MicroRNA signatures predict dysregulated vitamin D receptor and calcium pathways status in limb girdle muscle dystrophies (LGMD) 2A/2B.

miRNA expression profile and predicted pathways involved in selected limb-girdle muscular dystrophy (LGMD)2A/2B patients were investigated. A total of 187 miRNAs were dysregulated in all patients, with six miRNAs showing opposite regulation in LGMD2A versus LGMD2B patients. Silico analysis evidence: (1) a cluster of the dysregulated miRNAs resulted primarily involved in inflammation and calcium metabolism, and (2) two genes predicted as controlled by calcium-assigned miRNAs (Vitamin D Receptor gene and Guanine Nucleotide Binding protein beta polypeptide 1gene) showed an evident upregulation in LGMD2B patients, in accordance with miRNA levels. Our data support alterations in calcium pathway status in LGMD 2A/B, suggesting myofibre calcium imbalance as a potential therapeutic target. Copyright © 2016 John Wiley & Sons, Ltd.

Comparative FE biomechanical and microbial adhesion analyses on an implanted humerus.

Objective: In this study a multi fragment humeral fracture, treated with locking plate system implant, was investigate and compared with a healthy humerus by the mining of a Finite Element (FE) analysis. Locking plate implant, in this case AxSOS 3® Titanium produced by Stryker is the preferred solution in presence of multiple fracture or osteoporosis. Methods: Loading conditions were imposed by rotating of 52,5° respect the vertical axe, both the humeri (healthy and fractured), fixing distal end, and loading the top of bones with a vertical force of 543 N (Newton). This finite element analysis aimed to compare stability of implanted humerus, implant-bone interface, stress shielding, with those related to a healthy one. A microbial adhesion analysis was also performed on the implant's material. Results: Results obtained by FE analysis confirm a good agreement of the mechanical behavior of the models tested. The maximum values of the registered stressed, are of about 45 MPa (Mega Pascal) for the intact humerus and 113 MPa for the fractured one. Displacements, confirm higher values on the fractured humerus and no viable bacteria were found after microbial adhesion analyses.Conclusion: comparison between healthy and fractured humerus showed an optimal stability of the implant, when contact surfaces optimization and screws insertion are correctly performed.

Immunolocalization and activation of nuclear factor-kappaB in the sciatic nerves of rats with experimental autoimmune neuritis.

Recent data support an important role played by nuclear factor kappa B (NF-kappaB) in peripheral neuropathies. We investigated expression and activation of NF-kappaB in experimental autoimmune neuritis (EAN) in rat sciatic nerves removed after 7, 14 and 21 days after immunization. Immunoreactivity for the activated form of NF-kappaB was found in the nuclei of T cells and macrophages at days 14 and 21, and also in the nuclei of few Schwann cells and of vascular endothelial cells at all time points, especially during the peak stage. Western blot showed a single band corresponding to 65 kDa in all EAN animals. NF-kappaB DNA-binding activity was revealed by electrophoretic mobility shift assay. Our results support NF-kappaB activation in EAN during the induction stage as well as in the disease remission.

Clinical and molecular aspects of 30 patients with late-onset Pompe disease (LOPD): unusual features and response to treatment.

Pompe disease is a rare metabolic disorder, due to mutations in the gene encoding acid alpha-glucosidase (GAA), of which infantile and late-onset forms may occur. Aim of the work was to analyze clinical and laboratory data of a cohort of late-onset Pompe disease (LOPD) patients, collected during the last 15 years and to point out unusual phenotypic/genotypic features as well as enzyme replacement therapy (ERT) responses. We diagnosed 30 LOPD patients; at follow-up, they underwent motor, respiratory, cardiac and muscle MRI evaluations. Motor performances were tested by Walton Gardner-Medwin, GSGC and 6MWT tests. Respiratory function was assessed as FVC% in upright/supine position. LOPD presentations were represented by presymptomatic hyperCKemia (37%), proximal/axial muscle weakness (53%) and respiratory impairment (10%). Median diagnostic delay was 8.6 years (± 8.8). Atypical features were observed in 4 patients: marked distal muscle weakness and severe hearing loss at onset, as well as leukoencephalopathy and mesial temporal sclerosis during the disease course. By GAA sequence analysis, two causing mutations were detected in 22/30 patients, only one in the remaining 8 subjects. Overall, 29/30 patients harbored the common c.-32-13T>G mutation (2 were homozygous). Two new DNA variations were discovered (c.2395C>G, c.1771C>T). 14 patients received ERT for up to 60 months. Our study confirms LOPD clinical and genetic heterogeneity: atypical features may contribute to expand the clinical phenotype highlighting its multi-systemic nature. A timely diagnosis could allow early ERT start. An accurate follow-up is recommended to evaluate treatment responses.

Localization of vinculin and talin at perineurial cells of human sural nerve.

Immunolocalization of spectrin, vinculin, talin, desmin and titin was investigated in human sural nerve. No binding for spectrin and titin was seen in any structure of the nerve. Antibody against desmin immunostained sporadic epineurial vessels only. Endoneurial and epineurial vessels were intensely positive for vinculin and talin. We found expression of vinculin and talin at the perineurial cells, using immunocytochemistry and gold immunoelectron microscopy. Since vinculin and talin are known to be involved in cell-cell and cell-extracellular matrix transmembrane connections, we propose that they, possibly together with other cytoskeletal proteins, may be implicated in the permeability barrier property of the perineurium. In pathological conditions, perineurium plays an as yet unknown role. Future studies are needed to investigate expression of vinculin and talin in neuropathies.

Dp116, talin, vinculin and vimentin immunoreactivities following nerve transection.

The time course of the expression of Dp116, talin, vinculin and vimentin in rat sciatic nerve was investigated after experimental transection. Dp116 was still found at 5 days after experiment in some degenerating myelinated fibers of both proximal and distal stumps. The findings are consistent with the known preservation of electrical excitability of the distal nerve in the first days after injury. Some regenerating nerve fibers into the neuroma also expressed Dp116 at 25 and 40 days after nerve transection. Talin and vinculin markedly and diffusely immunostained the neuroma. Talin in the distal stump and vimentin in both proximal and distal stumps were found decreased during the time course of the experiment. Vinculin binding increased in the distal stump, due to a real overexpression or simply to a cross-reaction to degeneration products.

Perineurium talin immunoreactivity decreases in diabetic neuropathy.

We studied the immunolocalization of Dp116 (a 116 kDa protein product of the dystrophin gene), vinculin, talin, vimentin, desmin, spectrin and titin in the sural nerve biopsies of 25 patients with peripheral neuropathies of different origin. 4 patients presented with HMSN type 1, 4 with HMSN type 2, 2 with HNPP, 4 with CIDP, 5 with chronic axonal neuropathy of unknown origin, 3 with vasculitic neuropathy, 3 with diabetic neuropathy. Expression and localization of Dp116, vinculin, vimentin, desmin, spectrin and titin did not differ from normal control cases. Spectrin and titin immunoreactivities were absent and desmin was occasionally found in few epineurial vessels. A thin rim of Dp116 binding surrounded the outermost layer of myelin sheaths. Perineurium and epineurial vessels stained deeply for vinculin. Vimentin immunoreactivity was seen in all endoneurial, perineurial and epineurial cells. Immunoreactivity for talin was normally found at endoneurial and epineurial vessel walls, perineurial cells and epineurial fibroblasts in all the sural nerves except diabetic nerves. In the latter, whereas talin binding was normal in the vessel walls and epineurial fibroblasts, it was markedly reduced in the perineurium. On immunoblot, two bands at 235 and 190 kDa were found in the sural nerves with the antibody anti-talin, and both were reduced only in the patients with diabetic neuropathy. We postulate that decreased perineurium talin in diabetic polyneuropathy may be related to the known alterations of the tight junctions of the perineurial cells, which have been proposed to be a contributory factor to impaired permeability barrier properties.

Expression of cytoskeleton proteins in central core disease.

Despite advances in genetics the pathogenesis of central core disease (CCD) is still unknown. We studied muscles from 5 CCD patients by immunocytochemistry using monoclonal antibodies against various cytoskeletal proteins (dystrophin, spectrin, vinculin, desmin, vimentin, myosin heavy chain (MHC) of developmental, neonatal, adult slow and fast types). Dystrophin, spectrin and vinculin immunoreactivity was localized only at sarcolemma as in normal muscle. Vimentin was not present in myofibers. Only sporadic fibers were positive for developmental and neonatal MHC isoforms in adult CCD muscles. A 4-month-old patient had 5% of neonatal MHC-immunoreactive fibers, a finding similar to that of age-matched normal muscle. Desmin intermediate filaments were overexpressed in many core-fibers in extra-core regions, reduced or absent at cores, and greatly increased at the periphery of some cores. Moreover, irregular desmin-positive spots were seen within some cores. On the contrary, in neurogenic muscle atrophy patients, target lesions had increased desmin. These features indicate a possible role of desmin in the pathogenesis of cores, although we do not know if primary or secondary. In addition, they suggest that: (i) cores and targets may be manifestations of different processes; (ii) it is likely that core-fibers are not denervated fibers.

Myopathy as the persistently isolated symptomatology of primary autoimmune hypothyroidism.

Although disorders of thyroid function may cause a wide range of muscle disturbances, an overt myopathy has been rarely reported as an isolated clinical presentation of hypothyroidism. We observed 10 patients (5 males and 5 females) who had been referred to the department of neurology because of muscular fatigability, myalgia, cramps, or proximal weakness. Laboratory investigation showed that all patients had hypothyroidism due to Hashimoto's thyroiditis (atrophic variant in 9/10). Classic symptoms/signs of hypothyroidism such as lethargy, constipation, cold intolerance, myxedematous facies, and/or bradycardia were absent, as assessed independently by the three coauthoring thyroidologists. Muscular complaints improved greatly and then disappeared after substitutive levothyroxine treatment. Muscle biopsy revealed nonspecific changes. Nicotinamide adenine dinucleotide reductase (NADH-TR)-hyporeactive cores were present in two patients (10% and 90% of type 1 fibers). On electron microscopy, the core areas showed disorganized myofibrils, Z-band streaming, rod formation, and paucity of mitochondria and glycogen granules. Desmin intermediate filaments were overexpressed only in some cores. The similarity of the pattern of desmin expression between hypothyroid cores and target lesions of denervated fibers supports the hypothesis that, at least in some of our patients, myopathy was the result of an impaired nerve-mediated action of thyroid hormones on skeletal muscle. Our observations suggest that an isolated myopathy as the sole manifestation of hypothyroidism is not a rare event. We postulate that our cases may constitute a peculiar subgroup of Hashimoto's thyroiditis patients: (1) the strikingly abnormal F/M ratio of 1:1; (2) the relatively younger age; (3) the rarity of the goitrous variant; (4) the unusual finding of antithyroglobulin (Tg-Ab) > antithyroid peroxidase (TPO-Ab). Thorough evaluation of thyroid function is appropriate in patients with myopathy of uncertain origin.

Onset of hypothyroidism with polymyositis-like clinical features in elderly patients.

Impaired muscle function may be a predominant aspect of hypothyroidism and is virtually present in all patients with overt thyroid failure. Less common is the onset of hypothyroidism with clinical features mimicking a polymyositis. We have observed 3 patients, whose age ranged 63-68 years, presenting with muscle aches, cramps, proximal weakness and stiffness. Two patients had dysphagia. Serum creatine kinase (CK) and electromyography (EMC) were altered in two patients. Muscle biopsy showed type II atrophy, sporadic type I and type II grouping, "core-like" areas, and some myopathic changes such as central nuclei and muscle necrosis. No inflammatory changes were present. Immunohistochemistry of several muscle cytoskeletal proteins revealed increased desmin in "corelike" areas. Detection of serum thyroid hormone levels revealed very low triiodo-L-thyronine (T3) and thyroxine (T4), whereas thyroid-stimulating hormone (TSH) was greatly increased as well as serum anti-thyroglobulin, anti-peroxidase and anti-microsome antibodies. The patients were diagnosed having a hypothyroid myopathy due to Hashimoto thyroiditis. L-thyroxine treatment normalized clinical and hormone levels, but serum antibodies remained elevated. Muscle biopsy was fundamental to establish the correct diagnosis in our patients. Presence of over-expression of desmin in cores, as described in target lesions in neurogenic diseases, may suggest a nerve-mediated pathogenesis of hypothyroid myopathy.

[Pseudonecrosis waves: simulation of myocardial necrosis by retrograde P wave]. / Onde da "pseudonecrosi": simulazione di necrosi miocardica per onda P retrograda.

The Authors describe a case of a patient showing, during an episode of chest pain, an ecg-pattern of wide and tall "Q" wave simulating inferior myocardial infarction. In fact, a further ecg recorded during sinus rhythm denotes that the "Q" wave was a retrograde P wave generated by a nodal rhythm. The other known causes of "pseudonecrosis" are discussed.

[Topical treatment of peri-implantitis with metronidazole dental gel 25%. Clinical analysis and microbiological control]. / Trattamento topico delle perimplantiti con metronidazolo gel dentale 25%. Analisi clinica e controllo microbiologico.

BACKGROUND: Aim of this research is to verify the efficacy of metronidazole dental gel 25%, used as a topical antibiotic for the treatment of peri-implantitis. The efficacy of the medicine in the starting phase of the disease (mucositis), as well as during peri-implantitis involving bone-bearing loss has been evaluated. METHODS: Twenty patients were chosen as a sample for the study. After careful evaluation of the several protocols concerning peri-implantitis treatment, some diagnostic clinical parameters have been recorded for each patient and metronidazole dental gel 25% (Elyzol Cabon) has been administered in two applications at perimplants pocket (one after a week). For each patient 3 microbiological drawings were made for the evaluation of the bacterial population around the implant site. RESULTS: From the microbiological examination a decrease of Gram- and an increase of Gram+ which returned in almost all the patients to normal levels have been observed; moreover, in all the patients a gradual decrease of PMN, resolution index of the inflammatory process was obtained, confirmed also by a remarkable improvement of all the observed diagnostic parameters, except for the peri-implants bone radiotransparency which was unchanged. CONCLUSIONS: The research showed that the peri-implant diseases can be positively resolved by using the metronidazole dental gel 25% topical antibiotic. This drug led to a 60-70% decrease of Gram-, and 40-50% increase for Gram+, bringing these back to normal values in almost all the patients. Moreover, a good recovery of the peri-implants soft tissues has been observed.

Clinical and muscle magnetic resonance imaging study of an Italian family with autosomal dominant inclusion body myopathy not linked to known genetic loci.

The objective was to report a clinical, pathological and muscle magnetic resonance (MR) study of an Italian family with an autosomal dominant inclusion body myopathy (AD-IBM). Eight subjects (age range 20-56 years; 5 females and 3 males) belonging to four generations were studied. Onset of disturbances (distal weakness at lower limbs) ranged from 20 to 28 years. CK levels were increased to five times. Only in an early stage oedema of involved muscles has been demonstrated by muscle MR. Quadriceps femoris was characteristically spared; in the last phases a mild involvement of the vasti became evident with persistent sparing of the rectus femori. Rimmed vacuoles and hyperphosphorylated tau filaments were evident at muscle biopsy. Linkage analysis excluded the association of the disease to chromosome loci 14q11, 17p13.1, 2p13, 19p13. The study suggests that quadriceps sparing is a characteristic feature also of AD-IBM. This finding could represent a muscle-image hallmark helpful in diagnosis of autosomal dominant muscular disorders.

Immunocytochemistry of muscle cytoskeletal proteins in acid maltase deficiency.

Immunocytochemistry of dystrophin, spectrin, vinculin (sarcolemma-specific proteins), and desmin (an intermediate filament protein) were investigated in 5 patients with acid maltase deficiency (AMD). One patient had infant onset, 2 had childhood onset, and 2 had adult onset. All had a vacuolar myopathy with autophagic vacuoles containing glycogen and cytoplasmic degradation products. Dystrophin, spectrin, and vinculin were localized at the sarcolemma as in normal muscle fibers. Within the cytoplasm of many fibers, immunoreactivity for the three proteins was seen as single or multiple spots or as circular structures, which most likely corresponded to the limiting membrane of vacuoles. Desmin was overexpressed at the periphery of some vacuoles. It is plausible that, before exocytosis occurs, sarcolemma-specific proteins appear within the vacuole membrane. Vacuole immunolabeling frequently occurred in the patients with childhood and adult onset AMD, but very rarely occurred in the case with infant onset. We hypothesize that a reduced exocytosis rate might explain the infrequent vacuole immunolabeling and the early onset of the infant form of the disease.