No effective treatment exists for patients with X-linked myotubular myopathy (XLMTM), a fatal congenital muscle disease caused by deficiency of the lipid phosphatase, myotubularin. The Mtm1δ4 and ...Mtm1 p.R69C mice model severely and moderately symptomatic XLMTM, respectively, due to differences in the degree of myotubularin deficiency. Contractile function of intact extensor digitorum longus (EDL) and soleus muscles from Mtm1δ4 mice, which produce no myotubularin, is markedly impaired. Contractile forces generated by chemically skinned single fiber preparations from Mtm1δ4 muscle were largely preserved, indicating that weakness was largely due to impaired excitation contraction coupling. Mtm1 p.R69C mice, which produce small amounts of myotubularin, showed impaired contractile function only in EDL muscles. Short-term replacement of myotubularin with a prototypical targeted protein replacement agent (3E10Fv-MTM1) in Mtm1δ4 mice improved contractile function and muscle pathology. These promising findings suggest that even low levels of myotubularin protein replacement can improve the muscle weakness and reverse the pathology that characterizes XLMTM.
The clinical significance of diaphragm weakness in critically ill patients is evident: it prolongs ventilator dependency and increases morbidity, duration of hospital stay, and health care costs. The ...mechanisms underlying diaphragm weakness are unknown, but might include mitochondrial dysfunction and oxidative stress.
We hypothesized that weakness of diaphragm muscle fibers in critically ill patients is accompanied by impaired mitochondrial function and structure, and by increased markers of oxidative stress.
To test these hypotheses, we studied contractile force, mitochondrial function, and mitochondrial structure in diaphragm muscle fibers. Fibers were isolated from diaphragm biopsies of 36 mechanically ventilated critically ill patients and compared with those isolated from biopsies of 27 patients with suspected early-stage lung malignancy (control subjects).
Diaphragm muscle fibers from critically ill patients displayed significant atrophy and contractile weakness, but lacked impaired mitochondrial respiration and increased levels of oxidative stress markers. Mitochondrial energy status and morphology were not altered, despite a lower content of fusion proteins.
Critically ill patients have manifest diaphragm muscle fiber atrophy and weakness in the absence of mitochondrial dysfunction and oxidative stress. Thus, mitochondrial dysfunction and oxidative stress do not play a causative role in the development of atrophy and contractile weakness of the diaphragm in critically ill patients.
Duchenne muscular dystrophy (DMD) is a progressive muscle wasting disease caused by the absence of dystrophin, a membrane-stabilizing protein encoded by the
gene. Although mouse models of DMD provide ...insight into the potential of a corrective therapy, data from genetically homologous large animals, such as the dystrophin-deficient golden retriever muscular dystrophy (GRMD) model, may more readily translate to humans. To evaluate the clinical translatability of an adeno-associated virus serotype 9 vector (AAV9)-microdystrophin (μDys5) construct, we performed a blinded, placebo-controlled study in which 12 GRMD dogs were divided among four dose groups control, 1 × 10
vector genomes per kilogram (vg/kg), 1 × 10
vg/kg, and 2 × 10
vg/kg;
= 3 each, treated intravenously at 3 months of age with a canine codon-optimized microdystrophin construct, rAAV9-CK8e-c-μDys5, and followed for 90 days after dosing. All dogs received prednisone (1 milligram/kilogram) for a total of 5 weeks from day -7 through day 28. We observed dose-dependent increases in tissue vector genome copy numbers; μDys5 protein in multiple appendicular muscles, the diaphragm, and heart; limb and respiratory muscle functional improvement; and reduction of histopathologic lesions. As expected, given that a truncated dystrophin protein was generated, phenotypic test results and histopathologic lesions did not fully normalize. All administrations were well tolerated, and adverse events were not seen. These data suggest that systemically administered AAV-microdystrophin may be dosed safely and could provide therapeutic benefit for patients with DMD.
ACTA1 encodes skeletal muscle-specific α-actin, which polymerizes to form the thin filament of the sarcomere. Mutations in ACTA1 are responsible for approximately 30% of nemaline myopathy (NM) cases. ...Previous studies of weakness in NM have focused on muscle structure and contractility, but genetic issues alone do not explain the phenotypic heterogeneity observed in patients with NM or NM mouse models. To identify additional biological processes related to NM phenotypic severity, proteomic analysis was performed using muscle protein isolates from wild-type mice in comparison to moderately affected knock-in (KI) Acta1H40Y and the minimally affected transgenic (Tg) ACTA1D286G NM mice. This analysis revealed abnormalities in mitochondrial function and stress-related pathways in both mouse models, supporting an in-depth assessment of mitochondrial biology. Interestingly, evaluating each model in comparison to its wild-type counterpart identified different degrees of mitochondrial abnormality that correlated well with the phenotypic severity of the mouse model. Muscle histology, mitochondrial respiration, electron transport chain function, and mitochondrial transmembrane potential were all normal or minimally affected in the TgACTA1D286G mouse model. In contrast, the more severely affected KI.Acta1H40Y mice displayed significant abnormalities in relation to muscle histology, mitochondrial respirometry, ATP, ADP, and phosphate content, and mitochondrial transmembrane potential. These findings suggest that abnormal energy metabolism is related to symptomatic severity in NM and may constitute a contributor to phenotypic variability and a novel treatment target.
Using a targeted transcriptomics approach, we have analyzed resected brain tissue from a cohort of 53 pediatric epilepsy surgery cases, and have found that there is a spectrum of involvement of both ...the innate and adaptive immune systems as evidenced by the differential expression of immune-specific genes in the affected brain tissue. The specimens with the highest expression of immune-specific genes were from two Rasmussen encephalitis cases, which is known to be a neuro-immunological disease, but also from tuberous sclerosis complex (TSC), focal cortical dysplasia, and hemimegalencephaly surgery cases. We obtained T cell receptor (TCR) Vβ chain sequence data from brain tissue and blood from patients with the highest levels of T cell transcripts. The clonality indices and the frequency of the top 50 Vβ clonotypes indicated that T cells in the brain were clonally restricted. The top 50 Vβ clonotypes comprised both public and private (patient specific) clonotypes, and the TCR Vβ chain third complementarity region (CDR3) of the most abundant public Vβ clonotype in each brain sample was strikingly similar to a CDR3 that recognizes an immunodominant epitope in either human cytomegalovirus or Epstein Barr virus, or influenza virus A. We found that the frequency of 14 of the top 50 brain Vβ clonotypes from a TSC surgery case had significantly increased in brain tissue removed to control recurrent seizures 11 months after the first surgery. Conversely, we found that the frequency in the blood of 18 of the top 50 brain clonotypes from a second TSC patient, who was seizure free, had significantly decreased 5 months after surgery indicating that T cell clones found in the brain had contracted in the periphery after removal of the brain area associated with seizure activity and inflammation. However, the frequency of a public and a private clonotype significantly increased in the brain after seizures recurred and the patient underwent a second surgery. Combined single cell gene expression and TCR sequencing of brain-infiltrating leukocytes from the second surgery showed that the two clones were CD8 effector T cells, indicating that they are likely to be pathologically relevant.
The symptoms of Lyme disease are caused by inflammation induced by species of the
complex. The various presentations of Lyme disease in the population suggest that differences exist in the intensity ...and regulation of the host response to the spirochete. Previous work has described correlations between the presence of regulatory T cells and recovery from Lyme arthritis. However, the effects of Foxp3-expressing CD4
T cells existing prior to, and during,
infection have not been well characterized. Here, we used C57BL/6 "depletion of regulatory T cell" mice to assess the effects these cells have on the arthritis-resistant phenotype characteristic of this mouse strain. We showed that depletion of regulatory T cells prior to infection with
resulted in sustained swelling, as well as histopathological changes, of the tibiotarsal joints that were not observed in infected control mice. Additionally,
stimulation of splenocytes from these regulatory T cell-depleted mice resulted in increases in gamma interferon and interleukin-17 production and decreases in interleukin-10 production that were not evident among splenocytes of infected mice in which Treg cells were not depleted. Depletion of regulatory T cells at various times after infection also induced rapid joint swelling. Collectively, these findings provide evidence that regulatory T cells existing at the time of, and possibly after,
infection may play an important role in limiting the development of arthritis.
Neu-Laxova syndrome (NLS) is a rare lethal disorder with autosomal recessive inheritance and is characterized by multiple congenital anomalies. Our case of NLS presented with severe intrauterine ...growth restriction (IUGR), abnormal facial features, severe central nervous system malformations, skeletal muscle contractures, and the hallmark signs of NLS: ichthyotic skin and excessive subcutaneous tissue with edema. Additionally, testing amniotic fluid from a prior pregnancy with a fetus showing similar abnormalities revealed several regions of homozygosity; one of these regions involved chromosome 1p13.2-p11.2, where the
gene is located. Based on the pattern of findings on serial fetal ultrasounds, postmortem neonatal exams, gross and microscopic exams, radiographs, and genetic analysis in conjunction with the clinical history and the prior pregnancy with the above-described molecular alteration, a final diagnosis of NLS was made. This rare developmental disorder is characterized by heterogenous neuroectodermal defects. Fetal ultrasound in the second trimester can help diagnose it. It is postulated to be caused by loss-of-function mutations in the
(phosphoglycerate dehydrogenase),
(phosphoserine aminotransferase 1), and
(phosphoserine phosphatase) genes, which are responsible for de novo L-serine synthesis.
Intracranial extraskeletal mesenchymal chondrosarcoma is a rare, malignant variant of chondrosarcoma that is characterized by undifferentiated mesenchymal cells interspersed with pockets of mature ...hyaline cartilage.
In this study, we report a 23-year-old female patient who underwent multiple craniotomies for tumor resection, as well as adjuvant radiotherapy and chemotherapy. We review the literature for reported cases and discuss the histopathologic features, radiologic findings, therapeutic approaches, and outcomes associated with this rare tumor.
Intracranial extra-skeletal mesenchymal chondrosarcomas are very aggressive tumors, and their management should emphasize attempting gross total resection followed by adjuvant treatment modalities, including radiation therapy and/or chemotherapy.
The ability to extract somatic cells from a patient and reprogram them to pluripotency opens up new possibilities for personalized medicine. Induced pluripotent stem cells (iPSCs) have been employed ...to generate beating cardiomyocytes from a patient's skin or blood cells. Here, iPSC methods were used to generate cardiomyocytes starting from the urine of a patient with Duchenne muscular dystrophy (DMD). Urine was chosen as a starting material because it contains adult stem cells called urine-derived stem cells (USCs). USCs express the canonical reprogramming factors c-myc and klf4, and possess high telomerase activity. Pluripotency of urine-derived iPSC clones was confirmed by immunocytochemistry, RT-PCR and teratoma formation. Urine-derived iPSC clones generated from healthy volunteers and a DMD patient were differentiated into beating cardiomyocytes using a series of small molecules in monolayer culture. Results indicate that cardiomyocytes retain the DMD patient's dystrophin mutation. Physiological assays suggest that dystrophin-deficient cardiomyocytes possess phenotypic differences from normal cardiomyocytes. These results demonstrate the feasibility of generating cardiomyocytes from a urine sample and that urine-derived cardiomyocytes retain characteristic features that might be further exploited for mechanistic studies and drug discovery.
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•We derived induced pluripotent stem cells (iPSCs) from human urine.•Duchenne Muscular Dystrophy (DMD) disease specific iPSCs were derived from urine.•DMD iPSCs were directed to form beating cardiomyocytes.•Physiological differences were observed between DMD and normal cardiomyocytes.•Disease-specific cardiomyocytes can be exploited for drug discovery.
X-linked myotubular myopathy (XLMTM) is a life-threatening skeletal muscle disease caused by mutations in the MTM1 gene. XLMTM fibres display a population of nuclei mispositioned in the centre. In ...the present study, we aimed to explore whether positioning and overall distribution of nuclei affects cellular organization and contractile function, thereby contributing to muscle weakness in this disease. We also assessed whether gene therapy alters nuclear arrangement and function. We used tissue from human patients and animal models, including XLMTM dogs that had received increasing doses of recombinant AAV8 vector restoring MTM1 expression (rAAV8-cMTM1). We then used single isolated muscle fibres to analyze nuclear organization and contractile function. In addition to the expected mislocalization of nuclei in the centre of muscle fibres, a novel form of nuclear mispositioning was observed: irregular spacing between those located at the fibre periphery, and an overall increased number of nuclei, leading to dramatically smaller and inconsistent myonuclear domains. Nuclear mislocalization was associated with decreases in global nuclear synthetic activity, contractile protein content and intrinsic myofilament force production. A contractile deficit originating at the myofilaments, rather than mechanical interference by centrally positioned nuclei, was supported by experiments in regenerated mouse muscle. Systemic administration of rAAV8-cMTM1 at doses higher than 2.5 × 10
vg kg
allowed a full rescue of all these cellular defects in XLMTM dogs. Altogether, these findings identify previously unrecognized pathological mechanisms in human and animal XLMTM, associated with myonuclear defects and contractile filament function. These defects can be reversed by gene therapy restoring MTM1 expression in dogs with XLMTM.
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