Arthritis Research & Therapy

Skeletal muscle dysfunction is a major contributor to disability and incomplete functional recovery in patients with end-stage knee osteoarthritis (KOA), yet the spatial components of disease-associated molecular remodeling remain poorly understood. Here, we applied spatial transcriptomics to paired skeletal muscle biopsies obtained from the surgical (Sx) and contralateral (Ct) limbs of individuals undergoing total knee arthroplasty (TKA) to define the cellular architecture of periarticular muscle and determine how muscle inflammation susceptibility (MuIS) shapes local transcriptional programs. Integrated analysis of 27,087 spots obtained from 22 muscle histological cross-sections (11 Sx-Ct pairs) revealed seven spatially resolved transcriptional domains corresponding to slow and fast myofiber states, an extracellular matrix/fibroadipogenic-enriched domain, a pericyte/smooth muscle domain, and a satellite cell/myogenic-enriched domain. Despite advanced unilateral disease, the major annotated cellular compartments were similarly represented between Sx and Ct limbs. KOA-associated remodeling was reflected primarily by within-cluster transcriptional changes, with the most informative differences observed in fibroadipogenic, pericyte/smooth muscle, and satellite/myogenic domains. Within Sx, MuIS stratification identified a coordinated transcriptional program characterized by denervation- and regeneration-associated genes and altered contractile and metabolic features. Neighborhood analysis localized denervation-associated signals primarily to fast-myofiber-rich regions, while local adjacency patterns among the examined myofiber, fibroadipogenic, and pericyte/smooth muscle domains were broadly preserved. Our findings provide the first spatial transcriptomic analysis of periarticular skeletal muscle in end-stage KOA and identify MuIS as a distinct local transcriptional phenotype in diseased muscle.

Even though osteoarthritis is considered a disease of the whole joint, integrated analysis of articular calcified cartilage (ACC) behaviour with adjacent tissues remains a persistent limitation. Herein, we performed synchrotron X-ray computed tomography in intact loaded joints and coupled anatomical analysis with digital volume correlation to examine whether microarchitectural ACC features serve as predictive mechano-biomarkers of osteoarthritis. We reveal that male osteoarthritis-prone STR/Ort mice have thicker yet more porous ACC that contains larger but less spherical chondrocyte lacunae than their healthy parental-control CBA mice. This was linked to asymmetrical distribution of tensile and compressive strains generated under physiological loads in the ACC and underlying subchondral bone in tibial epiphyses of STR/Ort mice. Together these data suggest that murine ACC microarchitecture has a mechano-predictive role in age-related osteoarthritis.

Cartilage thickness on MRI is a structural biomarker of osteoarthritis (OA), yet anatomical variation and aging can mask early disease, including changes occurring 12-36 months before incident OA. We propose longitudinal local centile maps that use normative growth charts as prior information and integrate a knee's earlier scans to identify cartilage that is thinner or thicker than expected. We built the reference charts from 5,045 scans of 957 persistently radiographically normal knees in the Osteoarthritis Initiative. Under participant-separated nested cross-validation, longitudinal centile profiles achieved higher 12- and 24-month AUCs for predicting incident radiographic OA than regional PCA, radiomics, cross-sectional centile features, and raw-map deep features across most classifiers. These conventional descriptors added little when combined with centile profiles, and cross-sectional centiles added little beyond longitudinal centiles. Deep centile features achieved the best AUCs (0.954 [95% CI 0.931-0.974] at 12 months and 0.969 [0.949-0.985] at 24 months; balanced accuracy 0.917 and 0.931) and outperformed raw-map deep features in all 12 classifiers after correction for multiple comparisons. No representation consistently improved 36-month prediction. Longitudinal centiles transform cartilage thickness maps into reference-based deviation maps: deep longitudinal centile features provide the highest predictive accuracy, while regional longitudinal centile profiles provide interpretable prognosis with the second-highest accuracy.

Purpose: To determine whether a cobalt-chrome genetic reactivity (CoGR) score - originally developed to identify patients at greater risk of developing adverse reaction to metal-on-metal hips - is associated with poorer patient-reported outcomes after knee arthroplasty (KA). Methods: In a prospective multicentre study, patients at a minimum two years post all cobalt-chrome KA were invited to research clinics. Participants provided samples for blood metal ion measurements and DNA sequencing and completed Forgotten Joint Scores (FJS). Component alignment was assessed on AP and lateral plain radiographs. The association between CoGR and FJS was assessed using regression adjusted for time since surgery, age, gender, BMI and tested for modification by blood cobalt. In a case-control analysis, CoGR scores of revised patients with histologically confirmed adverse reactions to metal debris (and specifically aseptic lymphocyte-dominated vasculitis-associated lesions (ALVAL)) were compared with controls (revised patients with no signs of ARMD and unrevised patients with FJS of 75 or more) using Firth logistic regression. Results: 376 patients were included: 312 unrevised patients and 64 patients who underwent revision. Each one-unit increase in CoGR score was associated with a 9.8 point lower FJS (95% confidence interval (CI) 4.2 to 15.4 points; p < 0.001). This increased to a 17.5 (9.4 to 25.5) point reduction per CoGR score unit in patients with blood cobalt above the median level. Comparing 28 ARMD cases (12 with ALVAL) with 177 controls, each one-unit CoGR increase was associated with odds ratios of 1.90 (CI 1.34 to 2.75) for ARMD and 3.05 (CI 1.89 to 5.34) for ALVAL. Conclusions: A higher CoGR score was associated with worse patient-reported outcome and higher odds of histologically confirmed adverse reaction to metal debris at revision. Adverse immune responses may be a more significant factor in unexplained pain after KA than is generally recognised.

Objective: The principal diagnostic test for rheumatoid arthritis (RA) relies on the detection of anti-cyclic citrullinated peptide (CCP) antibodies. The CCP test exhibits moderate sensitivity and specificity but does not adequately detect a high proportion of cases or effectively distinguish RA from related conditions such as systemic lupus erythematosus (SLE). To discover improved diagnostic biomarkers for RA, we performed a comprehensive proteomic analysis of antibody-bound antigens present in RA, SLE, and healthy control (HC) sera and identified proteins associated with transforming growth factor-beta-1 (TGF-beta-1) signaling and assessed the sensitivity and specificity of antibodies against these antigens in comparison to CCP. Methods: Total antibodies were immunoprecipitated from pooled sera of HC individuals (n=3) and patients with clinically diagnosed RA (n=3) or SLE (n=3). Antibody-bound antigens were identified using multidimensional protein identification technology (MudPIT) shotgun proteomics. Candidate antigens associated with TGF-beta-1 signaling were selected for evaluation, and corresponding serum autoantibodies were assessed for their ability to classify RA using a POC lateral-flow immunochromatographic format. Results: Proteins present in antibody immunoprecipitations included TGF-beta-1-induced protein (TGFBI), thrombospondin 1 (TSP1), serum amyloid A1 (SAA), and fibronectin (FN). POC assays using these candidates, as well as TGF-beta-1 itself, as capture antigens were used to detect corresponding serum autoantibodies in 107 subjects with clinically diagnosed RA, 42 subjects with other rheumatic diseases (non-RA), and 121 HC subjects. Receiver operating characteristic curves were constructed using RA and HC groups to assess diagnostic performance, and optimal cutpoints identified using the Youden Index (maximizing the sum of sensitivity and specificity). CCP test results were determined using previously established thresholds. Using an analysis et of 169 subjects in the HC and RA groups who had values for TGFBI, SAA, and CCP, TGFBI autoantibodies achieved a sensitivity of 82%, specificity of 98.8%, positive likelihood ratio (PLR) of 65.6, and NLR of 0.2. SAA autoantibodies achieved a sensitivity of 92.1%, specificity of 85%, PLR of 6.1, and NLR of 0.1. Both outperformed CCP (sensitivity of 68.5%, specificity of 95%, PLR of 13.7, and NLR of 0.3). Using an analysis set of 120 subjects in the RA and non-RA groups who had values for TGFBI, SAA, and CCP, TGFBI and SAA autoantibodies exhibited sensitivities and specificities of 82 and 92.1% and 74.2 and 45.2%, respectively, compared to 68.5 and 74.2% for CCP. Autoantibodies to TGF-beta-1, TSP1, and FN exhibited only modest diagnostic performance. Conclusion: RA sera contain autoantibodies targeting multiple proteins associated with the TGF-beta-1 signaling pathway that is implicated in inflammatory processes in RA. Among the candidates tested, TGFBI and SAA autoantibodies exhibited the highest diagnostic performance in a POC lateral-flow format, supporting their potential utility as biomarkers for RA diagnosis. These findings also support further investigation of the TGF-beta-1 signaling pathway as a potential therapeutic target in RA.

Objective: Autoantibodies are central for pathology of rheumatoid arthritis (RA) and diabetes mellitus (DM) carrying high predictive and diagnostic value. Here, we explore the frequency and functional potential of DM-related antibodies (DMab) in RA. Methods: DMab targeting glutamic acid decarboxylase (GAD65ab), tyrosine phosphatase-like islet antigen 2 (IA2ab) and zinc transporter 8 (ZnT8ab) were measured in 326 DM-free RA patients (260 females, 66 males), using specific ELISAs. The DMab profile was analysed in relation to 30 clinical and serological variables. ZnT8+ cells were detailed by a public single-cell transcriptome in RA synovial tissue. Results: DMab production was more frequent in females, dominated by ZnT8ab (86%), followed by GAD65ab (26.4%) and IA2ab (7.0%). DMab-positive patients, and specifically ZnT8ab-positive women, had higher levels of insulin and HOMA-IR index and lower serum IL-6, IFN{gamma}, and IL-9. Multivariable logistic regression estimating ZnT8ab, IA2ab, GAD65ab production revealed that BDNF, insulin and DAS28 were positive predictors of ZnT8ab. Increasing ZnT8ab levels were inversely related to RAab in the mixed interaction model. CD14+ cell transcriptome identified association between an activated secretory cell type and increasing ZnT8ab levels. Additionally, ZnT8ab levels were in direct proportion with ZnT8 production in RA CD4+ T cells. Single-cell transcriptome of RA synovial tissue revealed ZnT8+ cells among the lubricin producing lining layer fibroblasts programmed for invasive behaviour. Conclusion: This study reveals frequent production of ZnT8ab in non-diabetic RA patients and identifies their antigenic target expressed in lining synovial fibroblasts of RA joints.

Individuals with fibrodysplasia ossificans progressiva (FOP), a rare genetic disorder caused by mutations in the bone morphogenetic protein receptor ACVR1 (also known as ALK2), experience progressive and severely debilitating endochondral heterotopic ossification (HO). Previous studies have identified fibro-adipogenic progenitors (FAPs) as a major cellular source of HO in FOP mouse models. Here, we used mRNA sequencing of FAPs isolated from injured muscle of FOP mice to capture the earliest gene expression changes associated with the transformation of muscle tissues into bone. Whereas the transcriptomes of mutant and wild-type FAPs were indistinguishable prior to injury and at 1 day post injury, dramatic dysregulation of gene expression was observed at 3 and 5 days after injury. Surprisingly, gene expression changes suggested early specification of osteochondral lineages by 3 days post injury, approximately 3 to 4 days before the appearance of histologically identifiable cartilage. We identified dysregulation of genes encoding components of major signaling pathways, cell cycle regulators, extracellular matrix components, and hypoxia response mediators, and showed that these gene expression changes are temporally associated with abnormal population growth of mutant FAPs. We further demonstrated that treatment of FOP mice with either an anti-activin A monoclonal antibody or the ACVR1 kinase inhibitor BLU-782 (IPN60130; fidrisertib) normalized the expression of the majority of dysregulated FAP genes. However, the relative efficacy of the drugs differed, and distinct sets of overlapping genes were normalized by the treatments. The substantial transcriptional changes caused by these drugs in FAPs from normally regenerating muscle were also enumerated and described. These findings define early transcriptional events associated with pathogenic FAP reprogramming and provide a molecular framework for understanding the shared and distinct effects of activin A and ACVR1 inhibition.

The mechanical features of synovial fibroblasts may encode clinically relevant information for the management of rheumatoid arthritis. Indeed, it remains poorly understood the links between cellular and microenvironment mechanics with patient stratification and therapeutic response. Here we show that combining organotypic synovial cultures with multimodal mechanophenotyping and transcriptomics identifies distinct ''soft'' and ''stiff'' fibroblast states. Healthy and rheumatoid arthritis fibroblasts were cultured in two or three dimensions (organotypic cell cultures), alone or with endothelial cells, and characterized by high-throughput nanoindentation, picobalance measurements, digital holographic microscopy, Brillouin microscopy and RNA sequencing. Three-dimensional culture reduced single-cell Young's modulus and total mass while increasing dry-mass surface density; endothelial co-culture increased fibroblast stiffness and the longitudinal modulus of intact organotypic cultures. Stiffness-based grouping revealed transcriptional programmes related to extracellular-matrix remodelling, cytoskeletal contractility, and TGF-beta, WNT, and Hippo signalling. Mapping these signatures to publicly available rheumatoid arthritis clinical transcriptomic datasets associated the ''stiff'' state with fibrotic and myeloid-fibrotic pathotypes and treatment-refractory programmes, whereas the ''soft'' state aligned with lymphoid pathotypes and rituximab- or tocilizumab-responsive programmes. These findings support mechanophenotype-associated transcriptional signatures as potential biomarkers for rheumatoid arthritis stratification.

Objectives. Before-and-after transcriptomics in rheumatic disease often yields no reproducible treatment signal, though disease-state stratification is robust. We asked whether exposure and response-prediction labels fall below the detection floor of the designs defining them, while matched disease-state labels do not. Methods. Eight public datasets were analysed (paired juvenile dermatomyositis n = 10; RA before/after methotrexate, n = 28 pairs; lupus nephritis n = 10; controlled exposures; a single-cell dose series). Response labels in current use (one benchmark trajectory, five published signatures) were audited in every analysable source cohort, using effect sizes (dz) against each design's exact minimum detectable effect. Results. Disease-state labels were detectable: the interferon signature declined in 10/10 juvenile dermatomyositis patients (p = 0.002; 59.5% of score variance). No exposure label cleared its design floor in any clinical cohort; a drug-matched methotrexate prior was nominally significant in juvenile dermatomyositis yet did not clear its floor (dz = -0.80; CI spanning it). Exposure became detectable only where prior-context and intensity were jointly favourable (multi-day target tissue dz = 1.24, q = 0.018; well-level pseudobulk dz = +3.70, floor 1.80). Audited response labels failed in 14 of 15 cohorts (93%; descriptive binomial 95% CI 68-100): eleven below their floors, three sign-reversed; the largest training cohort pooled to zero (dz = +0.07; I2 = 69%). Effect size ordered reported AUCs monotonically. Conclusion. Under conventional treatment intensities, exposure and response-prediction labels are either undetectable below the design floor or detected with a sign that does not replicate; disease-state labels are detectable. Part of the negative literature reflects a design floor.

Introduction: Across major phases of the COVID-19 pandemic (March 2020-April 2023), we compared the burden of severe COVID-19 among individuals with systemic autoimmune rheumatic diseases (SARDs) versus immunocompetent individuals with and without comorbidities. Methods: This population-based cohort study used administrative datasets to estimate crude and age- and sex-standardized SARS-CoV-2 and severe COVID-19 incidences among adults >18 years with SARDs, chronic comorbidities, or no comorbidities. Relative to the latter, we used Cox models to derive adjusted hazard ratios (aHRs) for infection, hospitalization, and intensive care unit admission or death (ICU/death) during the pre-vaccination (March-December 2020), vaccination pre-Omicron (December 2020-December 2021), and Omicron (December 2021-April 2023) periods. Results: We included 81,434 adults with SARDs, and 2,020,356 with comorbidities versus 3,144,700 without comorbidities. Despite similar adjusted infection rates, the SARDS cohort had higher risks of hospitalization and ICU/death during the pre-vaccination period (aHRs: 3.4, 95%CI:2.8-4.1; and 4.0, 95%CI:2.8-5.7, respectively), persisting through the Omicron period (aHRs: 4.5, 95%CI:4.0-4.9; and 4.4, 95%CI:3.6-5.4, respectively). Among SARDS, rheumatoid arthritis, psoriatic arthritis and systemic lupus erythematosus contributed highest burden, with comparable hospitalization and mortality rates among younger rheumatoid arthritis versus healthy older adults. Highly immunosuppressive rituximab- or mycophenolate-treated patients had highest hospitalization and ICU/death risks, particularly during the Omicron period (aHRs: 11.9, 95%CI:9.9-14.3; and 17.6, 95%CI:13.0-23.9, respectively). Conclusion: Individuals with SARDs, especially receiving highly immunosuppressive therapies, experienced elevated COVID-19 burden and severe outcome risks throughout the pandemic, warranting their prioritized consideration during future public health emergencies.

Chronic pain is frequently accompanied by anxiety and other affective disturbances, but the molecular and cellular changes linking peripheral injury to persistent alterations in brain function remain incompletely defined. The hippocampal extracellular matrix (ECM) regulates synaptic plasticity, cellular signaling, and neuroimmune interactions and may therefore contribute to pain-related central plasticity. We used a tibial fracture/cast immobilization model to examine temporal changes in hippocampal ECM, glial morphology, and affective behavior following peripheral injury. Behavioral testing was performed at 3, 7, and 20 weeks after injury, while high-dimensional morphometric analysis of microglia and astrocytes was performed at 1, 3, 7, and 20 weeks. Hippocampal ECM associated molecular signals were mapped at the chronic stage using decellularization coupled with MALDI-TOF mass spectrometry imaging. Injured mice developed mechanical hypersensitivity and reduced exploration in two anxiety related paradigms during the early and chronic stages, with these behavioral phenotypes no longer detected at 20 weeks. At 7 weeks, injury was associated with changes in hippocampal ECM-associated peptide signals, including reduced signals corresponding to four ECM-associated proteins. Microglia and astrocytes exhibited distinct, time-dependent changes in density and multidimensional morphology, with some alterations persisting after behavioral measures returned toward control levels. In vitro, BV2 microglia exhibited rigidity-dependent changes in morphology, phagocytic behavior, and calcium signaling. Together, these findings define a temporally evolving hippocampal ECM and glial phenotype following peripheral injury and identify matrix biomechanics as a candidate regulator of microglial function.

Clinical translation of mesenchymal stromal cell (MSC) therapies derived from primary tissue sources is limited by manufacturing inconsistencies driven by donor-to-donor variability, which reduces predictability of clinical response. Induced pluripotent stem cell (iPSC)-derived MSCs (iMSCs) offer a promising donor-independent, clonal alternative strategy that is also more amenable to engineering, but whether engineered iMSCs reproduce the potency attributes of primary MSCs remains unclear. Here, we benchmarked a doxycycline-inducible, hTERT-immortalized iMSC line engineered to overexpress TGFB1 (TGFB1-iMSCs), a key regulator of macrophage polarization, against multiple adipose tissue-derived MSC (MSC(AT)) donors across predefined immunomodulatory and angiogenic potency attributes. TGFB1-iMSCs were smaller and more circular with comparable or higher proliferative rates than primary MSC(AT) donors. Interestingly, they had a distinct angiogenic signature (EDIL3, EDN1, PDGFA), and nine differentially expressed microRNAs were predicted to target secretory trafficking and growth factor receptor signalling. Functionally, however, TGFB1-iMSCs secreted less VEGF and showed intermediate HUVEC tube formation, but matched or exceeded all primary MSC(AT) donors in a monocyte-macrophage transwell immunomodulatory assay. Notably, in a murine destabilization of the medial meniscus (DMM) model of post-traumatic osteoarthritis, a single intra-articular injection of TGFB1-iMSCs, but not MSC(AT), reduced total synovial macrophage numbers and lowered the MHCII/CD206 ratio at eight weeks, although it did not reduce cartilage degeneration, synovitis, or fibrosis. Together, these findings show that TGFB1-engineered iMSCs have a macrophage-directed potency profile distinct from that of primary MSC(AT). Immortalized iMSCs therefore provide a reproducible platform for mechanistically dissecting engineered MSC potency and its effects on the immune environment.

Objective: To determine how fibronectin fragment (Fn-f), a matrikine that induces an osteoarthritis (OA)-like phenotype, reshapes chondrocyte states and transcriptional programs at single-cell resolution. Design: Primary human ankle chondrocytes from two cadaveric donors without OA were treated with PBS or purified recombinant Fn-f for 18 hours and profiled by single-cell RNA sequencing. Transcriptional states were characterized by marker genes and functional enrichment, compared with classifications used for OA chondrocytes, and evaluated for Fn-f-dependent changes in abundance and transcriptional programs. Results: Five transcriptionally distinct populations were identified with features that matched previous classifications of effector, inflammatory, pre-hypertrophic/fibrochondrocyte, reparative, and homeostatic chondrocytes. Fn-f markedly redistributed cells among these states, enriching inflammatory chondrocytes while reducing effector and reparative populations. Within transcriptional states, Fn-f differentially altered matrix/cartilage, inflammatory, and stress/immediate-early programs. These responses did not uniformly correspond to assigned subtype identities, demonstrating that transcriptional programs can be dynamically deployed across chondrocyte states. Conclusions: Controlled perturbations of healthy chondrocytes complement observational OA atlases by resolving stimulus-dependent changes in chondrocyte state abundance and transcriptional programs. Discordance between subtype annotations and dynamic functional programs further highlights the need for harmonized definitions of chondrocytes across phenotypic states.

The pace of fracture repair declines with age, increasing the risk of delayed union and nonunion. Delayed union and nonunion are associated with significant morbidity and even mortality with few effective non-surgical interventions. Here, we identify cathelicidin antimicrobial peptide (CAMP) as an age-dependent regulator of bone repair. Single-cell RNA sequencing and immunohistochemistry demonstrated higher CAMP expression during fracture healing in young compared with aged mice. Global or myeloid lineage-depletion of Camp delayed fracture repair in young mice, whereas Camp depletion in aged mice increased callus bone formation. Heterochronic bone marrow transplantation using the conditional genetic knockout further implicated hematopoietic-lineage derived cells in these effects and demonstrated that myeloid-specific Camp expression is important in the rejuvenation of the pace of fracture healing. CAMP promotes osteogenic differentiation through P2X7 receptor (P2X7R)-dependent signaling, influencing the downstream regulation of NLRP3 expression. NLRP3 expression is also correlated with osteogenesis following Camp modulation in both young and old mice, supporting CAMP-P2X7R-NLRP3 signaling as a regulator of osteogenesis. Together, these findings identify CAMP as a hematopoietic-derived regulator of bone repair whose function is age-dependent, promoting fracture healing in young animals while constraining it in aged animals.

Objectives. Observational studies report large, conflicting associations between common drugs and hip or knee replacement. We tested them in the serious adverse event (SAE) tables of randomized trials on ClinicalTrials.gov. Methods. We included randomized drug trials with at least 500 participants. A language model classified arms from summaries without outcome counts (masked by instruction only). We pooled osteoarthritis-related SAEs by drug class as Mantel-Haenszel risk ratios (RRs). Because the plan followed a preliminary analysis of 72 trials, we report estimates rather than tests, checked by positive and negative controls and calibration. Results. The 881 placebo- or usual-care-controlled trials included 1.75 million participants and 9,408 osteoarthritis-related SAEs. The tables reproduced CANTOS's own canakinumab effect (RR 0.57, 95% CI 0.39 to 0.82), the anti-nerve growth factor excess (2.31, 1.82 to 2.92) and two of three modest effects specified before extraction: gallbladder disease with glucagon-like peptide-1 receptor agonists (GLP-1RAs; 1.36, 1.22 to 1.51) and ketoacidosis with sodium-glucose cotransporter-2 (SGLT2) inhibitors (1.96, 1.48 to 2.60). In the same trials, osteoarthritis-related SAEs did not differ detectably with GLP-1RAs (0.97, 0.86 to 1.10; calibrated 0.87, 0.54 to 1.41) or SGLT2 inhibitors (0.96, 0.82 to 1.12; calibrated 0.86, 0.53 to 1.40). Other proposed drugs gave imprecise estimates. Post hoc, 8 of 21 published observational estimates differed from randomized ones, in both directions (3 to 6 after calibration). Conclusions. Over about two years, GLP-1RAs and SGLT2 inhibitors had no detectable effect on osteoarthritis-related SAEs in trials that detected their known effects. Slow or modest structural effects remain possible.

Background: Chronic musculoskeletal pain (CMP) is a common and debilitating symptom of Long COVID (LC), which shares several clinical features with Fibromyalgia Syndrome (FMS). Aims: This longitudinal study investigated whether the symptom overlap between LC-CMP and FMS persists over time, whether patterns differ according to pre-COVID CMP history, and the extent to which neuropathic pain features are present. Methods: Individuals with LC completed questionnaires assessing the symptom-based component of the 2010 American College of Rheumatology criteria for FMS and the presence of neuropathic pain features (PainDetect Questionnaire, PDQ) at baseline, 6- and 12-month follow-ups. Participants were grouped according to pre-COVID CMP history. Group differences were analysed using mixed-effects logistic regression. Results: 240 individuals completed the baseline assessment at a mean of 2.61 years after LC onset, including 131 (100 females) with new-onset CMP and 94 (87 females) with pre-existing CMP. The prevalence of participants meeting the symptom-based criteria for FMS remained high across follow-up (67.5-78.6%), with 50.0-61.0% of those with complete follow-up data fulfilling criteria at all timepoints. No significant subgroup differences were observed (OR = 0.43, 95% CI [0.14 1.39], p = 0.160). Across follow-up, 22.2-36.1% of participants reported likely neuropathic pain features, with significantly higher odds in the pre-existing CMP group (OR = 3.17, 95% CI [1.22 8.18], p = 0.017). Conclusions: FMS-like symptoms remain highly prevalent several years after acute COVID-19 and persist over a further 12 months, suggesting that LC-CMP frequently represents a persistent long-term condition with substantial overlap with FMS. These findings support closer integration of LC and chronic pain rehabilitation services and add to the wider literature linking post-infectious processes with FMS-like symptoms.

Objective: To assess the accuracy of codes and algorithms used to identify selected connective tissue diseases (CTDs) in electronic health records (EHRs) and administrative databases. Methods: We searched MEDLINE, Embase, and CENTRAL databases for studies that validated case definitions in EHRs against a reference standard, including rheumatologist-confirmed diagnosis or clinical classification criteria. Title/abstract screening, full-text review, data extraction, and quality appraisal were independently performed in duplicate. Findings were synthesised narratively and through a bivariate random-effects meta-analysis of sensitivity and specificity. Results: A total of 38 studies were included. Systemic lupus erythematosus (SLE) was the most frequently studied disease, followed by systemic sclerosis (SSc). Across diseases, progressively restrictive algorithms generally improved positive predictive value (PPV), although the optimal strategy varied by disease. In SLE, multiple diagnostic codes provided the most favorable overall performance, whereas adding clinical data provided limited incremental benefit. In SSc, disease-specific clinical features, particularly Raynaud's phenomenon, improved case identification, while specialist or inpatient-based algorithms performed better for polymyositis/dermatomyositis, although evidence was limited. For SLE, meta-analysis yielded pooled sensitivity of 0.90 (95% Cis 0.70-0.97), specificity of 0.83 (95% CIs 0.54-0.95), and PPV of 0.75 (95% CIs 0.59-0.86). Evidence for Sjogren's Disease was limited to one study, and no studies evaluated MCTD/UCTD. Conclusions: Optimal EHR case-identification strategies vary by disease and should be tailored to disease-specific clinical and diagnostic characteristics rather than algorithm complexity alone. Future research should develop and externally validate robust, standardised algorithms in representative populations, using rigorous validation methods, and assess the added value of longitudinal EHR data, biomarkers, and computational phenotyping approaches.

To correct lower limb length discrepancies, orthopedic surgeons often perform limb lengthening procedures. These procedures involve directly lengthening a primary bone (e.g., femur), which also stretches the muscles and tendons spanning the elongation site. Limb lengthening procedures improve lower-limb length symmetry, often at the expense of chronic joint-level decrements. For example, knee joint range of motion and strength does not always return to pre-surgery values following femur lengthening. These joint-level decrements governed by altered muscle force-length characteristics, but it is unestablished how human muscles remodel during surgical limb lengthening. Here, we characterized bilateral human vastus lateralis fascicle force-length profiles following femur lengthening. At 2.2 {+/-} 1.7 years (Avg {+/-} SE) post-initial surgery, the lengthened limb's vastus lateralis fascicles were longer, effectively stiffer, and weaker than the contralateral limb. The lengthened limb's vastus lateralis fascicles were 38 mm longer (Avg) than that of the contralateral limb, even though the lengthened limb's femur was only 31 mm longer (Avg). The onset of vastus lateralis fascicle passive force production began at 22% shorter (Avg) operating lengths in lengthened versus contralateral limb muscles. Additionally, vastus lateralis peak active force production was 34% less (Avg) in the lengthened versus contralateral limbs, and in-series quadriceps tendon stiffness was not stiffer in lengthened limbs. These data suggest that active muscle tissues lengthen more than passive muscle tissues following limb lengthening, effectively creating stiffer muscles. Years after limb lengthening, patient muscles exhibit numerous differences in their force-length properties versus that of natural growth, likely contributing to impaired joint-function.

In the murine osteochondral unit, morphology of chondrocyte lacunae (CCL) within calcified articular cartilage and osteocyte lacunae (OCL) within the underlying subchondral bone reflects local biomechanical conditions that are altered in disease, making reliable discrimination of both lacunar populations important for studying osteochondral physiology and pathology. Histology is generally considered the gold standard for identifying CCL and OCL. However, it is inherently limited to two-dimensional assessment and a relatively small tissue section, restricting comprehensive characterisation of three-dimensional lacunar structure. Synchrotron-radiation micro-computed tomography (SR-microCT) enables volumetric quantification of lacunae; where classification is typically based on the position of each lacuna relative to the cement line separating calcified cartilage from bone, requiring manual delineation of this convoluted interface. Such approaches are labour-intensive, operator-dependent, and rely on access to specialised synchrotron infrastructure that is not widely available. To address these limitations, we present a fully automated, annotation-free pipeline for classifying OCL and CCL in the mouse osteochondral unit based on desktop micro-computed tomography (microCT), independent of cement line identification. The proposed unsupervised workflow requires neither tissue separation nor labelled training data, enabling objective and scalable discrimination of lacunar populations directly from volumetric image data. Seven morphometric and positional features per lacuna were extracted from SR-microCT and desktop microCT datasets and analysed using Uniform Manifold Approximation and Projection (UMAP). Validation of the pipeline outcome on SR-microCT against manual classification across specimens yielded very-high agreement, with Gwet's AC1, the Matthews correlation coefficient, and balanced accuracy each approaching 0.99, in both healthy and post-traumatic osteoarthritis specimens. Such a workflow can be readily adapted for the investigation of osteochondral interface in various other species.

Objectives: Patients with pediatric acute-onset neuropsychiatric syndrome (PANS) frequently report pain and fatigue. While some patients experience resolution of pain and fatigue following improvement in psychiatric symptoms, others develop persistent symptoms resembling fibromyalgia. Understanding these trajectories is critical for early identification and intervention in patients at risk for chronic pain or fatigue. In this study we characterize fibromyalgia symptoms in PANS and identify predictors of symptom progression following a PANS flare. Design: Prospective observational cohort study. Setting: Stanford University Immune Behavioral Health (IBH) Clinic in Menlo Park, California, USA. Participants: 204 consecutive patients with PANS evaluated by the IBH Clinic. The average (SD) age at first neuropsychiatric deterioration was 8.2 (3.3) years. Main outcome measures: We prospectively collected data on pain, fatigue, and other fibromyalgia symptoms using the 2016 American College of Rheumatology (ACR) Fibromyalgia Survey Questionnaire (FSQ), which includes the Widespread Pain Index (WPI) and Symptom Severity Scale (SSS). For the reference standard, clinician-documented diagnoses of pain syndromes were ascertained through a retrospective review of participants' electronic medical records. We report the prevalence of these symptoms in PANS patients and controls and evaluate factors associated with time to meeting ACR fibromyalgia criteria threshold scores following clinic presentation using a Cox proportional hazards model. Results: Among 204 patients with PANS (110 boys [54%]), eighty-five (42%) met ACR fibromyalgia criteria threshold scores at least once during follow-up (95% CI at 10 years follow-up time: 60.5 [46.7-74.5]). Twenty-eight of these 85 patients (13.7% of total cohort) progressed to meet full ACR fibromyalgia criteria (95% CI at 10 years follow-up time: 24.1 [15.6-36.2]). Compared with controls, patients with PANS reported substantially elevated rates of pain, daytime fatigue, waking unrefreshed, and cognitive symptoms (brain fog). Higher scores on the WPI and SSS at presentation were associated with a higher hazard of subsequently meeting ACR fibromyalgia criteria threshold scores. Conclusions: Fibromyalgia symptoms were common in this PANS cohort, with a notable subset developing symptoms meeting full ACR fibromyalgia criteria. These findings suggest that pain and fatigue may represent post-inflammatory sequelae of PANS and may overlap mechanistically with other post-infectious conditions. Recognizing these symptom patterns may inform clinical management, development of outcome measures, and future clinical trials in these patients, as well as support investigation into shared neuroimmune mechanisms underlying post-infectious pain and fatigue syndromes.