Existem diferenças nos parâmetros hematológicos e bioquímicos séricos entre fêmeas normais e portadoras do modelo experimental GRMD (Golden Retriever Muscular Dystrophy)?

A proposta deste estudo foi avaliar se existem alterações nos padrões hematológicos e bioquímicos de cadelas da raça Golden Retriever portadoras do gene da distrofia muscular progressiva em comparação aos valores obtidos em cadelas não portadoras de mesma raça e idade. Foram analisados 33 animais, distribuídos em dois grupos, um composto por 19 cadelas Golden Retrievers não portadoras (GRNP) e outro composto por 14 cadelas Golden Retrievers portadoras do gene da distrofia muscular (GRP). Os dois grupos foram submetidos aos mesmos testes hematológicos e bioquímicos, com a mesma frequência e durante o mesmo intervalo de tempo. Apesar de existir diferença estatisticamente significativa entre os grupos para alguns parâmetros hematológicos avaliados, todos os resultados obtidos estavam de acordo com os valores de referência utilizados. Na avaliação dos parâmetros bioquímicos séricos a dosagem de ALT no grupo GRNP ficou levemente acima da média, porém sem grandes significados clínicos A CK também apresentou níveis elevados no grupo GRP, devido à degeneração e necrose muscular característicos da doença, as alterações encontradas nessa análise já eram esperadas. Os demais parâmetros não se alteraram.

Alterações morfofuncionais renais em cães Golden Retriever Distróficos (GRMD)

A Golden Retriever Muscular Dystrophy (GRMD) é geneticamente homóloga à distrofia muscular de Duchenne (DMD) que acomete seres humanos. É uma doença genética que gera degeneração progressiva da musculatura esquelética. Considerando-se as intensas alterações musculares, é natural pensar em uma possível lesão renal decorrente da intensa lesão muscular. Foram avaliados seis cães machos da raça Golden Retriever afetados pela distrofia muscular (GRMD) e três cães machos clinicamente sadios. A concentração de creatinina foi determinada e as proteínas urinárias foram avaliadas por eletroforese em gel de poliacrilamida. Os resultados mostraram que a proteinúria patológica não está diretamente associada à Distrofia Muscular de Duchenne, porém diversos parâmetros apresentaram concentrações aumentadas para animais afetados, como a razão proteína/albumina, que foi maior em cães distróficos, podendo ser indício de microalbuminúria e conseqüente lesão renal precoce. Estes resultados visam embasar avaliações clínicas e futuros estudos considerando-se as patologias decorrentes ou associadas a esta doença genética.

Síndrome de Lowe: relato de cinco casos

INTRODUÇÃO: A síndrome de Lowe, ou distrofia oculocerebrorrenal (OCRL), tem herança recessiva ligada ao cromossomo X. Apresenta-se com catarata, glaucoma, atraso no desenvolvimento neuropsicomotor (DNPM), déficit cognitivo e síndrome de Fanconi. OBJETIVO: Descrever a evolução de cinco pacientes pediátricos atendidos no ambulatório de tubulopatias do Departamento de Nefrologia Pediátrica da Universidade Federal de São Paulo-Escola Paulista de Medicina Unifesp (Unifesp-EPM). MÉTODOS: Estudo retrospectivo de cinco pacientes masculinos atendidos no ambulatório de tubulopatias. RESULTADOS: A média de idade na primeira consulta foi de 76,5 meses; o tempo médio de acompanhamento, de 30,5 meses (mínimo de 8 meses e máximo de 53 meses). Os sintomas e os sinais clínicos incluíram catarata e nistagmo. Atraso no DNPM e déficit de peso e de estatura estiveram presentes em todos os casos, bem como poliúria, polidipsia, constipação, acidose metabólica, fosfatúria, bicarbonatúria, proteinúria, hipercalciúria e hiperuricosúria. Nefrocalcinose foi identificada em um paciente; litíase renal, em três; e redução do tamanho renal, em dois. Fraturas patológicas e raquitismo foram observados em dois pacientes; rarefação óssea e atraso na idade óssea, em todos os pacientes. Um deles apresentou redução no ritmo de filtração glomerular. Terapeuticamente, todos receberam álcalis, fósforo e reposição com vitamina D, além de orientação dietética para suas necessidades. CONCLUSÃO: Este estudo preconiza a necessidade do diagnóstico precoce e do acompanhamento médico e nutricional desses pacientes para evitar complicações relacionadas com distúrbios metabólicos.

Deletion Analysis of 15 exons located outside and inside a “Hot Spot” in the Duchenne Muscular Dystrophy gene in Duchenne’s Muscular dystrophy patients

Introduction. Duchenne and Becker Muscular Dystrophies (DMD/DMB) are X-linked recessive diseases characterized by progressive muscle weakness and wasting, loss of motor skills and death after the second decade of life. Deletions are the most prevalent mutations that affect the dystrophin gene, which spans 79 exons.Objective: Identify deletions on the dystrophin gene in 58 patients affected with DMD.Methods: Through multiplex PCR identify deletions on the dystrophin gene in 58 patients with DMD and observe the frequency of this mutation in our population.Results: We found deletions in 1.72% of patients (1 of 58 persons). Deletions were not the principal cause of disease in our population. It is possible that duplications and point mutations caused this illness in our patients.Conclusions: The frequency of deletions in the 15 exons analyzed from the dystrophin gene was low. The predominant types of mutation in our patients` samples were not deletions as has been observed in the literature worldwide, therefore, it is important to determine other types of mutations as are duplications and point mutations.

Muscle Protein Alterations in LGMD21 Patients With Different Mutations in the Fukutin-related Protein Gene

Fukutin-related protein (FKRP) is a protein involved in the glycosylation of cell surface molecules. Pathogenic mutations in the FKRP gene cause both the more severe congenital muscular dystrophy Type 1C and the milder Limb-Girdle Type 21 form (LGMD21). Here we report muscle histological alterations and the analysis of 11 muscle proteins: dystrophin, four sarcoglycans, calpain 3, dysferlin, telethonin, collagen VI, alpha-DG, and alpha 2-laminin, in muscle biopsies from 13 unrelated LGMD21 patients with 10 different FKRP mutations. In all, a typical dystrophic pattern was observed. In eight patients, a high frequency of rimmed vacuoles was also found. A variable degree of alpha 2-laminin deficiency was detected in 12 patients through immunofluorescence analysis, and 10 patients presented a-DG deficiency on sarcolemmal membranes. Additionally, through Western blot analysis, deficiency of calpain 3 and dystrophin bands was found in four and two patients, respectively. All the remaining proteins showed a similar pattern to normal controls. These results suggest that, in our population of LGMD21 patients, different mutations in the FKRP gene are associated with several secondary muscle protein reductions, and the deficiencies of alpha 2-laminin and alpha-DG on sections are prevalent, independently of mutation type or clinical severity.

Effect of Citrus aurantium l. essential oil on muscle regeneration in mdx mice

Duchenne muscular dystrophy (DMD) is a severe X-linked recessive disorder characterized by the progressive loss of muscular strength. Mdx mutant mice show a marked deficiency in dystrophin, which was related to muscle membrane stability. The aim of this study was to verify the possible protective anti-inflammatory effect of citrus oil on mdx muscle fibers. Thus, adult male and female mdx mice (014/06-CEEA) were divided into control and citrus-treated. After 60 days of treatment, one ml of blood was collected for creatine kinase (CK) test. Diaphragm, sternomastoideus, anterior tibial and gastrocnemius muscles were removed and processed according to histological routine methods. The observed alterations indicate a direct effect of citrus. Recent studies have improved the diagnosis of muscular diseases but with no definitions of efficient treatments. Intervention with several therapies is important to many patients presenting muscular dystrophy, which enables them to live longer and be more active, while there is no development of gene therapies.

Síndrome dolorosa complexa regional: epidemiologia, fisiopatologia, manifestações clínicas, testes diagnósticos e propostas terapêuticas

JUSTIFICATIVA E OBJETIVOS: A Síndrome Dolorosa Complexa Regional (SDCR), assim denominada a partir de 1994 pelo Consenso da Associação Internacional para o Estudo da Dor (AIED) e anteriormente denominada de várias formas, tais como Distrofia Simpático Reflexa, Causalgia, Algodistrofia ou Atrofia de Sudeck, é uma doença cuja compreensão dos limites clínicos, fisiopatologia e implicações de patogenia ainda é pobre. Disto resulta a enorme insatisfação não só para os pacientes como para os profissionais da saúde quanto aos métodos terapêuticos atualmente disponíveis. O objetivo deste trabalho é rever a literatura e atualizar um conjunto de informações com o intuito da melhor compreensão desta importante síndrome dolorosa. CONTEÚDO: Este é um trabalho de revisão da literatura nos diversos aspectos da SDCR, com ênfase em suas causas, definição e taxonomia, fisiopatologia, características clínicas, testes diagnósticos e propostas de tratamentos mais recentes. CONCLUSÕES: Poucos são os estudos controlados adequadamente, encobertos e aleatórios, publicados com grandes amostras, havendo muitas dúvidas sobre esta doença. Desta forma, ainda há enorme empirismo na sua terapêutica, e os resultados obtidos são insatisfatórios.

Efeito da inflamação do tornozelo sobre as características histológicas, a expressão gênica e níveis da creatina cinase nos músculos sóleo e tibial anterior de ratos diabéticos

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Diferenciação miogênica “in vitro” de células tronco mesenquimais murinas de diferentes origens

Muscular dystrophy refers to a group of more than 30 genetical disorders characterized by progressive weakness and degeneration of the skeletal muscle. No effective therapy is available at present. Recent studies have reported that the transplantation of stem cells can offer an important potential therapy for genetic diseases. Adult bone marrow mesenchymal stem cells have been identified as a nonhematopoietic stem cell population capable of self-renewal with the ability to differentiate into many cell lineages, including bone, fat, cartilage and connective tissue. Because of their similarity with muscle progenitor cells, when they are injected in affected individuals, they are able to migrate into areas of skeletal muscle degeneration and participate in the regeneration process. The adipose tissue represents an alternative source of MSCs that, as the MSCs derived from bone marrow, are capable of in vitro differentiation into osteogenic, adipogenic, myogenic and chondrogenic lineages. The objective of this project is to investigate the “in vitro” myogenic potential of mesenchymal stem cells derived from murine bone marrow and adipose tissue. Four experimental groups were analyzed: mice from lineages Lama2dy-2J/J and C57black and, C2C12 lineage cells and transformed C2C12 expressing the eGFP protein. MSCs cultures were obtained by flushing the bone marrow femurs and tibials with α-MEM or by the subcutaneous and inguinal fat from the mice. Their characterization was done by flow cytometry and in vitro differentiation. Muscle differentiation was studied through the analysis of the expression of transcriptional factors involved in muscle differentiation and/or the presence and amount of specific proteins from muscle differentiated cell. The pluripotency from bone marrow MSCs of the two lineages was evidenced and, in the muscular differentiation... (Complete abstract click electronic access below)

Thomsen or Becker myotonia? A novel autosomal recessive nonsense mutation in the CLCN1 gene associated with a mild phenotype

We describe a large Brazilian consanguineous kindred with 3 clinically affected patients with a Thomsen myotonia phenotype. They carry a novel homozygous nonsense mutation in the CLCN1 gene (K248X). None of the 6 heterozygote carriers show any sign of myotonia on clinical evaluation or electromyography. These findings confirm the autosomal recessive inheritance of the novel mutation in this family, as well as the occurrence of phenotypic variability in the autosomal recessive forms of myotonia. Muscle Nerve, 2012

Differential Expression of Genes Involved in the Degeneration and Regeneration Pathways in Mouse Models for Muscular Dystrophies

The genetically determined muscular dystrophies are caused by mutations in genes coding for muscle proteins. Differences in the phenotypes are mainly the age of onset and velocity of progression. Muscle weakness is the consequence of myofiber degeneration due to an imbalance between successive cycles of degeneration/regeneration. While muscle fibers are lost, a replacement of the degraded muscle fibers by adipose and connective tissues occurs. Major investigation points are to elicit the involved pathophysiological mechanisms to elucidate how each mutation can lead to a specific degenerative process and how the regeneration is stimulated in each case. To answer these questions, we used four mouse models with different mutations causing muscular dystrophies, Dmd (mdx) , SJL/J, Large (myd) and Lama2 (dy2J) /J, and compared the histological changes of regeneration and fibrosis to the expression of genes involved in those processes. For regeneration, the MyoD, Myf5 and myogenin genes related to the proliferation and differentiation of satellite cells were studied, while for degeneration, the TGF-beta 1 and Pro-collagen 1 alpha 2 genes, involved in the fibrotic cascade, were analyzed. The result suggests that TGF-beta 1 gene is activated in the dystrophic process in all the stages of degeneration, while the activation of the expression of the pro-collagen gene possibly occurs in mildest stages of this process. We also observed that each pathophysiological mechanism acted differently in the activation of regeneration, with distinctions in the induction of proliferation of satellite cells, but with no alterations in stimulation to differentiation. Dysfunction of satellite cells can, therefore, be an important additional mechanism of pathogenesis in the dystrophic muscle.

Muscle reorganisation through local injection of stem cells in the diaphragm of mdx mice

Background: The diaphragm is the major respiratory muscle affected by Duchenne muscular dystrophy (DMD) and is responsible for causing 80% of deaths. The use of mechanical forces that act on the body or intermittent pressure on the airways improves the quality of life of patients but does not prevent the progression of respiratory failure. Thus, diseases that require tissue repair, such as DMD, represent a group of pathologies that have great potential for cell therapy. The application of stem cells directly into the diaphragm instead of systemic application can reduce cell migration to other affected areas and increase the chances of muscle reorganisation. The mdx mouse is a suitable animal model for this research because its diaphragmatic phenotype is similar to human DMD. Therefore, the aim of this study was to assess the potential cell implantation in the diaphragm muscle after the xenotransplantation of stem cells. Methods: A total of 9 mice, including 3 control BALB/Cmice, 3 5-month-old mdx mice without stem cell injections and 3 mdx mice injected with stem cells, were used. The animals injected with stem cells underwent laparoscopy so that stem cells from GFP-labelled rabbit olfactory epithelium could be locally injected into the diaphragm muscle. After 8 days, all animals were euthanised, and the diaphragm muscle was dissected and subjected to histological and immunohistochemical analyses. Results: Both the fresh diaphragm tissue and immunohistochemical analyses showed immunopositive GFP labelling of some of the cells and immunonegativity of myoblast bundles. In the histological analysis, we observed a reduction in the inflammatory infiltrate as well as the presence of a few peripheral nuclei and myoblast bundles. Conclusion: We were able to implant stem cells into the diaphragm via local injection, which promoted moderate muscle reorganisation. The presence of myoblast bundles cannot be attributed to stem cell incorporation because there was no immunopositive labelling in this structure. It is believed that the formation of the bundles may have been stimulated by cellular signalling mechanisms that have not yet been elucidated.

Human Adipose-Derived Mesenchymal Stromal Cells Injected Systemically Into GRMD Dogs Without Immunosuppression Are Able to Reach the Host Muscle and Express Human Dystrophin

Duchenne muscular dystrophy (DMD), a lethal X-linked disorder, is the most common and severe form of muscular dystrophies, affecting I in 3,500 male births. Mutations in the DMD gene lead to the absence of muscle dystrophin and a progressive degeneration of skeletal muscle. The possibility to treat DMD through cell therapy has been widely investigated. We have previously shown that human adipose-derived stromal cells (hASCs) injected systemically in SJL mice are able to reach and engraft in the host muscle, express human muscle proteins, and ameliorate the functional performance of injected animals without any immunosuppression. However, before starting clinical trials in humans many questions still need to be addressed in preclinical studies, in particular in larger animal models, when available. The best animal model to address these questions is the golden retriever muscular dystrophy (GRMD) dog that reproduces the full spectrum of human DMD. Affected animals carry a mutation that predicts a premature termination codon in exon 8 and a peptide that is 5% the size of normal dystrophin. These dogs present clinical signs within the first weeks and most of them do not survive beyond age two. Here we show the results of local and intravenous injections of hASCs into GRMD dogs, without immunosuppression. We observed that hASCs injected systemically into the dog cephalic vein are able to reach, engraft, and express human dystrophin in the host GRMD dystrophic muscle up to 6 months after transplantation. Most importantly, we demonstrated that injecting a huge quantity of human mesenchymal cells in a large-animal model, without immunosuppression, is a safe procedure, which may have important applications for future therapy in patients with different forms of muscular dystrophies.

Motor function measure scale, steroid therapy and patients with Duchenne muscular dystrophy

Objective: To assess the evolution of motor function in patients with Duchenne muscular dystrophy (DMD) treated with steroids (prednisolone or deflazacort) through the Motor Function Measure (MFM), which evaluates three dimensions of motor performance (D1, D2, D3). Methods: Thirty-three patients with DMD (22 ambulant, 6 non-ambulant and 5 who lost the capacity to walk during the period of the study) were assessed using the MFM scale six times over a period of 18 months. Results: All the motor functions remained stable for 14 months in all patients, except D1 for those who lost their walking ability. In ambulant patients, D2 (axial and proximal motor capacities) motor functions improved during six months; an improvement in D3 (distal motor capacity) was noted during the total follow-up. D1 (standing posture and transfers) and total score were useful to predict the loss of the ability to walk. Conclusions: The use of the MFM in DMD patients confirms the benefits of the steroid treatment for slowing the progression of the disease.

Early transplantation of human immature dental pulp stem cells from baby teeth to golden retriever muscular dystrophy (GRMD) dogs: Local or systemic?

Background:The golden retriever muscular dystrophy (GRMD) dogs represent the best available animal model for therapeutic trials aiming at the future treatment of human Duchenne muscular dystrophy (DMD). We have obtained a rare litter of six GRMD dogs (3 males and 3 females) born from an affected male and a carrier female which were submitted to a therapeutic trial with adult human stem cells to investigate their capacity to engraft into dogs muscles by local as compared to systemic injection without any immunosuppression. Methods Human Immature Dental Pulp Stem Cells (hIDPSC) were transplanted into 4 littermate dogs aged 28 to 40 days by either arterial or muscular injections. Two non-injected dogs were kept as controls. Clinical translation effects were analyzed since immune reactions by blood exams and physical scores capacity of each dog. Samples from biopsies were checked by immunohistochemistry (dystrophin markers) and FISH for human probes. Results and Discussion We analyzed the cells' ability in respect to migrate, engraftment, and myogenic potential, and the expression of human dystrophin in affected muscles. Additionally, the efficiency of single and consecutive early transplantation was compared. Chimeric muscle fibers were detected by immunofluorescence and fluorescent in situ hybridisation (FISH) using human antibodies and X and Y DNA probes. No signs of immune rejection were observed and these results suggested that hIDPSC cell transplantation may be done without immunosuppression. We showed that hIDPSC presented significant engraftment in GRMD dog muscles, although human dystrophin expression was modest and limited to several muscle fibers. Better clinical condition was also observed in the dog, which received monthly arterial injections and is still clinically stable at 25 months of age. Conclusion Our data suggested that systemic multiple deliveries seemed more effective than local injections. These findings open important avenues for further researches.