Human multipotent adipose-derived stem cells restore dystrophin expression of Duchenne skeletal-muscle cells in vitro.

BACKGROUND INFORMATION: DMD (Duchenne muscular dystrophy) is a devastating X-linked disorder characterized by progressive muscle degeneration and weakness. The use of cell therapy for the repair of defective muscle is being pursued as a possible treatment for DMD. Mesenchymal stem cells have the potential to differentiate and display a myogenic phenotype in vitro. Since liposuctioned human fat is available in large quantities, it may be an ideal source of stem cells for therapeutic applications. ASCs (adipose-derived stem cells) are able to restore dystrophin expression in the muscles of mdx (X-linked muscular dystrophy) mice. However, the outcome when these cells interact with human dystrophic muscle is still unknown. RESULTS: We show here that ASCs participate in myotube formation when cultured together with differentiating human DMD myoblasts, resulting in the restoration of dystrophin expression. Similarly, dystrophin was induced when ASCs were co-cultivated with DMD myotubes. Experiments with GFP (green fluorescent protein)-positive ASCs and DAPI (4',6-diamidino-2-phenylindole)-stained DMD myoblasts indicated that ASCs participate in human myogenesis through cellular fusion. CONCLUSIONS: These results show that ASCs have the potential to interact with dystrophic muscle cells, restoring dystrophin expression of DMD cells in vitro. The possibility of using adipose tissue as a source of stem cell therapies for muscular diseases is extremely exciting.     

Human multipotent adipose-derived stem cells restore dystrophin expression of Duchenne skeletal-muscle cells in vitro

Background information. DMD (Duchenne muscular dystrophy) is a devastating X‐linked disorder characterized by progressive muscle degeneration and weakness. The use of cell therapy for the repair of defective muscle is being pursued as a possible treatment for DMD. Mesenchymal stem cells have the potential to differentiate and display a myogenic phenotype in vitro. Since liposuctioned human fat is available in large quantities, it may be an ideal source of stem cells for therapeutic applications. ASCs (adipose‐derived stem cells) are able to restore dystrophin expression in the muscles of mdx (X‐linked muscular dystrophy) mice. However, the outcome when these cells interact with human dystrophic muscle is still unknown. Results. We show here that ASCs participate in myotube formation when cultured together with differentiating human DMD myoblasts, resulting in the restoration of dystrophin expression. Similarly, dystrophin was induced when ASCs were co‐cultivated with DMD myotubes. Experiments with GFP (green fluorescent protein)‐positive ASCs and DAPI (4′,6‐diamidino‐2‐phenylindole)‐stained DMD myoblasts indicated that ASCs participate in human myogenesis through cellular fusion. Conclusions. These results show that ASCs have the potential to interact with dystrophic muscle cells, restoring dystrophin expression of DMD cells in vitro. The possibility of using adipose tissue as a source of stem cell therapies for muscular diseases is extremely exciting.     

Muscle Protein Alterations in LGMD2I 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 2I form (LGMD2I). Here we report muscle histological alterations and the analysis of 11 muscle proteins: dystrophin, four sarcoglycans, calpain 3, dysferlin, telethonin, collagen VI, α-DG, and α2-laminin, in muscle biopsies from 13 unrelated LGMD2I 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 α2-laminin deficiency was detected in 12 patients through immunofluorescence analysis, and 10 patients presented α-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 LGMD2I patients, different mutations in the FKRP gene are associated with several secondary muscle protein reductions, and the deficiencies of α2-laminin and α-DG on sections are prevalent, independently of mutation type or clinical severity. (J Histochem Cytochem 56:995–1001, 2008)     

Successful treatment of refractory chylous ascites due to follicular lymphoma with very low-dose radiotherapy

Chylous ascites is an extremely rare complication of lymphoma. Here, we discuss the case of a patient presenting with refractory chylous ascites due to a massive retroperitoneal follicular lymphoma, staged as IVB. The patient was unresponsive to chemoimmunotherapy, which prompted us to consider alternative treatment strategies. Low-dose radiotherapy was initiated and resulted in a marked decrease of the lymphadenopathy and complete regression of the peritoneal fluid. Low-dose radiotherapy represents a well-tolerated, highly effective treatment and should remain an important modality in cases of follicular lymphoma-associated chylous ascites.     

Mild and severe muscular dystrophy caused by a single gamma-sarcoglycan mutation.

Autosomal recessive muscular dystrophy is genetically heterogeneous. One form of this disorder, limb-girdle muscular dystrophy type 2C (LGMD 2C), is prevalent in northern Africa and has been shown to be associated with a single mutation in the gene encoding the dystrophin-associated protein gamma-sarcoglycan. The previous mutation analysis of gamma-sarcoglycan required the availability of muscle biopsies. To establish a mutation assay for genomic DNA, the intron-exon structure of the gamma-sarcoglycan gene was determined, and primers were designed to amplify each of the exons encoding gamma-sarcoglycan. We studied a group of Brazilian muscular dystrophy patients for mutations in the gamma-sarcoglycan gene. These patients were selected on the basis of autosomal inheritance and/or the presence of normal dystrophin and/or deficiency of alpha-sarcoglycan immunostaining. Four of 19 patients surveyed had a single, homozygous mutation in the gamma-sarcoglycan gene. The mutation identified in these patients, all of African-Brazilian descent, is identical to that seen in the North African population, suggesting that even patients of remote African descent may carry this mutation. The phenotype in these patients varied considerably. Of four families with an identical mutation, three have a severe Duchenne-like muscular dystrophy. However, one family has much milder symptoms, suggesting that other loci may be present that modify the severity of the clinical course resulting from gamma-sarcoglycan gene mutations.     

Characterization of the binding of a rat somatomedin to receptors in human placental cell membranes.

The somatomedins presumably initiate their growth promoting effects by first binding to specific cell surface receptors in responsive tissues. The specific and high affinity binding of [¹²⁵I]-rat somatomedin to human placental membranes was saturable and reversible with a dissociation constant of 4.5 × 10^?9 M calculated from Scatchard analysis of competitive binding experiments. Competition for [¹²⁵I]-rat somatomedin binding to placental receptors by other somatomedins and growth factors suggest a close structural relationship between rat somatomedin and the human somatomedin, insulin-like growth factor I.     

High proportion of new mutations and possible anticipation in Brazilian facioscapulohumeral muscular dystrophy families.

A gene responsible for facioscapulohumeral muscular dystrophy (FSHD) has been localized at 4q35. Subsequently, it was found that probe p13E-11 detects a polymorphic EcoRI fragment, usually > 28 kb, in normal individuals, whereas in sporadic and familial FSHD cases, an EcoRI fragment, usually < 28 kb, was found. Although these findings have been amply confirmed, several aspects are as yet either controversial or unsolved. In the present investigation, 34 Brazilian FSHD families were studied at the clinical and the molecular level for the following purposes: to assess the frequency of new mutations and their effect on estimates of biological fitness, to characterize FSHD-associated EcoRI fragments detected with probe p13E-11 in familial--as compared with isolated--FSHD cases, and to assess whether anticipation occurs in multigenerational families. Results from our study suggest that new mutations are apparently frequent for FSHD and may account for at least one-third of the cases, that somatic mosaicism may not be rare, and that biological fitness appeared to be reduced in FSHD, ranging from 0.6 to 0.82 by different estimates, with no difference in sexes. Interestingly, the size of the new EcoRI fragment is apparently smaller in more severely affected isolated patients. Moreover, the age at onset of clinical signs, as well as the age at ascertainment, in patients from multigenerational families suggests that anticipation occurs for FSHD in the majority of the families.     

Testicular juvenile granulosa cell tumor: a case report

BACKGROUND: Juvenile granulosa cell tumors of the testis are rare gonadal stromal tumors of the pediatric age. They represent the most common neoplasms of the testis in the first 6 months of life. A testicular cystic mass is detected, or it can appear as an abdominal or inguinal mass. Differential diagnosis for testicular tumors at this age includes teratoma, simple cyst, epidermoid cyst, lymphangioma and testicular torsion. Association with ambiguous genitalia and sex chromosome abnormalities has been reported. Orchiectomy alone is recommended, because no case of metastasis or recurrence has been reported. CASE: We report a case of a 3-month-old male infant with a testicular juvenile granulosa cell tumor mass initially evaluated by fine needle biopsy, which disclosed single or cohesive groups of vimentin, alpha-inhibin and S-100-positive spindle cells with regular nuclei and fine chromatin and inconspicuous nucleoli. Orchiectomy was performed, and histology revealed a juvenile granulosa cell tumor. CONCLUSION: Even though juvenile granulosa cell tumor is the most common neoplasm of the testis in the first 6 months of life, we found no reports describing its cytologic features. In this setting, fine needle aspiration cytology is a useful tool for initial and therapeutic management.     

Quantitative T2 Combined with Texture Analysis of Nuclear Magnetic Resonance Images Identify Different Degrees of Muscle Involvement in Three Mouse Models of Muscle Dystrophy: mdx,Largemyd and mdx/Largemyd

Quantitative nuclear magnetic resonance imaging (MRI) has been considered a promising non-invasive tool for monitoring therapeutic essays in small size mouse models of muscular dystrophies. Here, we combined MRI (anatomical images and transverse relaxation time constant—T2—measurements) to texture analyses in the study of four mouse strains covering a wide range of dystrophic phenotypes. Two still unexplored mouse models of muscular dystrophies were analyzed: The severely affected Large^myd mouse and the recently generated and worst double mutant mdx/Large^myd mouse, as compared to the mildly affected mdx and normal mice. The results were compared to histopathological findings. MRI showed increased intermuscular fat and higher muscle T2 in the three dystrophic mouse models when compared to the wild-type mice (T2: mdx/Large^myd: 37.6±2.8 ms; mdx: 35.2±4.5 ms; Large^myd: 36.6±4.0 ms; wild-type: 29.1±1.8 ms, p<0.05), in addition to higher muscle T2 in the mdx/Large^myd mice when compared to mdx (p<0.05). The areas with increased muscle T2 in the MRI correlated spatially with the identified histopathological alterations such as necrosis, inflammation, degeneration and regeneration foci. Nevertheless, muscle T2 values were not correlated with the severity of the phenotype in the 3 dystrophic mouse strains, since the severely affected Large^myd showed similar values than both the mild mdx and worst mdx/Large^myd lineages. On the other hand, all studied mouse strains could be unambiguously identified with texture analysis, which reflected the observed differences in the distribution of signals in muscle MRI. Thus, combined T2 intensity maps and texture analysis is a powerful approach for the characterization and differentiation of dystrophic muscles with diverse genotypes and phenotypes. These new findings provide important noninvasive tools in the evaluation of the efficacy of new therapies, and most importantly, can be directly applied in human translational research.