effectors in the skin, and AA has been associated both with atopy and with perturbations in intestinal homeostasis. This study aimed to investigate mechanisms driving AA by characterizing the circulating immunophenotype and faecal microbiome, and by stratifying AA to understand how identified signatures associated with heterogeneous clinical features of the condition. Flow cytometric analyses identified alterations in circulating B cells and CD4 T cells, while 16S sequencing identified changes in alpha and beta diversity in the faecal microbiome in AA. The proportions of transitional and naïve B cells were found to be elevated in AA, particularly in AA samples from individuals with >50% hair loss and those with comorbid atopy, which is commonly associated with extensive hair loss. Although significant changes in circulating CD8 T cells were not observed, we found significant changes in CD4+ populations. In individuals with

Data sharing: The PHOSP-COVID study protocol, consent form, definition, and derivation of clinical characteristics and outcomes, training materials, regulatory documents, information about requests for data access, and other relevant study materials are available online. UK Biobank information can be released once necessary approvals have been obtained. Other data (eg, the R code and protocol) will be made available on reasonable request to the corresponding author. Copyright © 2023 The Author(s). Background: Sleep disturbance is common following hospital admission both for COVID-19 and other causes. The clinical associations of this for recovery after hospital admission are poorly understood despite sleep disturbance contributing to morbidity in other scenarios. We aimed to investigate the prevalence and nature of sleep disturbance after discharge following hospital admission for COVID-19 and to assess whether this was associated with dyspnoea. Methods: CircCOVID was a prospective multicentre cohort substudy designed to investigate the effects of circadian disruption and sleep disturbance on recovery after COVID-19 in a cohort of participants aged 18 years or older, admitted to hospital for COVID-19 in the UK, and discharged between March, 2020, and October, 2021. Participants were recruited from the Post-hospitalisation COVID-19 study (PHOSP-COVID). Follow-up data were collected at two timepoints: an early time point 2–7 months after hospital discharge and a later time point 10–14 months after hospital discharge. Sleep quality was assessed subjectively using the Pittsburgh Sleep Quality Index questionnaire and a numerical rating scale. Sleep quality was also assessed with an accelerometer worn on the wrist (actigraphy) for 14 days. Participants were also clinically phenotyped, including assessment of symptoms (ie, anxiety [Generalised Anxiety Disorder 7-item scale questionnaire], muscle function [SARC-F questionnaire], dyspnoea [Dyspnoea-12 questionnaire] and measurement of lung function), at the early timepoint after discharge. Actigraphy results were also compared to a matched UK Biobank cohort (non-hospitalised individuals and recently hospitalised individuals). Multivariable linear regression was used to define associations of sleep disturbance with the primary outcome of breathlessness and the other clinical symptoms. PHOSP-COVID is registered on the ISRCTN Registry (ISRCTN10980107). Findings: 2320 of 2468 participants in the PHOSP-COVID study attended an early timepoint research visit a median of 5 months (IQR 4–6) following discharge from 83 hospitals in the UK. Data for sleep quality were assessed by subjective measures (the Pittsburgh Sleep Quality Index questionnaire and the numerical rating scale) for 638 participants at the early time point. Sleep quality was also assessed using device-based measures (actigraphy) a median of 7 months (IQR 5–8 months) after discharge from hospital for 729 participants. After discharge from hospital, the majority (396 [62%] of 638) of participants who had been admitted to hospital for COVID-19 reported poor sleep quality in response to the Pittsburgh Sleep Quality Index questionnaire. A comparable proportion (338 [53%] of 638) of participants felt their sleep quality had deteriorated following discharge after COVID-19 admission, as assessed by the numerical rating scale. Device-based measurements were compared to an age-matched, sex-matched, BMI-matched, and time from discharge-matched UK Biobank cohort who had recently been admitted to hospital. Compared to the recently hospitalised matched UK Biobank cohort, participants in our study slept on average 65 min (95% CI 59 to 71) longer, had a lower sleep regularity index (–19%; 95% CI –20 to –16), and a lower sleep efficiency (3·83 percentage points; 95% CI 3·40 to 4·26). Similar results were obtained when comparisons were made with the non-hospitalised UK Biobank cohort. Overall sleep quality (unadjusted effect estimate 3·94; 95% CI 2·78 to 5·10), deterioration in sleep quality following hospital admission (3·00; 1·82 to 4·28), and sleep regularity (4·38; 2·10 to 6·65) were associated with higher dyspnoea scores. Poor sleep quality, deterioration in sleep quality, and sleep regularity were also associated with impaired lung function, as assessed by forced vital capacity. Depending on the sleep metric, anxiety mediated 18–39% of the effect of sleep disturbance on dyspnoea, while muscle weakness mediated 27–41% of this effect. Interpretation: Sleep disturbance following hospital admission for COVID-19 is associated with dyspnoea, anxiety, and muscle weakness. Due to the association with multiple symptoms, targeting sleep disturbance might be beneficial in treating the post-COVID-19 condition. Funding: UK Research and Innovation, National Institute for Health Research, and Engineering and Physical Sciences Research Council. UK Research and Innovation, National Institute for Health Research, and Engineering and Physical Sciences Research Council.

Background Monoclonal antibodies targeting the interleukin (IL)-23p19 subunit are effective in the treatment of inflammatory bowel diseases (IBD), but have different molecular attributes that may translate to differences in clinical efficacy. Within this class, guselkumab (GUS) is a fully human IgG1 monoclonal antibody with a native Fc region, and risankizumab (RIS) is a humanised IgG1 antibody with a mutated Fc region. Binding of these therapeutic antibodies to Fcγ receptor (FcγR) I, or CD64, is of particular interest, as CD64+ IL-23-producing myeloid cells are increased in the inflamed colon in IBD and correlated with endoscopic disease severity (Chapuy L, et al. Mucosal Immunol. 2019; Chapuy et al. J Crohns Colitis. 2020). Here, we compared functional characteristics of the antigen-binding and Fc regions of GUS and RIS. Methods IL-23 binding affinity of GUS and RIS was compared in vitro using KinExA and surface plasmon resonance. In vitro cellular potency of GUS and RIS was measured by impact on IL-23-induced signal transducer and activator of transcription 3 (STAT3) phosphorylation in human peripheral blood mononuclear cells. Binding of GUS and RIS to FcγRs was assessed in cells transfected with individual FcγRs. In primary human “inflammatory” monocytes differentiated with granulocyte-macrophage colony-stimulating factor and interferon-γ (IFN-γ) followed by toll-like receptor stimulation to induce IL-23 production, binding of GUS and RIS to CD64 and capture of endogenously secreted IL-23 was assessed using flow cytometry. The potential impact of GUS binding to CD64 was assessed in IFN-γ primed monocytes using a human 41-plex cytokine bead assay. Results GUS and RIS displayed comparable picomolar binding affinity for IL-23 and equivalent high potency for inhibition of IL-23-induced STAT3 phosphorylation. GUS showed strongest binding to CD64 compared with other FcγRs, whereas RIS had negligible binding to any FcγR. GUS, but not RIS, showed dose-dependent Fc-mediated binding to CD64 in primary human “inflammatory” monocytes. Moreover, CD64-bound GUS was able to simultaneously capture IL-23 endogenously secreted from the same cells (Figure). GUS binding to CD64 on monocytes did not induce cytokine production. Conclusion GUS, but not RIS, can simultaneously bind CD64+ myeloid cells via its Fc region and neutralise IL-23 with high affinity and potency. Our in vitro data suggest a mechanistic benefit through enhanced localization of GUS within the inflamed colon, where CD64+ IL-23-producing myeloid cells are increased, and GUS can potently neutralise IL-23 at its source of production. These findings may contribute to differences in therapeutic profiles between antibodies.

The management of rheumatic and musculoskeletal diseases has transformed over two decades of exciting discoveries regarding pathogenesis and innovative drug development. The introduction of sophisticated immunomodulatory therapies has given renewed hope to many patients, but notable challenges remain within the field of rheumatology. Before the advent of biologic therapies, conventional synthetic DMARDs (csDMARDs) provided effective disease control for some patients, although a comprehensive understanding of the mechanisms by which these drugs exert their effects remained elusive. Reflecting upon the efficacy and mechanisms of action of csDMARDs can provide intriguing insights. First, contemplating past approaches brings our remarkable current approaches concerning pathogenesis-driven discovery and drug development into sharper focus. Second, increased understanding of the mode of action of these older drugs might in turn provide exciting opportunities for the understanding of disease and for the development of future therapies.

Background The genetic risk associated with rheumatoid arthritis (RA) includes genes regulating DNA methylation, one of the hallmarks of epigenetic re-programing, as well as many T-cell genes, with a strong MHC association, pointing to immunogenetic mechanisms as disease triggers leading to chronicity. The aim of our study was to explore DNA methylation in early, drug-naïve RA patients, towards a better understanding of early events in pathogenesis. Result Monocytes, naïve and memory CD4+ T-cells were sorted from 6 healthy controls and 10 RA patients. DNA methylation was assessed using a genome-wide Illumina 450K CpG promoter array. Differential methylation was confirmed using bisulfite sequencing for a specific gene promoter, ELISA for several cytokines and flow cytometry for cell surface markers. Differentially methylated (DM) CpGs were observed in 1047 genes in naïve CD4+ T-cells, 913 in memory cells and was minimal in monocytes with only 177 genes. Naive CD4+ T-cells were further investigated as presenting differential methylation in the promoter of > 500 genes associated with several disease-relevant pathways, including many cytokines and their receptors. We confirmed hypomethylation of a region of the TNF-alpha gene in early RA and differential expression of 3 cytokines (IL21, IL34 and RANKL). Using a bioinformatics package (DMRcate) and an in-house analysis based on differences in β values, we established lists of DM genes between health and RA. Publicly available gene expression data were interrogated to confirm differential expression of over 70 DM genes. The lists of DM genes were further investigated based on a functional relationship database analysis, which pointed to an IL6/JAK1/STAT3 node, related to TNF-signalling and engagement in Th17 cell differentiation amongst many pathways. Five DM genes for cell surface markers (CD4, IL6R, IL2RA/CD25, CD62L, CXCR4) were investigated towards identifying subpopulations of CD4+ T-cells undergoing these modifications and pointed to a subset of naïve T-cells, with high levels of CD4, IL2R, and CXCR4, but reduction and loss of IL6R and CD62L, respectively. Conclusion Our data provided novel conceptual advances in the understanding of early RA pathogenesis, with implications for early treatment and prevention.

Background: Interleukin (IL)-17 is the main Th-1 cytokine, produced by activated T-lymphocytes. The potential IL-17 value in rheumatoid arthritis (RA) pathogenesis consists of its independent inflammatory response induction and mediated stimulation of proinflammatory factors synthesis resulting in joint destruction. The aim of study was to determine the role of IL-17 in immuno-inflammatory/autoimmune reactions development and to reveal IL-17 serum level associations with clinical and immunological characteristics of RA. Methods: 50 patients with early RA (disease duration >, Russia), anti-CCP antibodies (Axies-Shield Diagnostic, UK) were revealed using ELISA immunoassay. Results: On the base of IL-17 serum level patients were divided in two groups: group1 (n = 28) were patients with normal IL-17 serum level and group2 (n = 22) were those with high IL-17 serum level. In the group2, the rate of patients' pain assessment by visual analogue scale (67.3 ± 7.2 vs 32.8 ± 4.6; P

Interleukin-33 (IL-33) is a novel member of IL-1 cytokine family. It can act both as a nuclear factor and as a soluble mediator; however, the precise role of IL-33 within the nucleus is still not clear. As a cytokine, IL-33 is suggested to function as an alarmin that is released upon endothelial or epithelial cell damage. As such, IL-33 targets multiple cell types thereby alerting the immune system to endogenous trauma such as physical stress or infection. However, a dysregulated release of IL-33 has a potential to drive distinct pathologies. In this review, we discuss the contribution of IL-33 to the pathophysiology of asthma, arthritis, obesity and atherosclerosis as well as the potential of IL-33 for therapeutic intervention.

In the last decade, basic science has unraveled in considerable detail the inflammatory and related processes ongoing in RA synovial membrane. This has translated to cytokine targeting therapies with some effect. How much more can be achieved? It may be argued that the order of improvement obtained thus far obviates further study. This ignores the potential to achieve a far greater magnitude of disease suppression. Our objective in innovating biologic therapies should now be routine achievement of American College of Rheumatology "70% responses" or disease remission and design of patient-specific therapy based on individual disease characteristics. We have not yet explored the potential contained in combination biologic approaches, particularly when different processes within the disease are targeted. Cocktails might target T cells, cytokines, and angiogenic factors, for example. These developments must also be seen in the context of the information soon to be available from the Human Genome Project. The impact of gene array analysis and similar techniques that facilitate simultaneous evaluation of thousands of gene activation events is also awaited. This, in turn, is likely to require considerable development in our use of information technology, because the volume of information will soon (or may already) be prohibitive. Finally, encompassing genomic and bioinformatic approaches should certainly challenge our basic diagnostic criteria such that it ultimately may be necessary to consider our clinical diagnoses on the basis not only of clinical phenotype but of genotype and biologic response profile. Through this rapid evolution, close communication between physician and scientist is essential.

The functional division of CD4(+) T cells into Th1 and Th2 subsets is generally accepted but the mechanisms leading to their preferential induction remain elusive. Cytokines are considered the main determining factors in the initial differentiation of precursor T cells into these distinct subsets. Thus, IL-12 drives Th1 cells whereas IL-4 drives Th2 cells. Recently IL-18, originally designated as IFN-gamma-inducing factor, has been reported to synergize with IL-12 in the induction of Th1 cells. We report here that IL-18 can also induce T cells to differentiate into Th2 cells, in the presence of TCR activation, either alone or together with IL-4. This effect of IL-18 is mediated primarily on CD4(+) T cells compared with CD8(+) T cells and is inhibited in the presence of IL-12. IL-18, however, has no effect on functionally committed Th2 cells.( )Moreover, the effect of IL-18 on Th2 cell development is differentially manifest in different mouse strains, suggesting profound underlying genetic influences. BALB/c mice infected with Leishmania major and treated with recombinant IL-18 developed exacerbated disease and enhanced Th2 response compared with untreated controls. These data therefore provide a novel mechanism for Th2 cell development. Thus, IL-18, a cytokine constitutively expressed by cells of the innate response, is capable of inducing Th2 cell differentiation in the absence of IL-4.

To determine whether lactoferrin can modify articular inflammation in murine models of autoimmune and septic arthritis.Collagen arthritis was induced in DBA/1 mice and Staphylococcus aureus septic arthritis in Swiss mice. Joints with established inflammation were injected periarticularly with 0.5 mg or 1 mg of human lactoferrin, and arthritis was monitored for 3 days.DBA/1 mice injected with lactoferrin showed significantly suppressed local inflammation for up to 3 days, achieving up to 71% of the effect of corticosteroid. Periarticular injection of 125I-lactoferrin confirmed that 25% of lactoferrin was retained in paws after 6 hours. Serum levels of interleukin-6, however, were not significantly reduced, suggesting a predominantly local antiinflammatory effect. Similarly, local, periarticular administration of lactoferrin into S aureus-infected Swiss mice significantly suppressed paw inflammation and did not enhance bacterial survival.Lactoferrin may have clinical utility in reducing articular inflammation, particularly in septic arthritis, in which antiinflammatory effects may be achieved without promoting bacterial survival.

Monocyte-derived cytokines are important mediators in synovitis and represent novel therapeutic targets. This study was undertaken to analyze their expression in synovial membrane (SM) of patients with psoriatic arthritis (PsA) compared with that in skin of patients with PsA and SM of patients with rheumatoid arthritis (RA).Multiple synovial biopsy samples (24 from patients with PsA, 20 from patients with RA, 5 from patients with osteoarthritis [OA]) and skin biopsy samples (lesional and perilesional skin from 25 PsA patients) were obtained. Standard leukocyte antigens, cytokines (tumor necrosis factor alpha [TNFalpha], interleukin-1apha [IL-1alpha], IL-1beta, IL-15, and IL-10) and the transcription factor nuclear factor KB (NF-kappaB; active p65 subunit) were localized and quantified immunohistochemically by light microscopy and digital image analysis.Sublining cellular infiltration, lymphoid aggregation, and vascularity were similar in PsA and RA SM. Lining layer thickness was greater in RA SM, associated with more CD68+ macrophages. In PsA SM, TNFalpha, IL-1alpha, IL-1beta, IL-15, and IL-10 were primarily localized to lining layer and perivascular macrophages, as were cells expressing the active subunit of NF-kappaB (p65). TNFalpha, IL-1p, and IL-15 expression in PsA lining layer was less than that in RA lining layer, likely reflecting lower macrophage numbers. In sublining areas, levels of TNFalpha and IL-15 were lower in PsA patients than in RA patients, whereas IL-lalpha and IL-1beta expression was equivalent. IL-10 was identified at similar levels in RA and PsA SM lining layer and sublining. Expression of NF-kappaB (p65) was equal in lining layer from both patient groups, but lower in PsA than RA sublining. Histologic findings did not correlate with clinical parameters of disease. Cytokine expression in skin did not correlate directly with that in SM. Cytokine expression was greater in PsA and RA SM than in OA SM.This study shows, for the first time, that monocyte-derived cytokines are found in PsA SM and demonstrates the relative paucity of the antiinflammatory cytokine IL-10 in PsA skin and SM. Significant divergence from RA SM expression was observed, despite similar clinical and demographic features in the 2 patient groups.

Mechanisms whereby T lymphocytes contribute to synovial inflammation in rheumatoid arthritis are poorly understood. Here we review data that indicate an important role for cell contact between synovial T cells, adjacent macrophages and fibroblast-like synoviocytes (FLS). Thus, T cells activated by cytokines, endothelial transmigration, extracellular matrix or by auto-antigens can promote cytokine, particularly TNF alpha, metalloproteinase production by macrophages and FLS through cell-membrane interactions, mediated at least through beta-integrins and membrane cytokines. Since soluble factors thus induced may in turn contribute directly to T cell activation, positive feedback loops are likely to be created. These novel pathways represent exciting potential therapeutic targets.

Cultures of neurons and non-neuronal cells were prepared from adult mouse dorsal root ganglia and maintained for periods of up to six weeks upon uncoated petri dish substrates. Modifications were made to both existing isolation procedures and culture medium to ensure high cell survival. Cell structure was observed using phase contrast microscopy for living cells and standard light microscopy for Palmgren silver stained preparations. Scanning and transmission electron microscopy revealed the fine structure of the cells and showed the close relationship of satellite cells with the surviving dark neurons as culture establishment progressed. Satellite cells were associated with both the cell bodies and the regenerated neuritic processes. The use of the cultures for future cytotoxicity testing is assessed.