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1.
Horm Metab Res ; 43(13): 919-30, 2011 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-22072432

RESUMO

Growth hormone (GH) is the main regulator of longitudinal growth before puberty, and treatment with human recombinant (rh) GH can increase muscle strength. Nevertheless, molecular mechanisms responsible remain mostly unknown. Many physiological effects of GH require hormone-mediated changes in gene expression. In an attempt to gain insight into the mechanism of GH action in muscle cells we evaluated the effects of rhGH on gene expression profile in a murine skeletal muscle cell line C2C12. The objective of the work was to identify changes in gene expression in the murine skeletal muscle cell line C2C12 after rGH treatment using microarray assays. C2C12 murine skeletal muscle cell cultures were differentiated during 4 days. After 16 h growing in serum-free medium, C2C12 myotubes were stimulated during 6 h with 500 ng/ml rhGH. Four independent sets of experiments were performed to identify GH-regulated genes. Total RNA was isolated and subjected to analysis. To validate changes candidate genes were analyzed by real-time quantitative polymerase chain reaction. One hundred and fifty-four differentially expressed genes were identified; 90 upregulated and 64 downregulated. Many had not been previously identified as GH-responsive. Real-time PCR in biological replicates confirmed the effect of rGH on 15 genes: Cish, Serpina3g, Socs2, Bmp4, Tnfrsf11b, Rgs2, Tgfbr3, Ugdh, Npy1r, Gbp6, Tgfbi, Tgtp, Btc, Clec3b, and Bcl6. This study shows modifications in the gene expression profile of the C2C12 cell line after rhGH exposure. In vitro and gene function analysis revealed genes involved in skeletal and muscle system as well as cardiovascular system development and function.


Assuntos
Regulação da Expressão Gênica , Hormônio do Crescimento Humano/metabolismo , Proteínas/genética , Animais , Linhagem Celular , Perfilação da Expressão Gênica , Humanos , Camundongos , Fibras Musculares Esqueléticas/metabolismo , Proteínas/metabolismo
2.
Eur J Neurol ; 18(7): 1021-3, 2011 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-21658164

RESUMO

BACKGROUND: In some cases, a definitive confirmation of dysferlinopathy cannot be achieved by DNA test, because the mutation is detected in one allele only. PATIENTS AND METHODS: DYSFERLIN expression in skeletal muscle and peripheral blood monocytes (PBM) was studied by Western blot in two unrelated adult patients. The comparative C(T) method (ΔΔC(T) ) was used to calculate relative changes in dysferlin mRNA determined from real-time quantitative PCR experiments. The dysferlin gene was studied by direct sequencing of cDNA and genomic DNA and by Multiplex Ligation-dependent Probe Amplification (MLPA) analysis. RESULTS: A comparable severe reduction in dysferlin was demonstrated in both skeletal muscle and PBM. The expression of dysferlin mRNA was significantly reduced. A novel mutation in exon 47 (c.5289G>C) of the dysferlin gene in the heterozygous state, causing an amino acid change (p.Glu1763Asp), was detected in both patients. The MLPA analysis did not reveal any deletion or duplication. CONCLUSIONS: Dysferlin and/or dysferlin mRNA abnormalities are diagnostic for dysferlinopathy when mutational analysis detects a mutation in one allele only. Analysis of dysferlin mRNA can be helpful for distinguishing symptomatic heterozygotes from such patients.


Assuntos
Proteínas de Membrana/genética , Monócitos/patologia , Proteínas Musculares/genética , Músculo Esquelético/patologia , Distrofia Muscular do Cíngulo dos Membros/diagnóstico , Distrofia Muscular do Cíngulo dos Membros/genética , Adulto , Alelos , Western Blotting , Análise Mutacional de DNA , Disferlina , Feminino , Heterozigoto , Humanos , Masculino , Monócitos/metabolismo , Músculo Esquelético/metabolismo , Linhagem , Mutação Puntual , RNA Mensageiro/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa
3.
Neuromuscul Disord ; 19(1): 21-5, 2009 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-19084402

RESUMO

We report two patients with a new phenotype of dysferlinopathy presenting as congenital muscular disease. Both patients showed weakness in proximal lower limbs and neck flexor muscles at birth. The presence of normal CK levels during the first years should be noted. Initial MRI showed no abnormalities but short-time-inversion-recovery (STIR) sequences revealed a striking myoedema in gastrocnemius and hamstring muscles at the age of 5. Muscle biopsy showed mild dystrophic features and the absence of dysferlin. Dysferlin gene (DYSF) analysis revealed a p.Ala927LeufsX21 mutation in a homozygous state in both siblings. This new phenotype widens the clinical spectrum of dysferlin myopathies.


Assuntos
Predisposição Genética para Doença/genética , Proteínas de Membrana/genética , Proteínas Musculares/genética , Músculo Esquelético/patologia , Doenças Musculares/congênito , Doenças Musculares/genética , Mutação/genética , Substituição de Aminoácidos/genética , Biópsia , Pré-Escolar , Creatina Quinase/análise , Creatina Quinase/metabolismo , Análise Mutacional de DNA , Disferlina , Edema/genética , Edema/patologia , Edema/fisiopatologia , Feminino , Marcadores Genéticos/genética , Testes Genéticos , Genótipo , Humanos , Imageamento por Ressonância Magnética , Masculino , Debilidade Muscular/genética , Debilidade Muscular/patologia , Debilidade Muscular/fisiopatologia , Músculo Esquelético/metabolismo , Músculo Esquelético/fisiopatologia , Doenças Musculares/fisiopatologia , Fenótipo
4.
Neuromuscul Disord ; 17(1): 69-76, 2007 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-17070050

RESUMO

Dysferlin protein is expressed in peripheral blood monocytes. The genomic analysis of the DYSF gene has proved to be time consuming because it has 55 exons. We designed a mutational screening strategy based on cDNA from monocytes to find out whether the mutational analysis could be performed in mRNA from a source less invasive than the muscle biopsy. We studied 34 patients from 23 families diagnosed with dysferlinopathy. The diagnosis was based on clinical findings and on the absence of protein expression using either immunohistochemistry or Western blot of skeletal muscle and/or monocytes. We identified 28 different mutations, 13 of which were novel. The DYSF mutations in both alleles were found in 30 patients and only in one allele in four. The results were confirmed using genomic DNA in 26/34 patients. This is the first report to furnish evidence of reliable mutational analysis using monocytes cDNA and constitutes a good alternative to genomic DNA analysis.


Assuntos
Proteínas de Membrana/metabolismo , Monócitos/metabolismo , Proteínas Musculares/metabolismo , Distrofias Musculares/genética , Mutação , Análise Mutacional de DNA/métodos , Disferlina , Saúde da Família , Feminino , Regulação da Expressão Gênica , Humanos , Masculino , Proteínas de Membrana/genética , Proteínas Musculares/genética , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Distrofias Musculares/patologia , RNA Mensageiro/genética
5.
Neurology ; 57(11): 2136-8, 2001 Dec 11.
Artigo em Inglês | MEDLINE | ID: mdl-11739845

RESUMO

Inflammation was detected in 9 of 13 patients with different phenotypes of dysferlin myopathy. Endomysial or perivascular infiltrates consisted of 11.1% +/- 6.6% CD8(+) cells, 40.6% +/- 22.8% CD4(+) cells, 36.7% +/- 23.7% macrophages, and no B cells. Major histocompatibility complex class I was not upregulated in normal muscle fibers. In young patients with sporadic proximal weakness, very high creatine kinase levels, necrotic fibers and inflammation in the muscle biopsy, a diagnosis of dysferlin myopathy should be considered.


Assuntos
Linfócitos T CD4-Positivos/patologia , Linfócitos T CD8-Positivos/patologia , Cromossomos Humanos Par 2 , Proteínas de Membrana , Proteínas Musculares/genética , Distrofias Musculares/genética , Miosite/genética , Adolescente , Adulto , Biópsia , Disferlina , Feminino , Humanos , Masculino , Músculo Esquelético/patologia , Distrofias Musculares/patologia , Miosite/patologia
6.
J Neuroimmunol ; 120(1-2): 78-83, 2001 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-11694322

RESUMO

Antidisialosyl antibodies have been previously associated to chronic and acute ataxic neuropathies. We studied the presence of these antibodies in nine patients with acute self-limiting ataxic neuropathy (ASLAN) using ELISA and TLC immunodetection. One patient showed serum IgG immunoreactivity against gangliosides GD3 and GQ1b. The patient's IgG was able to bind to the nodes of Ranvier on frozen human dorsal root. Our studies confirmed that antidisialosyl reactivity is associated to ataxic neuropathy and its specific binding to the dorsal root could explain the predominant sensory involvement. Nevertheless, the low incidence of this reactivity indicates that a different pathogenic mechanism should be involved in most ASLAN patients.


Assuntos
Ataxia/imunologia , Autoanticorpos/sangue , Gangliosídeos/imunologia , Doenças do Sistema Nervoso Periférico/imunologia , Nós Neurofibrosos/imunologia , Raízes Nervosas Espinhais/imunologia , Doença Aguda , Idoso , Idoso de 80 Anos ou mais , Ataxia/sangue , Autoanticorpos/imunologia , Ensaio de Imunoadsorção Enzimática , Feminino , Gangliosídeos/metabolismo , Humanos , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Imuno-Histoquímica , Incidência , Masculino , Pessoa de Meia-Idade , Doenças do Sistema Nervoso Periférico/sangue , Doenças do Sistema Nervoso Periférico/patologia , Nós Neurofibrosos/metabolismo , Nós Neurofibrosos/patologia , Raízes Nervosas Espinhais/metabolismo , Raízes Nervosas Espinhais/patologia
7.
J Neurol ; 249(11): 1525-8, 2002 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-12420092

RESUMO

Antidisialosyl antibodies were found in two out of 13 patients with chronic idiopathic ataxic neuropathy (CIAN) and not in 32 patients with different sensory neuropathies of known cause. This finding confirms the association of antidisialosyl antibodies and CIAN regardless of the absence of the M band. These antibodies may have pathogenic relevance; however, larger series are needed to establish their clinical significance.


Assuntos
Ataxia/imunologia , Autoanticorpos/imunologia , Gânglios Espinais/imunologia , Gangliosídeos/imunologia , Neurônios Aferentes/imunologia , Doenças do Sistema Nervoso Periférico/imunologia , Idoso , Ataxia/fisiopatologia , Autoanticorpos/metabolismo , Doença Crônica , Cisplatino/toxicidade , Feminino , Gânglios Espinais/patologia , Gânglios Espinais/fisiopatologia , Gangliosídeos/metabolismo , Humanos , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Imunoglobulina M/sangue , Imunoglobulina M/imunologia , Masculino , Pessoa de Meia-Idade , Neurônios Aferentes/metabolismo , Neurônios Aferentes/patologia , Polineuropatia Paraneoplásica/imunologia , Doenças do Sistema Nervoso Periférico/fisiopatologia , Síndrome de Sjogren/imunologia
8.
Med Clin (Barc) ; 116(20): 761-4, 2001 Jun 02.
Artigo em Espanhol | MEDLINE | ID: mdl-11440679

RESUMO

BACKGROUND: To study the presence of anti-GQ1b antibodies as a tool for the diagnosis of Miller-Fisher syndrome (MFS). PATIENTS AND METHOD: We studied 54 patients with probable diagnosis of MFS and 10 patients diagnosed as Guillain-Barré syndrome plus ophthalmoplegia (1 case), Bickerstaff's encephalitis (1 case), relapsing ophthalmoplegia (7 cases) and relapsing diplopia (1 case). Results were compared with 130 patients with other disimmune neuropathies. Antibodies were detected by ELISA and checked by thin layer chromatography. Campylobacter jejuni serology was studied using a complement fixation test. RESULTS: Diagnosis of MFS was confirmed in 38 patients. A 97.3% were positive for GQ1b, being all negative for Campylobacter jejuni serology. A second test after 4-5 weeks of nadir was negative in 84.2% (16/19), concomitant with clinical recovery. CONCLUSIONS: Anti-GQ1b antibodies are useful markers for the differential diagnosis of MFS, specially with some acute brainstem disorders. Testing must be performed during the first four weeks of clinical course. This correlation between the triad ataxia, arreflexia and ophthalmoplegia and anti-GQ1b antibodies confirms that they are highly specific of MFS.


Assuntos
Anticorpos/sangue , Gangliosídeos/imunologia , Síndrome de Miller Fisher/diagnóstico , Fatores de Crescimento Neural/imunologia , Biomarcadores/sangue , Diagnóstico Diferencial , Ensaio de Imunoadsorção Enzimática , Humanos , Síndrome de Miller Fisher/imunologia
9.
Neurology ; 75(4): 316-23, 2010 Jul 27.
Artigo em Inglês | MEDLINE | ID: mdl-20574037

RESUMO

BACKGROUND: The most frequent phenotypes of dysferlin myopathy are limb-girdle muscular dystrophy 2B (LGMD2B) and Miyoshi myopathy (MM). Our objective was to find clinical or MRI markers to differentiate phenotypes of dysferlin myopathy regardless of initial symptoms. METHODS: This retrospective study included 29 patients with confirmed mutations in the DYSF gene (14 MM, 12 LGMD2B, 1 asymptomatic hyperCKemia, and 2 symptomatic carriers). All underwent an annual clinical examination (Medical Research Council scale), functional status assessment, and creatine kinase, pulmonary, and cardiac testing. For research purposes, we performed lower limb MRI studies in all 29 patients to identify the pattern of muscle impairment and to quantify involvement. Statistical correlations between MRI findings and phenotype, disease duration, and functional status were determined. RESULTS: The mean clinical follow-up was 6.4 +/- 5.7 years. No significant differences were found in the rate of progression, functional prognosis, or mutations between patients with MM and patients with LGMD2B. The MRI pattern of muscle involvement was the same for patients with MM and patients with LGMD2B. The adductor magnus and gastrocnemius medialis were the first to be impaired in both phenotypes. The progression of muscle involvement correlated with clinical status. CONCLUSIONS: Splitting dysferlin myopathy into separate phenotypes does not reveal significant differences in terms of rate of progression, prognosis, genotype, or MRI pattern. The finding that proximal and distal muscles are already impaired in the MRI at onset in both MM and LGMD2B favors grouping all phenotypes under the term dysferlin myopathy.


Assuntos
Imageamento por Ressonância Magnética , Proteínas de Membrana/genética , Proteínas Musculares/genética , Distrofia Muscular do Cíngulo dos Membros/genética , Distrofia Muscular do Cíngulo dos Membros/patologia , Atividades Cotidianas , Adolescente , Adulto , Idoso , Criança , Estudos de Coortes , Progressão da Doença , Disferlina , Seguimentos , Genótipo , Humanos , Lactente , Perna (Membro)/patologia , Pessoa de Meia-Idade , Músculo Esquelético/patologia , Distrofia Muscular do Cíngulo dos Membros/fisiopatologia , Fenótipo , Prognóstico , Estudos Retrospectivos , Índice de Gravidade de Doença , Adulto Jovem
10.
Neurology ; 68(16): 1284-9, 2007 Apr 17.
Artigo em Inglês | MEDLINE | ID: mdl-17287450

RESUMO

OBJECTIVE: To describe two symptomatic dysferlin gene mutation carriers. METHODS: One patient had limb girdle weakness. His brother was diagnosed with limb girdle muscular dystrophy 2B with two mutations in the dysferlin gene (D625Y and E1734G). The second patient had distal weakness. He had two sons with Miyoshi myopathy with a homozygous mutation (G519R). We performed immunofluorescence (dystrophin, DAG proteins, dysferlin, caveolin-3), Western blot (dysferlin, caveolin-3, calpain-3), and real-time PCR (dysferlin) using skeletal muscle samples. We also studied dysferlin in peripheral blood monocytes (PBMs) by Western blot. RESULTS: In addition to the muscle weakness, both patients showed elevated creatine kinase and abnormal muscle MRI. They presented a mutation in only one allele after screening of the whole gene (skeletal muscle and monocyte mRNA and genomic DNA). A muscle biopsy specimen showed moderate dystrophic changes and patchy dysferlin expression in the sarcolemma. Western blot of both PBMs and skeletal muscle demonstrated a significant reduction in dysferlin. All the other proteins including caveolin-3 and calpain-3 were normal. Real-time PCR showed normal levels of dysferlin mRNA vs the patients' affected relatives. CONCLUSIONS: The diagnosis of symptomatic carriers of dysferlin mutations should be considered when a pathologic pattern of dysferlin protein is observed.


Assuntos
Predisposição Genética para Doença/genética , Proteínas de Membrana/genética , Proteínas Musculares/genética , Debilidade Muscular/genética , Debilidade Muscular/fisiopatologia , Doenças Musculares/genética , Doenças Musculares/fisiopatologia , Adulto , Creatina Quinase/metabolismo , Análise Mutacional de DNA , Disferlina , Feminino , Triagem de Portadores Genéticos/métodos , Marcadores Genéticos , Testes Genéticos , Heterozigoto , Humanos , Imageamento por Ressonância Magnética , Masculino , Proteínas de Membrana/deficiência , Pessoa de Meia-Idade , Proteínas Musculares/deficiência , Proteínas Musculares/metabolismo , Debilidade Muscular/diagnóstico , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Músculo Esquelético/fisiopatologia , Doenças Musculares/diagnóstico , Mutação/genética , Linhagem
11.
Neurology ; 61(12): 1814-6, 2003 Dec 23.
Artigo em Inglês | MEDLINE | ID: mdl-14694058

RESUMO

Two patients with chronic motor neuropathy, high antiganglioside antibody (AGA) titers, and a declining response to IV immunoglobulins were treated with rituximab at a standard dose. The drug was well tolerated and effectively eliminated peripheral B cells (CD20+), but AGA titers continued significantly high. No clinical improvement was detected during the 1-year follow-up.


Assuntos
Anticorpos Monoclonais/uso terapêutico , Doenças Autoimunes do Sistema Nervoso/tratamento farmacológico , Doenças Autoimunes do Sistema Nervoso/imunologia , Gangliosídeos/imunologia , Imunoglobulina M/sangue , Adulto , Anticorpos Monoclonais/efeitos adversos , Anticorpos Monoclonais Murinos , Antígenos CD20/imunologia , Autoanticorpos/sangue , Linfócitos B/efeitos dos fármacos , Doença Crônica , Progressão da Doença , Seguimentos , Humanos , Masculino , Pessoa de Meia-Idade , Rituximab , Tempo , Falha de Tratamento
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