A novel multitargeted self-assembling peptide-siRNA complex for simultaneous inhibition of SARS-CoV-2-host cell interaction and replication.

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.

Effects of sporadic inclusion body myositis on skeletal muscle fibre type specific morphology and markers of regeneration and inflammation.

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.

Short term treatment of secondary lymphedema with hyaluronidase injections reduces mouse hindlimb lymphedema.

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.

Sporadic late onset nemaline myopathy with concurrent dermatological symptoms responding to immunosuppressive treatment.

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.

Ryanodine receptor 1 related myasthenia like myopathy responsive to pyridostigmine.

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.

Lack of muscle stem cell proliferation and myocellular hypertrophy in sIBM patients following blood-flow restricted resistance training.

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.

Stone heart syndrome after prolonged cardioplegia induced cardiac arrest in open-heart surgery - a pilot study on pigs.

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.

Efficacy of bioreactor-activated bone substitute with bone marrow nuclear cells on fusion rate and fusion mass microarchitecture in sheep.

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.

Early-stage inflammation changes in supraspinatus muscle after rotator cuff tear.

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.

Efficacy of marine bioactive compound fucoidan for bone regeneration and implant fixation in sheep.

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.

Validation of a new rat model of urethral sphincter injury and leak point pressure measurements.

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.

Metabolic impairment of non-small cell lung cancers by mitochondrial HSPD1 targeting.

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.

The Effect of Botulinum Toxin Type A Injections on Stricture Formation, Leakage Rates, Esophageal Elongation, and Anastomotic Healing Following Primary Anastomosis in a Long- and Short-Gap Esophageal Atresia Model - A Protocol for a Randomized, Controlled, Blinded Trial in Pigs.

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.

High-intensity strength training in patients with idiopathic inflammatory myopathies: a randomised controlled trial protocol.

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.

The inflammatory response of the supraspinatus muscle in rotator cuff tear conditions.

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-ß 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.

C57BL/6J substrain differences in response to high-fat diet intervention.

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.

Comparison between stromal vascular fraction and adipose derived stem cells in a mouse lymphedema model.

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 × 106 SVF, the ASC group with 100 µl ml containing 1 × 106 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.

Spatial and phenotypic characterization of pancreatic cancer-associated fibroblasts after neoadjuvant treatment.

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-Rß, 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-Rß 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.

Statin use and peripheral nerve function-A prospective follow-up study.

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.