Daiji Obara

Efficacy of low-dose FK506 in the treatment of Myasthenia gravis--a randomized pilot study.

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.

Overexpression of α7 nicotinic acetylcholine receptor prevents G1‐arrest and DNA fragmentation in PC12 cells after hypoxia

We investigated the neuroprotective function of α7 nicotinic acetylcholine receptor (α7nAChR) after transient hypoxia (12 h) and reoxygenation (0–72 h), comparing rat pheochromocytoma (PC12) cells overexpressing FLAG‐tagged α7nAChR (α7pCMV 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 α7pCMV cells with nicotine. Inhibition of DNA fragmentation in the late phase after hypoxia was most remarkable in α7pCMV cells with nicotine, but, surprisingly, it was more remarkable in α7pCMV 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 α7pCMV cells, with or without nicotine. Furthermore, in α7pCMV 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 α7nAChR 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.

Effects of FK506 on myasthenia gravis patients with high interleukin‐2 productivity in peripheral blood mononuclear cells

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 (<1250 pg/mL, n = 10) levels of PBM IL‐2 production. Reduction in clinical severity and PBM IL‐2 production were significantly greater in the patients with elevated IL‐2 production than those with normal PBM IL‐2 production in the first month of treatment. Muscle Nerve 27: 245–248, 2003

Over-expression of α7 nicotinic acetylcholine receptor induces sustained ERK phosphorylation and N-cadherin expression in PC12 cells

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.

Dendritic cells in hyperplastic thymuses from patients with myasthenia gravis.

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

Increased expression of α7 nAChR after transient hypoxia in PC12 cells

We examined the effects of transient (6 h) hypoxia on nicotinic acetylcholine receptor (nAChR) subunit α7 expression in cultured PC12 cells, using RT-PCR and cytochemistry for α-bungarotoxin (αBTX) binding sites. The relative amount of α7 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 αBTX binding sites compared with before hypoxia. However, at 12 h and 24 h after hypoxia, αBTX binding sites remarkably increased, where-after cells resumed outgrowth of their neurites at 24–48 h. These findings suggested that nAChR subunit α7 was upregulated in both mRNA and protein levels in response to transient hypoxia/reoxygenation in PC 12 cells.

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

Immunohistochemical expression of natural killer T (NKT) cell–associated markers (Vα24 and CD56) and perforin in relation to CD44–highly positive (CD44high) 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, Vα24‐positive (Vα24+) and CD56‐positive (CD56+) cells were sparsely distributed in the medullary area only. In contrast, in hyperplastic MG thymus, Vα24+ and CD56+ cells were more frequent in connective tissue, appeared to have penetrated the thymic parenchyma, and most coexpressed CD44high. 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 CD44high 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 Vα24, 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.

Interleukin-2 Production by Peripheral Blood Mononuclear Cells from Patients with Myasthenia gravis

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 (<1,250 pg/ml) (n = 46, 61%). Significant characteristics of patients with elevated PBM IL-2 production included elevated serum levels of anti-acetylcholine receptor antibodies, severe generalized symptoms, thymic hyperplasia, and marked effects of thymectomy (p < 0.05). These findings suggest that elevated PBM IL-2 production can reflect functional abnormalities of T cells in some patients with MG, and that PBM IL-2 production should be considered as a candidate target of therapy.

Hypoxic condition interferes with phosphorylation of Akt at Thr308 in cultured rat pheochromocytoma-12 cells

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.