Roles of cytokines and T cells in the pathogenesis of myasthenia gravis.

Myasthenia gravis (MG) is characterized by muscle weakness and fatigue caused by the presence of autoantibodies against the acetylcholine receptor (AChR) or the muscle-specific tyrosine kinase (MuSK). Activated T, B and plasma cells, as well as cytokines, play important roles in the production of pathogenic autoantibodies and the induction of inflammation at the neuromuscular junction in MG. Many studies have focused on the role of cytokines and lymphocytes in anti-AChR antibody-positive MG. Chronic inflammation mediated by T helper type 17 (Th17) cells, the promotion of autoantibody production from B cells and plasma cells by follicular Th (Tfh) cells and the activation of the immune response by dysfunction of regulatory T (Treg ) cells may contribute to the exacerbation of the MG pathogenesis. In fact, an increased number of Th17 cells and Tfh cells and dysfunction of Treg cells have been reported in patients with anti-AChR antibody-positive MG; moreover, the number of these cells was correlated with clinical parameters in patients with MG. Regarding cytokines, interleukin (IL)-17; a Th17-related cytokine, IL-21 (a Tfh-related cytokine), the B-cell-activating factor (BAFF; a B cell-related cytokine) and a proliferation-inducing ligand (APRIL; a B cell-related cytokine) have been reported to be up-regulated and associated with clinical parameters of MG. This review focuses on the current understanding of the involvement of cytokines and lymphocytes in the immunological pathogenesis of MG, which may lead to the development of novel therapies for this disease in the near future.

Cardiac involvements in myasthenia gravis associated with anti-Kv1.4 antibodies.

BACKGROUND AND PURPOSE: There is no general consensus as to whether autoimmune myasthenia gravis (MG) is associated with heart diseases, despite the fact that myocarditis, a serious cardiac involvement treatable by immunotherapy, is a complication of MG. It has been observed previously that MG patients with clinically suspected myocarditis had anti-Kv1.4 antibodies. The purpose of this study was to disclose the association between anti-Kv1.4 antibodies and cardiac involvements in MG patients. METHODS: Anti-Kv1.4 antibody was detected by an immunoprecipitation assay using (35) S-labeled rhabdomyosarcome cellular extract as the antigen source. Cardiac findings including electrocardiography (ECG) and clinical features of clinically suspected myocarditis in MG patients with anti-Kv1.4 antibodies were investigated. Ultrasound echocardiography (UCG) of ex vivo chick embryos was performed to determine the suppressive effects of sera with or without anti-Kv1.4 antibodies on heart muscle functions. RESULTS: Seventy (10.8%) of 650 MG patients had anti-Kv1.4 antibodies and 60% of them had abnormal ECG findings with high frequencies of T-wave abnormality and QT prolongation. Clinically suspected myocarditis was found in eight MG patients with anti-Kv1.4 antibodies but in none of the MG patients without anti-Kv1.4 antibodies. Most patients showed rapid deterioration with lethal arrhythmias such as ventricular tachycardia, sick sinus syndrome, or complete atrial ventricular block and severe heart failure. It was concluded using UCG of ex vivo chick embryos that MG serum with anti-Kv1.4 antibodies suppressed heart muscle functions. CONCLUSION: It has been demonstrated that anti-Kv1.4 antibodies are possible markers for cardiac involvements in MG patients.

Taste disorders in myasthenia gravis: a multicenter cooperative study.

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±16.9years) 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.

Monitoring treatment with cyclosporine microemulsion in myasthenia gravis.

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.

Perivascular infiltrate of memory lymphocytes and mature dendritic cells in MG thymomas.

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.

NKT-associated markers and perforin in hyperplastic thymuses from patients with Myasthenia gravis.

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.

Comparison of Scheimpflug images of posterior capsule opacification and histological findings in rabbits and humans.

PURPOSE: To compare the posterior capsule opacification in Scheimpflug photographic images produced by an electronic anterior eye segment analysis system with the histopathological findings in rabbits and humans. SETTING: Department of Ophthalmology, Wakayama Medical College, Japan. METHODS: Opacified posterior capsules were photographed using the EAS-1000 system (Nidek) and were then extracted during vitreous surgery for proliferative diabetic retinopathy or proliferative vitreoretinopathy in 2 patients. In rabbits, phacoemulsification and aspiration (PEA) with intraocular lens (IOL) implantation was performed. The IOL was implanted in the bag or in the sulcus. After intervals of healing, the posterior capsule was photographed with the EAS-1000 and the animals were then killed. In both clinical and experimental specimens, the posterior capsule was processed for light microscopic histology and immunohistochemistry. RESULTS: Opacified human capsules were well imaged by the EAS-1000. Histology showed that lens epithelial cells proliferated with and without an accumulation of extracellular matrix. Details such as rolling of the capsulotomy edge were seen well. Regenerated lens fibers of Soemmering's ring were seen as a mass within the capsule. In the rabbit model, Scheimpflug images accurately represented the capsules as they appeared histologically. CONCLUSION: The EAS-1000 system provided faithful, relatively high-resolution images that corresponded to the histologic findings in the posterior capsules after PEA-IOL surgery in humans and rabbits.

[A case with trigeminal herpes zoster manifesting a long lesion of the spinal trigeminal nucleus and tract on MR T2-weighted image].

We reported a 53-year-old man with the right trigeminal herpes zoster with preceding neuralgia (preherpetic neuralgia) in the right upper cervical nerve area. He developed dysesthesia and scapular pain in the right second cervical nerve area. 5 days later, herpes zoster emerged in the area of the right maxillary division of trigeminal nerve. Furthermore, he developed paralysis on the right facial muscle on the 12th day after the onset of scapular pain. Neurological examination revealed decrease in superficial sensation accompanied by pain and dysesthesia in the areas innervated by the right maxillary division of trigeminal nerve and the right second cervical nerve, and the right peripheral facial nerve palsy. Any rash was not observed in the right second cervical nerve area throughout the course. The cerebrospinal fluid showed a mild mononuclear pleocytosis. The antibody titer for varicella zoster virus (VZV) was elevated in both cerebrospinal fluid and blood serum. T2-weighted magnetic resonance (MR) image revealed a continuously long high-signal lesion corresponding to the right spinal trigeminal nucleus and tract, extending from the lower pons to the second cervical segment of the spinal cord. This lesion could have resulted from a centripetal migration of VZV from the Gasser ganglion to the spinal trigeminal nucleus and tract, which was probably related to the preherpetic neuralgia in the upper cervical nerve area without rash.

Hypoxia-induced expression of C1q, a subcomponent of the complement system, in cultured rat PC12 cells.

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.

Increased expression of alpha7 nAChR after transient hypoxia in PC12 cells.

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.

Protective effect of nicotine through nicotinic acetylcholine receptor alpha 7 on hypoxia-induced membrane disintegration and DNA fragmentation of cultured 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.

PCR amplification in bisulfite methylcytosine mapping in the GC-rich promoter region of amyloid precursor protein gene in autopsy human brain.

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 degrees C) and 30 cycles at a lower annealing temperature (50 degrees C) using both the upper and lower primers. The present method likely can also be applied to other GC-rich genomic sequences.

Marked increase in CD44-highly positive cells in hyperplastic thymuses from patients with Myasthenia gravis.

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 methylation status of cytosines in a tau gene promoter region alters with age to downregulate transcriptional activity in human cerebral cortex.

Changes with age in the methylation status of cytosines in a promoter region of the tau 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 tau gene alters with age to decrease its transcriptional activity in the human cerebral cortex.

Changes with aging and ischemia in nicotinic acetylcholine receptor subunit alpha7 mRNA expression in postmortem human frontal cortex and putamen.

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 (P < 0.05). In the putamen, alpha7 subunit mRNA expression was significantly lower than that in the frontal cortex (P < 0.0001), 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 (P < 0.001). 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.

Local variation in expression of pro- and antithrombotic factors in vascular endothelium of human autopsy brain.

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.

Reduction with age in methylcytosine in the promoter region -224 approximately -101 of the amyloid precursor protein gene in autopsy human cortex.

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 (8%) (p<0.05). 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 (5%) (p<0.01). 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.

[The effect of lymphocytapheresis on multiple sclerosis].

We studied the effect of lymphocytapheresis (LCP) on the expanded disability status scale (EDSS) clinical score, lymphocyte subsets and Interleukin-2 (IL-2) production by peripheral blood mononuclear cell (PBM) in 5 patients with multiple sclerosis (MS). The EDSS clinical score significantly decreased after LCPs (p < 0.05). PBM IL-2 production and CD 4/8 ratio significantly decreased (p < 0.05, p < 0.05), and the number of neutrocytes and CD 11 b+CD 8+ (%) significantly increased immediately after LCP (p < 0.05, p < 0.05). Down-regulation of PBM IL-2 production and CD 4/8 ratio and up-regulation of CD 11 b+CD 8+ may account for therapeutic effect of LCP on MS. However, similar changes were observed in patients with CIDP and MG during immunoadsorbent therapy (IAT). It is possible that down-regulation of PBM IL-2 production and CD 4/8 ratio and up regulation of CD 11 b+CD 8+ and the number of neutrocytes may commonly result from apheresis therapy using extra-corporeal circulation.

Quantitation of nicotinic acetylcholine receptor subunits alpha 4 and beta 2 messenger RNA in postmortem human brain using a non-radioactive RT-PCR and CCD imaging system.

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 alpha 4 and beta 2 mRNA in postmortem human putamen in the present study, but is also applicable to mRNAs of other receptors and neurotransmitter precursor peptides.