Evaluation of Neuromuscular Diseases and Complaints by Quantitative Muscle MRI.

Background: Quantitative muscle MRI (qMRI) is a promising tool for evaluating and monitoring neuromuscular disorders (NMD). However, the application of different imaging protocols and processing pipelines restricts comparison between patient cohorts and disorders. In this qMRI study, we aim to compare dystrophic (limb-girdle muscular dystrophy), inflammatory (inclusion body myositis), and metabolic myopathy (Pompe disease) as well as patients with post-COVID-19 conditions suffering from myalgia to healthy controls. Methods: Ten subjects of each group underwent a 3T lower extremity muscle MRI, including a multi-echo, gradient-echo, Dixon-based sequence, a multi-echo, spin-echo (MESE) T2 mapping sequence, and a spin-echo EPI diffusion-weighted sequence. Furthermore, the following clinical assessments were performed: Quick Motor Function Measure, patient questionnaires for daily life activities, and 6-min walking distance. Results: Different involvement patterns of conspicuous qMRI parameters for different NMDs were observed. qMRI metrics correlated significantly with clinical assessments. Conclusions: qMRI metrics are suitable for evaluating patients with NMD since they show differences in muscular involvement in different NMDs and correlate with clinical assessments. Still, standardisation of acquisition and processing is needed for broad clinical use.

Proteomic studies in VWA1-related neuromyopathy allowed new pathophysiological insights and the definition of blood biomarkers.

Bi-allelic variants in VWA1, encoding Von Willebrand Factor A domain containing 1 protein localized to the extracellular matrix (ECM), were linked to a neuromuscular disorder with manifestation in child- or adulthood. Clinical findings indicate a neuromyopathy presenting with muscle weakness. Given that pathophysiological processes are still incompletely understood, and biomarkers are still missing, we aimed to identify blood biomarkers of pathophysiological relevance: white blood cells (WBC) and plasma derived from six VWA1-patients were investigated by proteomics. Four proteins, BET1, HNRNPDL, NEFM and PHGDH, known to be involved in neurological diseases and dysregulated in WBC were further validated by muscle-immunostainings unravelling HNRNPDL as a protein showing differences between VWA1-patients, healthy controls and patients suffering from neurogenic muscular atrophy and BICD2-related neuromyopathy. Immunostaining studies of PHGDH indicate its involvement in apoptotic processes via co-localisation with caspase-3. NEFM showed an increase in cells within the ECM in biopsies of all patients studied. Plasma proteomics unravelled dysregulation of 15 proteins serving as biomarker candidates among which a profound proportion of increased ones (6/11) are mostly related to antioxidative processes and have even partially been described as blood biomarkers for other entities of neuromuscular disorders before. CRP elevated in plasma also showed an increase in the extracellular space of VWA1-mutant muscle. Results of our combined studies for the first time describe pathophysiologically relevant biomarkers for VWA1-related neuromyopathy and suggest that VWA1-patient derived blood might hold the potential to study disease processes of clinical relevance, an important aspect for further preclinical studies.

Exome Sequencing and Optical Genome Mapping in Molecularly Unsolved Cases of Duchenne Muscular Dystrophy: Identification of a Causative X-Chromosomal Inversion Disrupting the DMD Gene.

Duchenne muscular dystrophy (DMD) is a severe progressive muscle disease that mainly affects boys due to X-linked recessive inheritance. In most affected individuals, MLPA or sequencing-based techniques detect deletions, duplications, or point mutations in the dystrophin-encoding DMD gene. However, in a small subset of patients clinically diagnosed with DMD, the molecular cause is not identified with these routine methods. Evaluation of the 60 DMD patients in our center revealed three cases without a known genetic cause. DNA samples of these patients were analyzed using whole-exome sequencing (WES) and, if unconclusive, optical genome mapping (OGM). WES led to a diagnosis in two cases: one patient was found to carry a splice mutation in the DMD gene that had not been identified during previous Sanger sequencing. In the second patient, we detected two variants in the fukutin gene (FKTN) that were presumed to be disease-causing. In the third patient, WES was unremarkable, but OGM identified an inversion disrupting the DMD gene (~1.28 Mb) that was subsequently confirmed with long-read sequencing. These results highlight the importance of reanalyzing unsolved cases using WES and demonstrate that OGM is a useful method for identifying large structural variants in cases with unremarkable exome sequencing.

Microscopic and Biochemical Hallmarks of BICD2-Associated Muscle Pathology toward the Evaluation of Novel Variants.

BICD2 variants have been linked to neurodegenerative disorders like spinal muscular atrophy with lower extremity predominance (SMALED2) or hereditary spastic paraplegia (HSP). Recently, mutations in BICD2 were implicated in myopathies. Here, we present one patient with a known and six patients with novel BICD2 missense variants, further characterizing the molecular landscape of this heterogenous neurological disorder. A total of seven patients were genotyped and phenotyped. Skeletal muscle biopsies were analyzed by histology, electron microscopy, and protein profiling to define pathological hallmarks and pathogenicity markers with consecutive validation using fluorescence microscopy. Clinical and MRI-features revealed a typical pattern of distal paresis of the lower extremities as characteristic features of a BICD2-associated disorder. Histological evaluation showed myopathic features of varying severity including fiber size variation, lipofibromatosis, and fiber splittings. Proteomic analysis with subsequent fluorescence analysis revealed an altered abundance and localization of thrombospondin-4 and biglycan. Our combined clinical, histopathological, and proteomic approaches provide new insights into the pathophysiology of BICD2-associated disorders, confirming a primary muscle cell vulnerability. In this context, biglycan and thrombospondin-4 have been identified, may serve as tissue pathogenicity markers, and might be linked to perturbed protein secretion based on an impaired vesicular transportation.

Quantitative muscle MRI captures early muscle degeneration in calpainopathy.

To evaluate differences in qMRI parameters of muscle diffusion tensor imaging (mDTI), fat-fraction (FF) and water T2 time in leg muscles of calpainopathy patients (LGMD R1/D4) compared to healthy controls, to correlate those findings to clinical parameters and to evaluate if qMRI parameters show muscle degeneration in not-yet fatty infiltrated muscles. We evaluated eight thigh and seven calf muscles of 19 calpainopathy patients and 19 healthy matched controls. MRI scans were performed on a 3T MRI including a mDTI, T2 mapping and mDixonquant sequence. Clinical assessment was done with manual muscle testing, patient questionnaires (ACTIVLIM, NSS) as well as gait analysis. Average FF was significantly different in all muscles compared to controls (p < 0.001). In muscles with less than 8% FF a significant increase of FA (p < 0.005) and decrease of RD (p < 0.004) was found in high-risk muscles of calpainopathy patients. Water T2 times were increased within the low- and intermediate-risk muscles (p ≤ 0.045) but not in high-risk muscles (p = 0.062). Clinical assessments correlated significantly with qMRI values: QMFM vs. FF: r = - 0.881, p < 0.001; QMFM versus FA: r = - 0.747, p < 0.001; QMFM versus MD: r = 0.942, p < 0.001. A good correlation of FF and diffusion metrics to clinical assessments was found. Diffusion metrics and T2 values are promising candidates to serve as sensitive early and non-invasive methods to capture early muscle degeneration in non-fat-infiltrated muscles in calpainopathies.

ANO5-related muscle diseases: From clinics and genetics to pathology and research strategies.

Anoctamin-5 (ANO5) is a multi-pass membrane protein localized to the sarcolemma and the sarcoplasmic reticulum. Mutations were linked to rare autosomal recessive muscle diseases. Here, we summarize the clinical spectrum, imaging data and molecular research findings as well as results of animal modeling, which significantly moved forward the understanding of mechanisms underlying ANO5-related muscle diseases. Given that precise histological information on inflammatory processes taking place in patient-derived muscle are still lacking, an (immuno)histological study on biopsies derived from six ANO5-patients was performed showing focal accumulation of necrotic fibers, mild fiber-size variances and myophagocytosis. In addition, MRI data of four ANO5-patients (including a 10-year follow-up in one patient) are presented and discussed in the context of previously published MRI-findings. Hence, data presented in this article combining a review of the literature with own myopathological findings address scientific trends and open questions on ANO5-related muscle diseases, which would be of significant interest for a wide neuromuscular diseases community. To conclude, a clear genotype-phenotype correlation does not exist, and ANO5-related muscle disorders might represent the next entity of a clinical continuum with varying degree of muscle cell pathologies. In addition, results of pre-clinical studies allowed the definition of suitable cell and animal models characterized by certain histological and functional pathologies resembling the human phenotype. These models might serve as suitable systems for testing of interventional concepts in future.

Small fiber involvement is independent from clinical pain in late-onset Pompe disease.

BACKGROUND: Pain occurs in the majority of patients with late onset Pompe disease (LOPD) and is associated with a reduced quality of life. The aim of this study was to analyse the pain characteristics and its relation to a small nerve fiber involvement in LOPD patients. METHODS: In 35 patients with LOPD under enzyme replacement therapy without clinical signs of polyneuropathy (19 females; 51 ± 15 years), pain characteristics as well as depressive and anxiety symptoms were assessed using the PainDetect questionnaire (PDQ) and the hospital anxiety and depression scale (HADS), respectively. Distal skin biopsies were analysed for intraepidermal nerve fiber density (IENFD) and compared to age- and gender-matched reference data. Skin biopsies from 20 healthy subjects served as controls to assure validity of the morphometric analysis. RESULTS: Pain was reported in 69% of the patients with an average intensity of 4.1 ± 1.1 on the numeric rating scale (NRS; anchors: 0-10). According to PDQ, neuropathic pain was likely in one patient, possible in 29%, and unlikely in 67%. Relevant depression and anxiety symptoms occurred in 31% and 23%, respectively, and correlated with pain intensity. Distal IENFD (3.98 ± 1.95 fibers/mm) was reduced in 57% of the patients. The degree of IENFD reduction did not correlate with the durations of symptoms to ERT or duration of ERT to biopsy. CONCLUSIONS: Pain is a frequent symptom in treated LOPD on ERT, though a screening questionnaire seldom indicated neuropathic pain. The high frequency of small nerve fiber pathology in a treated LOPD cohort was found regardless of the presence of pain or comorbid risk factors for SFN and needs further exploration in terms of clinical context, exact mechanisms and when developing novel therapeutic options for LOPD.

Robustness and stability of volume-based tractography in a multicenter setting.

Muscle diffusion tensor imaging (mDTI)-based tractography is a promising tool with which to detect subclinical changes in muscle injuries and to evaluate pathophysiology in neuromuscular diseases. Classic region of interest (ROI)-based tractography is very time-consuming and requires an examiner with extensive experience. (Semi)automatic approaches such as volume-based tractography (VBT) can diminish this problem but its robustness and stability are unknown. The aim of the current study was to assess the performance of VBT in a multicenter setting and to evaluate semiautomatic segmentation approaches in the analysis of VBT-derived data in terms of the comparability of the outcome measures. Five traveling volunteers underwent 3-T mDTI of seven calf muscles of both legs at six different MR sites. Tract properties and diffusion metrics were calculated using VBT. Within-subject coefficients of variance (wsCVs) and intraclass correlation coefficients (ICCs) were calculated to assess the multicenter reproducibility of tract properties such as tract density (TD), mean tract length, volume and tract propagation angle, and diffusion metrics such as fractional anisotropy, mean diffusivity, axial diffusivity (λ1 ) and radial diffusivity in traveling subjects. Furthermore, 50 individual datasets from five different centers (10 datasets per center) were pooled to assess the feasibility of VBT with manual and semiautomatic segmentation. To assess the differences of tract properties and diffusion metrics between segmentation approaches an ANOVA was performed, and ICC and Bland-Altman plots were analyzed. wsCVs and ICCs showed good reproducibility of the tract properties TD and volume, as well as diffusion metrics. ANOVA showed no significant differences between manual and semiautomatic approaches. ICCs were excellent (≥ 0.992) and Bland-Altman analysis did not reveal any systemic bias between the methods. Tract properties and diffusion metrics derived from VBT showed good comparability among centers. Semiautomatic approaches revealed excellent agreement with gold standard of manual segmentation. These findings suggest that pooling data from different centers to construct a reference database for tractography results is feasible using semiautomatic segmentation approaches.

Seroprevalence of Binding and Neutralizing Antibodies against 39 Human Adenovirus Types in Patients with Neuromuscular Disorders.

High pre-existing antibodies against viral vectors reduce their functionality and may lead to adverse complications. To circumvent this problem in future gene therapy approaches, we tested the seroprevalence of a large range of human adenovirus types in patients with neuromuscular disorders (NMDs) to find appropriate viral vector candidates for gene replacement therapy for NMDs. Binding and neutralizing antibodies against 39 human adenovirus types were tested in the sera of 133 patients with NMDs and 76 healthy controls aged 17-92 years. The influence of age, sex, and NMDs on antibody levels was analyzed. The seroprevalence of different adenoviruses in the cohort varied widely. The highest levels of binding antibodies were detected against HAdV-D27, -C1, -D24, -D70, -B14, -C6, -D13, -B34, and -E4, whereas the lowest reactivity was detected against HAdV-F41, -A31, -B11, -D75, -D8, -D65, -D26, -D80, and -D17. The highest neutralizing reactivity was observed against HAdV-B3, -C2, -E4, -C1, -G52, -C5, and -F41, whereas the lowest neutralizing reactivity was observed against HAdV-D74, -B34, -D73, -B37, -D48, -D13, -D75, -D8, -B35, and -B16. We detected no influence of sex and only minor differences between different age groups. Importantly, there were no significant differences between healthy controls and patients with NMDs. Our data show that patients with NMDs have very similar levels of binding and neutralizing antibodies against HAdV compared to healthy individuals, and we identified HAdV-A31, -B16, -B34, -B35, -D8, -D37, -D48, -D73, -D74, -D75, and -D80 as promising candidates for future vector development due to their low binding and neutralizing antibody prevalence.

High Inter-Rater Reliability of Manual Segmentation and Volume-Based Tractography in Healthy and Dystrophic Human Calf Muscle.

BACKGROUND: Muscle diffusion tensor imaging (mDTI) is a promising surrogate biomarker in the evaluation of muscular injuries and neuromuscular diseases. Since mDTI metrics are known to vary between different muscles, separation of different muscles is essential to achieve muscle-specific diffusion parameters. The commonly used technique to assess DTI metrics is parameter maps based on manual segmentation (MSB). Other techniques comprise tract-based approaches, which can be performed in a previously defined volume. This so-called volume-based tractography (VBT) may offer a more robust assessment of diffusion metrics and additional information about muscle architecture through tract properties. The purpose of this study was to assess DTI metrics of human calf muscles calculated with two segmentation techniques-MSB and VBT-regarding their inter-rater reliability in healthy and dystrophic calf muscles. METHODS: 20 healthy controls and 18 individuals with different neuromuscular diseases underwent an MRI examination in a 3T scanner using a 16-channel Torso XL coil. DTI metrics were assessed in seven calf muscles using MSB and VBT. Coefficients of variation (CV) were calculated for both techniques. MSB and VBT were performed by two independent raters to assess inter-rater reliability by ICC analysis and Bland-Altman plots. Next to analysis of DTI metrics, the same assessments were also performed for tract properties extracted with VBT. RESULTS: For both techniques, low CV were found for healthy controls (≤13%) and neuromuscular diseases (≤17%). Significant differences between methods were found for all diffusion metrics except for λ1. High inter-rater reliability was found for both MSB and VBT (ICC ≥ 0.972). Assessment of tract properties revealed high inter-rater reliability (ICC ≥ 0.974). CONCLUSIONS: Both segmentation techniques can be used in the evaluation of DTI metrics in healthy controls and different NMD with low rater dependency and high precision but differ significantly from each other. Our findings underline that the same segmentation protocol must be used to ensure comparability of mDTI data.

FLNC-Associated Myofibrillar Myopathy: New Clinical, Functional, and Proteomic Data.

OBJECTIVE: To determine whether a new indel mutation in the dimerization domain of filamin C (FLNc) causes a hereditary myopathy with protein aggregation in muscle fibers, we clinically and molecularly studied a German family with autosomal dominant myofibrillar myopathy (MFM). METHODS: We performed mutational analysis in 3 generations, muscle histopathology, and proteomic studies of IM protein aggregates. Functional consequences of the FLNC mutation were investigated with interaction and transfection studies and biophysics molecular analysis. RESULTS: Eight patients revealed clinical features of slowly progressive proximal weakness associated with a heterozygous c.8025_8030delCAAGACinsA (p.K2676Pfs*3) mutation in FLNC. Two patients exhibited a mild cardiomyopathy. MRI of skeletal muscle revealed lipomatous changes typical for MFM with FLNC mutations. Muscle biopsies showed characteristic MFM findings with protein aggregation and lesion formation. The proteomic profile of aggregates was specific for MFM-filaminopathy and indicated activation of the ubiquitin-proteasome system (UPS) and autophagic pathways. Functional studies revealed that mutant FLNc is misfolded, unstable, and incapable of forming homodimers and heterodimers with wild-type FLNc. CONCLUSIONS: This new MFM-filaminopathy family confirms that expression of mutant FLNC leads to an adult-onset muscle phenotype with intracellular protein accumulation. Mutant FLNc protein is biochemically compromised and leads to dysregulation of protein quality control mechanisms. Proteomic analysis of MFM protein aggregates is a potent method to identify disease-relevant proteins, differentiate MFM subtypes, evaluate the relevance of gene variants, and identify novel MFM candidate genes.

Quantitative Muscle-MRI Correlates with Histopathology in Skeletal Muscle Biopsies.

BACKGROUND: Skeletal muscle biopsy is one of the gold standards in the diagnostic workup of muscle disorders. By histopathologic analysis, characteristic features like inflammatory cellular infiltrations, fat and collagen replacement of muscle tissue or structural defects of the myofibers can be detected. In the past years, novel quantitative MRI (qMRI) techniques have been developed to quantify tissue parameters, thus providing a non-invasive diagnostic tool in several myopathies. OBJECTIVE: This proof-of-principle study was performed to validate the qMRI-techniques to skeletal muscle biopsy results. METHODS: Ten patients who underwent skeletal muscle biopsy for diagnostic purposes were examined by qMRI. Fat fraction, water T2-time and diffusion parameters were measured in the muscle from which the biopsy was taken. The proportion of fat tissue, the severity of degenerative and inflammatory parameters and the amount of type 1- and type 2- muscle fibers were determined in all biopsy samples. The qMRI-data were then correlated to the histopathological findings. RESULTS: The amount of fat tissue in skeletal muscle biopsy correlated significantly with the fat fraction derived from the Dixon sequence. The water T2-time, a parameter for tissue edema, correlated with the amount of vacuolar changes of myofibers and endomysial macrophages in the histopathologic analysis. No significant correlations were found for diffusion parameters. CONCLUSION: In this proof-of-principle study, qMRI techniques were related to characteristic histopathologic features in neuromuscular disorders. The study provides the basis for further development of qMRI methods in the follow-up of patients with neuromuscular disorders, especially in the context of emerging treatment strategies.

Chaperones in sporadic inclusion body myositis-Validation of proteomic data.

INTRODUCTION: Sporadic inclusion body myositis (sIBM) is characterized by myopathological features including rimmed vacuoles (RVs) and proteins associated with protein aggregation, autophagy, and inflammation. Previous proteomic studies of RV areas revealed an overrepresentation of several chaperones and subunits of the T-complex protein 1 (TCP-1), which is involved in prevention of protein aggregation. METHODS: To validate our proteomic findings, immunofluorescence analyses of selected chaperones and quantitative Western blot analysis of TCP-1 proteins were performed in five sIBM patients and five healthy controls. RESULTS: Immunofluorescence studies confirmed increased immunoreactivity for VCP, UNC45B, GRP-75, αB-crystallin, LAMP-2, Rab-7a, and TCP-1α and TCP-θ in RVs. Quantitative Western blot analysis revealed a significantly higher level of TCP-1 in sIBM muscle tissue when compared with healthy controls. DISCUSSION: Our study findings validate new insights in protein quality control and degradation processes that seem to be relevant in sIBM. These data provide an important basis for future functional and therapeutic studies.

Treadmill Training with HAL Exoskeleton-A Novel Approach for Symptomatic Therapy in Patients with Limb-Girdle Muscular Dystrophy-Preliminary Study.

Purpose: Exoskeletons have been developed for rehabilitation of patients with walking impairment due to neurological disorders. Recent studies have shown that the voluntary-driven exoskeleton HAL® (hybrid assistive limb) can improve walking functions in spinal cord injury and stroke. The aim of this study was to assess safety and effects on walking function of HAL® supported treadmill therapy in patients with limb-girdle muscular dystrophy (LGMD). Materials and Methods: Three LGMD patients received 8 weeks of treadmill training with HAL® 3 times a week. Outcome parameters were 10-meter walk test (10 MWT), 6-minute walk test, and timed-up-and-go test (TUG). Parameters were assessed pre and post training and 6 weeks later (follow-up). Results: All patients completed the therapy without adverse reactions and reported about improvement in endurance. Improvements in outcome parameters after 8 weeks could be demonstrated. Persisting effects were observed after 6 weeks for the 10 MWT and TUG test (follow-up). Conclusions: HAL® treadmill training in LGMD patients can be performed safely and enables an intensive highly repetitive locomotor training. All patients benefitted from this innovative method. Upcoming controlled studies with larger cohorts should prove its effects in different types of LGMD and other myopathies.

Proteomics of rimmed vacuoles define new risk allele in inclusion body myositis.

OBJECTIVE: Sporadic inclusion body myositis (sIBM) pathogenesis is unknown; however, rimmed vacuoles (RVs) are a constant feature. We propose to identify proteins that accumulate within RVs. METHODS: RVs and intact myofibers were laser microdissected from skeletal muscle of 18 sIBM patients and analyzed by a sensitive mass spectrometry approach using label-free spectral count-based relative protein quantification. Whole exome sequencing was performed on 62 sIBM patients. Immunofluorescence was performed on patient and mouse skeletal muscle. RESULTS: A total of 213 proteins were enriched by >1.5 -fold in RVs compared to controls and included proteins previously reported to accumulate in sIBM tissue or when mutated cause myopathies with RVs. Proteins associated with protein folding and autophagy were the largest group represented. One autophagic adaptor protein not previously identified in sIBM was FYCO1. Rare missense coding FYCO1 variants were present in 11.3% of sIBM patients compared with 2.6% of controls (p = 0.003). FYCO1 colocalized at RVs with autophagic proteins such as MAP1LC3 and SQSTM1 in sIBM and other RV myopathies. One FYCO1 variant protein had reduced colocalization with MAP1LC3 when expressed in mouse muscle. INTERPRETATION: This study used an unbiased proteomic approach to identify RV proteins in sIBM that included a novel protein involved in sIBM pathogenesis. FYCO1 accumulates at RVs, and rare missense variants in FYCO1 are overrepresented in sIBM patients. These FYCO1 variants may impair autophagic function, leading to RV formation in sIBM patient muscle. FYCO1 functionally connects autophagic and endocytic pathways, supporting the hypothesis that impaired endolysosomal degradation underlies the pathogenesis of sIBM. Ann Neurol 2017;81:227-239.

Diagnostic support for selected neuromuscular diseases using answer-pattern recognition and data mining techniques: a proof of concept multicenter prospective trial.

BACKGROUND: Diagnosis of neuromuscular diseases in primary care is often challenging. Rare diseases such as Pompe disease are easily overlooked by the general practitioner. We therefore aimed to develop a diagnostic support tool using patient-oriented questions and combined data mining algorithms recognizing answer patterns in individuals with selected neuromuscular diseases. A multicenter prospective study for the proof of concept was conducted thereafter. METHODS: First, 16 interviews with patients were conducted focusing on their pre-diagnostic observations and experiences. From these interviews, we developed a questionnaire with 46 items. Then, patients with diagnosed neuromuscular diseases as well as patients without such a disease answered the questionnaire to establish a database for data mining. For proof of concept, initially only six diagnoses were chosen (myotonic dystrophy and myotonia (MdMy), Pompe disease (MP), amyotrophic lateral sclerosis (ALS), polyneuropathy (PNP), spinal muscular atrophy (SMA), other neuromuscular diseases, and no neuromuscular disease (NND). A prospective study was performed to validate the automated malleable system, which included six different classification methods combined in a fusion algorithm proposing a final diagnosis. Finally, new diagnoses were incorporated into the system. RESULTS: In total, questionnaires from 210 individuals were used to train the system. 89.5 % correct diagnoses were achieved during cross-validation. The sensitivity of the system was 93-97 % for individuals with MP, with MdMy and without neuromuscular diseases, but only 69 % in SMA and 81 % in ALS patients. In the prospective trial, 57/64 (89 %) diagnoses were predicted correctly by the computerized system. All questions, or rather all answers, increased the diagnostic accuracy of the system, with the best results reached by the fusion of different classifier methods. Receiver operating curve (ROC) and p-value analyses confirmed the results. CONCLUSION: A questionnaire-based diagnostic support tool using data mining methods exhibited good results in predicting selected neuromuscular diseases. Due to the variety of neuromuscular diseases, additional studies are required to measure beneficial effects in the clinical setting.

Muscle imaging data in late-onset Pompe disease reveal a correlation between the pre-existing degree of lipomatous muscle alterations and the efficacy of long-term enzyme replacement therapy.

BACKGROUND: Late-onset Pompe disease (LOPD) is a metabolic myopathy caused by mutations in GAA and characterized by proximal muscle weakness and respiratory insufficiency. There is evidence from clinical studies that enzyme replacement therapy (ERT) with human recombinant alpha-glucosidase improves motor performance and respiratory function in LOPD. OBJECTIVE: We analyzed quantitative muscle MRI data of lower limbs to evaluate the effects of long-term ERT on muscle parameters. METHODS: Three symptomatic LOPD patients who received ERT for five years and four untreated presymptomatic LOPD patients were included in the study. T1-weighted MRI images were used to determine volumes of thigh and lower leg muscles. In addition, mean gray values of eight individual thigh muscles were calculated to assess the degree of lipomatous muscle alterations. RESULTS: We detected a decrease in thigh muscle volume of 6.7% (p < 0.001) and an increase in lower leg muscle volume of 8.2% (p = 0.049) after five years of ERT. Analysis of individual thigh muscles revealed a positive correlation between the degree of lipomatous muscle alterations at baseline and the increase of gray values after five years of ERT (R(2) = 0.68, p < 0.001). Muscle imaging in presymptomatic patients showed in one case pronounced lipomatous alteration of the adductor magnus muscle and mild to moderate changes in further thigh muscles. CONCLUSIONS: The results demonstrate that fatty muscle degeneration can occur before clinical manifestation of muscle weakness and suggest that mildly affected muscles may respond better to ERT treatment than severely involved muscles. If these findings can be validated by further studies, it should be discussed if muscle alterations detected by muscle MRI may be an objective sign of disease manifestation justifying an early start of ERT in clinically asymptomatic patients in order to improve the long-term outcome.

Human ß-defensin-3 correlates with muscle fibre degeneration in idiopathic inflammatory myopathies.

Sporadic inclusion body myositis (sIBM) and polymyositis (PM) are characterized by muscle inflammation, with sIBM showing additional degenerative alterations. In this study we investigated human beta defensins and associated TLRs to elucidate the role of the innate immune system in idiopathic inflammatory myopathies (IIM), and its association with inflammatory and degenerative alterations. Expression levels of human beta-defensin (HBD)-1, HBD-2, HBD-3 and TLR2, 3, 4, 7 and 9 were analysed by quantitative real-time PCR in skeletal muscle tissue. Localization of HBD-3, collagen 6, dystrophin, CD8-positive T-cells, CD-68-positive macrophages, ß-amyloid, the autophagy marker LC3, and TLR3 were detected by immunofluorescence and co-localization was quantified. HBD-3 and all TLRs except for TLR9 were overexpressed in both IIM with significant overexpression of TLR3 in sIBM. HBD-3 showed characteristic intracellular accumulations near deposits of ß-amyloid, LC3 and TLR3 in sIBM, and was detected in inflammatory infiltrations and macrophages invading necrotic muscle fibres in both IIM. The characteristic intracellular localization of HBD-3 near markers of degeneration and autophagy, and overexpression of endosomal TLR3 in sIBM hint at different pathogenetic mechanisms in sIBM compared with PM. This descriptive study serves as a first approach to the role of the innate immune system in sIBM and PM.

Anti-inflammatory modulation of immune response by probiotic Escherichia coli Nissle 1917 in human blood mononuclear cells.

Escherichia coli Nissle 1917 (EcN) bears a defect in its LPS biosynthesis leading to truncated variable oligosaccharide-antigen chains and a semi-rough phenotype. It is effectively inactivated by complement factors due to resolved serum resistance and is, therefore, safe as a probiotic strain, i.e. for the treatment of inflammatory gastrointestinal diseases. It is unknown whether the modification of LPS in EcN contributes to its probiotic properties. Purified LPS from EcN and wild-type LPS from uropathogenic E. coli W536 together with raw lysates of both strains were analyzed for their gene expression activity with human PBMCs measured by microarrays. Comparing the two LPS molecules and the two lysate variants with each other, respectively, no differences of transcriptional patterns were observed. However, when comparing LPS with lysate patterns, pro-inflammatory cytokine IL-12p40 was up-regulated by both LPS molecules and anti-inflammatory IL-10 by both lysates. The higher the lysate concentration, the higher IL-10 release from PBMCs, clearly exceeding LPS induced IL-12p40 release. Furthermore, inflammatory chemokine CCL24 (eotaxin) was down-regulated by lysates and quantitative real-time PCR revealed that EcN compared to wild-type LPS was 8 times stronger in down-regulation of CCL24. We conclude that truncated LPS may down-regulate CCL24-mediated inflammation and that EcN lysate contains as yet unidentified factors which preferably induce anti-inflammatory activity. Both effects may contribute to the probiotic properties of EcN.