Senescent fibro-adipogenic progenitors are potential drivers of pathology in inclusion body myositis.

Inclusion body myositis (IBM) is unique across the spectrum of idiopathic inflammatory myopathies (IIM) due to its distinct clinical presentation and refractoriness to current treatment approaches. One explanation for this resistance may be the engagement of cell-autonomous mechanisms that sustain or promote disease progression of IBM independent of inflammatory activity. In this study, we focused on senescence of tissue-resident cells as potential driver of disease. For this purpose, we compared IBM patients to non-diseased controls and immune-mediated necrotizing myopathy patients. Histopathological analysis suggested that cellular senescence is a prominent feature of IBM, primarily affecting non-myogenic cells. In-depth analysis by single nuclei RNA sequencing allowed for the deconvolution and study of muscle-resident cell populations. Among these, we identified a specific cluster of fibro-adipogenic progenitors (FAPs) that demonstrated key hallmarks of senescence, including a pro-inflammatory secretome, expression of p21, increased ß-galactosidase activity, and engagement of senescence pathways. FAP function is required for muscle cell health with changes to their phenotype potentially proving detrimental. In this respect, the transcriptomic landscape of IBM was also characterized by changes to the myogenic compartment demonstrating a pronounced loss of type 2A myofibers and a rarefication of acetylcholine receptor expressing myofibers. IBM muscle cells also engaged a specific pro-inflammatory phenotype defined by intracellular complement activity and the expression of immunogenic surface molecules. Skeletal muscle cell dysfunction may be linked to FAP senescence by a change in the collagen composition of the latter. Senescent FAPs lose collagen type XV expression, which is required to support myofibers' structural integrity and neuromuscular junction formation in vitro. Taken together, this study demonstrates an altered phenotypical landscape of muscle-resident cells and that FAPs, and not myofibers, are the primary senescent cell type in IBM.

Female sex and overweight are associated with a lower quality of life in patients with myasthenia gravis: a single center cohort study.

BACKGROUND: Myasthenia gravis (MG) affects individuals as a chronic autoimmune disease for many years. Commonly, chronic diseases significantly reduce the patients' quality of life. Aiming to improve the future quality of life in MG, this study assessed the factors impacting quality of life. As gender-specific medicine is becoming increasingly important, this study also focused on understanding gender differences in the outcome of MG. METHODS: The study is a combined monocentric, retrospective and prospective database analysis of patient records based on 2,370 presentations of 165 patients with clinically, serologically and/or electrophysiologically confirmed MG over an observation period of up to 47 years. The data collection included the following parameters: antibody status, disease severity, age, medication use, gender, and disease duration. In addition, a prospective survey was conducted on the quality of life using the Myasthenia gravis-specific 15-item Quality of Life scale (MG-QoL15) and on the activities of daily living using the MG-specific Activities of Daily Living scale (MG-ADL). RESULTS: Of the 165 patients, 85 were male (51.5%) and 80 were female (48.5%). The remaining baseline characteristics (e.g. age and antibody status) were consistent with other myasthenia gravis cohorts. A high body mass index (BMI) (p = 0.005) and a high disease severity (p < 0.001) were significantly associated with lower disease-specific quality of life. Additionally, the quality of life in women with MG was significantly reduced compared to male patients (19.7 vs. 13.0 points in the MG-QoL15, p = 0.024). Gender differences were also observable in terms of the period between initial manifestation and initial diagnosis and women were significantly more impaired in their activities of daily living (MG-ADL) than men (4.8 vs. 3.0 points, p = 0.032). CONCLUSION: Women with MG had significantly poorer disease specific quality of life compared to men as well as patients with a higher BMI. In order to improve the quality of life, gender-specific medicine and further investigation regarding a modification of the quality of life by lowering the BMI are essential and necessary. TRIAL REGISTRATION: Study approval by the Ethics Committee of the University Medical Center Göttingen was granted (number 6/5/18).

Description of Osmolyte Pathways in Maturing Mdx Mice Reveals Altered Levels of Taurine and Sodium/Myo-Inositol Co-Transporters.

Duchenne muscular dystrophy (DMD) is a genetic disorder characterized by progressive muscle degeneration. Osmotic stress participates to DMD pathology and altered levels of osmolyte pathway members have been reported. The goal of this study was to gain insight in osmoregulatory changes in the mdx mouse model by examining the expression of osmolyte pathway members, including taurine transporter (TauT), sodium myo-inositol co-transporter (SMIT), betaine GABA transporter (BGT), and aldose reductase (AR) in the skeletal muscles and diaphragm of mdx mice aged 4, 8, 12, and 26 weeks. Necrosis was most prominent in 12 week-old mdx mice, whereas the amount of regenerated fibers increased until week 26 in the tibialis anterior. TauT protein levels were downregulated in the tibialis anterior and gastrocnemius of 4 to 12 week-old mdx mice, but not in 26 week-old mice, whereas TauT levels in the diaphragm remained significantly lower in 26 week-old mdx mice. In contrast, SMIT protein levels were significantly higher in the muscles of mdx mice when compared to controls. Our study revealed differential regulation of osmolyte pathway members in mdx muscle, which points to their complex involvement in DMD pathogenesis going beyond general osmotic stress responses. These results highlight the potential of osmolyte pathway members as a research interest and future therapeutic target in dystrophinopathy.

Exploring the Therapeutic Potential of Ectoine in Duchenne Muscular Dystrophy: Comparison with Taurine, a Supplement with Known Beneficial Effects in the mdx Mouse.

Duchenne Muscular Dystrophy (DMD) is a debilitating muscle disorder that condemns patients to year-long dependency on glucocorticoids. Chronic glucocorticoid use elicits many unfavourable side-effects without offering satisfying clinical improvement, thus, the search for alternative treatments to alleviate muscle inflammation persists. Taurine, an osmolyte with anti-inflammatory effects, mitigated pathological features in the mdx mouse model for DMD but interfered with murine development. In this study, ectoine is evaluated as an alternative for taurine in vitro in CCL-136 cells and in vivo in the mdx mouse. Pre-treating CCL-136 cells with 0.1 mM taurine and 0.1 mM ectoine prior to exposure with 300 U/mL IFN-γ and 20 ng/mL IL-1ß partially attenuated cell death, whilst 100 mM taurine reduced MHC-I protein levels. In vivo, histopathological features of the tibialis anterior in mdx mice were mitigated by ectoine, but not by taurine. Osmolyte treatment significantly reduced mRNA levels of inflammatory disease biomarkers, respectively, CCL2 and SPP1 in ectoine-treated mdx mice, and CCL2, HSPA1A, TNF-α and IL-1ß in taurine-treated mdx mice. Functional performance was not improved by osmolyte treatment. Furthermore, ectoine-treated mdx mice exhibited reduced body weight. Our results confirmed beneficial effects of taurine in mdx mice and, for the first time, demonstrated similar and differential effects of ectoine.

[SARS-CoV-2 antibody response to the second COVID-19 vaccination in neuromuscular disease patients under immune modulating treatment]. / SARS-CoV-2-Antikörper-Antwort auf die zweite COVID-19-Impfung bei neuromuskulären Patienten unter immunmodulierender Therapie.

Successful vaccination (adequate elevation of anti-spike protein antibodies) is attributed with sufficient protection against a severe course of coronavirus disease 2019 (COVID-19). For patients with chronic inflammatory diseases (CID) and immunosuppression the success of vaccination is an ongoing scientific discourse. Therefore, we evaluated the antibody titer against the S1 antigen of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) 2 weeks after complete immunization in patients with an underlying neuromuscular disease (NMD), who presented to our neurological day clinic and outpatient department for regular infusions of immunoglobulins. The data show that patients with chronic autoimmune NMD and simultaneous immunosuppressive or immune modulating treatment show an antibody response after vaccination with both mRNA and vector vaccines. In comparison to healthy subjects there is a comparable number of seroconversions due to the vaccination. A correlation between immunoglobulin dose and vaccination response could not be found; however, in contrast, there was a significant reduction of specific antibody synthesis, especially for the combination of mycophenolate mofetil (MMF) and prednisolone.

Synergistic PXT3003 therapy uncouples neuromuscular function from dysmyelination in male Charcot-Marie-Tooth disease type 1A (CMT1A) rats.

Charcot-Marie-Tooth disease 1 A (CMT1A) is caused by an intrachromosomal duplication of the gene encoding for PMP22 leading to peripheral nerve dysmyelination, axonal loss, and progressive muscle weakness. No therapy is available. PXT3003 is a low-dose combination of baclofen, naltrexone, and sorbitol which has been shown to improve disease symptoms in Pmp22 transgenic rats, a bona fide model of CMT1A disease. However, the superiority of PXT3003 over its single components or dual combinations have not been tested. Here, we show that in a dorsal root ganglion (DRG) co-culture system derived from transgenic rats, PXT3003 induced myelination when compared to its single and dual components. Applying a clinically relevant ("translational") study design in adult male CMT1A rats for 3 months, PXT3003, but not its dual components, resulted in improved performance in behavioral motor and sensory endpoints when compared to placebo. Unexpectedly, we observed only a marginally increased number of myelinated axons in nerves from PXT3003-treated CMT1A rats. However, in electrophysiology, motor latencies correlated with increased grip strength indicating a possible effect of PXT3003 on neuromuscular junctions (NMJs) and muscle fiber pathology. Indeed, PXT3003-treated CMT1A rats displayed an increased perimeter of individual NMJs and a larger number of functional NMJs. Moreover, muscles of PXT3003 CMT1A rats displayed less neurogenic atrophy and a shift toward fast contracting muscle fibers. We suggest that ameliorated motor function in PXT3003-treated CMT1A rats result from restored NMJ function and muscle innervation, independent from myelination.

Treatment with human immunoglobulin G improves the early disease course in a mouse model of Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is a severe hereditary myopathy. Standard treatment by glucocorticosteroids is limited because of numerous side effects. The aim of this study was to test immunomodulation by human immunoglobulin G (IgG) as treatment in the experimental mouse model (mdx) of DMD. 2 g/kg human IgG compared to human albumin was injected intraperitoneally in mdx mice at the age of 3 and 7 weeks. Advanced voluntary wheel running parameters were recorded continuously. At the age of 11 weeks, animals were killed so that blood, diaphragm, and lower limb muscles could be removed for quantitative PCR, histological analysis and ex vivo muscle contraction tests. IgG compared to albumin significantly improved the voluntary running performance and reduced muscle fatigability in an ex vivo muscle contraction test. Upon IgG treatment, serum creatine kinase values were diminished and mRNA expression levels of relevant inflammatory markers were reduced in the diaphragm and limb muscles. Macrophage infiltration and myopathic damage were significantly ameliorated in the quadriceps muscle. Collectively, this study demonstrates that, in the early disease course of mdx mice, human IgG improves the running performance and diminishes myopathic damage and inflammation in the muscle. Therefore, IgG may be a promising approach for treatment of DMD. Two monthly intraperitoneal injections of human immunoglobulin G (IgG) improved the early 11-week disease phase of mdx mice. Voluntary running was improved and serum levels of creatine kinase were diminished. In the skeletal muscle, myopathic damage was ameliorated and key inflammatory markers such as mRNA expression of SPP1 and infiltration by macrophages were reduced. The study suggests that IgG could be explored as a potential treatment option for Duchenne muscular dystrophy and that pre-clinical long-term studies should be helpful.

Scanning for Therapeutic Targets within the Cytokine Network of Idiopathic Inflammatory Myopathies.

The idiopathic inflammatory myopathies (IIM) constitute a heterogeneous group of chronic disorders that include dermatomyositis (DM), polymyositis (PM), sporadic inclusion body myositis (IBM) and necrotizing autoimmune myopathy (NAM). They represent distinct pathological entities that, most often, share predominant inflammation in muscle tissue. Many of the immunopathogenic processes behind the IIM remain poorly understood, but the crucial role of cytokines as essential regulators of the intramuscular build-up of inflammation is undisputed. This review describes the extensive cytokine network within IIM muscle, characterized by strong expression of Tumor Necrosis Factors (TNFα, LTß, BAFF), Interferons (IFNα/ß/γ), Interleukins (IL-1/6/12/15/18/23) and Chemokines (CXCL9/10/11/13, CCL2/3/4/8/19/21). Current therapeutic strategies and the exploration of potential disease modifying agents based on manipulation of the cytokine network are provided. Reported responses to anti-TNFα treatment in IIM are conflicting and new onset DM/PM has been described after administration of anti-TNFα agents to treat other diseases, pointing to the complex effects of TNFα neutralization. Treatment with anti-IFNα has been shown to suppress the IFN type 1 gene signature in DM/PM patients and improve muscle strength. Beneficial effects of anti-IL-1 and anti-IL-6 therapy have also been reported. Cytokine profiling in IIM aids the development of therapeutic strategies and provides approaches to subtype patients for treatment outcome prediction.

Fatigue and associated factors in myasthenia gravis: a nationwide registry study.

Fatigue is commonly associated with myasthenia gravis (MG), but factors contributing to fatigue development in MG are incompletely understood. This nationwide cross-sectional registry study included 1464 patients diagnosed with autoimmune MG, recruited between February 2019 and April 2023. Frequency and severity of fatigue was assessed at study inclusion using the patient-reported Chalder Fatigue Questionnaire (CFQ). Frequency of fatigue was 59%. Fatigue severity strongly correlated with both patient-reported and physician-assessed MG outcome measures (MG-ADL, MG-QoL15, QMG and MGFA classes) and was associated with a history of myasthenic exacerbation and/or myasthenic crises and a delay in diagnosis of more than 1 year after symptom onset. Fatigue was more prevalent in women and coincided with symptoms of depression, anxiety, and sleep dissatisfaction. Differences in fatigue severity were observed between antibody (ab) subgroups, with highest fatigue severity in LRP4-ab-positive patients and lowest fatigue severity in AChR-ab-positive patients. Fatigue is a frequent and clinically highly relevant symptom of MG. Early diagnosis and prevention of MG crises may limit the long-term burden of fatigue in patients with MG.

Landscape analysis of available European data sources amenable for machine learning and recommendations on usability for rare diseases screening.

BACKGROUND: Patient registries and databases are essential tools for advancing clinical research in the area of rare diseases, as well as for enhancing patient care and healthcare planning. The primary aim of this study is a landscape analysis of available European data sources amenable to machine learning (ML) and their usability for Rare Diseases screening, in terms of findable, accessible, interoperable, reusable(FAIR), legal, and business considerations. Second, recommendations will be proposed to provide a better understanding of the health data ecosystem. METHODS: In the period of March 2022 to December 2022, a cross-sectional study using a semi-structured questionnaire was conducted among potential respondents, identified as main contact person of a health-related databases. The design of the self-completed questionnaire survey instrument was based on information drawn from relevant scientific publications, quantitative and qualitative research, and scoping review on challenges in mapping European rare disease (RD) databases. To determine database characteristics associated with the adherence to the FAIR principles, legal and business aspects of database management Bayesian models were fitted. RESULTS: In total, 330 unique replies were processed and analyzed, reflecting the same number of distinct databases (no duplicates included). In terms of geographical scope, we observed 24.2% (n = 80) national, 10.0% (n = 33) regional, 8.8% (n = 29) European, and 5.5% (n = 18) international registries coordinated in Europe. Over 80.0% (n = 269) of the databases were still active, with approximately 60.0% (n = 191) established after the year 2000 and 71.0% last collected new data in 2022. Regarding their geographical scope, European registries were associated with the highest overall FAIR adherence, while registries with regional and "other" geographical scope were ranked at the bottom of the list with the lowest proportion. Responders' willingness to share data as a contribution to the goals of the Screen4Care project was evaluated at the end of the survey. This question was completed by 108 respondents; however, only 18 of them (16.7%) expressed a direct willingness to contribute to the project by sharing their databases. Among them, an equal split between pro-bono and paid services was observed. CONCLUSIONS: The most important results of our study demonstrate not enough sufficient FAIR principles adherence and low willingness of the EU health databases to share patient information, combined with some legislation incapacities, resulting in barriers to the secondary use of data.

Patient experiences of interprofessional collaboration and intersectoral communication in rare disease healthcare in Germany - a mixed-methods study.

BACKGROUND: Rare diseases are often complex, chronic and many of them life-shortening. In Germany, healthcare for rare diseases is organized in expert centers for rare diseases. Most patients additionally have regional general practicioners and specialists for basic medical care. Thus, collaboration and information exchange between sectors is highly relevant. Our study focuses on the patient and caregiver perspective on intersectoral and interdisciplinary care between local healthcare professionals (HCPs) and centers for rare diseases in Germany. The aims were (1) to investigate patients' and caregivers' general experience of healthcare, (2) to analyse patients' and caregivers' perception of collaboration and cooperation between local healthcare professionals and expert centers for rare diseases and (3) to investigate patients' and caregivers' satisfaction with healthcare in the expert centers for rare diseases. RESULTS: In total 299 individuals of whom 176 were patients and 123 were caregivers to pediatric patients participated in a survey using a questionnaire comprising several instruments and constructs. Fifty participants were additionally interviewed using a semistructured guideline. Most patients reported to receive written information about their care, have a contact person for medical issues and experienced interdisciplinary exchange within the centers for rare diseases. Patients and caregivers in our sample were mainly satisfied with the healthcare in the centers for rare diseases. The qualitative interviews showed a rather mixed picture including experiences of uncoordinated care, low engagement and communication difficulties between professionals of different sectors. Patients reported several factors that influenced the organization and quality of healthcare e.g. engagement and health literacy in patients or engagement of HCPs. CONCLUSIONS: Our findings indicate the high relevance of transferring affected patients to specialized care as fast as possible to provide best medical treatment and increase patient satisfaction. Intersectoral collaboration should exceed written information exchange and should unburden patients of being and feeling responsible for communication between sectors and specialists. Results indicate a lack of inclusion of psychosocial aspects in routine care, which suggests opportunities for necessary improvements.

Provision of an explanation for the inefficacy of immunotherapy in sporadic inclusion body myositis: quantitative assessment of inflammation and ß-amyloid in the muscle.

OBJECTIVE: In sporadic inclusion body myositis (IBM), inflammation and accumulation of ß-amyloid-associated molecules cause muscle fiber damage. We undertook this study to determine why intravenous immunoglobulin (IVIG) and prednisone are not effective in sporadic IBM despite their effectiveness in other inflammatory myopathies. METHODS: Relevant inflammatory and degeneration- associated markers were assessed by quantitative polymerase chain reaction and immunohistochemistry in repeated muscle biopsy specimens from patients with sporadic IBM treated in a controlled study with IVIG and prednisone (n = 5) or with prednisone alone (n = 5). Functional effects were assessed in a muscle cell culture model. RESULTS: In muscle biopsy specimens, messenger RNA (mRNA) expression of the proinflammatory chemokines CXCL9, CCL3, and CCL4 and of the cytokines interferon-γ (IFNγ), transforming growth factor ß, interleukin-10 (IL-10), and IL-1ß was significantly reduced after treatment in both groups. No consistent changes were observed for tumor necrosis factor α, IL-6, inducible costimulator (ICOS), its ligand ICOSL, and perforin. Messenger RNA expression of the degeneration-associated molecule ubiquitin and the heat-shock protein αB-crystallin was also reduced, but no changes were noted for amyloid precursor protein (APP) or desmin. By immunohistochemistry, a significant down-modulation of chemokines was observed, but not of inducible nitric oxide (NO) synthase, nitrotyrosine, IL-1ß, APP, and ubiquitin; ß-amyloid was reduced in 6 of 10 patients. Pronounced staining of IgG was observed in the muscle after treatment with IVIG, indicating penetration of infused IgG into the muscle and a possible local effect. In muscle cells exposed to IFNγ plus IL-1ß, IgG and/or prednisone down-regulated mRNA expression of IL-1ß 2.5-fold. Accumulation of ß-amyloid, overexpression of αB-crystallin, and cell death were prevented. In contrast, NO-associated cell stress remained unchanged. CONCLUSION: IVIG and prednisone reduce some inflammatory and degenerative molecules in muscle of patients with sporadic IBM and in vitro, but do not sufficiently suppress myotoxic and cell stress mediators such as NO. The data provide an explanation for the resistance of sporadic IBM to immunotherapy and identify markers that may help to design novel treatment strategies.

Nitric oxide stress in sporadic inclusion body myositis muscle fibres: inhibition of inducible nitric oxide synthase prevents interleukin-1ß-induced accumulation of ß-amyloid and cell death.

Sporadic inclusion body myositis is a severely disabling myopathy. The design of effective treatment strategies is hampered by insufficient understanding of the complex disease pathology. Particularly, the nature of interrelationships between inflammatory and degenerative pathomechanisms in sporadic inclusion body myositis has remained elusive. In Alzheimer's dementia, accumulation of ß-amyloid has been shown to be associated with upregulation of nitric oxide. Using quantitative polymerase chain reaction, an overexpression of inducible nitric oxide synthase was observed in five out of ten patients with sporadic inclusion body myositis, two of eleven with dermatomyositis, three of eight with polymyositis, two of nine with muscular dystrophy and two of ten non-myopathic controls. Immunohistochemistry confirmed protein expression of inducible nitric oxide synthase and demonstrated intracellular nitration of tyrosine, an indicator for intra-fibre production of nitric oxide, in sporadic inclusion body myositis muscle samples, but much less in dermatomyositis or polymyositis, hardly in dystrophic muscle and not in non-myopathic controls. Using fluorescent double-labelling immunohistochemistry, a significant co-localization was observed in sporadic inclusion body myositis muscle between ß-amyloid, thioflavine-S and nitrotyrosine. In primary cultures of human myotubes and in myoblasts, exposure to interleukin-1ß in combination with interferon-γ induced a robust upregulation of inducible nitric oxide synthase messenger RNA. Using fluorescent detectors of reactive oxygen species and nitric oxide, dichlorofluorescein and diaminofluorescein, respectively, flow cytometry revealed that interleukin-1ß combined with interferon-γ induced intracellular production of nitric oxide, which was associated with necrotic cell death in muscle cells. Intracellular nitration of tyrosine was noted, which partly co-localized with amyloid precursor protein, but not with desmin. Pharmacological inhibition of inducible nitric oxide synthase by 1400W reduced intracellular production of nitric oxide and prevented accumulation of ß-amyloid, nitration of tyrosine as well as cell death inflicted by interleukin-1ß combined with interferon-γ. Collectively, these data suggest that, in skeletal muscle, inducible nitric oxide synthase is a central component of interactions between interleukin-1ß and ß-amyloid, two of the most relevant molecules in sporadic inclusion body myositis. The data further our understanding of the pathology of sporadic inclusion body myositis and may point to novel treatment strategies.

Why Temporal Inference Stimulation May Fail in the Human Brain: A Pilot Research Study.

Temporal interference stimulation (TIS) aims at targeting deep brain areas during transcranial electrical alternating current stimulation (tACS) by generating interference fields at depth. Although its modulatory effects have been demonstrated in animal and human models and stimulation studies, direct experimental evidence is lacking for its utility in humans (in vivo). Herein, we directly test and compare three different structures: firstly, we perform peripheral nerve and muscle stimulation quantifying muscle twitches as readout, secondly, we stimulate peri-orbitally with phosphene perception as a surrogate marker, and thirdly, we attempt to modulate the mean power of alpha oscillations in the occipital area as measured with electroencephalography (EEG). We found strong evidence for stimulation efficacy on the modulated frequency in the PNS, but we found no evidence for its utility in the CNS. Possible reasons for failing to activate CNS targets could be comparatively higher activation thresholds here or inhibitory stimulation components to the carrier frequency interfering with the effects of the modulated signal.

Genetic newborn screening and digital technologies: A project protocol based on a dual approach to shorten the rare diseases diagnostic path in Europe.

Since 72% of rare diseases are genetic in origin and mostly paediatrics, genetic newborn screening represents a diagnostic "window of opportunity". Therefore, many gNBS initiatives started in different European countries. Screen4Care is a research project, which resulted of a joint effort between the European Union Commission and the European Federation of Pharmaceutical Industries and Associations. It focuses on genetic newborn screening and artificial intelligence-based tools which will be applied to a large European population of about 25.000 infants. The neonatal screening strategy will be based on targeted sequencing, while whole genome sequencing will be offered to all enrolled infants who may show early symptoms but have resulted negative at the targeted sequencing-based newborn screening. We will leverage artificial intelligence-based algorithms to identify patients using Electronic Health Records (EHR) and to build a repository "symptom checkers" for patients and healthcare providers. S4C will design an equitable, ethical, and sustainable framework for genetic newborn screening and new digital tools, corroborated by a large workout where legal, ethical, and social complexities will be addressed with the intent of making the framework highly and flexibly translatable into the diverse European health systems.

Motor performance of young dystrophic mdx mice treated with long-circulating prednisolone liposomes.

For Duchenne muscular dystrophy (DMD), a common myopathy that leads to severe disability, no causal therapy is available. Glucocorticosteroids improve patients' muscle strength, but their long-term use is limited by negative side effects. Thus, pharmacological modifications of glucocorticosteroids are required to increase the efficacy by drug targeting. Liposomal encapsulation augments systemic half-life and local tissue concentrations of glucocorticosteroids and, at the same time, reduces systemic side effects. In this study, the efficacy of novel, long-circulating, polyethylene-glycol-coated liposomes encapsulating prednisolone was compared with free prednisolone in the treatment of mdx mice, a well-established animal model for DMD. Using an objective and sensitive computerized 24-hr detection system of voluntary wheel-running in single cages, we demonstrate a significant impairment of the running performance in mdx compared with black/10 control mice aged 3-6 weeks. Treatment with liposomal or free prednisolone did not improve running performance compared with saline control or empty liposomes. Histopathological parameters, including the rate of internalized nuclei and fiber size variation, and mRNA and protein expression levels of transforming growth factor (TGF)-ß and monocytes chemotactic protein (MCP)-1 also remained unchanged. Bioactivity in skeletal muscle of liposomal and free prednisolone was demonstrated by elevated mRNA expression of muscle ring finger protein 1 (MuRF1), a mediator of muscle atrophy, and its forkhead box transcription factors (Foxo1/3). Our data support the assessment of voluntary running to be a robust and reproducible outcome measure of skeletal muscle performance during the early disease course of mdx mice and suggest that liposomal encapsulation is not superior in treatment efficacy compared with conventional prednisolone. Our study helps to improve the future design of experimental treatment in animal models of neuromuscular diseases.

Simultaneous assessment of lung morphology and respiratory motion in retrospectively gated in-vivo microCT of free breathing anesthetized mice.

Retrospective gating (RG) is a well established technique in preclinical computed tomography (CT) to assess 3D morphology of the lung. In RG additional angular projections are recorded typically by performing multiple rotations. Consequently, the projections are sorted according to the expansion state of the chest and those sets are then reconstructed separately. Thus, the breathing motion artefacts are suppressed at a cost of strongly elevated X-ray dose levels. Here we propose to use the entire raw data to assess respiratory motion in addition to retrospectively gated 3D reconstruction that visualize anatomical structures of the lung. Using this RG based X-ray respiratory motion measurement approach, which will be referred to as RG based X-ray lung function measurement (rgXLF) on the example of the mdx mouse model of Duchenne muscle dystrophy (mdx) we accurately obtained both the 3D anatomical morphology of the lung and the thoracic bones as well as functional temporal parameters of the lung. Thus, rgXLF will remove the necessity for separate acquisition procedures by being able to reproduce comparable results to the previously established planar X-ray based lung function measurement approach in a single low dose CT scan.

Non-Invasive Optical Motion Tracking Allows Monitoring of Respiratory Dynamics in Dystrophin-Deficient Mice.

Duchenne muscular dystrophy (DMD) is the most common x-chromosomal inherited dystrophinopathy which leads to progressive muscle weakness and a premature death due to cardiorespiratory dysfunction. The mdx mouse lacks functional dystrophin protein and has a comparatively human-like diaphragm phenotype. To date, diaphragm function can only be inadequately mapped in preclinical studies and a simple reliable translatable method of tracking the severity of the disease still lacks. We aimed to establish a sensitive, reliable, harmless and easy way to assess the effects of respiratory muscle weakness and subsequent irregularity in breathing pattern. Optical respiratory dynamics tracking (ORDT) was developed utilising a camera to track the movement of paper markers placed on the thoracic-abdominal region of the mouse. ORDT successfully distinguished diseased mdx phenotype from healthy controls by measuring significantly higher expiration constants (k) in mdx mice compared to wildtype (wt), which were also observed in the established X-ray based lung function (XLF). In contrast to XLF, with ORDT we were able to distinguish distinct fast and slow expiratory phases. In mdx mice, a larger part of the expiratory marker displacement was achieved in this initial fast phase as compared to wt mice. This phenomenon could not be observed in the XLF measurements. We further validated the simplicity and reliability of our approach by demonstrating that it can be performed using free-hand smartphone acquisition. We conclude that ORDT has a great preclinical potential to monitor DMD and other neuromuscular diseases based on changes in the breathing patterns with the future possibility to track therapy response.

The Evolution of Complex Muscle Cell In Vitro Models to Study Pathomechanisms and Drug Development of Neuromuscular Disease.

Many neuromuscular disease entities possess a significant disease burden and therapeutic options remain limited. Innovative human preclinical models may help to uncover relevant disease mechanisms and enhance the translation of therapeutic findings to strengthen neuromuscular disease precision medicine. By concentrating on idiopathic inflammatory muscle disorders, we summarize the recent evolution of the novel in vitro models to study disease mechanisms and therapeutic strategies. A particular focus is laid on the integration and simulation of multicellular interactions of muscle tissue in disease phenotypes in vitro. Finally, the requirements of a neuromuscular disease drug development workflow are discussed with a particular emphasis on cell sources, co-culture systems (including organoids), functionality, and throughput.

Evidence of Two Novel LAMA2 Variants in a Patient With Muscular Dystrophy: Facing the Challenges of a Certain Diagnosis.

Background: Benefits and challenges resulting from advances in genetic diagnostics are two sides of the same coin. Facilitation of a correct and timely diagnosis is paralleled by challenges in interpretation of variants of unknown significance (VUS). Focusing on an individual VUS-re-classification pipeline, this study offers a diagnostic approach for clinically suspected hereditary muscular dystrophy by combining the expertise of an interdisciplinary team. Methods: In a multi-step approach, a thorough phenotype assessment including clinical examination, laboratory work, muscle MRI and histopathological evaluation of muscle was performed in combination with advanced Next Generation Sequencing (NGS). Different in-silico tools and prediction programs like Alamut, SIFT, Polyphen, MutationTaster and M-Cap as well as 3D- modeling of protein structure and RNA-sequencing were employed to determine clinical significance of the LAMA2 variants. Results: Two previously unknown sequence alterations in LAMA2 were detected, a missense variant was classified initially according to ACMG guidelines as a VUS (class 3) whereas a second splice site variant was deemed as likely pathogenic (class 4). Pathogenicity of the splice site variant was confirmed by mRNA sequencing and nonsense mediated decay (NMD) was detected. Combination of the detected variants could be associated to the LGMDR23-phenotype based on the MRI matching and literature research. Discussion: Two novel variants in LAMA2 associated with LGMDR23-phenotype are described. This study illustrates challenges of the genetic findings due to their VUS classification and elucidates how individualized diagnostic procedure has contributed to the accurate diagnosis in the spectrum of LGMD.