Kanji Yamamoto

Effective restoration of dystrophin-associated proteins in vivo by adenovirus-mediated transfer of truncated dystrophin cDNAs

A series of truncated dystrophin cDNAs (3.1–4.2 kbp) containing only three, three, two or one rod repeats with hinge 1 and 4 (named ΔDysAX2, AX11, AH3, M3, respectively) or no rod repeat retaining either hinge 1 or 4 (named ΔDysH1, H4, respectively) were constructed. These cDNAs were introduced into skeletal muscle of adult mdx mice using the adenovirus vector with a strong CAG promoter. ΔDysAX2, AX11, AH3 and ΔDysM3 expressed themselves successfully and recovered dystrophin‐associated proteins effectively. Especially 3.7 kbp cDNA for ΔDysM3 offers the possibility of an approach utilizing newly developed virus vectors, such as an adeno‐associated virus vector, toward gene therapy of Duchenne muscular dystrophy.

Hemiballism with striatal hyperintensity on T1-weighted MRI in diabetic patients: A unique syndrome

We report 3 diabetic patients who developed hemiballism without involvement of the subthalamic nucleus. Each patient exhibited vigorous, flinging, ballistic involuntary movements in the extremities and slight facial grimacing involving one side of the body. Although diabetes was poorly controlled in all 3, each patient was nonketotic at the onset of hemiballism. Magnetic resonance imaging (MRI), in these patients showed abnormalities in the striatum contralateral to the hemiballism that were characterized by an increase in intensity on T1-weighted images and a slight decrease in intensity on T2-weighted images, and these changes persisted for more than 2 months. The striatal lesions are presumed to have developed following mild ischemia in the territory of the lateral striate branches of the middle cerebral artery. This combination of hemiballism and striatal lesions in diabetic patients may constitute a unique syndrome.

Quantitative evaluation of expression of iron-metabolism genes in ceruloplasmin-deficient mice.

Aceruloplasminemia is an autosomal recessive disorder caused by mutations in the ceruloplasmin (CP) gene, and is characterized by a unique combination of neurovisceral iron overload and iron deficiency anemia. We generated CP-deficient (CP(-/-)) mice to investigate the functional involvement of CP in iron metabolism. The mice showed a marked iron overload in the liver and mild iron deficiency anemia. We examined the expression of iron-metabolism genes in the duodenum and liver using TaqMan RT-PCR. The divalent metal transporter 1 (DMT1), ferroportin 1 (FPN1), and hephaestin (HEPH) genes were not up-regulated in the duodenum from CP(-/-) mice. These data suggest that the mechanism of hepatic iron overload in aceruloplasminemia is quite different from that in hemochromatoses and atransferrinemia. In the liver, CP(-/-) mice showed no increase of gene expression for DMT1 and transferrin receptors (TFR and TFR2), indicating that none of the known pathways of iron uptake is activated in hepatocytes of CP(-/-) mice. This result supports the hypothesis that CP mainly acts to release iron from cells in the liver.

Feasibility of auxiliary partial orthotopic liver transplantation from living donors for patients with adult-onset type II citrullinemia

More than 20 patients with adult‐onset type II citrullinemia have undergone liver transplantation, showing dramatic therapeutic effects. In Japan, living donor liver transplantation is the standard technique of liver transplantation because of the rare availability of cadaveric donors. The feasibility of auxiliary partial orthotopic liver transplantation (APOLT) for adult‐onset type II citrullinemia to overcome the problem of a small‐for‐size graft in living donor liver transplantation has not been defined. We recently performed APOLT for patients with type II citrullinemia. Here, we present 2 patients: patient 1 was a 32‐year‐old man and patient 2 was a 43‐year‐old woman. Both patients suffered from hepatic encephalopathy, and laboratory data showed highly elevated plasma levels of ammonia and citrulline. In patient 1, the liver graft was obtained from a patient with familial amyloid polyneuropathy as a domino liver transplant. In patient 2, APOLT was performed after graft donation from her husband. The postoperative clinical courses of both patients were uneventful, and the neurological symptoms were completely resolved. The plasma concentrations of ammonia and citrulline normalized rapidly in both patients. APOLT can provide an adequate hepatocyte mass to correct the underlying enzyme deficiency in adult patients with type II citrullinemia. In addition, APOLT can be carried out safely to overcome the limitation of graft volume in living donor liver transplantation. (Liver Transpl 2004;10:550–554.)

Effective adenovirus-mediated gene expression in adult murine skeletal muscle

We established an efficient method for obtaining expression of a foreign marker gene transferred in vitro into myoblasts and in vivo into adult mouse skeletal muscles using adenovirus vector. After infection of the C2 myoblasts with the adenovirus vector containing the β‐actin promoter with cytomegalovirus (CMV) enhancer (CAG promoter) AxCALacZ, significantly greater number of cells express β‐galactosidase when compared with the adenovirus vector expressing the lacZ gene under the control of the SRα viral terminal repeat promoter (AxSRLacZL) or the myosin heavy chain (MHC) IIB promoter (AxMHCLacZ). We also injected AxCALacZ into the skeletal muscles of 5‐ to 6‐week‐old C57BL/10 mice and determined that more than 60% of their muscle fibers expressed the lacZ gene 7 days after injection. The CAG promoter may have application in the development of gene therapy for Duchenne muscular dystrophy (DMD) using adenovirus vector.

Immune response to adenovirus-delivered antigens upregulates utrophin and results in mitigation of muscle pathology in mdx mice.

The upregulation of endogenous utrophin in skeletal muscle may lead to a new approach to the treatment of Duchenne muscular dystrophy (DMD). We found that injection of an E1, E3-deleted adenovirus vector expressing beta-galactosidase (beta-Gal) or green fluorescent protein (GFP) into the skeletal muscle of neonatal dystrophin-deficient mdx mice alleviated dystrophic pathology. In the adenovirus-infected muscles, an evaluation of sarcolemma stability showed low permeability and immunohistochemistry revealed utrophin upregulation at the extrasynaptic sarcolemma of mature muscle fibers. This utrophin upregulation was concomitant with endomysial cellular infiltration from a host immune reaction. There was no evidence of active muscle regeneration. In normal C57BL/10 mice, utrophin was also upregulated in adenovirus-injected skeletal muscles, where upregulated utrophin often coexisted with dystrophin. FK506 and anti-CD4 antibody administration decreased utrophin expression in adenovirus-injected mdx muscles and prevented the dystrophic phenotype from being mitigated, suggesting that an immune reaction is involved in utrophin upregulation. This is the first report demonstrating the improvement of the dystrophic phenotype as a result of the acquired overexpression of endogenous utrophin. Our findings provide an important clue to understanding the mechanism of utrophin expression and the development of an effective treatment for DMD.

Sarcoidosis with high serum levels of vascular endothelial growth factor (VEGF), showing RS3PE-like symptoms in extremities

We report a patient with sarcoidosis who showed edema in the distal portion of all extremities, particularly the legs, as seen in remitting seronegative symmetrical synovitis with pitting edema (RS3PE). Magnetic resonance imaging demonstrated diffuse abnormal intensity in subcutaneous tissues of both legs, and skin biopsy led to a diagnosis of sarcoidosis. Vascular endothelial growth factor (VEGF) showed a high serum level, which decreased soon after starting oral prednisolone, in parallel with an improvement in the limb edema. In this patient VEGF as well as infiltration by sarcoid granuloma in the skin might have played an important role in the pathogenesis of RS3PE-like symptoms in the extremities. When painful pitting edema is seen predominantly in the distal portion of all extremities, sarcoidosis as well as RS3PE should be considered as a possible diagnosis.

Rituximab therapy in chronic inflammatory demyelinating polyradiculoneuropathy with anti-SGPG IgM antibody.

We report a patient with chronic inflammatory demyelinating polyradiculoneuropathy (CIDP) who showed high titers of anti-sulfated glucuronyl paragloboside (SGPG) IgM antibody without M-protein in serum. The patient was resistant to corticosteroids and immunosuppressants, but after administration of rituximab, clinical symptoms improved and the patient remained in a stable state for approximately 10 months. Rituximab may be a potent therapeutic option for refractory cases of CIDP irrespective of detectable M-protein in either serum or urine.

Efficacy of tacrolimus in treatment of polymyositis associated with myasthenia gravis

We report a patient with polymyositis (PM) associated with myasthenia gravis (MG). Both disorders had been controlled for around 15 years by oral prednisolone and a cholinesterase inhibitor following surgical removal of invasive thymoma and radiotherapy, but muscular weakness due to myalgia and an increase in serum levels of myogenic enzymes, mainly ascribable to the recurrence of PM, reappeared immediately after cessation of these drugs, which was done because the patient had multiple bone fractures and severe osteoporosis due to the long-term corticosteroid therapy. Oral tacrolimus was therefore tried, and produced an improvement in muscular symptoms in association with normalization of myogenic enzymes. PM associated with MG as in this patient might be the best indication for tacrolimus, considering its efficacy in MG, but this drug should also be actively considered as a therapeutic option in refractory cases of PM alone, particularly when either corticosteroids or other immunosuppressive agents are not usable.

Chronic inflammatory demyelinating polyradiculoneuropathy with autonomic involvement

We report a case of chronic acquired neuropathy predominantly affecting sensory and autonomic nerves. Investigations showed a demyelinating polyradiculoneuropathy with axonal degeneration and depletion of postganglionic noradrenergic fibers in the rectal mucosa. Intravenous immunoglobulin and corticosteroid administration were effective in alleviating symptoms and improving electrophysiological abnormalities. This neuropathy may be a novel variant of chronic inflammatory demyelinating polyradiculoneuropathy (CIDP), in which autoimmunoreactivity is directed not only against myelin but also against axon‐ or ganglion‐composing protein. Autonomic nerve involvement does not exclude a diagnosis of CIDP. Muscle Nerve 2004