Kiyokazu Kawabe

Protein and gene analyses of dysferlinopathy in a large group of Japanese muscular dystrophy patients

Mutations in the dysferlin gene cause muscular dystrophies called dysferlinopathy, which include limb-girdle muscular dystrophy type 2B (LGMD2B) and Miyoshi myopathy (MM). To clarify the frequency, clinicopathological and genetic features of dysferlinopathy in Japan, we performed protein and gene analyses of dysferlin. We examined a total of 107 unrelated Japanese patients, including 53 unclassified LGMD, 28 MM and 26 other neuromuscular disorders (ONMD). Expression of dysferlin protein was observed using immunohistochemistry (IHC) and mini-multiplex Western blotting (MMW), and mutation analysis was performed. We found a deficiency of dysferlin protein by both IHC and MMW in 19% of LGMD and 75% of MM patients, and mutations in the dysferlin gene were identified in this group alone. 19% of dysferlin-deficient patients had 3370G-->T missense mutation and 16% had 1939C-->G nonsense mutation. The patients with homozygous 3370G-->T mutation showed milder clinical phenotypes. Twenty-five percent of MM muscles had normal dysferlin protein contents that suggested the genetic heterogeneity of this disease. Altered immunolocalization of dysferlin was observed in not only primary dysferlinopathy, but also in the several diseased muscles with normal protein contents. This result implies the necessity of other protein(s) for proper membrane localization of dysferlin, or some roles of dysferlin in the cytoplasmic region.

Dysferlin mutation analysis in a group of Italian patients with limb‐girdle muscular dystrophy and Miyoshi myopathy

Mutations in the dysferlin gene (DYSF) on chromosome 2p13 cause distinct phenotypes of muscular dystrophy: limb‐girdle muscular dystrophy type 2B (LGMD2B), Miyoshi myopathy (MM), and distal anterior compartment myopathy, which are known by the term ‘dysferlinopathy’. We performed mutation analyses of DYSF in 14 Italian patients from 10 unrelated families with a deficiency of dysferlin protein below 20% of the value in normal controls by immunoblotting analysis. We identified 11 different mutations, including eight missense and three deletion mutations. Nine of them were novel mutations. We also identified a unique 6‐bp insertion polymorphism within the coding region of DYSF in 15% of Italian population, which was not observed in East Asian populations. The correlation between clinical phenotype and the gene mutations was unclear, which suggested the role of additional genetic and epigenetic factors in modifying clinical symptoms.

Cardiovascular Risk and Neuroradiological Profiles in Asymptomatic Vertebrobasilar Dolichoectasia

Background: Clinicoradiological variability of vertebrobasilar dolichoectasia (VBD) is known. Little is known about cardiovascular disease (CVD) risk and neuroradiological profiles of asymptomatic VBD. Methods: A total of 7,345 adults (5,534 men and 1,811 women) underwent physical checkup (PC) and brain magnetic resonance (MR) studies between 2004 and 2007. Asymptomatic VBD was diagnosed by neurological examination and MR angiography. Neuroradiological features were analyzed in VBD subjects. CVD risk factors were compared between VBD subjects and 5,000 controls matched by sex and age. Results: Ninety-six subjects (85 men and 11 women) had asymptomatic VBD. The detection rate was 1.3% and the male/female ratio 2.5. The mean age ± SD was 60.4 ± 10.6 years (60.0 ± 10.2 in men and 64.0 ± 13.1 in women). As compared to controls, the frequency of hypertension, obesity, smoking, dyslipidemia, diabetes mellitus and a family history of stroke or CVD was increased significantly in VBD subjects. The mean diameter ± SD of the basilar artery (BA) was 4.7 ± 0.2 mm. Only 4 subjects (4%) had a severe degree of elongation and lateral displacement of the BA. Contact of the vertebral artery with the rostral ventrolateral medulla (AMC) was found in 81 subjects: right AMC in 22 subjects and left AMC in 59 subjects. Frequency of hypertension was significantly higher in the left-AMC subjects (57%) than in subjects with right AMC (9%) and no AMC (5%). Other neuroradiological findings revealed small infarcts in 42 subjects, brainstem compression in 4, hydrocephalus in 4 and brain saccular aneurysm in 3. Conclusions: Asymptomatic VBD was detected in 1.3% of the Japanese PC group. Our data indicated male predominance, multiple CVD risk factors, neurovascular hypertension and small infarcts in asymptomatic VBD.

Serological Profiles of Urate, Paraoxonase-1, Ferritin and Lipid in Parkinson’s Disease: Changes Linked to Disease Progression

Background: Oxidative stress plays a role in the pathogenesis of neuronal death. Serum levels of urate or lipid were associated with the incidence of Parkinson’s disease (PD). Objective: We compared urate, paraoxonase-1 (PON1), iron, ferritin and lipid in sera of 119 PD patients and 120 healthy controls matched by age, sex and body mass index. We aimed to elucidate whether those serological data are correlated with disease progression. Results: Mean age (SD) of PD patients was 73.4 (8.7) years. Mean Yahr stage (SD) was 3.2 (0.9). Mean disease duration (SD) was 6.9 (5.1) years. Mean dose of L-DOPA (SD) was 355 (157) mg/day. As compared to controls, serum levels of total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), urate and PON1 activity were significantly reduced, and serum ferritin levels were significantly increased in male and female PD patients. Serum urate levels and PON1 activities were inversely related, and serum ferritin levels were correlated with Yahr stage and PD duration in men and women. Serum levels of TC and LDL-C were inversely related to Yahr stage or PD duration in female patients. Conclusions: Our studies indicated serological profiles of urate, PON1, ferritin, TC and LDL-C in PD patients. These serological changes were linked to PD progression. Metabolism of lipid, oxidant- and antioxidant-related substances may contribute to the pathogenesis and the progression of PD.

Expression of Activity-Dependent Neuroprotective Protein in the Immune System: Possible Functions and Relevance to Multiple Sclerosis

Background: Activity-dependent neuroprotector (ADNP) is a neuroprotective molecule containing an 8-amino acid peptide, NAPVSIPQ (NAP), that is sufficient for its neuroprotective effects. Objective: To assess the expression of ADNP in the human immune system in normal subjects and multiple sclerosis patients. MaterialsandMethods: ADNP expression was assessed in peripheral blood mononuclear cells (PBMCs) from healthy donors and multiple sclerosis (MS) patients using staining with anti-ADNP (NAP) antibodies and markers for T cells, B cells, monocytes and natural killer cells. ADNP mRNA was determined in peripheral blood from MS patients (n = 24) and matched controls (n = 21). Expression of activation markers CD69 and CD154 and of IFN-γ was assessed by flow cytometry in stimulated PBMCs. Effects of NAP on immune cell proliferation was assessed by tritiated thymidine incorporation. Results: Monocytes, B cells and T cells, but not regulatory (CD4+CD25+) T cells expressed ADNP. NAP peptide decreased the expression of CD69, CD154 and IFN-γ in PBMC and caused suppressed anti-CD3-/anti-CD28-stimulated PBMC proliferation. ADNP mRNA was reduced in MS compared to control peripheral blood. Conclusion: ADNP is expressed in many immune system cells. ADNP mRNA is reduced in PBMCs in MS. The peptide NAP, which plays an important role in neuroprotection, has potential immunomodulatory properties.

Pulse Wave Velocity Study in Middle-Aged Migraineurs at Low Cardiovascular Disease Risk

(Headache 2011;51:1239‐1244)

Olmesartan, an angiotensin II receptor blocker, restores cerebral hypoperfusion in elderly patients with hypertension.

We evaluated the effects of olmesartan, an angiotensin II receptor blocker (ARB), on cerebral blood flow (CBF) in elderly and hypertensive subjects. Ten subjects with first- or second-degree essential hypertension (mean age, 70.5 years) underwent brain single-photon emission tomography (SPECT) scanning with (99m)Tc-ethyl cysteinate dimer before and after a 24-week course of olmesartan. Mean systolic blood pressure (SBP) was 156.2+/-9.9 mm Hg, and mean diastolic blood pressure (DBP) was 89.1+/-5.5mm Hg. No subject had any abnormalities on neurologic examination or previous history of stroke or cardiovascular disease. Before olmesartan administration, the hypertensive subjects had approximately 15% less whole brain CBF compared with age-matched normotensive controls. Regional CBF was decreased by 11%-20% in the frontal, parietal, temporal, and posterior lobes. Olmesartan treatment significantly decreased SBP to 130.4+/-4.2mm Hg (P < .001) and DBP to 78.2+/-7.0mm Hg (P < .001). After 24 weeks of olmesartan treatment, CBF of whole brain and regional CBF of the frontal, parietal, and temporal lobe were similar to those of control subjects. Our brain SPECT data indicate that olmesartan restores brain hypoperfusion in elderly and hypertensive patients without organic damage. This ARB may have a favorable potential for cerebrovascular circulation, in addition to a blood pressure-lowering effect.

A case of Vernet syndrome with varicella zoster virus infection.

A 40-year-old man was admitted to our department, because of sudden onset of dysphagia, hoarseness, left neck pain and headache. There were no skin lesions. On neurological examination, there were paralysis of the left soft palate and constrictor muscles of the pharynx, weakness of the left sternocleidomastoid and left upper trapezius. In cerebrospinal fluid (CSF) examination, cell count and protein concentration were elevated. Antibody titer to varicella zoster virus (VZV) was elevated in both the serum and CSF. And VZV-DNA was detected by PCR from CSF. Gd enhanced MRI showed the nodular lesion at the left jugular foramen. The diagnosis of Vernets syndrome (VS) associated with VZV infection was made. The patients symptoms were immediately improved with 30 mg of prednisone and 3 g of varaciclovir daily for 14 days. Only a few cases of VS due to VZV have been reported previously. Our case is the first case that detected VZV-DNA in CSF by PCR.

Reciprocal Regulation of Substance P and IL-12/IL-23 and the Associated Cytokines, IFNγ/IL-17: A Perspective on the Relevance of This Interaction to Multiple Sclerosis

The neuropeptide substance P (SP) exhibits cytokine-like properties and exerts different effects in autoimmune inflammation. Various immune cells express SP and its neurokinin-1 receptor (NK1R) isoforms. A role for SP has been demonstrated in a number of autoimmune conditions, including multiple sclerosis (MS). In this work, we studied the role of SP and NK1R in human immune cells with a focus on their relationship with IL-12/IL-23 family cytokines and the associated IFN-γ/IL-17. AIMS: (1) To determine the role of SP mediated effects on induction of various inflammatory cytokines in peripheral blood mononuclear cells (PBMC); (2) to investigate the expression of SP and its receptor in T cells and the effects of stimulation with IL-12 and IL-23. Quantitative real-time PCR, flow cytometry, ELISA, promoter studies on PBMC and primary T cells from healthy volunteers, and Jurkat cell line. Treatment with SP significantly increased the expression of IL-12/IL-23 subunit p40, IL-23 p19 and IL-12 p35 mRNA in human PBMC. Expression of NK1R and SP in T cells was upregulated by IL-23 but a trend was observed with IL-12. The IL-23 effect likely involves IL-17 production that additionally mediates IL-23 effects. Mutual interactions exist with SP enhancing the cytokines IL-23 and IL-12, and SP and NK1R expression being differentially but potentially synergistically regulated by these cytokines. These findings suggest a proinflammatory role for SP in autoimmune inflammation. We propose a model whereby immunocyte derived SP stimulates Th1 and Th17 autoreactive cells migrating to the central nervous system (CNS), enhances their crossing the blood brain barrier and perpetuates inflammation in the CNS by being released from damaged nerves and activating both resident glia and infiltrating immune cells. SP may be a therapeutic target in MS.

Relationship between cervical cord 1H-magnetic resonance spectroscopy and clinoco-electromyographic profile in amyotrophic lateral sclerosis

Introduction: Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the loss of motor neurons, leading to limb paralysis and respiratory failure. Methods: C1–C3 cord 1H‐magnetic resonance spectroscopy (1H‐MRS) was performed in 19 patients with ALS and 20 controls. N‐acetylaspartate (NAA), choline‐containing compounds, creatine plus phosphocreatine (Cr), and myo‐Inositol (m‐Ins) were measured. ALS Functional Rating Scale‐Revised (ALSFRS) and forced vital capacity (FVC) were assessed. The rates of decline were calculated at 6 months before and after 1H‐MRS. Results: NAA/Cr and NAA/m‐Ins were decreased significantly, and m‐Ins/Cr was increased significantly in ALS patients compared with controls. NAA/Cr and NAA/m‐Ins were correlated with ALSFRS and FVC and inversely linked to the decline rates. NAA/Cr, NAA/m‐Ins, and m‐Ins/Cr were altered markedly in 9 patients with denervation and neurogenic changes in both C2 paraspinal and upper limb muscles. Conclusions: These metabolite ratios were associated with disease progression and ongoing denervation in neck and hand muscles. C1–C3 cord 1H‐MRS might reflect anterior horn cell damage causing neck/arm weakness and respiratory dysfunction in ALS patients. Muscle Nerve, 2013