Recessive RYR1 mutations in a patient with severe congenital nemaline myopathy with ophthalomoplegia identified through massively parallel sequencing

Nemaline myopathy (NM) is a group of congenital myopathies, characterized by the presence of distinct rod-like inclusions "nemaline bodies" in the sarcoplasm of skeletal muscle fibers. To date, ACTA1, NEB, TPM3, TPM2, TNNT1, and CFL2 have been found to cause NM. We have identified recessive RYR1 mutations in a patient with severe congenital NM, through high-throughput screening of congenital myopathy/muscular dystrophy-related genes using massively parallel sequencing with target gene capture. The patient manifested fetal akinesia, neonatal severe hypotonia with muscle weakness, respiratory insufficiency, swallowing disturbance, and ophthalomoplegia. Skeletal muscle histology demonstrated nemaline bodies and small type 1 fibers, but without central cores or minicores. Congenital myopathies, a molecularly, histopathologically, and clinically heterogeneous group of disorders are considered to be a good candidate for massively parallel sequencing.     

Development of mature cartilage constructs using novel three-dimensional porous scaffolds for enhanced repair of osteochondral defects

In this study, we successfully developed two types of volume-reduced three-dimensional scaffolds, including cushion- and cylinder-shape scaffolds, fabricated from chitosan-based hyaluronic acid hybrid polymer fibers. Using these scaffolds combined with a bioreactor system, we regenerated histologically and mechanically mature cartilage constructs. The final goal of this study was to clarify the ability of this engineered cartilage construct to induce cartilage repair in osteochondral defects. The mature cartilage constructs regenerated with two types of scaffolds were implanted into 5-mm diameter osteochondral defects in the patellar groove of rabbits. At 12 weeks after implantation, the reparative tissues consisted of hyaline-like cartilage with evidence of stable fusion to adjacent native cartilage and normal reconstitution of subchondral bone. The histological score of these tissues significantly outranked the value of untreated tissue. Biomechanically, compression modulus of reparative tissue at 12 weeks postoperatively was comparative to that of normal articular cartilage. Our results indicate that the implantation of constructs with mature cartilage have potential as a better approach for joint resurfacing.     

Dectin-1 limits autoimmune neuroinflammation and promotes myeloid cell-astrocyte crosstalk via Card9-independent expression of Oncostatin M

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Development of murine hepatic sinusoidal endothelial cells characterized by the expression of hyaluronan receptors.

Endothelial cells (ECs) display distinct structural and functional characteristics depending on the tissue and developmental stage; however, the development of tissue-specific ECs remains poorly understood. Here, we describe the development of hepatic sinusoids in mice based on the expression of hyaluronan receptors Stab2 and Lyve-1. Flk-1(+) cells in and around the liver bud begin to express Stab2 at embryonic day (E) 9.5, before the formation of vascular lumen. Hepatic sinusoidal endothelial cells (HSECs) begin to express Lyve-1 at E10.5, and both markers continue to be expressed in HSECs thereafter. Although HSECs and lymphatic ECs (LECs) are known to share functional and phenotypic characteristics, we clearly show that HSECs can be distinguished from LECs by the expression of molecular markers and higher endocytotic activity. Our results provide new insight into the development of tissue-specific ECs and phenotypic criteria to distinguish HSECs from other types of ECs, including LECs.     

Results of immunotherapy for patients with unexplained primary recurrent abortions--prospective non-randomized cohort study

PROBLEM: The present study was conducted to examine the efficacy of immunotherapy for unexplained primary recurrent aborters using paternal lymphocytes. METHOD OF STUDY: Two hundred and twenty-eight recurrent aborters were prospectively followed up regarding immunotherapy. Of the 228 patients, 165 underwent immunotherapy using freshly prepared paternal lymphocytes and pregnancy outcome was analyzed. No mixed lymphocyte culture reaction-blocking antibodies (MLR-BAbs) were observed in these patients prior to vaccinations. Pregnancy outcome was also analyzed in such as those patients positive for MLR-BAbs and who did not undergo immunotherapy, and in patients negative for MLR-BAbs and who had become pregnant without immunotherapy. RESULTS: Of the 140 newly pregnant patients after immunotherapy, the pregnancy continued successfully in 110 (78.6%), and the pregnancy continued successfully in 24 of 32 patients (75.0%) who were positive for MLR-BAbs. The success rate of pregnancy was 30.0% in 18 non-immunized patients. Thus, the success rate was significantly higher among patients with immunotherapy and patients positive for MLR-BAbs than in non-immunized patients, negative for MLR-BAbs. CONCLUSION: Immunotherapy using paternal lymphocytes is considered to be effective for unexplained primary recurrent aborters negative for MLR-BAbs.     

Myopathy phenotype of transgenic mice expressing active site-mutated inactive p94 skeletal muscle-specific calpain, the gene product responsible for limb girdle muscular dystrophy type 2A

A defect of the gene for p94 (calpain 3), a skeletal muscle-specific calpain, is responsible for limb girdle muscular dystrophy type 2A (LGMD2A), or 'calpainopathy', which is an autosomal recessive and progressive neuromuscular disorder. To study the relationships between the physiological functions of p94 and the etiology of LGMD2A, we created transgenic mice that express an inactive mutant of p94, in which the active site Cys129 is replaced by Ser (p94:C129S). Three lines of transgenic mice expressing p94:C129S mRNA at various levels showed significantly decreased grip strength. Sections of soleus and extensor digitorum longus (EDL) muscles of the aged transgenic mice showed increased numbers of lobulated and split fibers, respectively, which are often observed in limb girdle muscular dystrophy muscles. Centrally placed nuclei were also frequently found in the EDL muscle of the transgenic mice, whereas wild-type mice of the same age had almost none. There was more p94 protein produced in aged transgenic mice muscles and it showed significantly less autolytic degradation activity than that of wild-type mice. Although no necrotic-regenerative fibers were observed, the age and p94:C129S expression dependence of the phenotypes strongly suggest that accumulation of p94:C129S protein causes these myopathy phenotypes. The p94:C129S transgenic mice could provide us with crucial information on the molecular mech-anism of LGMD2A.     

Preclinical evaluation of a hollow fiber silicone membrane oxygenator for extracorporeal membrane oxygenator application

A silicone membrane hollow fiber oxygenator applicable for use as an extracorporeal membrane oxygenator (ECMO) has been developed in our laboratory. This silicone hollow fiber displays astonishing mechanical stability, is barely compressible or stretchable, and assembles easily while maintaining good gas permeability. The priming volume is 140 cc with a surface area of 0.8 m2. This study evaluated the gas transfer performances and biocompatibility of the oxygenator under ECMO and CPB conditions. In vitro studies that were performed at a blood flow rate of 2 L/min, and revealed O2 and CO2 gas transfer rates of 82.35 +/- 0.56 ml/m2/L/min and 38.72 +/- 2.88 ml/m2/L/min, respectively. The commercially available Kolobow (Avecor 1500) oxygenator was used as the control, and had O2 and CO2 gas transfer rates of 53.8 +/- 0.5 ml/m2/L/min and 24.7 +/- 2.0 ml/m2/L/min. To evaluate blood trauma, Normalized Index of Hemolysis (NIH) was measured according to American Society of Testing and Materials (ASTM) standards. The NIH findings were 0.0112 g/100L at a blood flow of 1 L/min, and 0.0152 g/100L at 5 L/min. Three ex vivo experiments, using a blood flow rate of 1 L/min, were performed with venoarterial bypass, and O2 transfer rate and CO2 transfer rate of the oxygenators were well maintained. This indicates that this preclinical silicone membrane hollow fiber oxygenator has superior efficiency, less blood trauma, and is smaller when compared with the only clinically available Kolobow oxygenator.     

Extrapleural pneumonectomy of malignant pleural mesothelioma

We analyzed 7 patients with malignant pleural mesothelioma who underwent extrapleural pneumonectomy. Six men and a woman had a mean age of 65 years old. The postoperative mortality rate was 14% (1 death) and morbidity, 43% (3 cases). According to staging of International Mesothelioma Interest Group, 2 patients had stage I disease, 1 did stage II, 3 did stage III and 1 did stage IV. Local recurrences were found in 3 patients and metastasis in 2. In patients with local recurrences, 2 had irradiation with chemotherapy and 1, irradiation. In patients with recurrences of metastasis, 1 had chemotherapy and 1, supportive care. Seven patients with extrapleural pneumonectomy and 10 without surgery had median survivals of 16 months and 10 months, 1-year survival rates of 71% and 40% and 2-year survival rates of 57% and 0% respectively (p=0.071). Extrapleural pneumonectomy with adjuvant therapy could be effective treatment for malignant pleural mesothelioma.     

Clinical features and course of aortitis syndrome in Japanese women older than 40 years

The clinical features and course of aortitis syndrome were studied in 11 women older than 40 years of age. The patients were Japanese women, mean age 57 +/- 6 years old, who were followed for 6.9 +/- 3.8 years. Data from 24 young patients were used for comparison. In the older patients, systemic hypertension (73%), calcification of the aorta (73%), left ventricular hypertrophy (92%) and cardiomegaly (82%) were frequent, whereas the erythrocyte sedimentation rate was normal in 5 patients and only slightly accelerated in 6. C-reactive protein was positive in 2. The incidence of cardiac involvement and inflammatory signs was significantly different from findings in the young patients. Aortic regurgitation (AR) (55%) was significantly more frequent and renal artery stenosis was not observed. Other arterial lesions revealed a pattern similar to those seen in the young patients. An irregular luminal surface, kinking and calcification were present in the lesions in the older patients. The survival rate at 5 years was 80%. Five of 6 patients with AR had congestive heart failure, 4 of whom died. One died after a stroke. Thus, aortitis syndrome in older patients has a long course. There is usually an associated AR, renal artery stenosis is rare and other arterial lesions do not change a great deal. The prognosis may be good, but depends on the association of AR.     

Changes in lipid peroxide and oxygen radical scavengers in cerulein-induced acute pancreatitis. Imbalance between the offense and defense systems

The role of free radicals in the development of cerulein-induced pancreatitis was evaluated by measuring the activity of the endogenous scavengers, superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSHpx), as indicators of the defense system, and the level of lipid peroxide (LPO) in the pancreas, as an indicator of the offense system. Acute pancreatitis was induced by 5 hourly intraperitoneal administrations of cerulein (50 micrograms/kg body weight), in 0.9% NaCl, to mice. The presence of acute pancreatitis was confirmed by changes in serum amylase levels and in typical microscopical features. Regarding the changes in the levels of endogenous scavengers, the SOD level was decreased significantly from a basal level of 52.6 +/- 3.94 to 43.1 +/- 2.79 mU/micrograms DNA at 6 h (p less than 0.01) to 38.8 +/- 5.18 mU/micrograms DNA at 9 h (p less than 0.05) and to 31.7 +/- 3.10 mU/micrograms DNA at 12 h (p less than 0.01) after the first intraperitoneal cerulein injection. The CAT level also decreased significantly from a basal level of 7.80 +/- 0.27 to 5.86 +/- 0.46 mU/micrograms DNA at 9 h (p less than 0.01) and to 4.52 +/- 0.21 mU/microgram DNA at 12 h (p less than 0.01). GSHpx increased from a basal level of 6.80 +/- 0.43 to 7.58 +/- 0.50 mU/micrograms DNA at 9 h and to 10.2 +/- 0.52 mU/micrograms DNA at 12 h after the first intraperitoneal cerulein injection.(ABSTRACT TRUNCATED AT 250 WORDS)