Noritoshi Shibuya

CDR3 Spectratyping Analysis of the TCR Repertoire in Myasthenia Gravis

Because myasthenia gravis (MG) is an autoimmune disease mediated by Abs specific for the acetylcholine receptor, helper T cells play a role in Ab production. In this study, we have performed large-scale cross-sectional and longitudinal TCR studies by CDR3 spectratyping using PBL and thymus tissues from MG patients. We found that there was no preferential usage of any particular TCR β-chains that was identical among MG patients. However, the longitudinal study clearly demonstrated that one or more TCR Vβ expansions persisted frequently in MG patients. Importantly, persistent TCR expansions correlated with clinical severity and high anti-acetylcholine receptor Ab titer. Finally, examinations of T cells expressing CXCR5, i.e., follicular B-helper T cells, revealed that spectratype expansions in MG patients were detected mainly in the CD4+ CXCR5+ T cell populations, whereas CD8+ T cells were the major source of clonal expansion in healthy subjects. These findings suggest that persistent clonal expansions of T cells in MG patients are associated with the development and maintenance of MG. Close examination of pathogenic T cells in MG provides useful information to elucidate the pathogenesis and to estimate the disease status.

Serum factor blocks neuromuscular transmission in myasthenia gravis Electrophysiologic study with intracellular microelectrodes

We studied the effects of normal and myasthenic sera on the miniature endplate potential (MEPP) and resting membrane potential (RP) of rat muscle in vitro by conventional intracellular microelectrode techniques. Normal sera had little or no effect on either the amplitude or frequency of MEPP or RP. On the other hand, MEPP amplitude was reduced in each of nine muscles during exposure to myasthenic sera; five of these muscles showed a significant difference, by students t-test, from the values in a control solution. The half decay time of diminished MEPP remained unchanged. MEPP frequency and RP were not affected by myasthenic sera. The reduced amplitude of the MEPP was almost completely restored when the muscle was washed with a control solution for more than 30 minutes. These observations indicate that myasthenic sera contain factors that bind reversibly with the acetylcholine receptor and reduce postsynaptic responses to acetylcholine.

Thymic myoid cells as a myasthenogenic antigen and antigen-presenting cells

We investigated immune property of a myoid cell line, established from Fisher rat thymus. Immunization of syngeneic rats with the myoid cells induced anti-rat acetylcholine receptor (AChR). Implantation of them into the thymus failed to induce typical thymic pathology of human myasthenia gravis (MG) or anti-AChR responses. We also demonstrated that the myoid cells were able to present exogenous antigens to T cells and induce antigen-specific T cell proliferation. These results suggest that myoid cells have the potential antigenicity to induce anti-AChR and the functions of antigen-presenting cells, but their expansion in the thymus may not directly cause MG.

Cytapheresis with a filter for selective removal of CD4+ T cells in experimental autoimmune encephalomyelitis

Experimental autoimmune encephalomyelitis (EAE) is a major animal model of human multiple sclerosis (MS). CD4+ T cells are thought to play a pivotal role in the patho genesis of EAE and MS. In order to investigate the depletio n of CD4+ T cells from the systemic circulation as an effective strategy for the treatment of MS, we performed extracorporeal CD4+ T cell adsorption, using a filter to which anti-CD4+ antibody is immobilized as a ligand, in adoptively transferred EAE. Rats treated with CD4+ T cell removal filter (C D4RF) exhibited milder clinical signs of EAE and earlier recovery than those receiving sham treatment. Moreover, the thymic cells from EAE rats treated with C D4RF exhibited a suppressed proliferative response and IFN-g production to myelin basic protein. These results suggest that depletion of CD4+ T cells from the systemic circulation by extracorporeal treatment is a potentially useful strategy for treatment of acute phase and relapsing MS.

Development of a Device for Selective Removal of CD4+ T Cells

Abstract: To control antigen (Ag)‐specific immune cells is important in the treatment of autoimmune diseases. In particular, controlling the immune response of autoimmune T cells is effective in the treatment of these diseases. The development of a device that can remove CD4+ T cells specifically by extracorporeal circulation is now in progress, with the aim to deplete autoimmune T cells. We developed a removal material made of polypropylene non‐woven fabrics with anti human CD4 monoclonal antibody immobilized on the surface. Using a column packed with the removal material, we succeeded in removing CD4 + T cells specifically from peripheral whole blood by direct perfusion. Moreover, CD4+ T cells can be specifically removed even from blood with lower surface antigen density by in vitro activation.

Paramyotonia congenita due to a de novo mutation: A case report

A Japanese man with a negative family history of paramyotonia congenita (PMC) was evaluated for symptoms of cold‐induced weakness and stiffness. Exercise testing revealed findings characteristic of PMC, and a genetic analysis was therefore performed. A well‐known sodium channel mutation for PMC (T1313M) was identified in the patient, but was absent in his biological parents. These data demonstrate the occurrence of a de novo mutation, suggesting that evaluation for PMC should be performed in patients with typical symptoms even if the family history is negative. Muscle Nerve 28: 232–235, 2003

Selective Adsorption of Human CD4+ T Cells

Abstract:  The pathogenesis of most autoimmune diseases directly involves CD4+ helper T cells. To remove CD4+ T cells selectively from the circulation, we designed a new column in which an anti‐CD4 monoclonal antibody was immobilized on the activated substance. Nearly 90% of CD4+ T cells were selectively adsorbed from whole blood with a single passage through the column in vitro, resulting in depletion of the antigen‐specific T cell responses. We conclude that this new column would be potentially useful for treatment of T cell‐mediated autoimmune diseases.

Effect of incomplete ligation of peripheral nerves on neuromuscular transmission

Abstract Myasthenia-like neuromuscular fatigue has been successfully produced in the gastrocnemius muscle of the rat after incomplete ligation of the sciatic nerve. Electrophysiological characteristics of this experimental model of myasthenia were: (1) waning of the amplitude of the evoked muscle potential on repetitive nerve stimulation even at fairly low frequencies, (2) the existence of post-tetanic exhaustion, (3) a positive edrophonium test, and (4) changes that were similar to those of ACh resynthesis block after hemicholinium administration. It can be anticipated from the appearance of the myasthenic phenomenon that the lower motor neuron exerts an influence over mechanisms of synaptic transmission at neuromuscular junctions. This suggests the possibility that some abnormality of the lower motor neuron may play a role in producing the neuromuscular transmission failure in myasthenia gravis.

Effects of long-term administration of ambenonium chloride on motor end-plate fine structure and acetylcholine receptor in rat

Ambenonium chloride was administered orally in a dosage of 6 mg/kg/day to rats for 14-360 days. Motor end-plate fine structure and junctional AChR were quantitatively analyzed and red (soleus) and white (EDL) muscle fibers. In treated animals, degeneration and simplification of postsynaptic folds and widening of synaptic clefts were often observed in soleus end-plates, but infrequently in EDL end-plates. On the other hand, the postsynaptic AChR was reduced markedly in both soleus and EDL end-plates. No presynaptic changes were observed. These results show that long-term administration of Anti-ChE agents in myasthenia gravis may have an adverse effect on neuromuscular transmission.

MICROELECTROPHYSIOLOGIC STUDIES ON THE NATURE OF NEUROMUSCULAR TRANSMISSION BLOCK CAUSED BY PERIPHERAL NERVE INJURY

Neuromuscular transmission block due to an injury of the peripheral nerve appears in the early stages of reinnervation. In microelectrode methods, miniature end-plate potentials (MEPPs) were of normal amplitude but of decreased frequency. The quantum content of the end-plate potential (EPP) decreased significantly, while the quantum size of the EPP was within normal limit. The time course of the EPPs was remarkably prolonged despite the normal time course of MEPPs. Abnormal end-plate responses returned to normal after day 40. Neuromuscular transmission block in the early stages of reinnervation could best be explained by a failure of impulse to invade the axon terminal and by the decreased number of acetylcholine (ACh) quanta readily available for release from the nerve terminals. It is likely that the metabolism of ACh and calcium ions is not appropriate in immature nerve terminals.