R-spondin 2 promotes acetylcholine receptor clustering at the neuromuscular junction via Lgr5.

At the neuromuscular junction (NMJ), acetylcholine receptor (AChR) clustering is mediated by spinal motor neuron (SMN)-derived agrin and its receptors on the muscle, the low-density lipoprotein receptor-related protein 4 (LRP4) and muscle-specific receptor tyrosine kinase (MuSK). Additionally, AChR clustering is mediated by the components of the Wnt pathway. Laser capture microdissection of SMNs revealed that a secreted activator of Wnt signaling, R-spondin 2 (Rspo2), is highly expressed in SMNs. We found that Rspo2 is enriched at the NMJ, and that Rspo2 induces MuSK phosphorylation and AChR clustering. Rspo2 requires Wnt ligands, but not agrin, for promoting AChR clustering in cultured myotubes. Leucine-rich repeat-containing G-protein coupled receptor 5 (Lgr5), an Rspo2 receptor, is also accumulated at the NMJ, and is associated with MuSK via LRP4. Lgr5 is required for Rspo2-mediated AChR clustering in myotubes. In Rspo2-knockout mice, the number and density of AChRs at the NMJ are reduced. The Rspo2-knockout diaphragm has an altered ultrastructure with widened synaptic clefts and sparse synaptic vesicles. Frequency of miniature endplate currents is markedly reduced in Rspo2-knockout mice. To conclude, we demonstrate that Rspo2 and its receptor Lgr5 are Wnt-dependent and agrin-independent regulators of AChR clustering at the NMJ.

Reactions of community members regarding community health workers' activities as a measure of the impact of a training program in Amazonas, Brazil.

OBJECTIVES: The aim of this study was to evaluate the impact of community health worker (CHW) training on recognition and satisfaction regarding the performance of CHWs among members of the community in Amazonas, Brazil, which is a resource-poor area underserved with regard to medical health-care accessibility. METHODS: Baseline and endline surveys concerning recognition and satisfaction with respect to CHW performance among members of the community were conducted by interview using a questionnaire before and after implementation of a program to strengthen community health projects in Manicoré, Amazonas, Brazil. One of the components of the project was CHW refresher training, which focused on facilitating adequate use of health-care services and providing primary health care, including health guidance. The baseline survey was performed in February 2004 at the beginning of the project, and the endline survey was performed in February 2006 at the end of the project. There were 82 and 120 CHWs working in Manicoré at the times of the baseline and endline surveys, respectively. Statistical analysis was performed to determine the significance of changes in experience with CHW activities, expected functions of CHWs, and satisfaction regarding the performance of CHWs between the baseline and endline surveys. In addition, qualitative analysis was conducted to evaluate the acceptability, feasibility, and sustainability of CHW refresher training. RESULTS: Overall recognition and level of satisfaction regarding CHW performance among members of the community were improved from the baseline to the endline survey, regardless of type of residential area, such as town and/or remote area. Members of the community came to not expect CHWs to "provide strong medicine" (P < 0.001) and "provide injections" (P < 0.001), and came to appreciate "go to hospital with a sick person" (P = 0.031) as a function and role of CHWs. CONCLUSIONS: The results of the present study indicated that steady approaches to motivate and support CHWs in resource-limited settings could improve performance of CHWs and satisfaction of people in the community regarding the activities of CHWs to sustain their health.

Effects of Vibration Therapy on Immobilization-Induced Hypersensitivity in Rats.

BACKGROUND: Cast immobilization induces mechanical hypersensitivity, which disturbs rehabilitation. Although vibration therapy can reduce various types of pain, whether vibration reduces immobilization-induced hypersensitivity remains unclear. OBJECTIVE: The purpose of this study was to investigate the preventive and therapeutic effects of vibration therapy on immobilization-induced hypersensitivity. DESIGN: The experimental design of the study involved conducting behavioral, histological, and immunohistochemical studies in model rats. METHODS: Thirty-five Wistar rats (8 weeks old, all male) were used. The right ankle joints of 30 rats were immobilized by plaster cast for 8 weeks, and 5 rats were used as controls. The immobilized rats were divided randomly into the following 3 groups: (1) immobilization-only group (Im, n=10); (2) vibration therapy group 1, for which vibration therapy was initiated immediately after the onset of immobilization (Im+Vib1, n=10); and (3) vibration therapy group 2, for which vibration therapy was initiated 4 weeks after the onset of immobilization (Im+Vib2, n=10). Vibration was applied to the hind paw. The mechanical hypersensitivity and epidermal thickness of the hind paw skin were measured. To investigate central sensitization, calcitonin gene-related peptide (CGRP) expression in the spinal cord and dorsal root ganglion (DRG) was analyzed. RESULTS: Immobilization-induced hypersensitivity was inhibited in the Im+Vib1 group but not in the Im+Vib2 group. Central sensitization, which was indicated by increases in CGRP expression in the spinal cord and the size of the area of CGRP-positive neurons in the DRG, was inhibited in only the Im+Vib1 group. Epidermal thickness was not affected by vibration stimulation. LIMITATIONS: A limitation of this study is that the results were limited to an animal model and cannot be generalized to humans. CONCLUSIONS: The data suggest that initiation of vibration therapy in the early phase of immobilization may inhibit the development of immobilization-induced hypersensitivity.

Upregulation of interleukin-1ß/transforming growth factor-ß1 and hypoxia relate to molecular mechanisms underlying immobilization-induced muscle contracture.

INTRODUCTION: In this study we investigated the molecular mechanism underlying muscle contracture in rats. METHODS: The rats were divided into immobilization and control groups, and soleus muscles of the right and left sides were selected for analyses. RESULTS: The levels of CD11b and α-SMA protein, IL-1ß, and TGF-ß1 mRNA, and type I and III collagen protein and mRNA were significantly greater in the immobilization group than in the control group at all time-points. HIF-1α mRNA levels were significantly higher in the immobilization group at 4 weeks. Moreover, HIF-1α, α-SMA, and type I collagen levels were significantly higher at 4 weeks than at 1 and 2 weeks in the immobilization group. CONCLUSIONS: In the early stages of immobilization, upregulation of IL-1ß/TGF-ß1 via macrophages may promote fibroblast differentiation that could affect muscle contracture. The soleus muscle became hypoxic in the later stages of immobilization, suggesting that hypoxia influences the progression of muscle contracture.

Neuromuscular disease. DOK7 gene therapy benefits mouse models of diseases characterized by defects in the neuromuscular junction.

The neuromuscular junction (NMJ) is the synapse between a motor neuron and skeletal muscle. Defects in NMJ transmission cause muscle weakness, termed myasthenia. The muscle protein Dok-7 is essential for activation of the receptor kinase MuSK, which governs NMJ formation, and DOK7 mutations underlie familial limb-girdle myasthenia (DOK7 myasthenia), a neuromuscular disease characterized by small NMJs. Here, we show in a mouse model of DOK7 myasthenia that therapeutic administration of an adeno-associated virus (AAV) vector encoding the human DOK7 gene resulted in an enlargement of NMJs and substantial increases in muscle strength and life span. When applied to model mice of another neuromuscular disorder, autosomal dominant Emery-Dreifuss muscular dystrophy, DOK7 gene therapy likewise resulted in enlargement of NMJs as well as positive effects on motor activity and life span. These results suggest that therapies aimed at enlarging the NMJ may be useful for a range of neuromuscular disorders.

[Increased rate of serum uric acid levels in Yusho sufferers].

We measured serum uric acid levels in Yusho sufferers annually from 2007 to 2012 in Nagasaki prefecture. We observed an increased rate of serum uric acid levels in 38.2% of the male and 5.5% of the female sufferers. There was no relation among serum uric acid value, Body Mass Index, liver function, blood polychlorinated biphenyls and hypersensitive C reactive protein. We conclude that it is unclear if blood polychlorinated biphenyls may play a role in the increase of serum uric acid levels in Yusho sufferers.

Immobilization-induced hypersensitivity associated with spinal cord sensitization during cast immobilization and after cast removal in rats.

This study examined mechanical and thermal hypersensitivity in the rat hind paw during cast immobilization of the hind limbs for 4 or 8 weeks and following cast removal. Blood flow, skin temperature, and volume of the rat hind paw were assessed in order to determine peripheral circulation of the hind limbs. Sensitization was analyzed by measuring the expression of the calcitonin gene-related peptide (CGRP) in the spinal dorsal horn following cast immobilization. Two weeks post immobilization, mechanical and thermal sensitivities increased significantly in all rats; however, peripheral circulation was not affected by immobilization. Cast immobilization for 8 weeks induced more serious hypersensitivity compared to cast immobilization for 4 weeks. Moreover, CGRP expression in the deeper lamina layer of the spinal dorsal horn increased in the rats immobilized for 8 weeks but not in those immobilized for 4 weeks. These findings suggest that immobilization-induced hypersensitivity develops during the immobilization period without affecting peripheral circulation. Our results also highlight the possibility that prolonged immobilization induces central sensitization in the spinal cord.

Antibodies against the main immunogenic region of the acetylcholine receptor correlate with disease severity in myasthenia gravis.

OBJECTIVE: We developed an assay that detects autoantibodies against the main immunogenic region (MIR) located at the extracellular end of the nicotinic acetylcholine receptor (AChR) α subunit, and investigated its clinical relevance in myasthenia gravis (MG). METHODS: In this retrospective cohort study, we measured MIR antibody (Ab) titres in sera obtained before treatment and analysed their associations with clinical parameters in 102 MG patients from two neurological centres. MIR Ab titres were determined using a modified competition immunoprecipitation assay in the presence or absence of monoclonal antibody 35. RESULTS: 11 of 23 (47.8%) ocular type and 66 of 72 (91.7%) generalised type MG patients were positive for the presence of MIR Abs, defined as a titre >16.8% (3 SDs above the mean for 70 healthy controls). A significantly higher MIR Ab titre (p<0.001) was shown in generalised type (47.9±19.2%) rather than in ocular type MG patients (16.4±8.4%). Bivariate regression analysis using both titre levels of MIR Ab and routine AChR binding Ab as variables revealed MIR Abs to be an exclusive indicator positively associated with disease severity (Myasthenia Gravis Foundation of America classification, p<0.0001; Quantitative MG score, p=0.008), the presence of bulbar symptoms (p<0.0001) and thymoma (p=0.016), and negatively associated with ocular MG (p<0.0001). CONCLUSIONS: MIR Ab titre levels show much better correlations with factors related to disease severity compared with AChR binding Ab titres. The MIR Ab assay may be useful for predicting MG symptom severity, especially for discriminating between ocular and generalised types of MG.

A case of Japanese spotted fever complicated with central nervous system involvement and multiple organ failure.

Japanese spotted fever (JSF), first reported in 1984, is a rickettsial disease characterized by high fever, rash, and eschar formation. A 61-year-old man was admitted to a local hospital in Nagasaki City, Japan, after several days of high fever and generalized skin erythema. His condition deteriorated and laboratory findings indicated disseminated intravascular coagulation (DIC). The patient was transferred to our hospital with mental disturbance and status epilepticus. Treatment included minocycline, and new quinolone. Definitive diagnosis was made with a serological test showing increased antibody levels against Rickettsia japonica. Rickettsial infections are rare, but should be seriously considered for the differential diagnosis of aseptic meningitis and encephalitis, as they show no response to conventional antibiotic treatment.

Protein-anchoring strategy for delivering acetylcholinesterase to the neuromuscular junction.

Acetylcholinesterase (AChE) at the neuromuscular junction (NMJ) is anchored to the synaptic basal lamina via a triple helical collagen Q (ColQ). Congenital defects of ColQ cause endplate AChE deficiency and myasthenic syndrome. A single intravenous administration of adeno-associated virus serotype 8 (AAV8)-COLQ to Colq(-/-) mice recovered motor functions, synaptic transmission, as well as the morphology of the NMJ. ColQ-tailed AChE was specifically anchored to NMJ and its amount was restored to 89% of the wild type. We next characterized the molecular basis of this efficient recovery. We first confirmed that ColQ-tailed AChE can be specifically targeted to NMJ by an in vitro overlay assay in Colq(-/-) mice muscle sections. We then injected AAV1-COLQ-IRES-EGFP into the left tibialis anterior and detected AChE in noninjected limbs. Furthermore, the in vivo injection of recombinant ColQ-tailed AChE protein complex into the gluteus maximus muscle of Colq(-/-) mice led to accumulation of AChE in noninjected forelimbs. We demonstrated for the first time in vivo that the ColQ protein contains a tissue-targeting signal that is sufficient for anchoring itself to the NMJ. We propose that the protein-anchoring strategy is potentially applicable to a broad spectrum of diseases affecting extracellular matrix molecules.

[Histochemical findings of and fine structural changes in motor endplates in diseases with neuromuscular transmission abnormalities].

We herein review the histochemical findings and fine structural changes of motor endplates associated with diseases causing neuromuscular transmission abnormalities. In anti-acetylcholine receptor (AChR) antibody-positive myasthenia gravis (MG), type 2 fiber atrophy is observed, and the motor endplates show a reduction in the nerve terminal area, simplification of the postsynaptic membrane, decreased number of acetylcholine receptors, and deposition of immune complexes. In anti-MuSK antibody-positive MG, the fine structure shows a decrease in the postsynaptic membrane length, but the secondary synaptic cleft is preserved. There is no decrease in the number of AChRs, and there are no deposits of immune complexes at the motor endplates. Patients with Lambert-Eaton myasthenic syndrome show type 2 fiber atrophy, their motor endplates show a decrease in both the mean postsynaptic area and postsynaptic membrane length in the brachial biceps muscle. Congenital myasthenic syndrome with episodic apnea is characterized only by small-sized synaptic vesicles; the postsynaptic area is preserved. In subjects with congenital myasthenic syndrome with acetylcholinesterase deficiency, quantitative electron microscopy reveals a significant decrease in the nerve terminal size and presynaptic membrane length; further, the Schwann cell processes extend into the primary synaptic cleft, and partially or completely occlude the presynaptic membrane. The postsynaptic folds are degenerated, and associated with pinocytotic vesicles and labyrinthine membranous networks. Patients with slow-channel congenital myasthenia syndrome show type 1 fiber predominance, and their junctional folds are typically degenerated with widened synaptic space and loss of AChRs. Patients with AChR deficiency syndrome caused by recessive mutations in AChR subunits also show type 1 fiber predominance, and while most junctional folds are normal, some are simplified and have smaller than normal endplates. Rapsin and MuSK mutations cause type 1 fiber predominance, and the small postsynaptic area is associated with AChR decrease.

[Differences in the bone mineral density in patients with Kanemi Yusho treated before and after the age of 18 years].

This study examined patients with Kanemi Yusho. The patients' height, weight, and bone mineral density were measured. The density of the distal end of the radius was measured using dual energy X-ray absorptiometry and the calcaneum was measured with ultrasound. We also measured urine levels of cross-linked N-telopeptides of type I collagen, serum tartrate-resistant acid phosphatase 5b, serum bone-specific alkaline phosphatase, serum Ca, serum P and blood PCB level. The patient group that took PCBs when they were 0 to 18 years old (such patients were 42 to 60 years old at the time of the study) showed no correlation between the bone density of the radius and calcaneum in spite of treatment received when they were over 18 years of age (> 60 years of age at the time of the study). The bone mineral density in Kanemi Yusho was not different from the control group. The levels of only serum bone-specific alkaline phosphatase were correlated with the bone mineral density of the radius and calcaneum in patients treated when they were over 18 years of age (currently over 60 years old). PCBs might have had an effect on bone density and bone metabolism.

False-positive accumulation of metaiodobenzylguanidine in a case with acute intermittent porphyria.

We report a 36-year-old woman presenting with hypertensive encephalopathy followed by bulbar palsy and quadriplegia. After an extensive screening for secondary causes of hypertension, the patient was suspected of having pheochromocytoma due to increased levels of catecholamines in the plasma and the urine, and positive (131)I-metaiodobenzylguanidine (MIBG) accumulation in the gallbladder. However, MIBG accumulation was not reproducible without any tumors accompanying this accumulation in the gallbladder. A diagnosis of acute intermittent porphyria was finally confirmed based on the characteristic pictures, increased urinary excretion of porphobilinogen, and identification of a heterozygous missense mutation of R173W in the hydroxymethylbilane synthase gene. This case highlights a pitfall in utilizing MIBG to detect a source of excessive catecholamine and also suggests the importance of having a complete clinical history and extensive work-up of any possible differential diagnosis. We also review the potential mechanism by which false-positive MIBG accumulation occurs.

Rheumatoid vasculitis of crural muscles confirmed by muscle biopsy in the absence of inflammatory myopathy: histologic and MRI study.

A 60-year-old man who had been diagnosed as rheumatoid arthritis admitted to our hospital by dysesthesia on his legs with edema. Nerve conduction velocity test led to diagnosis of mononeuritis multiplex. Magnetic resonance imaging (MRI) of lower legs showed high intensity in slow tau inversion recovery. Typical vasculitis with neutrophil-dominant cell infiltration was observed by muscle biopsy without inflammatory myopathy or fascitis. Diagnosis was made by rheumatoid vasculitis found in crural muscles. Intravenous cyclophosphamide with oral tacrolimus effectively improved dysesthesia with reduction of inflammatory response.

[Bone mineral density, PCB, PCQ and PCDF in Yusho].

We measured bone mineral density of the distal end of radius with dual energy X-ray absorptiometry, serum cross-linked N-telopeptides of type I collagen, serum bone-specific alkaline phosphatase, serum Ca, serum P, blood PCB level, blood PCQ level and blood PCDF level in Yusho. As a result, the osteoporosis group (< 70% of the young adult mean [YAM] bone mineral density [BMD]) was observed in 7.1% of the studied male subjects. And, the moderate group (> or = 70% and < 80% of YAM BMD), 16.1%, the normal (> or = 80% of YAM BMD) group was 76.8%. Also, 42.3% of all female tested subjects observed in osteoporosis group. The moderate group, 19.2%, the normal group was 38.5%. There was no difference in PCB blood level, PCQ, PCDF for men and women in osteoporosis group, moderate group, and in the normal group. Serum cross-linked N-telopeptides of type I collagen increased in the male osteoporosis group, but serum bone-specific alkaline phosphatase did not change. This study was inconclusive since the results did not determine the influence that PCB, PCQ, PCDF gave to bone density and bone metabolism.

Low-level laser irradiation promotes the recovery of atrophied gastrocnemius skeletal muscle in rats.

Low-level laser (LLL) irradiation promotes proliferation of muscle satellite cells, angiogenesis and expression of growth factors. Satellite cells, angiogenesis and growth factors play important roles in the regeneration of muscle. The objective of this study was to examine the effect of LLL irradiation on rat gastrocnemius muscle recovering from disuse muscle atrophy. Eight-week-old rats were subjected to hindlimb suspension for 2 weeks, after which they were released and recovered. During the recovery period, rats underwent daily LLL irradiation (Ga-Al-As laser; 830 nm; 60 mW; total, 180 s) to the right gastrocnemius muscle through the skin. The untreated left gastrocnemius muscle served as the control. In conjunction with LLL irradiation, 5-bromo-2-deoxyuridine (BrdU) was injected subcutaneously to label the nuclei of proliferating cells. After 2 weeks, myofibre diameters of irradiated muscle increased in comparison with those of untreated muscle, but did not recover back to normal levels. Additionally, in the superficial region of the irradiated muscle, the number of capillaries and fibroblast growth factor levels exhibited significant elevation relative to those of untreated muscle. In the deep region of irradiated muscle, BrdU-positive nuclei of satellite cells and/or myofibres increased significantly relative to those of the untreated muscle. The results of this study suggest that LLL irradiation can promote recovery from disuse muscle atrophy in association with proliferation of satellite cells and angiogenesis.

Immobilization-induced cartilage degeneration mediated through expression of hypoxia-inducible factor-1alpha, vascular endothelial growth factor, and chondromodulin-I.

Immobilization results in thinning of the articular cartilage and cartilage degeneration, although the exact mechanisms are not clear yet. Hypoxia is thought to contribute to the degeneration of articular cartilage. We investigated the roles of hypoxia inducible factor (HIF)-1alpha, vascular endothelial growth factor (VEGF), and the newly cloned antiangiogenic factor, chondromodulin-I (ChM-1), in cartilage degeneration in immobilized joints. Male Wistar rats (n = 30, 12-week-old) were divided randomly into the control group (n = 10), immobilization group (n = 10), and continuous passive motion (CPM) group (n = 10). In the immobilization group, the ankle joints were fixed in full plantar flexion with plaster casts for 4 weeks. In the CPM group, the ankle casts were removed during the immobilization period and the ankle joints were subjected to CPM. Significant thinning of the articular cartilage was noted in the immobilization group but not in the control or CPM group. In the immobilized group, vascular channels were found in the area between the calcified cartilage zone and the subchondral bone. The densities of HIF-1alpha-and VEGF-immunostained cells were higher in the immobilized group than the other two groups. In contrast, low expression of ChM-1 was detected in the articular cartilage of the immobilized group compared with the control and CPM group. Our results showed that immobilization induces thinning of the articular cartilage and appearance of vascular channel, in areas with balanced expression of HIF-1alpha/VEGF and ChM-1.

Effects of therapeutic ultrasound on joint mobility and collagen fibril arrangement in the endomysium of immobilized rat soleus muscle.

This study examined effects of therapeutic ultrasound on joint mobility and collagen fibril arrangement in the endomysium of immobilized rat soleus muscle. Twenty-two male Wistar rats were divided randomly into control (n = 5) and experimental groups (n = 17). In the experimental group, bilateral ankle joints of each rat were fixed in full plantar flexion with a plaster cast over a 4-wk period. Five animals in the experimental group were immobilized throughout the 4-wk (immobilization group) period, whereas the remaining rats in the experimental group were randomly divided into the ultrasound (US, n = 6) and sham (n = 6) treatment groups. Under anesthesia, continuous ultrasonic energy (frequency, 1 MHz; intensity, 1.0 W/cm(2)) was delivered to the triceps surae muscle of the US group for 15 min per d, 6 d per wk over the 4-wk immobilization period. Ultrasonic energy was not delivered to the triceps surae muscle in sham animals; only the transducer head was moved. Ankle joint mobility on dorsiflexion in the immobilization, sham and US groups was significantly smaller than that of the control group, whereas in the US group, this parameter was significantly greater than in the immobilization and sham groups. Collagen fibril arrangement in the endomysium of the control and US groups was longitudinal to the axis of the muscle fibers; in contrast, it was circumferential in the immobilization and sham groups. Our findings revealed that joint immobilization induces decreased joint mobility and collagen fibril movement in the endomysium; furthermore, ultrasound treatment can prevent these changes. We hypothesized that therapeutic ultrasound during the immobilization process may inhibit deterioration of muscle contracture.

[Overview: MuSK/Dok-7].

MuSK/Dok-7 mediate the clustering of acetylcholine receptor (AChR) during synapse formation and are expressed at the mature neuromuscular junction. These proteins are deeply associated with myasthenia gravis (MG) and congenital myasthenic syndrome (CMS). Compared with MG patients with AChR antibodies, those with muscle-specific tyrosine kinase (MuSK) antibodies are more likely to present oculobulbar than limb weakness, myasthenic crisis and muscle wasting. None have thymoma, so the indication for thymectomy should be investigated. MuSK antibodies do not appear to cause complement-mediated morphological motor endplate damage, but how they cause myasthenic symptoms is unclear. As the results, the three types of MG presently characterized by known antibody targets are classified into 1) AChR antibody-positive, 2) MuSK antibody -positive, and 3) double seronegative type which the above-mentioned antibodies are negative. In 2006, MuSK-interacting cytoplasmic protein termed Dok-7 has been found. Subsequently, mutations in Dok-7 as a cause of CMS were identified, providing evidence for a crucial role of Dok-7 in maintaining synaptic structure. Their effect on MuSK/Dok -7 function needs to be explored.