Tetsuya Kanai

Serum cytokine and chemokine profiles in patients with myasthenia gravis

Myasthenia gravis (MG) is an autoimmune‐mediated inflammatory disease of the neuromuscular junction. Previous studies of animal MG models have suggested important roles of cytokines in MG pathogenesis, but adequate studies on cytokines in human MG are lacking. Using a multiplex suspension array system, we measured the serum levels of 27 cytokines/chemokines in 47 anti‐acetylcholine receptor antibody‐positive patients with MG and 20 normal controls (NC) to investigate the contribution of cytokines/chemokines toward MG pathogenesis. Correlations between clinical parameters and cytokine/chemokine levels in patients with MG were also examined. The serum levels of interleukin (IL)‐15 (mean ± standard deviation: 6·85 ± 6·97 pg/ml) and vascular endothelial growth factor (VEGF) (96·21 ± 71·60 pg/ml) significantly increased, whereas IL‐4 levels (3·57 ± 0·86 pg/ml) decreased in patients with MG compared with NC (IL‐15: 4·42 ± 1·55 pg/ml; VEGF: 63·51 ± 32·95 pg/ml; IL‐4: 4·15 ± 0·81 pg/ml, P < 0·05). In addition, eight cytokines (IL‐4, IL‐8, IL‐15, eotaxin, macrophage inflammatory protein‐1α, macrophage inflammatory protein‐1β, VEGF and IL‐1b) were significantly changed among MG patients with thymoma, MG patients without thymoma and NC (P < 0·05). Some cytokines, such as IL‐4, IL‐15, and VEGF, may play roles in the pathogenesis of MG.

HLA-DRB1*14 and DQB1*05 are associated with Japanese anti-MuSK antibody-positive myasthenia gravis patients

BACKGROUND Myasthenia gravis (MG) is an autoimmune disorder presumed to be associated with genetic susceptibility. This study aims to determine whether HLA is associated with MG in Japanese patients. METHODS We included 58 MG patients with anti-acetylcholine receptor antibody (AChR+MG) and 14 MG patients with muscle-specific receptor tyrosine kinase (MuSK+MG) and determined HLA-A, B, DRB1 and -DQB1 alleles using polymerase chain reaction with sequence-specific oligonucleotide and primers. AChR+MG was classified into the three subgroups: early-onset MG (EOMG; n=11), late-onset MG (LOMG; n=20), and thymoma-associated MG (n=27). Healthy volunteers (n=100) served as controls. RESULTS A significant positive association was observed between MuSK+MG with the DRB1*14 [57.1%, MuSK+MG vs. 18.0%, healthy controls (HC); odds ratio (OR): 6.1] and DQB1*05 [78.6%, MuSK+MG vs. 30.0%, HC; odds ratio (OR): 8.5]. We found a negative association between LOMG and DQB1*04 [5.0%, LOMG vs. 37.0%, HC; OR: 0.09]. There was no association between other MG subgroups and HLA alleles. CONCLUSION HLA-DRB1*14 and DQB1*05 were associated with MuSK+MG, therefore these alleles may play important roles in developing MuSK+MG across the races.

Changes in inflammatory cytokine networks in myasthenia gravis

Myasthenia gravis (MG) is an autoimmunological inflammatory disorder of the neuromuscular junction. Inflammation could be a key player for understanding the pathogenesis of MG. We measured the serum levels of 24 inflammatory cytokines in 43 patients with anti-acetylcholine receptor antibody-positive MG and 25 healthy controls. In patients with MG, serum levels of a proliferation-inducing ligand (APRIL), IL-19, IL-20, IL-28A and IL-35 were significantly increased as compared with controls (p < 0.05). Among them, IL-20, IL-28A and IL-35 were significantly decreased after treatment (p < 0.05). In clinical subtype analyses, APRIL and IL-20 were increased in patients with late-onset MG and IL-28A levels were increased in patients with thymoma-associated MG compared with healthy controls (p < 0.01). The results of the present study demonstrate both anti-inflammatory and inflammatory cytokines are upregulated in MG, reflecting the importance of cytokine-mediated inflammation and its regulation in MG pathophysiology.

Transient global amnesia with a hippocampal lesion followed by transient epileptic amnesia

Transient epileptic amnesia (TEA) is a seizure disorder characterized by brief, recurrent attacks of amnesia in middleaged or elderly subjects, often occurring on waking, with favorable response to anticonvulsant medication [1]. While this syndrome is becoming increasingly recognized, its association with transient global amnesia (TGA) is not fully understood. It remains unknown whether TEA is a separate entity from recurrent TGA, or whether TGA can be a cause of TEA. We report a patient initially presenting with TGA and a hippocampal lesion on magnetic resonance imaging (MRI), followed by TEA episodes, suggesting that this damaged region associated with TGA may be an epileptic focus resulting in TEA.

Serum high mobility group box 1 is upregulated in myasthenia gravis

Objective High mobility group box 1 (HMGB1) functions as an inflammatory mediator and is implicated in the pathogenesis of various autoimmune diseases. Our primary aim is to determine whether HMGB1 is involved in the pathogenesis of myasthenia gravis (MG). Methods Serum HMGB1 levels of 60 patients with anti-acetylcholine receptor (AChR) antibody-positive MG without immunosuppressive treatment and of 10 patients with anti-muscle-specific receptor tyrosine kinase (MuSK) antibody-positive MG were compared with those in 40 controls. We also investigated the potential correlation between serum HMGB1 levels and the clinical variables in patients with MG. Results Serum HMGB1 levels in patients with anti-AChR antibody-positive MG were higher than those in controls (7.80±7.47 vs 4.13±2.55 ng/mL, p=0.004) and were decreased after treatment (p=0.051). Although not significant, patients with anti-MuSK antibody-positive MG showed higher serum HMGB1 levels than the controls (p=0.178). There were correlations between serum HMGB1 levels and phenotypes of anti-AChR antibody-positive MG: patients with generalised MG showed higher HMGB1 levels than those of patients with ocular MG (p=0.059) and controls (p=0.002); patients with thymoma showed higher HMGB1 levels than those without thymoma (p=0.094) and controls (p=0.001). Conclusions Serum HMGB1 is elevated in patients with MG and may play a key role in the inflammation of the neuromuscular junction.

Increased serum peroxiredoxin 5 levels in myasthenia gravis.

Extracellular peroxiredoxin 5 (PRX5) is known to be an inflammatory mediator. The serum PRX5 levels of 40 patients with anti-acetylcholine receptor antibody-positive MG and those of 40 controls were measured. PRX5 levels in patients with MG were higher than those in the controls (P=0.045). Thymoma-associated MG patients showed higher PRX5 levels than late-onset MG patients and controls (P<0.05). There were significant associations between the serum PRX5 levels and high mobility group box 1 levels. PRX5 elevation in MG could be related to the neuromuscular junction breakdown and plays a pivotal role in the pathogenic inflammation of MG.

Adequate tacrolimus concentration for myasthenia gravis treatment

A single, oral dose of 3 mg/day tacrolimus, approved for myasthenia gravis (MG) treatment in Japan, was shown to reduce steroid dose and anti‐acetylcholine receptor (AChR) antibody titers as well as to improve MG symptoms. However, no studies have investigated the association between tacrolimus concentration and its clinical efficacy in MG. In this study, we aimed to determine the optimal tacrolimus concentration for MG treatment.

Vertebral body infarction revealed by diffusion-weighted magnetic resonance imaging

Vertebral body infarction (VBI) is infrequent, but evidence of VBI by magnetic resonance imaging (MRI) may serve as an indication of spinal cord ischemic stroke [1]. Diffusion-weighted MRI (DWI) is also useful, especially for early detection of spinal cord infarction (SCI) [2–4]; however, VBI manifestations on DWI have not been reported in detail. We describe a case of SCI accompanied by VBI that was detected and monitored by DWI. A 43-year-old woman noticed she could not move both legs properly the night before hospital examination; her condition worsened over the next 4 h until she could not walk unaided. The patient’s medical history included adultonset Still’s disease, dyslipidemia, hypertension, and allergies to cats and NSAIDs. On examination, we observed hypoalgesia of bilateral T8–T10 dermatomes, loss of proprioception and vibratory sense in lower limbs, and urinary retention. Brain CT was normal and CSF analysis revealed only mild elevation of total protein (48 mg/dl). Serum showed lupus anticoagulant positivity, but other blood tests were normal, including those for antinuclear antibodies, protein C activity, protein S activity, and anti-cardiolipin antibodies. A spinal DWI acquired 12 h after symptom onset revealed a slightly hyperintense lesion in the spinal cord at T6–T8 (Fig. 1a) with low signal on an apparent diffusion coefficient (ADC) map (Fig. 1b). In addition, a T2-weighted MR image revealed a hyperintensity in the dorsocaudal T8 vertebral body (Fig. 1c), which was also hyperintense on T1-weighted MR images and probably corresponded to fat deposition. These scans showed no evidence of aortic dissection. She was tentatively diagnosed with posterior spinal artery infarction and hospitalized. Intravenous steroid (methylprednisolone 1 g/day for 5 days) and antithrombotic therapy (argatroban 60 mg daily for the first 2 days and 20 mg per day for the next 5 days) was started. Despite treatment, she complained of occasional aggravated deep sensation and was assessed by follow-up MRI, 2 (Fig. 1d–f), 5 (Fig. 1g–j), and 18 days (Fig. 1k–m) after symptom onset. On DWI, the spinal cord lesion became more prominent after 2 days, and a new hyperintense lesion was observed in the dorsal T8 vertebral body after 5 days (Fig. 1g, arrow). The vertebral lesion was also high intensity on an ADC map (Fig. 1h, arrow) and on T2 (Fig. 1i, j, arrows), while the T1-weighted MR image revealed an isointensity signal. On T2 axial images, the spinal cord hyperintensity was located in the territory of the posterior spinal arteries (Fig. 1j). The clinical and MRI findings indicating spinal cord infarction made us consider that the new vertebral lesion was caused by the same etiology. After intravenous steroid and antithrombotic therapy, she was able to walk with a cane and was transferred to a rehabilitation center. The utility of DWI for early detection of SCI has been confirmed [2–4], but VBI manifestations on DWI have not been described in detail. VBI typically appears as a hyperintensity region on T2 and as an isoor low-intensity region on T1. The lesion contour may appear as characteristic triangles or take other various shapes, and the lesion is usually located at the same level or slightly below the SCI focus [1, 5–11]. In our case, VBI appeared as a T2 high-intensity and T1 iso-intensity lesion 5 days after symptom onset and was located near the lower end of SCI. VBI also appeared as a high-intensity area on DWI and an ADC map. The lesion had a higher ADC value (0.60 9 10 mm/s) than adjacent M. Kobayashi (&) T. Kanai Department of Neurology, Asahi General Hospital, 1326 I, Asahi, Chiba 289-2511, Japan e-mail: ma-ko@pg7.so-net.ne.jp

Predictive score for oral corticosteroid-induced initial worsening of seropositive generalized myasthenia gravis

BACKGROUND Initial worsening of symptoms after the start of corticosteroid administration is a major concern in the treatment of myasthenia gravis (MG). However, the risk factors or specific patient backgrounds related to this issue have not been fully understood. We aimed to determine the risk factors and developed a scoring system for predicting initial worsening in generalized MG. METHODS We enrolled 62 generalized MG patients with anti-acetylcholine receptor antibody. Initial worsening was defined as an increment of three points in the Quantitative MG score within 2 weeks after the start of steroid treatment. A multivariate logistic regression model was used to determine the risk factors, and predictive scores were assigned. Bootstrap resampling was applied to evaluate the risk score models internal validity. RESULTS Steroid-induced initial worsening occurred in 26% of MG patients and was correlated with thymoma-associated or early-onset MG (p = 0.018), initial prednisolone doses ≥40 mg/day (p = 0.029), and upper limb weakness (p = 0.039). Stepwise multivariate logistic regression identified these three clinical factors for predicting initial worsening in MG. A predictive score of 0-3 points had a bootstrapping area under the curve of 0.770 (0.625-0.878). CONCLUSIONS Our scoring system based on three clinical characteristics can predict the likelihood of steroid-induced initial worsening in MG.

A Novel Fusion Protein, AChR-Fc, Ameliorates Myasthenia Gravis by Neutralizing Antiacetylcholine Receptor Antibodies and Suppressing Acetylcholine Receptor-Reactive B Cells

Most patients with myasthenia gravis (MG) have elevated levels of autoantibodies against the nicotinic acetylcholine receptor (AChR) at the neuromuscular junction, which leads to muscle weakness. We developed a fusion protein, AChR-Fc, as a novel therapeutic biomolecule for patients with MG and examined its efficacy. AChR-Fc was expressed by Chinese hamster ovary cells and purified. We examined the neutralizing activity and cellular cytotoxicity of AChR-Fc using anti-AChR antibody-producing hybridoma cells and serum samples from 16 patients with MG. The effects of AChR-Fc in vivo were also examined using rat MG models. AChR-Fc bound to anti-AChR antibodies and exhibited cytotoxicity against patient-derived antibody-producing B cells. Additionally, a dose-dependent improvement in the clinical signs of disease was observed in a rat MG model. AChR-Fc can diminish signs of MG by neutralizing anti-AChR antibodies and enhancing cytotoxicity against autoantibody-producing B cells. Thus, AChR-Fc can be a novel therapeutic biomolecule for patients with MG.