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The success of rotator cuff tendon repair relies on both tendon healing and muscle recovery. The objective of this descriptive study was to investigate the regenerative potential of the supraspinatus muscle in rotator cuff tear conditions by quantifying the expression of Pax7, MyoD, and myogenin, basic factors that regulate myogenesis. Muscle biopsies were collected from thirty-three patients aged 34 to 73 years who underwent surgery for a rotator cuff tear affecting the supraspinatus muscle. Among these patients, twenty-seven percent were women, and the age of the lesions ranged from 2 to 72 months post-initial trauma. Biopsies were harvested from the supraspinatus muscle at the end closest to the tendon, and control biopsies were harvested from the ipsilateral deltoid muscle. The densities of immunohistochemically stained Pax7 + , MyoD + , and myogenin + nuclei/mm 2 were used to estimate the myogenic potential of the muscle. Adjustments were made for patient age and lesion age. We found increased density of MyoD + and myogenin + cells in supraspinatus muscles compared to deltoid muscles ( p < 0.001 and p = 0.003, respectively). Regression analyses that combined the density of positive nuclei with patient age showed a continuous increase in Pax7 with age but also a reduction of MyoD and myogenin in older patients. When combined with lesion age, there was a decline in the density of all myogenic markers after an initial rise. Pax7 density continued to be higher in supraspinatus compared to the deltoid muscle, but the density of MyoD and myogenin terminally dropped to a density lower than in the deltoid. Our findings suggest that the supraspinatus muscle in tear conditions showed signs of initial activation of muscle regeneration. When compared to the unaffected deltoid muscle, an apparent reduction in capacity to progress to full muscle fiber maturity was also demonstrated. This pattern of inhibited myogenesis seemed to increase with both patient age and lesion age. Our results on muscle regenerative capacity indicate that younger patients with rotator cuff tears have better chances of muscle recovery and may benefit from early surgical reconstruction.

Background: Rotator cuff tears (RCT) are a common musculoskeletal condition, especially in the aging population. The prevalence of rotator cuff tears varies based on factors like age, occupation, and activity level. In the general population, the prevalence of rotator cuff tears is estimated to be around 20 to 25%. Rotator cuff tears (RCT) have an impact in patients' pain level, shoulder function, sleep disturbance, and quality of life. Primary tendon surgery is in mostly cases necessary. This study aimed to examine if treatment of rotator cuff lesions with implantation of micro-fragmented adipose tissue can improve patients' reported pain and function compared to conventional surgery., Methods: The study is a prospective superiority parallel-group single-center randomized controlled trial including 30 patients between 40 and 69 years of age in Denmark. Patients will be allocated 1:1 ratio to reconstruction of the supraspinatus tendon with an injection of micro-fragmented adipose tissue into the related muscle (stem cell treatment) or the standard of care (SOC), which is conventional surgery. Patients, project assistants, physicians, and outcome adjudicators are not blinded to randomization due to practical constraints. The radiologist and the statistician performing the analysis will be blinded. The primary outcome will be the Oxford shoulder score at 12 months post-surgery., Discussion: This study will assess whether adding micro-fragmented adipose tissue therapy to conventional rotator cuff tear treatment can enhance recovery, accelerate return to daily activities, and improve functional outcomes. The research will also determine if this minimally invasive procedure could be standardized for routine patient care., Trial Registration: ClinicalTrials.gov NCT06505135. Registered on July 10, 2024., (© 2024. The Author(s).)

Myositis is associated with reduced quality of life, which is accompanied by significant impairments in muscle endurance and strength, altogether representing cardinal traits in patients with myositis. This randomised controlled trial aimed to investigate the effect of high-intensity resistance training on quality of life in patients with myositis. Thirty-two patients with established, stable myositis were randomised to 16 weeks of high-intensity resistance training (intervention group) or 16 weeks of usual care (control group). Primary outcome was quality of life assessed as the change in the physical component summary score (PCS) of the Short Form-36 health questionnaire from baseline to post-intervention. Secondary outcomes included functional capacity measures, such as functional index 3, and International Myositis Assessment and Clinical Studies Group (IMACS) disease activity and damage core set measures, including manual muscle testing 8 (MMT8). The primary outcome PCS showed an improvement in favour of high-intensity resistance training with a between-group difference of 5.33 (95% CI 0.61; 10.05) (p = 0.03). Additionally, functional index 3 showed a between-group difference indicating greater gains with high-intensity resistance training 11.49 (95% CI 3.37; 19.60) (p = 0.04), along with a between-group improvement in MMT8 1.30 (95% CI 0.09; 2.51) (p = 0.04). High-intensity resistance training for 16 weeks effectively improved quality of life in patients with myositis. Clinical measures of muscle endurance and muscle strength were also found to improve with high-intensity resistance training, while patients stayed in disease remission. Consequently, progressively adjusted high-intensity resistance training is feasible and causes no aggravation of the disease, while benefitting patients with myositis.Clinical trial registration: Clinicaltrials.gov ID: NCT04486261- https://clinicaltrials.gov/study/NCT04486261 ., (© 2024. The Author(s).)

Sporadic inclusion body myositis (sIBM) is a subgroup of idiopathic inflammatory myopathies characterised by progressive muscle weakness and skeletal muscle inflammation. Quantitative data on the myofibre morphology in sIBM remains scarce. Further, no previous study has examined fibre type association of satellite cells (SC), myonuclei number, macrophages, capillaries, and myonuclear domain (MD) in sIBM patients. Muscle biopsies from sIBM patients (n = 18) obtained previously (NCT02317094) were included in the analysis for fibre type-specific myofibre cross-sectional area (mCSA), SCs, myonuclei and macrophages, myonuclear domain, and capillarisation. mCSA (p < 0.001), peripheral myonuclei (p < 0.001) and MD (p = 0.005) were higher in association with type 1 (slow-twitch) than type 2 (fast-twitch) fibres. Conversely, quiescent SCs (p < 0.001), centrally placed myonuclei (p = 0.03), M1 macrophages (p < 0.002), M2 macrophages (p = 0.013) and capillaries (p < 0.001) were higher at type 2 fibres compared to type 1 fibres. In contrast, proliferating (Pax7 + /Ki67 + ) SCs (p = 0.68) were similarly associated with each fibre type. Type 2 myofibres of late-phase sIBM patients showed marked signs of muscle atrophy (i.e. reduced mCSA) accompanied by higher numbers of associated quiescent SCs, centrally placed myonuclei, macrophages and capillaries compared to type 1 fibres. In contrast, type 1 fibres were suffering from pathological enlargement with larger MDs as well as fewer nuclei and capillaries per area when compared with type 2 fibres. More research is needed to examine to which extent different therapeutic interventions including targeted exercise might alleviate these fibre type-specific characteristics and countermeasure their consequences in impaired functional performance., (© 2024. The Author(s).)

Effective therapeutics are necessary for managing severe COVID-19 disease despite the availability of vaccines. Small interfering RNA (siRNA) can silence viral genes and restrict SARS-CoV-2 replication. Cell-penetrating peptides is a robust method for siRNA delivery, enhancing siRNA stability and targeting specific receptors. We developed a peptide HE25 that blocks SARS-CoV-2 replication by various mechanisms, including the binding of multiple receptors involved in the virus's internalization, such as ACE2, integrins and NRP1. HE25 not only acts as a vehicle to deliver the SARS-CoV-2 RNA-dependent RNA polymerase siRNA into cells but also facilitates their internalization through endocytosis. Once inside endosomes, the siRNA is released into the cytoplasm through the Histidine-proton sponge effect and the selective cleavage of HE25 by cathepsin B. These mechanisms effectively inhibited the replication of the ancestral SARS-CoV-2 and the Omicron variant BA.5 in vitro . When HE25 was administered in vivo , either by intravenous injection or inhalation, it accumulated in lungs, veins and arteries, endothelium, or bronchial structure depending on the route. Furthermore, the siRNA/HE25 complex caused gene silencing in lung cells in vitro . The SARS-CoV-2 siRNA/HE25 complex is a promising therapeutic for COVID-19, and a similar strategy can be employed to combat future emerging viral diseases., Competing Interests: The authors declare no competing interests., (© 2024 The Author(s).)

Objective: To investigate the potential associations between functional capacity, muscle strength, body composition, and disease-related measures and quality of life in patients with myositis., Methods: Baseline measures of functional capacity (functional index 3 (FI3), 2-minute walk test (2MWT), timed up and go (TUG) and 30-s sit-to-stand (30-STS)), muscle strength (incl. leg and handgrip strength), maximal leg extensor power, body composition (appendicular lean mass, fat percentage/mass) and disease-related measures (disease activity & damage core sets) were examined for their associations with quality of life (physical- and mental component summary scores, Short Form 36 questionnaire (SF-36)) by means of Spearman's correlation analysis., Results: A total of 32 patients with myositis were included. Positive correlations between SF-36 physical component summary score (PCS) and FI3, 30-STS, TUG, 2MWT, leg extensor power, leg strength, bench press strength, and handgrip strength were observed. In contrast, fat percentage and fat mass correlated negatively with PCS. In disease-related measures, Extramuscular global assessment, health assessment questionnaire, physician global damage, and patient global damage scores were negatively associated with SF-36 PCS. No correlations to the mental component summary score of SF-36 were observed., Conclusion: All measures of functional capacity were positively related to the SF-36 physical component summary score, indicating higher functional capacity positively affects quality of life in patients with myositis. Health assessment questionnaire and patient global damage scores demonstrated the strongest correlations with SF-36 physical component summary scores, further supporting these patient-reported outcomes as viable monitoring tools in patients with myositis., (© 2024 The Authors. International Journal of Rheumatic Diseases published by Asia Pacific League of Associations for Rheumatology and John Wiley & Sons Australia, Ltd.)

Lymphedema is a common complication following breast cancer treatment with axillary lymphadenectomy and radiotherapy. Currently, there is no curative treatment for this disease, hence there is a need for new therapeutic suggestions. The aim of this study was to investigate the effect of hyaluronidase (HYAL) injections after inducing hindlimb lymphedema in 36 female C57BL/6 mice. HYAL injections were administered every second day for 14 days in three groups: (1) HYAL for 1 week followed by saline for 1 week, (2) HYAL for 2 weeks, and (3) saline injections for 2 weeks. Volume of the lymphedema limb was weekly assessed with micro-computed tomography (μ-CT) scans for a total course of 6 weeks. Lymph vessel morphometry was assessed in the end of the study after staining cross-sections of the hindlimb for anti-LYVE-1 blindly. Lymphatic function was assessed by lymphoscintigraphy to assess lymphatic clearance. There was a significant reduction of the volume of lymphedema in mice treated with HYAL-7 compared with mice treated with HYAL-14 (p < 0.05) and saline (p < 0.05). No differences were detected in lymph vessel morphometry and the lymphoscintigraphy between groups. Short-term treatment with HYAL-7 might be a potential therapeutic suggestion for secondary lymphedema induced in mouse hindlimbs. In the future, clinical studies are needed to investigate the potential of HYAL treatment in human beings.

Background: Sporadic late onset nemaline myopathy is a rare, progressive muscle disease, presenting in adulthood, mainly affecting proximal limb and bulbar muscles. Muscle biopsies show characteristic nemaline rods. The putative mechanism is considered immune-related. Other manifestations aside from neuromuscular symptoms have not been described previously., Case Presentation: We present a case with atypical sporadic late onset nemaline myopathy (SLONM) of a non-HIV, non-MGUS subtype, where skin manifestations preceded neuromuscular symptoms, and a residual thymus with the histology of thymic follicular hyperplasia was detected during the diagnostic workup. Thorough dermatological investigations could not explain the skin presentations. Muscle biopsy revealed variation in fiber diameter, ragged-red and COX-negative fibers associated with discrete fibrosis. Electron microscopy detected atrophic muscle fibres with disorganization of the myofibrils, nemaline rods and abnormal mitochondria. Single-fiber EMG suggested signs of a neuromuscular transmission defect, EMG showed signs of myopathy. Analyses of antibodies associated with myasthenia gravis were negative. The patient showed improvement after intravenous immunoglobulin treatment regarding both the skin and the muscle symptoms., Conclusions: Our case highlights the heterogeneity of SLONM with its varied spectrum of presentation. A unique combination of dermatological symptoms and SLONM could be seen with skin lesions as primary presenting symptoms. An association can be considered between the different manifestations, presumably based on immune etiology, where immunosuppressive therapy has been beneficial., (© 2023. The Author(s).)

Objective: To describe neurophysiological abnormalities in Long COVID and correlate quantitative electromyography (qEMG) and single fiber EMG (sfEMG) results to clinical scores and histopathology., Methods: 84 patients with non-improving musculoskeletal Long COVID symptoms were examined with qEMG and sfEMG. Muscle biopsies were taken in a subgroup., Results: Mean motor unit potential (MUP) duration was decreased in ≥ 1 muscles in 52 % of the patients. Mean jitter was increased in 17 % of the patients in tibialis anterior and 25 % in extensor digitorum communis. Increased jitter was seen with or without myopathic qEMG. Low quality of life score correlated with higher jitter values but not with qEMG measures. In addition to our previously published mitochondrial changes, inflammation, and capillary injury, we show now in muscle biopsies damage of terminal nerves and motor endplate with abundant basal lamina material. At the endplate, axons were present but no vesicle containing terminals. The post-synaptic cleft in areas appeared atrophic with short clefts and coarse crests., Conclusions: Myopathic changes are common in Long COVID. sfEMG abnormality is less common but may correlate with clinical scores. sfEMG changes may be due to motor endplate pathology., Significance: These findings may indicate a muscle pathophysiology behind fatigue in Long COVID., (Copyright © 2023 International Federation of Clinical Neurophysiology. Published by Elsevier B.V. All rights reserved.)

Disease causing variants in the Ryanodine receptor 1 (RYR1) gene are a common cause for congenital myopathy and for malignant hyperthermia susceptibility. We report a 17 year old boy with congenital muscle weakness progressing to a myasthenia like myopathy with muscle weakness, fatigability, ptosis, and ophthalmoplegia. Muscle biopsy showed predominance and atrophy of type 1 fibers. Whole-exome trio sequencing revealed three variants in the RYR1-gene in the patient: c.6721C > T,p.(Arg2241*) and c.2122G > A,p.(Asp708Asn) in cis position, and the c.325C > T,p.(Arg109Trp) variant in trans. Treatment with pyridostigmine improved symptoms. This case supports that a myasthenia like phenotype is part of the phenotypic spectrum of RYR1 related disorders, and that treatment with pyridostigmine can be beneficial for patients with this phenotype., Competing Interests: Declaration of competing interest There exist no conflicts of interest., (Copyright © 2023. Published by Elsevier Masson SAS.)

Introduction:  Treatment for long-gap esophageal atresia (LGEA) aims at achieving primary anastomosis with minimal tension. Previous studies have shown that intramural injections with botulinum toxin type-A (BTX-A) from the adventitial side can increase the elongation of the piglet and rat esophagus before bursting, and that this effect is dose and time dependent. Our aim was to determine if endoscopic injections would be feasible, safe, and with an effect on the mechanical properties of the esophagus., Methods:  Twenty-two male piglets (5.15 kg) were randomized into two groups, one receiving 2 units/kg BTX-A, the other equal volume 0.9% NaCl. On day 3, the esophagus was harvested and tested in a stretch-tension machine to evaluate elongation and maximum load, followed by histological examination., Results:  No adverse effects to the procedure were observed. No statistically significant difference in elongation or maximum load before bursting between the treatment and placebo group was found. In histopathological analysis, inflammation and abscess formation were observed with no statistically significant difference between the two groups., Conclusion:  Endoscopic placement of BTX-A injections in the piglet esophagus was safe and feasible but did not result in any difference in the mechanical properties or histology of the esophagus., Competing Interests: None declared., (Thieme. All rights reserved.)

Objectives: To investigate Stone Heart Syndrome (SHS) as consequence of prolonged ischemic arrest in an experimental study on pigs in regards to onset of SHS and pathological changes. Outcomes defined as aortic cross clamp (ACC) time until onset of SHS and cellular changes characterized by SHS., Methods: Eight pigs were included to undergo normothermic cardioplegia induced cardiac arrest ranging from 80 to 240 minutes of ACC. Duration of ACC was defined as time from initiation of aortic cross clamping until cessation. Normothermic, cardioplegic solution administered directly into the arterial system, though in a reduced dose compared to clinical practice. Myocardial contracture evaluated by palpation of the myocardium. Biopsies were collected from the left ventricle just after the induction of cardiac arrest and after reperfusion. Biopsies were evaluated for pathological changes indicative of SHS by electron microscopy., Results: Six pigs completed the full trial, while two were lost to bleeding. Pigs undergoing 80 to 120 minutes of ACC regained heart rhythm either spontaneously or after defibrillation. Pigs undergoing more than 180 minutes of ACC had contracted hearts with no electrocardiographic response indicating the development of SHS. Electron microscopy findings after ACC of 80 to 120 minutes showed no or low degrees of cellular changes, whereas pig hearts with more than 180 minutes of ACC showed severe mitochondrial changes, endothelial damage, and shortening of sarcomeres consistent with SHS., Conclusion: Development of SHS in pigs was ACC time dependent and solely avoided when ACC was limited to a maximum of 120 minutes., Competing Interests: Conflict of interest The authors declare no conflict of interest., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background and Purpose: Among post-COVID-19 symptoms, fatigue is reported as one of the most common, even after mild acute infection, and as the cause of fatigue, myopathy diagnosed by electromyography has been proposed in previous reports. This study aimed to explore the histopathological changes in patients with post-COVID-19 fatigue., Methods: Sixteen patients (mean age = 46 years) with post-COVID-19 complaints of fatigue, myalgia, or weakness persisting for up to 14 months were included. In all patients, quantitative electromyography and muscle biopsies analyzed with light and electron microscopy were taken., Results: Muscle weakness was present in 50% and myopathic electromyography in 75%, and in all patients there were histological changes. Muscle fiber atrophy was found in 38%, and 56% showed indications of fiber regeneration. Mitochondrial changes, comprising loss of cytochrome c oxidase activity, subsarcollemmal accumulation, and/or abnormal cristae, were present in 62%. Inflammation was found in 62%, seen as T lymphocytes and/or muscle fiber human leukocyte antigen ABC expression. In 75%, capillaries were affected, involving basal lamina and cells. In two patients, uncommon amounts of basal lamina were found, not only surrounding muscle fibers but also around nerves and capillaries., Conclusions: The wide variety of histological changes in this study suggests that skeletal muscles may be a major target of SARS-CoV-2, causing muscular post-COVID-19 symptoms. The mitochondrial changes, inflammation, and capillary injury in muscle biopsies can cause fatigue in part due to reduced energy supply. Because most patients had mild-moderate acute affection, the new variants that might cause less severe acute disease could still have the ability to cause long-term myopathy., (© 2022 The Authors. European Journal of Neurology published by John Wiley & Sons Ltd on behalf of European Academy of Neurology.)

Bioreactors have been used for bone graft engineering in pre-clinical investigations over the past 15 years. The ability of bioreactor-incubated bone marrow nuclear cells (BMNCs) to enhance bone-forming potential varies significantly, and the three-dimensional (3D) distribution of BMNCs within the scaffold is largely unknown. The aims of this study were (1) to investigate the efficacy of a carbonated hydroxyapatite (CHA) with/without BMNCs on spine fusion rate and fusion mass microarchitecture using a highly challenging two-level posterolateral spine fusion without instrumentation; and (2) to evaluate 3D distribution of BMNCs within scaffolds characterized by immunohistochemistry. Fusion rate and fusion mass were quantified by micro-CT, microarchitectural analysis, and histology. While the homogenous 3D distribution of BMNCs was not observed, BMNCs were found to migrate towards a substitute core. In the autograft group, the healing rate was 83.3%, irrespective of the presence of BMNCs. In the CHA group, also 83.3% was fused in the presence of BMNCs, and 66.7% fused without BMNCs. A significant decrease in the fusion mass porosity (p = .001) of the CHA group suggested the deposition of mineralized bone. The autograft group revealed more bone, thicker trabeculae, and better trabecular orientation but less connection compared to the CHA group. Immunohistochemistry confirmed the ability of bioreactors to incubate a large-sized substitute coated with viable BMNCs with the potential for proliferation and differentiation. These findings suggested that a bioreactor-activated substitute is comparable to autograft on spine fusion and that new functional bone regeneration could be achieved by a combination of BMNCs, biomaterials, and bioreactors., (© 2022 Wiley Periodicals LLC.)

Background: Rotator cuff (RC) tendon tear leads to impaired shoulder function and pain. The supraspinatus (SS) tendon is most often affected, but the biological response of the SS muscle to SS tendon tear is largely unknown. This study aimed to investigate time-dependent muscle inflammation, degeneration, fatty infiltration, and regeneration in experimental SS tear conditions., Methods: Forty-five C57BL/6 mice were subjected to SS tendon tear and allowed to recover for 1, 3, 5, 7, 14, or 28 days. The extent of muscle damage was examined using histologic, flow cytometric, proteomic, and chemiluminescence analyses., Results: We found that muscle inflammation peaked around day 5 with increased monocyte infiltration and increased cytokine levels in the ipsilateral compared to the contralateral SS muscle. Bioinformatics analysis of proteomics on mice that survived 5 days after RC tendon tear revealed upregulated proteins involved in "neutrophil activation involved in immune response" and "extracellular matrix organization," whereas "skeletal muscle tissue development and contraction" and "respiratory electron transport chain" were among the most downregulated. Histologic analysis of collagen showed increased collagen accumulation and fatty infiltration of the ipsilateral SS over time. Finally, we observed time- and lesion-dependent changes in satellite cell and fibro-adipogenic progenitor populations., Conclusion: Altogether, we demonstrate that the SS muscle shows severe signs of acute inflammation, early degeneration, and fatty infiltration, as well as reduced regenerative potential following SS tendon tear., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Sporadic inclusion body myositis (sIBM) is characterised by skeletal muscle inflammation, progressive muscle loss and weakness, which is largely refractory to immunosuppressive treatment. Low-load blood-flow restricted (BFR) training has been shown to evoke gains in myofibre cross sectional area (mCSA) in healthy adults. This could partially be due to the activation and integration of muscle satellite cells (SC) resulting in myonuclei addition. Consequently, this study investigated the effect of 12-weeks lower limb low-load BFR resistance training in sIBM patients on SC and myonuclei content, myofibre size and capillarization. Muscle biopsies from sIBM patients randomised to 12-weeks of low-load BFR resistance training (n = 11) or non-exercising controls (CON) (n = 9) were analysed for SC and myonuclei content, myofibre size and capillarization using three-colour immunofluorescence microscopy and computerised quantification procedures. No between-group differences (time-by-group interactions) or within-groups changes were observed for resident SCs (Pax7 + /Six1 + ), proliferating SCs (Pax7 + / Ki67 + ), myonuclei (Six1 + ), type 1 mCSA or capillary number (CD31 + ). However, a time-by-group interaction for type 2 mCSA was observed (p = 0.04). Satellite cell content, myonuclei number, mCSA and capillary density remained unaffected following 12-weeks low-load BFR resistance training, indicating limited myogenic capacity and satellite cell plasticity in long-term sIBM patients., Competing Interests: Declaration of Competing Interest ANJ was employed by Orphazyme A/S, who is developing pharmaceuticals for patients affected by sporadic inclusion body myositis. Orphazyme A/S does not have any financial nor scientific interest in the data presented in this article., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

The need for a substitute for allograft and autograft is rising as bone graft surgeries exceed available supplies. We investigated the efficacy of the low-molecular weight marine bioactive compound fucoidan (FUC) on bone regeneration and implant fixation in seven female sheep, as FUC has shown great promise as a bone substitute. Titanium implants were inserted bilaterally in the distal femurs to test three hydroxyapatite/fucoidan (HA/FUC) groups and compared to allograft. The HA was coated with either 500 or 1500 μg of FUC, obtained by microwave-assisted chemical extraction, or 500 μg of FUC obtained by an enzyme-assisted extraction method. The concentric 2-mm gap around the implant was filled with either one of the HA/FUCs or allograft from the donor sheep. After 12 weeks, implant-bone blocks were harvested and divided into three parts for mechanical push-out testing, immunohistochemistry, and micro-CT and histomorphometry. Pronounced bone formations were observed by micro-CT and histomorphometry in all groups, but higher bone volume fractions were seen in the allograft group compared to the three HA/FUC groups. The trabecular thickness, trabecular separation, and architectural anisotropy were all significantly higher in the allograft group compared to the three HA/FUC groups. In conclusion, adequate bone formation was observed in all groups, although the bone formation was significantly greater in the allograft group. Also, no significant differences existed in the shear mechanical properties between groups, suggesting that the combination of HA and FUC can achieve a similar fixation strength to allograft in this model., (© 2021 Wiley Periodicals LLC.)

Aims: In vivo experiments were performed to establish and validate a rat model of urethral sphincter injury and to develop a method for leak point pressure (LPP) measurements performed repeatedly in the same animal., Methods: Twenty-four Sprague-Dawley female rats underwent bladder and epidural catheter implantation. Five days later, cystometry was performed using continuous infusion. Anesthesia with isoflurane, ketamine-xylazine (KX) or fentanyl-fluanisone-midazolam (FFM) was used. After three micturition cycles, intrathecal bupivacaine was administered leading to the suppression of reflex bladder contractions. LPP measurements were performed using vertical tilt. After the initial LPP measurement, animals underwent partial resection of the striated urethral sphincter. The effect was evaluated 6 weeks after surgery, by repeating the LPP measurement in the same animal., Results: Ten out of 19 animals showed full micturition cycles under isoflurane, and all 9 animals under KX anesthesia. No significant difference in micturition pressures (Mean ± SEM; 30.1 ± 2.3 vs. 26.8 ± 1.6 mmHg) and LPP (31.0 ± 2.4 vs. 28.0 ± 0.9 mmHg) was observed between isoflurane and KX groups, respectively. Reflex micturition was suppressed with FFM. Bupivacaine led to overflow incontinence in all cases. Sphincter injury caused fibrotic changes and a significant increase in LPP (26.4 ± 2.3 before vs. 46.9 ± 4.6 mmHg after injury, p  < 0.05)., Conclusions: KX anesthesia preserves bladder contractions. Intrathecal bupivacaine eliminates reflex micturition, allowing for repeated LPP measurements in the same animal. Resection of striated sphincter resulted in increased LPP 6 weeks post injury. The site of urethral sphincter resection healed with fibrosis.

Background: Esophageal atresia (EA) is a congenital malformation affecting 1:3000-4500 newborns. Approximately 15% have a long-gap EA (LGEA), in which case a primary anastomosis is often impossible to achieve. To create continuity of the esophagus patients instead have to undergo lengthening procedures or organ interpositions; methods associated with high morbidity and poor functional outcomes. Esophageal injections of Botulinum Toxin Type A (BTX-A) could enable primary anastomosis and mitigate stricture formation through decreased tissue tension., Methods and Analysis: In this randomized controlled blinded animal trial, 24 pigs are divided into a long- or short-gap EA group (LGEA and SGEA, respectively) and randomized to receive BTX-A or isotonic saline injections. In the LGEA group, injections are given endoscopically in the esophageal musculature. After seven days, a 3 cm esophageal resection and primary anastomosis is performed. In the SGEA group, a 1 cm esophageal resection and primary anastomosis is performed, followed by intraoperative injections of BTX-A or isotonic saline. After 14 days, stricture formation, presence of leakage, and esophageal compliance is assessed using endoscopic and manometric techniques, and in vivo and ex vivo contrast radiography. Tissue elongation is evaluated in a stretch-tension test, and the esophagus is assessed histologically to evaluate anastomotic healing., Ethics and Dissemination: The study complies with the ARRIVE guidelines for animal studies and has been approved by the Danish Animal Experimentation Council. Results will be published in peer-reviewed journals and presented at national and international conferences., Highlights: The optimal management of long-gap esophageal atresia remains controversialPrimary anastomosis could improve functional outcomes and reduce complicationsBotulinum Toxin Type A decreases tissue tension and could facilitate anastomosisReduced tension could further abate the risk for anastomotic stricture and leakageWe present a model to evaluate the method in long- and short-gap esophageal atresia., Competing Interests: The authors have no competing interests to declare., (Copyright: © 2021 The Author(s).)

Background: The identification of novel targets is of paramount importance to develop more effective drugs and improve the treatment of non-small cell lung cancer (NSCLC), the leading cause of cancer-related deaths worldwide. Since cells alter their metabolic rewiring during tumorigenesis and along cancer progression, targeting key metabolic players and metabolism-associated proteins represents a valuable approach with a high therapeutic potential. Metabolic fitness relies on the functionality of heat shock proteins (HSPs), molecular chaperones that facilitate the correct folding of metabolism enzymes and their assembly in macromolecular structures., Methods: Gene fitness was determined by bioinformatics analysis from available datasets from genetic screenings. HSPD1 expression was evaluated by immunohistochemistry from formalin-fixed paraffin-embedded tissues from NSCLC patients. Real-time proliferation assays with and without cytotoxicity reagents, colony formation assays and cell cycle analyses were used to monitor growth and drug sensitivity of different NSCLC cells in vitro. In vivo growth was monitored with subcutaneous injections in immune-deficient mice. Cell metabolic activity was analyzed through extracellular metabolic flux analysis. Specific knockouts were introduced by CRISPR/Cas9., Results: We show heat shock protein family D member 1 (HSPD1 or HSP60) as a survival gene ubiquitously expressed in NSCLC and associated with poor patients' prognosis. HSPD1 knockdown or its chemical disruption by the small molecule KHS101 induces a drastic breakdown of oxidative phosphorylation, and suppresses cell proliferation both in vitro and in vivo. By combining drug profiling with transcriptomics and through a whole-genome CRISPR/Cas9 screen, we demonstrate that HSPD1-targeted anti-cancer effects are dependent on oxidative phosphorylation and validated molecular determinants of KHS101 sensitivity, in particular, the creatine-transporter SLC6A8 and the subunit of the cytochrome c oxidase complex COX5B., Conclusions: These results highlight mitochondrial metabolism as an attractive target and HSPD1 as a potential theranostic marker for developing therapies to combat NSCLC., (© 2021. The Author(s).)

Introduction: Idiopathic inflammatory myopathies (IIMs) are rare diseases characterised by non-suppurative inflammation of skeletal muscles and muscle weakness. Additionally, IIM is associated with a reduced quality of life. Strength training is known to promote muscle hypertrophy and increase muscle strength and physical performance in healthy young and old adults. In contrast, only a few studies have examined the effects of high intensity strength training in patients with IIM and none using a randomised controlled trial (RCT) set-up. Thus, the purpose of this study is to investigate the effects of high-intensity strength training in patients affected by the IIM subsets polymyositis (PM), dermatomyositis (DM) and immune-mediated necrotising myopathy (IMNM) using an RCT study design., Methods and Analysis: 60 patients with PM, DM or IMNM will be included and randomised into (1) high-intensity strength training or (2) Care-as-Usual. The intervention period is 16 weeks comprising two whole-body strength exercise sessions per week. The primary outcome parameter will be the changes from pre training to post training in the Physical Component Summary measure in the Short Form-36 health questionnaire. Secondary outcome measures will include maximal lower limb muscle strength, skeletal muscle mass, functional capacity, disease status (International Myositis Assessment and Clinical Studies Group core set measures) and questionnaires assessing physical activity levels and cardiovascular comorbidities. Furthermore, blood samples and muscle biopsies will be collected for subsequent analyses., Ethics and Dissemination: The study complies with the Helsinki Declaration II and is approved by The Danish Data Protection Agency (P-2020-553). The study is approved by The Danish National Committee on Health Research Ethics (H-20030409). The findings of this trial will be submitted to relevant peer-reviewed journals. Abstracts will be submitted to international conferences., Trial Registration Number: NCT04486261., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Background: Rotator cuff (RC) disorders involve a spectrum of shoulder conditions from early tendinopathy to full-thickness tears leading to impaired shoulder function and pain. The pathology of RC disorder is, nonetheless, still largely unknown. Our hypothesis is that a supraspinatus (SS) tendon tear leads to sustained inflammatory changes of the SS muscle along with fatty infiltration and muscle degeneration, which are threshold markers for poor RC muscle function. The aim of this study was to determine the extent of this muscle inflammation in conjunction with lipid accumulation and fibrosis in RC tear conditions., Methods: We used proteomics, histology, electrochemiluminescence immunoassay, and quantitative polymerase chain reaction analyses to evaluate inflammatory and degenerative markers and fatty infiltration in biopsies from 22 patients undergoing surgery with repair of a full-thickness SS tendon tear., Results: Bioinformatic analysis showed that proteins involved in innate immunity, extracellular matrix organization, and lipid metabolism were among the most upregulated, whereas mitochondrial electronic transport chain along with muscle fiber function was among the most downregulated. Histologic analysis confirmed changes in muscle fiber organization and the presence of inflammation and fatty infiltration. Inflammation appeared to be driven by a high number of infiltrating macrophages, accompanied by elevated matrix metalloprotease levels and changes in transforming growth factor-β and cytokine levels in the SS compared with the deltoid muscle., Conclusions: We demonstrated massive SS muscle inflammation after the tendon tear combined with fatty infiltration and degeneration. The regulation of tissue repair is thus extremely complex, and it may have opposite effects at different time points of healing. Inhibition or stimulation of muscle inflammation may be a potential target to enhance the outcome of the repaired torn RC., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Oxaliplatin-induced peripheral neuropathy negatively affects the quality of life for patients with gastrointestinal cancers and may cause neuropathic pain. Measures of peripheral nerve structure or function, such as intraepidermal nerve fiber density (IENFD) during treatment could reduce neuropathy severity through individualized dose reduction., Objective: The aim was to evaluate the predictive values of IENFD, quantitative sensory testing (QST), and nerve conduction studies (NCS) for significant neuropathy and neuropathic pain., Methods: Fifty-five patients were examined prospectively before, during, and six months following treatment using skin biopsies, QST and NCS. Clinically significant neuropathy six months after treatment was defined as reduced Total Neuropathy Score of more than five and neuropathic pain was assessed according to International Association for the Study of Pain criteria., Results: Thirty patients had a clinically significant neuropathy, and 14 had neuropathic pain. Vibration detection threshold (VDT) before treatment was correlated with clinically significant neuropathy six months after treatment (OR 0.54, p = 0.01) and reductions in cold detection threshold (CDT) after 25% of treatment (OR 1.38, p = 0.04) and heat pain threshold (HPT) after 50% of treatment (OR 1.91, p = 0.03) with neuropathic pain. Cut off values of 5 for baseline VDT and changes of more than -0.05 °C and -0.85 °C in CDT and HPT were estimated. Sensitivity and specificity was low to moderate. There was no correlation between changes in IENFD or NCS and significant neuropathy or neuropathic pain., Conclusions: Vibration detection thresholds and thermal detection thresholds may be useful for prediction of clinically significant and painful neuropathy, respectively. However, low to moderate sensitivity and specificity may limit the predictive value in clinical practice.

Introduction:  Anastomosis with minimal tension is desirable in long-gap esophageal atresia. Prior studies in piglets showed that intraesophageal injection of botulinum toxin type A (BTX-A) results in significant esophageal elongation. Our aim was to determine the BTX-A dose, number of injections, and time necessary to elicit maximal response., Materials and Methods:  Adult male Wistar rats ( n  = 48) were randomly assorted into five groups. Four treatment groups received 2 or 4 U/kg of BTX-A, delivered using two or four injections, and a control group received 0.9% NaCl. Esophagus was removed 6 or 24-hours postinjection and tested ex vivo using a stretch tension device. Subsequently, an optimal dose and time following injection was used to study the effects of BTX-A on anastomotic healing in vivo. Rats ( n  = 12) received an intraesophageal injection of BTX-A or 0.9% NaCl, followed by resection of 0.5 cm of esophagus and end-to-end anastomosis. Rats were observed for 9 days, and esophagus was removed for gross and histological evaluation., Results:  The largest effect on elongation was recorded in the BTX-A (2 U/kg) 24 hour, four injection group. In the anastomosis study, stricture formation was observed in all animals in the control group. Absence of esophageal stricture was found in three out of four animals in the treatment group macroscopically and histologically., Conclusion:  We found that BTX-A exerts a positive effect on stretch characteristics of esophageal tissue in rats at 2 U/kg via four-injection delivery and 24-hour waiting period. This study suggests that BTX-A might improve anastomotic healing., Competing Interests: None declared., (Thieme. All rights reserved.)

Background: Lymphedema is one of the most common complications following breast cancer. Axillary lymph node dissection and radiotherapy are two well-known risk factors resulting in either removal or damage to the lymph nodes. As stem cells are known for their regenerative capabilities, they could theoretically repair/restore the damaged lymph vessels leading to a decrease in lymphedema. Methods: We evaluated the treatment of SVF and ASC on a mouse lymphedema model. Forty-five mice were allocated into three groups containing 15 mice each. The SVF group was injected with 100 μl containing 1 × 10 6 SVF, the ASC group with 100 μl ml containing 1 × 10 6  ASC and the NS with 100 μl ml of NS. Volumes of the mice were assessed weekly by μCT hindlimb volumetry for a total of 8 weeks. Lymph vessel morphometry was assessed by cross-sections of both hindlimbs stained for anti-LYVE1. Lymphatic function was assessed by lymphatic clearance. Results: The volume change between the groups was non-significant throughout all 8 weeks. The immunohistochemistry showed a statistically significant difference between the hindlimbs in ASC vs. NS group p  = 0.032, 95% CI [-2121, -103]. Conclusion: The volume of the hindlimbs showed that treatment with SVF or ASC yielded very similar results compared to the control group when assessed after 8 weeks. In week two the biggest difference between ASC and NS was seen but the difference diminished during the 8 weeks. The secondary outcomes showed that the lymph vessel lumen decreased when treated with ASC compared to the control group. Lymphoscintigraphy yielded non-significant results.

C57BL/6J-related mouse strains are widely used animal models for diet-induced obesity (DIO). Multiple vendors breed C57BL/6J-related substrains which may introduce genetic drift and environmental confounders such as microbiome differences. To address potential vendor/substrain specific effects, we compared DIO of C57BL/6J-related substrains from three different vendors: C57BL/6J (Charles Rivers), C57BL/6JBomTac (Taconic Bioscience) and C57BL/6JRj (Janvier). After local acclimatization, DIO was induced by either a high-fat diet (HFD, 60% energy from fat) or western diet (WD, 42% energy from fat supplemented with fructose in the drinking water). All three groups on HFD gained a similar amount of total body weight, yet the relative amount of fat percentage and mass of inguinal- and epididymal white adipose tissue (iWAT and eWAT) was lower in C57BL/6JBomTac compared to the two other C57BL/6J-releated substrains. In contrast to HFD, the three groups on WD responded differently in terms of body weight gain, where C57BL/6J was particularly prone to WD. This was associated with a relative higher amount of eWAT, iWAT, and liver triglycerides. Although the HFD and WD had significant impact on the microbiota, we did not observe any major differences between the three groups of mice. Together, these data demonstrate significant differences in HFD- and WD-induced adiposity in C57BL/6J-related substrains, which should be considered in the design of animal DIO studies.

Pancreatic ductal adenocarcinoma (PC) is characterized by a highly fibrotic desmoplastic stroma. Subtypes of cancer-associated fibroblasts (CAFs) have been identified in chemotherapy-naïve PC (CTN-PC), but their precise functions are still unclear. Our knowledge regarding the properties of CAFs in the regressive stroma after neoadjuvant treatment (NAT) of PC (NAT-PC) is particularly limited. We aimed to examine the marker phenotypic properties of CAFs in the regressive stroma of PC. Surgical specimens from patients with CTN-PC (n=10) and NAT-PC (n=10) were included. Juxtatumoural, peripheral, lobular, septal, peripancreatic, and regressive stromal compartments were manually outlined using digital imaging analysis (DIA) for area quantification. The compartment-specific expression of CD271, cytoglobin, DOG-1, miR-21, osteonectin, PDGF-Rβ, and tenascin C was evaluated by immunohistochemistry or in situ hybridization, using manual scoring and automated DIA. The area fraction of the regressive stroma was significantly higher in NAT-PC than in CTN-PC (P=0.0002). CD271 (P<0.01), cytoglobin (P<0.05), DOG1 (P<0.05), miR-21 (P<0.05), and tenascin C (P<0.05) exhibited significant differences in their expression profiles between the juxtatumoural compared to the peripheral and regressive stroma. PDGF-Rβ expression was significantly higher in juxtatumoural than in peripheral CAFs (P<0.05). Our data provide further support of the concept of stromal heterogeneity and phenotypic different CAF subtypes in PC. CAFs in the regressive stroma of NAT-PC show a marker phenotype similar to some (namely, peripheral) and different from other (namely, juxtatumoural) previously defined CAF subtypes. It may be hypothesized that phenotypic CAF subtypes, at least in part, also may share functional properties. Studies examining the precise functional characteristics of CAF subtypes in PC are needed.

Preclinical and clinical proof-of-concept studies have suggested the effectiveness of pharmacological modulation of inflammatory cytokines in ischemic stroke. Experimental evidence shows that targeting tumor necrosis factor (TNF) and interleukin (IL)-1 holds promise, and these cytokines are considered prime targets in the development of new stroke therapies. So far, however, information on the cellular expression of TNF and IL-1 in the human ischemic brain is sparse.We studied 14 cases of human post-mortem ischemic stroke, representing 21 specimens of infarcts aged 1 to > 8 days. We characterized glial and leukocyte reactions in the infarct/peri-infarct (I/PI) and normal-appearing tissue (NAT) and the cellular location of TNF, TNF receptor (TNFR)1 and TNFR2, IL-1α, IL-1β, and IL-1 receptor antagonist (IL-1Ra). The immunohistochemically stained tissue sections received a score reflecting the number of immunoreactive cells and the intensity of the immunoreactivity (IR) in individual cells where 0 = no immunoreactive cells, 1 = many intermediately to strongly immunoreactive cells, and 2 = numerous and intensively immunoreactive cells. Additionally, we measured blood TNF, TNFR, and IL-1 levels in surviving ischemic stroke patients within the first 8 h and again at 72 h after symptom onset and compared levels to healthy controls.We observed IL-1α and IL-1β IR in neurons, glia, and macrophages in all specimens. IL-1Ra IR was found in glia, in addition to macrophages. TNF IR was initially found in neurons located in I/PI and NAT but increased in glia in older infarcts. TNF IR increased in macrophages in all specimens. TNFR1 IR was found in neurons and glia and macrophages, while TNFR2 was expressed only by glia in I/PI and NAT, and by macrophages in I/PI. Our results suggest that TNF and IL-1 are expressed by subsets of cells and that TNFR2 is expressed in areas with increased astrocytic reactivity. In ischemic stroke patients, we demonstrate that plasma TNFR1 and TNFR2 levels increased in the acute phase after symptom onset compared to healthy controls, whereas TNF, IL-1α, IL-1β, and IL-1Ra did not change.Our findings of increased brain cytokines and plasma TNFR1 and TNFR2 support the hypothesis that targeting post-stroke inflammation could be a promising add-on therapy in ischemic stroke patients.

Purpose: To examine the association between use of statins and risk of deterioration of peripheral nerve function., Methods: We prospectively followed patients who initiated statin treatment and compared them with statin never-users (non-users). At the time of inclusion and at 1-year follow-up, participants underwent tests for peripheral nerve function (ie nerve conduction studies, quantitative sensory testing), skin biopsies and ratings of symptoms and signs of neuropathy. We selected five tests of nerve function and the intraepidermal nerve fibre density (IENFD) a priori as primary outcomes. We used linear regression to test for differences between statin users and non-users with Holm-Bonferroni-corrected statistical significance level of .05., Results: Comparisons were based on 57 statin users and 46 non-users. Changes in nerve function test results during follow-up were not uniform with regard to direction and were statistically not significant with the exception of IENFD (change in IENFD: statin users 1 fibre/mm vs. non-statin users -2 fibres/mm; P-value = .006). None of the participants developed overt peripheral neuropathy. However, five statin users developed neuropathy-like symptoms and a post hoc analysis showed a significant decrease in vibration sensitivity compared to asymptomatic statin users., Conclusion: Statin use was not clearly associated with increased risk of deterioration of peripheral nerve function analysed at a group level. However, given the sample size limitations of our study and the findings of our post hoc analysis, we cannot preclude that peripheral nerve function may be affected in some individuals exposed to statins., (© 2019 Nordic Association for the Publication of BCPT (former Nordic Pharmacological Society).)

Background and Purpose: Assessment of the severity of chronic peripheral neuropathy during oxaliplatin treatment is based on symptoms. Efforts to adjust the total dose of oxaliplatin to prevent severe neuropathy can be complicated by the worsening of neuropathy symptoms following treatment. Objective measures of the structure and function of peripheral nerves during early phases of treatment may aid in determining the optimal oxaliplatin dose in individual patients. Intraepidermal nerve fibre density (IENFD) has been suggested as an early marker of peripheral neuropathy., Methods: Sixty patients were examined before treatment and following 25% and 50% of the total planned oxaliplatin dose. Fifty-five of them were also examined at completion of chemotherapy and 6 months later. IENFD in skin biopsies from the distal leg, nerve conduction studies and quantitative sensory testing at the dorsum of the foot were performed. Forty-six healthy subjects were examined at baseline and after 6 and 52 weeks for comparison., Results: Intraepidermal nerve fibre density was not reduced during treatment. Sural nerve amplitude and conduction velocity, vibration detection thresholds, mechanical detection threshold and cold detection threshold were significantly reduced during treatment. Compared to reference values and spontaneous changes in healthy subjects, the largest proportions of patients with deterioration were found for vibration detection thresholds followed by nerve conduction studies, mechanical detection threshold, cold detection threshold and IENFD., Conclusions: Significant changes were most pronounced for measures of large nerve fibre function, especially vibration sensation. Skin biopsies do not seem to provide a clinically relevant objective measure of peripheral nerve deterioration during oxaliplatin treatment., (© European Academy of Neurology 2019.)

Cerebral choline metabolism is crucial for normal brain function, and its homoeostasis depends on carrier-mediated transport. Here, we report on four individuals from three families with neurodegenerative disease and homozygous frameshift mutations (Asp517Metfs*19, Ser126Metfs*8, and Lys90Metfs*18) in the SLC44A1 gene encoding choline transporter-like protein 1. Clinical features included progressive ataxia, tremor, cognitive decline, dysphagia, optic atrophy, dysarthria, as well as urinary and bowel incontinence. Brain MRI demonstrated cerebellar atrophy and leukoencephalopathy. Moreover, low signal intensity in globus pallidus with hyperintensive streaking and low signal intensity in substantia nigra were seen in two individuals. The Asp517Metfs*19 and Ser126Metfs*8 fibroblasts were structurally and functionally indistinguishable. The most prominent ultrastructural changes of the mutant fibroblasts were reduced presence of free ribosomes, the appearance of elongated endoplasmic reticulum and strikingly increased number of mitochondria and small vesicles. When chronically treated with choline, those characteristics disappeared and mutant ultrastructure resembled healthy control cells. Functional analysis revealed diminished choline transport yet the membrane phosphatidylcholine content remained unchanged. As part of the mechanism to preserve choline and phosphatidylcholine, choline transporter deficiency was implicated in impaired membrane homeostasis of other phospholipids. Choline treatments could restore the membrane lipids, repair cellular organelles and protect mutant cells from acute iron overload. In conclusion, we describe a novel childhood-onset neurometabolic disease caused by choline transporter deficiency with autosomal recessive inheritance., (© The Author(s) (2019). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Background: Sporadic inclusion body myositis (sIBM) is clinically characterised by progressive proximal and distal muscle weakness and impaired physical function while skeletal muscle tissue displays abnormal cellular infiltration of T cells, macrophages, and dendritic cells. Only limited knowledge exists about the effects of low-load blood flow restriction exercise in sIBM patients, and its effect on the immunological responses at the myocellular level remains unknown. The present study is the first to investigate the longitudinal effects of low-load blood flow restriction exercise on innate and adaptive immune markers in skeletal muscle from sIBM patients., Methods: Twenty-two biopsy-validated sIBM patients were randomised into either 12 weeks of low-load blood flow restriction exercise (BFRE) or no exercise (CON). Five patients from the control group completed 12 weeks of BFRE immediately following participation in the 12-week control period leading to an intervention group of 16 patients. Muscle biopsies were obtained from either the m. tibialis anterior or the m. vastus lateralis for evaluation of CD3-, CD8-, CD68-, CD206-, CD244- and FOXP3-positive cells by three-colour immunofluorescence microscopy and Visiopharm-based image analysis quantification. A linear mixed model was used for the statistical analysis., Results: Myocellular infiltration of CD3 - /CD8 + expressing natural killer cells increased following BFRE (P < 0.05) with no changes in CON. No changes were observed for CD3 + /CD8 - or CD3 + /CD8 + T cells in BFRE or CON. CD3 + /CD244 + T cells decreased in CON, while no changes were observed in BFRE. Pronounced infiltration of M1 pro-inflammatory (CD68 + /CD206 - ) and M2 anti-inflammatory (CD68 + /CD206 + ) macrophages were observed at baseline; however, no longitudinal changes in macrophage content were observed for both groups., Conclusions: Low-load blood flow restriction exercise elicited an upregulation in CD3 - /CD8 + expressing natural killer cell content, which suggests that 12 weeks of BFRE training evokes an amplified immune response in sIBM muscle. However, the observation of no changes in macrophage or T cell infiltration in the BFRE-trained patients indicates that patients with sIBM may engage in this type of exercise with no risk of intensified inflammatory activity.

There is a growing recognition that myogenic stem cells are influenced by their microenvironment during regeneration. Several interstitial cell types have been described as supportive for myoblasts. In this role, both the pericyte as a possible progenitor for mesenchymal stem cells, and interstitial cells in the endomysium have been discussed. We have applied immunohistochemistry on normal and pathological human skeletal muscle using markers for pericytes, or progenitor cells and found a cell type co-expressing CD10, CD34, CD271, and platelet-derived growth factor receptor α omnipresent in the endomysium. The marker profile of these cells changed dynamically in response to muscle damage and atrophy, and they proliferated in response to damage. The cytology and expression profile of the CD10+ cells indicated a capacity to participate in myogenesis. Both morphology and indicated function of these cells matched properties of several previously described interstitial cell types. Our study suggests a limited number of cell types that could embrace many of these described cell types. Our study indicate that the CD10+, CD34+, CD271+, and platelet-derived growth factor receptor α+ cells could have a supportive role in human muscle regeneration, and thus the mechanisms by which they exert their influence could be implemented in stem cell therapy.

Objective: To determine interrater variability in diagnosing individual muscle biopsy abnormalities and diagnosis., Methods: We developed a scoring tool to analyze consensus in muscle biopsy reading of an ad hoc workgroup of international experts. Twenty-four samples from patients with suspected idiopathic inflammatory myopathy (IIM) were randomly selected, providing sections that were stained with standard histologic and immunohistochemical methods. Sections were made available on an online platform, and experts were queried about myopathologic features within 4 pathologic domains: muscle fibers, inflammation, connective tissue, and vasculature. A short clinical presentation of cases was included, and experts were asked to give a tentative diagnosis of polymyositis, dermatomyositis, inclusion-body myositis, antisynthetase syndrome-related myositis, immune-mediated necrotizing myopathy, nonspecific myositis, or other disease. Fleiss κ values, scoring interrater variability, showed the highest agreement within the muscle fiber and connective tissue domains., Results: Despite overall low κ values, moderate agreement was achieved for tentative diagnosis, supporting the idea of using holistic muscle biopsy interpretation rather than adding up individual features., Conclusion: The assessment of individual pathologic features needs to be standardized and harmonized and should be measured for sensitivity and specificity for subgroup classification. Standardizing the process of diagnostic muscle biopsy reading would allow identification of more homogeneous patient cohorts for upcoming treatment trials., (© 2019 American Academy of Neurology.)

Introduction: Standardised scoring systems for rheumatoid arthritis (RA) joint disease activity include Larsen score for radiographs, rheumatoid arthritis magnetic resonance imaging score (RAMRIS) for MRI and using the European League Against Rheumatisms-Outcome Measures in Rheumatology (EULAR-OMERACT) score for ultrasound (US) images. The aim of this prospective study was to investigate the relationship between histological synovitis and radiological synovitis, assessed by conventional X-ray, US and MRI of the wrist radiocarpal joint., Methods: 20 patients with treatment naive early RA (ERA) and 20 with long-standing RA (LRA) were enrolled in a 6-month prospective study. Patients with RA underwent US-guided synovial biopsy, X-ray and US of the wrist at enrolment and 6 months. MRI at baseline and also at 6 months for the ERA group, and scored with the RAMRIS system. X-ray was scored by Larsen score and US by the EULAR-OMERACT system. Synovial biopsy inflammation was determined by the Krenn score., Results: In the ERA group at baseline, Krenn score was correlated strongly with both US combined score (r = 0.77 p < 0.001) and MRI synovitis score (r = 0.85 p < 0.001), while uncorrelated at 6 months. In the LRA group at baseline, these scores correlated strongly (r = 0.83, p < 0.001) to moderately (r = 0.61, p = 0.002), and persisted at 6 months for US score (r = 0.81 p < 0.001). For all patients with RA, change in Krenn score between baseline and 6 months was correlated with both change in US combined score (r = 0.65, p < 0.001) and change in MRI synovitis score (r = 0.50, p = 0.03)., Conclusion: The MRI RAMRIS synovitis score and EULAR-OMERACT US scoring system are sensitive measures of histological synovitis in LRA and ERA. After 6 months, this correlation persists in the established RA group, but not in the ERA group. Overall, decreases in MRI/US synovitis are associated with reductions in histological synovitis. The study validates the use of MRI RAMRIS and EULAR-OMERACT US scores as surrogate markers of histological synovitis in established RA and early untreated RA., Competing Interests: Competing interests: None declared.

Background: Lymphedema is a common and debilitating complication following cancer treatment with surgical lymph node excision and radiotherapy. Currently there are no curative treatments for lymphedema. Animal models that intended to replicate the disease have been inadequate, making a troublesome transition from experimental therapeutic studies into the clinic. It is therefore imperative to establish an experimental animal model that can reliably replicate clinical lymphedema., Methods: To discover the optimal method of lymphedema induction, surgical lymph ablation and irradiation or silicone splint emplacement were combined in 8 experimental groups (n = 4). In total, 32 mice served in this study and were followed for 8 weeks after surgery. Outcomes included micro-computed tomography hind limb volumetry, lymphatic clearance measured with technetium Tc 99m (Tc) human serum albumin lymphoscintigraphy and lymph vessel ectasia quantified with LYVE-1 immunohistochemistry., Results: All trialed models but one resulted in only transient lymphedema or lasting lymphedema with adverse morbidity. Combined surgical lymph obstruction with 2 fractions of 10-Gy irradiation successfully induced lasting lymphedema without adverse events. Over the 8 weeks' follow-up, limb volumes were significantly increased at all time points (P < 0.001), lymph drainage was impaired (P < 0.001), and lymph vessels were ectatic (P < 0.001), when compared with the unoperated limbs., Conclusions: The presented model of acquired lymphedema is a reduction and refinement of previous works and can transpose to future observational and interventional studies. In addition, it is shown how Tc-HSA lymphoscintigraphy can quantify lymphatic clearance, which can prove insightful in therapeutic studies aiming to enhance lymphatic drainage.

Bag3opathy is a rare myofibrillar myopathy (MFM) caused by a mutation in the Bcl-2 associated-athanogene-3 gene. Less than twenty patients have been described, almost all with severe cardiac involvement. We present a 26-year-old man with a c.626C>T (p.Pro209Leu) mutation in the Bcl-2 associated-athanogene-3 gene (BAG3). Our patient presented with problems running before he turned 10 and rapidly progressing, proximal muscle weakness and rigidity of the neck and back. Muscle biopsy showed Z-disc streaming, vacuoles, which is typical findings of Bag3opathy, as well as accumulation of filamentous materials. He rapidly developed respiratory insufficiency necessitating assisted ventilation, and became wheelchair bound by age 13. The progression of his muscle disease is characteristic of Bag3opathy, but unlike other reported cases, he had no evidence of cardiac involvement at age 25 years, despite serial Holter monitoring, ECG and echocardiographs. This case illustrates that counseling of patients with BAG3 myopathy should not predict an inevitable occurrence of cardiomyopathy., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Brain calcification of especially the basal ganglia characterizes primary familial brain calcification (PFBC). PFBC is a rare neurodegenerative disorder with neuropsychiatric and motor symptoms, and only symptomatic treatment is available. Four PFBC-associated genes are known; approximately 40% of patients carry mutations in the gene SLC20A2, which encodes the type III sodium-dependent inorganic phosphate transporter PiT2. To investigate the role of PiT2 in PFBC development, we studied Slc20a2-knockout (KO) mice using histology, microcomputed tomography, electron microscopy, and energy-dispersive X-ray spectroscopy. Slc20a2-KO mice showed histologically detectable nodules in the brain already at 8 weeks of age, which contained organic material and were weakly calcified. In 15-week-old mice, the nodules were increased in size and number and were markedly more calcified. The major minerals in overt calcifications were Ca and P, but Fe, Zn, and Al were also generally present. Electron microscopy suggested that the calcifications initiate intracellularly, mainly in pericytes and astrocytes. As the calcification grew, they incorporated organic material. Furthermore, endogenous IgG was detected around nodules, suggesting local increased blood-brain barrier permeabilities. Nodules were found in all 8-week-old Slc20a2-KO mice, but no prenatal or marked postnatal lethality was observed. Thus, besides allowing for the study of PFBC development, the Slc20a2-KO mouse is a potential solid preclinical model for evaluation of PFBC treatments., (Copyright © 2018 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Collectin liver 1 (CL-L1, alias collectin 10) and collectin kidney 1 (CL-K1, alias collectin 11) are oligomeric pattern recognition molecules associated with the complement system, and mutations in either of their genes may lead to deficiency and developmental defects. The two collectins are reportedly localized and synthesized in the liver, kidneys, and adrenals, and can be found in the circulation as heteromeric complexes (CL-LK), which upon binding to microbial high mannose-like glycoconjugates activates the complement system via the lectin activation pathway. The tissue distribution of homo- vs. heteromeric CL-L1 and -K1 complexes, the mechanism of heteromeric complex formation and in which tissues this occurs, is hitherto incompletely described. We have by immunohistochemistry using monoclonal antibodies addressed the precise cellular localization of the two collectins in the main human tissues. We find that the two collectins have widespread and almost identical tissue distribution with a high expression in epithelial cells in endo-/exocrine secretory tissues and mucosa. There is also accordance between localization of mRNA transcripts and detection of proteins, showing that local synthesis likely is responsible for peripheral localization and eventual formation of the CL-LK complexes. The functional implications of the high expression in endo-/exocrine secretory tissue and mucosa is unknown but might be associated with the activity of MASP-3, which has a similar pattern of expression and is known to potentiate the activity of the alternative complement activation pathway.

Low testosterone levels in aging men are associated with insulin resistance. Mitochondrial dysfunction, changes in glycogen metabolism, and lipid accumulation are linked to insulin resistance in skeletal muscle. In this randomized, double-blinded, placebo-controlled study, we investigated the effects of six-month testosterone replacement therapy (TRT) and strength training (ST) on mitochondrial, glycogen, and lipid droplet (LD) content in skeletal muscle of aging men with subnormal bioavailable testosterone (BioT) levels. Mitochondrial, glycogen, and LD volume fractions in muscle biopsies were estimated by transmission electron microscopy. Insulin sensitivity (insulin-stimulated Rd) and body composition were assessed by euglycemic-hyperinsulinemic clamp and dual X-ray absorptiometry, respectively. TRT significantly increased total testosterone levels, BioT, and lean body mass (LBM) (p < 0.05), whereas percent body fat decreased (p < 0.05), and insulin sensitivity was unchanged. Baseline mitochondrial volume fraction correlated inversely with percent body fat (ρ = -0.43; p = 0.003). Δ-mitochondrial fraction correlated positively with Δ-total testosterone (ρ = 0.70; p = 0.02), and Δ-glycogen fraction correlated inversely with Δ-LBM (ρ = -0.83; p = 0.002) during six-month TRT, but no significant changes were observed in mitochondrial, glycogen, and LD volume fractions during TRT and ST. In conclusion, in this exploratory small-scale study, the beneficial effects of six-month TRT on total testosterone, LBM, and percent body fat were not followed by significant changes in fractions of mitochondria, glycogen, or lipid in skeletal muscle of aging men with lowered testosterone levels. Six-month ST or combined three-month ST+TRT did not change intramyocellular mitochondria, glycogen, and LD fractions compared to placebo. However, further studies with a larger sample size are needed., (© 2018 American Society of Andrology and European Academy of Andrology.)