AMPK inhibits voltage-gated calcium channel-current in rat chromaffin cells.

Metabolic changes are critical in the regulation of Ca2+ influx in central and peripheral neuroendocrine cells. To study the regulation of L-type Ca2+ channels by AMPK we used biochemical reagents and ATP/glucose-concentration manipulations in rat chromaffin cells. AICAR and Compound-C, at low concentration, significantly induce changes in L-type Ca2+ channel-current amplitude and voltage dependence. Remarkably, an overlasting decrease in the channel-current density can be induced by lowering the intracellular level of ATP. Accordingly, Ca2+ channel-current density gradually diminishes by decreasing the extracellular glucose concentration. By using immunofluorescence, a decrease in the expression of CaV1.2 is observed while decreasing extracellular glucose, suggesting that AMPK reduces the number of functional Ca2+ channels into the plasma membrane. Together, these results support for the first time the dependence of metabolic changes in the maintenance of Ca2+ channel-current by AMPK. They reveal a key step in Ca2+ influx in secretory cells.

Skin biopsy is useful for the antemortem diagnosis of neuronal intranuclear inclusion disease.

BACKGROUND: Neuronal intranuclear inclusion disease (NIID) is a progressive neurodegenerative disease characterized by eosinophilic hyaline intranuclear inclusions in neuronal and somatic cells. Because of the variety of clinical manifestations, antemortem diagnosis of NIID is difficult. METHODS: Seven skin biopsy samples from patients with familial NIID were evaluated histochemically, and the results were compared with those of skin samples from normal control subjects and from patients with other neurologic diseases. We also examined skin biopsy samples from patients with NIID by electron microscopy. RESULTS: In NIID skin biopsy samples, intranuclear inclusions were observed in adipocytes, fibroblasts, and sweat gland cells. These inclusions were stained with both anti-ubiquitin and anti-SUMO1 antibodies. Electron microscopy revealed that the features of the intranuclear inclusions in adipocytes, fibroblasts, and sweat gland cells were identical to those of neuronal cells. Approximately 10% of adipocytes showed intranuclear inclusions. No intranuclear inclusions were identified in the skin samples from normal control subjects and patients with other neurologic diseases. CONCLUSIONS: Skin biopsy is an effective and less invasive antemortem diagnostic tool for NIID.

Interferon alfa treatment for Sjogren's syndrome associated neuropathy.

Treatment response to interferon alfa (IFNalpha) is described in three consecutive cases of two forms of Sjogren's syndrome associated neuropathy (SSN)-two with sensory ataxic ganglionopathy and one with sensorimotor neuropathy with demyelinating features. All responded well to IFNalpha in terms of neuropathic symptoms, sicca symptoms, antibody titres, and findings in salivary gland biopsy specimens. IFNalpha thus showed promise in treating both SSN and the underlying Sjogren's syndrome.

Transgenic mouse models of spinal and bulbar muscular atrophy (SBMA).

Spinal and bulbar muscular atrophy (SBMA) is a late-onset motor neuron disease characterized by proximal muscle atrophy, weakness, contraction fasciculations, and bulbar involvement. Only males develop symptoms, while female carriers usually are asymptomatic. A specific treatment for SBMA has not been established. The molecular basis of SBMA is the expansion of a trinucleotide CAG repeat, which encodes the polyglutamine (polyQ) tract, in the first exon of the androgen receptor (AR) gene. The pathologic hallmark is nuclear inclusions (NIs) containing the mutant and truncated AR with expanded polyQ in the residual motor neurons in the brainstem and spinal cord as well as in some other visceral organs. Several transgenic (Tg) mouse models have been created for studying the pathogenesis of SBMA. The Tg mouse model carrying pure 239 CAGs under human AR promoter and another model carrying truncated AR with expanded CAGs show motor impairment and nuclear NIs in spinal motor neurons. Interestingly, Tg mice carrying full-length human AR with expanded polyQ demonstrate progressive motor impairment and neurogenic pathology as well as sexual difference of phenotypes. These models recapitulate the phenotypic expression observed in SBMA. The ligand-dependent nuclear localization of the mutant AR is found to be involved in the disease mechanism, and hormonal therapy is suggested to be a therapeutic approach applicable to SBMA.

[A patient with lymphoma of mucosa-associated lymphoid tissue (MALT) and adenocarcinoma in the same tumor of the lung].

A 60-year-old woman with no symptoms was found to have a mass shadow in the left lower lobe of the lung on chest radiography. Open lung biopsy and left lower lobectomy were performed. Histopathological study of the specimen revealed two distinct neoplasms. One tumor was a low grade B-cell lymphoma of mucosa-associated lymphoid tissue (MALT) of the lung, while the other was an adenocarcinoma. The two neoplasms were admixed to form a composite tumor.

[Bronchiolitis obliterans organizing pneumonia after adjuvant thoracic radiotherapy for breast cancer: a case report].

We report a case of recurrent cough and migratory pulmonary infiltrates in a 55-year-old woman after adjuvant thoracic radiotherapy for breast cancer. The pulmonary infiltrates were initially limited to the area adjacent to the irradiated breast, but later migrated to the opposite lung. The diagnosis of bronchiolitis obliterans organizing pneumonia (BOOP) was made using a transbronchial biopsy, which disclosed intraluminal fibrosis in the distal airspace, together with a radiographic appearance typical of BOOP. This case was assumed to be in a series of reported cases of BOOP primed by radiotherapy.

The role of lymphotoxin in pathogenesis of polymyositis.

Polymyositis (PM) is a cell-mediated autoimmune disease. Perforin (PF), Fas ligand (FasL) and TNF-alpha are considered to be important factors in cytotoxic T lymphocyte-mediated cell injury, and several studies have established a role of lymphotoxin (LT) in T helper type 1 (Th1)-induced cell-mediated autoimmune diseases. In the present study, to determine how LT, PF and FasL are involved in the pathogenesis of PM, we used immunohistochemical staining (IHC), reverse transcription polymerase chain reaction (RT-PCR), and in situ hybridization (ISH) on muscle specimens from patients with PM, amyotrophic lateral sclerosis (ALS), myotonic dystrophy (MyD) and controls (NC). There were many mononuclear cells (MNCs) immunoreactive for LT and some for PF and FasL within the fasciculus in PM muscles. On the other hand, only few or no LT-, PF- and FasL-positive cells were detected in MyD, ALS and NC muscles. The results of mRNA expression of these three molecules with RT-PCR were consistent with those using IHC methods. The number of MNCs positive for LT with ISH was far higher in PM compared to MyD, ALS and NC (P < 0.05 or 0.01). The MNCs located in the connective tissue or in the vicinity of necrotizing or non-necrotizing muscles were mainly LT mRNA and CD4 positive, while MNCs invading the non-necrotic fibers were mainly LT mRNA and CD8 positive. Our results indicated that the expression of LT was up-regulated in PM, and LT plays an important role in muscle injury and orchestrating the inflammatory reaction in PM.

Reduced expression of DMAHP/SIX5 gene in myotonic dystrophy muscle.

In myotonic dystrophy (DM), the expansion of CTG triplet repeats in the 3'-untranslated region of DM-protein kinase (DMPK) is a causal gene mutation. However, the pathogenic molecular mechanism of CTG repeat expansion for DM phenotypic expression is unclear. To investigate this issue, we examined the influence of CTG repeat expansion on the expression levels of DMPK gene and 3'-flanking DM locus-associated homeodomain protein (DMAHP)/SIX5 gene in the muscles of DM patients. We isolated RNA from muscle tissues of six DM patients and six controls, and performed a competitive reverse transcriptional polymerase chain reaction (RT-PCR) assay. The total mRNA level of DMAHP/SIX5 was significantly lower in DM than in controls, but the DMPK mRNA level was unchanged. Our results suggest that CTG repeat expansion influences the expression of genes other than DMPK to cause the DM phenotype.

[CNS cryptococcosis with idiopathic CD4+ T lymphocytopenia].

A 33-year-old Japanese man, with a history of recurrent skin cryptococcosis, was admitted complaining of fever and severe headache for 3 weeks. He had no known risk factors for human immunodeficiency virus (HIV) infection. Cerebrospinal fluid examination revealed an elevated opening pressure of 32 cm H2O, cell counts of 884/mm3, a total protein value of 184 mg/dl, a glucose level of 16 mg/dl, and demonstrated a positive India ink stain for fungus. Cultures grew Cryptococcus neoformans. Hematological studies showed a persistently low CD4+ cell count (30/mm3) and a low CD4/CD8 ratio of 0.1. He has been repeatedly seronegative (ELISA and Western blot) for HIV-1 and HIV-2. He responded to fluconazole, and was given itraconazole as secondary prophylaxis because of persistent low CD4 counts. To our knowledge this is the first patient with idiopathic CD4+ T lymphocytopenia associated with CNS cryptococcosis in Japan. CD4 counts should be part of the initial work up for patients with CNS cryptococcosis.

Tumor necrosis factor-alpha expression in muscles of polymyositis and dermatomyositis.

We evaluated the expression of tumor necrosis factor-alpha (TNF-alpha) mRNA in muscle biopsy specimens from patients with polymyositis (PM) and dermatomyositis (DM) to clarify its role in the pathogenesis of PM and DM. We performed non-radioactive in situ hybridization studies for TNF-alpha combined with immunohistochemistry for cell type-specific markers on muscles from ten PM and five DM patients. TNF-alpha-positive infiltrating cells present in the endomysium and perimysium were found in all PM and DM muscles. The frequency of TNF-alpha-positive cells against total infiltrating cells was similar among PM and DM (27.1 +/- 7.4% in PM and 28.5 +/- 13.6% in DM). However, TNF-alpha/CD8-positive lymphocytes and TNF-alpha-positive macrophages invading the non-necrotic muscle fiber were observed only in PM but not in DM. TNF-alpha was more highly expressed in PM and DM than was previously thought, and it was suggested that TNF-alpha plays a role in muscle fiber degeneration in PM.

Expression of HLA-DR and its enhancing molecules in muscle fibers in polymyositis.

Polymyositis (PM) is an autoimmune inflammatory muscle disease of unknown cause in which cellular immunity is thought to play an important pathogenic role. Class II major histocompatibility complex (class II MHC: human leukocyte antigen (HLA)-DR operates as a cofactor of antigen presentation in immunological responses. There has been a major debate over whether muscle fibers themselves synthesize and express HLA-DR molecules and play a role in antigen presentation in PM pathogenesis. In this study, we demonstrated that most muscle fibers from patients with PM synthesized and expressed HLA-DR molecules on their surface. Human leukocyte antigen-DR expression was highly specific to PM. In addition, class II transactivator (CIITA), human leukocyte antigen DM (HLA-DM), and invariant chain (Ii), which are indispensable for expression of mature HLA-DR molecules and for antigen processing and presentation, were co-expressed. One of the cytokines that could induce this expression is interferon-gamma (IFN-gamma), released by activated lymphocytes. Our results indicate that in PM muscle fibers synthesize and express HLA-DR molecules and may contribute to the inflammatory responses together with lymphocytes.

Late-onset familial amyloid polyneuropathy type I (transthyretin Met30-associated familial amyloid polyneuropathy) unrelated to endemic focus in Japan. Clinicopathological and genetic features.

Clinicopathological and genetic features were assessed on 35 Japanese families affected by late-onset familial amyloid polyneuropathy type I (transthyretin Met30-associated familial amyloid polyneuropathy, FAP TTR Met30) whose siblings were unrelated to endemic Japanese foci. In these patients (50 years or older), the most common initial symptom was paraesthesias in the legs. Autonomic symptoms were generally mild and did not seriously affect daily activities. The male-to-female ratio was extremely high (10.7 : 1). A family history was evident in only 11 out of 35 families, and other patients were apparently sporadic. The rate of penetrance was very low. Symptomatic siblings of familial cases showed a late age of onset, male preponderance and clinical features similar to those of the probands. Asymptomatic carriers, predominantly female, were detected relatively late in life. The geographical distribution of these late-onset, FAP TTR Met30 cases was scattered throughout Japan. In three autopsy cases and 20 sural nerve biopsy specimens, neurons in sympathetic and sensory ganglia were relatively preserved. Amyloid deposition was seen in the peripheral nervous system, particularly in the sympathetic ganglia, dorsal root ganglia and proximal nerve trunks such as sciatic nerve. These abnormalities were milder than those seen in typical early-onset FAP TTR Met30, as observed in two Japanese endemic foci of this disease. While axonal degeneration was prominent in myelinated fibres, resulting in severe fibre loss, unmyelinated fibres were relatively preserved. Our cases of late-onset FAP TTR Met30 showed features distinct from those of typical early-onset FAP TTR Met30 that occurred in the two Japanese endemic foci. Factors responsible for clinicopathological differences between these two forms of FAP TTR Met30 need to be identified.

Tissue-specific somatic mosaicism in spinal and bulbar muscular atrophy is dependent on CAG-repeat length and androgen receptor--gene expression level.

The factors influencing the tissue-specific pattern of somatic mosaicism in CAG-repeat diseases have not yet been fully resolved. We performed a detailed analysis of the degree of somatic mosaicism in various tissues from 20 patients with spinal and bulbar muscular atrophy (SBMA), including 4 who were deceased. The most outstanding feature was the prominent somatic mosaicism observed in the cardiac and skeletal muscles, composed predominantly of postmitotic cells, and in the skin, prostate, and testis. The CNS tissues, liver, and spleen showed the least mosaicism. The tissue distribution of somatic mosaicism in patients with SBMA was markedly different from that in patients with Huntington disease (HD) and from that in patients with dentatorubral-pallidoluysian atrophy (DRPLA). The degree of somatic mosaicism correlated with the CAG-repeat number but not with age at examination. Furthermore, tissues with a higher mosaicism level corresponded well to those with a higher expression level of androgen receptor protein. The tissue-specific pattern of somatic mosaicism related not only to cell composition with different cell turnover rates but to repeat size and gene expression levels, and postnatal cell division is unlikely to be a major cause of somatic mosaicism probably because of the relative stability of CAG repeat in SBMA.

Expression of GDNF and GDNFR-alpha mRNAs in muscles of patients with motor neuron diseases.

The mRNA expression levels of GDNF, GDNFR-alpha and RET were examined in the muscles of amyotrophic lateral screlosis (ALS) and X-linked spinal and bulbar muscular atrophy (SBMA). GDNF mRNA levels were significantly elevated to variable extent in the diseased muscles compared to control muscles, although they were not specific to the type of the diseases. The diseased muscles also have a different expression pattern of GDNF mRNA isoforms from controls. GDNF mRNA expression, however, tended to reduce in advanced muscle pathology. On the other hand, GDNFR-alpha mRNA levels were not changed significantly on expression levels in the diseased muscles. In situ hybridization study revealed that GDNF and GDNFR-alpha mRNAs were localized in subsarcolemmal space of muscle cells. RET mRNA was not detected in control nor diseased muscles. These results suggest that the elevated muscle GDNF acts as a trophic signal for motor neurons of motor neuron diseases, implying a possible therapeutic implication of GDNF to this type of diseases.

[A patient of polymyositis with severe myocardial damage and conduction block].

A patient with polymyositis manifesting severe myocardial damage and conduction block is described. A 57-year-old man presented dysarthria, dysphagia, proximal-dominant muscle weakness and wasting of the extremities. Muscle biopsy revealed degeneration and regeneration of muscle fibers and infiltration of mononuclear cells. After admission, muscle weakness rapidly progressed and mechanical ventilation was needed for respiratory failure. Simultaneously, cardiac symptom developed and resulted in bradycardia and trifascicular conduction block, which required a pacemaker. Echocardiogram revealed diffuse hypokinesia, ventricular enlargement and thickened wall. Marked elevations of serum CK-MB, cardiac myosin light chain I and cardiac troponin T were observed. High dose administration of methylprednisolone resulted in improvement of muscular and cardiac symptoms, and prevented complete heart block. Immediate and high dose of steroid therapy was considered to be effective for severe myocarditis in polymyositis.

[A case of advanced gastric cancer successfully treated by UFT and CDDP followed by surgical resection proving cancer cell disappearance in the stomach].

A patient with advanced gastric cancer responded remarkably to UFT combined with CDDP. UFT was administered orally for 28 consecutive days at a dose of 400 mg/m2, and CDDP was injected intravenously for a day at a dose of 100 mg/m2. Surgical resection proved the histological disappearance of cancer cells in stomach.

[Apoptosis and myopathies].

Recently a new type of cell death, apoptosis, was reported in the biological and many other fields. In an attempt to determine whether the Fas-Fas-ligand mediated apoptosis operates in the pathologic process of human myopathies, we examined the expression of Fas antigen, the synthesis of Fas-ligand mRNA, and the presence of chromosomal DNA fragmentation in the muscles from patients with various human muscle disorders. The Fas antigen of a whole molecule that can transduces the apoptotic signal into the cytoplasm was expressed on the muscle fibers of patients with various muscle wasting diseases. Its expression on muscle fibers was not disease specific, and the frequency of it becomes high according to the extent of the alteration of the muscle pathology. However, there was no evidence of operating the apoptotic process, that is no DNA fragmentation by TUNEL method. Furthermore, no Fas-ligand synthesis was detected in the diseased muscle tissue by RT-PCR method. Our data suggest that the expression of Fas antigen on muscle fibers in diseased muscles might be related to unknown biological functions other than "apoptosis" in the process of muscle fiber injury.

Expression of Fas antigen is not associated with apoptosis in human myopathies.

To determine whether the Fas-Fas-ligand mediated apoptosis occurs in the pathologic process of human muscle diseases, we examined the expression of Fas antigen, Fas-ligand messenger RNA, and chromosomal DNA fragmentation in the muscle cells of patients with a variety of human muscle disorders. The present study demonstrated that the Fas antigen of a whole molecule was expressed on the muscle fibers of patients with muscle wasting diseases. However, the apoptotic process did not occur in the muscle cells, and there was no evidence of Fas-ligand synthesis in the diseased muscle tissue. Our data suggest that the expression of Fas antigen on fibers in diseased muscle is related to unknown biological functions other than "apoptosis" in the process of muscle fiber injury.

[Fas antigen is expressed in human diseased muscles, but does not link to apoptosis].

In an attempt to determine whether the Fas-Fas-ligand mediated apoptosis occurs in the pathologic process of human myopathies, we examined the expression of Fas antigen, Fas-ligand mRNA, and chromosomal DNA fragmentation in the muscle cells of several human muscle disorders. The Fas antigen of a whole molecule was expressed on the muscle fibers of patients with muscle wasting diseases. However, there was no evidence of an apoptotic process, nor Fas-ligand synthesis in the diseased muscle tissue. Therefore, the expression of Fas antigen on muscle fibers in diseased muscle might be related to unknown biological functions other than "apoptosis" in the process of muscle fiber injury.

[Long-term maintenance therapy of multifocal motor neuropathy by weekly administration of human immunoglobulin].

High-dose intravenous immunoglobulin therapy for multifocal motor neuropathy (MMN) exhibits a beneficial, but temporary effect in most cases. We experienced a patient with MMN having a high titer of anti-GM1 ganglioside antibody. He was weekly administered human immunoglobulin, and his muscle strength was successfully improved and maintained at the improved level after high-dose human immunoglobulin infusion. The pattern of improvement and maintenance by weekly administration of human immunoglobulin was variable among the muscles; e.g. only a minimal improvement was observed in the most severely involved brachioradial muscles. Successive measurements with a hand-held dynamometer revealed in detail the changes in the strength of each muscle during treatment. Anti-GM1 ganglioside antibody titers did not significantly change during the treatment, but a slight improvement in motor conduction amplitudes and velocities was observed in mildly affected nerves. Our findings suggest that weekly administration of human immunoglobulin is a safe and effective therapy for MMN and that the anti-GM1 ganglioside antibody titer may not be a good indicator for this therapy.