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Introduction: Recent research suggests that synovial fluid neutrophil-to-lymphocyte ratio (SF-NLR) is a superior diagnostic for pyogenic septic arthritis (SA) in adults compared with synovial fluid white blood cell count (SF-WBC) ≥50,000 cells/μL or ≥90% polymorphonuclear leukocytes (SF-%PMN). Other research also indicates that the neutrophil-to-lymphocyte ratio in the blood (B-NLR) may be of diagnostic significance. However, it is not known whether these findings extend to the pediatric population., Methods: Medical records at a large urban tertiary-care children's hospital were queried for emergency department visits between 2012 and 2023, where synovial fluid (SF) analysis was performed to evaluate for SA of the hip or knee. Patients 18 years old and above were excluded. The "conventional composite test" (CCT) for SA was considered positive if SF analysis showed any of the following: (1) SF-WBC ≥50,000 cells/μL, (2) ≥90% PMNs, or (3) organisms reported on gram stain. Patients with aspirate and/or operating room (OR) cultures (or supplemental testing, ie, nucleic acid identification) revealing an offending organism were considered to have culture-positive septic arthritis (CPSA). The remaining patients were considered culture-negative (CN). Serum and SF test data were analyzed to assess their diagnostic utility in identifying CPSA. Receiver operating characteristic (ROC) curves were examined to compare the predictive value of SF-NLR and B-NLR versus conventional indicators of SA., Results: A total of 394 patients met the inclusion criteria. In all, 58.6% (n=231) were male, 67.5% (n=266) involved the knee, and 20.1% (n=79) had CPSA. Those with CPSA had higher ESR and CRP compared with CN patients ( P <0.01). Bivariate testing did not show a difference in SF-NLR or B-NLR between those with CPSA and CN patients ( P =0.93 and 0.37, respectively). The CCT showed 91% sensitivity and 35% specificity using conventional thresholds. ROC analysis showed that SF-WBC was superior to SF-NLR and B-NLR in the diagnosis of CPSA (AUC=0.71 vs. 0.50 and 0.53, respectively; both P <0.01). Among CCT (+) patients who ended up culture negative, Lyme testing was positive in 48.8% (100/205)., Conclusion: In contrast to adults, SF-NLR and B-NLR were not found to be strong diagnostic indicators of SA of the hip or knee in pediatric patients. This may be because competing diagnoses in children come with systemic inflammatory responses similar to that seen in pyogenic SA, while noninfectious conditions that might represent the major alternate diagnoses in adults do not increase systemic inflammatory markers as significantly. Given the high incidence of Lyme disease seen among patients in this study, this topic should be further studied at pediatric centers outside Lyme-endemic areas to better understand the generalizability of these findings., Significance: Despite excitement regarding SF-NLR and B-NLR as diagnostics for adult SA, these criteria appear less useful in the diagnosis of pyogenic SA in pediatric patients in Lyme-endemic areas., Competing Interests: The authors declare no conflicts of interest., (Copyright © 2024 Wolters Kluwer Health, Inc. All rights reserved.)

Objective: Synovial fluid (SF)-derived T cells are frequently studied as a proxy for investigating the synovial tissue (ST) T cell infiltrate in inflammatory arthritis. However, because ST is the primary site of inflammatory activity, there is debate as to whether SF provides a true reflection of the ST T cell population., Methods: In this study, we used single-cell RNA sequencing paired with single-cell T cell receptor (TCR) sequencing to directly compare memory T cells from paired samples of SF and ST from six patients with inflammatory arthritis to investigate their similarity in terms of TCR repertoire and T cell subset composition., Results: The TCR repertoires of SF and ST T cells were strikingly similar, particularly for CD8+ T cells. A median of 49% of the total CD8+ TCR repertoire in SF was shared with ST, compared with 20% shared with blood. Similarly, 47% of the ST CD8+ TCR repertoire was shared with SF compared to 25% with blood. Furthermore, once the effect of collagenase digestion on gene expression by ST T cells had been accounted for, the frequencies of specific CD8+ and CD4+ T cell subsets were, in general, similar in SF and ST and were distinct from blood., Conclusion: Our results suggest that T cells migrate and equilibrate between the SF and ST and maintain similar phenotypes in both sites. We conclude that SF is an appropriate proxy for investigating the T cell infiltrate in inflamed synovium, particularly in terms of investigating the TCR repertoire., (© 2024 The Author(s). Arthritis & Rheumatology published by Wiley Periodicals LLC on behalf of American College of Rheumatology.)

Introduction: Falsely elevated synovial white blood cell (WBC) counts using automated hematology analyzers have been reported particularly in the setting of joint arthroplasty. We evaluated the implementation of a laboratory workflow based on Sysmex XN-1000-automated cell counting and scattergram interpretation., Methods: WBC and differential were measured for 76 synovial fluid samples (29 native joints and 47 with joint arthroplasties) with Sysmex XN-1000 and manual methods. All scattergrams were evaluated for possible incorrect WBC and/or differential according to our implemented workflow. A specific finding was the "banana-shape" scattergram, which indicates possible interferences. The European Bone & Joint Infection Society (EBJIS) criteria were applied to identify possible prosthetic joint infections (PJIs) in patients with joint arthroplasties., Results: Correlation between automated and manual WBC counts, calculated for samples with WBC count <50 000/μL, was higher for native joints (r = 0.938) compared with patients known with arthroplasty (r = 0.906). Scattergrams classified as OK showed overall a higher correlation compared with scattergrams, which were interpreted as NOT OK. "Banana-shape" scattergrams (n = 19) showed falsely elevated automated WBC count, and the patterns were mainly seen in prosthesis patients (17/19 [89%]). Six of 47 (13%) patients with joint arthroplasties were reclassified from "confirmed" to "unlikely" PJI according to the EBJIS criteria., Conclusion: Our workflow based on scattergram interpretation resulted in accurate WBC counts in synovial fluid using automated/and or manual methods. It is important to identify the presence of "banana-shape" scattergrams to avoid overestimated automated WBC counts. Overall, automated synovial WBC count can be used, even for patients with arthroplasty, but after visual inspection of the scattergram to exclude possible interferences., (© 2024 John Wiley & Sons Ltd.)

Background: Acute bacterial arthritis (ABA) is a serious, pediatric infection that can result in motor comorbidities. Normally, a joint fluid white blood cell (WBC) count of 50,000 or more cells/mm 3 is used to make a presumptive diagnosis of ABA. This study evaluated the utility of the joint fluid WBC count for diagnosing pediatric ABA confirmed by a positive culture result., Methods: Patients with ABA between March 2010 and March 2023 at Tokyo Metropolitan Children's Medical Center were included. ABA was confirmed by positive joint fluid culture results for a pathogenic organism. Patients with negative results and those without a joint fluid WBC count were excluded. Electronic medical records were retrospectively reviewed for demographic data, timing of arthrocentesis, culture results and the joint fluid WBC count., Results: Ninety-five patients with ABA were identified; of these, 22 were included. The median age was 5 years [interquartile range (IQR): 2-10 years]. Males comprised 55% of the population. The median joint fluid WBC count was 19,575 (IQR: 6806-47,388) cells/mm 3 , and 23% of the patients had 50,000 cells/mm 3 or more. The median time from symptom onset to arthrocentesis was 3 days (IQR: 2-5 days). The isolated organisms were methicillin-susceptible Staphylococcus aureus (50%), methicillin-resistant S. aureus (9%), Streptococcus pyogenes (27%), Streptococcus pneumoniae (5%), Klebsiella pneumoniae (5%) and Salmonella spp. (5%)., Conclusions: Most of the patients with ABA confirmed by positive results of a joint fluid culture had a joint fluid WBC count of less than 50,000 cells/mm 3 ., Competing Interests: The authors have no funding or conflicts of interest to disclose., (Copyright © 2024 Wolters Kluwer Health, Inc. All rights reserved.)

Introduction: Distinguishing between septic arthritis and crystal arthropathy flares can be challenging. The purpose of this study was to determine how the presence of synovial crystals affects the diagnostic criteria of septic arthritis., Methods: A retrospective review identified patients undergoing joint aspirations to rule out native septic arthritis. Differences between septic arthritis presenting with and without synovial crystals were analyzed. A receiver-operating characteristic curve was plotted for laboratory markers to determine the area under the curve, or diagnostic accuracy, for septic arthritis and to evaluate thresholds that maximized sensitivity and specificity., Results: There were 302 joint aspirations in 267 patients. Septic arthritis was diagnosed in 17.9% (54/302). Patients with synovial crystals were less likely to have septic arthritis (4.2% [5/119] vs. 26.8% [49/183], P < 0.0001). Septic arthritis in patients with no synovial crystals was associated with fever and a higher synovial white blood cell (WBC) count, synovial polymorphonuclear cell percentage (PMN%), serum WBC, and C-reactive protein (CRP) ( P < 0.05). Septic arthritis in patients with synovial crystals was only associated with inability to bear weight and a higher synovial WBC and CRP ( P < 0.05). Synovial PMN% was considered nondiagnostic of septic arthritis (area under the curve 0.56) in patients with crystals while synovial WBC and CRP had acceptable (0.76) and excellent (0.83) diagnostic utility, respectively. The WBC and CRP value thresholds that maximized sensitivity and specificity for septic arthritis were greater in patients with crystals (21,600 vs. 17,954 cells/μL and 125 vs. 69 mg/L, respectively)., Discussion: The presence of synovial crystals reduced the likelihood of septic arthritis and altered the laboratory diagnostic criteria. PMN% was nondiagnostic in the setting of synovial crystals., (Copyright © 2024 by the American Academy of Orthopaedic Surgeons.)

Introduction: Periprosthetic joint infection (PJI) is a devastating complication of hip hemiarthroplasty (HHA) that is not well-represented in the literature. Therefore, this study aimed to evaluate diagnostic markers for identifying PJI in patients after HHA and compare them with the most recent 2018 International Consensus Meeting on Musculoskeletal Infection criteria., Methods: A total of 98 patients (64 PJIs, 65.3%) were analyzed. Patients were identified by relevant Current Procedural Terminology and International Classification of Diseases-9/10 codes from 2000 to 2021 across a single healthcare system. Preoperative or intraoperative synovial fluid nucleated cell (NC) count, synovial polymorphonuclear (PMN) percentage, serum erythrocyte sedimentation rate (ESR), serum C-reactive protein (CRP), and serum white blood cell count were compared with Student t -test between aseptic and septic cohorts. Diagnostic utility and laboratory cutoff values were determined using receiver-operating characteristic curves and Youden index, respectively., Results: Mean values were significantly higher in the septic cohort for synovial NC count (120,992.2 versus 1,498.0 cells/μL, P < 0.001), synovial PMN percentage (91.3% versus 56.2%, P < 0.001), serum ESR (75.6 versus 36.3 mm/hr, P < 0.001), serum CRP (20.2 versus 125.8 mg/L, P < 0.001), and serum white blood cell count (8.5 versus 11.5 cells/μL, P < 0.001). Synovial NC count, synovial PMN percentage, and serum CRP had excellent PJI discriminatory ability with an area under the curve of 0.99, 0.90, and 0.93, respectively. Optimal cutoffs were 2,700 cells/μL for synovial NC count (100% sensitivity and 94% specificity), 81.0% for synovial PMN percentage (96% sensitivity and 89% specificity), 52.0 mm/hr for serum ESR (75% sensitivity and 80% specificity), and 40.0 mg/L for serum CRP (85% sensitivity and 92% specificity)., Conclusion: Our findings support the continued use of routine serum and synovial fluid tests for diagnosing PJI in HHA patients. Optimal cutoff values for both synovial fluid biomarkers were very close in alignment with the 2018 International Consensus Meeting criteria., Level of Evidence: Level III, retrospective comparative study., (Copyright © 2024 by the American Academy of Orthopaedic Surgeons.)

Background: Biomarkers that predict the treatment response in patients with knee osteoarthritis are scarce. This study aimed to investigate the potential role of synovial fluid cell counts and their ratios as biomarkers of primary knee osteoarthritis., Methods: This retrospective study investigated 96 consecutive knee osteoarthritis patients with knee effusion who underwent joint fluid aspiration analysis and received concomitant intra-articular corticosteroid injections and blood tests. The monocyte-to-lymphocyte ratio (MLR) and neutrophil-to-lymphocyte ratio (NLR) were calculated. After 6 months of treatment, patients were divided into two groups: the responder group showing symptom resolution, defined by a visual analog scale (VAS) score of ≤ 3, without additional treatment, and the non-responder group showing residual symptoms, defined by a VAS score of > 3 and requiring further intervention, such as additional medication, repeated injections, or surgical treatment. Unpaired t-tests and univariate and multivariate logistic regression analyses were conducted between the two groups to predict treatment response after conservative treatment. The predictive value was calculated using the area under the receiver operating characteristic curve, and the optimal cutoff value was determined., Results: Synovial fluid MLR was significantly higher in the non-responder group compared to the responder group (1.86 ± 1.64 vs. 1.11 ± 1.37, respectively; p = 0.02). After accounting for confounding variables, odds ratio of non-responder due to increased MLR were 1.63 (95% confidence interval: 1.11-2.39). The optimal MLR cutoff value for predicting patient response to conservative treatment was 0.941., Conclusions: MLR may be a potential biomarker for predicting the response to conservative treatment in patients with primary knee osteoarthritis., (© 2024. The Author(s).)

Background: Graphic type differentiation of cell count data of synovial aspirates is a new method for the diagnosis of early and late periprosthetic joint infection., Objective: The aim of the study was to analyse if the same 6 LMNE-types can be differentiated in the new Yumizen H500 cell counter as it was the case for the old cell counter ABX Pentra XL 80 of previous publications, to verify if the erythrocyte and thrombocyte curves of the new device give additional information and to calculate the difference of cell count in LMNE-type I and III (with abrasion) in the cell counter and in the manual counting chamber (Neubauer improved)., Methods: 450 aspirates of 152 total hip arthroplasties and 298 knee arthroplasties obtained for the diagnosis of periprosthetic joint infection were analysed with the Yumizen H500., Results: All LMNE-matrices of the 450 aspirates could assigned to one of the six LMNE-types. There were 76 LMNE-type I, 72 LMNE-type II, 14 LMNE-type III, 241 LMNE-type IV, 36 LMNE-type V and 12 LMNE-type VI. The erythrocyte and thrombocyte distribution curves were very helpful for differentiation of hematoma and infection. The cell count in the manual counting procedure was lower than in the cell counter: for the LMNE-type I (abrasion type) the median of the difference was 925/μL (median) and for the LMNE-type III (combined type of infection and abrasion) 3570/μL (median)., Conclusion: The described graphic type differentiation is a new and helpful method for differentiation of hematoma and early PJI as well as abrasion and late PJI.

Background: Cartilage injuries are currently the most prevalent joint disease. Previous studies have emphasized the use of stem cells as the effective treatment for regenerating cartilage damage., Objective: In this study, considering the difficulties of the cellular therapy method, it was hypothesized that human synovial fluid-derived mesenchymal stem cell (hSFMSC) exosomes as a SC source could be used to treat these injuries as a safer and cell-free therapeutic alternative procedure due to its direct relevance to cartilage regeneration. Moreover, this study aimed to determine the miRNA and target genes required for the formation of SC treatment combined with gene therapy in order to reveal the mechanism of cartilage regeneration and increase its effectiveness., Methods: MSCs were characterized by flow cytometry, and immunocytochemical and differentiation analyses were done. To characterize functionally isolated exosomes, in vitro uptake analysis was performed. RT-qPCR was used to examine in terms of the advantages of cellular and cell-free therapy, mature human chondroblasts derived by differentiation from hSF-MSCs and human chondrocyte profiles were compared in order to demonstrate the above profile of hSF-MSCs and exosomes isolated from them, and the effectiveness of SC therapy in repairing cartilage damage., Results: According to our findings, the expression level of hsa-miR-155-5p was found to be considerably higher in chondrocytes differentiated from human synovial fluid MSCs than in mature human chondrocytes. These findings were also supported by the TGF-signalling pathway and chondrogenesis marker genes., Conclusion: It was concluded that hSF-MSCs and exosomes can be used in the treatment of cartilage damage, and hsa-miR-155-5p can be used as a target miRNA in a new gene therapy approach because it increases the therapeutic effect on cartilage damage., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Objectives: The aim of this study was to describe clinical and diagnostic findings in cats with bone and joint disease associated with histoplasmosis. Methods: Medical records from between 2011 and 2017 were reviewed. Inclusion criteria required: (1) diagnosis of histoplasmosis by cytology, histology, urine or serum Histoplasma antigen testing, or culture; and (2) lameness or joint effusion as a presenting complaint or physical examination finding. Results: Twenty-five cases met the inclusion criteria. Four had incomplete records, but available data were included when applicable. Lameness was a presenting complaint in 17/21 cats and was the only complaint in 9/21 cats. Initial diagnosis was made by cytology in 22/25 cats and by culture, urine antigen and necropsy in one case each. Diagnostic cytology samples included synovial fluid (n = 13), lymph node (n = 5), skin (n = 2), lung (n = 1) and bone (n = 1). Two additional cases had synovial fluid examined but no organisms present. Inflammation was present in all synovial fluid samples examined. Biopsy was obtained in two cats and histologic diagnoses included osteomyelitis with no infectious organisms identified and severe lymphoplasmacytic synovitis suggestive of feline periosteal proliferative polyarthritis. Histoplasma urine antigen test was positive in 7/12 cats. Conclusions and relevance: Inflammatory arthritis is common in cats with histoplasmosis, with lameness a common presenting complaint. Organisms are found in synovial fluid cytology in most cases. If not, appropriate additional diagnostics must be pursued. [ABSTRACT FROM AUTHOR]

Diagnosis of bone and joint infections (BJI) relies on microbiological culture which has a long turnaround time and is challenging for certain bacterial species. Rapid molecular methods may alleviate these obstacles. Here, we investigate the diagnostic performance of IS-pro, a broad-scope molecular technique that can detect and identify most bacteria to the species level. IS-pro additionally informs on the amount of human DNA present in a sample, as a measure of leukocyte levels. This test can be performed in 4 h with standard laboratory equipment. Residual material of 591 synovial fluid samples derived from native and prosthetic joints from patients suspected of joint infections that were sent for routine diagnostics was collected and subjected to the IS-pro test. Bacterial species identification as well as bacterial load and human DNA load outcomes of IS-pro were compared to those of culture. At sample level, percent positive agreement (PPA) between IS-pro and culture was 90.6% (95% CI 85.7- to 94%) and negative percent agreement (NPA) was 87.7% (95% CI 84.1 to 90.6%). At species level PPA was 80% (95% CI 74.3 to 84.7%). IS-pro yielded 83 extra bacterial detections over culture for which we found supporting evidence for true positivity in 40% of the extra detections. Missed detections by IS-pro were mostly related to common skin species in low abundance. Bacterial and human DNA signals measured by IS-pro were comparable to bacterial loads and leukocyte counts reported by routine diagnostics. We conclude that IS-pro showed an excellent performance for fast diagnostics of bacterial BJI., Competing Interests: The authors declare a conflict of interest. M.P.B., L.P., and A.-X.v.d.S. are employees of inbiome. A.E.B. is co-owner of inbiome. A.E.B. and P.S. are co-inventors of the Molecular Culture test technology. All other authors report no potential conflicts of interest.

(1) Background: Synovial fluid (SF) from knee joints with osteoarthritis (OA) has increased levels of phospholipids (PL). We have reported earlier that TGF-ß and IGF-1 stimulate fibroblast-like synoviocytes (FLS) to synthesize increased amounts of PLs. The current study examined whether IL-1ß induces the release of PLs in FLS and the underlying mechanism. (2) Methods: Cultured human OA FLS were treated with IL-1ß alone and with pathway inhibitors or with synthetic liver X receptor (LXR) agonists. Cholesterol hydroxylases, ABC transporters, apolipoproteins (APO), LXR, sterol regulatory binding proteins (SREBPs), and 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR) were analyzed by RT-PCR, Western blot, and ELISA. The release of radiolabeled PLs from FLS was determined, and statistical analysis was performed using R (N = 5-9). (3) Results: Like synthetic LXR agonists, IL-1ß induced a 1.4-fold greater release of PLs from FLS. Simultaneously, IL-1ß upregulated the level of the PL transporter ABCA1 and of cholesterol hydroxylases CH25H and CYP7B1. IL-1ß and T0901317 stimulated the expression of SREBP1c, whereas only T0901317 enhanced SREBP2, HMGCR, APOE, LXRα, and ABCG1 additionally. (4) Conclusions: IL-1ß partially controls PL levels in OA-SF by affecting the release of PLs from FLS. Our data show that IL-1ß upregulates cholesterol hydroxylases and thus the formation of oxysterols, which, as natural agonists of LXR, increase the level of active ABCA1, in turn enhancing the release of PLs.

Aryl-hydrocarbon receptor (AhR) is a ligand-activated transcription factor and regulates differentiation and function of various immune cells such as dendritic cells, Th17, and regulatory T cells. In recent studies, it was reported that AhR is involved in bone remodeling through regulating both osteoblasts and osteoclasts. However, the roles and mechanisms of AhR activation in human osteoclasts remain unknown. Here we show that AhR is involved in human osteoclast differentiation. We found that AhR expressed highly in the early stage of osteoclastogenesis and decreased in mature osteoclasts. Kynurenine (Kyn), formylindolo[3,4-b] carbazole (FICZ), and benzopyrene (BaP), which are AhR agonists, inhibited osteoclast formation and Kyn suppressed osteoclast differentiation at an early stage. Furthermore, blockade of AhR signaling through CH223191, an AhR antagonist, and knockdown of AhR expression reversed Kyn-induced inhibition of osteoclast differentiation. Overall, our study is the first report that AhR negatively regulates human osteoclast differentiation and suggests that AhR could be good therapeutic molecule to prevent bone destruction in chronic inflammatory diseases such as rheumatoid arthritis (RA).

Infection by (re-)emerging RNA arboviruses including Chikungunya virus (CHIKV) and Mayaro virus primarily cause acute febrile disease and transient polyarthralgia. However, in a significant subset of infected individuals, debilitating arthralgia persists for weeks over months up to years. The underlying immunopathogenesis of chronification of arthralgia upon primary RNA-viral infection remains unclear. Here, we analysed cell-intrinsic responses to ex vivo arthritogenic alphaviral infection of primary human synovial fibroblasts isolated from knee joints, one the most affected joint types during acute and chronic CHIKV disease. Synovial fibroblasts were susceptible and permissive to alphaviral infection. Base-line and exogenously added type I interferon (IFN) partially and potently restricted infection, respectively. RNA-seq revealed a CHIKV infection-induced transcriptional profile that comprised upregulation of expression of several hundred IFN-stimulated and arthralgia-mediating genes. Single-cell virus-inclusive RNA-seq uncovered a fine-tuned switch from induction to repression of cell-intrinsic immune responses depending on the abundance of viral RNA in an individual cell. Specifically, responses were most pronounced in cells displaying low-to-intermediate amounts of viral RNA and absence of virus-encoded, fluorescent reporter protein expression, arguing for efficient counteraction of innate immunity in cells expressing viral antagonists at sufficient quantities. In summary, cell-intrinsic sensing of viral RNA that potentially persists or replicates at low levels in synovial fibroblasts and other target cell types in vivo may contribute to the chronic arthralgia induced by alphaviral infections. Our findings might advance our understanding of the immunopathophysiology of long-term pathogenesis of RNA-viral infections.

Objectives: A number of immune populations have been implicated in psoriatic arthritis (PsA) pathogenesis. This study used mass cytometry (CyTOF) combined with transcriptomic analysis to generate a high-dimensional dataset of matched PsA synovial fluid (SF) and blood leucocytes, with the aim of identifying cytokine production ex vivo in unstimulated lymphoid and myeloid cells., Methods: Fresh SF and paired blood were either fixed or incubated with protein transport inhibitors for 6 hours. Samples were stained with two CyTOF panels: a phenotyping panel and an intracellular panel, including antibodies to both T cell and myeloid cell secreted proteins. Transcriptomic analysis by gene array of key expanded cell populations, single-cell RNA-seq, ELISA and LEGENDplex analysis of PsA SF were also performed., Results: We observed marked changes in the myeloid compartment of PsA SF relative to blood, with expansion of intermediate monocytes, macrophages and dendritic cell populations. Classical monocytes, intermediate monocytes and macrophages spontaneously produced significant levels of the proinflammatory mediators osteopontin and CCL2 in the absence of any in vitro stimulation. By contrast minimal spontaneous cytokine production by T cells was detected. Gene expression analysis showed the genes for osteopontin and CCL2 to be among those most highly upregulated by PsA monocytes/macrophages in SF; and both proteins were elevated in PsA SF., Conclusions: Using multiomic analyses, we have generated a comprehensive cellular map of PsA SF and blood to reveal key expanded myeloid proinflammatory modules in PsA of potential pathogenic and therapeutic importance., Competing Interests: Competing interests: SC and AM are employees of UCB. HA-M received unrestricted research support from UCB. PB has received research support from Regeneron, Benevolent AI and GSK., (© Author(s) (or their employer(s)) 2021. No commercial re-use. See rights and permissions. Published by BMJ.)

Purpose: Contradicting definitions of periprosthetic joint infection (PJI) are in use. Joint aspiration is performed before total hip arthroplasty (THA) revision. This study investigated the influence of PJI definition on PJI prevalence at THA revision. Test quality of prerevision aspiration was evaluated for the different PJI definitions., Methods: 256 THA revisions were retrospectively classified to be infected or not infected. Classification was performed according to the 4 different definitions proposed by the Musculoskeletal Infection Society (MSIS), the Infectious Diseases Society of America (IDSA), the International Consensus Meeting (ICM), and the European Bone and Joint Infection Society (EBJIS). Only chronic PJIs were included., Results: PJI prevalence at revision significantly correlated with the applied PJI definition ( p = 0.01, Cramer's V = 0.093). PJI prevalence was 20.7% for the MSIS, 25.4% for the ICM, 28.1% for the IDSA, and 32.0% for the EBJIS definition. For synovial fluid white blood cell count, the best ROC-AUC for predicting PJI was 0.953 in combination with the MSIS definition., Conclusion: PJI definition significantly influences the rate of diagnosed PJIs at THA revision. Synovial fluid white blood cell count is a reliable means to rule out PJI. In cases with a borderline high synovial white blood cell count before THA revision as the only sign of chronic PJI, an extended diagnostic work-up should be considered., Competing Interests: No benefits in any form have been received or will be received from a commercial party related directly or indirectly to the subject of this article., (Copyright © 2021 Sebastian Philipp Boelch et al.)

Fibroblast-like synoviocytes (FLS) play an important pathological role in persistent inflammatory joint diseases such as rheumatoid arthritis (RA). These cells have primarily been characterized in the RA synovial membrane. Here we aim to phenotypically and functionally characterize cultured synovial fluid-derived FLS (sfRA-FLS). Paired peripheral blood mononuclear cells (PBMC) and sfRA-FLS from patients with RA were obtained and monocultures of sfRA-FLS and autologous co-cultures of sfRA-FLS and PBMC were established. The in situ activated sfRA-FLS were CD34-, CD45-, Podoplanin+, Thymocyte differentiation antigen-1+. SfRA-FLS expressed uniform levels of NFкB-related pathway proteins and secreted several pro-inflammatory cytokines dominated by IL-6 and MCP-1. In a co-culture model with autologous PBMC, the ICAM-1 and HLA-DR expression on sfRA-FLS and secretion of IL-1β, IL-6, and MCP-1 increased. In vivo, human sfRA-FLS were cartilage invasive both at ipsilateral and contralateral implantation site. We conclude that, sfRA-FLS closely resemble the pathological sublining layer FLS subset in terms of surface protein expression, cytokine production and leukocyte cross-talk potential. Further, sfRA-FLS are comparable to tissue-derived FLS in their capabilities to invade cartilage at implantation sites but also spread tissue destruction to a distant site. Collectively, sfRA-FLS can serve as a an easy-to-obtain source of pathological sublining FLS in RA., (© 2021. The Author(s).)

The role of resident cells such a synoviocytes and chondrocytes in intra-articular inflammation is well-characterized, however the in vivo gene expression patterns of cells (predominantly leukocytes) in the synovial fluid (SF) of an inflamed joint have never previously been investigated. The aim of this study was to investigate gene expression in SF leukocytes from the inflamed joint cavity after intra-articular lipopolysaccharide (LPS) injection in horses to improve our understanding of the temporal regulation of the intra-articular inflammatory response. Gene expression was investigated in SF samples available from six horses 2, 4, 8 16 and 24 h after experimental induction of inflammation in the radiocarpal joint by lipopolysaccharide (LPS) injection. Leukocytic expression of 43 inflammation-related genes was studied using microfluidic high throughput qPCR (Fluidigm®). Expression of 26 genes changed significantly over the 24 h study period, including pro- and anti-inflammatory genes such as interleukin (IL)1, IL6, tumor necrosis factor (TNF), cyclooxygenase 2 (COX2), IL1 receptor antagonist (IL1RN), IL10, and superoxide dismutase 2 (SOD2), chemokine genes, apoptosis-related genes, and genes related to cartilage turnover (matrix metalloproteinase 8 and tissue inhibitor of metalloproteinase 1). The inflammatory responses appeared to be regulated, as an early increase (at 2 h) in expression of the pro-inflammatory genes IL1, IL6, TNF and COX2 was rapidly followed by increased expression (at 4 h) of several anti-inflammatory genes (IL10, IL1RN and SOD2). Similarly, both pro- and anti-apoptotic gene expression as well as expression of chondrodegenerative and chondroprotective genes were activated in SF leukocytes. Thus, the inflammatory response in leukocytes infiltrating the joint in the acute stage of arthritis was well orchestrated in this single-hit LPS-induced arthritis model. This study is the first to describe gene expression patterns in SF-derived leukocytes in vivo during severe joint inflammation, and the results thus expand our knowledge of basic inflammatory mechanisms in the early local response in an inflamed joint., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

Objectives: Direct inhibition of M1 polarization of synovial macrophages may be a useful therapeutic treatment for OA and OA-associated synovitis. Frugoside (FGS) is a cardiac glycoside compound isolated and extracted from Calotropis gigantea. Cardiac glycosides possess interesting anti-inflammatory potential. However, the corresponding activity of FGS has not been reported. Therefore, our aim was to find direct evidence of the effects of FGS on synovial macrophage M1 polarization and OA control., Methods: Collagenase was used to establish an experimental mouse OA model (CIOA) with considerable synovitis. Then, FGS was intra-articular administered. The mRNA and protein levels of iNOS were analysed by real-time PCR and Western blotting in vitro. Immunohistochemical and immunofluorescence staining were used to measure the expression of F4/80, iNOS, Col2α1 and MMP13 in vivo. The levels of pro-inflammatory cytokines in FGS-treated M1 macrophage culture supernatants were analysed by flow cytometry., Results: FGS attenuates synovial inflammation and delays the development of OA in CIOA mice. Further results demonstrate that FGS inhibits macrophage M1 polarization in vitro and in vivo, which subsequently decreases the secretion of IL-6 and TNF-α, in turn delaying cartilage and extracellular matrix (ECM) degradation and chondrocyte hypertrophy. FGS inhibits macrophage M1 polarization by partially downregulating miR-155 levels., Conclusion: This study demonstrates that intra-articular injection of FGS is a potential strategy for OA prevention and treatment, even at an early stage of disease progression. This is a novel function of FGS and has promising future clinical applications., (© The Author(s) 2021. Published by Oxford University Press on behalf of the British Society for Rheumatology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

The relationship between TLR4 and inflammation-related diseases has been paid more and more attention. The studies have shown that TLR4/NF-κB signaling pathway plays an important role in the transmission of inflammatory signals. A large number of pro-inflammatory factors, chemokines, adhesion factors, TLR4 and its ligands interact with each other, and jointly promote the development of diseases. In this work, 8 target compounds were synthesized to screen the inhibitory activity of TLR4 in vitro. The results of TLR4 inhibition test in vitro showed that the double-ring conjugated enones had a good inhibitory activity, and the IC 50 value of compound 4f was 0.56 ± 0.10 μM, and it was superior to the positive control methotrexate. To further study the anti-inflammatory effect and mechanism of double-ring conjugated enones by using LPS induced rat synovial cell inflammation model. The results of the mechanism test showed that compound 4f could effectively promote the apoptosis of rat synovial cells, and the mechanism might be related to the up-regulation of the expression of apoptosis-related protein Caspase-3. In addition, compound 4f could significantly inhibit the increase of inflammatory factors TNF-α, IL-1β and IL-6 in rat synovial cells induced by LPS, showing a good anti-inflammatory activity. In the TLR4/NF-κB signaling pathway test of rat synovial cells, compound 4f can effectively regulate the expression levels of TLR4, MyD88, NF-κB and IκB related proteins in TLR4/NF-κB signaling pathway, which may be due to its inhibition of LPS-induced inflammation in rat synovial cells. At the same time, it inhibits the abnormal proliferation of cells and its important mechanism promoted of apoptosis., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Background: Imbalance between apoptosis and autophagy in fibroblast-like synoviocytes (FLS) is one of the pathogenic mechanisms responsible for their abnormal proliferation in rheumatoid arthritis (RA). Methotrexate (MTX) demonstrated limited efficacy in amending this imbalance in fluid-derived (fd)-FLS. The active compound of black tea Theaflavin 3,3'-digallate (TF3) may be effective in restoring apoptosis-autophagy imbalance in (fd)-FLS. The combined effect of MTX + TF3 upon the same is yet to be elucidated., Objective: To evaluate the effect of MTX + TF3 on fd-FLS to induce apoptosis and inhibit autophagy through Endoplasmic Reticulum (ER) stress-mediated pathways., Methods: FLS from synovial fluid of 11 RA and 10 osteoarthritis patients were cultured after treatment with MTX/TF3 or a combination of MTX (125 nM) and TF3(10 µM) and the following parameters were evaluated. C-reactive protein, cytokines (TNF-α, IL-6), angiogenic markers were quantified by ELISA. fd-FLS viability was determined by MTT assay and apoptosis by flow cytometry. ER stress markers were estimated by RT-PCR (IRE1A, spliced-XBP-1) and immunoblotting (Grp78, Hsp70, CHOP, HIF-1α). Immunoblot studies were done to evaluate apoptotic (Bcl-2, Bax, Caspases) and autophagic (Beclin1, LC3b, p62) proteins., Results: MTX (IC 25 ) and TF3 (IC 50 ) both in single doses could down-regulate the levels of pro-inflammatory and angiogenic markers. Combinatorial treatment modulated autophagosomal proteins in fd-FLS and induced apoptosis by regulating ER stress response., Conclusion: Disruption in homeostasis between apoptosis and autophagy in fd-FLS might be an underlying phenomenon in the progression of pathophysiology in RA. Co-administration of MTX + TF3 successfully restored the homeostasis by inducing apoptosis., (© 2021. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

Background: Although synovial fluid can be used to diagnose periprosthetic joint infections (PJI) effectively, only the cutoff values adopted at the time of PJI diagnosis have been standardized, and few data are currently available about effectiveness of synovial fluid examination before definitive reimplantation., Questions/purposes: We asked: (1) What are the most appropriate thresholds for synovial fluid leukocyte counts (WBC) and neutrophil percentage (PMN percentage) in a patient group undergoing definitive reimplantation after an uninterrupted course of antibiotic therapy for chronic PJI? (2) What is the predictive value of our synovial WBC and PMN percentage threshold compared with previously proposed thresholds?, Methods: In all, 101 patients with PJI were evaluated for inclusion from January 2016 to December 2018. Nineteen percent (19 of 101) of patients were excluded because of the presence of a chronic inflammatory disease, acute/late hematogenous infection, low amount of synovial fluid for laboratory investigations or infection persistence after spacer placement, and adequate antibiotic therapy. Finally, 81% (82 of 101) of patients with a median (range) age of 74 years (48 to 92) undergoing two-stage revision for chronic TKA infection, who were followed up at our institution for a period 96 weeks or more, were included in this study. The patients did not discontinue antibiotic treatment before reimplantation and were treated for 15 days after reimplantation if intraoperative cultures were negative. No patient remained on suppressive treatment after reimplantation. Synovial fluid was aspirated aseptically with a knee spacer in place to evaluate the cell counts before reimplantation. Thirteen percent (11 of 82) of patients had persistent or recurrent infection, defined as continually elevated erythrocyte sedimentation rate or C-reactive protein levels coupled with local signs and symptoms or positive cultures. The synovial fluid WBC counts and PMN percentage from the 11 patients with persistent or recurrent PJI were compared with the 71 patients who were believed to be free of PJI. Receiver operating characteristic (ROC) curve analyses assessed the predictive value of the parameters, and the areas under the curves (AUCs) were evaluated. The sensitivities, specificities, and positive and negative predictive values were determined for the WBC count and PMN percentage. Patients with persistent or recurrent infection had higher median WBC counts (471 cells/µL versus 1344 cells/µL; p < 0.001) and PMN percentage (36% versus 61%; p < 0.001) than did patients believed to be free of PJI., Results: ROC curve analysis identified the best threshold values to be a WBC count of 934 cells/µL or more (sensitivity 0.82 [95% CI 0.71 to 0.89], specificity 0.82 [95% CI 0.71 to 0.89]) as well as a PMN percentage of at least 52% (sensitivity 0.82 [95% CI 0.71 to 0.89] and specificity 0.78 [95% CI 0.67 to 0.86]. We found no difference between the AUCs for the WBC count and the PMN percentage (0.87 [95% CI 0.79 to 0.96] versus 0.84 [95% CI 0.73 to 0.95]. Comparing the sensitivities and specificities of the synovial fluid WBC count and PMN percentage proposed by other authors, we find that a PMN percentage more than 52% showed better predictive value than previously reported., Conclusion: Based on our findings, we believe that patients with WBC counts of at least 934 and PMN percentage of 52% or more should not undergo reimplantation but rather a repeat debridement, as their risk of persistent or recurrent PJI appears prohibitively high. The accuracy of the proposed cutoffs is better than previously reported., Level of Evidence: Level III, diagnostic study., Competing Interests: Each author certifies that neither he or she, nor any member of his or her immediate family, has funding or commercial associations (consultancies, stock ownership, equity interest, patent/licensing arrangements, etc.) that might pose a conflict of interest in connection with the submitted article. All ICMJE Conflict of Interest Forms for authors and Clinical Orthopaedics and Related Research® editors and board members are on file with the publication and can be viewed on request., (Copyright © 2021 by the Association of Bone and Joint Surgeons.)

Inflammatory monocyte-derived dendritic cells (Mo-DCs) have been described in several chronic inflammatory disorders, such as rheumatoid arthritis (RA), and are suspected to play a detrimental role by fueling inflammation and skewing adaptive immune responses. However, the characterization of their phenotype is still limited, as well as the comprehension of the factors that govern their differentiation. Here, we show that inflammatory Mo-DCs generated in vitro expressed a large and atypical panel of C-type lectin receptors, including isoforms of CD209 and CD206, CD303 and CD207, as well as intracellular proteins at their surfaces such as the lysosomal protein CD208. Combination of these markers allowed us to identify cells in the synovial fluid of RA patients with a close phenotype of inflammatory Mo-DCs generated in vitro. Finally, we found in coculture experiments that RA synoviocytes critically affected the phenotypic differentiation of monocytes into Mo-DCs, suggesting that the crosstalk between infiltrating monocytes and local mesenchymal cells is decisive for Mo-DCs generation.

Objective: To compare immune cell phenotype and function in psoriatic arthritis (PsA) versus psoriasis in order to better understand the pathogenesis of PsA., Methods: In-depth immunophenotyping of different T cell and dendritic cell subsets was performed in patients with PsA, psoriasis, or axial spondyloarthritis and healthy controls. Subsequently, we analyzed cells from peripheral blood, synovial fluid (SF), and skin biopsy specimens using flow cytometry, along with high-throughput transcriptome analyses and functional assays on the specific cell populations that appeared to differentiate PsA from psoriasis., Results: Compared to healthy controls, the peripheral blood of patients with PsA was characterized by an increase in regulatory CD4+ T cells and interleukin-17A (IL-17A) and IL-22 coproducing CD8+ T cells. One population specifically differentiated PsA from psoriasis: i.e., CD8+CCR10+ T cells were enriched in PsA. CD8+CCR10+ T cells expressed high levels of DNAX accessory molecule 1 and were effector memory cells that coexpressed skin-homing receptors CCR4 and cutaneous lymphocyte antigen. CD8+CCR10+ T cells were detected under inflammatory and homeostatic conditions in skin, but were not enriched in SF. Gene profiling further revealed that CD8+CCR10+ T cells expressed GATA3, FOXP3, and core transcriptional signature of tissue-resident memory T cells, including CD103. Specific genes, including RORC, IFNAR1, and ERAP1, were up-regulated in PsA compared to psoriasis. CD8+CCR10+ T cells were endowed with a Tc2/22-like cytokine profile, lacked cytotoxic potential, and displayed overall regulatory function., Conclusion: Tissue-resident memory CD8+ T cells derived from the skin are enhanced in the circulation of patients with PsA compared to patients with psoriasis alone. This may indicate that aberrances in cutaneous tissue homeostasis contribute to arthritis development., (© 2021 The Authors. Arthritis & Rheumatology published by Wiley Periodicals LLC on behalf of American College of Rheumatology.)

Aims: It remains difficult to diagnose early postoperative periprosthetic joint infection (PJI) following total knee arthroplasty (TKA). We aimed to validate the optimal cutoff values of ESR, CRP, and synovial fluid analysis for detecting early postoperative PJI in a large series of primary TKAs., Methods: We retrospectively identified 27,066 primary TKAs performed between 2000 and 2019. Within 12 weeks, 169 patients (170 TKAs) had an aspiration. The patients were divided into two groups: those evaluated ≤ six weeks, or between six and 12 weeks postoperatively. The 2011 Musculoskeletal Infection Society (MSIS) criteria for PJI diagnosis in 22 TKAs. The mean follow-up was five years (two months to 17 years). The results were compared using medians and Mann-Whitney U tests and thresholds were analyzed using receiver operator characteristic curves., Results: Within six weeks, the median CRP (101 mg/l vs 35 mg/l; p = 0.011), synovial WBCs (58,295 cells/μl vs 2,121 cells/μl; p ≤ 0.001), percentage of synovial neutrophils (91% vs 71% (p < 0.001), and absolute synovial neutrophil count (ANC) (50,748 cells/μl vs 1,386 cells/μl (p < 0.001) were significantly higher in infected TKAs. Between six and 12 weeks, the median CRP (85 mg/l vs 5 mg/l (p < 0.001)), ESR (33 mm/hr vs 14 mm/hr (p = 0.015)), synovial WBCs (62,247 cells/μl vs 620 cells/μl (p < 0.001)), percentage of synovial neutrophils (93% vs 54% (p < 0.001)), and ANC (55,911 cells/μl vs 326 cells/μl (p < 0.001)) were also significantly higher in infected TKAs. Optimal thresholds at ≤ six weeks were: CRP ≥ 82 mg/l (sensitivity 70%, specificity 77%), synovial WBCs ≥ 8,676 cells/μl (83%, 90%), percentage of synovial neutrophils ≥ 88% (67%, 78%), and ANC ≥ 8,346 cells/μl (83%, 91%). Between six and 12 weeks, thresholds were: CRP ≥ 34 mg/l (90%, 93%), synovial WBCs ≥ 1,983 cells/μl (80%, 85%), percentage of synovial neutrophils ≥ 76% (80%, 81%), and ANC ≥ 1,684 cells/μl (80%, 87%)., Conclusion: Early PJI after TKA should be suspected within six weeks if the CRP is ≥ 82 mg/l, synovial WBCs are ≥ 8,676 cells/μl, the percentage of synovial neutrophils is ≥ 88%, and/or the ANC is ≥ 8,346 cells/μl. Between six and 12 weeks, thresholds include a CRP of ≥ 34 mg/l, synovial WBC of ≥ 1,983 cells/μl, a percentage of synovial neutrophils of ≥ 76%, and/or an ANC of ≥ 1,684 cells/μl. Cite this article: Bone Joint J  2021;103-B(6 Supple A):177-184.

Objective: Human and in vivo animal research implicates inflammation following articular fracture as contributing to post-traumatic arthritis. However, relevant immune cell subsets present following injury are currently undefined. Immunophenotyping human and murine synovial fluid may help to identify immune cell populations that play key roles in the response to articular fracture., Methods: Immunophenotyping by polychromatic flow cytometry was performed on human and mouse synovial fluid following articular fracture. Specimens were collected in patients with closed ankle fracture at the time of surgical fixation and from C57BL/6 mice with closed articular knee fracture. Immune cells were collected from injured and uninjured joints in mice via a novel cell isolation method. Whole blood samples were also collected. Immunohistochemistry (IHC) was performed on mouse synovial tissue to assess for macrophages and T cells., Results: Following intra-articular fracture, the prominent human synovial fluid immune cell subset was CD3+ T cells, containing both CD4+ and CD8+ T cells. In mice, infiltration of CD45+ immune cells in synovial fluid of the fractured limb was dominated by CD19+ B cells and CD3+ T cells at 7 days after intra-articular fracture. We also detected adaptive immune cells, including macrophages, NK cells, dendritic cells and monocytes. Macrophage and T cell findings were supported by IHC of murine synovial tissue., Conclusions: Determining specific cell populations that mediate the immune response is essential to elucidating the chain of events initiated after injury and may be an important step in identifying potential immune signatures predictive of PTA susceptibility or potential therapeutic targets., Competing Interests: Declaration of competing interest The authors have no financial or personal relationships that could potentially influence or bias this work and conclusions., (Copyright © 2021 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.)

Short-chain fatty acids are gut-bacteria-derived metabolites that execute important regulatory functions on adaptive immune responses, yet their influence on inflammation driven by innate immunity remains understudied. Here, we show that propionate treatment in drinking water or upon local application into the joint reduced experimental arthritis and lowered inflammatory tissue priming mediated by synovial fibroblasts. On a cellular level, incubation of synovial fibroblasts with propionate or a physiological mixture of short-chain fatty acids interfered with production of inflammatory mediators and migration and induced immune-regulatory fibroblast senescence. Our study suggests that propionate mediates its alleviating effect on arthritis by direct abrogation of local arthritogenic fibroblast function.

Optical diffraction tomography (ODT) enables imaging of unlabeled intracellular components by measuring the three-dimensional (3D) refractive index (RI). We aimed to detect intracellular monosodium urate (MSU) crystals in synovial leukocytes derived from gout patients using ODT. The 3D RI values of the synthetic MSU crystals, measured by ODT, ranged between 1.383 and 1.440. After adding synthetic MSU crystals to a macrophage, RI tomograms were reconstructed using ODT, and the reconstructed RI tomograms discerned intracellular and extracellular MSU crystals. We observed unlabeled synthetic MSU crystal entry into the cytoplasm of a macrophage through time-lapse imaging. Furthermore, using gout patient-derived synovial leukocytes, we successfully obtained RI tomogram images of intracellular MSU crystals. The 3D RI identification of MSU crystals was verified with birefringence through polarization-sensitive ODT measurements. Together, our results provide evidence that this novel ODT can identify birefringent MSU crystals in synovial leukocytes of patients with gout.

Objective: Juvenile idiopathic arthritis (JIA) is the most common rheumatic disease in childhood. The predominant subtypes, oligoarticular and polyarticular JIA, are traditionally considered to be autoimmune diseases with a central role for T cells and autoantibodies. Mounting evidence suggests an important role for neutrophils in JIA pathogenesis. We undertook this study to investigate the phenotypic features of neutrophils present in the blood and inflamed joints of patients., Methods: JIA synovial fluid (SF) and parallel blood samples from JIA patients and healthy children were collected. SF-treated neutrophils from healthy donors and pleural neutrophils from patients with pleural effusion were investigated as controls for SF exposure and extravasation. Multicolor flow cytometry panels allowed for in-depth phenotypic analysis of neutrophils, focusing on the expression of adhesion molecules, activation, and maturation markers and chemoattractant receptors. Multiplex technology was used to quantify cytokines in plasma and SF., Results: SF neutrophils displayed an activated, hypersegmented phenotype with decreased CD62L expression, up-regulation of adhesion molecules CD66b, CD11b, and CD15, and down-regulation of CXCR1/2. An elevated percentage of CXCR4-positive neutrophils was detected in SF from patients. Pleural neutrophils showed less pronounced maturation differences. Strikingly, significant percentages of SF neutrophils showed a profound up-regulation of atypical neutrophil markers, including CXCR3, intercellular adhesion molecule 1, and HLA-DR., Conclusion: Our data show that neutrophils in inflamed joints of JIA patients have an activated phenotype. This detailed molecular analysis supports the notion that a complex intertwining between these innate immune cells and adaptive immune events drives JIA., (© 2020, American College of Rheumatology.)

Adverse reactions to metal debris in relation to metal-on-metal hip arthroplasty has been heavily discussed in the literature. In contrast, few cases have been reported in the context of total knee arthroplasty. A 77-year-old woman presented with a painful total knee arthroplasty. At the time of revision surgery, intra-articular cream-coloured fluid and material was found in association with a well-fixed prosthesis. Synovial and capsular samples were obtained for histological assessment and a diagnosis of aseptic lymphocytic vasculitis associated lesion was confirmed. The patient went on to have an uncomplicated recovery following a two-stage revision to a constrained knee prosthesis.

The current limitations in evaluating synovial fluid (SF) components in health and disease and between species are due in part to the lack of data on normal SF, because of low availability of SF from healthy articular joints. Our study aimed to quantify species-dependent differences in phospholipid (PL) profiles of normal knee SF obtained from equine and human donors. Knee SF was obtained during autopsy by arthrocentesis from 15 and 13 joint-healthy human and equine donors, respectively. PL species extracted from SF were quantitated by mass spectrometry whereas ELISA determined apolipoprotein (Apo) B-100. Wilcoxon's rank sum test with adjustment of scores for tied values was applied followed by Holm´s method to account for multiple testing. Six lipid classes with 89 PL species were quantified, namely phosphatidylcholine, lysophosphatidylcholine, sphingomyelin, phosphatidylethanolamine, plasmalogen, and ceramide. Importantly, equine SF contains about half of the PL content determined in human SF with some characteristic changes in PL composition. Nutritional habits, decreased apolipoprotein levels and altered enzymatic activities may have caused the observed different PL profiles. Our study provides comprehensive quantitative data on PL species levels in normal human and equine knee SF so that research in joint diseases and articular lubrication can be facilitated., Competing Interests: The authors have declared that no competing interests exist.

This study elucidates the mechanism of CCL25 and CCR9 in rheumatoid arthritis (RA). RA synovial fluid (SF) expresses elevated levels of CCL25 compared to OA SF and plasma from RA and normal. CCL25 was released into RA SF by fibroblasts (FLS) and macrophages (MΦs) stimulated with IL-1β and IL-6. CCR9 is also presented on IL-1β and IL-6 activated RA FLS and differentiated MΦs. Conversely, in RA PBMCs neither CCL25 nor CCR9 are impacted by 3-month longitudinal TNF inhibitor therapy. CCL25 amplifies RA FLS and monocyte infiltration via p38 and ERK phosphorylation. CCL25-stimulated RA FLS secrete potentiated levels of IL-8 which is disrupted by p38 and ERK inhibitors. CCL25 polarizes RA monocytes into nontraditional M1 MΦs that produce IL-8 and CCL2. Activation of p38 and ERK cascades are also responsible for the CCL25-induced M1 MΦ development. Unexpectedly, CCL25 was unable to polarize RA PBMCs into effector Th1/Th17 cells. Consistently, lymphokine like RANKL was uninvolved in CCL25-induced osteoclastogenesis; however, this manifestation was regulated by osteoclastic factors such as RANK, cathepsin K (CTSK), and TNF-α. In short, we reveal that CCL25/CCR9 manipulates RA FLS and MΦ migration and inflammatory phenotype in addition to osteoclast formation via p38 and ERK activation., (© 2021 Wiley-VCH GmbH.)

We have been studying mesenchymal stem cells (MSCs) in synovial fluid and the intra-articular injection of synovial MSCs in osteoarthritis (OA) knees. Here, mainly based on our own findings, we overview the characteristics of endogenous MSCs in the synovial fluid of OA knees and their mode of action when injected exogenously into OA knees. Many MSCs similar to synovial MSCs were detected in the synovial fluid of human OA knees, and their number correlated with the radiological OA grade. Our suspended synovium culture model demonstrated the release of MSCs from the synovium through a medium into a non-contacting culture dish. In OA knees, endogenous MSCs possibly mobilize in a similar manner from the synovium through the synovial fluid and act protectively. However, the number of mobilized MSCs is limited; therefore, OA progresses in its natural course. Synovial MSC injections inhibited the progression of cartilage degeneration in a rat OA model. Injected synovial MSCs migrated into the synovium, maintained their MSC properties, and increased the gene expressions of TSG-6, PRG-4, and BMP-2. Exogenous synovial MSCs can promote anti-inflammation, lubrication, and cartilage matrix synthesis in OA knees. Based on our findings, we have initiated a human clinical study of synovial MSC injections in OA knees.

A previously healthy 53-year-old man was hospitalised for 12 days due to COVID-19 with shortness of breath. A few days after discharge from hospital, the patient developed fever and severe pain in several joints in the lower extremities. The pain was so severe that the patient was unable to stand on his feet. Synovial fluid from the right-side knee contained a high number of polynuclear cells and a few mononuclear cells. Microscopy, culture and PCR tests for bacterial infection were all negative. Furthermore, the patient tested negative for rheumatoid factor, anti-cyclic citrullinated peptide and human leukocyte antigen (HLA)-B27. Thus, the condition was compatible with reactive arthritis. The condition improved markedly after a few days' treatment with non-steroid anti-inflammatory drugs and prednisolone., Competing Interests: Competing interests: None declared., (© BMJ Publishing Group Limited 2021. No commercial re-use. See rights and permissions. Published by BMJ.)

Osteoarthritis (OA) is a significant cause of pain in both humans and horses with a high socio-economic impact. The horse is recognized as a pertinent model for human OA. In both species, regenerative therapy with allogeneic mesenchymal stem cells (MSCs) appears to be a promising treatment but, to date, no in vivo studies have attempted to compare the effects of different cell sources on the same individuals. The objective of this study is to evaluate the ability of a single blinded intra-articular injection of allogeneic bone-marrow (BM) derived MSCs and umbilical cord blood (UCB) derived MSC to limit the development of OA-associated pathological changes compared to placebo in a post-traumatic OA model applied to all four fetlock joints of eight horses. The effect of the tissue source (BM vs. UCB) is also assessed on the same individuals. Observations were carried out using clinical, radiographic, ultrasonographic, and magnetic resonance imaging methods as well as biochemical analysis of synovial fluid and postmortem microscopic and macroscopic evaluations of the joints until Week 12. A significant reduction in the progression of OA-associated changes measured with imaging techniques, especially radiography, was observed after injection of bone-marrow derived mesenchymal stem cells (BM-MSCs) compared to contralateral placebo injections. These results indicate that allogeneic BM-MSCs are a promising treatment for OA in horses and reinforce the importance of continuing research to validate these results and find innovative strategies that will optimize the therapeutic potential of these cells. However, they should be considered with caution given the low number of units per group.

Purpose: To assess whether point-of-care devices designed for collecting cellular components from blood or bone marrow could be used to isolate viable stem cells from synovial fluid., Methods: Male and female patients older than 18 years old with either an acute, anterior cruciate ligament (ACL) injury or knee osteoarthritis (OA) with a minimum estimated 20 mL of knee effusion volunteered. Ten patients with an ACL injury and 10 patients with OA were enrolled. Two milliliters of collected synovial effusion were analyzed and cultured for cellular content. The remaining fluid was combined with whole blood and processed using a buffy-coat based platelet-rich plasma (PRP) processing system. Specimens were analyzed for cell counts, colony-forming unit (CFU) assays, differentiation assays, and flow cytometry., Results: ACL effusion fluid contained 42.1 ± 20.7 CFU/mL and OA effusion fluid contained 65.4 ± 42.1 CFU/mL. After PRP processing, the counts in ACL-PRP were 101.6 ± 66.1 CFU/mL and 114.8 ± 73.4 CFU/mL in the OA-PRP. Cells showed tri-lineage differentiation potential when cultured under appropriate parameters. When analyzed with flow cytometry, >95% of cells produced with culturing expressed cell surface markers typically expressed by known stem cell populations, specifically CD45-, CD73+, CD29+, CD44+, CD105+, and CD90+., Conclusions: Multipotent viable stem cells can be harvested from knee synovial fluid, associated with an ACL injury or OA, and concentrated with a buffy coat-based PRP-processing device., Clinical Relevance: PRP devices can be used to harvest stem cells from effusion fluids. Methods to use effusion fluid associated with an ACL injury and OA should be investigated further., (Copyright © 2020 Arthroscopy Association of North America. Published by Elsevier Inc. All rights reserved.)

Macrophages continuously survey their environment in search of pathogens or apoptotic corpses or debris. Targets intended for clearance expose ligands that initiate their phagocytosis ("eat me" signals), while others avoid phagocytosis by displaying inhibitory ligands ("don't eat me" signals). We report that such ligands can be obscured by the glycosaminoglycans and glycoproteins that coat pathogenic as well as malignant phagocytic targets. In addition, a reciprocal barrier of self-synthesized or acquired glycocalyx components on the macrophage surface shrouds phagocytic receptors, curtailing their ability to engage particles. The coating layers of macrophages and their targets hinder phagocytosis by both steric and electrostatic means. Their removal by enzymatic means is shown to markedly enhance phagocytic efficiency. In particular, we show that the removal of mucins, which are overexpressed in cancer cells, facilitates their clearance. These results shed light on the physical barriers that modulate phagocytosis, which have been heretofore underappreciated. VIDEO ABSTRACT., Competing Interests: Declaration of Interests C.R.B is a co-founder and scientific advisory board member of Lycia Therapeutics, Palleon Pharmaceuticals, Enable Bioscience, Redwood Biosciences (a subsidiary of Catalent), and InterVenn Biosciences, and a member of the board of directors of Eli Lilly & Company., (Crown Copyright © 2020. Published by Elsevier Inc. All rights reserved.)

Objective: To evaluate the role of immune cells and their effector cytokines in the pathogenesis and progression of knee osteoarthritis (OA) in matched OA synovial fluid (SF) and synovial tissue samples., Methods: Cells from matched samples of synovial tissue and SF acquired from individuals undergoing total knee replacement for OA (n = 39) were characterized for immune cell-associated surface markers and intracellular cytokine expression using polychromatic flow cytometry. Additional individuals with radiographic knee OA (Kellgren/Lawrence severity grades ≥1) who had available etarfolatide (inflammatory cell) imaging (n = 26) or baseline and 3-year data on progression of radiographic knee OA (n = 85) were also assessed. SF cytokine concentrations in all cohorts were evaluated for associations with synovial tissue and SF cell phenotypes and severity of radiographic knee OA., Results: Macrophages (predominant in the synovial tissue, 53% of total cells) and neutrophils (predominant in the SF, 26% of total cells) were the major immune cell populations identified in the OA knee joints, exhibiting expression of or association with transforming growth factor β1 (TGFβ1) and elastase, respectively, in the SF. Expression levels of TGFβ1 and elastase were significantly associated with severity of radiographic knee OA. Baseline SF concentrations of TGFβ1 and elastase along with radiographic knee OA severity scores were predictive of knee OA progression, with areas under the receiver operating characteristic curves of 0.810 (for TGFβ1), 0.806 (for elastase), and 0.846 (for both TGFβ1 and elastase combined), with greater stability of prediction when both markers were utilized., Conclusion: Our findings demonstrate the hitherto underappreciated role of neutrophils in the sterile inflammatory process and progression of OA. Two soluble mediators, SF elastase and TGFβ1, are strong predictors of knee OA progression, reflecting a synergistic role of neutrophil and macrophage populations in the pathogenesis and worsening of OA that could potentially be utilized to identify patients who may have a greater risk of more rapid disease progression., (© 2020, American College of Rheumatology.)

Background: The diagnosis of periprosthetic joint infection (PJI) in the early postoperative period remains a challenge. Although studies have established that serum C-reactive protein (CRP) and synovial markers may be useful, recent studies have suggested that the current thresholds used may lack sensitivity. The purpose of this study was to examine the role of serum CRP, erythrocyte sedimentation rate (ESR), synovial fluid white blood-cell (WBC) count, and polymorphonuclear neutrophil (PMN) percentage in the diagnosis of acute postoperative PJI and to identify the optimal threshold., Methods: This multicenter study included patients who were investigated for possible PJI within 90 days of an index arthroplasty. This study included 197 patients from 4 institutions who underwent total joint arthroplasty from 2000 to 2017. Of these patients, 123 were confirmed to have PJI, and 74 were ruled out as not having PJI (non-infected group). Analyses of receiver operating characteristic (ROC) curves and the area under the curve were performed to determine the value of each test and optimal cutoff values., Results: The optimal cutoff value was 6,130 cells/μL for synovial fluid WBC count (91% sensitivity and 83% specificity), 39.8 mg/L for serum CRP (91% sensitivity and 87% specificity), 39.5 mm/hr for ESR (76% sensitivity and 67% specificity), and 79.5% for PMN percentage (95% sensitivity and 59% specificity). Reducing the acute period from 90 days to 30 days or 45 days made little difference in most threshold values. However, the optimal cutoff for synovial fluid WBC count was almost twice as high (10,170 cells/μL) when using a 30-day definition instead of a 90-day definition., Conclusions: The calculated cutoffs in our study were substantially lower than the thresholds used by the Musculoskeletal Infection Society. The calculated values of this study should be used, as previous cutoffs may be too high and lack sensitivity. In addition, it appears that the threshold values, at least for some of the tests, change as the duration since the index arthroplasty lengthens. A continuum of threshold values that is dependent on the number of days since the index arthroplasty may need to be used for the diagnosis of acute PJI., Level of Evidence: Diagnostic Level III. See Instructions for Authors for a complete description of levels of evidence.

There is interest in novel synovial fluid biomarkers for the detection of periprosthetic joint infection (PJI). Here, we assessed the diagnostic accuracy of 23 simple or sophisticated synovial fluid biomarkers for periprosthetic hip or knee infection detection. One hundred seven subjects were studied, 57 of whom had aseptic failure (AF) and 50 PJI. The following synovial fluid biomarkers were tested using spectrophotometric assays, immunoassays, lateral flow tests, or test strips: leukocyte count, monocyte percentage, lymphocyte percentage, neutrophil percentage, C-reactive protein (CRP), glucose, lactate, granulocyte-macrophage colony-stimulating factor, interferon-γ, interleukin-1β (IL-1β), IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12p70, IL-13, IL-17A, IL-23, tumor necrosis factor-α, α-defensin, and leukocyte esterase. The best-performing synovial fluid biomarkers to differentiate PJI from AF-that is, those with highest area under the curve compared to all other biomarkers-were leukocyte count, percent neutrophils and percent monocytes, CRP, and α-defensin (P < .0001)., (© 2020 Orthopaedic Research Society. Published by Wiley Periodicals LLC.)

Background: Mesenchymal stem cells (MSCs) in synovial fluid increase after traumatic meniscus injuries. However, MSC kinetics in synovial fluid may differ for knees with degenerative meniscus injuries. Furthermore, the combination of surgical repair and synovial MSC transplantation has been found to improve clinical symptoms in patients with degenerative meniscus injury, and in this treatment, only the operation procedure without MSC transplantation might increase MSCs in synovial fluid; if so, soluble factors in synovial fluid will be involved. The purpose is this study was to examine whether MSCs exist in synovial fluid of knees with degenerative meniscus injury, to investigate whether MSCs in synovial fluid increase after harvest of synovium and meniscus repair, and to explore what soluble factors in synovial fluids affect the number of MSCs in synovial fluid., Methods: Subjects were 7 patients with degenerative meniscus injury who underwent meniscal repair and synovial MSC transplantation. Synovial fluid (Pre) was aspirated from knees before harvest of synovium and meniscus repair. After 2 weeks, synovial fluid (Post) was aspirated again before transplantation of synovial MSCs. A half volume of the synovial fluid was plated and cultured for 2 weeks to count the colony formation. The other half was used for antibody array analysis, and the correlation coefficients between the signal intensity and colony number were measured in 503 factors. Factors with high correlation coefficients were verified by migration assay., Results: While cell colonies derived from synovial fluid (Pre) were hardly observed, greater numbers of colonies from synovial fluid (Post) were demonstrated. Of the 503 factors, calcitonin gene-related peptide (CGRP) and hepatocyte growth factor (HGF) had high correlation coefficients between colony number and expression level. Both CGRP and HGF promoted migration of synovial fluid MSCs., Conclusions: MSCs in synovial fluid were hardly seen in knees with degenerated meniscus injury. They significantly increased 2 weeks after harvest of synovium and meniscus repair. Both CGRP and HGF in synovial fluid can possibly induce MSCs from synovium into synovial fluid. Graphical abstract.

Background: Synovial fibroblasts (SFs) act as key effector cells mediating synovial inflammation and joint destruction in rheumatoid arthritis (RA). Fibroblast growth factor 2 (FGF2) and its receptors (FGFRs) play important roles in RASF-mediated osteoclastogenesis. Pentraxin 3 (PTX3) is a soluble pattern recognition receptor with nonredundant roles in inflammation and innate immunity. PTX3 is produced by various cell types, including SFs and is highly expressed in RA. However, the role of PTX3 in FGF2-induced osteoclastogenesis in RA and the underlying mechanism have been poorly elucidated., Methods: We first determined the expression of FGF2 and RANKL in synovial tissue and synovial fluid of RA patients. We then examined the effect of PTX3 on RASF osteoclastogenesis induced by endogenous and exogenous FGF2 in isolated RASF cells treated with FGF2 and/or recombinant PTX3 (rPTX3). Thirdly, we analyzed the effect of PTX3 on FGF2 binding to FGFR-1 and HSPG receptors on RASFs. Lastly, we evaluated joint morphology after injection of rPTX3 into collagen-induced arthritis (CIA) mice., Results: FGF2 was confirmed to be highly expressed in both synovial tissue and synovial fluid of RA patients. FGF2 promoted cell proliferation and increased the expressions of RANKL and ICAM-1 and RANKL/OPG to induce osteoclastogenesis in RASF, while anti-FGF2 neutralized this effect. PTX3 significantly inhibited FGF2-induced RASF cell growth and osteoclastogenesis by preventing the interaction of 125 I-FGF2 and FGFRs on the same cells. In addition, administration of rPTX3 significantly ameliorated cartilage and bone destruction in mice with CIA., Conclusions: PTX3 exhibited an inhibitory effect on the autocrine and paracrine stimulation of FGF2 on SFs, and ameliorated bone destruction in CIA mice. PTX3 may be implicated in bone destruction in RA, which may provide theoretical evidence and potential therapeutic targets for RA treatment., (Copyright © 2020 The Author(s). Published by Elsevier Masson SAS.. All rights reserved.)

Joint diseases are conditions with an often progressive and generally painful nature affecting the patient's quality of life and, in some cases, requiring a prompt diagnosis in order to start the treatment urgently. Synovial fluid (SF) laboratory testing is an important part of a diagnostic evaluation of patients with joint diseases. Laboratory testing of SF can provide valuable information in establishing the diagnosis, be a part of a patient's follow-up and treatment with the purpose of improving the patient's health and quality of life. Synovial fluid laboratory testing is rarely performed in Croatian medical biochemistry laboratories. Consequently, procedures for SF laboratory testing are poorly harmonized. This document is the second in the series of recommendations prepared by the members of the Working group for extravascular body fluid samples of the Croatian Society of Medical Biochemistry and Laboratory Medicine. It addresses preanalytical, analytical, and postanalytical issues and the clinical significance of tests used in SF laboratory testing with the aim of improving the value of SF laboratory testing in the diagnosis of joint diseases and assisting in the achievement of national harmonization. It is intended for laboratory professionals and all medical personnel involved in synovial fluid collection and testing., Competing Interests: Potential conflict of interest: None declared., (Croatian Society of Medical Biochemistry and Laboratory Medicine.)

Accumulating evidence suggests that miR-483-3p is implicated in maintaining biological properties in human cancers. However, its biological roles in rheumatoid arthritis (RA) remain unknown. miR-483-3p levels in synovial tissue samples and fibroblast-like synoviocytes (FLSs) were determined using quantitative real-time PCR. The CCK-8 assay and EdU staining were performed to assess cell proliferation in RA FLSs after transfection with miR-483-3p mimics or inhibitor. Flow cytometry with Annexin V-FITC staining or PI staining was performed to assess apoptosis or cell cycle progression in RA FLSs, respectively. miR-483-3p was upregulated in RA, which markedly promoted cell proliferation, induced the G0/G1-to-S phase transition, and suppressed apoptosis in RA FLSs, whereas miR-483-3p silencing yielded opposite results. Moreover, insulin growth factor 1 (IGF-1) was detected as a direct miR-483-3p target. IGF-1 silencing partially restored cell proliferation, the G0/G1-to-S phase transition, and apoptosis suppression in RA FLSs via miR-483-3p inhibition. Our results showed that miR-483-3p promotes RA FLSs proliferation by targeting IGF-1, suggesting a potential strategy for diagnostic and treatment strategy for RA., (Copyright © 2020. Published by Elsevier Masson SAS.)

Resistin is a cysteine-rich adipokine that promotes the release of inflammatory cytokines, particularly interleukin 1 beta (IL-1β) and tumor necrosis factor alpha (TNF-α), which are critical pro-inflammatory mediators in osteoarthritis (OA) pathogenesis. We describe evidence of significantly higher levels of resistin, IL-1β, and TNF-α expression in OA knee synovial tissue compared with that from non-OA knees. Resistin-induced enhancement of IL-1β and TNF-α expression in human OA synovial fibroblasts (OASFs) were attenuated by MEK and ERK inhibitors, as well as their respective siRNAs. Our data reveal that resistin enhances the expression of TNF-α and IL-1β in OASFs by inhibiting miR-149 expression via MEK and ERK signaling. Our findings elucidate the inter-relationships between resistin and pro-inflammatory mediators during OA pathogenesis and could help to facilitate the development of synovium-targeted therapy in OA., (© 2020 Federation of American Societies for Experimental Biology.)

Osteoarthritis (OA) is a chronic joint disease afflicting a substantial portion of the world's population with no currently available cure. Mesenchymal stem cell (MSC)-based therapies have been observed to have a mild beneficial effect in OA but the mechanism behind their action remains unclear. This study aimed to identify the lymphocytic response to a xenogeneic human umbilical cord-derived MSC-based cell therapy. A unilateral medial meniscal release model was employed in an ovine model of post-traumatic OA, with the contralateral limb employed as the control. A dose of 1.0 × 10 7 MSCs was administered to a subset of the OA group as well as to a normal sham-operated group. Synovial fluid was aspirated periodically for 13 weeks for flow cytometry analysis. At the termination of the study the stifle joints were collected and analyzed for potential pathologic changes. Cell therapy induced a transient influx of CD4 + leukocytes; there was a similar significant increase in the proportion of CD4 + CD25 + and CD4 + CD25 hi leukocytes in response to cell therapy, the latter being a subset that may be composed of regulatory T cells. There was no significant effect of the cell therapy treatment on the proportion of synovial fluid-derived CD8 + cells or BAQ44A + B cells. iNOS expression of intimal lining macrophages was evident but reduced in the cell therapy OA group suggesting macrophage phenotype transformation. There were no inflammatory or histological changes that could be attributed to the cell therapy. Cell therapy induced chemotaxis of CD4 + cells to the joint but these cells were not associated with pathological changes, despite their expression of activation markers (CD25 + )., (Copyright © 2020. Published by Elsevier B.V.)

BACKGROUND Rheumatoid arthritis (RA) is an inflammatory disorder that is present in approximately 1% of the world's population. This study was aimed to investigate the effect of retinoic acid-platinum (II) complex [RT-Pt(II)] on rheumatoid arthritis (RA) and to explore the mechanism involved. MATERIAL AND METHODS MH7A cell viability was determined by MTT assay and apoptosis was assessed using FACSCalibur flow cytometry. RT-PCR and Western blot assays were used for assessment of mRNA and proteins levels. RESULTS Treatment of rheumatoid arthritis with RT-Pt(II) significantly reduced the levels of IL‑1ß, IL-6, IL-8, MMP-1, and MMP-13 in synovial fluid of mice in a dose-dependent manner. The expression of iNOS and COX-2 mRNA and protein in rheumatoid arthritis rats was also significantly inhibited by treatment with RT-Pt(II). The TNF-alpha-induced proliferation of MH7A cells was alleviated by RT-Pt(II) treatment in a concentration-dependent manner. Moreover, RT-Pt(II) treatment induced apoptosis and caused arrest of cell cycle in MH7A cells. The activation of MEK/NF-kappaB pathway was downregulated by RT-Pt(II) treatment in MH7A cells. CONCLUSIONS In summary, the present study demonstrated that RT-Pt(II) inhibits TNF-alpha-induced inflammatory response, suppresses cell viability, and induces apoptosis in rheumatoid arthritis synovial cells. Moreover, RT-Pt(II) exhibited its effect through targeting the MEK/NF-kappaB pathway. Therefore, RT-Pt(II) can be used for the development of treatments for rheumatoid arthritis.

Background: Rheumatoid arthritis, like many inflammatory diseases, is characterized by episodes of quiescence and exacerbation (flares). The molecular events leading to flares are unknown., Methods: We established a clinical and technical protocol for repeated home collection of blood in patients with rheumatoid arthritis to allow for longitudinal RNA sequencing (RNA-seq). Specimens were obtained from 364 time points during eight flares over a period of 4 years in our index patient, as well as from 235 time points during flares in three additional patients. We identified transcripts that were differentially expressed before flares and compared these with data from synovial single-cell RNA-seq. Flow cytometry and sorted-blood-cell RNA-seq in additional patients were used to validate the findings., Results: Consistent changes were observed in blood transcriptional profiles 1 to 2 weeks before a rheumatoid arthritis flare. B-cell activation was followed by expansion of circulating CD45-CD31-PDPN+ preinflammatory mesenchymal, or PRIME, cells in the blood from patients with rheumatoid arthritis; these cells shared features of inflammatory synovial fibroblasts. Levels of circulating PRIME cells decreased during flares in all 4 patients, and flow cytometry and sorted-cell RNA-seq confirmed the presence of PRIME cells in 19 additional patients with rheumatoid arthritis., Conclusions: Longitudinal genomic analysis of rheumatoid arthritis flares revealed PRIME cells in the blood during the period before a flare and suggested a model in which these cells become activated by B cells in the weeks before a flare and subsequently migrate out of the blood into the synovium. (Funded by the National Institutes of Health and others.)., (Copyright © 2020 Massachusetts Medical Society.)

Objective: The NLRP3 inflammasome is closely linked to the pathophysiology of a wide range of inflammatory diseases. This study was undertaken to identify small molecules that directly bind to NLRP3 in order to develop pharmacologic interventions for NLRP3-related diseases., Methods: A structure-based virtual screening analysis was performed with ~62,800 compounds to select efficient NLRP3 inhibitors. The production of caspase 1-p10 and interleukin-1β (IL-1β) was measured by immunoblotting and enzyme-linked immunosorbent assay to examine NLRP3 inflammasome activation. Two gouty arthritis models and an air pouch inflammation model induced by monosodium urate monohydrate (MSU) crystal injection were used for in vivo experiments. Primary synovial fluid cells from gout patients were used to determine the relevance of NLRP3 inflammasome inhibition in human gout., Results: Beta-carotene (provitamin A) suppressed the NLRP3 inflammasome activation induced by various activators, including MSU crystals, in mouse bone marrow-derived primary macrophages (P < 0.05). Surface plasmon resonance analysis demonstrated the direct binding of β-carotene to the pyrin domain (PYD) of NLRP3 (K D = 3.41 × 10 -6 ). Molecular modeling and mutation assays revealed the interaction mode between β-carotene and the NLRP3 PYD. Inflammatory symptoms induced by MSU crystals were attenuated by oral administration of β-carotene in gouty arthritis mouse models (P < 0.05), correlating with its suppressive effects on the NLRP3 inflammasome in inflamed tissues. Furthermore, β-carotene reduced IL-1β secretion from human synovial fluid cells isolated from gout patients (P < 0.05), showing its inhibitory efficacy in human gout., Conclusion: Our results present β-carotene as a selective and direct inhibitor of NLRP3, and the binding of β-carotene to NLRP3 PYD as a novel pharmacologic strategy to combat NLRP3 inflammasome-driven diseases, including gouty arthritis., (© 2020, American College of Rheumatology.)

Aims: In patients with a "dry" aspiration during the investigation of prosthetic joint infection (PJI), saline lavage is commonly used to obtain a sample for analysis. The aim of this study was to investigate prospectively the impact of saline lavage on synovial fluid analysis in revision arthroplasty., Methods: Patients undergoing revision hip (THA) or knee arthroplasty (TKA) for any septic or aseptic indication were enrolled. Intraoperatively, prior to arthrotomy, the maximum amount of fluid possible was aspirated to simulate a dry tap (pre-lavage) followed by the injection with 20 ml of normal saline and re-aspiration (post-lavage). Pre- and post-lavage synovial white blood cell (WBC) count, percent polymorphonuclear cells (%PMN), and cultures were compared., Results: A total of 78 patients had data available for analysis; 17 underwent revision THA and 61 underwent revision TKA. A total of 16 patients met modified Musculoskeletal Infection Society (MSIS) criteria for PJI. Pre- and post-lavage %PMNs were similar in septic patients (87% vs 85%) and aseptic patients (35% vs 39%). Pre- and post-lavage synovial fluid WBC count were far more disparate in septic (53,553 vs 8,275 WBCs) and aseptic (1,103 vs 268 WBCs) cohorts. At a cutoff of 80% PMN, the post-lavage aspirate had a sensitivity of 75% and specificity of 95%. At a cutoff of 3,000 WBCs, the post-lavage aspirate had a sensitivity of 63% and specificity of 98%. As the post-lavage synovial WBC count increased, the difference between pre- and post-lavage %PMN decreased (mean difference of 5% PMN in WBC < 3,000 vs mean difference 2% PMN in WBC > 3,000, p = 0.013). Of ten positive pre-lavage fluid cultures, only six remained positive post-lavage., Conclusion: While saline lavage aspiration significantly lowered the synovial WBC count, the %PMN remained similar, particularly at WBC counts of > 3,000. These findings suggest that in patients with a dry-tap, the %PMN of a saline lavage aspiration has reasonable sensitivity (75%) for the detection of PJI. Cite this article: Bone Joint J 2020;102-B(6 Supple A):138-144.