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Cell selectivity in succinate receptor SUCNR1/GPR91 signaling in skeletal muscle.
Succinate is released by skeletal muscle during exercise and activates SUCNR1/GPR91. Signaling of SUCNR1 is involved in metabolite-sensing paracrine communication in skeletal muscle during exercise. However, the specific cell types responding to succinate and the directionality of communication are unclear. We aim to characterize the expression of SUCNR1 in human skeletal muscle. De novo analysis of transcriptomic datasets demonstrated that SUCNR1 mRNA is expressed in immune, adipose, and liver tissues, but scarce in skeletal muscle. In human tissues, SUCNR1 mRNA was associated with macrophage markers. Single-cell RNA sequencing and fluorescent RNAscope demonstrated that in human skeletal muscle, SUCNR1 mRNA is not expressed in muscle fibers but coincided with macrophage populations. Human M2-polarized macrophages exhibit high levels of SUCNR1 mRNA and stimulation with selective agonists of SUCNR1 triggered Gq- and Gi-coupled signaling. Primary human skeletal muscle cells were unresponsive to SUCNR1 agonists. In conclusion, SUCNR1 is not expressed in muscle cells and its role in the adaptive response of skeletal muscle to exercise is most likely mediated via paracrine mechanisms involving M2-like macrophages within the muscle.NEW & NOTEWORTHY Macrophages but not skeletal muscle cells respond to extracellular succinate via SUCNR1/GPR91.
GM-CSF Primes Proinflammatory Monocyte Responses in Ankylosing Spondylitis.
Objectives: GM-CSF is a pro-inflammatory cytokine with multiple actions predominantly on myeloid cells. Enhanced GM-CSF expression by lymphocytes from patients with Ankylosing Spondylitis (AS) has recently been described, however, its potential pathogenic role(s) in AS are unknown. Methods: The effects of GM-CSF on TNF, IL-23, and CCL17 production by blood, PBMCs and isolated CD14+ monocytes from AS patients and healthy controls (HCs) were studied using ELISA. Serum CCL17 and GM-CSF and T cell GM-CSF production were studied in AS patients including pre-and on TNFi therapy. Results: GM-CSF markedly increased TNF production by LPS-stimulated whole blood, peripheral blood mononuclear cells (PBMC) and purified monocytes from AS patients, with 2 h GM-CSF exposure sufficient for monocyte "priming." Blocking of GM-CSF significantly reduced the production of TNF by whole blood from AS patients but not HCs. GM-CSF priming increased IL-23 production from LPS-stimulated AS and HC whole blood 5-fold, with baseline and stimulated IL-23 levels being significantly higher in AS whole blood. GM-CSF also stimulated CCL17 production from AS and HC blood and CCL17 levels were elevated in AS plasma. GM-CSF could be detected in plasma from 14/46 (30%) AS patients compared to 3/18 (17%) HC. Conclusion: We provide evidence that GM-CSF primes TNF and IL-23 responses in myeloid cells from AS patients and HC. We also show CCL17 levels, downstream of GM-CSF, were elevated in plasma samples of AS patients. Taken together these observations are supportive of GM-CSF neutralization as a potential novel therapeutic approach for the treatment of AS.
Genome-wide classification of epigenetic activity reveals regions of enriched heritability in immune-related traits.
Epigenetics underpins the regulation of genes known to play a key role in the adaptive and innate immune system (AIIS). We developed a method, EpiNN, that leverages epigenetic data to detect AIIS-relevant genomic regions and used it to detect 2,765 putative AIIS loci. Experimental validation of one of these loci, DNMT1, provided evidence for a novel AIIS-specific transcription start site. We built a genome-wide AIIS annotation and used linkage disequilibrium (LD) score regression to test whether it predicts regional heritability using association statistics for 176 traits. We detected significant heritability effects (average |τ∗|=1.65) for 20 out of 26 immune-relevant traits. In a meta-analysis, immune-relevant traits and diseases were 4.45× more enriched for heritability than other traits. The EpiNN annotation was also depleted of trans-ancestry genetic correlation, indicating ancestry-specific effects. These results underscore the effectiveness of leveraging supervised learning algorithms and epigenetic data to detect loci implicated in specific classes of traits and diseases.
The outcomes of acute periprosthetic joint infection following unicompartmental knee replacement managed with early debridement, Antibiotics, and implant retention.
BACKGROUND: Periprosthetic joint infection (PJI) following unicompartmental knee replacement (UKR) is an uncommon, yet serious, complication. There is a paucity of evidence regarding the effectiveness of Debridement-Antibiotics-and-Implant-Retention (DAIR) in this setting. The aim of this study is to investigate the effectiveness of DAIR for acute UKR PJI. METHOD: Between 2006 and 2019, 5195 UKR were performed at our institution. Over this period, sixteen patients underwent DAIR for early, acute PJI. All patients met MSIS PJI diagnostic criteria. The median age at DAIR was 67 years (range 40-73) and 12 patients were male (75.0%). The median time to DAIR was 24 days (range 6-60). Patients were followed up for a median of 6.5 years (range1.4-10.5) following DAIR. RESULTS: 0.3% (16/5195) of UKR in our institution had a DAIR within 3 months. 15 of 16 patients (93.8%) were culture positive, with the most common organism MSSA (n = 8, 50.0%). Patients were treated with an organism-specific intravenous antibiotic regime for a median of 6 weeks, followed by oral antibiotics for a median duration of 6 months. The Kaplan-Meier survivor estimate for revision for PJI was 57% (95%CI: 28-78%) at five years, and survivor estimate for all cause revision 52% (95%CI: 25-74%).The median Oxford Knee Score for patients with a viable implant at final follow-up was 45 points (range 39-46). CONCLUSION: Early, acute PJI after UKR is rare. DAIR had a moderate success rate, with infection-free survivorship of 57% at 5 years. Those successfully treated with DAIR had excellent functional outcome and implant survival.
Radiology reporting of incidental osteoporotic vertebral fragility fractures present on CT studies: results of UK national re-audit.
AIM: To describe a UK-wide re-audit of the 2019 Royal College of Radiologists (RCR) audit evaluating patient-related data and organisational infrastructure in the radiological reporting of vertebral fragility fractures (VFFs) on computed tomography (CT) studies and to assess the impact of a series of RCR interventions, initiated to raise VFF awareness, on reporting practice and outcomes. MATERIALS AND METHODS: Patient specific and organisational questionnaires largely replicated those utilised in 2019. The patient questionnaire involved retrospective analysis of between 50 and 100 consecutive, non-traumatic CT studies which included the thoracolumbar spine. All RCR radiology audit leads were invited to participate. Data collection commenced from 1 April 2022. RESULTS: Data were supplied by 129/194 (67%) departments. One thousand five hundred and eighty-six of 7,316 patients (21.7%) had a VFF on auditor review. Overall improvements were demonstrated in key initial/provisional reporting results; comment on spine/bone (93.2%, 14.4% improvement, p<0.0002); fracture severity assessment (34.7%, 8.5% improvement, p=0.0007); use of recommended terminology (67.8%, 7.5% improvement, p=0.0034); recommendations for further management (11.7%, 9.1% improvement, p<0.0002). CONCLUSIONS: The 2022 national re-audit confirms improvements in diagnostic performance and practice in VFF reporting. Continuing work is required to build on this improvement and to further embed best practice.
Chimeric antigens displaying GPR65 extracellular loops on a soluble scaffold enabled the discovery of antibodies, which recognized native receptor.
GPR65 is a proton-sensing G-protein coupled receptor associated with multiple immune-mediated inflammatory diseases, whose function is relatively poorly understood. With few reagents commercially available to probe the biology of receptor, generation of an anti-GPR65 monoclonal antibody was desired. Using soluble chimeric scaffolds, such as ApoE3, displaying the extracellular loops of GPR65, together with established phage display technology, native GPR65 loop-specific antibodies were identified. Phage-derived loop-binding antibodies recognized the wild-type native receptor to which they had not previously been exposed, generating confidence in the use of chimeric soluble proteins to act as efficient surrogates for membrane protein extracellular loop antigens. This technique provides promise for the rational design of chimeric antigens in facilitating the discovery of specific antibodies to GPCRs.
Establishment of a Triple Quadrupole HPLC-MS Quantitation Method for Dystrophin Protein in Mouse and Human Skeletal Muscle.
Duchenne muscular dystrophy (DMD) is the most common type of neuromuscular disease caused by mutations in the DMD gene encoding dystrophin protein. To quantitively assess human dystrophin protein in muscle biopsy samples, it is imperative to consistently detect as low as 0.003% of the dystrophin protein relative to the total muscle protein content. The quantitation of dystrophin protein has traditionally been conducted using semiquantitative immunoblotting or immunohistochemistry; however, there is a growing need to establish a more precise quantitative method by employing liquid chromatography-mass spectrometry (LC-MS) to measure dystrophin protein. In this study, a novel quantification method was established using a mouse experiment platform applied to the clinical quantification of human dystrophin protein. The method using a spike-in approach with a triple quadrupole LC-MS quantitated the amount of dystrophin in wild-type and human DMD transgenic mice but not in DMD-null mice. In conclusion, we established a quantitating method of dystrophin using HPLC-LC-MS with a novel spike-in approach. These results indicate that our methodology could be applied to several LC-MS devices to enable the accurate measurement of dystrophin protein in patients with DMD.