Objective To investigate effect of CD4+CD25+Foxp3+Tregs in the treatment of autoimmune myositis (EAM) in mice and explore the possible mechanisms. Methods Mouse models of EAM were divided randomly into model group and treatment group, and the latter received infusion of CD4 + CD25 + Foxp3 + Tregs separated from normal mouse spleen by magnetic activated cell sorting. The changes of muscle pathology was observed, and the expression of PD-1 and CTLA-4 in spleen CD4+ CD25+ Foxp3+ Tregs was analyzed using flow cytometry; peripheral blood IL-10 and TGF-β levels were tested using double antibody sandwich ELISA. Results Compare with the model group, the mice in the treatment group showed significantly reduced muscular inflammatory cell infiltration, increased blood levels of IL-10 and TGF-β (P<0.05), and increased expression of PD-1 and CTLA-4 in spleen CD4+CD25+Foxp3+Tregs (P<0.05). Conclusion CD4+CD25+Foxp3+Tregs reinfusion produces therapeutic effect in mice with EAM by increasing peripheral blood IL-10 and TGF-βlevels and PD-1 and CTLA-4 expressions in spleen CD4+CD25+Foxp3+Tregs.

Objective To investigate effect of CD4+CD25+Foxp3+Tregs in the treatment of autoimmune myositis (EAM) in mice and explore the possible mechanisms. Methods Mouse models of EAM were divided randomly into model group and treatment group, and the latter received infusion of CD4 + CD25 + Foxp3 + Tregs separated from normal mouse spleen by magnetic activated cell sorting. The changes of muscle pathology was observed, and the expression of PD-1 and CTLA-4 in spleen CD4+ CD25+ Foxp3+ Tregs was analyzed using flow cytometry; peripheral blood IL-10 and TGF-β levels were tested using double antibody sandwich ELISA. Results Compare with the model group, the mice in the treatment group showed significantly reduced muscular inflammatory cell infiltration, increased blood levels of IL-10 and TGF-β (P<0.05), and increased expression of PD-1 and CTLA-4 in spleen CD4+CD25+Foxp3+Tregs (P<0.05). Conclusion CD4+CD25+Foxp3+Tregs reinfusion produces therapeutic effect in mice with EAM by increasing peripheral blood IL-10 and TGF-βlevels and PD-1 and CTLA-4 expressions in spleen CD4+CD25+Foxp3+Tregs.

Objective To investigate the clinicopathological features of tubular aggregate myopathy.Methods Eight patients as experimental group were diagnosed with tubular aggregate myopathy in Department of Neurology,People' s Liberation Army General Hospital,between March 2000 and March 2013.The data were retrospectively analyzed.Enzyme histochemical techniques and electron microscopy were taken to observe the muscle structures.Results The detection rate of tubular aggregate was 0.374％ (8/2 137).All of the 8 patients with tubular aggregate myopathy were male.Five patients presented with episodes of muscle weakness,while 3 patients presented chronic progressive muscle weakness.The main clinical features of all patients were muscle weakness.The creatine kinase level was mildly elevated in 4 patients,while it was normal in the other 4 patients.Electromyogram showed myogenic damage in 5 patients and normal in 3 patients.All of the 8 patients denied family history.By light microscopy,hematoxylin-eosin staining showed that tubular aggregates were multiple basophilic subsarcolemmal substance.Tubular aggregate stained red with modified Gomori trichrome stain,reacted intensely for nicotinamide adenine dinucleotidetetrazolium reductase,and remained unstained on oil red O,periodic acid Schiff,sudan black B,acid phosphatas,adenosine triphosphatase.By electron microscopy,tubular aggregates were densely packed tubules predominantly in the subsarcolemmal region.Conclusion Enzyme histochemical staining and electron microscopy show special features of tubular aggregate myopathy,are the critical techniques for the diagnosis of the disease.

Objective To analyze and summarize the clinical , pathological features of 16 patients with centronuclear myopathy.Methods All of the 16 patients performed clinical examination and sporadic patients and a proband with family history ( n=6 ) performed serum creatine kinase , electromyography and open muscle biopsies , and the specimens were used for a standard series of histological and histochemical stainings.The clinical and pathological features of these patients were analyzed.Results The proportion of centronuclear myopathy in suspected myopathy cases was 0.127%(6/4 724) in our department.The onset time ranged from infancy to adulthood.The common initial symptoms included mild to moderate weakness of lower limbs and bilateral ptosis ( n =4 ).The other symptoms were facial weakness ( n =1 ) and ophthalmoplegia (n=1).There were 12 patients performing distal weakness exceeding proximal weakness . One family with autosomal dominant inheritance of 11 patients had a later age of onset than the sporadic ones and manifested bilateral ptosis , bilateral lower limbs weakness , especially in distal muscle.Muscle biopsies showed pronouncedly increased amount of fibers with centrally placed nuclei with predominant type Ⅰfibers and a clear perinuclear halo surrounding the centrally placed nuclei and an appearance of spoke of a wheel in some fibers.Conclusions This series of centronuclear myopathy patients manifest clinical heterogeneity.Muscle biopsies show features of centralized nuclei pronounced increase , type Ⅰfibers predominance , etc.These can provide evidences for the diagnosis of the disease.

Objective To study the clinical and pathological features of nemaline myopathy(NM) in 12 cases.Methods Clinical manifestations and pathological features of muscle-biopsy specimens were summarized and analyzed retrospectively in 12 NM cases.Results In 12 cases,7 patients with typical congenital type exhibited lower or four limbs weakness as the first symptom and benign course.Three patients with childhood onset type exhibited lower limbs weakness and progressive course,and this type of patient might have muscle atrophy.Two patients with adult onset type exhibited four limbs and throat muscle weakness,rapidly progressive course and obvious muscle atrophy,and one patient had already shown acute respiratory failure.High arched feet and elongated face were observed.Creatin kinase value in all patients was normal or mildly elevated,and all electromyography showed myogenic changes.In light microscopy,the nemaline bodies were observed in more than half muscle fibers,especially in type 1 fibers.All patients showed type 1 predominance and atrophy.Modified Gomori trichrome stains showed characteristic purplecolored rods.Muscle electron microscopy showed high electron dense nemaline bodies around nucleus and disorganized myofibrillar apparatus such as broken myofilaments,irregular myofibril and Z lines.Nemaline bodies under electron microscopy may be part of myofibril or high electron-dense bodies with no structure.Conclusions The 12 patients in this study with NM are divided into 3 types,of which adult onset type is the most severe one.The key diagnosis is based on the appearance of nemaline bodies in more than half of the muscle fibers and the muscle electron microscopy observation.

OBJECTIVETo investigate the expression of C5b-9 in the skeletal muscle blood vessels in patients with necrotizing myopathy and explore its role in the pathogenesis of this disease.

METHODSThe expression of C5b-9 and MHC-I in the skeletal muscular fibers and blood vessels in 4 patients with necrotizing myopathy was detected using enzymohistochemistry and immunohistochemistry.

RESULTSFocal or dispersive necrotic muscle fibers with obvious phagocytosis were observed in all the 4 patients. No inflammatory cell infiltration was found in the perimysium or perivascular regions. HE staining showed a decreased number of local small blood vessels, and the some small blood vessels showed thickened vascular walls. Immunohistochemistry detected prominent C5b-9 expression in the necrotic muscle fibers and the blood vessels, and diffuse strong C5b-9 expression was found in the vascular walls, vascular endothelial cells and the smooth muscle layer. No MHC-I deposition was detected in the muscular fibers and blood vessels.

CONCLUSIONC5b-9 contributes to the pathogenesis of necrotizing myopathy mediated by pathologies in the blood vessels.

Aged ; Complement Membrane Attack Complex ; metabolism ; Female ; Humans ; Male ; Middle Aged ; Muscle, Skeletal ; blood supply ; Muscular Diseases ; blood ; pathology ; Necrosis

ObjectiveTo investigate the clinical,myopathological and molecular changes in two Chinese families with oculopharyngodistal myopathy ( OPDM).MethodsWe performed muscle biopsy and histopathologic study on the probands of two families,and further examined molecular genetic testing on PABPN1 and GNE gene. Results Family 1 included 3 affected brothers in the same generation and family 2 involved 4 patients in 2 generations. Dysarthria rather than external ophthalmoplegia was the prominent oculopharyngeal symptoms for Chinese patients. No intranuclear inclusions were observed in ultrastructural examination.The number of GCG repeats in the PABPN1 gene was within normal range and no mutations were identified in the GNE gene.ConclusionsFamily 1 is the first publication on autosomal recessive OPDM in China.The age of onset of two families was comparable with Japanese patients and the pattern of muscle involvement was different. OPDM is a distinct phenotypical,histological,and genetic entity.

Objective To explore the expression of myostatin mRNA,a negative regulator of muscle growth,in the skeletal muscle of Duchenne muscular dystrophy(DMD)patients.Methods A semi-quantitative reverse transcription polymerase chain reaction was performed to evaluate the expression of myostatin in the skeletal muscle of 7 DMD patients and 4 healthy controls.Results The level of myostatin gene expression in the skeletal muscle of DMD patients was higher than that of healthy controls(0.56 ± 0.16 vs 0.34 ± 0.15,Z =-2.268,P =0.023).Conclusions The myostatin gene expression was increased in the DMD patients compared to the healthy controls.Enhanced expression of myostatin in the skeletal muscle might be involved in the pathogenesis of DMD.

OBJECTIVETo construct and investigate the cell model of a novel mutation R675Q in the skeletal muscle Na channel type 4 alpha subunit gene (SCN4A) identified from a Chinese family with normokalemic periodic paralysis.

METHODScDNA encoding the adult isoform of SCN4A was used as a template for in vitro site-directed mutagenesis by PCR method. The mutated plasmid was transiently transfected into HEK-293 cells by calcium phosphate precipitation. Twenty four and 48 hours after transfection, the expression level of SCN4A was detected by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot. Whole cell voltage-clamp recording was used to study the current of sodium channels.

RESULTSThe site-mutagenesis of the plasmid was confirmed by sequencing. The expression of SCN4A gene was significantly elevated 24 h and 48 h after transfection. The relative current of R675Q is smaller than that of wide type before reaching peak current under the same test voltage, but larger than that of wild type current after reaching peak current. They both had the largest peak current under 0 mV test pulse.

CONCLUSIONA cell model of normokalemic periodic paralysis was successfully constructed. The R675Q mutation of the SCN4A gene enhances the activation and inactivation of the sodium channel, and the S4 transmembrane segment may have intimate relationship with the attack of weakness in normoKPP patients.

Asian Continental Ancestry Group ; genetics ; Base Sequence ; Cell Line ; Electric Conductivity ; Family ; Humans ; Models, Biological ; Mutation ; NAV1.4 Voltage-Gated Sodium Channel ; Paralyses, Familial Periodic ; genetics ; metabolism ; pathology ; Patch-Clamp Techniques ; Plasmids ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Sodium Channels ; genetics ; metabolism ; Transfection

96% 7-10 days after culture. Immunohistochemistry of Ⅷ factor polyclone antibody showed positive in cytomembrane and cytoplasm, but negative in cell nucleus. These were cells with blood-brain barrier. CONCLUSION: Ideal endothelial cells of blood-brain barrier can be cultured by the improved cultural method of double filtering.