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SummaryIn order to define the role of angiotensin II (AngII) receptor subtypes, AT1 and AT2, in platelet activation, we examined the effects of AngII and receptor antagonists on both aggregability and phosphorylation status of protein kinase C (PKC) isoforms in human platelets obtained from 56 healthy volunteers. AngII promoted both spontaneous and agonist (collagen and ADP) stimulated platelet aggregation at concentrations of 10 nM or less, but the promotion effects were lost at 100 nM. Antagonism of AT1 receptor inhibited the promotion effects of AngII at 10 nM or less. On the other hand, antagonism of AT2 receptor enhanced platelet aggregability modestly with AngII at 10 nM or less, and markedly with 100 nM AngII. Furthermore, with 10 nM AngII, phospho-PKCα/βII expression in platelets was increased after collagen stimulation and was inhibited by antagonism of AT1 receptor. With 100 nM AngII, expression levels of phospho-PKCα/ βII remained low even after collagen stimulation but were markedly enhanced by antagonism of AT2 receptor. These findings suggest that at 10 nM or below, AngII promotes aggregability and PKC phosphorylation in human platelets through the AT1 receptor, which can be inhibited by AT1 receptor antagonists, but at higher concentrations, the promotion effects were lost through the opposing action of the AT2 receptor. The present study may provide an additional mechanism for AT1 receptor antagonism, which would provide clinical benefit to patients with stroke or cardiovascular disease accompanied by hypertension.

We examined interleukin-2 (IL-2) production by phytohemagglutinin (PHA)-stimulated peripheral blood mononuclear cells (PBMs) from 75 untreated myasthenia gravis (MG) patients and 48 control patients. Patients with MG consisted of those with elevated PBM IL-2 production (>1,250 pg/ml; mean + 2SD of the controls) (n = 29, 39%) and those with normal PBM IL-2 production (

Background and purpose: The purpose of the present study was to investigate the prevalence and clinical characteristics of taste disorders in patients with myasthenia gravis (MG). Methods: We studied 371 Japanese patients with MG (127 men and 244 women; mean age, 56.6 ± 16.9 years) consecutively evaluated between May and September 2010 in six neurological centers comprising the East Japan MG Study Group. Ninety-three patients (25%) had thymoma. We interviewed all patients to determine whether they had taste disorders during the clinical course of MG and then further evaluated the patients with MG, who reported having taste disorders, using a questionnaire. Results: Taste disorders were observed in 16 (4.3%) of the 371 patients with MG. We concluded that taste disorders in 2.4% of patients with MG excluding other factors were associated with MG itself. All patients had thymoma with seropositivity for anti-acetylcholine receptor antibodies. Thymoma tended to be advanced, and four patients with Masaoka stage IVa required radiation therapy or chemotherapy. Five patients noticed taste disorders 2–3 months before the onset of MG. Sweet taste loss was more common than salty, bitter, and sour taste loss. Conclusions: This was the first systematic survey of taste disorders in patients with MG by a multicenter study. Taste disorders were more common in the present sample of patients with MG than in the general population.

Phosphorylation of Akt induced under hypoxic or ischemic conditions has been reported only for residue Ser 473 . We examined whether Akt can be phosphorylated at Thr 308 , another phosphorylation site on Akt, and can exhibit neuroprotective effects under conditions of hypoxia /reoxygenation, comparing pheochromocytoma-12 (PC12) cells transfected with constitutively active Akt (Myr-pCMV cells) and those transfected with pCMV vector only (pCMV cells). Expression levels of Akt phosphorylated at Ser 473 were 2.1-fold higher in Myr-pCMV cells compared with pCMV cells, before the onset of hypoxia, which were increased transiently during hypoxia, and then decreased gradually during reoxygenation. In contrast, Akt phosphorylated at Thr 308 was not detected in pCMV cells under any conditions but was expressed in Myr-pCMV cells prior to hypoxia, followed by an immediate decrease during hypoxia and a further decline during reoxygenation. However, G1-arrest of the cell cycle observed at 12 h after hypoxia in pCMV cells was prevented in Myr-pCMV cells. These findings suggest that hypoxia activates Akt by phosphorylation at Ser 473 only, which is sufficient to elicit a neuroprotective function against hypoxic neuronal damage.

Over-expression of alpha7 nicotinic acetylcholine receptor (alpha7nAChR) in PC12 cells, independent of agonistic stimulation, induces marked neurite outgrowth and high capacity for migration and adherence (differentiation-like transformation), and increases tolerance against cell damage. In the present study, we investigated the effects of alpha7nAChR over-expression and nicotine on ERK phosphorylation and N-cadherin expression by comparing 3 groups of cells: PC12 cells transfected with alpha7 subunit cDNA (alpha7pCMV cells); untransfected PC12 cells exposed to 50 microM nicotine (PC12 cells+nicotine); and PC12 cells transfected with vector only (pCMV cells). alpha7 subunit protein was detected in alpha7pCMV cells at 24 to 72 h after transfection. alpha7pCMV cells exhibited sustained expression of phospho-ERKs (p42 and p44) at 24 to 72 h after transfection, and differentiation-like transformation at 72 h after transfection. PC12 cells+nicotine exhibited transient expression of phospho-ERKs at 48 h after addition of nicotine, but did not exhibit differentiation-like transformation. Neither ERK phosphorylation nor differentiation-like transformation was observed in pCMV cells. Expression of surface N-cadherin increased at 72 h after transfection on alpha7pCMV cells, but did not increase on PC12 cells+nicotine or pCMV cells. These findings suggest that, in PC12 cells, over-expression of alpha7nAChR induces sustained activation of ERK, which probably promotes N-cadherin expression and differentiation-like transformation.

The aim of this study was to elucidate the effectiveness of oral prednisolone (PSL) according to dosing regimen in 472 patients with myasthenia gravis (MG).We compared the clinical characteristics and PSL treatment between 226 patients who achieved minimal manifestations (MM) or better and 246 patients who remained improved (I) or worsened, according to the MG Foundation of America postintervention status.Achievement of MM or better at peak PSL dose (odds ratio 12.25, P 0.0001) and combined use of plasma exchange/plasmapheresis (PE/PP) and/or intravenous immunoglobulin (IVIg) (odds ratio 1.92, P = 0.04) were associated positively, and total PSL dose during the past year (odds ratio 0.17, P = 0.03) was associated negatively with present MM or better status.Higher PSL dose and longer PSL treatment do not ensure better outcome. In the absence of a good response, the PSL dose should be decreased by combining with modalities such as PE/PP or IVIg.

Background and Purpose Myasthenia gravis (MG) is often categorized into thymoma-associated MG, early-onset MG with onset age

Immunohistochemical expression of natural killer T (NKT) cell-associated markers (Valpha24 and CD56) and perforin in relation to CD44-highly positive (CD44(high)) cells was studied in hyperplastic thymuses from patients with myasthenia gravis (MG) whose symptoms dramatically improved after thymectomy and compared with non-MG control thymuses. In the control thymuses, Valpha24-positive (Valpha24(+)) and CD56-positive (CD56(+)) cells were sparsely distributed in the medullary area only. In contrast, in hyperplastic MG thymus, Valpha24(+) and CD56(+) cells were more frequent in connective tissue, appeared to have penetrated the thymic parenchyma, and most coexpressed CD44(high). Perforin-positive cells were not present in the control thymus, but were in the connective tissue and perilobular cortical areas in the hyperplastic MG thymus. Most of these perforin-positive cells were CD44(high) and were located near blood vessels. They appeared to have migrated directly from the vascular system and penetrated the thymic parenchyma. Some perforin-positive cells coexpressed Valpha24, CD56, or both. These findings suggest that in this particular type of MG thymus, NKT-like cells may have increased via a CD44- and perforin-mediated mechanism, leading to an imbalance in the immune system that favored an antibody-mediated autoimmunity against the acetylcholine receptor.

Presently, myasthenia gravis (MG) is seldom lethal. However, full remission without immune treatment is not common, and many patients still find it difficult to maintain daily activities. To both determine factors affecting health-related quality of life (QOL) and propose an appropriate treatment target for patients with MG, we evaluated 640 consecutive patients with MG seen at 11 neurological centers. Two-year follow-up data were obtained for 282 patients. Correlations between detailed clinical facotrs and the Japanese version of the 15-item MG-specific QOL scale were analyzed.In the cross-sectional analysis of 640 MG patients, multivariate regression revealed that disease severity as evaluated by MG Composite (p < 0.0001), but not quantitative MG score, total dose of oral prednisolone (p = 0.002), but not current dose, and Cushingoid appearance index (p = 0.0004) showed significant negative effects on QOL. Achieving status of Minimal Manifestations or better with prednisolone ≤ 5 mg/day was found to exert a major positive impact on QOL in both the cross-sectional and 2-year follow-up patient samples and can be recommended as a treatment target.

We examined the effects of transient (6 h) hypoxia on nicotinic acetylcholine receptor (nAChR) subunit alpha7 expression in cultured PC12 cells, using RT-PCR and cytochemistry for alpha-bungarotoxin (alphaBTX) binding sites. The relative amount of alpha7 subunit mRNA compared with that before hypoxia decreased to 84% immediately after hypoxia, but then began to increase at 6 h after hypoxia, reaching 171% at 12 h. After this point, it decreased again to 81% at 48 h. Until 6 h after hypoxia, cells appeared to shorten their neurites and form aggregates, without any accompanying remarkable change in alphaBTX binding sites compared with before hypoxia. However, at 12 h and 24 h after hypoxia, alphaBTX binding sites remarkably increased, whereafter cells resumed outgrowth of their neurites at 24-48 h. These findings suggested that nAChR subunit alpha7 was upregulated in both mRNA and protein levels in response to transient hypoxia/reoxygenation in PC12 cells.

To investigate the effect of nicotine on hypoxic neuronal damage, cultured PC12 cells were exposed to hypoxia for 9 h and then reoxygenated for 72 h. The cells were stained by propidium iodide (PI), a marker of cell membrane disintegration and the TUNEL method, which indicates DNA fragmentation. In control cultures, the ratio of PI-positive cells to total cells progressively increased during and after exposure to hypoxia, constituting 39% of total cells at 72 h posthypoxia. This increase in PI-positive cells was completely inhibited by nicotine until 12 h posthypoxia, and was partially and dose-dependently inhibited thereafter. The ratio of TUNEL-positive cells to total cells started to increase at 24 h posthypoxia and reached 36% at 72 h in control cultures. This ratio was also dose-dependently inhibited by nicotine. These inhibitory effects of nicotine on the increase in PI-positive and TUNEL-positive cells were abolished by the addition to the medium of alpha-bungarotoxin, an antagonistic ligand for nicotinic acetylcholine receptor (AChR) alpha7. These findings suggest that nicotine inhibits, through AChR alpha7, hypoxia-induced cell membrane disintegration and DNA fragmentation of cultured PC12 cells exposed to hypoxia.

Changes with age in the methylation status of cytosines in a promoter region of the τ gene were investigated in autopsy human cerebral cortex, using the bisulfite method, polymerase chain reaction (PCR) and direct sequencing of PCR products. While the total number of methylcytosines decreased with age, the changes in methylation status differed among transcription factor binding sites. Cytosines in the AP2-binding sites were never methylated in any of the cases studied at any age. Methylcytosines in the binding sites for Sp1, a transcriptional activator, significantly increased with age, whereas those in the binding sites for GCF, a repressor of GC-rich promoters, significantly decreased with age. These findings suggest that the methylation status of cytosines in the promoter region of the τ gene alters with age to decrease its transcriptional activity in the human cerebral cortex.

Age-related changes in nicotinic acetylcholine receptor (nAChR) subunit alpha7 messenger RNA (mRNA) expression in postmortem human frontal cortex and putamen of controls and status lacunaris patients were investigated using nonradioactive reverse transcription(RT)-PCR. In the frontal cortex of control brains, alpha7 subunit mRNA significantly decreased with age (P0.05). In the putamen, alpha7 subunit mRNA expression was significantly lower than that in the frontal cortex (P0.0001), and showed no significant correlation with age. However, in cases with status lacunaris in the putamen, alpha7 subunit mRNA expression was significantly higher compared with controls (P0.001). The reduction in alpha7 nAChR in the frontal cortex with age may decrease functional cholinergic synapses and cortical activity, and play a role in the cognitive impairments associated with normal aging. The functional significance of the upregulation of alpha7 nAChR mRNA in ischemic conditions remains to be determined.

Methylation status of cytosines and its changes with age in the promoter region (-226 approximately -101) of the amyloid precursor protein (APP) was analyzed using bisulfite genomic sequencing in the cerebral cortex of human autopsy brain. Cytosines at 13 locations were methylated in at least one of the cases studied. Methylcytosines at these locations was more frequent in cases/=70 years old (26%) than in cases70 years old (8%) (p0.05). Cytosines at -207, -204, -200, and -182 are frequently methylated, and the frequency of methylcytosine in these locations was significantly higher in cases/=70 years old (55%) than cases70 years old (5%) (p0.01). These cytosines constituted one of the 9-bp-long GC-rich elements (GGGCGC G/A GG) or an 11-bp inverted repeat (GGCCGT CGGCC). The present findings indicate that some cytosines, particularly those at -207 approximately -182, in the promoter region of the APP gene are frequently methylated and suggest that their demethylation with age may have some significance in the development of Abeta deposition in the aged brain. The relative importance of these elements in the total promoter activity of the APP gene remains to be definitively established.

We present a simple and rapid procedure for quantifying mRNA in the brain after RT-PCR, in which the intensity of the ethidium bromide luminescence of PCR products is measured directly from electrophoretic gels by a highly sensitive CCD camera combined with an image analyzing computer system (Gel Doc 1000 system). The CCD camera allows the mRNA in the ethidium bromide-stained PCR-amplified bands to be quantified in a broad exponential range of PCR cycles. The proposed protocol enables standard curves to be constructed to examine the relationship between the number of reaction cycles and amplified log intensity and between the amount of sample RNA for RT-PCR and amplified intensity. The method was applied to nicotinic acetylcholine receptor (nAChR) subunits α4 and β2 mRNA in postmortem human putamen in the present study, but is also applicable to mRNAs of other receptors and neurotransmitter precursor peptides. Themes: Neurotransmitters, modulators, transporters, and receptors Topics: Acetylcholine receptors: nicotinic

Cerebral blood flow and oxygen metabolism were examined in two patients with mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes (MELAS) using positron emission tomography (PET). Regional cerebral blood flow (rCBF), regional cerebral oxygen metabolic rate (rCMRO2) and regional oxygen extraction fraction (rOEF) were determined with the steady-state technique using oxygen-15-labeled tracers (15O2, C15O2 and C15O). Case 1, a 45-year-old woman, presented with abrupt onset of fluent aphasia. T2-weighted magnetic resonance imaging (MRI) showed a high signal intensity lesion in the left temporoparietal region. The first PET study on day 16 showed increased rCBF and decreased rCMRO2 in the temporal region. In the second PET study, on day 35, rCBF in the temporal region had decreased. Case 2 was a 19-year-old male; the second son of Case 1. He complained of transient blurring of vision, and then generalized tonic-clonic convulsion occurred. A PET study six days before this stroke-like episode demonstrated increased rCBF in both frontal lobes and putamen, where MRI showed lesions after the episode. Focal hyperemia of the lesion antedated and lasted for at least sixteen days after the stroke-like episode in these MELAS patients. These stroke-like episodes appear to be the result of metabolic dysfunction in neural tissue, although the role of an ischemic vascular event cannot be ruled out.

Age-related changes in nicotinic acetylcholine receptor (nAChR) subunit alpha4 and beta2 messenger RNA (mRNA) expression in the postmortem human frontal cortex and hippocampus was investigated using the reverse transcription-polymerase chain reaction (RT-PCR). In the frontal cortex, both alpha4 and beta2 subunit mRNA expression decreased with age. In the hippocampus, alpha4 subunit mRNA expression was unaltered, while beta2 subunit mRNA expression significantly decreased with age. These findings suggest that nAChR transcription decreases during aging with differing vulnerability between subunits and brain regions, which could in part contribute to the reduction in cognitive functions seen in the elderly.

Gamma-aminobutyric acid (GABA)/substance P (SP) neurons and GABA/enkephalin (Enk) neurons in the striatum exert opposing influence on the regulation of movement. The loss of GABA/SP neurons results in hypokinetic disorders (parkinsonism), whereas the loss of GABA/Enk neurons results in hyperkinetic disorders (e.g. chorea). The present study determined age-related changes in the beta-preprotachykinin (the precursor of SP) and preproenkaphalin (the precursor of Enk) messenger RNA (mRNA) ratio in the postmortem human putamen using the reverse transcription-polymerase chain reaction (RT-PCR). The ratio of beta-preprotachykinin to preproenkephalin mRNA expression decreased with age. The reduction in the beta-preprotachykinin/preproenkephalin mRNA ratio was more marked in cases with multiple small infarcts (status lacunaris) in the putamen. These findings may in part explain the susceptibility of the elderly, particularly of those with ischemic changes in the striatum to hypokinetic disorders.

In thymomas associated with myasthenia gravis (MG), the authors found that perivascular infiltrates of memory lymphocytes and mature dendritic cells (DCs) were more frequent in patients with early improvement after thymectomy than in patients without response to thymectomy. Although these findings may be limited to particular types of thymoma, thymectomy may interrupt the recruitment of mature DCs in thymus and export of activated T cells to extra-thymic tissues, thereby improving the disease.

The aim of this study was to determine factors affecting health-related quality of life (HRQOL) and to propose appropriate treatment targets for patients with myasthenia gravis (MG).We evaluated 640 consecutive patients with MG seen at 11 neurological centers. Two-year follow-up data were obtained for 282 patients. Correlations between detailed clinical factors and the Japanese version of the 15-item MG-specific QOL scale score were analyzed.In a cross-sectional analysis of 640 MG patients, multivariate regression revealed that disease severity, as evaluated by the MG Composite (P0.0001), total dose of oral prednisolone during the last year (P=0.002), and Cushingoid appearance index (P=0.0004), showed significant negative effects on HRQOL, but the quantitative MG score and current prednisolone dose did not.Achieving minimal manifestations (MM) status or better with prednisolone ≤ 5 mg/day was found to exert a major positive impact on HRQOL in both the cross-sectional and 2-year follow-up patient samples and can be recommended as a treatment target.

Many patients with myasthenia gravis (MG) still find it difficult to maintain daily activities due to chronic residual fatigability and long-term side effects of oral corticosteroids, since full remission is not common. Our analysis demonstrated that disease severity, oral corticosteroids, and depressive state are the major factors negatively associated with QOL, and that QOL of MM status patients takingor = 5 mg prednisolne/day is identically good as that seen in CSR and is a target of treatment. In order to achieve early MM or better status with prednisolneor = 5 mg/day, we advocate the early aggressive treatment strategy that can achieve early improvement by performing an aggressive therapy using combined treatment with plasmapheresis and high-dose intravenous methylprednisolone and then maintain an improved status using low-dose oral corticosteroids and calcineurin inhibitors.

We produced a Japanese translation of the 15-item myasthenia gravis (MG)-specific quality of life (QOL) scale (MG-QOL15), assessed its reliability and validity, and examined clinical factors affecting the self-perceived QOL in MG. Consecutive 327 patients with MG seen at six neurological centers were evaluated. All patients completed an MG-QOL15 Japanese version (MG-QOL15-J), the Beck Depression Inventory-Second Edition (BDI-II), and a generic health-related QOL questionnaire, the SF-36. Disease severity was determined according to the MG Foundation of America (MGFA) quantitative MG score and the MG composite. The MG-QOL15-J exhibited adequate internal reliability, test-retest repeatability, and concurrent validity with SF-36, disease severity, and known-patient groups categorized by MGFA postintervention status. Multivariate analysis revealed severity, dose of oral corticosteroids, and BDI-II as independent factors negatively affecting QOL. The MG-QOL15-J is anticipated to be a valuable clinical measure of QOL in Japanese patients with MG.

The advent of effective immune treatment has meant that myasthenia gravis (MG) is most often not lethal. However, many MG patients still find it difficult to maintain daily activities due to chronic residual fatigability and long-term side effects of medication, since full remission without immune treatment is not common. Our analysis demonstrated that disease severity, dose of oral corticosteroids, and depressive state are the major independent factors negatively associated with self-reported QOL (MG-QOL15-J score). It is noteworthy that oral corticosteroid, the first-line agent for MG, is negatively associated with patients' QOL. When the analysis took into account MGFA postintervention status and dose of oral prednisolne (PSL), the MG-QOL15-J score of MM status patients taking ≤ 5 mg PSL per day is identically low (i.e., just as good QOL) as that seen in CSR and is a target of treatment. In order to veer away from high-dose oral corticosteroids and to achieve early MM or better status with PSL ≤ 5 mg/day, we advocate the early aggressive treatment strategy that can achieve early improvement by performing an aggressive therapy using combined treatment with plasmapheresis and high-dose intravenous methylprednisolone and then maintain an improved clinical status using low-dose oral corticosteroids and calcineurin inhibitors (cyclosporine microemulsion and tacrolimus). The early stages of MG are susceptible to treatment with calcineurin inhibitors. When using cyclosporine microemulsion for MG, blood concentrations 2 h after administration (C2) correlate with clinical improvement and immediately before administration (C0) with side effects (increased serum creatinine and/or hypertension). Monitoring of C2 and C0 levels is useful to estimate efficacy and safety of the drug.

Introduction: Our study aim was to produce a Japanese translation of the 15-item Myasthenia Gravis Quality-of-Life Scale (MG-QOL15), assess its reliability and validity, and examine clinical factors affecting self-perceived QOL in MG. Methods: We evaluated 327 consecutive patients with MG seen at six neurological centers. All patients completed the Japanese version of the MG-QOL15 (MG-QOL15-J), the Beck Depression Inventory—second edition (BDI-II), and a generic health-related QOL questionnaire, the SF-36. Disease severity was determined according to the Myasthenia Gravis Foundation of America (MGFA) quantitative MG score and the MG composite. Results: The MG-QOL15-J exhibited adequate internal reliability, test–retest repeatability, and concurrent validity with SF-36, disease severity, and known-patient groups categorized by MGFA post-intervention status. Multivariate analysis revealed severity, dose of oral corticosteroids, and BDI-II as independent factors negatively affecting QOL. Conclusion: The MG-QOL15-J is anticipated to be a valuable clinical measure of QOL in Japanese patients with MG. Muscle Nerve 46: 166–173, 2012

Thymic abnormalities were first noticed at autopsies of patients with myasthenia gravis (MG) more than 100 years ago. The thymus is believed to play an important role in the pathogenesis of MG, an autoimmune disease mediated by antibodies against the acetylcholine receptor (AChR) of skeletal muscles. Production of these antibodies in B cells is T cell dependent. T cells potentially specific for AChR are probably generated in the thymus via nontolerogenic thymopoiesis by an aberrant function of thymic epithelial cells. However, generation of these AChR-specific T cells is not the cause of MG, because these cells are also found in healthy individuals. The pathogenetic step in MG involves the activation of these potentially AChR-specific T cells; this activation is the trigger to develop the disease and a therapeutic target. The intra-thymic activation of AChR-specific T cells is probably limited to particular types of MG patients: those with early-onset MG in whom the thymus exhibits lymphofollicular hyperplasia (TLFH) and a few patients in whom MG is associated with a thymoma. The majority of thymomas and atrophic thymuses of patients with late-onset MG, an increasingly common condition, do not exhibit this T cell-activation process. In this paper, we review the available literature on thymic changes (TLFH, thymoma, and atrophic thymus) and the relationship of these changes to the pathogenesis of MG.

Immunological characteristics of myasthenia gravis (MG) with late-onset have not been fully elucidated. We examined several autoantibodies and HLA-DRB1 genotyping in 260 Japanese MG patients. Sixty-two MG patients had thymoma. The others were divided into early-onset and late-onset groups separated by an age of 50 years. The ocular form was more frequent in late-onset compared to early-onset group. Seropositivity of anti-muscle-specific tyrosine kinase antibody was 2–3% in acetylcholine receptor-seronegative patients. HLA-DRB1 genotyping failed to detect statistical differences in specific alleles between each group and healthy controls. The immunological profiles in late-onset MG were different from early-onset in Japan.

Objective To investigate the clinical, histological, and immunological features of patients with myasthenia gravis (MG) who also developed myocarditis and/or myositis. Design Observational and retrospective case series. Setting Keio University, Hanamaki General Hospital, Kanazawa University, Nagasaki University, and Juntendo University. Patients A cohort of 8 patients with MG with clinically defined inflammatory myopathies. Interventions Clinical and histological features were described. Serological analyses included MG-related antistriational autoantibodies (those to titin, ryanodine receptor, muscular voltage-gated potassium channel Kv1.4) and myositis-specific autoantibodies. Results Of 924 patients with MG, 8 (0.9%) had inflammatory myopathies. The mean (SD) onset age of MG was 55.3 (10.3) years. All patients showed severe symptoms with bulbar involvement; 5 patients had myasthenic crisis and 4 had invasive thymoma. Myocarditis was found in 3 patients and myositis in 6. Myocarditis, developing 13 to 211 months after the MG onset, was characterized by heart failure and arrhythmias. Myositis, developing before or at the same time as MG, affected limb and paraspinal muscles. Histological findings of skeletal muscles showed CD8 + lymphocyte infiltration. Seven patients had 1 of these antistriational autoantibodies but not myositis-specific autoantibodies. Immunomodulatory therapy was required for all patients and was effective for both MG and inflammatory myopathies, although 1 patient died. Conclusions Heart and skeletal muscles are autoimmune targets in some patients with MG. This autoimmunity has a broad clinical spectrum with antistriational autoantibodies. Published online September 14, 2009 (doi:10.1001/archneurol.2009.229).

Purpose: To examine whether the monitoring of cyclosporine (CsA) blood concentrations is of benefit in CsA microemulsion pre-concentrate (MEPC) therapy for myasthenia gravis (MG). Methods: We measured CsA blood concentrations both 2 h after administration (C2) and immediately before administration (C0) and examined associations with changes to clinical parameters in 20 MG patients treated with CsA MEPC in an unblinded, 6-month prospective open trial. Results: Initial dose of CsA MEPC (4.7 ± 0.5 mg/kg/day) provided both high C2 levels and safe C0 levels. Disease severity, daily dose of prednisolone, acetylcholine receptor–antibody titre levels and levels of interleukin-2 production by peripheral blood mononuclear cells were significantly reduced following treatment with CsA MEPC. A significant correlation existed between C2 levels following the initial dose and clinical improvement in responder MG patients. C0 levels were significantly higher in patients who exhibited increased serum creatinine or hypertension compared with patients free from side effects. Body mass index of individual patients was significantly correlated with C0 level, and may thus offer a useful marker to predict C0 levels. Discussion: CsA MEPC was effective at suppressing symptoms and T-cell-dependent pathogenesis of MG, and monitoring of C2 and C0 levels can be useful to estimate efficacy and safety of the drug.

A rare variant transthyretin that has a leucine-for-valine substitution at position 30 was reported in a sporadic case of type 1 familial amyloid polyneuropathy (FAP). We found the same substitution in members of a Japanese family with FAP. Three individuals in this family had a guanine-to-cytosine mutation at the first base of codon 30 in exon 2. This family shows a direct link between a valine-to-leucine substitution at position 30 and type 1 FAP.

Objectives To investigate the autoantibody status of patients with myasthenia gravis (MG) and to evaluate its usefulness for disease classification. Design Retrospective cohort study of patients with MG, who have autoantibodies to receptors and ion channels expressed at neuromuscular junctions and in muscles that impair neuromuscular transmission. One of the autoantibodies studied was a recently identified, novel, MG-specific autoantibody to a voltage-gated potassium (Kv) channel, Kv1.4. Setting Keio University Hospital, Tokyo, and Iwate Medical University Hospital, Morioka. Patients Two hundred nine patients with MG. Main Outcome Measures Anti-Kv1.4 antibody was measured by an immunoprecipitation assay with sulfur 35–labeled extract from rhabdomyosarcoma cells. Antititin antibody was detected with a commercially available enzyme-linked immunosorbent assay. Results Anti–acetylcholine receptor, anti-Kv1.4, and antititin antibodies were detected in 150 (72%), 26 (12%), and 50 (24%) of the 209 patients with MG, respectively. All of the patients who were positive for anti-Kv1.4 or antititin antibody were seropositive for the anti–acetylcholine receptor antibody. They were classified into 4 groups based on their status in regard to 3 MG-related autoantibodies: anti-Kv1.4, antititin, and anti–acetylcholine receptor. Clinical associations were found between anti-Kv1.4 and bulbar involvement, myasthenic crisis, thymoma, and concomitant myocarditis and/or myositis; between antititin and older-onset MG; between anti–acetylcholine receptor alone and younger-onset MG; and between seronegativity and ocular MG. In addition, patients with MG in the anti-Kv1.4 group had more severe manifestations of disease than those in the other 3 groups. Conclusion Classification of patients with MG based on autoantibody status may be useful in defining clinical subsets.

In order to determine whether antigen-specific T-cell activation by dendritic cells (DCs) is accelerated in thymuses exhibiting lymphofollicular hyperplasia (TLFH) among patients with early-onset myasthenia gravis (EOMG), we investigated the expression levels of phosphorylated protein kinase C (PKC)theta and the local relationship between the presence of phosphorylated PKCtheta and the homing receptor CD44 or CD83, a marker for mature DCs, in samples taken from EOMG patients with early improvement following thymectomy, in remnant thymuses from late-onset MG patients, and in non-MG control thymuses. Antigen-specific T-cell activation was markedly accelerated in TLFH from EOMG patients. Activated T cells and adjacent DCs appeared to be components of a CD44(high) cell population circulating from the blood to the thymus. Although there is no convincing evidence that thymectomy is of benefit in MG, in some EOMG patients with early improvement following thymectomy, blockade of CD44-associated circulation mechanisms is probably the cause for the early benefits of thymectomy and is a potential alternative to thymectomy.

A 77-year-old woman developed progressive dysesthesia, hypesthesia and weakness in four extremities immediately after improvement of herpes zoster in the left Th10 dermatome area. Examination of the cerebrospinal fluid (CSF) showed an increase in protein concentrations. Evidence of demyelinating polyneuropathy was demonstrated by nerve conduction studies. Her hypesthesia and weakness in the extremities were gradually improved following intravenous immunoglobulin therapy (IVIg). Varicella zoster virus (VZV) titer levels in CSF well correlated both with neurological symptoms and CSF protein concentrations. VZV DNA in the CSF was not detectable. These findings suggested autoimmune Guillain-Barré syndrome (GBS) associated with herpes zoster. An interesting finding in the present patient is that one day after the completion of IVIg, when the neurological symptoms in the extremities were apparently ameliorating, the herpes zoster eruptions again emerged in the left L3 dermatome area. By treatment with intravenous acyclovir, the vesicular eruptions were improved. We assume that IVIg might suppress the immune response against VZV and promote the recurrence of eruptions.

We reported a rare case with hypothyroid myopathy after interferon-alpha (IFN-alpha) therapy. A 59-year-old man complained of his weakness in proximal part of upper and lower extremities which started at 1 month and progressed during 6 months after IFN-alpha therapy for chronic hepatitis C, but he did not complain of any other symptoms. Blood chemistry showed an elevated level of CK (1,843 IU/l; normal range 43-170 IU/l) and increased myoglobin (250 ng/ml; normal range60 ng/ml). Thyroid function tests revealed an elevated level of TSH (148.7 microIU/ml; normal range, 0.4-4.1 microIU/ml), and decreased levels of free T3 (0.56 pg/ml; normal range, 2.27-3.90 pg/ml) and free T4 (0.24 ng/ml; normal range, 0.95-1.74 ng/ml). Blocking type TSH receptor antibody titer was elevated (75.4%; normal range15%) while other types of antithyroid antibody were not detected in his serum. Muscle biopsy from his quadriceps femoris muscle showed non-specific mild myopathic changes. His weakness was completely ameliorated and serum CK levels were normalized by thyroid hormone administration alone, confirming the diagnosis of hypothyroid myopathy. However, even after total amelioration of his hypothyroidism, blocking type TSH receptor antibody titer remained elevated. These findings may suggest that IFN-alpha fostered an autoreactivity arising from HCV infection to cause the autoimmune thyroid disease.

To determine the efficacy of low-dose FK506 in the treatment of myasthenia gravis (MG), untreated de novo patients were randomly selected to receive treatment with (n = 18) or without (n = 16) FK506, and were evaluated for 1 year after treatment with limitation of daily dose of prednisolone. Low-dose FK506 reduced the duration of early-phase therapy in hospital (p < 0.05) and the need for combined therapy with plasmapheresis and high-dose intravenous methylprednisolone or high-dose intravenous methylprednisolone alone (p < 0.05). It also reduced the daily dose of prednisolone (p < 0.05) required to maintain minimal manifestations of MGFA postintervention status. None of the patients exhibited significant side effects up to 1 year after treatment. These findings suggest that low-dose FK506 is safe and efficacious for the treatment of de novo MG patients.

To investigate the role of protein kinase C delta (PKC delta) in angiotensin II-induced facilitation mechanisms of hypoxic neuronal damage and whether candesartan, an AT1 receptor antagonist, can suppress these mechanisms, we performed in vitro experiments which were free from vascular components using PC12 cells under hypoxic (12 h)/reoxygenation (0-48 h) conditions. Angiotensin II apparently increased the basal expression level of PKC delta phosphorylated at Ser(643) before hypoxia, promoted the cleavage of PKC delta to its catalytic fragment, and fostered the progression of DNA fragmentation after hypoxia. Candesartan inhibited both phosphorylation and cleavage of PKC delta and suppressed the angiotensin II-induced facilitation of DNA fragmentation under hypoxic/reoxygenation conditions. However, PD123319, an AT2 receptor antagonist, influenced neither PKC delta nor the angiotensin II-induced facilitation of DNA fragmentation. Furthermore, in PC12 cells expressing the ATP-binding mutant of PKC delta (PKC delta(K376R)) acting as a dominant-negative protein, both phosphorylation and cleavage of PKC delta were attenuated and DNA fragmentation was markedly suppressed regardless of the presence of angiotensin II. These findings suggest that angiotensin II-induced facilitation of DNA fragmentation under hypoxic conditions is mediated by PKC delta, and the mechanisms can be suppressed by the candesartan mediated blockade of the AT1 receptor.

To investigate the role of dendritic cells (DCs) in the hyperplastic myasthenia gravis (MG) thymus, we studied the frequency and distribution of three mature DC phenotypes (CD83+CD11c+, CD86+CD11c+, and HLA-DR+CD11c+) in samples from patients with MG whose symptoms dramatically improved following thymectomy and in non-MG control thymuses. In hyperplastic MG thymuses, mature DCs were much more numerous in nonmedullary areas, such as the subcapsular/outer cortex; around the germinal centers; and in extralobular connective tissue, particularly around blood vessels. Mature DCs strongly coexpressed CD44 and appeared to be components of a CD44–highly positive (CD44high) cell population migrating from the vascular system. Furthermore, in the hyperplastic MG thymus, the expression of secondary lymphoid-tissue chemokine (SLC) markedly increased especially around extralobular blood vessels, where the CD44high cell population accumulated. These findings suggest that DCs may migrate into the hyperplastic thymus from the vascular system via mechanisms that involve CD44 and SLC. DCs may present self-antigens, thereby promoting the priming and/or boosting of potentially autoreactive T cells against the acetylcholine receptor. Muscle Nerve 27: 582–589, 2003

We compared the early effects of FK506 on clinical severity, interleukin-2 (IL-2) production by phytohemagglutinin-stimulated peripheral blood mononuclear cells (PBMs), and serum levels of acetylcholine receptor antibodies between myasthenia gravis (MG) patients with elevated (>1250 pg/ml, n = 9) or normal (

We investigated the neuroprotective function of alpha7 nicotinic acetylcholine receptor (alpha7nAChR) after transient hypoxia (12 h) and reoxygenation (0-72 h), comparing rat pheochromocytoma (PC12) cells overexpressing FLAG-tagged alpha7nAChR (alpha7pCMV cells) and control PC12 cells (non-transfected or transfected with vector only) in medium with and without nicotine. Plasma membrane degradation in the early phase after hypoxia was inhibited in PC12 cells with nicotine, and more profoundly in alpha7pCMV cells with nicotine. Inhibition of DNA fragmentation in the late phase after hypoxia was most remarkable in alpha7pCMV cells with nicotine, but, surprisingly, it was more remarkable in alpha7pCMV cells without nicotine than in PC12 cells with nicotine. G1-arrest of the cell cycle, observed in control PC12 cells at 12 h after hypoxia, preceding DNA fragmentation, was not evident in alpha7pCMV cells, with or without nicotine. Furthermore, in alpha7pCMV cells with and without nicotine, the basal expression levels of total Akt were approximately 1.5-fold higher, and the up-regulation of Akt phosphorylated at Ser473 after hypoxia was strikingly enhanced, compared with control PC12 cells. These findings suggest that alpha7nAChR functions constitutively in PC12 cells, that its overexpression raises tolerance against G1-arrest and DNA fragmentation after hypoxia, and that it can be considered a candidate target for treatment against hypoxia-induced acute membrane degradation and delayed DNA fragmentation in neurons.

This study investigated the effect of exposure to hypoxia on the expression of C1q mRNA and protein in cultured PC12 cells. PC12 cells expressed neither C1q mRNA nor protein before hypoxia. However, the cells expressed C1q mRNA immediately after hypoxia, and then A, B, and C chains of C1q and higher molecular weight C1q proteins during reoxygenation. Under the same experimental conditions, cell membrane disintegration began during hypoxia, whereas DNA fragmentation initiated during reoxygenation later than C1q protein expression. These results suggest that in response to hypoxia, PC12 cells per se express C1q mRNA and protein in the early phase before initiation of DNA fragmentation in the absence of any influence of other cellular components. These findings may be relevant for the pathogenesis and treatment of stroke.

Alterations in DNA 5-methyldeoxycytidine pattern influence gene expression for certain mammalian genes in development, differentiation, carcinogenesis, and aging. Detection of DNA methylation at the promoter region, which generally represses transcription activity, is one important element in studying changes in molecular expression with aging and age-associated disorders. Bisulfite genomic sequencing is a useful method for mapping methylated cytosines. However, PCR amplification for bisulfite-treated DNA does not yield a sufficient amount of products that have a sufficient level of specificity, especially in the GC-rich sequences usually seen at the promoter regions of house keeping genes. We present a method for increasing the sensitivity and specificity of PCR amplification in bisulfite methylcytosine mapping in an extremely GC-rich promoter region of amyloid precursor protein (APP) gene from the cerebral cortex of human autopsy brain. The PCR used consists of two cycles using the lower primer alone to amplify the sense sequence, and then eight cycles at a theoretical annealing temperature (60°C) and 30 cycles at a lower annealing temperature (50°C) using both the upper and lower primers. The present method likely can also be applied to other GC-rich genomic sequences. Theme: Cellular and molecular biology Topic: Gene structure and function: general.

To investigate the role of the thymus in the pathogenesis of myasthenia gravis (MG), immunohistochemical expression of CD44, CD45R0, B7-1, and IL-2 was studied in: (1) hyperplastic thymuses of patients with MG whose symptoms markedly improved after thymectomy, (2) remnant thymuses of patients with MG whose symptoms did not respond to thymectomy, and (3) non-MG control thymus. Lymphocytes strongly expressing CD44, a marker for homing lymphocytes and activated memory lymphocytes in adults, were much more frequently observed in hyperplastic MG thymuses than in remnant thymuses and non-MG control thymuses. These CD44-highly positive cells in hyperplastic MG thymuses were for the most part located in the subcapsular and cortical areas but also occasionally in medullary areas. Some of these CD44-highly positive cells coexpress CD45R0. CD44-highly positive cells were located in the vicinity of blood vessels and thus appeared to have migrated directly from extralobular blood vessels. B7-1-positive cells and interleukin (IL)-2-positive cells were also more abundant in the MG patients than in controls and were localized in the proximity of CD44-highly positive cells. These findings suggest that mature T and B cells recirculate into hyperplastic MG thymus via CD44-associated mechanisms and are activated there.

The expression of tissue factor (TF), tissue factor pathway inhibitor (TFPI), von Willebrand factor (vWF), endothelial nitric oxide (NO) synthase (eNOS), tissue plasminogen activator (tPA), its inhibitor (PAI-1), and myosin, an indicator of local shear stress, was examined in the endothelium of cerebral vessels according to vessel size and location in human autopsy brains, using immunohistochemistry. Expression of TF, vWF, eNOS, tPA/PAI-1, and myosin was much greater in intracerebral perforating arteries and the microvasculature than the pial and carotid arteries. Expression of all antigens studied was normally faint or negative in the pial and carotid arteries. However, TF, vWF, myosin, tPA, and PAI-1 were strongly expressed in the endothelium of the inner wall of the carotid bifurcation where flowing blood collides, but not in the outer wall. In the endothelium of arteries with fibrillary hyperplasia, vWF, myosin, eNOS, tPA, and PAI-1 were strongly expressed. Within the brain, microvascular expression of TFPI was very faint or negative, whereas that of vWF was intense throughout all brain regions. However, expression of TF and myosin was more intense in the basal gray matter and white matter than in the cortex. eNOS was expressed more strongly in the basal gray matter and cortex than the white matter, whereas tPA and PAI-1 expression was more intense in the white matter than the gray matter. In addition to intrinsic properties of individual vessels, these local variations in expression of pro- and antithrombotic factors in cerebral vessels may in part be due to differences in hemorheological and humoral environments to which they are exposed, and may result in local difference in vulnerability to ischemia. The present findings may in part account for the propensity of thrombus generation in the carotid inner wall, an usual source of artery-to-artery microemboli, frequent development of lacunar (small) infarcts in deep brain regions, and diffuse white matter lesions as seen in Binswanger’s leukoencephalopathy.